U.S. patent application number 11/238171 was filed with the patent office on 2006-05-18 for soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases.
This patent application is currently assigned to Halozyme, Inc.. Invention is credited to Louis H. Bookbinder, Tyler M. Dylan, Gregory I. Frost, Michael F. Haller, Gilbert A. Keller, Anirban Kundu.
Application Number | 20060104968 11/238171 |
Document ID | / |
Family ID | 36927767 |
Filed Date | 2006-05-18 |
United States Patent
Application |
20060104968 |
Kind Code |
A1 |
Bookbinder; Louis H. ; et
al. |
May 18, 2006 |
Soluble glycosaminoglycanases and methods of preparing and using
soluble glycosaminogly ycanases
Abstract
The invention relates to the discovery of novel soluble neutral
active Hyaluronidase Glycoproteins (sHASEGPs), methods of
manufacture, and their use to facilitate administration of other
molecules or to alleviate glycosaminoglycan associated pathologies.
Minimally active polypeptide domains of the soluble, neutral active
sHASEGP domains are described that include asparagine-linked sugar
moieties required for a functional neutral active hyaluronidase
domain. Included are modified amino-terminal leader peptides that
enhance secretion of sHASEGP. The invention further comprises
sialated and pegylated forms of a recombinant sHASEGP to enhance
stability and serum pharmacokinetics over naturally occurring
slaughterhouse enzymes. Further described are suitable formulations
of a substantially purified recombinant sHASEGP glycoprotein
derived from a eukaryotic cell that generate the proper
glycosylation required for its optimal activity.
Inventors: |
Bookbinder; Louis H.; (San
Diego, CA) ; Kundu; Anirban; (San Diego, CA) ;
Frost; Gregory I.; (Del Mar, CA) ; Haller; Michael
F.; (San Diego, CA) ; Keller; Gilbert A.;
(Belmont, CA) ; Dylan; Tyler M.; (San Diego,
CA) |
Correspondence
Address: |
DLA PIPER RUDNICK GRAY CARY US, LLP
4365 EXECUTIVE DRIVE
SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
Halozyme, Inc.
San Diego
CA
|
Family ID: |
36927767 |
Appl. No.: |
11/238171 |
Filed: |
September 27, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11065716 |
Feb 23, 2005 |
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11238171 |
Sep 27, 2005 |
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10795095 |
Mar 5, 2004 |
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11065716 |
Feb 23, 2005 |
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60452360 |
Mar 5, 2003 |
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Current U.S.
Class: |
424/94.61 ;
435/200 |
Current CPC
Class: |
A61P 25/00 20180101;
C12N 9/2408 20130101; Y02A 50/466 20180101; A61K 35/768 20130101;
Y02A 50/30 20180101; A61P 29/00 20180101; A61P 9/04 20180101; A61K
38/47 20130101; A61P 43/00 20180101; A61K 38/465 20130101; A61P
9/14 20180101; A61P 27/02 20180101; A61P 35/00 20180101; C12N
9/2474 20130101; A61K 47/60 20170801; C12N 7/00 20130101; A61K
39/395 20130101; A61K 39/3955 20130101; A61K 47/6811 20170801; A61P
9/00 20180101; A61P 17/02 20180101; A61K 2039/505 20130101; A61P
9/10 20180101; A61K 39/3955 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/094.61 ;
435/200 |
International
Class: |
A61K 38/47 20060101
A61K038/47; C12N 9/24 20060101 C12N009/24 |
Claims
1. A substantially purified glycoprotein, comprising a neutral
active soluble hyaluronidase polypeptide and at least one N-linked
sugar moiety, wherein the N-linked sugar moiety is covalently
attached to an asparagine residue of the polypeptide.
2. The polypeptide of claim 1, wherein the polypeptide is selected
from of: (a) a polypeptide that comprises a sequence of amino acids
encoded by the sequence that includes at least about 74% amino acid
sequence identity with the sequence of amino acids set forth in SEQ
ID NO. 1; (b) a polypeptide that comprises a sequence of amino
acids encoded by the sequence of nucleotides set forth in SEQ ID
NO. 6; (c) a polypeptide that comprises a sequence of amino acids
encoded by a sequence of nucleotides that hybridizes along at least
85% of its full-length under conditions of high stringency to the
sequence of nucleotides set forth in SEQ ID NO. 6; or (d) a
polypeptide that is encoded by a sequence of nucleotides that is a
splice variant of the sequence of nucleotides that comprises the
sequence set forth in SEQ ID NO. 50.
3. The polypeptide of claim 1, wherein said sugar moiety is
covalently attached to an asparagine residue selected from amino
acids 82, 166, 235, 254, 368, 393 or 490 as set forth in SEQ ID NO.
1.
4. The polypeptide of claim 1, wherein said sugar moiety is
covalently linked to said polypeptide through a PNGase sensitive
bond.
5. The polypeptide of claim 1, wherein said sugar moiety is of the
high mannose type.
6. The polypeptide of claim 1, wherein said sugar moiety is of the
complex type.
7. The polypeptide of claim 1, wherein said sugar moiety is a
hybrid type.
8. The polypeptide of claim 1, wherein said sugar moiety further
comprises at least one terminated with sialic acid.
9. The polypeptide of claim 1, wherein the polypeptide consists
essentially of the hyaluronidase domain of human hyaluronidase
glycoprotein (sHASEGP) or a catalytically active portion
thereof.
10. The polypeptide of claim 1, wherein the hyaluronidase portion
of the polypeptide comprises of the hyaluronidase domain of sHASEGP
or a catalytically active portion thereof.
11. The polypeptide of claim 1, wherein the polypeptide is modified
with a polymer.
12. The polypeptide of claim 11, wherein the polymer is PEG or
dextran.
13. A polypeptide, consisting essentially of a hyaluronidase domain
or a catalytically active portion thereof of a human hyaluronidase
protein.
14. The polypeptide of claim 13, wherein the hyaluronidase is
super-sialated.
15. The polypeptide of claim 13, wherein the hyaluronidase domain
comprises the sequence of amino acids set forth as amino acids
1-450 of SEQ ID NO. 3.
16. The polypeptide of claim 13, wherein the hyaluronidase domain
comprises the sequence of amino acids set forth as amino acids
1-438 of SEQ ID NO. 3.
17. The polypeptide of claim 13, wherein the hyaluronidase domain
comprises the sequence of amino acids set forth as amino acids
1-459 of SEQ ID NO. 3.
18. The polypeptide of claim 13, wherein the polypeptide is
modified with a polymer.
19. The polypeptide of claim 18, wherein the polymer is PEG or
dextran.
20. (canceled)
21. (canceled)
22. The polypeptide of claim 1, wherein the sHASEGP is a human
polypeptide.
23. The polypeptide of claim 1, wherein the polypeptide is a
soluble polypeptide as set forth in SEQ ID NO.4.
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. The polypeptide of claim 1, wherein a signal sequence capable
of directing the polypeptide outside the cell is operably attached
to the sHASEGP polypeptide.
31. An isolated soluble human hyaluronidase glycoprotein (sHASEGP),
wherein the potency of the sHASEGP is greater than 40,000 USP
Units/mg protein.
32. The sHASEGP of claim 31, wherein the hyaluronidase is
sialated.
33. (canceled)
34. (canceled)
35. The sHASEGP of claim 31, wherein the polypeptide is modified
with a polymer.
36. The sHASEGP of claim 35, wherein the polymer is PEG or
dextran.
37. (canceled)
38. A nucleic acid molecule, encoding the polypeptide of claim
1.
39. A nucleic acid molecule, comprising a sequence selected from:
(a) a sequence as set forth in SEQ ID NO.6; (b) a sequence that
hybridizes under high stringency to at least about 70% of the
full-length to the sequence of nucleotides set forth in SEQ ID NO.
6; (c) a sequence of nucleotides that encodes the polypeptide set
forth in SEQ ID No. 1, 3, 4, 5 and 50; (d) a sequence that is a
splice variant of (a), (b) or (c); (e) a sequence that encodes the
hyaluronidase domain or a catalytically active portion thereof that
includes a sequence of nucleotides having at least about 60%
sequence identity with the sequence set forth in SEQ ID No. 6 or
50; and (f) a sequence comprising degenerate codons of (a), (b),
(c), (d) or (e).
40. A vector having a sequence as set forth in SEQ ID NO: 51.
41. A vector comprising the nucleic acid molecule of claim 38.
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. An isolated cell, comprising the vector of claim 41.
48. (canceled)
49. (canceled)
50. (canceled)
51. The cell of claim 47 that is a mammalian cell.
52. An antibody that binds to the sHASEGP of claim 1, wherein said
antibody is free of reactivity to bovine, ovine or bacterial
hyaluronidase.
53. A transgenic non-human animal, wherein an endogenous gene that
encodes a polypeptide of claim 1 has been disrupted by homologous
recombination or insertional mutagenesis of the animal or an
ancestor thereof.
54. A method for producing a soluble polypeptide that contains
sHASEGP, comprising: introducing a nucleic acid as set forth in SEQ
ID NO. 6 operably linked to a promoter into a cell that
incorporates N-linked sugar moieties into sHASEGP; culturing the
cell under conditions whereby the encoded polypeptide is expressed
by the cell; and recovering the expressed polypeptide.
55. A method for producing a soluble polypeptide that contains
sHASEGP, comprising: introducing a nucleic acid that encodes the
polypeptide of claim 1 operably linked to a suitable promoter into
a cell capable of incorporating N-linked sugar moieties into
sHASEGP; culturing the cell under conditions whereby the encoded
polypeptide is expressed by the cell; and recovering the expressed
polypeptide.
56. (canceled)
57. (canceled)
58. (canceled)
59. A method for producing a soluble polypeptide that contains
sHASEGP, comprising: culturing the cell of claim 47 under
conditions whereby the encoded polypeptide is expressed by the
cell; and recovering the expressed polypeptide.
60. A method of ex vivo gene therapy, comprising: introducing, in
vitro, a nucleic acid encoding the polypeptide of claim 1 and
operably linked to a suitable promoter into a cell capable of
incorporating N-linked sugar moieties into sHASEGP; thereby
generating a genetically modified cell containing the nucleic acid;
and administering the cell comprising the nucleic acid to the
subject, whereby the nucleic acid encoding the sHASEGP is
transferred to the subject.
61. (canceled)
62. (canceled)
63. A method of preparing a mammalian oocyte for in vitro
fertilization said method comprising: contacting an oocyte with a
sHASEGP of claim 1 in amount effective for removing the cumulus
matrix.
64. The method of claim 62, wherein the cumulus is effectively
removed without manipulation of the oocyte with a narrow bore
pipette.
65. A method for generating a sHASEGP polypeptide comprising:
contacting the polypeptide of claim 1 with glycosyltransferase
enzymes capable of introducing said N-linked sugar moieties into
polypeptide, thereby generating sHASEGP.
66. (canceled)
67. A composition, comprising a substantially purified sHASEGP
glycoprotein polypeptide and a suitable pharmaceutical carrier.
68. A composition, comprising the polypeptide of claim 1 and a
suitable pharmaceutical carrier.
69. A method for treating a subject having an excess of sHASEGP
substrate, comprising: administering an amount of sHASEGP
polypeptide of claim 1 sufficient to remove said sHASEGP
substrate.
70. The method of claim 69, wherein said excess substrate is
produced from a scar tissue.
71. The method of claim 70, wherein said scar tissue is a glial
scar resulting from spinal cord injury.
72. (canceled)
73. The method of claim 70, wherein said scar tissue is a keloid
scar.
74. (canceled)
75. A method for increasing the diffusion of a therapeutic
substance or other molecule or macromolecular complex (each an
Agent) smaller than about 0.5 microns in diameter in a subject,
comprising: administering to a subject a sHASEGP polypeptide in an
amount sufficient to open or to form channels smaller than about
0.5 microns in diameter and an Agent, whereby the diffusion of the
Agent is increased.
76. The method of claim 75, wherein said sHASEGP polypeptide is
injected at a dose between 0.1 and 1500 Units.
77. The method of claim 75 wherein said sHASEGP polypeptide is
mixed with the therapeutic substance or other Agent prior to
injection.
78. A kit, comprising: (a) a sHASEGP polypeptide at a dose between
0.1 and 1500 Units/ml in an acceptable carrier; (b) at least one
therapeutic substance or other molecule or macromolecular complex
(each an Agent) in an acceptable carrier; and (c) optionally,
instructions for delivering therapeutic substances or other
Agents.
79. The kit of claim 78, wherein the sHASEGP polypeptide and the
therapeutic substance or other Agent are provided as a mixture.
80. A method of treating a cardiovascular disorder comprising:
administration of a sHASEGP polypeptide of claim 1 to a subject in
an amount sufficient to remove excess glycosaminoglycans.
81. The method of claim 80, wherein said cardiovascular disorder is
selected from a myocardial infarct, congestive heart failure or
arteriosclerosis.
82. A method of treating a tumor comprising: administration of a
sHASEGP polypeptide of claim 1 to a subject in an amount sufficient
to remove excess glycosaminoglycans.
83. A method of delivering a molecule to a tissue containing excess
amounts of glycosaminoglycan, comprising: administering a sHASEGP
polypeptide of claim 1 to a tissue in an amount sufficient to
degrade glycosaminoglycans sufficiently to open channels less than
about 500 nm in diameter; and administering a molecule to the
tissue comprising the degraded glycosaminoglycans.
84. The method of claim 83, wherein the polypeptide is modified
with a polymer.
85. The method of claim 84, wherein the polymer is PEG or
dextran.
86. The method of claim 83, wherein the sHASEGP polypeptide is
administered prior to, simultaneously with or following
administration of the molecule.
87. (canceled)
88. (canceled)
89. A pharmaceutical composition, comprising, substantially
purified soluble hyaluronidase glycoprotein (sHASEGP) polypeptide
of claim 1 and a pharmaceutical carrier.
90. A pharmaceutical composition, comprising the substantially
purified sHASEGP polypeptide of claim 1.
91. The pharmaceutical composition of claim 89, further comprising
a pharmaceutically active or pharmacologic agent.
92. The pharmaceutical composition of claim 91, wherein the
pharmaceutically active or pharmacologic agent is selected from the
group of agents consisting of a chemotherapeutic agent, an
analgesic agent, an anti-inflammatory agent, an antimicrobial
agent, an amoebicidal agent, a trichomonocidal agent, an
anti-parkinson agent, an anti-malarial agent, an anticonvulsant
agent, an anti-depressant agent, an antiarthritics agent, an
anti-fungal agent, an antihypertensive agent, an antipyretic agent,
an anti-parasitic agent, an antihistamine agent, an
alpha-adrenargic agonist agent, an alpha blocker agent, an
anesthetic agent, a bronchial dilator agent, a biocide agent, a
bactericide agent, a bacteriostatic agent, a beta adrenergic
blocker agent, a calcium channel blocker agent, a cardiovascular
drug agent, a contraceptive agent, a decongestant agent, a diuretic
agent, a depressant agent, a diagnostic agent, an electrolyte
agent, a hypnotic agent, a hormone a gent, a hyperglycemic agent, a
muscle relaxant agent, a muscle contractant agent, an ophthalmic
agent, a parasympathomimetic agent, a psychic energizer agent, a
sedative agent, a sleep inducer, a sympathomimetic agent, a
tranquilizer agent, a urinary agent, a vaginal agent, a viricide
agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an
angiotensin converting enzyme inhibitor agent, a polypeptide, a
protein, a nucleic acid, a drug, or an organic molecule.
93. (canceled)
94. (canceled)
95. (canceled)
96. (canceled)
97. (canceled)
98. (canceled)
99. (canceled)
100. (canceled)
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107. (canceled)
108. (canceled)
109. (canceled)
110. (canceled)
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116. (canceled)
117. (canceled)
118. (canceled)
119. (canceled)
120. (canceled)
121. (canceled)
122. (canceled)
123. A combination comprising, a sterile vial and the
pharmaceutical composition of claim 89 wherein the composition is
contained in the vial.
124. (canceled)
125. (canceled)
126. (canceled)
127. A kit comprising the combination of claim 123 and at least one
of: (a) a packaging material; or (b) instructions for using the kit
for use of the pharmaceutical composition.
128. (canceled)
129. A combination comprising, a sterile syringe containing the
pharmaceutical composition of claim 89.
130. (canceled)
131. (canceled)
132. A combination comprising, a sterile syringe containing the
pharmaceutical composition of claim 110.
133. (canceled)
134. (canceled)
135. The combination of claim 132, further comprising a second
syringe containing a pharmaceutically effective agent.
136. The combination of claim 132, wherein the pharmaceutically
effective or pharmacologic agent is selected from a
chemotherapeutic agent, an analgesic agent, an anti-inflammatory
agent, an antimicrobial agent, an amoebicidal agent, a
trichomonocidal agent, an anti-parkinson agent, an anti-malarial
agent, an anticonvulsant agent, an anti-depressant agent, an
antiarthritics agent, an anti-fungal agent, an antihypertensive
agent, an antipyretic agent, an anti-parasitic agent, an
antihistamine agent, an alpha-adrenargic agonist agent, an alpha
blocker agent, an anesthetic agent, a bronchial dilator agent, a
biocide agent, a bactericide agent, a bacteriostatic agent, a beta
adrenergic blocker agent, a calcium channel blocker agent, a
cardiovascular drug agent, a contraceptive agent, a decongestant
agent, a diuretic agent, a depressant agent, a diagnostic agent, an
electrolyte agent, a hypnotic agent, a hormone agent, a
hyperglycemic agent, a muscle relaxant agent, a muscle contractant
agent, an ophthalmic agent, a parasympathomimetic agent, a psychic
energizer agent, a sedative agent, a sleep inducer, a
sympathomimetic agent, a tranquilizer agent, a urinary agent, a
vaginal agent, a viricide agent, a vitamin agent, a non-steroidal
anti-inflammatory agent, or an angiotensin converting enzyme
inhibitor agent, a polypeptide, a protein, a nucleic acid, a drug,
or an organic molecule.
137. The combination of claim 135, wherein the second syringe
comprises a viscoelastic.
138. A kit comprising the combination of claim 132 and one or more
of the following: (a) packaging material; and (b) instructions for
using the kit for the use of the pharmaceutical composition.
139. (canceled)
140. A method for the treatment of a pathologic accumulation of
glycosaminoglycans, comprising: administration of a recombinant
sHASEGP, wherein said sHASEGP is free of bovine, ovine or bacterial
enzymes, in an amount sufficient to ameliorate or reduce the
accumulated glycosaminoglycan.
141. The method of claim 140, wherein the accumulation is selected
from cardiovascular, cerebral, paraphimosis, myxedema,
scleromyxedema and lymphedema.
142. A method of facilitating revascularization of necrotic tissue,
comprising administration of a recombinant sHASEGP in an amount
sufficient to induce the growth of neovasculature into said
necrotic tissue.
143. A method for removal of glycosaminoglycans from the vitreous
humor comprising; administration of a sHASEGP.
144. (canceled)
145. (canceled)
146. (canceled)
147. (canceled)
148. (canceled)
149. (canceled)
150. (canceled)
151. (canceled)
152. (canceled)
153. (canceled)
154. A process for producing a substantially purified neutral
active soluble hyaluronidase polypeptide and at least one N-linked
sugar moiety, wherein the N-linked sugar moiety is covalently
attached to an asparagine residue of the polypeptide, comprising
growing Chinese Hamster Ovary cells in a suitable growth media and
in the presence of 0.1-1 mM Sodium Butyrate under conditions
suitable for production of the polypeptide.
155. A process for purifying a sHASEGP, said process comprising;
contacting sHASEGP containing media under low ionic strength with
an anion exchange resin at neutral pH, and eluting said sHASEGP
with about 400 mM of a salt; contact of said sHASEGP with
hydrophobic interaction chromatography resin in the presence of
about 0.5M ammonium sulfate; contact of said sHASEGP in ammonium
sulfate with phenyl boronate resin; eluting said sHASEGP in low
salt at neutral pH; contact said sHASEGP with Hydroxyapatite resin,
and elution of said sHASEGP with about 100 mM Na Phosphate,
resulting in a sHASEGP wherein said sHASEGP is substantially
purified.
156. A method of inducing liquefaction of vitreous humor to treat a
disorder of a mammalian eye comprising contacting the vitreous
humor with an amount of a protein of claim 1 effective to liquefy
said vitreous humor whereby the disorder is treated.
157. (canceled)
158. (canceled)
159. A method of reducing intraocular pressure in the eye of a
subject comprising administering to a subject in need thereof,
human sHASEGP characterized as being substantially free of bovine,
ovine or bacterial enzymes; being greater than about 40,000 USP
Units/mg protein; and thereby reducing intraocular pressure in the
subject.
160. (canceled)
161. (canceled)
162. A method of promoting delivery of a sHASEGP or other
glycosaminoglycanase (GAG Enzyme) into a first tissue within a
mammal, comprising introducing a volume of liquid comprising the
GAG Enzyme into the first tissue and allowing the volume of liquid
and catalytic activity of the GAG Enzyme to promote delivery of the
GAG Enzyme into the tissue.
163. The method of claim 162, wherein the volume of liquid is
sufficient to cause localized distension of the first tissue at or
near the site of introduction of the volume of liquid.
164. The method of claim 162, further comprising introducing one or
more additional pharmacologic or other agents (A-X) into the first
tissue.
165. The method of claim 164, wherein A-X is selected from the
group consisting of a detectable molecule or other diagnostic
agent, an anesthetic or other tissue-modifying agent, a
pharmacologic or pharmaceutically effective agent, and a cosmetic
or other esthetic agent.
166. The method of claim 164, wherein A-X is a pharmacologic or
pharmaceutically effective agent.
167. (canceled)
168. (canceled)
169. (canceled)
170. (canceled)
171. (canceled)
172. (canceled)
173. (canceled)
174. (canceled)
175. (canceled)
176. (canceled)
177. (canceled)
178. (canceled)
179. (canceled)
180. (canceled)
181. (canceled)
182. (canceled)
183. (canceled)
184. (canceled)
185. (canceled)
186. (canceled)
187. (canceled)
188. (canceled)
189. (canceled)
190. (canceled)
191. (canceled)
192. (canceled)
193. (canceled)
194. (canceled)
195. (canceled)
196. (canceled)
197. (canceled)
198. (canceled)
199. (canceled)
200. (canceled)
201. (canceled)
202. (canceled)
203. (canceled)
204. The method of claim 164, wherein A-X is selected from the
group consisting of a chemotherapeutic or anticancer agent, an
analgesic agent, an anti-inflammatory agent, an antimicrobial
agent, an amoebicidal agent, a trichomonocidal agent, an
anti-parkinson agent, an anti-malarial agent, an anticonvulsant
agent, an anti-depressant agent, an antiarthritics agent, an
anti-fungal agent, an antihypertensive agent, an antipyretic agent,
an anti-parasitic agent, an antihistamine agent, an
alpha-adrenargic agonist agent, an alpha blocker agent, an
anesthetic agent, a bronchial dilator agent, a biocide agent, a
bactericide agent, a bacteriostatic agent, a beta adrenergic
blocker agent, a calcium channel blocker agent, a cardiovascular
drug agent, a contraceptive agent, a cosmetic or esthetic agent, a
decongestant agent, a diuretic agent, a depressant agent, a
diagnostic agent, an electrolyte agent, a hypnotic agent, a hormone
agent, a hyperglycemic agent, a muscle relaxant agent, a muscle
contractant agent, an ophthalmic agent, a parasympathomimetic
agent, a psychic energizer agent, a sedative agent, a sleep
inducer, a sympathomimetic agent, a tranquilizer agent, a urinary
agent, a vaginal agent, a viricide agent, a vitamin agent, a
non-steroidal anti-inflammatory agent, or an angiotensin converting
enzyme inhibitor agent.
205. The method of claim 164, wherein A-X is selected from the
group consisting of Adalimumabs (e.g. HUMIRA.TM.), Agalsidase Betas
(e.g. FABRAZYME.TM.), Aldesleukins (PROLEUKIN.TM. IL-2), Alefacepts
(e.g. AMEVIVE.TM.), Ampicillins (e.g. UNASYN.TM. Injection),
Anakinras (e.g. KINERET.TM.), Antipoliomyelitic Vaccines (e.g.
PEDIARIX.TM.), Anti-Thymocytes (e.g. THYMOGLOBULIN.TM.),
Azithromycins (e.g. ZITHROMAX.TM. IV), Becaplermins (e.g.
REGRANEX.TM.), Caspofungins (e.g. CANCIDAS.TM.), Cefazolins (e.g.
ANCEF.TM. and CEFAZOLIN.TM.), Cefepimes (e.g. MAXIPIME.TM.),
Cefotetans (e.g. CEFOTAN.TM.), Ceftazidimes (e.g. FORTAZ.TM.),
Ceftriaxones (e.g. ROCEFIN.TM.), Cetuximabs (e.g. ERBITUX.TM.),
Cilastatins (e.g. PRIMAXIN.TM. IV), Clavulanic Acids (e.g. in
conjunction with Amoxicillins such as in AUGMENTIN.TM.),
Clindamycins (e.g. CLEOCIN.TM.), Darbepoetin Alfas (e.g.
ARANESP.TM.), Deaclizumabs (e.g. ZENAPAX.TM.), Diphtheria Toxoids
(typically in combinations e.g. DAPTACEL.TM., INFANRIX.TM. and
PEDIARIX.TM.), Efalizumabs (e.g. RAPTIVA.TM.), Epinephrines (e.g.
EPIPEN.TM.), Erythropoietin Alphas (e.g. EPOGEN.TM. And
PROCRIT.TM.), Etanercepts (e.g. ENBREL.TM.), Filgrastims (e.g.
NEUPOGEN.TM.), Fluconazoles (e.g. DIFLUCAN.TM. Injection),
Follicle-Stimulating Hormones such as Follitropin Alphas (e.g.
GONAL-F.TM.) and Follitropin Betas (e.g. FOLLISTIM.TM.),
Fosphenytoins (e.g. CEREBYX.TM.), Fluconazoles (e.g. DIFLUCAN.TM.),
Gadodiamides (e.g. OMNISCAN.TM.), Gadopentetates (e.g.
MAGNEVIST.TM.), Gatifloxacins (e.g. TEQUIN.TM.), Glatiramers (e.g.
COPAXONE.TM.), GM-CSF's (e.g. LEUKINE.TM.), Goserelins (e.g.
ZOLADEX.TM.), Granisetrons (e.g. KYTRIL.TM.), Haemophilus Influenza
B's (e.g. COMVAX.TM. and HibTITER.TM.), Haloperidols (e.g.
HALDOL.TM.), Hepatitis A Vaccines (e.g. HAVRIX.TM., TWINRIX.TM. and
VAQTA.TM.), Hepatitis B Vaccines (e.g. recombinants COMVAX.TM.,
ENGERIX.TM.-B, RECOMBIVAX.TM.-HB, TWINRIX.TM., and non-recombinants
BAYHEP.TM.-B and NABI.TM.-HB), Ibritumomab Tiuxetans (e.g.
ZEVALIN.TM.), Inmunoglobulins (including mixtures of
immunoglobulins such as GAMMAGARD.TM. and the like, as well as any
of a variety of purified immunoglobulins), Influenza Virus Vaccines
(e.g. FLUMIST.TM.), Infliximabs (e.g. REMICADE.TM.), Insulins (e.g.
HUMALOG.TM., HUMALOG.TM. MIX 75/25.TM., HUMULIN.TM. products (incl.
50/50, 70/30, Regular, NPH, Ultra and Ultralente), and
NOVOLIN.TM.), Insulin Glargines (e.g. LANTUS.TM.), Interferon
Alfa-2a's (ROFERAN.TM.-A), Interferon Alfa-2b's (e.g.
INTRON-A.TM.), Interferon Alfacon-1's (e.g. INFERGEN.TM.),
Interferon Alfa-n3s (e.g. ALFERON N.TM.), Interferon Betas (e.g.
BETASERON.TM. And BETAFERON.TM.), Interferon Beta-1a's (e.g.
AVONEX.TM. And REBIF.TM.), Interferon Gammas (e.g. ACTIMMUNE.TM.),
Iodixanols (e.g. VISIPAQUE.TM.), Iohexols (OMNIPAQUE.TM.),
Iopamidols, Ioversols (e.g. OPTIRAY.TM.), Ketorolacs (e.g.
TORADOL.TM.), Laronidases (e.g. ALDURAZYME.TM.), Levofloxacins
(e.g. LEVAQUIN.TM.), Lidocaines, Linezolids (e.g. ZYVOX.TM.),
Lorazepams (e.g. ATIVAN.TM.), Measles Vaccines (e.g.
ATTENUVAX.TM.), Measles-Mumps-Rubella Virus Vaccines (e.g.
M-M-R.TM. II), Medroxyprogesterones (e.g. DEPO-PROVERA.TM.),
Meropenems (e.g. MERREM.TM. IV), Methylprednisolones (e:g.
SOLU-MEDROL.TM.), Midazolams (e.g. VERSED.TM.), Morphines (e.g.
ASTRAMORPH/PF.TM.), Octreotides (e.g. SANDOSTATIN.TM.), Omalizumabs
(e.g. XOLAIR.TM.), Ondansetrons (e.g. ZOFRAN.TM.), Palivizumabs
(e.g. SYNAGIS.TM.), Pantoprazoles (e.g. PROTONIX.TM.),
Pegaspargases (e.g. ONCASPAR.TM.), Pegfilgrastims (e.g.
NEULASTA.TM.), Peg-Interferon Alfa-2a's (e.g. PEGASYS.TM.),
Peg-Interferon Alfa-2b's (e.g. PEG-INTRON.TM.), Pegvisomants (e.g.
SOMAVERT.TM.), Pertussis vaccines, Piperacillins (e.g. ZOSYN.TM.),
Pneumococcal Vaccines (e.g. PNEUMOVAX.TM. 23) and Pneumococcal
Conjugate Vaccines (e.g. PREVNAR.TM.), Promethazines (e.g.
PHENERGAN.TM.), Reteplases (e.g. RETAVASE.TM.), Somatropins (e.g.
GENOTROPIN.TM., HUMATROPE.TM., NORDITROPIN.TM., NUTROPIN.TM.,
SAIZEN.TM., SEROSTIM.TM., ZORBTIVE.TM.), Sulbactams, Sumatriptans
(e.g. IMITREX.TM.), Tazobactams, Tenecteplases (e.g. TNKASE.TM.),
Tetanus Purified Toxoids, Ticarcillins (e.g. TIMENTIN.TM.),
Tositumomabs (e.g. BEXXAR.TM.), Triamcinolone Acetonides,
Vancomycins (e.g. VANCOCIN.TM.), Varicella vaccines (e.g.
VARIVAX.TM.), and other vaccines.
206. The method of claim 164, wherein A-X is an antibiotic agent or
combination of agents selected from the group consisting of
Aminoglycosides; Amphenicols; Ansamycins; Carbacephems;
Carbapenems; Cephalosporins or Cephems; Cephamycins; Clavams;
Cyclic lipopeptides; Diaminopyrimidines; Ketolides; Lincosamides;
Macrolides; Monobactams; Nitrofurans; Oxacephems; Oxazolidinones;
Penems, thienamycins and miscellaneous beta-lactams; Penicillins;
Polypeptides antibiotics; Quinolones; Sulfonamides; Sulfones;
Tetracyclines; and other antibiotics (such as Clofoctols, Fusidic
acids, Hexedines, Methenamines, Nitrofurantoins Nitroxolines,
Ritipenems, Taurolidines, Xibomols).
207. (canceled)
208. The method of claim 162, wherein the GAG Enzyme is a
sHASEGP.
209. The method of claim 208, wherein the sHASEGP is a human
sHASEGP.
210. The method of claim 209, wherein the human sHASEGP is a PH20
sHASEGP.
211. The method of claim 210, wherein the sHASEGP is a soluble
polypeptide as set forth in SEQ ID NO. 4.
212. The method of claim 208, wherein the sHASEGP is a pegylated or
super-sialated sHASEGP.
213. The method of claim 211, wherein the sHASEGP is a pegylated
human PH20 sHASEGP.
214. (canceled)
215. (canceled)
216. (canceled)
217. (canceled)
218. (canceled)
219. (canceled)
220. (canceled)
221. (canceled)
222. A method of increasing the permeability of a target tissue of
a mammal, comprising introducing a sHASEGP into the tissue.
223. The method of claim 222, wherein the sHASEGP is introduced
into the tissue by injection of the sHASEGP into the tissue or into
a nearby tissue.
224. The method of claim 222, wherein the sHASEGP is introduced
into the tissue by prior introduction of the sHASEGP into the
bloodstream supplying the tissue.
225. The method of claim 224, wherein the prior introduction into
the bloodstream is mediated by intravenous injection or infusion of
the sHASEGP.
226. The method of claim 224, wherein the prior introduction into
the bloodstream is mediated by non-intravenous parenteral
administration of the sHASEGP.
227. The method of claim 222, further comprising introducing A-X
into the tissue.
228. The method of claim 227, wherein A-X is introduced into the
tissue by injection into the tissue or into a nearby tissue.
229. The method of claim 227, wherein A-X is introduced into the
tissue by prior introduction of the PH20 into the bloodstream
supplying the tissue.
230. The method of claim 229, wherein the prior introduction into
the bloodstream is mediated by intravenous injection or infusion of
the molecule or macromolecular complex.
231. The method of claim 229, wherein the prior introduction into
the bloodstream is mediated by non-intravenous parenteral
administration of the molecule or macromolecular complex.
232. The method of claim 227, wherein the step of introducing A-X
into the tissue is initiated within three hours following the step
of introducing the sHASEGP into the tissue.
233. The method of claim 227, wherein the step of introducing A-X
into the tissue is initiated within twelve hours following the step
of introducing the sHASEGP into the first tissue.
234. The method of claim 227, wherein A-X is a molecule selected
from the group consisiting of a small molecule, a protein, and a
nucleic acid.
235. The method of claim 227, wherein A-X is a macromolecular
complex comprising combinations of molecules selected from the
group consisting of small molecules, proteins, nucleic acids,
lipids and amphiphilic molecules.
236. The method of claim 227, wherein A-X is selected from the
group consisting of a detectable molecule or other diagnostic
agent, an anesthetic or other tissue-modifying agent, a
pharmacologic or pharmaceutically effective agent, and a cosmetic
or other esthetic agent.
237. The method of claim 227, wherein A-X is a pharmacologic or
pharmaceutically effective agent.
238. The method of claim 227, wherein A-X is selected from the
group consisting of a chemotherapeutic or anticancer agent, an
analgesic agent, an anti-inflammatory agent, an antimicrobial
agent, an amoebicidal agent, a trichomonocidal agent, an
anti-parkinson agent, an anti-malarial agent, an anticonvulsant
agent, an anti-depressant agent, an antiarthritics agent, an
anti-fungal agent, an antihypertensive agent, an antipyretic agent,
an anti-parasitic agent, an antihistamine agent, an
alpha-adrenargic agonist agent, an alpha blocker agent, an
anesthetic agent, a bronchial dilator agent, a biocide agent, a
bactericide agent, a bacteriostatic agent, a beta adrenergic
blocker agent, a calcium channel blocker agent, a cardiovascular
drug agent, a contraceptive agent, a cosmetic or esthetic agent, a
decongestant agent, a diuretic agent, a depressant agent, a
diagnostic agent, an electrolyte agent, a hypnotic agent, a hormone
agent, a hyperglycemic agent, a muscle relaxant agent, a muscle
contractant agent, an ophthalmic agent, a parasympathomimetic
agent, a psychic energizer agent, a sedative agent, a sleep
inducer, a sympathomimetic agent, a tranquilizer agent, a urinary
agent, a vaginal agent, a viricide agent, a vitamin agent, a
non-steroidal anti-inflammatory agent, or an angiotensin converting
enzyme inhibitor agent.
239. The method of claim 227, wherein A-X is an anticancer agent or
combination of agents selected from the group consisting of
Aclacinomycins, Actinomycins, Adriamycins, Ancitabines,
Anthramycins, Azacitidines, Azaserines, 6-Azauridines, Bisantrenes,
Bleomycins, Cactinomycins, Carmofurs, Carmustines, Carubicins,
Carzinophilins, Chromomycins, Cisplatins, Cladribines, Cytarabines,
Dactinomycins, Daunorubicins, Denopterins,
6-Diazo-5-Oxo-L-Norleucines, Doxifluridines, Doxorubicins,
Edatrexates, Emitefurs, Enocitabines, Fepirubicins, Fludarabines,
Fluorouracils, Gemcitabines, Idarubicins, Loxuridines, Menogarils,
6-Mercaptopurines, Methotrexates, Mithramycins, Mitomycins,
Mycophenolic Acids, Nogalamycins, Olivomycines, Peplomycins,
Pirarubicins, Piritrexims, Plicamycins, Porfiromycins,
Pteropterins, Puromycins, Retinoic Acids, Streptonigrins,
Streptozocins, Tagafurs, Tamoxifens, Thiamiprines, Thioguanines,
Triamcinolones, Trimetrexates, Tubercidins, Vinblastines,
Vincristines, Zinostatins, And Zorubicins.
240. The method of claim 227, wherein A-X is an antibody or other
protein having anti-cancer or chemotherapeutic activity.
241. The method of claim 227, wherein A-X is an antibiotic agent or
combination of agents selected from the group consisting of
Aminoglycosides; Amphenicols; Ansamycins; Carbacephems;
Carbapenems; Cephalosporins or Cephems; Cephamycins; Clavams;
Cyclic lipopeptides; Diaminopyrimidines; Ketolides; Lincosamides;
Macrolides; Monobactams; Nitrofurans; Oxacephems; Oxazolidinones;
Penems, thienamycins and miscellaneous beta-lactams; Penicillins;
Polypeptides antibiotics; Quinolones; Sulfonamides; Sulfones;
Tetracyclines; and other antibiotics (such as Clofoctols, Fusidic
acids, Hexedines, Methenamines, Nitrofurantoins Nitroxolines,
Ritipenems, Taurolidines, Xibomols).
242. The method of claim 222, wherein the sHASEGP is a human
sHASEGP.
243. The method of claim 242, wherein the human sHASEGP is a PH20
sHASEGP.
244. The method of claim 243, wherein the sHASEGP is a soluble
polypeptide as set forth in SEQ ID NO. 4.
245. The method of claim 222, wherein the sHASEGP is a pegylated or
super-sialated sHASEGP.
246. The method of claim 244, wherein the sHASEGP is a pegylated
human PH20 sHASEGP.
247. A method of ameliorating the consequences of stroke or other
CNS injury in a mammal, comprising introducing a sHASEGP into a
mammal following said stroke or CNS injury.
248. (canceled)
249. (canceled)
250. (canceled)
251. (canceled)
252. (canceled)
253. (canceled)
254. (canceled)
255. (canceled)
256. The polypeptide of any one of claims 12, 19 or 158, wherein
the polymer is PEG.
257. The polypeptide of claim 256, wherein the polypeptide
comprises at least about three PEG moieties per polypeptide.
258. The polypeptide of claim 256, wherein said PEG moieties are
branched.
259. The polypeptide of claim 256, wherein said PEG moieties are
selected from the group consisting of mPEG-SBA (5 kDa), mPEG-SBA
(20 kDa), mPEG-SBA 30 kDa), mPEG-SMB (20 kDa) mPEG-SMB (30 kDa),
mPEG-butyrldehyde (30 kDa), mPEG-SPA (20 kDa), mPEG-SPA (30 kDa),
mPEG2-NHS (10 kDa branched), mPEG2-NHS (20 kDa branched), mPEG-NHS
(40 kDa branched), mPEG2-NHS (60 kDa branched), PEG-NHS-biotin (5
kDa biotinylated), PEG-p-nitrophenyl-carbonate (30 kDa) and
PEG-propionaldehyde (30 kDa).
260. A method of preparing a PEGylated polypeptide, comprising
reacting a polypeptide of any one of claims 12, 19 or 158 with a
molar excess of a PEG reagent selected from the group consisting of
a PEG-succinimidyl butanoate, a PEG-succinimidyl methylbutanoate, a
PEG-succinimidyl proprionate, a PEG-N-hydroxysuccinimide, a
PEG-aldehyde, a PEG-carbonate, a PEG-propionaldehyde and a
PEG-maleimide.
261. The method of claim 260 wherein the PEG reagent is selected
from the group consisting of mPEG-SBA (5 kDa), mPEG-SBA (20 kDa),
mPEG-SBA 30 kDa), mPEG-SMB (20 kDa) mPEG-SMB (30 kDa),
mPEG-butyrldehyde (30 kDa), mPEG-SPA (20 kDa), mPEG-SPA (30 kDa),
mPEG2-NHS (10 kDa branched), mPEG2-NHS (20 kDa branched), mPEG-NHS
(40 kDa branched), mPEG2-NHS (60 kDa branched), PEG-NHS-biotin (5
kDa biotinylated), PEG-p-nitrophenyl-carbonate (30 kDa) and
PEG-propionaldehyde (30 kDa).
262. A PEGylated polypeptide produced according to the method of
claim 260.
263. A PEGylated polypeptide produced according to the method of
claim 261.
264. A PEGylated hyaluronidase, wherein the hyaluronidase comprises
at least about three PEG moieties per polypeptide.
265. The hyaluronidase of claim 264, wherein said PEG moieties are
branched.
266. The hyaluronidase of claim 264, wherein said PEG moieties are
selected from the group consisting of mPEG-SBA (5 kDa), mPEG-SBA
(20 kDa), mPEG-SBA 30 kDa), mPEG-SMB (20 kDa) mPEG-SMB (30 kDa),
mPEG-butyrldehyde (30 kDa), mPEG-SPA (20 kDa), mPEG-SPA (30 kDa),
mPEG2-NHS (10 kDa branched), mPEG2-NHS (20 kDa branched), mPEG-NHS
(40 kDa branched), mPEG2-NHS (60 kDa branched), PEG-NHS-biotin (5
kDa biotinylated), PEG-p-nitrophenyl-carbonate (30 kDa) and
PEG-propionaldehyde (30 kDa).
267. The hyaluronidase of claim 264, wherein the hyaluronidase is a
PEGylated sHASEGP.
268. The hyaluronidase of claim 264, wherein the hyaluronidase is a
PEGylated rHuPH20.
269. The hyaluronidase of claim 264, wherein the hyaluronidase is a
PEGylated animal-derived or bacterial-derived hyaluronidase.
270. A method of preparing a-PEGylated hyaluronidase, comprising
reacting a hyaluronidase with a molar excess of a PEG reagent
selected from the group consisting of a PEG-succinimidyl butanoate,
a PEG-succinimidyl methylbutanoate, a PEG-succinimidyl proprionate,
a PEG-N-hydroxysuccinimide, a PEG-aldehyde, a PEG-carbonate, a
PEG-propionaldehyde and a PEG-maleimide.
271. The method of claim 270 wherein the PEG reagent is selected
from the group consisting of mPEG-SBA (5 kDa), mPEG-SBA (20 kDa),
mPEG-SBA 30 kDa), mPEG-SMB (20 kDa) mPEG-SMB (30 kDa),
mPEG-butyrldehyde (30 kDa), mPEG-SPA (20 kDa), mPEG-SPA (30 kDa),
mPEG2-NHS (10 kDa branched), mPEG2-NHS (20 kDa branched), mPEG-NHS
(40 kDa branched), mPEG2-NHS (60 kDa branched), PEG-NHS-biotin (5
kDa biotinylated), PEG-p-nitrophenyl-carbonate (30 kDa) and
PEG-propionaldehyde (30 kDa).
272. A PEGylated hyaluronidase produced according to the method of
claim 270.
273. A PEGylated hyaluronidase produced according to the method of
claim 271.
274. A method of delivering a molecule to a tissue containing
excess amounts of glycosaminoglycan, comprising: administering a
PEGylated hyaluronidase of claim 264 to a tissue in an amount
sufficient to degrade glycosaminoglycans sufficiently to open
channels less than about 500 nm in diameter; and administering a
molecule to the tissue comprising the degraded
glycosaminoglycans.
275. A pharmaceutical composition, comprising a PEGylated
hyaluronidase of claim 264 and a pharmaceutical carrier.
276. A method for increasing the diff-usion of a therapeutic
substance or other molecule or macromolecular complex (each an
Agent) smaller than about 0.5 microns in diameter in a subject,
comprising: administering to a subject a PEGylated hyaluronidase of
claim 264 in an amount sufficient to open or to form channels
smaller than about 0.5 microns in diameter and an Agent, whereby
the diffusion of the Agent is increased.
277. The method of claim 276, wherein the Agent is selected from
the group of agents consisting of a chemotherapeutic agent, an
analgesic agent, an anti-inflammatory agent, an antimicrobial
agent, an amoebicidal agent, a trichomonocidal agent, an
anti-parkinson agent, an anti-malarial agent, an anticonvulsant
agent, an anti-depressant agent, an antiarthritics agent, an
anti-fungal agent, an antihypertensive agent, an antipyretic agent,
an anti-parasitic agent, an antihistamine agent, an
alpha-adrenargic agonist agent, an alpha blocker agent, an
anesthetic agent, a bronchial dilator agent, a biocide agent, a
bactericide agent, a bacteriostatic agent, a beta adrenergic
blocker agent, a calcium channel blocker agent, a cardiovascular
drug agent, a contraceptive agent, a decongestant agent, a diuretic
agent, a depressant agent, a diagnostic agent, an electrolyte
agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a
muscle relaxant agent, a muscle contractant agent, an ophthalmic
agent, a parasympathomimetic agent, a psychic energizer agent, a
sedative agent, a sleep inducer, a sympathomimetic agent, a
tranquilizer agent, a urinary agent, a vaginal agent, a viricide
agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an
angiotensin converting enzyme inhibitor agent, a polypeptide, a
protein, a nucleic acid, a drug, or an organic molecule.
278. A kit, comprising: (a) a PEGylated hyaluronidase of claim 264
at a dose between 0.1 and 1500 Units/ml in an acceptable carrier;
(b) at least one therapeutic substance or other molecule or
macromolecular complex (each an Agent) in an acceptable carrier;
and (c) optionally, instructions for delivering therapeutic
substances or other Agents.
279. A method for removal of glycosaminoglycans from the vitreous
humor comprising; administration of a PEGylated hyaluronidase of
claim 264.
280. A method of inducing liquefaction of vitreous humor to treat a
disorder of a mammalian eye comprising contacting the vitreous
humor with an amount of a PEGylated hyaluronidase of claim 264
effective to liquefy said vitreous humor whereby the disorder is
treated.
281. A method of ameliorating the consequences of stroke or other
CNS injury in a mammal, comprising introducing a PEGylated
hyaluronidase of claim 264 into a mammal following said stroke or
CNS injury.
282. A method of introducing one or more pharmacologic or other
agents (A-X) into a tissue of the body comprising administering a
PEGylated hyaluronidase of claim 264 to said tissue prior to or
coincident with administering said agent.
283. A method of claim 282, wherein A-X is selected from the group
consisting of a detectable molecule or other diagnostic agent, an
anesthetic or other tissue-modifying agent, a pharmacologic or
pharmaceutically effective agent, and a cosmetic or other esthetic
agent.
284. The method of claim 282, wherein A-X is an anti-cancer agent
or an anti-infective agent.
285. The method of claim 282, wherein A-X is a blood-modifying
pharmacologic agent
286. The method of claim 282, wherein A-X is a molecule or
macromolecular complex of between about 20 nm and 200 nm in
diameter.
287. The method of claim 282, wherein A-X is selected from the
group consisting of Adalimumabs (e.g. HUMIRA.TM.), Agalsidase Betas
(e.g. FABRAZYME.TM.), Aldesleukins (PROLEUKIN.TM. IL-2), Alefacepts
(e.g. AMEVIVE.TM.), Ampicillins (e.g. UNASYN.TM. Injection),
Anakinras (e.g. KINERET.TM.), Antipoliomyelitic Vaccines (e.g.
PEDIARIX.TM.), Anti-Thymocytes (e.g. THYMOGLOBULIN.TM.),
Azithromycins (e.g. ZITHROMAX.TM. IV), Becaplermins (e.g.
REGRANEX.TM.), Caspofungins (e.g. CANCIDAS.TM.), Cefazolins (e.g.
ANCEF.TM. and CEFAZOLIN.TM.), Cefepimes (e.g. MAXIPIME.TM.),
Cefotetans (e.g. CEFOTAN.TM.), Ceftazidimes (e.g. FORTAZ.TM.),
Ceftriaxones (e.g. ROCEFIN.TM.), Cetuximabs (e.g. ERBITUX.TM.),
Cilastatins (e.g. PRIMAXIN.TM. IV), Clavulanic Acids (e.g. in
conjunction with Amoxicillins such as in AUGMENTIN.TM.),
Clindamycins (e.g. CLEOCIN.TM.), Darbepoetin Alfas (e.g.
ARANESP.TM.), Deaclizumabs (e.g. ZENAPAX.TM.), Diphtheria Toxoids
(typically in combinations e.g. DAPTACEL.TM., INFANRIX.TM. and
PEDIARIX.TM.), Efalizumabs (e.g. RAPTIVA.TM.), Epinephrines (e.g.
EPIPEN.TM.), Erythropoietin Alphas (e.g. EPOGEN.TM. And
PROCRIT.TM.), Etanercepts (e.g. ENBREL.TM.), Filgrastims (e.g.
NEUPOGEN.TM.), Fluconazoles (e.g. DIFLUCAN.TM. Injection),
Follicle-Stimulating Hormones such as Follitropin Alphas (e.g.
GONAL-F.TM.) and Follitropin Betas (e.g. FOLLISTIM.TM.),
Fosphenytoins (e.g. CEREBYX.TM.), Fluconazoles (e.g. DIFLUCAN.TM.),
Gadodiamides (e.g. OMNISCAN.TM.), Gadopentetates (e.g.
MAGNEVIST.TM.), Gatifloxacins (e.g. TEQUIN.TM.), Glatiramers (e.g.
COPAXONE.TM.), GM-CSF's (e.g. LEUKINE.TM.), Goserelins (e.g.
ZOLADEX.TM.), Granisetrons (e.g. KYTRIL.TM.), Haemophilus Influenza
B's (e.g. COMVAX.TM. and HibTITER.TM.), Haloperidols (e.g.
HALDOL.TM.), Hepatitis A Vaccines (e.g. HAVRIX.TM., TWINRIX.TM. and
VAQTA.TM.), Hepatitis B Vaccines (e.g. recombinants COMVAX.TM.,
ENGERIX.TM.-B, RECOMBIVAX.TM.-HB, TWINRIX.TM., and non-recombinants
BAYHEP.TM.-B and NABI.TM.-HB), Ibritumomab Tiuxetans (e.g.
ZEVALIN.TM.), Immunoglobulins (including mixtures of
immunoglobulins such as GAMMAGARD.TM. and the like, as well as any
of a variety of purified immunoglobulins), Influenza Virus Vaccines
(e.g. FLUMIST.TM.), Infliximabs (e.g. REMICADE.TM.), Insulins (e.g.
HUMALOG.TM., HUMALOG.TM. MIX 75/25.TM., HUMULIN.TM. products (incl.
50/50, 70/30, Regular, NPH, Ultra and Ultralente), and
NOVOLIN.TM.), Insulin Glargines (e.g. LANTUS.TM.), Interferon
Alfa-2a's (ROFERAN.TM.-A), Interferon Alfa-2b's (e.g.
INTRON-A.TM.), Interferon Alfacon-1's (e.g. INFERGEN.TM.),
Interferon Alfa-n3s (e.g. ALFERON N.TM.), Interferon Betas (e.g.
BETASERON.TM. And BETAFERON.TM.), Interferon Beta-la's (e.g.
AVONEX.TM. And REBIF.TM.), Interferon Gamrnas (e.g. ACTIMMUNE.TM.),
Iodixanols (e.g. VISIPAQUE.TM.), Iohexols (OMNIPAQUE.TM.),
Iopamidols, Ioversols (e.g. OPTIRAY.TM.), Ketorolacs (e.g.
TORADOL.TM.), Laronidases (e.g. ALDURAZYME.TM.), Levofloxacins
(e.g. LEVAQUIN.TM.), Lidocaines, Linezolids (e.g. ZYVOX.TM.),
Lorazepams (e.g. ATIVAN.TM.), Measles Vaccines (e.g.
ATTENUVAX.TM.), Measles-Mumps-Rubella Virus Vaccines (e.g.
M-M-R.TM. II), Medroxyprogesterones (e.g. DEPO-PROVERA.TM.),
Meropenems (e.g. MERREM.TM. IV), Methylprednisolones (e.g.
SOLU-MEDROL.TM.), Midazolams (e.g. VERSED.TM.), Morphines (e.g.
ASTRAMORPH/PF.TM.), Octreotides (e.g. SANDOSTATIN.TM.), Omalizumabs
(e.g. XOLAIR.TM.), Ondansetrons (e.g. ZOFRAN.TM.), Palivizumabs
(e.g. SYNAGIS.TM.), Pantoprazoles (e.g. PROTONIX.TM.),
Pegaspargases (e.g. ONCASPAR.TM.), Pegfilgrastims (e.g.
NEULASTA.TM.), Peg-Interferon Alfa-2a's (e.g. PEGASYS.TM.),
Peg-Interferon Alfa-2b's (e.g. PEG-INTRON.TM.), Pegvisomants (e.g.
SOMAVERT.TM.), Pertussis vaccines, Piperacillins (e.g. ZOSYN.TM.),
Pneumococcal Vaccines (e.g. PNEUMOVAX.TM. 23) and Pneumococcal
Conjugate Vaccines (e.g. PREVNAR.TM.), Promethazines (e.g.
PHTNERGAN.TM.), Reteplases (e.g. RETAVASE.TM.), Somatropins (e.g.
GENOTROPIN.TM., HUMATROPE.TM., NORDITROPIN.TM., NUTROPIN.TM.,
SAIZEN.TM., SEROSTIM.TM., ZORBTIVE.TM.), Sulbactams, Sumatriptans
(e.g. IMITREX.TM.), Tazobactams, Tenecteplases (e.g. TNKASE.TM.),
Tetanus Purified Toxoids, Ticarcillins (e.g. TIMENTIN.TM.),
Tositumomabs (e.g. BEXXAR.TM.), Triamcinolone Acetonides,
Vancomycins (e.g. VANCOCIN.TM.), Varicella vaccines (e.g.
VARIVAX.TM.), and other vaccines.
288. The method of claim 282, wherein A-X is an antibiotic agent or
combination of agents selected from the group consisting of
Aminoglycosides; Amphenicols; Ansamycins; Carbacephems;
Carbapenems; Cephalosporins or Cephems; Cephamycins; Clavams;
Cyclic lipopeptides; Diaminopyrimidines; Ketolides; Lincosamides;
Macrolides; Monobactams; Nitrofurans; Oxacephems; Oxazolidinones;
Penems, thienamycins and miscellaneous beta-lactams; Penicillins;
Polypeptides antibiotics; Quinolones; Sulfonamides; Sulfones;
Tetracyclines; and other antibiotics (such as Clofoctols, Fusidic
acids, Hexedines, Methenamines, Nitrofurantoins Nitroxolines,
Ritipenems, Taurolidines, Xibomols).
289. The method of claim 282, wherein A-X is an anticancer agent or
combination of agents selected from the group consisting of
Aclacinomycins, Actinomycins, Adriamycins, Ancitabines,
Anthramycins, Azacitidines, Azaserines, 6-Azauridines, Bisantrenes,
Bleomycins, Cactinomycins, Carmofurs, Carmustines, Carubicins,
Carzinophilins, Chromomycins, Cisplatins, Cladribines, Cytarabines,
Dactinomycins, Daunorubicins, Denopterins,
6-Diazo-5-Oxo-L-Norleucines, Doxifluridines, Doxorubicins,
Edatrexates, Emitefurs, Enocitabines, Fepirubicins, Fludarabines,
Fluorouracils, Gemcitabines, Idarubicins, Loxuridines, Menogarils,
6-Mercaptopurines, Methotrexates, Mithramycins, Mitomycins,
Mycophenolic Acids, Nogalamycins, Olivomycines, Peplomycins,
Pirarubicins, Piritrexims, Plicamycins, Porfiromycins,
Pteropterins, Puromycins, Retinoic Acids, Streptonigrins,
Streptozocins, Tagafurs, Tamoxifens, Thiamiprines, Thioguanines,
Triamcinolones, Trimetrexates, Tubercidins, Vinblastines,
Vincristines, Zinostatins, and Zorubicins.
Description
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a continuation-in-part of U.S. Ser. No.
11/065,716 filed Feb. 23, 2005, which is a continuation-in-part of
U.S. Ser. No. 10/795,095, filed Mar. 5, 2004, which claims priority
under 35 U.S.C. .sctn.119(e) to U.S. Ser. No. 60/452,360, filed
Mar. 5, 2003, each of which is herein incorporated by reference in
its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention is generally related to
Glycosaminoglycanase enzymes, including Neutral-Active, Soluble
Hyaluronidase Glycoproteins (sHASEGPs), and portions thereof,
particularly Hyaluronidase domains. More specifically, the
invention is related to chemical modifications, pharmaceutical
compositions, expression plasmids, methods for manufacture and
therapeutic methods using the Glycosaminoglycanases (and domains
thereof and the encoding nucleic acid molecules) for the
therapeutic modification of glycosaminoglycans in the treatment of
disease and for use to increase diffusion of other molecules such
as injected molecules in an animal.
[0004] 2. Background Information
[0005] Glycosaminoglycans (GAGs) are complex linear polysaccharides
of the extracellular matrix (ECM). GAGs are characterized by
repeating disaccharide structures of an N-substituted hexosamine
and an uronic acid (in the case of hyaluronan (HA), chondroitin
sulfate (CS), chondroitin (C), dermatan sulfate (DS), heparan
sulfate (HS), and heparin (H)), or a galactose (in the case of
keratan sulfate (KS)). Except for HA, all exist covalently bound to
core proteins. The GAGs with their core proteins are structurally
referred to as proteoglycans (PGs).
[0006] Hyaluronan (HA) is found in mammals predominantly in
connective tissues, skin, cartilage, and in synovial fluid.
Hyaluronan is also the main constituent of the vitreous of the eye.
In connective tissue, the water of hydration associated with
hyaluronan creates hydrated matrices between tissues. Hyaluronan
plays a key role in biological phenomena associated with cell
motility including rapid development, regeneration, repair,
embryogenesis, embryological development, wound healing,
angiogenesis, and tumorigenesis (Toole 1991 Cell Biol. Extracell.
Matrix, Hay (ed), Plenum Press, New York, 1384-1386; Bertrand et
al. 1992 Int. J. Cancer 52:1-6; Knudson et al, 1993 FASEB J.
7:1233-1241). In addition, hyaluronan levels correlate with tumor
aggressiveness (Ozello et al. 1960 Cancer Res. 20:600-604; Takeuchi
et al. 1976, Cancer Res. 36:2133-2139; Kimata et al. 1983 Cancer
Res. 43:1347-1354).
[0007] HA is found in the extracellular matrix of many cells,
especially in soft connective tissues. HA has been assigned various
physiological functions, such as in water and plasma protein
homeostasis (Laurent TC et al (1992) FASEB J 6: 2397-2404). HA
production increases in proliferating cells and may play a role in
mitosis. It has also been implicated in locomotion and cell
migration. HA seems to play important roles in cell regulation,
development, and differentiation (Laurent et al, supra).
[0008] HA has been used in clinical medicine. Its tissue protective
and rheological properties have proved useful in ophthalmic surgery
(e.g. to protect the corneal endothelium during cataract surgery).
Serum HA is diagnostic of liver disease and various inflammatory
conditions, such as rheumatoid arthritis. Interstitial edema caused
by accumulation of HA may cause dysfunction in various organs
(Laurent et al, supra).
[0009] Hyaluronan protein interactions also are involved in the
structure of the extracellular matrix or "ground substance".
[0010] Hyaluronidases are a group of generally neutral- or
acid-active enzymes found throughout the animal kingdom.
Hyaluronidases vary with respect to substrate specificity, and
mechanism of action.
[0011] There are three general classes of hyaluronidases:
[0012] 1. Mammalian-type hyaluronidases, (EC 3.2.1.35) which are
endo-beta-N-acetylhexosaminidases with tetrasaccharides and
hexasaccharides as the major end products. They have both
hydrolytic and transglycosidase activities, and can degrade
hyaluronan and chondroitin sulfates (CS), generally C4-S and
C6-S.
[0013] 2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan
and, and to various extents, CS and DS. They are
endo-beta-N-acetylhexosaminidases that operate by a beta
elimination reaction that yields primarily disaccharide end
products.
[0014] 3. Hyaluronidases (EC 3.2.1.36) from leeches, other
parasites, and crustaceans are endo-beta-glucuronidases that
generate tetrasaccharide and hexasaccharide end products through
hydrolysis of the beta 1-3 linkage.
[0015] Mammalian hyaluronidases can be further divided into two
groups: neutral-active and acid-active enzymes. There are six
hyaluronidase-like genes in the human genome, HYAL1, HYAL2, HYAL3,
HYAL4, HYALP1 and PH20/SPAM1. HYALP1 is a pseudogene, and HYAL3 has
not been shown to possess enzyme activity toward any known
substrates. HYAL4 is a chondroitinase and exhibits little activity
towards hyaluronan. HYAL1 is the prototypical acid-active enzyme
and PH20 is the prototypical neutral-active enzyme. Acid-active
hyaluronidases, such as HYAL1 and HYAL2 generally lack catalytic
activity at neutral pH (i.e. pH 7). For example, HYAL1 has little
catalytic activity in vitro over pH 4.5 (Frost et al Anal
Biochemistry, 1997). HYAL2 is an acid-active enzyme with a very low
specific activity in vitro.
[0016] The hyaluronidase-like enzymes can also be characterized by
those which are generally locked to the plasma membrane via a
glycosylphosphatidyl inositol anchor such as human HYAL2 and human
PH20 (Danilkovitch-Miagkova, et al. Proc Natl Acad Sci USA. Apr.
15, 2003; 100(8):4580-5, Phelps et al., Science 1988), and those
which are generally soluble such as human HYAL1 (Frost et al,
Biochem Biophys Res Commun. Jul. 9, 1997; 236(1):10-5). However,
there are variations from species to species: bovine, PH20 for
example is very loosely attached to the plasma membrane and is not
anchored via a phospholipase sensitive anchor (Lalancette et al,
Biol Reprod. August 2001; 65(2):628-36.). This unique feature of
bovine hyaluronidase has permitted the use of the soluble bovine
testes hyaluronidase enzyme as an extract for clinical use
(Wydase.TM., Hyalase.TM.). Other PH20 species are lipid anchored
enzymes that are generally not soluble without the use of
detergents or lipases. For example, human PH20 is anchored to the
plasma membrane via a GPI anchor. Attempts to make human PH20 DNA
constructs that would not introduce a lipid anchor into the
polypeptide resulted in either a catalytically inactive enzyme, or
an insoluble enzyme (Arming et al Eur J Biochem. Aug. 1, 1997;
247(3):810-4). Naturally occurring macaque sperm hyaluronidase is
found in both a soluble and membrane bound form. While the 64 kDa
membrane bound form possesses enzyme activity at pH 7.0, the 54 kDa
form is only active at pH 4.0 (Cherr et al, Dev Biol. Apr. 10,
1996; 175(1):142-53.). Thus, soluble forms of PH20 are often
lacking enzyme activity under neutral conditions.
[0017] Chondroitinases are enzymes found throughout the animal
kingdom. These enzymes degrade glycosaminoglycans through an
endoglycosidase reaction. Specific examples of known
Chondroitinases include: Chondroitinase ABC (derived from Proteus
vulgaris; Japanese Patent Application Laid-open No 6-153947, T.
Yamagata, H. Saito, O. Habuchi, and S. Suzuki, J. Biol. Chem., 243,
1523 (1968), S. Suzuki, H. Saito, T. Yamagata, K. Anno, N. Seno, Y.
Kawai, and T. Furuhashi, J. Biol. Chem., 243, 1543 (1968));
Chondroitinase AC (derived from Flavobacterium heparinum; T.
Yamagata, H. Saito, O. Habuchi, and S. Suzuki, J. Biol. Chem., 243,
1523 (1968)); Chondroitinase AC II (derived from Arthrobacter
aurescens; K. Hiyama, and S. Okada, J. Biol. Chem., 250, 1824
(1975), K. Hiyama and S. Okada, J. Biochem. (Tokyo), 80, 1201
(1976)); Hyaluronidase ACIII (derived from Flavobacterium sp.
Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, Keiichi Yoshida, Kiyoshi
Morikawa, and Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1989));
Chondroitinase B (derived from Flavobacterium heparinum; Y. M.
Michelacci and C. P. Dietrich, Biochem. Biophys. Res. Commun., 56,
973 (1974), Y. M. Michelacci and C. P. Dietrich, Biochem. J., 151,
121 (1975), Kenichi Maeyama, Akira Tawada, Akiko Ueno, and Keiichi
Yoshida, Seikagaku, 57, 1189 (1985)); Chondroitinase C (derived
from Flavobacterium sp. Hp102; Hirofumi Miyazono, Hiroshi Kikuchi,
Kelichi Yoshida, Kiyoshi Morikawa, and Kiyochika Tokuyasu,
Seikagaku, 61, 1023 (1989)); and the like.
[0018] Glycoproteins are composed of a polypeptide chain covalently
bound to one or more carbohydrate moieties. There are two broad
categories of glycoproteins that possess carbohydrates coupled
though either N-glycosidic or O-glycosidic linkages to their
constituent protein. The N- and O-linked glycans are attached to
polypeptides through asparagine-N-acetyl-D-glucosamine and serine
(threonine)-N-acetyl-D-galactosamine linkages, respectively.
Complex N-linked oligosaccharides do not contain terminal mannose
residues. They contain only terminal N-acetylglucosamine,
galactose, and/or sialic acid residues. Hybrid oligosaccharides
contain terminal mannose residues as well as terminal
N-acetylglucosamine, galactose, and/or sialic acid residues.
[0019] With N-linked glycoproteins, an oligosaccharide precursor is
attached to the amino group of asparagine during peptide synthesis
in the endoplasmic reticulum. The oligosaccharide moiety is then
sequentially processed by a series of specific enzymes that delete
and add sugar moieties. The processing occurs in the endoplasmic
reticulum and continues with passage through the cis-, medial- and
trans-Golgi apparatus.
SUMMARY OF THE INVENTION
[0020] Provided herein are soluble glycosaminoglycanase enzymes
particularly members of the soluble neutral-active Hyaluronidase
Glycoprotein family (also referred to herein as sHASEGPs),
preferred members being human soluble neutral-active Hyaluronidase
Glycoproteins, particularly the human soluble PH-20 Hyaluronidase
Glycoproteins (also referred to herein as rHuPH20s). The soluble
neutral-active Hyaluronidases provided herein are each a sHASEGP
family member, designated herein as a sHASEGP. The soluble
Hyaluronidase domain, and uses thereof are also provided. While a
number of uses and applications of soluble glycosaminoglycanases
(sometimes referred to herein as GAG Enzymes) are illustrated
herein using rHuPH20 as an exemplary glycosaminoglycanase (e.g. in
promoting the delivery of pharmacologics and other agents within
tissues of the mammalian body), it will be appreciated by those of
skill in the art that other glycosaminoglycanases such as those
described herein or others known in the art can be applied to a
number of such applications. Presently preferred soluble
glycosaminoglycanases, such as sHASEGPs, exhibit some hyaluronidase
activity and can exhibit other glycosaminoglycanase activities as
well. Human soluble glycosaminoglycanases are presently preferred
for applications in which the enzyme is to be employed within the
human body, including many medical applications as described and
illustrated herein.
[0021] One aspect of the invention is based upon the discovery that
a soluble, neutral-active hyaluronidase activity can be produced
with high yield in a mammalian expression system by introducing
nucleic acids that lack a narrow region encoding amino acids in the
carboxy terminus of the human PH20 cDNA. Additional modifications
of the sHASEGP to enhance secretion by use of non-native leader
peptides are also provided. Further provided are methods to modify
the sHASEGP to prolong its half life by way of masking the protein
with polyethylene glycol (PEG) and/or post-translational
modifications to native glycosylation. Previous attempts to
generate soluble neutral-active human hyaluronidase glycoproteins
were unsuccessful. It was concluded that truncations of the human
hyaluronidase polypeptide resulted in both a loss of neutral
enzymatic activity, and an inability of cells to secrete the
recombinant protein in mammalian expression systems (Arming, et al
Eur J Biochem Aug. 1, 1997; 247 (3):810-4). It is critical to
generate neutral-acting secreted sHASEGP for commercial production
and therapeutic utility as a hyaluronidase. The invention,
disclosed herein, overcomes these and other challenges.
[0022] The invention further comprises a catalytically active human
sHASEGP glycoprotein wherein the sHASEGP possesses at least one
N-linked sugar moiety. The studies shown herein demonstrate that
human PH20 requires N-linked glycans for catalytic activity,
whereas bovine and bee venom hyaluronidases remain active without
such N-linked glycans. A human hyaluronidase PH20 domain devoid of
N-linked moieties is catalytically inactive. Thus classic
recombinant DNA technology does not permit the production of a
catalytically active human sHASEGP, unlike bee venom hyaluronidase,
which can be produced in E. coli.
[0023] The invention includes methods and cells for generation of
an N-linked sHASEGP glycoprotein polypeptide, by using of a cell
capable of introducing said N-linked sugar moieties or by
introduction of said N-linked moieties on a sHASEGP polypeptide.
Methods of identifying properly glycosylated sHASEGPs are further
disclosed.
[0024] Catalytically active PEGylated and/or super-Sialated sHASEGP
glycoproteins are also provided. PEGylated and/or super-sialated
sHASEGPs possess greater serum half-lives compared to naturally
occurring non-sialated bovine and ovine testes sHASEGPs, and are
thus preferable for both enzyme stability and use as drugs or
adjuvants in circumstances in which prolonged half-lives are
desirable (such as is commonly the case with intravenous delivery).
The invention provides methods for the preparation of PEGylated
and/or super-Sialated sHASEGPs, compositions and uses thereof.
Without being restricted to a particular application or mechanism
of action, it is generally considered that the attachment of PEG
moieties to sHASEGPs and other glycosaminoglycanases can be used to
effectively shield the molecules, decreasing its relative
sensitivity to proteases and potentially also decreasing the extent
and rate of its clearance and elimination from the body. Applying
these principles and techniques to other glycosaminoglycanases,
PEGylated, super-sialated and/or otherwise modified versions of
such other glycosaminoglycanases can likewise be generated and used
in the context of methods and techniques as described and
illustrated herein (e.g. techniques for promoting the dispersion of
pharmacologics and other agents within tissues of the body).
[0025] Proteins encoded by naturally GPI anchor deficient sHASEGP
splice variants are also provided.
[0026] Further provided are compositions of the sHASEGP comprising,
a soluble sHASEGP glycoprotein with a metal ion, wherein the metal
ion is Calcium, Magnesium or Sodium. sHASEGPs are generally
optimally active in the presence of said metals. Formulations
consisting of sHASEGP in the presence of said metal ions are also
provided.
[0027] Modifications of sHASEGPs and other glycosaminoglycanases to
further prolong their half life are provided. Chemical
modifications of a sHASEGPs and other glycosaminoglycanases with
polymers such as polyethylene glycol and dextran are provided. Such
modifications shield sHASEGPs and other glycosaminoglycanases from
removal from circulation and the immune system as well as
glycosylation receptors for mannose and asialoglycoprotein. Further
provide are methods to link to specific functional groups such as
glycosylation sites, positively charged amino acids and
cysteines.
[0028] Assays for identifying effectors, such as compounds,
including small molecules, and conditions, such as pH, temperature
and ionic strength, that modulate the activation, expression or
activity of sHASEGPs, are also provided herein. In exemplary
assays, the effects of test compounds on the ability of a
Hyaluronidase domain of sHASEGP to cleave a known substrate,
typically a glycosaminoglycan or proteoglycan, are assessed.
Agents, generally compounds, particularly small molecules, that
modulate the activity of the Hyaluronidase domain are candidate
compounds for modulating the activity of the sHASEGP. The
Hyaluronidase domains can also be used to produce
Hyaluronidase-specific antibodies with function-perturbing
activity. The Hyaluronidase domains provided herein include, but
are not limited to, the N-terminal glycsoyl-hydrolase domain with
C-terminal truncated portions thereof that exhibit catalytic
activity in vitro.
[0029] Nucleic acid molecules encoding the proteins and
Hyaluronidase domains are also provided. Nucleic acid molecules
that encode a soluble Hyaluronidase domain or catalytically active
portions thereof and also those that encode the full-length sHASEGP
are provided. Nucleic acid encoding an exemplary Hyaluronidase
domain and downstream nucleic acid is set forth in SEQ ID No. 6;
and the Hyaluronidase domain of an exemplary sHASEGP is set forth
in SEQ ID No. 1 (amino acids 35-464). The protein sequence and
encoding nucleic acid sequence of an exemplary full-length sHASEGP
are set forth in SEQ ID Nos. 1 and 6.
[0030] Also provided are nucleic acid molecules that hybridize to
such sHASEGP-encoding nucleic acid along their full-length or along
at least about 70%, 80% or 90% of the full-length and encode the
Hyaluronidase domain or portion thereof are provided. Hybridization
is generally effected under conditions of at least low, generally
at least moderate, and often high stringency.
[0031] The isolated nucleic acid fragment is DNA, including genomic
or cDNA, or is RNA, or can include other components, such as
protein nucleic acid or other nucleotide analogs. The isolated
nucleic acid may include additional components, such as
heterologous or native promoters, enhancers and other
transcriptional and translational regulatory sequences, these genes
may be linked to other genes, such as reporter genes or other
indicator genes or genes that encode indicators.
[0032] Also provided is an isolated nucleic acid molecule that
includes the sequence of molecules that is complementary to the
nucleotide sequence encoding sHASEGP or the portion thereof.
[0033] Also provided are fragments thereof or oligonucleotides that
can be used as probes or primers and that contain at least about 10
to 16 nucleotides, typically at least about 20 nucleotides, and
generally less than 1000, typically less than about 100
nucleotides, set forth in SEQ ID NO. 6 (or the complement thereof);
or contain at least about 30 nucleotides (or the complement
thereof) or contain oligonucleotides that hybridize along their
full-length (or at least about 70, 80 or 90% thereof) to any such
fragments or oligonucleotides. The length of the fragments are a
function of the purpose for which they are used and/or the
complexity of the genome of interest. Generally probes and primers
contain less than about 50, 150 or 500 nucleotides.
[0034] Also provided are plasmids containing any of the nucleic
acid molecules provided herein. Cells containing the plasmids are
also provided. Such cells include, but are not limited to,
bacterial cells, yeast cells, fungal cells, plant cells, insect
cells and animal cells.
[0035] Also provided are enhanced mammalian expression systems
using signal leaders capable of efficient secretion of sHASEGPs. An
example of such efficient secretory leader peptide amino acid
sequence and fusion protein with a sHASEGP is found in SEQ ID Nos.
43 and 46.
[0036] Also provided is a method of producing sHASEGPs by growing
the above-described cells under conditions whereby the sHASEGP is
expressed by the cells, and recovering the expressed sHASEGP
polypeptide or glycoprotein. Methods for isolating nucleic acid
encoding other sHASEGPs are also provided.
[0037] Also provided are cells, generally eukaryotic cells, such as
mammalian cells and yeast cells, in which a sHASEGP polypeptide is
expressed on the surface of the cells. Such cells can be used in
drug screening assays to identify compounds that modulate the
activity of the sHASEGP polypeptide. These assays, including in
vitro binding assays, and transcription based assays in which
signal transduction mediated directly or indirectly, such as via
activation of pro-growth factors, by the sHASEGP is assessed.
[0038] Also provided are peptides encoded by such nucleic acid
molecules. Included among those polypeptides is the sHASEGP
Hyaluronidase domain or a polypeptide with amino acid changes such
that the specificity and/or Hyaluronidase activity remains
substantially unchanged. In particular, a substantially purified
mammalian sHASEGP glycoprotein is provided that comprises a
secreted neutral-active enzyme.
[0039] The invention also includes a Hyaluronidase catalytic domain
and may additionally include other domains. The sHASEGP may form
homodimers and can also form heterodimers with some other protein,
such as a membrane-bound protein. Also provided is a substantially
purified glycoprotein including a sequence of amino acids that has
at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
to an exemplary sHASEGP where the percentage identity is determined
using standard algorithms and gap penalties that maximize the
percentage identity.
[0040] Splice variants of sHASEGPs, particularly those with a
catalytically active Hyaluronidase domains, are contemplated
herein.
[0041] In other embodiments, substantially purified polypeptides
that include a Hyaluronidase domain of a sHASEGP polypeptide or a
catalytically active portion thereof, but that do not include the
entire sequence of amino acids set forth in SEQ ID No. 1 are
provided. Among these are polypeptides that include a sequence of
amino acids that has at least 70%, 80%, 85%, 90%, 95% or 100%
sequence identity to SEQ ID No. 1 or 3.
[0042] In a specific embodiment, a nucleic acid that encodes a
eukaryotic Hyaluronidase glycoprotein, designated a eukaryotic
sHASEGP is provided. In particular, the nucleic acid includes the
sequence of nucleotides set forth in SEQ ID No. 6, particularly set
forth as nucleotides 106-1446 of SEQ ID NO. 6, or a portion thereof
that encodes a catalytically active polypeptide.
[0043] Also provided are nucleic acid molecules that hybridize
under conditions of at least low stringency, generally moderate
stringency, more typically high stringency to the SEQ ID NO. 6 or
degenerates thereof.
[0044] In one embodiment, the isolated nucleic acid fragment
hybridizes to a nucleic acid molecule containing the nucleotide
sequence set forth in SEQ ID No: 6 (or degenerates thereof) under
high stringency conditions. A full-length sHASEGP is set forth in
SEQ ID No. 1 and is encoded by SEQ ID NO. 6 or degenerates
thereof.
[0045] Also provided are muteins of the Hyaluronidase domain of
sHASEGPs, particularly muteins in which one or more Cys residues in
the Hyaluronidase domain that are free (i.e., do not form disulfide
linkages with any other Cys residue in the Hyaluronidase domain)
are substituted with another amino acid, typically, although not
necessarily, with a conservative amino acid substitution or a
substitution that does not eliminate the activity, and muteins in
which a specific glycosylation site (or sites) is eliminated.
[0046] sHASEGP polypeptides, including, but not limited to splice
variants thereof, and nucleic acids encoding sHASEGPs, and domains,
derivatives and analogs thereof are provided herein. Single chain
secreted Hyaluronidase glycoproteins that have an N-terminus
functionally equivalent to that generated by activation of a signal
peptidase to form sHASEGP are also provided. There are seven
potential N-linked glycosylation sites at N82, N166, N235, N254,
N368, N393, N490 of the human PH20 sHASEGP exemplified in SEQ ID
NO: 1. Disulfide bonds form between the Cys residues C60-C351 and
Cys residues C224 to C238 to form the core Hyaluronidase domain.
However, additional cysteines are required in the carboxy terminus
for neutral enzyme catalytic activity such that the sHASEGP domain
from amino acids 36 to Cys 464 in SEQ ID No.1 comprises the
minimally active human PH20 sHASEGP hyaluronidase domain. Thus,
N-linked glycosylation site N-490 is not required for proper
sHASEGP activity. As will be appreciated by those of skill in the
art, minor changes can be made to such compositions as described
and illustrated herein without substantially eliminating or in some
cases without substantially reducing (or potentially even
improving) their useful activity, and can thus be similarly
employed in various applications as described herein.
[0047] N-linked glycosylation of some sHASEGPs (such as the sHASEGP
comprising the amino acid sequence of SEQ ID No.1) can be very
important for their catalytic activity and stability. While
altering the type of glycan modifying a glycoprotein can have
dramatic affects on a protein's antigenicity, structural folding,
solubility, and stability, most enzymes are not thought to require
glycosylation for optimal enzyme activity. Such sHASEGPs are thus
unique in this regard, in that removal of N-linked glycosylation
can result in near complete inactivation of the Hyaluronidase
activity. For such sHASEGPs, the presence of N-linked glycans is
critical for generating an active enzyme. Protein expression
systems suitable for the introduction of critical N-linked
glycosylation residues on sHASEGPs are included. Additionally, the
introduction of deglycosylated sHASEGP polypeptide in the presence
of extracts capable of introducing N-linked glycans are included.
In one aspect of the invention, complex glycosylation capped with
sialation is described whereas others capped with free mannose
residues are contemplated as well. Preferably, sialic acid residues
are found in the terminal residues of N-linked glycosylation on
sHASEGPs.
[0048] N-linked oligosaccharides fall into several major types
(oligomannose, complex, hybrid, sulfated), all of which have (Man)
3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn
residues that fall within -Asn-Xaa-Thr/Ser- sequences (where Xaa is
not Pro). Glycosylation at an -Asn-Xaa-Cys- site has been reported
for coagulation protein C. N-linked sites are often indirectly
assigned by the appearance of a "blank" cycle during sequencing.
Positive identification can be made after release of the
oligosaccharide by PNGase F, which converts the glycosylated Asn to
Asp. After PNGase F release, N-linked oligosaccharides can be
purified, for example by using Bio-Gel P-6 chromatography, with the
oligosaccharide pool subjected to preparative high pH anion
exchange chromatography (HPAEC) (Townsend et al., (1989) Anal.
Biochem. 182, 1-8). Certain oligosaccharide isomers can be resolved
using HPAEC. Fucose residues will shift elution positions earlier
in the HPAEC chromatogram, while additional sialic acid residues
will increase the retention time. Concurrent treatment of
glycoproteins whose oligosaccharide structures are known (e.g.,
bovine fetuin, a-1 acid glycoprotein, ovalbumin, RNAse B,
transferrin) can facilitate assignment of the oligosaccharide
peaks. The collected oligosaccharides can be characterized by a
combination of compositional and methylation linkage analyses
(Waegheet al., (1983) Carbohydr Res. 123, 281-304.), with anomeric
configurations assigned by NMR spectroscopy (Van Halbeek (1993) in
Methods Enzymol 230).
[0049] In the case of some sHASEGPs, exemplified by the human
sHASEGP rHuPH20 described herein, the sHASEGP can comprise both
N-glycosidic and O-glycosidic linkages. For example, rHuPH20 (as
produced in the DG44 CHO line 3D3, as described in the Examples
below) has been discovered to have O-linked oligosaccharides as
well as N-linked oligosaccharides. Glycosidic modifications of such
sHASEGPs (for example, modifications comprising one or more
additions, removals or alterations of such N-linked and/or O-linked
oligosaccharides can be used to generate glycosidic variant
sHASEGPs exhibiting altered pharmacokinetic and/or pharmacodynamic
profiles that make them desirable for particular applications. By
way of illustration, but without being restricted to a particular
mechanism of action, removal or alteration of (such as by capping
or otherwise de-exposing) one or more N-linked and/or O-linked
oligosaccharides that are involved in binding to cellular or other
receptors can be used to alter the extent and/or rate at which a
sHASEGP binds to or is taken up by a particular tissue, which can
in turn be used to alter for example its pharmacokinetic profile
(e.g., by increasing its serum half life or otherwise altering its
bioabsorption).
[0050] Formulations of sHASEGPs are also provided. sHASEGPs may be
formulated in lyophilized forms and stabilized solutions for
example. Formulations containing specific metal ions, such as
calcium, magnesium, or sodium, are useful for optimal activity at
neutral pH. In addition to stabilized solution formulations, slow
release formulations are contemplated herein for extended removal
of glycosaminoglycans or extended promotion of spreading or
diffusion of agents such as pharmacologics. Also provided herein
are kits providing for pre-packaged syringes of sHASEGPs for the
administration of small volumes of sHASEGP for intraocular surgical
procedures and other small volume procedures. Balanced salt
formulations for ex vivo use in artificial reproductive technology
procedures are also provided.
[0051] Methods for the use of glycosaminoglycanases including
sHASEGPs in the removal of glycosaminoglycans are also provided.
Glycosaminoglycanases including sHASEGPs open channels in the
interstitial space through degradation of glycosaminoglycans that
generally permit the diffusion of molecules less than about 500 nm
in size. These channels can remain relatively open for a period of
24-48 hours depending on dose and formulation. Such channels can be
used to facilitate the diffusion of exogenously added molecules
such as fluids, small molecules, proteins, nucleic acids and gene
therapy vectors and other molecules less than about 500 nm in size.
In addition, without being restricted to a particular theory or
mechanism of action, it is believed that the formation of such
channels can facilitate bulk fluid flow within an interstitial
space, which can in turn promote the dispersion or movement of a
solute (such as a detectable molecule or other diagnostic agent, an
anesthetic or other tissue-modifying agent, a pharmacologic or
pharmaceutically effective agent, or a cosmetic or other esthetic
agent) that is effectively carried by the fluid in a process
sometimes referred to herein as "convective transport" or simply
convection. Such convective transport can substantially exceed the
rate and cumulative effects of molecular diffusion and can thus
cause the therapeutic or other administered molecule to more
rapidly and effectively perfuse a tissue. Furthermore, when a
molecule such as a therapeutic or other agent (such as a small
molecule drug or a larger molecule or complex) is co-formulated or
co-administered with a sHASEGP (or other glycosaminoglycanase) and
both are injected into a relatively confined local site, such as a
site of non-intravenous parenteral administration (e.g.,
intradermal, subcutaneous, intramuscular, or into or around other
internal tissues, organs or other relatively confined spaces within
the body), then the fluid associated with the administered dose can
both provide a local driving force (i.e. hydrostatic pressure) as
well as lower impedance to flow (by opening channels within the
interstitial matrix)--both of which would tend to increase fluid
flow, and with it convective transport of the therapeutic agent or
other molecule contained within the fluid. As discussed and
illustrated in more detail herein, and as will be appreciated by
those of skill in the art, these aspects of the use of sHASEGPs and
other glycosaminoglycanases can have substantial utility for
improving the bioavailability as well as manipulating other
pharmacokinetic and/or pharmacodynamic characteristics of
co-formulated or co-administered agents.
[0052] sHASEGPs and other glycosaminoglycanases can also be used to
remove excess glycosaminoglycans such as those that occur following
ischemia reperfusion, inflammation, arteriosclerosis, edema,
cancer, spinal cord injury and other forms of scarring. In some
instances, sHASEGPs and other glycosaminoglycanases can be
delivered systemically by intravenous infusion. This can be helpful
when local access is not readily available such as the heart or
brain or in the case of disseminated neoplasm wherein the disease
is through the body. For such intravenous or other intravascular
applications, Super-Sialated sHASEGPs are often preferable to
increase serum half-life and distribution over native hyaluronidase
enzymes that lack terminal sialic acids.
[0053] In some circumstances, such as spinal cord injury and other
nervous system disorders, glaucoma and certain other disorders of
the eye, as well as various autoimmune, inflammatory conditions,
cancers and other chronically progressive diseasesand conditions,
and cosmetic treatments, sustained delivery is preferred. As will
be appreciated by those of skill in the art, a number of different
compositions and techniques have been developed to provide
slow-release or sustained-release formulations or depots of
molecules of interest.
[0054] In other indications, a single short acting dose is
preferable. Temporary removal of glycosaminoglycans can be used to
enhance the delivery of solutions and drugs into and/or through
interstitial spaces. This can be useful for the diffusion of
anesthesia and for the administration of therapeutic fluids,
molecules and proteins. Subcutaneous, intradermal and intramuscular
administration of molecules in the presence of sHASEGPs (and/or
other glycosaminoglycanases) also facilitate their systemic
distribution more rapidly. Such methods are very useful when
intravenous access is not available or where more rapid systemic
delivery of molecules is needed. By way of illustration, delivery
of other large molecules such as Factor VIII, that are poorly
bioavailable upon subcutaneous administration, may be injected with
sHASEGPs to increase their availability.
[0055] Uses of sHASEGPs for enzymatic removal of the cumulus matrix
surrounding oocytes are also provided. The removal of the cumulus
matrix using a purified sHASEGP without the toxic contaminants of
extract derived hyaluronidase permits more gentle recover of the
oocyte with greater viabilities. Moreover, sHASEGPs can be
manufactured without the use of cattle extracts or other organisms
that carry viruses and other pathogens such as transmissible
spongiform encephalopathies.
[0056] Injections of small volumes of sHASEGP for intraocular use
may also be used for small spaces. For example, sHASEGPs and/or
other glycosaminoglycanases may be injected into the anterior
chamber of the eye to remove excess viscoelastic substrates that
are administered during surgery. Intraocular injection of sHASEGPs
can also be used to reduce intraocular pressure in glaucoma, to
dissolve vitreous aggregates, or "floaters", to clear vitreous
hemorrhage, for the treatment of macular degeneration, to promote
vitreo retinal detachment in diabetic retinopathy and to be mixed
with other enzymes to promote reshaping of the cornea along with
corrective lenses. As described herein, sHASEGPs and/or other
glycosaminoglycanases can also be injected into the eye or into
surrounding periorbital spaces as a form of "spreading agent," i.e.
to promote the delivery of diagnostic, anesthetic or pharmacologic
agents to sites within and/or around the target tissue. For
example, injection of a sHASEGP concomitant with or in temporal
proximity to injection of diagnostic, anesthetic or pharmacologic
agents into the sub-Tenon's or episcleral space can be used to
promote transcleral delivery of the pharmacologic to interior
portions of the eye (such as the choroid, retina and vitreous
body). The episcleral space is a lymph-containing space between the
Fascia Bulbi (capsule of Tenon) and the sclera which is a
relatively tough protective tissue surrounding the choroid and
retina and the interior of the eye. The Sub-Tenon's or episcleral
space, which is sometimes referred to as the periscleral lymph
space, is generally also continuous with the subdural and
subarachnoid cavities and is traversed by bands of connective
tissue extending from the fascia to the sclera. As will be
appreciated by those of skill in the art, the ability to promote
delivery of compositions across the protective scleral layer
covering the eye allows for a great variety of pharmacologic and
other agents to be conveniently and efficiently delivered to
underlying tissues within the eye, such as the choroid, retina or
vitreous humor. By applying compositions of the present invention
to such in vivo delivery routes even relatively difficult to reach
tissues, such as those at the back of the eye, can be treated by
minimally invasive means. It will be recognized that in some
instances, the use of a longer lasting sHASEGP such as a
pegylated-sHASEGP will be desirable.
[0057] It is important to note that while animal-derived
hyaluronidases can and have been applied to treat certain
conditions of the eye, and as a spreading factor to promote the
delivery of anesthetics and other therapeutic or pharmacologic
agents, several important considerations apply to the use of
animal-derived enzymes and potentially either limit their use or
introduce safety concerns or additional risks when they are used
for treating humans. Among these considerations is the concern that
proteins and other impurities present in the formulations can lead
to immunogenic and/or other problems when applied to human tissues
in vivo. In addition, to the extent that non-human animal enzymes
are employed, even purified enzymes can themselves further
contribute to immunogenicity since they are of non-human origin.
Further in that regard, the potential for degradative enzymes such
as hyaluronidases to effectively promote their own dispersal (as
described herein), could increase the probability that immunogenic
versions or formulations of such animal-derived products are
contacted by the immune system.
[0058] Concerns with animal-derived and/or impure factors are also
of increased significance in a number of situations; for example,
situations in which the target tissue has already been subjected to
surgical or other procedures, situations in which the agent is
being introduced into a relatively immune-privileged space (such as
portions of the eye, the heart, the nervous system and many other
tissues and organs that are not typically exposed to the external
environment), and/or situations in which the patient is
immuno-compromised or otherwise at elevated risk associated with
infection. Use of such animal-derived formulations may thus be
avoided in a number of applications in which the risk of infection
or other complications undercut their potential utility. In those
situations and in others, the use of recombinantly-prepared
sHASEGPs of the present invention can provide applications that are
both highly effective and have favorable safety profiles, and thus
improve and expand the conditions and situations in which these
enzymes are beneficially used.
[0059] In the context of non-intravenous parenteral injections
(such as intradermal, subcutaneous, intramuscular and other
injections into spaces other than the vasculature), a sHASEGP
(and/or another glycosaminoglycanase) and another agent (e.g. a
co-formulation or a mixture comrising a sHASEGP and another agent
such as a diagnostic agent, an anesthetic agent, a pharmacologic
agent, an esthetic agent, or combinations therefo) in a volume of
liquid (e.g. a pharmaceutical excipient or other solution) can be
introduced into a site or sites within the body by injection or
infusion. Without wishing to be bound by theory, it is believed
that several forces can be brought into motion to enhance delivery
of the pharmacologic or other agent (the extent of which depends in
part on the particular composition, volume and site of
administration for example). These driving forces can include an
increase in hydrostatic pressure as a volume of fluid is
effectively forced into a contained space (such as the sub-Tenon's
space referred to above, or a site of intradermal, subcutaneous,
intramuscular or other non-IV parenteral injection), a subsequent
increase in convective transport of solutes (or convection) as
fluid flow is increased down its pressure gradient (and dissolved
molecules or macromolecular complexes are carried with it), as well
as an increase in diffusion and/or permeation mediated by
degradation of glycosaminoglycans and concomitant channel formation
within the downstream intercellular matrix or interstitial
space.
[0060] In the case of sHASEGPs being injected into the anterior
chamber of the eye to remove excess viscoelastic substrates such as
glycosaminoglycan-containing formulations (which are commonly used
as opthalmosurgical aids in a variety of anterior segment
procedures), it will be appreciated by those of skill, in view of
the teachings of the present application, that sHASEGP can be used
to supplement or to obviate the need for post-surgical procedures
such as irrigation and aspiration, which are commonly used to
reduce the amount of applied viscoelastic remaining in the eye
after surgery is completed. Viscoelastics are commonly used during
anterior segment surgeries to maintain a deep anterior chamber
allowing for more efficient manipulations and potentially
protecting the corneal endothelium and other tissues during
surgery. A variety of viscoelastics containing hyaluronate and/or
chondroitin sulfate are used in surgeries such as anterior eye
segment procedures (see, e.g., Provisc.TM., Viscoat.TM. and
Duovisc.TM. (available from Alcon Laboratories, Inc.,
www.alconlabs.com); Healon.TM., Healon 5.TM. and Healon GV.TM.
(available from Advanced Medical Optics, www.healon.com);
Amvisc.TM. and Amvisc.TM. Plus (available from Bausch & Lomb,
www.bausch.com); I-Visc.TM., I-Visc.TM. Plus, I-ViSc.TM. 18, and
I-Visc.TM. Phaco (available from I-MED Pharma, www.imedpharma.com);
LA LON.TM. (available from General Innovations,
www.generaltrade.net)). However, as is known in the art, retained
viscoelastic in the anterior chamber following surgical procedures
such as cataract surgery can lead to compromised outflow from the
anterior chamber (potentially by reducing outflow through the
trabecular meshwork) which can cause elevations in intraocular
pressure (IOP spikes) that can lead to glaucoma.
[0061] Thus sHASEGPs of the present invention can be introduced,
for example, by injection into the anterior chamber of the eye as a
"viscoelastic antidote" for previously-applied viscoelastic
formulations containing glycosaminglycans such as hyaluronan. For
use as such a viscoelastic antidote, the sHASEGP will generally be
introduced by post-surgical injection into the anterior chamber
(with or without prior irrigation or aspiration to remove some
portion of the previously applied viscoelastic) following anterior
segment procedures such as cataract surgery and intraocular lens
implantation (e.g. by phacoemulsification or extracapsular
surgery), glaucoma filtration surgery (trabeculectomy), corneal
transplant surgery (e.g. penetrating keratoplasty), post-traumatic
eye surgery, and the like. As will be appreciated by those of skill
in the art, the amount of a sHASEGP applied in such a context will
depend, inter alia, on its specific activity as well as the amount
of viscoelastic used and whether or not some of the viscoelastic is
first physically removed (e.g. by irrigation and aspiration). Each
application will thus generally be optimized for the particular
circumstances, as will be appreciated by those of skill in the art
in view of the descriptions and illustrations provided herein.
[0062] Some viscoelastics, such as ViscoatTm and Duovisc.TM.
(available from Alcon Labs) comprise both hyaluronate and
chondroitin sulfate. Advantageously, many sHASEGPs of the present
invention are enzymatically active both as hyaluronidases and as
chondroitinases, making them particularly suitable for such dual
formulation viscoelastics.
[0063] Hyaluronidases including sHASEGPs can also be combined with
one or more additional glycosaminoglycanases (such as
chondroitinases) for applications such as the foregoing or to
further open channels within interstitial spaces for other
applications such as those described and illustrated herein.
[0064] To the extent that viscoelastics are also used in other
procedures, sHASEGPs of the present invention can likewise be used
in conjunction with or as replacement of techniques for reducing or
eliminating amounts of applied viscoelastic, i.e. as a viscoelastic
antidote.
[0065] In applications of sHASEGPs as spreading agents in tissues
such as those of the eye, a formulation comprising one or more
sHASEGPs as described herein is typically applied to a target
tissue prior to or concomitant with application of an anesthetic,
diagnostic, pharmacologic or other agent that is desired to be
delivered into or to the vicinity of the target tissue. In some
contexts, sHASEGPs can be used to promote delivery of a
pharmacologic or other agent across a first tissue to target one or
more tissues distal to the first. By way of illustration,
introduction of sHASEGPs into the episcleral space surrounding the
eye prior to or concomitantly with introduction of a pharmacologic
or other agent into the episcleral space can be used to promote
delivery of the agent across the protective scieral tissue and into
the choroid, the retina and the vitreous body. In this manner, a
number of pharmacologic and/or other agents which are useful for
treating conditions of the choroid, retina and/or vitreous can be
delivered relatively locally to the target tissues but at the same
time using a relatively non-invasive approach such as a Sub-Tenon's
injection. Analogous situations exist in other situations in which
sHASEGPs can be applied to one tissue or location in the body in
order to facilitate the delivery of a pharmacologic or other agent
within that tissue or location and/or to distal sites, as described
and illustrated herein.
[0066] While the extent and rate of dispersal of a co-formulated or
co-administered agent depends in part on the tissue and agent
involved, such disperal can generally be increased by, inter alia,
increasing the amount of sHASEGP applied, employing a sHASEGP
having higher specific activity, employing a sHASEGP that is more
resistant to degradation or other inactivation (such as a
PEGylated, super-sialated or other such modified sHASEGP),
providing a sustained release or depot formulation of the sHASEGP,
and other such approaches to increasing the effective activity
and/or duration of the applied sHASEGP. In addition, where it is
desired that the sHASEGP faciliate the dispersion of an agent into
a tissue or location distal to, e.g., a site of injection, then
providing the sHASEGP and/or agent in a significant volume of
liquid (such as would partially fill or even slightly distend the
site of injection), can be used to further promote dispersion
through a process of convective transport, as described and
illustrated herein.
[0067] As will be appreciated by those of skill in the art,
sHASEGPs can thus be used to effectively promote delivery of a
number of anesthetics, diagnostics, pharmacologics and/or other
agents to the posterior segments of the eye for treating conditions
such as retinal detachments, retinal vein occlusions, proliferative
retinopathies, diabetic retinopathies, inflammatory conditions
(such as uveitis, choroiditis, retinitis and the like), as well as
degenerative diseases, vascular diseases and various tumors. Again
as will be appreciated by those of skill in the art, a variety of
pharmacologic or pharmaceutically effective agents can be usefully
applied to treating such posterior segment conditions and diseases,
including, by way of illustration, anesthetic and pharmacologic
agents such as those described and illustrated below.
[0068] Co-formulations or co-administrations of sHASEGP with other
substances may also be envisioned for injectable pens for small
volume or rapid subcutaneous administration. Examples such as
Epipen.TM., insulin, and other fluids can be formulated. The
methods of the invention include administration of the sHASEGP
polypeptide or pharmaceutical compositions containing sHASEGP prior
to, simultaneously with or following administration of other
therapeutic molecules. The sHASEGP may be administered at a site
different from the site of administration of the therapeutic
molecule or the sHASEGP may be administered at a site the same as
the site of administration of the therapeutic molecule.
[0069] Without wishing to be bound by theory, it is believed that
the ability of sHASEGPs and other glycosaminoglycanases to cause
the degradation of a portion of the glycosaminoglycans in the
interstitial spaces between cells results in a temporary opening up
of channels within the interstitium, which in turn tends to
increase interstitial fluid flow and to concomitantly facilitate
the diffusion and/or convective solute transport (convection) of
dissolved components within the interstitial fluid (such as
anesthetics, drugs and other pharmacologic agents, labels and
diagnostic agents, and the like). As will be appreciated by those
of skill in the art, sHASEGPs of the present invention can be
applied to enhance the bioavailability (and potentially improve
other pharmacokinetic and/or pharmacodynamic properties) of a
number of pharnacologic and other agents that are useful for
treating or diagnosing various disease conditions or otherwise
modifying one or more tissues in vivo. Illustrative categories of
such agents include: anti-cancer agents, anti-infectives,
anesthetics, anti-inflammatories, cytokines, antibodies and other
proteins, nucleic acids, macromolecular complexes, and numerous
other molecules and pharmacologic agents which modify cellular or
other physiologic activities, including various categories of
agents (and exemplary members thereof) described herein and in the
art.
[0070] While the diffusion and convective transport of even small
molecules can be enhanced by opening interstitial channels and
increasing fluid flow, in the case of larger pharmacologic or other
agents, such as many biotherapeutics (including antibodies and
other proteins, large nucleic acids, macromolecular complexes (such
as liposomes and other macromolecular carriers), as well as gene
therapy vectors and the like)), the size of the molecules and the
presence of interstitial components such as glycosaminoglycans
substantially impairs the diffusion and/or convection of the
agents. Several consequences potentially limiting the agent's
usefulness can result. For example, the pharmacokinetics of the
agent can be effectively impaired by a slowing of absorption and
thus distribution of the agent. In addition, trapping of a portion
of the agent at or near the site of administration both limits its
bioavailability and can also cause toxicity as a result of a
potentially sustained and high local dose. In the latter regard,
local toxicity that may be associated with painful or other side
effects is a problem with many large biomolecules that are
administered by non-intravenous injection, such as by subcutaneous,
intradermal or intramuscualar injection. As a result, a number of
pharmacologic agents have pharacokinetic (PK) and/or
pharmacodynamic (PD) profiles that can be enhanced by
co-formulating the agent with a sHASEGP (or other
glycosaminoglycanase) and/or co-administering the agent with a
sHASEGP (or other glycosaminoglycanase), which may be provided
before, coincident with or after the agent, and administered at the
same or a different site, which parameters would be the subject of
optimization in standard models (such as animal models typically
used to assess the pharmacokinetics and pharmacodynamics of the
agent). Any of a variety of therapeutics and pharmacologics as well
as other agents (such as cosmetic or esthetic formulations) that
are typically administered by parenteral administration can thus be
enhanced using sHASEGPs.
[0071] While intravascular administration, particularly by
intravenous (IV) injection, can generally provide for rapid
bioavailability, IV injections are often inconvenient, associated
with risks or inapplicable (and even where practicable can be
associated with less than optimal overall PK/PD profiles). Since
sHASEGPs can be applied to enhance the bioavailability of agents
administered by non-IV parenteral routes (such as subcutaneous,
intradermal, intramuscular and other routes delivering agents into
the interstitium), agents that have been delivered by IV injection
can be reformulated for delivery by non-IV parenteral routes. Such
reformulations can provide substantial benefits such as allowing
use of agents in which IV administration is inconvenient,
inapplicable, unsafe, too expensive or otherwise limited. The
reformulation of IV agents as non-IV parenterals can also allow
patients to self-administer the agents, and can allow dosing to be
optimized (e.g. better tailored to the agent's PK/PD parameters) by
avoiding the need for administration by intravenous injection.
[0072] As will be appreciated by those of skill in the art, PK/PD
parameters that can be improved by using sHASEGPs (and/or other
glycosaminoglycanases) include such measures as Cmax (the maximal
concentration of agent achieved following absorption in, e.g., the
bloodstream), Tmax (the time required to achieve maximal
concentraion), T1/2 (the time required for the concentration to
fall by half), Cmin (the minimal concentration of agent following
metabolism and excretion), AUC (area under the curve of
concentration versus time, a measure of the overall amount of
bioavailability), concentrations in various tissues of interest
(including, e.g., the rate of achieving desired concentrations, the
overall levels, and the duration of maintaining desired levels),
and Emax (the maximal effect achieved). By way of illustration,
reformulating a drug or other pharmacologic agent with a sHASEGP or
co-administering the agent with a sHASEGP (which can be introduced
locally or systemically, and before, together with or after the
agent) can be used for example to increase an agent's Cmax,
decrease its Tmax, decrease its T1/2, increase its AUC and/or
increase its Emax. As will be appreciated, the ability to readily
and proportionately manipulate such key PK and PD parameters (e.g.
by applying more or less units of sHASEGP and altering the timing
and/or location of administration) enables the improvement of a
number of a pharmacologic and other agents. Evaluating the levels
of these parameters can be performed in models such as animal
models used for PK/PD measurements of the agent, and
co-formulations and/or co-administrations that enhance the relative
efficacy to safety profile thus selected for a particular
application.
[0073] Among non-IV parenterals, sHASEGPs (and/or other
glycosaminoglycanases) can also be used to allow the agents to be
administered by more convenient routes, and/or with greater
efficiency. By way of illustration (and without limitation), by
reformulating and/or co-administering agents with sHASEGP (either
local or systemic and either before, coincident with or after
administration of the agent) agents that are typically administered
by subcutaneous injection can instead be administered by
intradermal injection, and agents that are typically administered
by intramuscular injection can instead be administered by
subcutaneous or intradermal injection. Alternatively, agents can be
administered by the same route, but with improved pharmacokinetics
and/or pharmacodynamics using sHASEGPs.
[0074] The use of sHASEGPs (and/or other glycosaminoglycanases) to
reformulate IV drugs and other agents as non-IV parenterals also
enables delivery of the agents using any of a variety of new
injection devices designed to ease and/or speed delivery, and to
facilitate self-administration. Such devices include, for example,
ultra-sharp and microneedle devices (such as those being developed
by Becton Dickinson and others) as well as needle-free injection
devices (such as Biojector.TM. and other devices available from
Bioject; IntraJect.TM. and other devices available from Aradigm;
Medijector.TM. devices and the like). A number of devices (such as
the Biojector) are particularly useful for facilitating intradermal
injections which tend to require more experience when using
standard needles (due to the potential for penetrating the dermis
during needle placement, and delivering the agent to an underlying
tissue site such as the subcutaneous layer). For some pharmacologic
agents, such as vaccines for example (e.g. a DNA-based or other
vaccine), it can be particularly advantageous to deliver the agent
into the dermal as opposed to sub-dermal layers since there tends
to be a relatively high concentration of antigen-presenting cells
(APCs) localized within the dermis.
[0075] In the context of those agents that are typically delivered
by intradermal injection (whether by standard needles or other
newer devices), or those many other agents which are not currently
but could be delivered by intradermal injection, the local
co-introduction of a sHASEGP (and/or other glycosaminoglycanase)
can be used to enhance delivery of the pharmacologic or other agent
delivered and to promote its dispersal or spreading within the
dermis. In the case of a vaccine, where the dermis may be the
primary target tissue, given the local abundance of APCs, the
sHASEGP (and/or other glycosaminoglycanase) can thus facilitate
dispersal of the vaccine within the target tissue, thereby
increasing the probability and extent of interactions between a
vaccine and APCs (which can significantly potentiate the generation
of an immunomodulatory response). In the case of other agents, the
dermis may not be the primary target tissue but is rather a tissue
into which a dose of the agent is introduced from which it is
desired that the agent be absorbed into another tissue, typically
the bloodstream. In the latter case, the bloodstream may itself be
the target tissue of interest (e.g. for blood-modulatory factors as
described herein and in the art), or the bloodstream may itself be
a delivery tissue by which the agent is transported to a distal
target tissue (e.g. a tissue in the body supplied by the
bloodstream). In either of these latter cases (in which delivery is
intradermal but it is desired that the agent be delivered to the
bloodstream), the sHASEGP (and potentially a volume of liquid in
which it is injected) can facilitate dispersal of the agent first
within the dermis and thence into the vasculature draining the
dermis and eventually into the larger blood supply), as described
and illustrated herein.
[0076] The use of sHASEGPs to enhance the pharmacokinetics and/or
pharmacodynamics of other therapeutic or pharmacologic agents can
also be applied to agents delivered by routes other than non-IV
parenteral administration. For example, without wishing to be bound
by theory, it is believed that the presence of sHASEGPs in
interstitial spaces within the body (which can be achieved by local
and/or systemic administration of sHASEGPs) tends to promote
interstitial channels and an increase in fluid flow within the
interstitial space that in turn facilitates both diffusion and
convective transport of agents dissolved with the interstitial
fluid (such as pharmacologic and other agents). By way of
illustration, agents that are administered directly into the
bloodstream (e.g. by intravenous injection), or orally (and thus
enter the bloodstream after uptake from the gastrointestinal tract
for example), can still be subject to constraints in the
post-absorption i.e. distrubution phase of their pharmacokinetics.
Without wishing to be bound by theory, it is believed that sHASEGPs
can enhance delivery to target cells by increasing interstitial
permeability and fluid flow and thus increasing the diffusion
and/or convection of agents within the interstitial space (which
effectively forms the intermedium between almost all pharmacologic
delivery routes and the target cells).
[0077] In addition, it is believed that changes in oncotic pressure
brought about by the administration of sHASEGPs can contribute to
enhancing the delivery of pharmacologic agents. Again, without
wishing to be bound by theory, the presence of sHASEGP and
concomitant degradation along the interstitial side of a
vascularized tissue, would tend to decrease the oncotic pressure
within the interstitial space which in turn would enhance fluid
filtration from the vasculature into the interstitial space.
[0078] The potential to decrease interstitial pressure is
considered to be particularly important in the context of many
cancers in which interstitial pressure within the tumor (tumor
interstitial pressure of TIF) is elevated. High TIF can result in
relative impedance to fluid flow from the vasculature toward the
center of a tumor, thereby limiting the amount of an anti-cancer
agent that effectively reaches a tumor, particulary sites that are
deeper within a tumor mass. The introduction of sHASEGPs to tumor
interstitia would thus tend to enhance the delivery of locally
delivered as well as systemically available anti-cancer agents
which can more readily penetrate the tumor when interstitial
oncotic pressure is reduced and diffusion and/or convective
transport increased. Measurements of TIF and hydraulic conductivity
(K) within tumors (e.g. using tagged molecules such as albumin
labeled with Evan's Blue dye) can be used to quantitatively assess
the effect of various concentrations of sHASEGPs on the fluid
dynamics of tumors in vivo, as illusrated herein and/or in the
art.
[0079] Further exacerbating the reduced fluid dynamics within many
tumors, and highlighting an additional potential benefit of
applying sHASEGPs to tumors, is the fact that many tumors exhibit
accumulation of glycosaminoglycans, particularly hyaluronan (which
may be due to the fact that lymphatics, which are the predomint
route for hyaluronan catabolism, are impaired or lacking in many
tumors). Such excess hyaluronan can contribute to impeding
hydraulic conductivity. The introduction of sHASEGPs can thus be
used to counteract the accumulation of glycosaminoglycans in many
tumors, improving hydraulic conductivity within the tumor and
effectively rendering them more susceptible to anti-tumor agents
(whether locally or systemically delivered).
[0080] As will be appreciated by those of skill in the art, the
principles described herein can also be applied to numerous other
pharmacologics and other agents that are desired to be delivered to
sites within the body, such as for the prevention, diagnosis and/or
treatment of disease or to otherwise modulate physiological
functions. Illustrative classes of such agents (and exemplary
members thereof) are provided herein, and many more are known in
the art or under development.
[0081] In addition to their use in potential improvements and/or
reformulations of a variety of parenterally-administered
pharmacologics and/or agents, sHASEGPs can also be usefully
employed in connection with non-parenteral agents (such as agents
formulated as pills, liquids or other forms for ingestion and
typical absorption through the gastrointestinal tract). For
example, since most non-parenteral drugs must ultimately reach
cells within the interstitium in order to exert their desired
effects, the use of sHASEGPs to enhance diffusion and/or convective
transport within the interstitium (either systemically or locally
(e.g., by local or targeted administration of sHASEGP)) can be
applied to improve the extent and/or rate at which non-parenterals
(as well as parenterals) reach desired target cells.
[0082] In addition, a number of agents that are typically
administered non-parenterally (e.g. orally) may be reformulated, or
their active ingredients reformulated, for use in parenteral
administrations that are rendered more effective and/or safe in
combination with sHASEGPs. To illustrate this broadly applicable
approach, the ability of sHASEGPs to facilitate targeted delivery
to particular tissues (such as the ability of sHASEGPs to provide
for transcleral delivery to tissues within the posterior of the
eye) can be used to deliver any of a variety of agents (including
agents previously administered systemically) directly and
preferentially to a localized site of interest within the body.
This can not only provide for more desirable and/or more rapidly
achieved concentrations at the site of interest, but can
substantially reduce potential problems and limitations associated
with systemic administration in which concentrations at undesired
sites may equal or even exceed concentrations at the desired target
site (potentially triggering undesired side effects as well as
wasting agent). In some cases, agents that are not widely used or
are not used for certain indications or in certain patients, for
example, may be effectively applied to benefit additional patients
by being co-formulated or co-administered with sHASEGPs as
described and illustrated herein.
[0083] As will be appreciated by those of skill in the art, the
ability to employ sHASEGPs to enhance, speed and/or target
biodistribution of co-formulated and/or co-administered agents, and
to manipulate other aspects of their pharmacokinetics or
pharmacodynamics (e.g. in order to improve their risk:benefit
profile or facilitate their use by patients, family or health care
professionals), provides a major opportunity for improving drugs
and other agents that are used treat, diagnose or prevent
diseases.
[0084] Without being limited to a particular set of applications,
glycosaminoglycanases, including sHASEGPs as described herein (as
well as other hyaluronidases and other glycosaminoglycanases) can
be used to effectively achieve bolus or bolus-like delivery of any
number of pharmacologic and other agents by non-intravenous
parenteral routes, as well as by other routes of administration
(e.g. by enhancing the delivery of agents into and/or through a
target tissue after they leave the bloodstream (whether they were
introduced into the bloodstream directly (such as by IV
administration) or indirectly (such as by oral or non-IV parenteral
administration)).
[0085] Without being restricted to a specific mechanism of action
or aspect thereof, it is believed that the ability of these enzymes
to temporarily degrade components of the interstitial matrix
between cells (which accounts for a substantial portion of the
fluid space in the body and a correspondingly large space that must
be traversed in order for pharmacologics and other agents to reach
most target cells) can significantly promote the delivery of agents
to target cells by one or more of several potentially synergistic
means. First, while the bulk of the interstitial fluid exhibits
some degree of flow, that flow is often restrained or impeded by
glycosaminoglycans such as hyaluronan and other interstitial
components. The ability of glycosaminoglycanases including sHASEGPs
(as well as other hyaluronidases and other glycosaminoglycanases)
to open channels within such interstitial spaces, as described and
illustrated herein, can be used to decrease impedance and increase
the extent and rate of "downstream" flow resulting from any given
"upstream" pressure. Second, in the case of non-IV parenteral
injections of sHASEGPs (or other glycosaminoglycanase) and another
pharmacologic or other agent, the volume of the non-IV parenteral
injection can be used to increase the upstream driving pressure or
pressure head (e.g. by increasing the hydrostatic pressure within a
confined space), which can further promote flow. Third, since the
volume of fluid comprising the introduced sHASEGP (or other
glycosaminoglycanase) and other agent is effectively driven down
the increased pressure gradient (i.e. the increased gradient
created by elevating the hydrostatic pressure associated within the
injected "bolus" and simultaneously decreasing the interstitial
pressure within the surrounding tissue), solutes within the bolus
can be effectively carried (e.g. by convective transport) into the
adjacent tissue. This injected fluid or bolus can thus be
efficiently and relatively quickly moved into the adjacent tissue
and deliver whatever pharmacologic or other agent was introduced at
or near the same site of introduction (e.g. by co-formulating or
co-administering the agent in combination with the sHASEGP or other
glycosaminoglycanase).
[0086] By way of illustration, using glycosaminoglycanases such as
sHASEGPs to open channels within interstitial spaces, as described
and illustrated herein, can be used to increase interstitial flow
into and through tissues such as tumors and others in which flow is
typically impeded and interstitial pressures elevated. Illustrative
examples of these principles as described herein include the use of
sHASEGPs to reduce the interstitial pressure within a tumor in
vivo.
[0087] Increased flow can likewise be applied to normal tissue to,
e.g., allow an anesthetic, diagnostic, cosmetic or other esthetic,
or a pharmacologic or other agent to be delivered into a tissue
(e.g. to modify the tissue or introduce a depot formulation into a
tissue). Increased flow can likewise be applied to a first or
proximal tissue to promote delivery of an agent through the tissue
of introduction to a distal tissue, which distal tissue may itself
by the desired target tissue or a route to the desired target
tissue. Illustrative examples of these principles as described
herein include, for example: (i) the use of sHASEGPs introduced
into the episcleral space surrounding the eye to promote the
delivery of agents across the sclera in order to target distal
target tissues within the interior of the eye such as the retina
and choroid; and (ii) the use of sHASEGPs introduced intradermally
to promote the delivery of agents through the subdermal layers and
into the bloodstream, which can itself be the target tissue (e.g.
for blood-modifying agents) or a route by which the agent(s) can be
delivered to other target tissues within the body
[0088] By way of further illustration of these techniques and
compositions for promoting delivery of pharmacologics and other
agents to desired tissues within the body, the following additional
examples are provided. By means such as described and illustrated
herein, the pharmacokinetic and/or pharmacodynamic profiles of
pharmacologic agents, and the absorption, distribution and/or
effectiveness profiles of other agents, can be enhanced or
otherwise modified. In addition, agents that were essentially or
practically limited to delivery by one route (such as by IV
injection) can be delivered by other safer, less intrusive, less
costly, and/or more convenient routes.
[0089] By way of further illustration and without being limited to
a particular type of application, a volume (V) of liquid (L)
comprising a sHASEGP or other glycosaminoglycanase (GAG Enzyme) can
be introduced into a tissue site in the body (a Dosing Tissue). By
means as described and illustrated herein, the GAG Enzyme can be
delivered into and to some extent through the Dosing Tissue.
Without being restricted to a particular mechanism of action, the
GAG Enzyme can be effectively carried into the Dosing Tissue by
convective transport by the volume of liquid (L). The convective
transport can in turn be promoted in part by the hydrostatic
pressure associated with L (which can provide a driving pressure)
and in part by the ability of the GAG Enzyme to open channels
within the interstitial spaces of the Dosing Tissue (which can
reduce downstream impedance to flow and thereby lower the resisting
pressure). A fluid-driving pressure differential can thus be
created and to the extent desired it can be increased, for example,
by one or both of the following: (i) introducing and if desired
increasing a driving hydrostatic pressure (e.g. by introducing L
into a confined space and if desired increasing V); and (ii)
reducing the downstream impedance to flow (e.g. by introducing GAG
Enzyme, and if desired increasing its effectiveness by for example
increasing its concentration, increasing its resistance to
degradation (e.g. by PEGylation, super-sialation or other
modification), and/or increasing its delivery by convective
transport (e.g. by increasing V)).
[0090] In certain embodiments (e.g. in which increased extent or
rate of delivery is desired), V is a volume sufficient to
temporarily distend the site within the Dosing Tissue at which the
GAG Enzyme is introduced. As will be appreciated by those of skill
in the art, the volume (V) that can be accommodated and the
resulting extent of distension (D) depends on the particular
tissue. For purposes of illustration and not limitation, the extent
to which distention occurs varies, for example, from situations in
which (i) the Dosing Tissue is a relatively large and/or rapidly
moving fluid-filled space (such as the bloodstream in connection
with intravenous or intraarterial delivery), in which case L can be
large (e.g. many ml if desired) but D tends to be relatively
minimal; (ii) the Dosing Tissue is a relatively smaller and/or less
rapidly moving fluid-filled space (such as cerebrospinal fluid in
connection with intrathecal delivery), in which case L can still be
relatively large but D may be more than minimal depending on the
precise site and volume of injection); (iii) the Dosing Tissue is a
relatively smaller but somewhat distensible space (such as the
episcleral space surrounding the eye for purposes of illustration);
or (iv) the Dosing Tissue is a relatively more dense and/or more
confined space with higher resisting pressure (such as the dermis
in connection with intradermal delivery for purposes of
illustration).
[0091] As will be appreciated by those of skill in the art, V can
range from volumes of less than 0.1 ml to volumes of greater than
100 ml, for many applications being in the range of about 0.5 ml to
20 ml, and for many in the range of 1 ml to 10 ml, frequently from
2 to 5 ml; but can be varied for particular situations as
illustrated herein. V can also be specifically optimized within
such ranges for a particular application as desired; e.g. by
comparing standard pharmacokinetic and/or pharmacodynamic profiles
over a range of test volumes.
[0092] As will be appreciated by those of skill in the art in view
of these teachings, by combining the rapidly acting and
reinitiating channel-opening activity of the GAG Enzyme (which both
promotes and is promoted by its own delivery), together with a
delivery system that both promotes and is promoted by the catalytic
activity, a multiply synergistic drug delivery system can thus be
generated; as further described and illustrated herein.
[0093] In a first illustrative aspect, a volume of liquid (V)
comprising a sHASEGP or other glycosaminoglycanase (GAG Enzyme) and
one or more pharmacologic or other agents (Agent(s) (such as
detectable molecules or other diagnostic agents, anesthetics or
other tissue-modifying agents, pharmacologics or pharmaceutically
effective agents, cosmetics or other esthetic agents, and other
agents desired to be introduced into one or more tissues within the
body) is introduced into a first tissue site in the body (a Dosing
Tissue) which is itself a desired target tissue (a Target Tissue)
for the agent. By means as described and illustrated herein, the
Agent(s) are delivered into and throughout the Target Tissue to a
greater extent and/or rate than would occur in the absence of the
GAG Enzyme.
[0094] As will be appreciated by those of skill in the art in
connection with this and the other illustrative aspects herein, the
Dosing Tissue can be any of a variety of tissues that can be
readily reached from outside of the body (e.g. using various routes
of administration as described and illustrated herein and/or as
known in the art) or from inside the body (e.g. in connection with
surgery or using any of a variety of non-surgical or
minimally-invasive approaches and devices to reach tissues within
the body). Such Dosing Tissues include those described and
illustrated herein as well as others known in the art.
[0095] As will likewise be appreciated by those of skill in the art
in connection with this and the other illustrative aspects herein,
the Target Tissue can be any of a variety of tissues to which it is
desired that a GAG Enzyme and potentially other Agent(s) are
delivered (e.g. (i) tissues that are the subject of efforts to
diagnose, prevent or treat a disease condition (whether
intrinsically caused such as an inherited or developed disease
condition or extrinsically caused such as an infection); (ii)
tissues that are desired to be anesthetized or otherwise modulated
(e.g. in the context of surgery and/or other procedures); (iii)
tissues in which it is desired to trigger an immune response (such
as associated with vaccination); (iv) tissues which are to serve as
a depot site for releasing agent(s) to other tissues; (v) tissues
to be modified for esthetic or cosmetic purposes; and (vi) tissue
or sites in the body to be modified for other purposes). Such
Target Tissues include those described and illustrated herein as
well as others known in the art.
[0096] As will likewise be appreciated by those of skill in the art
in connection wth the forgoing and with other illustrative aspects
herein, the Agent(s) can be any of a variety of molecules,
macromolecules and macromolecular complexes, that are or can be
used for altering or modulating conditions within the body (e.g.
(i) for purposes of diagnosing, preventing or treating a disease
condition (whether intrinsically caused such as an inherited or
developed disease condition or extrinsically caused such as an
infection) affecting the Target Tissue and/or adjacent tissues;
(ii) for purposes of anesthetizing or otherwise modulating the
physiology of the Target Tissue; (iii) for purposes of triggering
an immune response as associated with vaccination; (iv) for
purposes of using the Target Tissue as a depot site for releasing
agent(s) to other tissues; (v) for esthetic or cosmetic purposes;
and/or (vi) for other purposes served by delivering a GAG Enzyme
and potentially other Agent(s) to a tissue or other site in the
body). Such Agents include the various classes of agents and
members thereof described and illustrated herein, as well as other
members of such classes of agents, and other classes of agents (and
members thereof) known in the art.
[0097] In a second illustrative aspect, a volume of liquid (V)
comprising a sHASEGP or other glycosaminoglycanase (GAG Enzyme) and
one or more pharmacologic or other agents (Agent(s)) is introduced
into a tissue site in the body (Dosing Tissue) which is adjacent or
proximal to a tissue (Tissue B) which is itself a desired target
tissue. By means as described and illustrated herein, the Agent(s)
are delivered from Tissue A to the target Tissue B to a greater
extent and/or rate than would occur in the absence of the GAG
Enzyme.
[0098] In a third illustrative aspect, a volume of liquid (V)
comprising a sHASEGP (or other glycosaminoglycanase) (GAG Enzyme)
and one or more pharmacologic or other agents (Agent(s)) is
introduced into a tissue site in the body (Dosing Tissue) which is
proximal to one or more tissues (Intervening Tissues) located
between Tissues A and another tissue or tissues (Distal Tissues)
which are themselves desired target tissues or include a desired
target tissue (Target Tissues). By means as described and
illustrated herein, the Agent(s) are delivered from Tissue A and
through one or more Intervening Tissues to reach one or more Target
Tissues to a greater extent and/or rate than would occur in the
absence of the GAG Enzyme(s).
[0099] In a fourth illustrative aspect, a volume of liquid (V)
comprising a sHASEGP (or other glycosaminoglycanase) (GAG Enzyme)
and one or more pharmacologic or other agents (Agent(s)) is
introduced into a tissue site in the body (Dosing Tissue) which is
proximal to one or more delivery tissues, such as the bloodstream,
lymph or cerebrospinal fluid (Delivery Tissues), which are desired
target tissues or include a desired target tissue. By means as
described and illustrated herein, the Agent(s) are delivered from
Tissue A to one or more Delivery Tissues to a greater extent and/or
rate than would occur in the absence of the GAG Enzyme(s).
[0100] In a fifth illustrative aspect, a volume of liquid (V)
comprising a sHASEGP (or other glycosaminoglycanase) (GAG Enzyme)
and one or more pharmacologic or other agents (Agent(s)) is
introduced into a tissue site in the body (Dosing Tissue) which is
proximal to one or more delivery tissues, such as the bloodstream,
lymph or cerebrospinal fluid (Delivery Tissues), which deliver
fluid to one or more desired target tissues (Target Tissues) within
the body. By means as described and illustrated herein, the
Agent(s) are delivered from Tissue A to one or more Delivery
Tissues and from there to one or more downstream Target Tissues to
a greater extent and/or rate than would occur in the absence of the
GAG Enzyme(s).
[0101] In a sixth illustrative aspect, a volume of liquid (V)
comprising a sHASEGP (or other glycosaminoglycanase) (GAG Enzyme)
together with one or more pharmacologic or other agents (Agent(s))
is introduced into one or more delivery tissues, such as the
bloodstream, lymph or cerebrospinal fluid (Delivery Tissues) which
supplies fluid to one or more desired Target Tissues (e.g. via the
bloodstream supplying a Target Tissue). By means as described and
illustrated herein, the Agent(s) permeate the Target Tissue to a
greater extent and/or rate than would occur in the absence of the
GAG Enzyme(s).
[0102] In a seventh illustrative aspect, a volume of liquid (V)
comprising a sHASEGP (or other glycosaminoglycanase) (GAG Enzyme)
is introduced into a tissue site in the body (Tissue A) which is a
desired target tissue or near a desired target tissue (Target
Tissue), and one or more pharmacologic or other agents (Agent(s))
are introduced into the body by another route of administration
(such as by oral delivery or intravenous injection) which allows
the Agent(s) to reach the Target Tissue (e.g. via the bloodstream
supplying the Target Tissue, following absorption or injection into
the blood). By means as described and illustrated herein, the
Agent(s) permeate the Target Tissue to a greater extent and/or rate
than would occur in the absence of the GAG Enzyme(s).
[0103] In an eighth illustrative aspect, a volume of liquid (V)
comprising a sHASEGP (or other glycosaminoglycanase) (GAG Enzyme)
is introduced into a tissue site or sites within the body (Tissue
Site(s)) which has an elevated interstitial pressure. By means as
described and illustrated herein, the interstitial pressure within
the Tissue Site(s) is reduced by the degradative activity of the
GAG Enzyme(s).
[0104] In a ninth illustrative aspect, a volume of liquid (V)
comprising a sHASEGP (or other glycosaminoglycanase) (GAG Enzyme)
is introduced into a tissue site or sites within the body (Tissue
Site(s)) which has an excess of hyaluronan or other
glycosaminoglycan. By means as described and illustrated herein,
the excess of hyaluronan or other glycosaminoglycan within the
Tissue Site(s) is reduced by the degradative activity of the GAG
Enzyme(s).
[0105] In an tenth illustrative aspect, a volume of liquid (V)
comprising a sHASEGP (or other glycosaminoglycanase) (GAG Enzyme)
is introduced into a tissue site or sites within the body (Tissue
Site(s)) which are at or near a desired access point for
hypodermoclysis (a Clysis Site). By means as described and
illustrated herein, fluids can then be delivered into and through
the Clysis Site(s) to a greater extent and/or rate than would occur
in the absence of the GAG Enzyme(s).
[0106] As will be appreciated by those of skill in view of the
detailed descriptions and illustrative embodiments provided herein,
any of a large variety of agents can be co-introduced (e.g. by
co-formulation or co-administration with the GAG Enzyme) and any of
a variety of GAG Enzymes can be employed at concentrations in
ranges such as those provided herein but typically optimized for
the particular tissue and objective involved. The volume of liquid
(V) applied in the above illustrative aspects will generally depend
on the nature of the site of introduction and will likewise
typically be optimized for the particular tissue and objective
involved. As described and illustrated herein, in the case of
non-IV parenteral administrations, V can be increased (within the
particular constraints of the tissue site) in order to provide an
enhanced hydrostatic driving pressure which, coupled with decreased
impedance associated with degradation of hyaluronan or other
glycosaminoglycan, can effectively cause the volume of liquid to
function as a bolus--carrying any solute it contains into the
downstream interstitial space and potentially beyond.
[0107] Hence, provided herein are a variety of techniques employing
hyaluronidases (and/or other glycosaminoglycanases) to promote the
delivery of pharmacologic and other agents to sites within the
body, as well as a family of eukaryotic secreted neutral active
hyaluronidase glycoproteins designated sHASEGPs, and functional
domains thereof, especially Hyaluronidase (or catalytic) domains
thereof, muteins and other derivatives and analogs thereof. Also
provided herein are nucleic acids encoding the sHASEGPs.
Additionally provided are formulations, co-formulations and
therapeutic uses of said sHASEGPs (including by co-administration
with other agents) to treat, prevent or diagnose disease, and for
use as tissue modifying enzymes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0108] FIG. 1 is a vector map of sHASEGP Vector HZ24.
DETAILED DESCRIPTION OF THE INVENTION
[0109] A. Definitions
[0110] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the invention (s) belong. All patents,
patent applications, published applications and publications,
Genbank sequences, websites and other published materials referred
to throughout the entire disclosure herein, unless noted otherwise,
are incorporated by reference in their entirety. In the event that
there is a plurality of definitions for terms herein, those in this
section prevail.
[0111] Where reference is made to a URL or other such identifier or
address, it understood that such identifiers can change and
particular information on the internet can come and go, but
equivalent information can be found by searching the internet.
Reference thereto evidences the availability and public
dissemination of such information.
[0112] As used herein, the abbreviations for any protective groups,
amino acids and other compounds, are, unless indicated otherwise,
in accord with their common usage, recognized abbreviations, or the
IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972)
Biochem. 11: 942-944).
[0113] As used herein, eukaryotic Hyaluronidase refers to a diverse
family of glycosaminoglycan endoglucosaminidases, wherein a
glutamate residue in the Hyaluronidase hydrolyzes the beta 1,4
linkages of hyaluronan and chondroitin sulfates through an
acid-base catalytic mechanism.
[0114] Of particular interest are soluble neutral-active
hyaluronidases or sHASEGPs of mammalian, including human, origin.
Those of skill in this art recognize that, in general, single amino
acid substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson et al.,
(1987) Molecular Biology of the Gene, 4th Edition, The
Benjamin/Cummings Pub. co., p. 224).
[0115] As used herein, membrane anchored HASEGP, refers to a family
of membrane anchored Hyaluronidases that share common structural
features as described herein. As described and illustrated herein,
hyaluronidases (i.e. glycosaminoglycanases capable of breaking down
hyaluronan, preferably those exhibiting at least some activity in
the ranges of neutral pH) that are normally membrane anchored can
be converted to soluble HASEGPs or sHASEGPs by removing or
otherwise modifying one or more of the regions that are associated
with anchoring the hyaluronidase in the membrane.
[0116] As used herein, soluble hyaluronidase refers to a
polypeptide characterized by its solubility under physiologic
conditions. Soluble HASEGP can be distinguished for example by its
partitioning into the aqueous phase of a Triton X-114 solution
warmed to 37C (Bordier et al J Biol Chem. Feb. 25, 1985;
256(4):1604-7). Lipid anchored HASEGP on the other hand will
partition into the detergent rich phase, but will partition into
the detergent poor or aqueous phase following treatment with
Phospholipase-C.
[0117] Thus, reference, for example, to "sHASEGP" encompasses all
glycoproteins encoded by the sHASEGP gene family, including but not
limited to: Human sHASEGP, mouse sHASEGP, or an equivalent molecule
obtained from any other source or that has been prepared
synthetically or that exhibits the same activity. Sequences of
encoding nucleic acid molecules and the encoded amino acid
sequences of exemplary sHASEGPs and/or domains thereof are set
forth, for example in SEQ ID NO: 4. The term also encompasses
sHASEGP with amino acid substitutions that do not substantially
alter activity of each member and also encompasses splice variants
thereof. Suitable substitutions, including, although not
necessarily, conservative substitutions of amino acids, are known
to those of skill in this art and can be made without eliminating
the biological activity, such as the catalytic activity, of the
resulting molecule.
[0118] As used herein, a sHASEGP, whenever referenced herein,
includes at least one or all of or any combination of: a
polypeptide encoded by the sequence of nucleotides set forth in SEQ
ID NO. 6 or by a sequence of nucleotides that includes nucleotides
that encode amino acids 1-509 of SEQ ID No. 1; a polypeptide
encoded by a sequence of nucleotides that hybridizes under
conditions of low, moderate or high stringency to the sequence of
nucleotides set forth in SEQ ID NO. 6; a polypeptide that includes
the sequence of amino acids set forth as amino acids 1-509 of SEQ
ID No. 1; a polypeptide that includes a sequence of amino acids
having at least about 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity with the sequence of amino acids set forth in
SEQ ID No. 1 or as amino acids 1448 of SEQ ID No. 4.
[0119] In particular, the sHASEGP polypeptide, with the
Hyaluronidase domains as indicated in SEQ ID No. 4 is provided. The
polypeptide is a single or two chain polypeptide. Smaller portions
thereof that retain Hyaluronidase activity are also provided. The
Hyaluronidase domains from sHASEGPs vary in size and constitution,
including insertions and deletions in surface loops. Thus, for
purposes herein, the catalytic domain is a portion of a sHASEGP, as
defined herein, and is homologous to a domain of other
hyaluronidase like sequences, such as HYAL1, HYAL2, HYAL3, which
have been previously identified; it was not recognized, however,
that an isolated single chain form of the human Hyaluronidase
domain could function in in vitro assays. The Aspartate and
Glutamate residues necessary for activity are present in conserved
motifs.
[0120] As used herein, a "neutral hyaluronidase domain of a soluble
sHASEGP" refers to a beta 1,4 endoglucosaminidase domain of a
sHASEGP that exhibits Hyaluronidase activity at neutral pH, is
soluble under conditions as described and shares homology and
structural features with the hyaluronidase glycosyl-hydrolase
family domains but contains additional sequences in the carboxy
terminus that are required for neutral activity. Hence it is at
least the minimal portion of the domain that exhibits Hyaluronidase
activity as assessed by standard in vitro assays and remains
soluble. Contemplated herein are such Hyaluronidase domains and
catalytically active portions thereof. Also provided are truncated
forms of the Hyaluronidase domain that include the smallest
fragment thereof that acts catalytically as a single chain form. As
used herein and in the art, neutral or neutral-active refers to a
protein exhibiting activity at neutral pH (e.g. exhibiting activity
at about pH 7) and which is therefore active at a pH range
characteristic of many physiological tissues. As will also be
appreciated by those of skill in the art, a protein generally
exhibits a range of activity around its pH optimum. The pH optimum
of a neutral active protein will typically be within one to several
pH units above or below pH 7, but its range of activity may extend
over many pH units.
[0121] A Hyaluronidase domain of an sHASEGP, whenever referenced
herein, includes at least one or all of or any combination of or a
catalytically active portion of: an N-linked glycoprotein
polypeptide that includes the sequence of amino acids set forth in
SEQ ID No. 1; a polypeptide encoded by a sequence of nucleotides
that hybridizes under conditions of low, moderate or high
stringency to the sequence of nucleotides set forth in SEQ ID NO.
6; a polypeptide that includes the sequence of amino acids set
forth in SEQ ID No. 1; a polypeptide that includes a sequence of
amino acids having at least about 70%, 75%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% sequence identity with the sequence of amino acids
set forth in SEQ ID No. 1; and/or a Hyaluronidase domain of a
polypeptide encoded by a splice variant of the sHASEGP.
[0122] Thus, for purposes herein, the Hyaluronidase domain is a
portion of a sHASEGP, as defined herein, and is homologous to a
domain of other sHASEGPs. As with the larger class of enzymes of
the hyaluronidase family, the sHASEGP catalytic domains share a
high degree of amino acid sequence identity. The Asp and Glu
residues necessary for activity are present in conserved
motifs.
[0123] By active form is meant a form active in vivo and/or in
vitro. As described herein, the Hyaluronidase domain also can exist
as a soluble secreted glycoprotein. It is shown herein that, at
least in vitro, the single chain forms of the sHASEGPs and the
catalytic domains or enzymatically active portions thereof
(typically C-terminal truncations) exhibit Hyaluronidase activity.
Hence provided herein are isolated forms of the Hyaluronidase
domains of sHASEGPs and their use in in vitro drug screening assays
for identification of agents that modulate the activity
thereof.
[0124] As used herein, the catalytically active domain of a sHASEGP
refers to the neutral active endoglucosaminidase domain as defined
by activity in vitro towards a glycosaminoglycan substrate.
[0125] sHASEGPs of interest include those that are active against
chondroitin sulfates and chondroitin sulfate proteoglycans (CSPG's)
in vivo and in vitro; and those that are active against hyaluronan.
As used herein, a human sHASEGP is one encoded by nucleic acid,
such as DNA, present in the genome of a human, including all
allelic variants and conservative variations as long as they are
not variants found in other mammals.
[0126] As used herein, nucleic acid encoding a Hyaluronidase domain
or catalytically active portion of a sHASEGP" shall be construed as
referring to a nucleic acid encoding only the recited single chain
Hyaluronidase domain or active portion thereof, and not the other
contiguous portions of the sHASEGP as a continuous sequence.
[0127] As used herein, "disease" or "disorder" refers to a
pathological condition in an organism resulting from, e.g.,
infection or genetic defect, and characterized by identifiable
symptoms.
[0128] As used herein, a splice variant refers to a variant
produced by differential processing of a primary transcript of
genomic nucleic acid, such as DNA, that results in more than one
type of mRNA. Splice variants of sHASEGPs are provided herein.
[0129] As used herein, the Hyaluronidase domain of a sHASEGP
protein refers to the Hyaluronidase domain of a sHASEGP that
exhibits neutral endoglucosaminidase activity. Hence it is at least
the minimal portion of the protein that exhibits
endoglucosaminidase activity as assessed by standard assays in
vitro. Exemplary human Hyaluronidase domains include at least a
sufficient portion of sequences of amino acids set forth in SEQ ID
No. 4 to exhibit endoglucosaminidase activity.
[0130] Also contemplated are nucleic acid molecules that encode a
polypeptide that has endoglucosaminidase activity in an in vitro
Hyaluronidase assay and that have at least 70%, 75%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% sequence identity with the full-length of a
Hyaluronidase domain of an sHASEGP polypeptide, or that hybridize
along their full-length or along at least about 70%, 80% or 90% of
the full-length to a nucleic acids that encode a Hyaluronidase
domain, particularly under conditions of moderate, generally high,
stringency.
[0131] For the Hyaluronidase domains of PH20 for example, residues
within the N-terminal region can be critical yet not sufficient for
activity. By application of techniques such as shown herein one can
generate Hyaluronidase domains of the sHASEGP that are
catalytically active. By way of illustration, while the
Hyaluronidase domain of human PH20 generally requires the
N-terminal amino acids thereof for activity; the C-terminus portion
can be truncated until the last Cysteine residue, yet requires
additional amino acids to be optimally active. The amount that can
be removed can be determined empirically by testing the polypeptide
for Hyaluronidase activity in an in vitro assay that assesses
catalytic cleavage.
[0132] Hence smaller portions of the Hyaluronidase domains,
particularly the single chain domains, thereof that retain
Hyaluronidase activity are contemplated. Such smaller versions
generally are C-terminal truncated versions of the Hyaluronidase
domains. The Hyaluronidase domains vary in size and constitution,
including insertions and deletions in surface loops. Such domains
exhibit conserved structure, including at least one structural
feature, such as the proton donor, and/or other features of
Hyaluronidase domains of endoglucosaminidases. Thus, for purposes
herein, the Hyaluronidase domain is a single chain portion of a
sHASEGP, as defined herein, but is homologous in its structural
features and retention of sequence of similarity or homology the
Hyaluronidase domain of other hyaluronidase-like sequences. The
glycoprotein exhibits Hyaluronidase activity as a single chain.
[0133] As used herein, by homologous means about greater than 25%
nucleic acid sequence identity, such as 25% 40%, 60%, 70%, 80%, 90%
or 95%. If necessary the percentage homology will be specified. The
terms "homology" and "identity" are often used interchangeably. In
general, sequences are aligned so that the highest order match is
obtained (see, e.g.: Computational Molecular Biology, Lesk, A. M.,
ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D. W., ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part/, Griffin,
A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; and Sequence Analysis Primer, Gribskov, M. and
Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et
al. (1988), Slam J Applied Math 48]:1073).
[0134] By sequence identity, the numbers of conserved amino acids
is determined by standard alignment algorithms programs, and are
used with default gap penalties established by each supplier.
Substantially homologous nucleic acid molecules would hybridize
typically at moderate stringency or at high stringency all along
the length of the nucleic acid or along at least about 70%, 80% or
90% of the full-length nucleic acid molecule of interest. Also
contemplated are nucleic acid molecules that contain degenerate
codons in place of codons in the hybridizing nucleic acid
molecule.
[0135] Whether any two nucleic acid molecules have nucleotide
sequences that are at least, for example, 80%, 85%, 90%, 95%, 96%,
97%, 98% or 99% "identical" can be determined using known computer
algorithms such as the "FASTA" program, using for example, the
default parameters as in Pearson et al (1988) Proc. Nati. Acad.
Sci. USA 85: 2444 (other programs include the GCG program package
(Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)),
BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J MOLEC BIOL 215: 403
(1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic
Press, San Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied
Math 48: 1073). For example, the BLAST function of the National
Center for Biotechnology Information database can be used to
determine identity. Other commercially or publicly available
programs include, DNASTAR "MEGALIGN" PROGRAM (Madison, Wis.) and
the University of Wisconsin Genetics Computer Group (UWG) "Gap"
program (Madison Wis.)). Percent homology or identity of proteins
and/or nucleic acid molecules can be determined, for example, by
comparing sequence information using a GAP computer program e.g.
Needleman et al. (1970), J Mol Biol. 48: 443, as revised by Smith
and Waterman Adv. Appl. Math (1981) 2:482). Briefly, the GAP
program defines similarity as the number of aligned symbols (i.e.,
nucleotides or amino acids) that are similar, divided by the total
number of symbols in the shorter of the two sequences. Default
parameters for the GAP program can include: (1) a unary comparison
matrix (containing a value of 1 for identities and 0 for
non-identities) and the weighted comparison matrix of Gribskov et
al (1986) Nucl. Acids Res. 14: 6745, as described by Schwartz and
Dayhoff, eds., Atlas Of Protein Sequence And Structure, National
Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty
of 3.0 for each gap and an additional 0.10 penalty for each symbol
in each gap; and (3) no penalty for end gaps. Therefore, as used
herein, the term "identity" represents a comparison between a test
and a reference polypeptide or polynucleotide.
[0136] As used herein, the term at least "90% identical to" refers
to percent identities from 90 to 99.99 relative to the reference
polypeptides. Identity at a level of 90% or more is indicative of
the fact that, assuming for exemplification purposes a test and
reference polynucleotide length of 100 amino acids are compared. No
more than 10% (i.e., 10 out of 100) amino acids in the test
polypeptide differ from that of the reference polypeptides. Similar
comparisons can be made between a test and reference
polynucleotides. Such differences can be represented as point
mutations randomly distributed over the entire length of an amino
acid sequence or they can be clustered in one or more locations of
varying length up to the maximum allowable, e.g. 10/100 amino acid
difference (approximately 90% identity). Differences are defined as
nucleic acid or amino acid substitutions, or deletions. At the
level of homologies or identities above about 85-90%, the result
should be independent of the program and gap parameters set; such
high levels of identity can be assessed readily, often without
relying on software.
[0137] As used herein, primer refers to an oligonucleotide
containing two or more deoxyribonucleotides or ribonucleotides,
typically more than three, from which synthesis of a primer
extension product can be initiated. Experimental conditions
conducive to synthesis include the presence of nucleoside
triphosphates and an agent for polymerization and extension, such
as DNA polymerase, and a suitable buffer, temperature and pH.
[0138] As used herein, animals include any animal, such as, but are
not limited to, goats, cows, deer, sheep, rodents, pigs and humans.
Non-human animals, exclude humans as the contemplated animal. The
sHASEGPs provided herein are from any source, animal, plant,
prokaryotic and fungal. Preferred sHASEGPs are of animal origin,
including mammalian origin, and more preferably are of human origin
in the case of their use in humans.
[0139] As used herein, genetic therapy involves the transfer of
heterologous nucleic acid, such as DNA, into certain cells, target
cells, of a mammal, particularly a human, with a disorder or
conditions for which such therapy is sought. The nucleic acid, such
as DNA, is introduced into the selected target cells in a manner
such that the heterologous nucleic acid, such as DNA, is expressed
and a therapeutic product encoded thereby is produced.
[0140] Alternatively, the heterologous nucleic acid, such as DNA,
can in some manner mediate expression of DNA that encodes the
therapeutic product, or it can encode a product, such as a peptide
or RNA that in some manner mediates, directly or indirectly,
expression of a therapeutic product. Genetic therapy can also be
used to deliver nucleic acid encoding a gene product that replaces
a defective gene or supplements a gene product produced by the
mammal or the cell in which it is introduced. The introduced
nucleic acid can encode a therapeutic compound, such as a growth
factor inhibitor thereof, or a tumor necrosis factor or inhibitor
thereof, such as a receptor therefore, that is not normally
produced in the mammalian host or that is not produced in
therapeutically effective amounts or at a therapeutically useful
time. The heterologous nucleic acid, such as DNA, encoding the
therapeutic product can be modified prior to introduction into the
cells of the afflicted host in order to enhance or otherwise alter
the product or expression thereof. Genetic therapy can also involve
delivery of an inhibitor or repressor or other modulator of gene
expression.
[0141] As used herein, heterologous nucleic acid is nucleic acid
that encodes proteins (or, if DNA, encodes RNA that encodes
proteins) that are not normally produced in vivo by the cell in
which it is expressed or that mediates or encodes mediators that
alter expression of endogenous nucleic acid, such as DNA, by
affecting transcription, translation, or other regulatable
biochemical processes. Heterologous nucleic acid, such as DNA, can
also be referred to as foreign nucleic acid, such as DNA. Any
nucleic acid, such as DNA, that one of skill in the art would
recognize or consider as heterologous or foreign to the cell in
which is expressed is herein encompassed by heterologous nucleic
acid; heterologous nucleic acid includes exogenously added nucleic
acid that is also expressed endogenously. Examples of heterologous
nucleic acid include, but are not limited to, nucleic acid that
encodes traceable marker proteins, such as a protein that confers
drug resistance, nucleic acid that encodes therapeutically
effective substances, such as anti-cancer agents, enzymes and
hormones, and nucleic acids (such as DNA and RNA), that indirectly
or directly encode other types of proteins, such as antibodies.
Antibodies that are encoded by heterologous nucleic acid can be
secreted or expressed on the surface of the cell in which the
heterologous nucleic acid has been introduced.
[0142] Heterologous nucleic acid is generally not endogenous to the
cell into which it is introduced, but has been obtained from
another cell or prepared synthetically.
[0143] Generally, although not necessarily, such nucleic acid
encodes RNA and/or proteins that are not normally produced by the
cell in which it is expressed.
[0144] As used herein, a therapeutically effective product is a
product that is encoded by heterologous nucleic acid, typically
DNA, that, upon introduction of the nucleic acid into a host, a
product is expressed that ameliorates or eliminates the symptoms,
manifestations of an inherited or acquired disease or that cures
the disease.
[0145] As used herein, recitation that a glycoprotein consists
essentially of the Hyaluronidase domain means that the only sHASEGP
portion of the polypeptide is a Hyaluronidase domain or a
catalytically active portion thereof. The polypeptide can
optionally, and generally will, include additional
non-sHASEGP-derived sequences of amino acids.
[0146] As used herein, domain refers to a portion of a molecule,
e.g., glycoproteins or the encoding nucleic acids that is
structurally and/or functionally distinct from other portions of
the molecule.
[0147] As used herein, Hyaluronidase refers to an enzyme catalyzing
hydrolysis of glycosaminoglycans including hyaluronans.
[0148] For clarity reference to Hyaluronidase refers to all forms,
and particular forms will be specifically designated. For purposes
herein, the Hyaluronidase domain includes the membrane bound and
soluble forms of a sHASEGP protein.
[0149] As used herein, nucleic acids include DNA, RNA and analogs
thereof, including protein nucleic acids (PNA) and mixture thereof.
Nucleic acids can be single or double-stranded. When referring to
probes or primers, optionally labeled with a detectable label, such
as a fluorescent dye or radiolabel, single-stranded molecules are
generally contemplated. Such molecules are typically of a length
such that their target is statistically unique or of low copy
number (typically less than 5, generally less than 3) for probing
or priming a library. As is known in the art, generally a probe or
primer contains at least 14, 16, 20 or 30 residues of contiguous or
nearly-contiguous sequence complementary to or identical a gene of
interest. Probes and primers can be 10, 20, 30, 50, 100 or more
nucleic acids long.
[0150] As used herein, nucleic acid encoding a fragment or portion
of a sHASEGP refers to a nucleic acid encoding only the recited
fragment or portion of sHASEGP, and not the other contiguous
portions of the sHASEGP.
[0151] As used herein, operative linkage of heterologous nucleic to
regulatory and effector sequences of nucleotides, such as
promoters, enhancers, transcriptional and translational stop sites,
and other signal sequences refers to the relationship between such
nucleic acid, such as DNA, and such sequences of nucleotides. For
example, operative linkage of heterologous DNA to a promoter refers
to the functional (and typically physical) relationship between the
DNA and the promoter such that the transcription of such DNA is
initiated from the promoter by an RNA polymerase that specifically
recognizes, binds to and transcribes the DNA in reading frame.
Thus, operatively linked or operationally associated refers to the
functional relationship of nucleic acid, such as DNA, with
regulatory and effector sequences of nucleotides, such as
promoters, enhancers, transcriptional and translational stop sites,
and other signal sequences. In order to optimize expression and/or
in vitro transcription, it can be necessary to remove, add or alter
5' Untranslated portions of the clones to eliminate extra,
potential inappropriate alternative translation initiation i.e.
start) codons or other sequences that can interfere with or reduce
expression, either at the level of transcription or translation.
Alternatively, consensus ribosome binding sites (see, e.g., Kozak
J. Biol. Chem. 266: 19867-19870 (1991) can be inserted immediately
5' of the start codon and can enhance expression. The desirability
of (or need for) such modification can be empirically
determined.
[0152] As used herein, a sequence complementary to at least a
portion of an RNA, with reference to antisense oligonucleotides,
means a sequence having sufficient complementarity to be able to
hybridize with the RNA, generally under moderate or high stringency
conditions, forming a stable duplex; in the case of double-stranded
sHASEGP antisense nucleic acids, a single strand of the duplex DNA
(or dsRNA) can thus be tested, or triplex formation can be assayed.
The ability to hybridize depends on the degree of complementarity
and the length of the antisense nucleic acid. Generally, the longer
the hybridizing nucleic acid, the more base mismatches with a
sHASEGP encoding RNA it can contain and still form a stable duplex
(or triplex, as the case can be). One skilled in the art can
ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex.
[0153] For purposes herein, amino acid substitutions can be made in
any of sHASEGPs and Hyaluronidase domains thereof provided that the
resulting protein exhibits Hyaluronidase activity. Amino acid
substitutions contemplated include conservative substitutions, such
as those set forth in Table 1, and other modifications that do not
eliminate proteolytic activity. As described herein, substitutions
that alter properties of the proteins, such as removal of cleavage
sites and other such sites are also contemplated; such
substitutions are generally non-conservative, but can be readily
effected by those of skill in the art.
[0154] Suitable conservative substitutions of amino acids are known
to those of skill in this art and can be made generally without
eliminating (and preferably without substantially altering) the
biological activity, for example enzymatic activity, of the
resulting molecule. Those of skill in this art recognize that, in
general, single amino acid substitutions in non-essential regions
of a polypeptide do not substantially alter biological activity
(see, e. g., Watson et al. Molecular Biology of the Gene, 4th
Edition, 1987, The Benjamin/Cummings Pub. co., p. 224). Also
included within the definition, is the catalytically active
fragment of a sHASEGP, particularly a single chain Hyaluronidase
portion. Conservative amino acid substitutions are made, for
example, in accordance with those set forth in TABLE 1 as
follows:
[0155] TABLE 1 Original residue Conservative substitution Ala (A)
Gly; Ser, Abu Arg (R) Lys, Orn Asn (N) Gln; His Cys (C) Ser Gln (Q)
Asn Glu (E) ASP Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val
; Met; Nle ; Nva Leu (L); Val ; Met; Nle ; Nva Lys (K) Arg; Gln ;
Glu Met (M) Leu; Tyr; Ile; NLe Val Orn Lys; Arg Phe (F) Met; Leu;
Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V)
ILE; Leu; Met; Nle; Nva. Other substitutions are also permissible
and can be determined empirically or in accord with known
conservative substitutions.
[0156] As used herein, Abu is 2-aminobutyric acid; Nie is
norleucine; Nva is norvaline; Orn is ornithine. As used herein, the
amino acids, which occur in the various amino acid sequences
appearing herein, are identified according to their well-known,
three-letter or one-letter abbreviations. The nucleotides, which
occur in the various DNA fragments, are designated with the
standard single-letter designations used routinely in the art.
[0157] As used herein, a probe or primer based on a nucleotide
sequence disclosed herein, includes at least 10, 14, typically at
least 16 contiguous sequence of nucleotides of SEQ ID NO. 6, and
even more preferably at least 20, 30, 50 or 100 contiguous sequence
of nucleotides of SEQ ID NO. 6. The length of the probe or primer
for unique hybridization is a function of the complexity of the
genome of interest.
[0158] As used herein, amelioration of the symptoms of a particular
disorder by administration of a particular pharmaceutical
composition refers to any lessening, whether permanent or
temporary, lasting or transient that can be attributed to or
associated with administration of the composition.
[0159] As used herein, antisense polynucleotides refer to synthetic
sequences of nucleotide bases complementary to mRNA or the sense
strand of double-stranded DNA. Admixture of sense and antisense
polynucleotides under appropriate conditions leads to the binding
of the two molecules, or hybridization. When these polynucleotides
bind to (hybridize with) mRNA, inhibition of protein synthesis
(translation) occurs. When these polynucleotides bind to
double-stranded DNA, inhibition of RNA synthesis (transcription)
occurs.
[0160] The resulting inhibition of translation and/or transcription
leads to an inhibition of the synthesis of the protein encoded by
the sense strand. Antisense nucleic acid molecule typically contain
a sufficient number of nucleotides to Specifically bind to a target
nucleic acid, generally at least 5 contiguous nucleotides, often at
least 14 or 16 or 30 contiguous nucleotides or modified nucleotides
complementary to the coding portion of a nucleic acid molecule that
encodes a gene of interest, for example, nucleic acid encoding a
single chain Hyaluronidase domain of an sHASEGP.
[0161] As used herein, an array refers to a collection of elements,
such as antibodies, containing three or more members. An
addressable array is one in which the members of the array are
identifiable, typically by position on a solid phase support.
Hence, in general the members of the array are immobilized on
discrete identifiable loci on the surface of a solid phase.
[0162] As used herein, antibody refers to an immunoglobulin,
whether natural or partially or wholly synthetically produced,
including any derivative thereof that retains the specific binding
ability the antibody. Hence antibody includes any protein having a
binding domain that is homologous or substantially homologous to an
immunoglobulin-binding domain. Antibodies include members of any
immunoglobulin claims, including IgG, IgM, IgA, IgD and IgE.
[0163] As used herein, antibody fragment refers to any derivative
of an antibody that is less then full-length, retaining at least a
portion of the full-length antibody's specific binding ability.
Examples of antibody fragments include, but are not limited to Fab,
Fab', F(AB)2, single chain Fvs (scFV), FV, dsFV diabody and Fd
fragments. The fragment can include multiple chains linked
together, such as by disulfide bridges. An antibody fragment
generally contains at least about 50 amino acids and typically at
least 200 amino acids.
[0164] As used herein, an Fv antibody fragment is composed of one
variable heavy domain (VH) and one variable light domain linked by
noncovalent interactions.
[0165] As used herein, a dsFV refers to an Fv with an engineered
intermolecular disulfide bond.
[0166] As used herein, an F (AB)2 fragment is an antibody fragment
that results from digestion of an immunoglobulin with pepsin at pH
4.0-4.5; it can be recombinantly expressed to produce the
equivalent fragment.
[0167] As used herein, Fab fragments are antibody fragments that
result from digestion of an immunoglobulin with papain; they can be
recombinantly expressed to produce the equivalent fragment.
[0168] As used herein, scFVs refer to antibody fragments that
contain a variable light chain V, and variable heavy chain (VH)
covalently connected by a polypeptide linker in any order. The
linker is of a length such that the two variable domains are
bridged without substantial interference. Included linkers are
(Gly-Ser) n residues with some Glu or Lys residues dispersed
throughout to increase solubility.
[0169] As used herein, humanized antibodies refer to antibodies
that are modified to include human sequences of amino acids so that
administration to a human does not provoke an immune response.
Methods for preparation of such antibodies are known. For example,
to produce such antibodies, the hybridoma or other prokaryotic or
eukaryotic cell, such as an E. coli or a CHO cell, that expresses
the monoclonal antibody are altered by recombinant DNA techniques
to express an antibody in which the amino acid composition of the
non-variable region is based on human antibodies. Computer programs
have been designed to identify such regions.
[0170] As used herein, diabodies are dimeric scFV; diabodies
typically have shorter peptide linkers than ScFVs, and they
generally dimerize.
[0171] As used herein, production by recombinant means by using
recombinant DNA methods means the use of the well known methods of
molecular biology for expressing proteins encoded by cloned
DNA.
[0172] As used herein the term assessing is intended to include
quantitative and qualitative determination in the sense of
obtaining an absolute value for the activity of an sHASEGP, or a
domain thereof, present in the sample, and also of obtaining an
index, ratio, percentage, visual or other value indicative of the
level of the activity. Assessment can be direct or indirect and the
chemical species actually detected need not of course be the
proteolysis product itself but can for example be a derivative
thereof or some further substance.
[0173] As used herein, biological activity refers to the in vivo
activities of a compound or physiological responses that result
upon in vivo administration of a compound, composition or other
mixture. Biological activity, thus, encompasses therapeutic effects
and pharmaceutical activity of such compounds, compositions and
mixtures. Biological activities can be observed in in vitro systems
designed to test or use such activities. Thus, for purposes herein
the biological activity of a luciferase is its oxygenase activity
whereby, upon oxidation of a substrate, light is produced.
[0174] As used herein, functional activity refers to a polypeptide
or portion thereof that displays one or more activities associated
with a full-length protein.
[0175] Functional activities include, but are not limited to,
biological activity, catalytic or enzymatic activity, antigenicity
(ability to bind to or compete with a polypeptide for binding to an
anti-polypeptide antibody), immunogenicity, ability to form
multimers, the ability to Specifically bind to a receptor or ligand
for the polypeptide.
[0176] As used herein, a conjugate refers to the compounds provided
herein that includes one or more sHASEGPs, including a sHASEGP,
particularly single chain Hyaluronidase domains thereof, and one or
more targeting agents. These conjugates include those produced by
recombinant means as fusion proteins, those produced by chemical
means, such as by chemical coupling, through, for example, coupling
to sulfhydryl groups, and those produced by any other method
whereby at least one sHASEGP, or a domain thereof, is linked,
directly or indirectly via linker (s) to a targeting agent.
[0177] As used herein, a targeting agent is any moiety, such as a
protein or effective portion thereof, that provides specific
binding of the conjugate to a cell surface receptor, which, can
internalize the conjugate or sHASEGP portion thereof. A targeting
agent can also be one that promotes or facilitates, for example,
affinity isolation or purification of the conjugate; attachment of
the conjugate to a surface; or detection of the conjugate or
complexes containing the conjugate.
[0178] As used herein, an antibody conjugate refers to a conjugate
in which the targeting agent is an antibody.
[0179] As used herein, derivative or analog of a molecule refers to
a portion derived from or a modified version of the molecule.
[0180] As used herein, an effective amount of a compound for
treating a particular disease is an amount that is sufficient to
ameliorate, or in some manner reduce the symptoms associated with
the disease. Such amount can be administered as a single dosage or
can be administered according to a regimen, whereby it is
effective. The amount can cure the disease but, typically, is
administered in order to ameliorate the symptoms of the disease.
Repeated administration can be required to achieve the desired
amelioration of symptoms.
[0181] As used herein equivalent, when referring to two sequences
of nucleic acids means that the two sequences in question encode
the same sequence of amino acids or equivalent proteins. When
equivalent is used in referring to two proteins or peptides, it
means that the two proteins or peptides have substantially the same
amino acid sequence with only amino acid substitutions (such, as
but not limited to, conservative changes such as those set forth in
Table 1, above) that do not substantially alter the activity or
function of the protein or peptide. When equivalent refers to a
property, the property does not need to be present to the same
extent (e. g., two peptides can exhibit different rates of the same
type of enzymatic activity), but the activities are usually
substantially the same. Complementary, when referring to two
nucleotide sequences, means that the two sequences of nucleotides
are capable of hybridizing, typically with less than 25%, 15%, 5%
or 0% mismatches between opposed nucleotides. If necessary the
percentage of complimentarity will be specified. Typically the two
molecules are selected such that they will hybridize under
conditions of high stringency.
[0182] As used herein, an agent that modulates the activity of a
protein or expression of a gene or nucleic acid either decreases or
increases or otherwise alters the activity of the protein or, in
some manner up-or down-regulates or otherwise alters expression of
the nucleic acid in a cell.
[0183] As used herein, inhibitor of the activity of an sHASEGP
encompasses any substance that prohibits or decrease production,
post-translational modification (s), maturation, or membrane
localization of the sHASEGP or any substance that interferes with
or decreases the proteolytic efficacy of thereof, particularly of a
single chain form in an in vitro screening assay.
[0184] As used herein, a method for treating or preventing
neoplastic disease means that any of the symptoms, such as the
tumor, metastasis thereof, the vascularization of the tumors or
other parameters by which the disease is characterized are reduced,
ameliorated, prevented, placed in a state of remission, or
maintained in a state of remission. It also means that the
hallmarks of neoplastic disease and metastasis can be eliminated,
reduced or prevented by the treatment. Non-limiting examples of the
hallmarks include uncontrolled degradation of the basement membrane
and proximal extracellular matrix, migration, division, and
organization of the endothelial cells into new functioning
capillaries, and the persistence of such functioning
capillaries.
[0185] As used herein, pharmaceutically acceptable salts, esters or
other derivatives of the conjugates include any salts, esters or
derivatives that can be readily prepared by those of skill in this
art using known methods for such derivatization and that produce
compounds that can be administered to animals or humans without
substantial toxic effects and that either are pharmaceutical active
or are prodrugs.
[0186] As used herein, a prodrug is a compound that, upon in vivo
administration, is metabolized or otherwise converted to the
biologically, pharmaceutically or therapeutically active form of
the compound. To produce a prodrug, the pharmaceutical active
compound is modified such that the active compound is regenerated
by metabolic processes. The prodrug can be designed to alter the
metabolic stability or the transport characteristics of a drug, to
mask side effects or toxicity, to improve the flavor of a drug or
to alter other characteristics or properties of a drug. By virtue
of knowledge of pharmacodynamic processes and drug metabolism in
vivo, those of skill in this art, once a pharmaceutically active
compound is known, can design prodrugs of the compound (see, e. g.,
Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford
University Press, New York, pages 388-392).
[0187] As used herein, a drug identified by the screening methods
provided herein refers to any compound that is a candidate for use
as a therapeutic or as a lead compound for the design of a
therapeutic. Such compounds can be small molecules, including small
organic molecules, peptides, peptide mimetics, antisense molecules
or dsRNA, such as RNAi, antibodies, fragments of antibodies,
recombinant antibodies and other such compounds that can serve as
drug candidates or lead compounds.
[0188] As used herein, a peptidomimetic is a compound that mimics
the conformation and certain stereochemical features of the
biologically active form of a particular peptide. In general,
peptidomimetics are designed to mimic certain desirable properties
of a compound, but not the undesirable properties, such as
flexibility, that lead to a loss of a biologically active
conformation and bond breakdown. Peptidomimetics may be prepared
from biologically active compounds by replacing certain groups or
bonds that contribute to the undesirable properties with
bioisosteres. Bioisosteres are known to those of skill in the art.
For example the methylene bioisostere CH2S has been used as an
amide replacement in enkephalin analogs (see, e.g. Spatola (1983)
pp. 267-357 in Chemistry and Biochemistry of Amino Acids, Peptides,
and Proteins, Weistein, Ed. volume 7, Marcel Dekker, New York).
Morphine, which can be administered orally, is a compound that is a
peptidomimetic of the peptide endorphin. For purposes herein,
cyclic peptides are included among pepidomimetics.
[0189] As used herein, a promoter region or promoter element refers
to a segment of DNA or RNA that controls transcription of the DNA
or RNA to which it is operatively linked. The promoter region
includes specific sequences that are sufficient for RNA polymerase
recognition, binding and transcription initiation.
[0190] This portion of the promoter region is referred to as the
promoter. In addition, the promoter region includes sequences that
modulate this recognition, binding and transcription initiation
activity of RNA polymerase. These sequences can be cis acting or
can be responsive to trans acting factors. Promoters, depending
upon the nature of the regulation, can be constitutive or
regulated. Exemplary promoters contemplated for use in prokaryotes
include the bacteriophage T7 and T3 promoters.
[0191] As used herein, a receptor refers to a molecule that has an
affinity for a given ligand. Receptors can be naturally occurring
or synthetic molecules. Receptors can also be referred to in the
art as anti-ligands. As used herein, the receptor and anti-ligand
are interchangeable. Receptors can be used in their unaltered state
or as aggregates with other species. Receptors can be attached,
covalently or noncovalently, or in physical contact with, to a
binding member, either directly or indirectly via a specific
binding substance or linker. Examples of receptors, include, but
are not limited to: antibodies, cell membrane receptors surface
receptors and internalizing receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants such as on
viruses, cells, or other materials], drugs, polynucleotides,
nucleic acids, peptides, factors, lectins, sugars, polysaccharides,
cells, cellular membranes, and organelles.
[0192] Examples of receptors and applications using such receptors,
include but are not restricted to: a) enzymes: specific transport
proteins or enzymes essential to survival of microorganisms, which
could serve as targets for antibiotic [ligand] selection; b)
antibodies: identification of a ligand-binding site on the antibody
molecule that combines with the epitope of an antigen of interest
can be investigated; determination of a sequence that mimics an
antigenic epitope can lead to the development of vaccines of which
the immunogen is based on one or more of such sequences or lead to
the development of related diagnostic agents or compounds useful in
therapeutic treatments such as for auto-immune diseases c) nucleic
acids: identification of ligand, such as protein or RNA, binding
sites; d) catalytic polypeptides: polymers, including polypeptides,
that are capable of promoting a chemical reaction involving the
conversion of one or more reactants to one or more products; such
polypeptides generally include a binding site specific for at least
one reactant or reaction intermediate and an active functionality
proximate to the binding site, in which the functionality is
capable of chemically modifying the bound reactant (see, e. g.,
U.S. Pat. No. 5,215,899); e) hormone receptors: determination of
the ligands that bind with high affinity to a receptor is useful in
the development of hormone replacement therapies; for example,
identification of ligands that bind to such receptors can lead to
the development of drugs to control blood pressure; and f) opiate
receptors: determination of ligands that bind to the opiate
receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0193] As used herein, sample refers to anything that can contain
an analyte for which an analyte assay is desired. The sample can be
a biological sample, such as a biological fluid or a biological
tissue. Examples of biological fluids include urine, blood, plasma,
serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,
mucus, sperm, amniotic fluid or the like. Biological tissues are
aggregate of cells, usually of a particular kind together with
their intercellular substance that form one of the structural
materials of a human, animal, plant, bacterial, fungal or viral
structure, including connective, epithelium, muscle and nerve
tissues. Examples of biological tissues also include organs,
tumors, lymph nodes, arteries and individual cells.
[0194] As used herein: stringency of hybridization in determining
percentage mismatch is as follows: 1) high stringency:
0.1.times.SSPE, 0.1% SDS, 65.degree. C. 2) medium stringency:
0.2.times.SspE, 0.1% SDS, 50.degree. C. 3 low stringency:
1.0.times.SspE, 0.1% SDS, 50.degree. C. Those of skill in this art
know that the washing step selects for stable hybrids and also know
the ingredients of SspE (see, e. g., Sambrook, E. F. Fritsch, T.
Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold spring
Harbor Laboratory Press 1989 Vol 3, p. B. 13, see, also, numerous
catalogs that describe commonly used laboratory solutions). SspE is
pH 7.4 phosphate-buffered 0.18 NaCl. Further, those of skill in the
art recognize that the stability of hybrids is determined by TmT
which is a function of the sodium ion concentration and temperature
(Tm=81.5.degree. C.-16.6+0.41 (% G+C)-600/L)) so that the only
parameters in the wash conditions critical to hybrid stability are
sodium ion concentration in the SspE (or SSC) and temperature.
[0195] It is understood that equivalent stringencies can be
achieved using alternative buffers, salts and temperatures. By way
of example and not limitation, procedures using conditions of low
stringency are as follows (see also Shilo and Weinberg, Proc. Natl.
Acad Sci USA 78: 6789-6792 (1981)): Filters containing DNA are
pretreated for 6 hours at 40 C in a solution containing 35%
formamide, 5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1%
PVP, 0.1% Ficoll 1% BSA, and 500 .mu.g/ml Denatured Salmon sperm
DNA (10.times.) SSC is 1.5 M sodium chloride, and 0.15 M sodium
citrate, adjusted to a pH of 7).
[0196] Hybridizations are carried out in the same solution with the
following modifications: 0.02% PVP, 0.02% Ficoll 0.2% BSA, 100 VG/M
sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20.times.106 cpm
32P-labled probe is used. Filters are incubated in hybridization
mixture for 18-20 hours at 40 C and then washed for 1.5 hours at 55
C in a solution containing 2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5
mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh
solution and incubated an additional 1.5 hours at 60 C. Filters are
blotted dry and exposed for autoradiography. If necessary, filters
are washed for a third time at 65-68 C and reexposed to film. Other
conditions of low stringency which can be used are well known in
the art e.g. as employed for cross-species hybridizations).
[0197] By way of example and not way of limitation, procedures
using conditions of moderate stringency include, for example, but
are not limited to, procedures using such conditions of moderate
stringency are as follows: Filters containing DNA are pretreated
for 6 hours at 55 C in a solution containing 6.times.SSC, 5.times.
Denhart's solution, 0.5% SDS and 100 .mu.g/ml denatured salmon
sperm DNA. Hybridizations are carried out in the same solution and
5-2.times.106 32P labeled probe is used. Filters are incubated in
hybridization mixture for 18-20 hours at 55 C and then washed twice
for 30 minutes at 60 C in a solution containing 1.times.SSC and
0.1% SDS. Filters are blotted dry and exposed for autoradiography.
Other conditions of moderate stringency that can be used are well
known in the art. Washing of filters is done at 37 C for 1 hour in
a solution containing 2.times.SSC, 0.1% SDS.
[0198] By way of example and not way of limitation, procedures
using conditions of high stringency are as follows:
Prehybridization of filters containing DNA is carried out for 8
hours to overnight at 65 C in buffer composed of 6.times.SSC, 50 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA,
and 500 .mu.g/ml denatured salmon sperm DNA. Filters are hybridized
for 48 hours at 65.degree. C. in prehybridization mixture
containing 100 .mu.g/ml denatured salmon sperm DNA and
5-20.times.106 CPM 32P labeled probe. Washing of filters is done at
37 C for 1 hour in a solution containing 2.times.SSC, 0.01% PVP,
0.01% Ficoll, and 0.01% BSA. This is followed by a wash in
0.1.times.SSC at 50 C for 45 minutes before autoradiography. Other
conditions of high stringency that can be used are well known in
the art.
[0199] The term substantially identical or substantially homologous
or similar varies with the context as understood by those skilled
in the relevant art and generally means at least 60% or 70%,
preferably means at least 80%, 85% or more preferably at least 90%,
and most preferably at least 95% identity.
[0200] As used herein, substantially identical to a product means
sufficiently similar so that the property of interest is
sufficiently unchanged so that the substantially identical product
can be used in place of the product.
[0201] As used herein, substantially pure means sufficiently
homogeneous to appear free of readily detectable impurities as
determined by standard methods of analysis, such as thin layer
chromatography (TLC), gel electrophoresis and high performance
liquid chromatography (HPLC), used by those of skill in the art to
assess such purity, or sufficiently pure such that further
purification would not detectably alter the physical and chemical
properties, such as enzymatic and biological activities, of the
substance. Methods for purification of the compounds to produce
substantially chemically pure compounds are known to those of skill
in the art. A substantially chemically pure compound can, however,
be a mixture of stereoisomers or isomers. In such instances,
further purification might increase the specific activity of the
compound.
[0202] As used herein, target cell refers to a cell that expresses
a sHASEGP (in the context of sHASEGP production as described
herein), or to a cell that is targeted by a sHASEGP or
co-formulated or co-administered agent in vivo (in the context of
using sHASEGPs to promote the delivery of pharmacologics and other
agents to target cells as described herein).
[0203] As used herein, test substance (or test compound) refers to
a chemically defined compound (e.g., organic molecules, inorganic
molecules, organic/inorganic molecules, proteins, peptides, nucleic
acids, oligonucleotides, lipids, polysaccharides, saccharides, or
hybrids among these molecules such as glycoproteins, etc.) or
mixtures of compounds (e.g., a library of test compounds, natural
extracts or culture supernatants, etc.) whose effect on an sHASEGP,
particularly a single chain form that includes the Hyaluronidase
domain or a sufficient portion thereof for activity, as determined
by an in vitro method, such as the assays provided herein.
[0204] As used herein, the terms a therapeutic agent, therapeutic
regimen, radioprotectant or chemotherapeutic mean conventional
drugs and drug therapies, including vaccines, which are known to
those skilled in the art. Radiotherapeutic agents are well known in
the art.
[0205] As used herein, treatment means any manner in which the
symptoms of a condition, disorder or disease are ameliorated or
otherwise beneficially altered.
[0206] Treatment also encompasses any pharmaceutical use of the
compositions herein.
[0207] As used herein, vector (or plasmid) refers to discrete
elements that are used to introduce heterologous nucleic acid into
cells for either expression or replication thereof. The vectors
typically remain episomal, but can be designed to effect
integration of a gene or portion thereof into a chromosome of the
genome. Also contemplated are vectors that are artificial
chromosomes, such as yeast artificial chromosomes and mammalian
artificial chromosomes. Selection and use of such vehicles are well
known to those of skill in the art. An expression vector includes
vectors capable of expressing DNA that is operatively linked with
regulatory sequences, such as promoter regions, that are capable of
effecting expression of such DNA fragments. Thus, an expression
vector refers to a recombinant DNA or RNA construct, such as a
plasmid, a phage, recombinant virus or other vector that, upon
introduction into an appropriate host cell, results in expression
of the cloned DNA. Appropriate expression vectors are well known to
those of skill in the art and include those that are repliable in
eukaryotic cells and/or prokaryotic cells and those that remain
episomal or those which integrate into the host cell genome.
[0208] As used herein, protein binding sequence refers to a protein
or peptide sequence that is capable of specific binding to other
protein or peptide sequences generally, to a set of protein or
peptide sequences or to a particular protein or peptide
sequence.
[0209] As used herein, epitope tag refers to a short stretch of
amino acid residues corresponding to an epitope to facilitate
subsequent biochemical and immunological analysis of the epitope
tagged protein or peptide. Epitope tagging is achieved by including
the sequence of the epitope tag to the protein-encoding sequence in
an appropriate expression vector. Epitope tagged proteins can be
affinity purified using highly specific antibodies raised against
the tags.
[0210] As used herein, metal binding sequence refers to a protein
or peptide sequence that is capable of specific binding to metal
ions generally, to a set of metal ions or to a particular metal
ion.
[0211] As used herein, a combination refers to any association
between two or among more items.
[0212] As used herein, a composition refers to any mixture. It can
be a solution, a suspension, liquid, powder, a paste, aqueous,
non-aqueous or any combination thereof.
[0213] As used herein, fluid refers to any composition that can
flow. Fluids thus encompass compositions that are in the form of
semi-solids, pastes, solutions, aqueous mixtures, gels, lotions,
creams and other such compositions.
[0214] As used herein, a cellular extract refers to a preparation
or fraction which is made from a lysed or disrupted cell.
[0215] As used herein, an agent is said to be randomly selected
when the agent is chosen randomly without considering the specific
sequences involved in the association of a protein alone or with
its associated substrates, binding partners, etc. An example of
randomly selected agents is the use a chemical library or a peptide
combinatorial library, or a growth broth of an organism or
conditioned medium.
[0216] As used herein, an agent is said to be rationally selected
or designed when the agent is chosen on a non-random basis that
takes into account the sequence of the target site and/or its
conformation in connection with the agent's action. As described in
the Examples, there are proposed binding sites for Hyaluronidase
and (catalytic) sites in the glycoprotein having SEQ ID NO: 1 or
SEQ ID NO: 4. Agents can be rationally selected or rationally
designed by utilizing the peptide sequences that make up these
sites. For example, a rationally selected peptide agent can be a
peptide whose amino acid sequence is identical to the ATP or
calmodulin binding sites or domains.
[0217] Oligosaccharides are considered to have a reducing end and a
non-reducing end, whether or not the saccharide at the reducing end
is in fact a reducing sugar. In accordance with accepted
nomenclature, oligosaccharides are depicted herein with the
non-reducing end on the left and the reducing end on the right. All
oligosaccharides described herein are described with the name or
abbreviation for the non-reducing saccharide (e.g., Gal), followed
by the configuration of the glycosidic bond (.alpha. or. beta.),
the ring bond, the ring position of the reducing saccharide
involved in the bond, and then the name or abbreviation of the
reducing saccharide (e.g., GIcNAc). The linkage between two sugars
may be expressed, for example, as 2,3, 2.fw darw.3, or (2,3). Each
saccharide is a pyranose.
[0218] As used herein, N-linked sugar moiety refers to an
oligosaccharide attached to a sHASEGP via the amide nitrogen of Asn
residues. N-linked oligosaccharides fall into several major types
(oligomannose, complex, hybrid, sulfated), all of which have (Man)
3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn
residues that fall within--Asn-Xaa-Thr/Ser- sequences (where Xaa is
not Pro). N-linked sites are often indirectly assigned by the
appearance of a "blank" cycle during sequencing. Positive
identification can be made after release of the oligosaccharide by
PNGase F, which converts the glycosylated Asn to Asp. After PNGase
F release, N-linked oligosaccharides can be purified using Bio-Gel
P-6 chromatography, with the oligosaccharide pool subjected to
preparative high pH anion exchange chromatography (HPAEC) (Townsend
et al., (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide
isomers can be resolved using HPAEC. Fucose residues will shift
elution positions earlier in the HPAEC chromatogram, while
additional sialic acid residues will increase the retention time.
Concurrent treatment of glycoproteins whose oligosaccharide
structures are known (e.g., bovine fetuin, a-1 acid glycoprotein,
ovalbumin, RNAse B, transferrin) can facilitate assignment of the
oligosaccharide peaks. The collected oligosaccharides can be
characterized by a combination of compositional and methylation
linkage analyses (Waegheet al., (1983) Carbohydr Res. 123,
281-304.), with anomeric configurations assigned by NMR
spectroscopy (Van Halbeek (1993) in Methods Enzymol 230).
[0219] Alternatively, oligosaccharides can be identified by
fluorescence assisted carbohydrate electrophoresis (FACE)
Callewaert et al. (2001) Glycobiology 11, 275-281.
[0220] As used herein, the term "sialic acid" refers to any member
of a family of nine-carbon carboxylated sugars. The most common
member of the sialic acid family is N-acetylneuraminic acid
(2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic
acid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member
of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in
which the N-acetyl group of NeuAc is hydroxylated. A third sialic
acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano
et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al.
(1990) J. Biol. Chem. 265: 21811-21819. Also included are
9-substituted sialic acids such as a 9-O-C.sub.1-C.sub.6
acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,
9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of
the sialic acid family, see, e.g., Varki (1992) Glycobiology 2:
2540; Sialic Acids: Chemistry, Metabolism and Function, R. Schauer,
Ed. (Springer-Verlag, N.Y. (1992)). The synthesis and use of sialic
acid compounds in a sialation procedure is disclosed in
international application WO 92/16640, published Oct. 1, 1992.
[0221] As used herein, PNGase refers to an Asparagine Peptide
specific N-glycosidase F such as the Flavobacterium maningoseptum
peptide-N-glycosidase F. PNGASE enzymes are characterized by their
specificity towards N-linked rather than O-linked oligosaccharides.
Characterization of PNGASE efficacy can be defined by both SDS PAGE
electrophoresis, or fluorescent assisted carbohydrate
electrophoresis.
[0222] As used herein substantially terminated Sialation refers to
N-linked oligosaccharides terminating with sialic acid residue as a
terminal sugar. Terminal sialic acids can be identified by FACE
analysis of released carbohydrates following treatment with
neuraminidase.
[0223] The circulatory lifetime of glycoproteins in the blood is
highly dependent on the composition and structure of its N-linked
carbohydrate groups. This fact is of direct relevance for
therapeutic glycoproteins that are intended to be administered
parenterally. In general, maximal circulatory half-life of a
glycoprotein requires that its N-linked carbohydrate groups
terminate in the sequence NeuAc-Gal-GlcNAc. Without the terminal
sialic acid (NeuAc), the glycoprotein is rapidly cleared from the
blood by a mechanism involving the recognition of the underlying
N-acetylgalactosamine (GalNAc) or galactose (Gal) residues (Goochee
et al. (1991) Biol/Technology 9: 1347-1355). For this reason,
ensuring the presence of terminal sialic acid on N-linked
carbohydrate groups of therapeutic glycoproteins is an important
consideration for their commercial development.
[0224] Circulating glycoproteins are exposed to sialidase(s) (or
neuraminidase) which can remove terminal sialic acid residues.
Typically the removal of the sialic acid exposes galactose
residues, and these residues are recognized and bound by
galactose-specific receptors in hepatocytes (reviewed in Ashwell
and Harford (1982) Ann. Rev. Biochem. 51:531). Liver also contains
other sugar-specific receptors which mediate removal of
glycoproteins from circulation. Specificities of such receptors
also include N-acetylglucosamine, mannose, fucose and
phosphomannose. Glycoproteins cleared by the galactose receptors of
hepatocytes undergo substantial degradation and then enter the
bile; glycoproteins cleared by the mannose receptor of Kupffer
cells enter the reticuloendothelial system (reviewed in Ashwell and
Harford (1982) Ann. Rev. Biochem. 51:53).
[0225] As used herein Neutral Active refers to a sHASEGP
glycoprotein with catalytic activity towards a glycosaminoglycan
substrate in vitro at a PH between 5 and 8 under conditions of salt
less than 150 mM and buffering strength less than 50 mM.
[0226] As used herein, a stabilized solution refers to a sHASEGP
that retains greater than 60% of its initial activity after storage
at room temperature for 30 days.
[0227] As used herein unless otherwise specified, a unit is
expressed in turbidity reducing units (TRU). One TRU is defined as
the amount of hyaluronidase activity required to reduce the
turbidity of an acidified solution of hyaluronic acid and is
equivalent to the U.S.P. National Formulary (NF XIII) units (NFU).
The ELISA-like enzyme assay described herein can be related to the
TRU, the NFU, and U.S.P. unit through a standard curve of a sample
of hyaluronidase (e.g., USP or WHO standard) standardized through
the U.S.P. Therefore, the enzyme activities determined by the
ELISA-like enzyme assay are actually relative TRU, since enzyme
activity is not actually measured using the turbidometric assay
(Dorfman et al., 1948, J. Biol. Chem. 172:367).
[0228] As used herein, potency is defined by the amount of sHASEGP
protein required to degrade substrate in vitro based upon a
Turbidity Reducing Unit or Relative Turbidity Reducing Unit.
[0229] As used herein, specific activity refers to Units of
activity per mg protein. The amount of sHASEGP protein is defined
by the absorption of a solution of sHASEGP at 280 nm assuming a
molar extinction coefficient of approximately 1.7, in units of
M.sup.-1 cm.sup.-1.
[0230] Polyethylene glycol (PEG) has been widely used in
biomaterials, biotechnology and medicine primarily because PEG is a
biocompatible, nontoxic, nonimmunogenic and water-soluble polymer
(Zhao and Harris, ACS Symposium Series 680: 458-72, 1997). In the
area of drug delivery, PEG derivatives have been widely used in
covalent attachment (i.e., "PEGylation") to proteins to reduce
immunogenicity, proteolysis and kidney clearance and to enhance
solubility (Zalipsky, Adv. Drug Del. Rev. 16:157-82, 1995).
Similarly, PEG has been attached to low molecular weight,
relatively hydrophobic drugs to enhance solubility, reduce toxicity
and alter biodistribution. Typically, PEGylated drugs are injected
as solutions.
[0231] A closely related application is synthesis of crosslinked
degradable PEG networks or formulations for use in drug delivery
since much of the same chemistry used in design of degradable,
soluble drug carriers can also be used in design of degradable gels
(Sawhney et al., Macromolecules 26: 581-87, 1993). It is also known
that intermacromolecular complexes can be formed by mixing
solutions of two complementary polymers. Such complexes are
generally stabilized by electrostatic interactions
(polyanion-polycation) and/or hydrogen bonds (polyacid-polybase)
between the polymers involved, and/or by hydrophobic interactions
between the polymers in an aqueous surrounding (Krupers et al.,
Eur. Polym J. 32:785-790, 1996). For example, mixing solutions of
polyacrylic acid (PAAc) and polyethylene oxide (PEO) under the
proper conditions results in the formation of complexes based
mostly on hydrogen bonding. Dissociation of these complexes at
physiologic conditions has been used for delivery of free drugs
(i.e., non-PEGylated). In addition, complexes of complementary
polymers have been formed from both homopolymers and
copolymers.
[0232] In one aspect, the polyethylene glycol has a molecular
weight ranging from about 3 kD to about 50 kD, and preferably from
about 5 kD to about 30 kD. Covalent attachment of the PEG to the
drug (known as "PEGylation") may be accomplished by known chemical
synthesis techniques. For example, in one aspect of the present
invention, the PEGylation of protein may be accomplished by
reacting NHS-activated PEG with the protein under suitable reaction
conditions.
[0233] While numerous reactions have been described for PEGylation,
those that are most generally applicable confer directionality,
utilize mild reaction conditions, and do not necessitate extensive
downstream processing to remove toxic catalysts or bi-products. For
instance, monomethoxy PEG (mPEG) has only one reactive terminal
hydroxyl, and thus its use limits some of the heterogeneity of the
resulting PEG-protein product mixture. Activation of the hydroxyl
group at the end of the polymer opposite to the terminal methoxy
group is generally necessary to accomplish efficient protein
PEGylation, with the aim being to make the derivatised PEG more
susceptible to nucleophilic attack. The attacking nucleophile is
usually the epsilon-amino group of a lysyl residue, but other
amines can also react (e.g. the N-terminal alpha-amine or the ring
amines of histidine) if local conditions are favorable. A more
directed attachment is possible in proteins containing a single
lysine or cysteine. The latter residue can be targeted by
PEG-maleimide for thiol-specific modification. Alternatively, PEG
hydrazide can be reacted with periodate oxidized sHASEGP and
reduced in the presence of NaCNBH.sub.3. More specifically,
PEGylated CMP sugars can be reacted with sHASEGP in the presence of
appropriate glycosyl-transferases. One technique is the
"PEGylation" technique where a number of polymeric molecules are
coupled to the polypeptide in question. When using this technique
the immune system has difficulties in recognizing the epitopes on
the polypeptide's surface responsible for the formation of
antibodies, thereby reducing the immune response. For polypeptides
introduced directly into the circulatory system of the human body
to give a particular physiological effect (i.e. pharmaceuticals)
the typical potential immune response is an IgG and/or IgM
response, while polypeptides which are inhaled through the
respiratory system (i.e. industrial polypeptide) potentially may
cause an IgE response (i.e. allergic response). One of the theories
explaining the reduced immune response is that the polymeric
molecule(s) shield(s) epitope(s) on the surface of the polypeptide
responsible for the immune response leading to antibody formation.
Another theory or at least a partial factor is that the heavier the
conjugate is, the more reduced immune response is obtained.
[0234] In the case of human sHASEGPs being used in the human body,
a preferred combination as described herein, the tendency for the
sSHASEGP to elicit an immune response can be significantly reduced
as compared to, for example, non-human animal-derived enzymes such
as bovine or ovine hyaluronidases (i.e. even if those
animal-derived enzymes could be completely purified away from other
animal-derived proteins, which is difficult to impossible to
achieve in practice). Although human sHASEGPs thus represent a
substantial and very important improvement over animal-derived
products, they can for many uses be improved still further by
post-translational modifications (PTMs) such as PEGylation to
enhance their resistance to degradation or elimination and/or to
improve other properties making them even more beneficial for
particular applications.
[0235] For example, although preferred compositions for humans
(such as the human sHASEGPs) exhibit reduced immunogenicity
compared to animal-derived hyaluronidases or non-human sHASEGPs,
PEGylation of such human sHASEGPs can be used for example to extend
their half life in the bloodstream and/or in other tissues within
the body. Without being restricted to a particular mechanism of
action, it is generally considered that the attachment of PEG
moieties to sHASEGPs can be used to effectively shield the
molecule, decreasing its relative sensitivity to proteases and
potentially also decreasing the extent and rate of its clearance
and elimination from the body.
[0236] The polymeric molecules coupled to the polypeptide may be
any suitable polymeric molecule with a molecular weight as defined
according to the invention, including natural and synthetic
homopolymers, such as polyols (i.e. poly-OH), polyamines (i.e.
poly-NH.sub.2) and polycarboxyl acids (i.e. poly-COOH), and further
heteropolymers i.e. polymers comprising one or more different
coupling groups e.g. a hydroxyl group and amine groups.
[0237] Examples of suitable polymeric molecules include polymeric
molecules selected from the group comprising polyalkylene oxides
(PAO), such as polyalkylene glycols (PAG), including polypropylene
glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylene
glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole
(CDI-PEG) branched polyethylene glycols (PEGs), polyvinyl alcohol
(PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino
acids, polyethylene-co-maleic acid anhydride, polystyrene-co-maleic
acid anhydride, dextrans including carboxymethyl-dextrans, heparin,
homologous albumin, celluloses, including methylcellulose,
carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose
carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of
chitosan, starches such as hydroxyethyl-starches and
hydroxypropyl-starches, glycogen, agaroses and derivatives thereof,
guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin,
alginic acid hydrolysates and bio-polymers.
[0238] Preferred polymeric molecules are non-toxic polymeric
molecules such as (m)polyethylene glycol (mPEG) which further
requires a relatively simple chemistry for its covalent coupling to
attachment groups on the enzyme's surface.
[0239] Generally seen polyalkylene oxides (PAO), such as
polyethylene oxides, such as PEG and especially mPEG, are the
preferred polymeric molecules, as these polymeric molecules, in
comparison to polysaccharides such as dextran, pullulan and the
like, have few reactive groups capable of cross-linking, which is
undesirable.
[0240] Improvements in PEGylation and other types of
post-translational modifications (PTMs) have been extensive and
there are now a large number of PTM techniques and reagents known
in the art, and new ones are regularly being developed. Techniques
and reagents for PEGylation include, for example: (i) specialized
linkers and coupling chemistries (see, e.g., the reagents and
technologies reviewed by Harris, J. M., Adv. Drug Deliv. Rev. 54:
459-476 (2002) and the primary references reviewed therein); (ii)
branched PEGs which effectively allow additional PEG groups to be
attached to a single conjugation site (see, e.g., Veronese, F. M.
et al., Bioorg. Med. Chem. Lett. 12: 177-180 (2002)); (iii)
site-specific PEGylation, including site-specific monoPEGylation
(see, e.g., Chapman, A. P. et al., Nature Biotech. 17: 780-783
(1999); and (iv) site-directed enzymatic PEGylation (e.g. using a
transglutaminase reaction as described by Sato, H., Adv. Drug
Deliv. Rev. 54: 487-504 (2002)). There are also additional
technologies and reagents available from an increasing numbr of
commercial suppliers (see, e.g., Nektar/Shearwater
(www.nektar.com), Sunbio (www.sunbio.com and
www.sunbio.com/peg-shop), Celares GmbH (www.celares.com), and
others).
[0241] B. Tissue Expression Profiles sHASEGP.
[0242] While previously thought to be testis specific, human
sHASEGP is expressed in multiple tissues in humans when using more
sensitive techniques such as RT-PCR. The sHASEGP transcript is
found in medulla (brain), microvascular endothelium, prostate,
breast, retina, pooled human melanocyte, fetal heart, and pregnant
uterus. sHASEGP is also expressed in germ cell tumors. RT-PCR based
detection of sHASEGP transcripts is generally required to detect
levels in tissues other than testis.
[0243] C. Assays for sHASEGP Enzyme Activity
[0244] Turbidometric Mircotiter Assay for Hyaluronidase
Activity
[0245] Hyaluronidase activity can be detected by way of a modified
turbidometric assay in acidified serum solution. The reagents
required are as follows: TABLE-US-00001 UV sterilized 2X-deionized
water or Braun R5000-01 sterile water for irrigation Hylumed
Medical - Sodium Genzyme Advanced 4876 Hyaluronate, High Molecular
Weight Biomaterials HA Hyaluronidase Reference Standard USP 31200
Potassium Acetate, Granular, USP, JTBaker 2914-01 ACS Acetic Acid,
Glacial, 99+ % Sigma A-6283 Sodium Phosphate Monobasic Mallinkrodt
7774 Monohydrate, USP Granular Sodium Phosphate Dibasic Mallinkrodt
7771 Anhydrous, USP Sodium Chloride, Crystals, GR, EMScience
SX0420-5 ACS Gelatin Hydrolysate Enzymatic Sigma G-0262 Horse
Serum, Donor Herd, cell Sigma H-1270 culture tested, Hybridoma
culture tested, USA Origin Human Serum Albumin 20% Griffols
Hydrochloric Acid, ACS Reagent Sigma H-7020 Calcium Chloride,
Dihydrate, JTBaker 1336-01 Granular, USP, -FCC
[0246] The following reagents are prepared: Acetate Buffer
Solution--14.0 g of potassium acetate and 25.0 mL of glacial acetic
acid in water to make 1000 mL. Phosphate Buffer Solution--2.5 g of
sodium phosphate monobasic, 1.0 g of anhydrous sodium phosphate
dibasic, and 8.2 g of sodium chloride in water to make 1000 mL.
Enzyme Diluent Stock Solution--500 mL of Phosphate Buffer Solution
with 500 mL of water. Enzyme Diluent Working Solution--33 mg of
hydrolyzed gelatin in 50 mL of Enzyme Diluent Stock
Solution--prepared within 2 hours of use. Sample Stabilization
Buffer Solution ("SSB" Soln.)--125 microliters (pL) of a 20% Human
Serum Albumin Solution and 50 .mu.L of a 1 M Calcium Chloride
solution in 50 mL of Enzyme Diluent Working Solution, and mix
thoroughly. Serum Stock Solution--Dilute 1 volume of Horse Serum
with 9 volumes of Acetate Buffer Solution. Adjust with 4 N
hydrochloric acid to a pH of 3.1 and allow the solution to stand at
room temperature for 18 to 24 hrs. Store the solution at 4 .degree.
C., and use within 30 days. Serum Working Solution--10 mL of the
Serum Stock Solution in 30 mL of the Acetate Buffer Solution,
adjusted to room temperature. Hyaluronic Acid Stock
Solution--Sodium Hyaluronic Acid to a concentration 5.0 mg/mL in
water. Hyaluronic Acid Working Solution--0.75 mL of the Hyaluronic
Acid Stock Solution in 4.25 mL of the Phosphate Buffer Solution.
Standard Stock Solution--One container of USP Reference Standard
Hyaluronidase to a concentration 1000 Units/mL in water, aliquoted
into 50 .mu.L portions, and stored at -20.degree. C. Standard
Working Solution--40 .mu.L of Standard Stock Solution in 960 .mu.L
of cold Enzyme Diluent Working Solution to obtain a solution having
a known concentration of 40 Units/mL, prepared immediately before
use in the assay.
[0247] All enzyme samples are diluted in a "Low Protein Binding"
96-well plate according to the following guidelines:
[0248] a) The range of maximum sensitivity of this assay is between
10-30 Units/mL. To minimize the number of times an assay must be
repeated in order to get results that are within range, first
determine the approximate number of total units/mL for the sample,
and then choose a (whole number) dilution such that the final
concentration is approximately 20 Units/mi.
[0249] b) Minimum Sample volumes needed to perform assay are as
follows: FPLC Fractions=50 .mu.L, Tissue Culture Supematants=1 mL,
Purified/Concentrated/Final Step Material=10 .mu.L.
[0250] c) For samples with serial dilutions, 1:10 dilutions in the
"Low Protein Binding" 96-well plate are made in triplicate by
pipetting 360 .mu.L of the "SSB" Solution and 40 .mu.L of Sample
into each well.
[0251] For preparation of USP Standard prepare the USP Standard
Curve in the "Low Protein Binding" 96-well plate as follows:
TABLE-US-00002 USP Standard Curve: Enzyme Standard Final Diluent
Working Conc.(in Wells: Standard: Soln.(in .mu.L): Soln.(in .mu.L):
Units/mL): A1-A3 St01 0 100 40 B1-B3 St02 20 80 32 C1-C3 St03 40 60
24 D1-D3 St04 60 40 16 E1-E3 St05 80 20 8 F1-F3 St06 90 10 4 G1-G3
St07 100 0 0
[0252] For preparation of the Hyaluronic Acid Control in columns
1-3, prepare the H.A. Control in the "Flat Bottom" 96-well plate is
prepared as follows: TABLE-US-00003 H.A. Controls: Hyaluronic Acid
Enzyme Diluent Wells: Control: Working Soln.(in .mu.L): Working
Soln.(in .mu.L): H1-H3 Co01 0 60
[0253] The Reaction Plate: 30 .mu.L per well of Hyaluronic Acid
Working Solution is pippetted using a 50 .mu.L 8-channel transfer
pipette into a "Flat Bottom" 96-well microtiter plate leaving wells
H1-H3 empty. 60 .mu.L/well of Enzyme Diluent Working Solution is
pipetted into wells H1-H3 of the same plate as the HA control.
[0254] Serum Working Solution: 40 mL of Serum Working Solution is
dispensed into a transfer basin and next to the Heat Block.
[0255] Pre-warming stage: Once both plates have been prepared, the
Low Protein Binding 96-Well plate containing the diluted samples,
standards, controls and the Flat Bottom 96-well plate containing
the Hyaluronic Acid Working Solution are placed onto a heat block
and allow them to warm for 5 min. at 37.degree. C.
[0256] The Reaction is initiated by the addition of Enzyme to
Substrate: 30 .mu.L from the enzyme plate into all of the wells in
column #1 of the 96-Well flat bottom plate (containing the
substrate) using a 5-50 .mu.L 8-channel pipette. The
Enzyme/Substrate reaction mixture is aspirated 5 times (drawing the
solution up and down with the transfer during the first 15 seconds
to ensure complete sample mixing. After mixing the enzyme and
substrate, the tips are ejected and a new set of tips loaded on the
transfer pipettor for the next column. A timer is restarted, and at
time (t)=0:30, this process is repeated for column 2. At the next
30 second interval (t)=1:00, this is repeated process for column 3.
This process is repeated moving from left to right across the
plate, every 30 seconds until all of the wells contain both enzyme
and substrate.
[0257] Stopping the reaction: When timer reaches 6 minutes
(t)=6:00, 240 .mu.L of the Serum Working Solution is pippetted into
each well, using a 50-300 .mu.L 8-channel transfer pipette, into
column 1 of the 96-well flat bottom plate from the adjacent 50 mL
Reagent Reservoir. The mixture is aspirated 3 times (drawing the
solution up and down with the transfer Pipettor) during the first
10 seconds to ensure complete mixing. The process is repeated every
30 seconds, proceeding from column's 1 to 12 . Upon completion of
the last column (column 12), the reaction plate is removed from the
heat block and place the plate onto the read tray of the plate
reader at 640 nM. A linear curve fit is generated from the standard
curve that permits extrapolation of test samples.
[0258] Alternative Assays for Hyaluronidase
[0259] Biotinylated Hyaluronan Microtiter Assay
[0260] The free carboxyl groups on glucuronic acid residues of
Hyaluronan are biotinylated in a one step reaction using
biotin-hydrazide (Pierce), Sulfo NHS (Pierce) and 1-Ethyl
dimethylaminopropyl-carbodiimide (Sigma). This biotinylated HA
substrate is covalently coupled to a 96 well microtiter plate in a
second reaction. At the completion of the enzyme reaction, residual
substrate is detected with an avidin-peroxidase reaction that can
be read in a standard ELISA plate reader. As the substrate is
covalently bound to the microtiter plate, artifacts such as
pH-dependent displacement of the biotinylated substrate does not
occur. The sensitivity permits rapid measurement of Hyaluronidase
activity from cultured cells and biological samples with an
inter-assay variation of less than 10%.
[0261] The specific activity of hyaluronidase is expressed in
turbidity reducing units (TRU). One TRU is defined as the amount of
hyaluronidase activity required to reduce the turbidity of an
acidified solution of hyaluronic acid and is equivalent to the
U.S.P./National Formulary (NF XIII) units (NFU). The ELISA-like
enzyme assay used for purification is related to the TRU, the NFU,
and U.S.P. unit through a standard curve of a sample of
hyaluronidase (e.g., USP) standardized through the U.S.P.
Therefore, the enzyme activities determined by the ELISA-like
enzyme assay are actually relative TRU, since enzyme activity is
not actually measured using the turbidometric assay (Dorfman et
al., 1948, J. Biol. Chem. 172:367).
[0262] Many Hyaluronidase assays have been based upon the
measurement of the generation of new reducing N-acetylamino groups
(Bonner and Cantey, Clin. Chim. Acta 13:746-752, 1966), or loss of
viscosity (De Salegui et al., Arch. Biochem. Biophys. 121:548-554,
1967) or turbidity (Dorfman and Ott, J. Biol. Chem. 172:367, 1948).
With purified substrates all of these methods suffice for
determination of the presence or absence of endoglucosamidic
activity.
[0263] Substantially purified glycosaminoglycan substrates can also
be used for in a Gel Shift Assay. Glycosaminoglycans are mixed with
recombinant sHASEGP to test for endoglucosidase activity that
results in a shift in substrate mobility within the gel.
Chondroitin-4 and 6 sulfate, dermatan sulfate, heparan-sulfate can
be obtained from Sigma Chemical. Human umbilical cord Hyaluronan
can be obtained from ICN. Each test substrate is diluted to 0.1
mg/ml in a buffer range from pH 3.5-7.5. 10 ul samples of purified
sHASEGP or conditioned media from sHASEGP expressing cells as well
as are mixed with 90 ul of test substrate in desired buffer and
incubated for 3 hours at 37 C. Following incubation samples are
neutralized with sample buffer (Tris EDTA PH 8.0, Bromophenol Blue
and glycerol) followed by electrophoresis. Glycosaminoglycans are
detected by staining the gels in 0.5% Alcian Blue in 3% Glacial
Acetic Acid overnight followed by destaining in 7% Glacial Acetic
Acid. Degradation is determined by comparison substrate mobility in
the presence and absence of enzyme.
[0264] Hyaluronidase activity can also be detected by substrate gel
zymography (Guentenhoner et al., 1992, Matrix 388-396). In this
assay a sample is applied to a SDS-PAGE gel containing hyaluronic
acid and the proteins in the sample separated by electrophoresis.
The gel is then incubated in an enzyme assay buffer and
subsequently stained to detect the hyaluronic acid in the gel.
Hyaluronidase activity is visualized as a cleared zone in the
substrate gel.
[0265] D. Identification and Isolation sHASEGP of Polypeptide
Genes.
[0266] The sHASEGP polypeptide gene and/or domains thereof, can be
obtained by methods well known in the art for DNA isolation. Any
method known to those of skill in the art for identification of
nucleic acids that encode desired genes can be used. Any method
available in the art can be used to obtain a full-length (i. e.,
encompassing the entire coding region) cDNA or genomic DNA clone
encoding a sHASEGP polypeptide. For example, the polymerase chain
reaction (PCR) can be used to amplify a sequence that is expressed
in normal tissues, e.g., nucleic acids encoding a sHASEGP
polypeptide (SEQ. Nos: 1 and 2), in a genomic or cDNA library.
Oligonucleotide primers that hybridize to sequences at the 3' and
5' termini of the identified sequences can be used as primers to
amplify by PCR sequences from a nucleic acid sample (RNA or DNA
generally a cDNA library, from an appropriate source (e. testis,
prostate, breast).
[0267] PCR can be carried out, e. g., by use of a Perkin-Elmer
Cetus thermal cycler and Taq polymerase (Gene Amp). The DNA being
amplified can include mRNA or cDNA or genomic DNA from any
eukaryotic species. One can choose to synthesize several different
degenerate primers, for use in the PCR reactions.
[0268] It is also possible to vary the stringency of hybridization
conditions used in priming the PCR reactions, to amplify nucleic
acid homologs (e. g., to obtain sHASEGP polypeptide sequences from
species other than humans or to obtain human sequences with
homology to sHASEGP polypeptide) by allowing for greater or lesser
degrees of nucleotide sequence similarity between the known
nucleotide sequence and the nucleic acid homolog being isolated.
For cross-species hybridization, low stringency to moderate
stringency conditions are used. For same species hybridization,
moderately stringent to highly stringent conditions are used. The
conditions can be empirically determined.
[0269] After successful amplification of the nucleic acid
containing all or a portion of the identified sHASEGP polypeptide
sequence or of a nucleic acid encoding all or a portion of a
sHASEGP polypeptide homolog, that segment can be molecularly cloned
and sequenced, and used as a probe to isolate a complete cDNA or
genomic clone. This, in turn, permits the determination of the
gene's complete nucleotide sequence, the analysis of its
expression, and the production of its protein product for
functional analysis. Once the nucleotide sequence is determined, an
open reading frame encoding the sHASEGP polypeptide gene protein
product can be determined by any method well known in the art for
determining open reading frames, for example, using publicly
available computer programs for nucleotide sequence analysis. Once
an open reading frame is defined, it is routine to determine the
amino acid sequence of the protein encoded by the open reading
frame. In this way, the nucleotide sequences of the entire sHASEGP
polypeptide genes as well as the amino acid sequences of sHASEGP
polypeptide proteins and analogs can be identified.
[0270] Any eukaryotic cell potentially can serve as the nucleic
acid source for the molecular cloning of the sHASEGP polypeptide
gene. The nucleic acids can be isolated from vertebrate, mammalian,
human, porcine, bovine, feline, avian, equine, canine, as well as
additional primate sources, insects, plants and other organisms.
The DNA can be obtained by standard procedures known in the art
from cloned DNA (e. g., a DNA "library"), by chemical synthesis, by
cDNA cloning, or by the cloning of genomic DNA, or fragments
thereof, purified from the desired cell (see, e. g., Sambrook et
al. 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York;
Glover, D. M. Ed., 1985, DNA Cloning: A Practical Approach, MRL
Press, Ltd., Oxford, U. K. Vol. 1,11. Clones derived from genomic
DNA can contain regulatory and intron DNA regions in addition to
coding regions; clones derived from cDNA will contain only exon
sequences. For any source, the gene is cloned into a suitable
vector for propagation thereof.
[0271] In the molecular cloning of the gene from genomic DNA, DNA
fragments are generated, some of which will encode the desired
gene.
[0272] The DNA can be cleaved at specific sites using various
restriction enzymes.
[0273] Alternatively, one can use DNAse in the presence of
manganese to fragment the DNA, or the DNA can be physically
sheared, for example, by sonication. The linear DNA fragments then
can be separated according to size by standard techniques,
including but not limited to, agarose and polyacrylamide gel
electrophoresis and column chromatography.
[0274] Once the DNA fragments are generated, identification of the
specific DNA fragment containing the desired gene can be
accomplished in a number of ways.
[0275] For example, a portion of the sHASEGP polypeptide (of any
species) gene (e. g., a PCR amplification product obtained as
described above or an oligonucleotide having a sequence of a
portion of the known nucleotide sequence) or its specific RNA, or a
fragment thereof be purified and labeled, and the generated DNA
fragments can be screened by nucleic acid hybridization to the
labeled probe (Benton and Davis, Science 196: 180 (1977); Grunstein
and Hogness, Proc. Natl. Acad. Sci. U.S.A. 72: 3961 (1975)). Those
DNA fragments with substantial homology to the probe will
hybridize. It is also possible to identify the appropriate fragment
by restriction enzyme digestion (s) and comparison of fragment
sizes with those expected according to a known restriction map if
such is available or by DNA sequence analysis and comparison to the
known nucleotide sequence of sHASEGP polypeptide. Further selection
can be carried out on the basis of the properties of the gene.
Alternatively, the presence of the gene can be detected by assays
based on the physical, chemical, or- immunological properties of
its expressed product. For example, cDNA clones, or DNA clones
which hybrid-select the proper mRNA can be selected which produce a
protein that, e.g., has similar or identical electrophoretic
migration, isoelectric focusing behavior, proteolytic digestion
maps, antigenic properties, Hyaluronidase activity. If an
anti-sHASEGP polypeptide antibody is available, the protein can be
identified by binding of labeled antibody to the putatively sHASEGP
polypeptide synthesizing clones, in an ELISA (enzyme-linked
immunosorbent assay)--type procedure.
[0276] Alternatives to isolating the sHASEGP polypeptide genomic
DNA include, but are not limited to, chemically synthesizing the
gene sequence from a known sequence or making cDNA to the mRNA that
encodes the sHASEGP polypeptide.
[0277] For example, RNA for cDNA cloning of the sHASEGP polypeptide
gene can be isolated from cells expressing the protein. The
identified and isolated nucleic acids then can be inserted into an
appropriate cloning vector. A large number of vector-host systems
known in the art can be used. Possible vectors include, but are not
limited to, plasmids or modified viruses, but the vector system
must be compatible with the host cell used. Such vectors include,
but are not limited to, bacteriophages such as lambda derivatives,
or plasmids such as pBR322 or pUC plasmid derivatives or the
Bluescript vector (Stratagene, La Jolla, Calif.). The insertion
into a cloning vector can, for example, be accomplished by ligating
the DNA fragment into a cloning vector that has complementary
cohesive termini.
[0278] If the complementary restriction sites used to fragment the
DNA are not present in the cloning vector, the ends of the DNA
molecules can be enzymatically modified. Alternatively, any site
desired can be produced by ligating nucleotide sequences (linkers)
onto the DNA termini; these ligated linkers can include specific
chemically synthesized oligonucleotides encoding restriction
endonuclease recognition sequences. In an alternative method, the
cleaved vector and sHASEGP polypeptide gene can be modified by
homopolymeric tailing.
[0279] Recombinant molecules can be introduced into host cells via
transformation, transfection, infection, electroporation, calcium
precipitation and other methods, so that many copies of the gene
sequence are generated.
[0280] In specific embodiments, transformation of host cells with
recombinant DNA molecules that incorporate the isolated sHASEGP
polypeptide gene, cDNA, or synthesized DNA sequence enables
generation of multiple copies of the gene.
[0281] Thus, the gene can be obtained in large quantities by
growing transformants, isolating the recombinant DNA molecules from
the transformants and, when necessary, retrieving the inserted gene
from the isolated recombinant DNA.
[0282] E. Vectors, Plasmids and Cells that Contain Nucleic Acids
Encoding A Shasegp Polypeptide or Hyaluronidase Domain Thereof and
Expression of Shasegp Polypeptides Vectors and Cells.
[0283] For recombinant expression of one or more of the sHASEGP
polypeptides, the nucleic acid containing all or a portion of the
nucleotide sequence encoding the sHASEGP polypeptide can be
inserted into an appropriate expression vector i.e., a vector that
contains the necessary elements for the transcription and
translation of the inserted protein coding sequence. The necessary
transcriptional and translational signals can also be supplied by
the native promoter for sHASEGP genes, and/or their flanking
regions.
[0284] Also provided are vectors that contain nucleic acid encoding
the sHASEGPs that can be introduced into an expression system
capable of producing a soluble neutral active sHASEGP.
[0285] Cells containing the vectors are also provided. The cells
include eukaryotic and prokaryotic cells, and the vectors suitable
for use therein.
[0286] Eukaryotic cells, including dihydrofolate reductase
deficient Chinese Hamster Ovary Cells (DG44), containing the
vectors are provided. Suitable cells include yeast cells, fungal
cells, plant cells, insect cells and animal cells. The cells are
used to produce a sHASEGP polypeptide or Hyaluronidase domain
thereof by (a) growing the above-described cells under conditions
whereby the encoded sHASEGP polypeptide or Hyaluronidase domain of
the sHASEGP polypeptide is expressed by the cell, and then (b)
recovering the expressed Hyaluronidase domain protein. In the
exemplified embodiments, the Hyaluronidase domain is secreted into
the medium.
[0287] In one embodiment, the vectors include a sequence of
nucleotides that encodes a polypeptide that has Hyaluronidase
activity and contains all or a portion of only the Hyaluronidase
domain, or multiple copies thereof, of a sHASEGP protein are
provided. Also provided are vectors that comprise a sequence of
nucleotides that encodes the Hyaluronidase domain and additional
portions of a sHASEGP protein up to and including a full-length
sHASEGP protein, as well as multiple copies thereof, are also
provided. The vectors can be selected for expression of the sHASEGP
protein or Hyaluronidase domain thereof in the cell or such that
the sHASEGP protein is expressed as a secreted protein.
Alternatively, the vectors can include signals necessary for
secretion of encoded proteins. When the Hyaluronidase domain is
expressed the nucleic acid is linked to nucleic acid encoding a
secretion signal, such as the Saccharomyces cerevisiae a mating
factor signal sequence or a portion thereof, or the native signal
sequence.
[0288] In order to generate a soluble, neutral active sHASEGP,
cells capable of introducing N-linked glycosylation are required.
In the preferred embodiment, mammalian Chinese Hamster Ovary cells
deficient in dihydrofolate reductase such as DG44, are
electroporated with a plasmid encoding a strong mammalian promoter,
such as CMV, nucleic acid encoding a sHASEGP followed by an
internal ribosomal entry site, the mouse dihydrofolate reductase
gene and the SV40 polyadenylation sequence as shown in SEQ ID NO
51. Such cells are then cultured in chemically defined medium in
the absence of hypoxanthine and thymidine, followed by further gene
amplification with increasing concentrations of methotrexate.
[0289] A variety of host-vector systems can be used to express the
protein coding sequence. These include but are not limited to
mammalian cell systems infected with virus e.g. vaccinia virus,
adenovirus, etc.); insect cell systems infected with virus (e.g.
baculovirus); microorganisms such as yeast containing yeast
vectors; or bacteria transformed with bacteriophage, DNA, plasmid
DNA, or cosmid DNA. The expression elements of vectors vary in
their strengths and specificities. Depending on the host-vector
system used, any one of a number of suitable transcription and
translation elements can be used. Note that bacterial expression of
sHASEGP DNA will not result in a catalytically active sHASEGP per
se, but when combined with proper glycosylation machinery can be
artificially glycosylated as such.
[0290] Any methods known to those of skill in the art for the
insertion of nucleic acid fragments into a vector can be used to
construct expression vectors containing a chimeric gene containing
appropriate transcriptional/translational control signals and
protein coding sequences. These methods can include in vitro
recombinant DNA and synthetic techniques and in vivo recombinants
(genetic recombination). Expression of nucleic acid sequences
encoding sHASEGP polypeptide, or domains, derivatives, fragments or
homologs thereof, can be regulated by a second nucleic acid
sequence so that the genes or fragments thereof are expressed in a
host transformed with the recombinant DNA molecule (s). For
example, expression of the proteins can be controlled by any
promoter/enhancer known in the art. In a specific embodiment, the
promoter is not native to the genes for sHASEGP polypeptide.
Promoters which can be used include but are not limited to the SV40
early promoter (Bernoist and Chambon, Nature 290: 304-310 (1981)
the promoter contained in the 3' long terminal repeat of Rous
sarcoma virus (Yamamoto et al., Ce//22: 787-797 (1980) the herpes
thymidine kinase promoter (Wagner et al. Proc. Natl. Acad. Sci. USA
78: 1441-1445 (1981) the regulatory sequences of the
metallothionein gene (Brinster et al., Nature 296: 39-42 (1982));
prokaryotic expression vectors such as the .beta.-Lactamase
promoter (Villa-Kamaroffet al., Proc. Natl. Acad. Sci. USA 75:
3727-3731 1978)) Or The TAC Promoter Deboer et al., Proc. Natl.
Acad. Sci. USA 80: 21-25 (1983)); see also "Useful Proteins from
Recombinant Bacteria": in Scientific American 242: 79-94 (1980));
plant expression vectors containing the opaline synthetase promoter
(Herrar-Estrella et al., Nature 303: 209-213 (1984)) or the
cauliflower mosaic virus 35S RNA promoter (Garder et al., Nucleic
Acids RES. 9: 2871 (1981)), and the promoter of the photosynthetic
enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et AL.,
Nature 310: 115-120 (1984)); promoter elements from yeast and other
fungi such as the Gal4 promoter, the alcohol dehydrogenase
promoter, the phosphoglycerol kinase promoter, the alkaline
phosphatase promoter, and the following animal transcriptional
control regions that exhibit tissue specificity and have been used
in transgenic animals: elastase I gene control region which is
active in pancreatic acinar cells (Swift Et Al., Cell 38: 639-646
(1984); Ornitz Et Al., Cold Spring Harbor Symp. Quant. Biol. 50:
399409 (1986); Macdonald, Hepatology 7: 425-515 (1987)); insulin
gene control region which is active in pancreatic beta cells
(Hanahan et AL., Nature 315: 115-122 (1985)), immunoglobulin gene
control region which is active in lymphoid cells (Grosschedl et
AL., Cell 38: 647-658 (1984); Adams et al., Nature 318: 533-538
(1985); Alexander et AL., Mol. Cell Biol. 7: 1436-1444 (1987)),
mouse mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et AL., CELL 45:
485-495 (1986)), albumin gene control region which is active in
liver (PINCKERT et AL., Genes and Devel. 1: 268-276 (1987)),
alpha-fetoprotein gene control region which is active in liver
(Krumlauf et AL., Mol. Cell. Biol. 5: 1639-1648 (1985); Hammer et
AL., Science 235: 53-58 1987)), alpha-1 antitrypsin gene control
region which is active in liver (Kelsey et al., Genes And Devel. 1:
161-171 (1987)), beta globin gene control region which is active in
myeloid cells (Mogram et al., Nature 315: 338-340 (1985); Kollias
et AL., CE//46: 89-94 (1986)), myelin basic protein gene control
region which is active in oligodendrocyte cells of the brain
(Readhead et al., Cell 48: 703-712 (1987)), myosin light chain-2
gene control region which is active in skeletal muscle (Sani,
Nature 314: 283-286 (1985)), and gonadotrophic releasing hormone
gene control region which is active in gonadotrophs of the
hypothalamus (Mason et al., Science 234: 1372-1378 (1986)).
[0291] In a specific embodiment, a vector is used that contains a
promoter operably linked to nucleic acids encoding a sHASEGP
polypeptide, or a domain, fragment, derivative or homolog, thereof,
one or more origins of replication, and optionally, one or more
selectable markers (e. G., an antibiotic resistance gene).
[0292] Specific initiation signals may also be required for
efficient translation of a sHASEGP sequence. These signals include
the ATG initiation codon and adjacent sequences. In cases where
sHASEGP, its initiation codon and upstream sequences are inserted
into the appropriate expression vector, no additional translational
control signals may be needed. However, in cases where only coding
sequence, or a portion thereof, is inserted, exogenous
transcriptional control signals including the ATG initiation codon
must be provided. Furthermore, the initiation codon must be in the
correct reading frame to ensure transcription of the entire insert.
Exogenous transcriptional elements and initiation codons can be of
various origins, both natural and synthetic. The efficiency of
expression may be enhanced by the inclusion of enhancers
appropriate to the cell system in use (Scharf D et al (1994)
Results Probl Cell Differ 20:125-62; Bittner et al (1987) Methods
in Enzymol 153:516-544).
[0293] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be important for
correct insertion, folding and/or function. Different host cells
such as CHO (DG44, DXB11 CHO-K1), HeLa, MDCK, 293, W138, etc have
specific cellular machinery and characteristic mechanisms for such
post-translational activities and may be chosen to ensure the
correct modification and processing of the introduced, foreign
protein.
[0294] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express sHASEGP may be transformed using expression
vectors which contain viral origins of replication or endogenous
expression elements and a selectable marker gene. Following the
introduction of the vector, cells may be allowed to grow for 1-2
days in an enriched media before they are switched to selective
media. The purpose of the selectable marker is to confer resistance
to selection, and its presence allows growth and recovery of cells
that successfully express the introduced sequences. Resistant
clumps of stably transformed cells can be proliferated using tissue
culture techniques appropriate to the cell type.
[0295] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler M et al (1977) Cell
11:223-32) and adenine phosphoribosyltransferase (Lowy I et al
(1980) Cell 22:817-23) genes which can be employed in TK- or
APRT-cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection; for
example, DHFR which confers resistance to methotrexate (Wigler M et
al (1980) Proc Natl Acad Sci 77:3567-70); npt, which confers
resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin F et al (1981) J Mol Biol 150:1-14) and als or
pat, which confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively (Murry, supra). Additional
selectable genes have been described, for example, trpB, which
allows cells to utilize indole in place of tryptophan, or hisD,
which allows cells to utilize histinol in place of histidine
(Hartman S C and R C Mulligan (1988) Proc Natl Acad Sci 85:8047-5
1). Recently, the use of visible markers has gained popularity with
such markers as anthocyanins, beta glucuronidase and its substrate,
GUS, and luciferase and its substrate, luciferin, being widely used
not only to identify transformants, but also to quantify the amount
of transient or stable protein expression attributable to a
specific vector system (Rhodes C A et al (1995) Methods Mol Biol
55:121-131).
[0296] Identification of Trandsformants Containing the
Polynucleotide Sequence
[0297] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of an active sHASEGP should be confirmed. For
example, if the sHASEGP is inserted within a marker gene sequence,
recombinant cells containing sHASEGP can be identified by the
absence of marker gene function. Alternatively, a marker gene can
be placed in tandem with a sHASEGP sequence under the control of a
single promoter. Expression of the marker gene in response to
induction or selection usually indicates expression of the tandem
sHASEGP as well. Detection of a properly glycosylated neutral
active sHASEGP can be determined by way of testing the conditioned
media for sHASEGP enzyme activity under appropriate conditions.
[0298] Purification of SHASEGP
[0299] Host cells transformed with a sHASEGP nucleotide sequence
may be cultured under conditions suitable for the expression and
recovery of the encoded protein from cell culture. The protein
produced by a recombinant cell is preferably secreted but may be
contained intracellularly depending on the sequence and/or the
vector used. As will be understood by those of skill in the art,
expression vectors containing sHASEGP can be designed with signal
sequences that facilitate direct secretion of sHASEGP through a
prokaryotic or eukaryotic cell membrane. Other recombinant
constructions may join sHASEGP to nucleotide sequence encoding a
polypeptide domain which will facilitate purification of soluble
proteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53; cf
discussion of vectors infra containing fusion proteins).
[0300] sHASEGP may also be expressed as a recombinant protein with
one or more additional polypeptide domains added to facilitate
protein purification. Such purification facilitating domains
include, but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle
Wash.). The inclusion of a cleavable linker sequences such as
Factor XA or enterokinase (Invitrogen, San Diego Calif.) between
the purification domain and sHASEGP is useful to facilitate
purification. One such expression vector provides for expression of
a fusion protein compromising a sHASEGP and contains nucleic acid
encoding 6 histidine residues followed by thioredoxin and an
enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilized metal ion affinity
chromatography as described in Porath et al (1992) Protein
Expression and Purification 3: 263-28 1) while the enterokinase
cleavage site provides a means for purifying the chemokine from the
fusion protein.
[0301] In addition to recombinant production, fragments of sHASEGP
may be produced by direct peptide synthesis using solid-phase
techniques (see, e.g., Stewart et al (1969) Solid-Phase Peptide
Synthesis, W H Freeman Co, San Francisco; Merrifield J (1963) J Am
Chem Soc 85:2149-2154). In vitro protein synthesis may be performed
using manual techniques or by automation. Automated synthesis may
be achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin Elmer, Foster City Calif.) in accordance with
the instructions provided by the manufacturer. Various fragments of
sHASEGP may be chemically synthesized separately and combined using
chemical methods to produce the full-length molecule.
[0302] Expression vectors containing the coding sequences, or
portions thereof, of a sHASEGP polypeptide, is made, for example,
by subcloning the coding portions into the EcoRI restriction site
of each of the three PGEX vectors (glutathione S-transferase
expression vectors (Smith and Johnson, Gene 7: 3140 (1988)). This
allows for the expression of products in the correct reading frame.
Exemplary vectors and systems for expression of the Hyaluronidase
domains of the sHASEGP polypeptides include the well-known Pichia
vectors (available, for example, from Invitrogen, San Diego,
Calif.), particularly those designed for secretion of the encoded
proteins. The protein can also be expressed cytoplasmically, such
as in the inclusion bodies. One exemplary vector is described in
the examples.
[0303] Plasmids for transformation of E. coli cells, include, for
example, the pET expression vectors (see, U.S. Pat. No. 4,952,496;
available from Novagen, Madison, Wis.; see, also literature
published by Novagen describing the system).
[0304] Such plasmids include pET 11a, which contains the T71ac
promoter, T7 terminator, the inducible E. coli lac operator, and
the lac repressor gene; pET 12A-C, which contains the T7 promoter,
T7 terminator, and the E. COLI OMPT secretion signal; and pET 15B
and PET 19B (Novagen, Madison, Wis.), which contain a His-Tag
leader sequence for use in purification with a His column and a
thrombin cleavage site that permits cleavage following purification
over the column; the T7-lac promoter region and the T7
terminator.
[0305] The vectors are introduced into host cells, such as Pichia
cells and bacterial cells, such as E. coli, and the proteins
expressed therein. Exemplary Pichia strains include, for example,
GS115. Exemplary bacterial hosts contain chromosomal copies of DNA
encoding T7 RNA polymerase operably linked to an inducible
promoter, such as the LACUV promoter (see, U.S. Pat. No.
4,952,496). Such hosts include, but are not limited to, the
lysogenic E. coli strain BL21 (DE3).
[0306] The sHASEGP domains, derivatives and analogs can be produced
by various methods known in the art. For example, once a
recombinant cell expressing a sHASEGP polypeptide, or a domain,
fragment or derivative thereof, is identified, the individual gene
product can be isolated and analyzed. This is achieved by assays
based on the physical and/or functional properties of the protein,
including, but not limited to, radioactive labeling of the product
followed by analysis by gel electrophoresis, immunoassay,
cross-linking to marker-labeled product, and assays of proteolytic
activity.
[0307] The sHASEGP polypeptides can be isolated and purified by
standard methods known in the art (either from natural sources or
recombinant host cells expressing the complexes or proteins),
including but not restricted to column chromatography (E.g., ion
exchange, affinity, gel exclusion, reversed-phase high pressure and
fast protein liquid), differential centrifugation, differential
solubility, or by any other standard technique used for the
purification of proteins.
[0308] In one embodiment, a sHASEGP can be purified to homogeneity
from the chemically defined conditioned media of HZ24 transfected
and methotrexate amplified DG44 cells by 1) tangential flow
diafiltration, 2) binding and elution from anion exchange
chromatography, 3) flow through phenyl sepharose chromatography, 4)
binding and elution from phenylboronate chromatography and 4)
binding and elution with hydroxyapatite chromatography.
[0309] Functional properties can be evaluated using any suitable
assay known in the art.
[0310] Alternatively, once a sHASEGP polypeptide or its domain or
derivative is identified, the amino acid sequence of the protein
can be deduced from the nucleotide sequence of the gene that
encodes it. As a result, the protein or its domain or derivative
can be synthesized by standard chemical methods known in the art
(e.G. see Hunkapiller et al, Nature 310: 105-111 (1984)) followed
by glycosylation in vitro.
[0311] Manipulations of sHASEGP polypeptide sequences can be made
at the protein level. Also contemplated herein are sHASEGP
polypeptide proteins, domains thereof, derivatives or analogs or
fragments thereof, which are differentially modified during or
after translation, e. g., by glycosylation, acetylation,
phosphorylation, amidation, pegylation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to an
antibody molecule or other cellular ligand.
[0312] Any of numerous chemical modifications can be carried out by
known techniques, including but not limited to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8,
NABH4, acetylation, formylation, oxidation, reduction, metabolic
synthesis in the presence of tunicamycin and other such agents.
[0313] In addition, domains, analogs and derivatives of a sHASEGP
polypeptide can be chemically synthesized. For example, a peptide
corresponding to a portion of a sHASEGP polypeptide, which includes
the desired domain or which mediates the desired activity in vitro
can be synthesized by use of a peptide synthesizer.
[0314] Furthermore, if desired, nonclassical amino acids or
chemical amino acid analogs can be introduced as a substitution or
addition into the sHASEGP polypeptide sequence. Non-classical amino
acids include but are not limited to the D-isomers of the common
amino acids, a-amino isobutyric acid, 4-aminobutyric acid, Abu,
2-aminobutyric acid, E-ABU, e-Ahx, 6-amino hexanoic acid, Aib,
2-amino isobutyric acid, 3-amino propionoic acid, ornithine,
norleucine, norvaline, hydroxyproline, sarcosine, citrulline,
cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, .beta.-alanine, fluoro-amino acids, designer
amino acids such as .beta.-methyl amino acids, ca-methyl amino
acids, na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be d (dextrorotary) or 1
(levorotary).
[0315] In cases where natural products are suspected of being
mutant or are isolated from new species, the amino acid sequence of
the sHASEGP polypeptide isolated from the natural source, as well
as those expressed in vitro, or from synthesized expression vectors
in vivo or in vitro, can be determined from analysis of the DNA
sequence, or alternatively, by direct sequencing of the isolated
protein. Such analysis can be performed by manual sequencing or
through use of an automated amino acid sequenator.
[0316] Modifications--A variety of modifications of the sHASEGP
polypeptides and domains are contemplated herein. A
sHASEGP-encoding nucleic acid molecule can be modified by any of
numerous strategies known in the art (Sambrook ET A/. (1990),
Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.). The sequences can be cleaved
at appropriate sites with restriction endonuclease (s), followed by
further enzymatic modification if desired, isolated, and ligated in
vitro. In the production of the gene encoding a domain, derivative
or analog of sHASEGP, care should be taken to ensure that the
modified gene retains the original translational reading frame,
uninterrupted by translational stop signals, in the gene region
where the desired activity is encoded.
[0317] Additionally, the sHASEGP-encoding nucleic acid molecules
can be mutated in vitro or in vivo, to create and/or destroy
translation, initiation, and/or termination sequences, or to create
variations in coding regions and/or form new restriction
endonuclease sites or destroy pre-existing ones, to facilitate
further in vitro modification. Also, as described herein muteins
with primary sequence alterations, such as replacements of Cys
residues and elimination or addition of glycosylation sites are
contemplated; the sHASEGP of SEQ ID No. 1 has seven potential
glycosylation sites. Such mutations can be effected by any
technique for mutagenesis known in the art, including, but not
limited to, chemical mutagenesis and in vitro site-directed
mutagenesis (Hutchinson et al., j. Biol. Chem. 253: 6551-6558
(1978)), use of TABE Linkers (Pharmacia). In one embodiment, for
example, a sHASEGP polypeptide or domain thereof is modified to
include a fluorescent label. In other specific embodiments, the
sHASEGP polypeptide is modified to have a heterobifunctional
reagent, such heterobifunctional reagents can be used to crosslink
the members of the complex.
[0318] In addition, domains, analogs and derivatives of a sHASEGP
can be chemically synthesized. For example, a peptide corresponding
to a portion of a sHASEGP, which includes the desired domain or
which mediates the desired activity in vitro can be synthesized by
use of a peptide synthesizer. Furthermore, if desired, nonclassical
amino acids or chemical amino acid analogs can be introduced as a
substitution or addition into the sHASEGP sequence. Non-classical
amino acids include but are not limited to the D-isomers of the
common amino acids, a-amino isobutyric acid, 4-aminobutyric acid,
Abu, 2-aminobutyric acid, S-ABU, e-Ahx, 6-amino hexanoic acid, Aib,
2-amino isobutyric acid, 3-amino propionoic acid, ornithine,
norleucine, norvaline, hydroxyproline, sarcosine, citrulline,
cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, B-alanine, fluoro-amino acids, designer amino
acids such as ti-methyl amino acids, ca-methyl amino acids,
na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be d (dextrorotary) or l
(levorotary).
[0319] F. Generation of a functionally Active Glycosylated Shasegp
with N-Linked Sugar Moieties.
[0320] Properly N-glycosylated human sHASEGP is required to
generate a catalytically stable protein. N-linked glycosylation of
sHASEGP can be achieved through various techniques. Glycosylation
of sHASEGP can be achieved by introducing nucleic acids encoding
sHASEGP into cells of eukaryotic origin capable of proper N-linked
glycosylation or alternatively, by contacting sHASEGP polypeptide
with cell free extracts or purified enzymes capable of introducing
the desired N-linked sugar moieties.
[0321] Selection of an expression System
[0322] Eukaryotic cell expression systems vary in the extent and
type of glycosylation they introduce into an ectopically expressed
polypeptide. CHO cells are, for example, highly efficient at the
introduction of N-linked glycosylation into an active sHASEGP
polypeptide.
[0323] Additional eukaryotic expression systems that introduce
N-linked glycosylation to generate a functional sHASEGP product can
be tested by introducing a human sHASEGP expression plasmid into
said cells and testing for neutral activity. Proper N-linked
glycosylation can be determined by way of FACE analysis of PNGASE
released oligosaccharides. Glycosylation profiles of catalytically
active sHASEGPs are further provided herein. Verification of
glycosylation can also be made by treatment of sHASEGP from said
cells with PNGASE-F or by growth of such cells in tunicamycin
following introduction of sHASEGP encoding nucleic acids.
[0324] N-glycosylation of sHASEGP polypeptide in vitro. The sHASEGP
polypeptide can be N-glycosylated by contact of sHASEGP polypeptide
with cell-free extracts containing activity capable of transferring
N-linked sugars to sHASEGP polypeptide such as canine microsomal
membranes or through coupled transcription and translation as is
commercially available (Promega Madison Wis.).
[0325] Oligosaccharides are considered to have a reducing end and a
non-reducing end, whether or not the saccharide at the reducing end
is in fact a reducing sugar. In accordance with accepted
nomenclature, oligosaccharides are depicted herein with the
non-reducing end on the left and the reducing end on the right. All
oligosaccharides described herein are described with the name or
abbreviation for the non-reducing saccharide (e.g., Gal), followed
by the configuration of the glycosidic bond (.alpha. or. beta.),
the ring bond, the ring position of the reducing saccharide
involved in the bond, and then the name or abbreviation of the
reducing saccharide (e.g., GlcNAc). The linkage between two sugars
may be expressed, for example, as 2,3, 2.fw darw.3, or (2,3). Each
saccharide is a pyranose.
[0326] As used herein, N-linked sugar moiety refers to an
oligosaccharide attached to a sHASEGP via the amide nitrogen of Asn
residues. N-linked oligosaccharides fall into several major types
(oligomannose, complex, hybrid, sulfated), all of which have (Man)
3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn
residues that fall within--Asn-Xaa-Thr/Ser- sequences (where Xaa is
not Pro). N-linked sites are often indirectly assigned by the
appearance of a "blank" cycle during sequencing. Positive
identification can be made after release of the oligosaccharide by
PNGase F, which converts the glycosylated Asn to Asp. After PNGase
F release, N-linked oligosaccharides can be purified using Bio-Gel
P-6 chromatography, with the oligosaccharide pool subjected to
preparative high pH anion exchange chromatography (HPAEC) (Townsend
et al., (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide
isomers can be resolved using HPAEC. Fucose residues will shift
elution positions earlier in the HPAEC chromatogram, while
additional sialic acid residues will increase the retention time.
Concurrent treatment of glycoproteins whose oligosaccharide
structures are known (e.g., bovine fetuin, a-1 acid glycoprotein,
ovalbumin, RNAse B, transferrin) can facilitate assignment of the
oligosaccharide peaks. The collected oligosaccharides can be
characterized by a combination of compositional and methylation
linkage analyses (Waegheet al., (1983) Carbohydr Res. 123,
281-304.), with anomeric configurations assigned by NMR
spectroscopy (Van Halbeek (1993) in Methods Enzymol 230).
[0327] Alternatively, oligosaccharides can be identified by
fluorescence assisted carbohydrate electrophoresis (FACE)
Callewaert et al. (2001) Glycobiology 11, 275-281.
[0328] G. Detection and Characterization of N-Linked Sugar Moieties
on sHASEGP
[0329] Determining whether a protein is in fact glycosylated is the
initial step in glycoprotein glycan analysis. Polyacrylamide gel
electrophoresis in the presence of sodium dodecyl sulfate
(SDS-PAGE) has become the method of choice as the final step prior
to protein sequencing. Glycosylated proteins often migrate as
diffuse bands by SDS-PAGE. A marked decrease in bandwidth and
change in migration position after treatment with
peptide-N4-(N-acetyl-D-glucosaminyl) asparagine amidase (PNGase F)
is considered diagnostic of N-linked glycosylation. If the other
types of glycosylation are predominant other approaches must be
used. Lectin blotting methods provide an approach that is
independent of the class of glycosylation (N versus O). Lectins,
carbohydrate-binding proteins from various plant tissues, have both
high affinity and narrow specificity for a wide range of defined
sugar epitopes found on glycoprotein glycans (Cummings, R. D.
(1994) Methods in Enzymol. 230, 66-86.). When conjugated with
biotin or digoxigenin, they can be easily identified on membrane
blots through a colorimetric reaction utilizing avidin or
anti-digoxigenin antibodies conjugated with alkaline phosphatase
(Haselbeck, et al. (1993) Methods in Mol. Biol. 14, 161-173.),
analogous to secondary antibody-alkaline phosphatase reactions
employed in Western blotting. Screening with a panel of lectins
with well-defined specificity can provide considerable information
about a glycoprotein's carbohydrate complement. Importantly, the
color development amplification is sufficiently high that 10-50 ng
of a glycoprotein can easily be seen on a membrane blot of an
SDS-PAGE. Although lectins exhibit very high affinity for their
cognate ligands, some do reveal significant avidity for
structurally related epitopes. Thus, it is important to carefully
note the possibility of cross-reactivity when choosing a panel of
lectins, and apply those with the highest probability of
individually distinguishing complex, hybrid and high mannose
N-linked glycans from O-linked structures.
[0330] Monosaccharide analysis can also be used to determine
whether sHASEGP is glycosylated and as in the case of lectin
analysis provides additional information on structural features.
Quantitative monosaccharide composition analysis i) identifies
glycosylated proteins, ii) gives the molar ratio of individual
sugars to protein, iii) suggests, in some cases, the presence of
oligosaccharide classes, iv) is the first step in designing a
structural elucidation strategy, and v) provides a measure of
production consistency for recombinant glycoprotein therapeutics.
In recent years high-pH anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD) has been extensively used to
determine monosaccharide composition (Townsend, et al. (1995) in
Carbohydrate Analysis: High-performance liquid chromatography and
capillary electrophoresis (Z. El Rassi ed.). pp. 181-209.). More
recently, fluorophore-based labeling methods have been introduced
and many are available in kit form. A distinct advantage of
fluorescent methods is an increase in sensitivity (50-fold). One
potential disadvantage is that different monosaccharides may
demonstrate different selectivity for the fluorophore during the
coupling reaction, either in the hydrolysate or in the external
standard mixture. However, the increase in sensitivity and the
ability to identify which monosaccharides are present from a small
portion of the total amount of available glycoprotein, as well as
the potential for greater sensitivity using laser induced
fluorescence makes this approach attractive.
[0331] Monosaccharide composition analysis of small amounts of
sHASEGP is best performed on PVDF (PSQ) membranes, after either
electroblotting (Weitzhandleret al, (1993) J. Biol. Chem. 268,
5121-5130.) or if smaller aliquots are to be analyzed on dot blots.
PVDF is an ideal matrix for carbohydrate analysis since neither
mono- or oligosaccharides bind to the membrane, once released by
either acid or enzymatic hydrolysis.
[0332] FACE analysis is an efficient means of detecting
glycosylation profiles of sHASEGPs. FACE.TM. N-Linked
Oligosaccharide Profiling (Prozyme) with 30% oligosaccharide gels
is one such mechanism. Oligosaccharides cleaved from 100 .mu.g of
glycoproteins by enzymatic digestion with N-Glycanase (a.k.a.
PNGase), labeled using the fluorophore ANTS, and separated by
electrophoresis can be used for detection of sHASEGP glycosylation
profiles. The relative positions of the oligosaccharide bands are
determined by running the sample and dilutions of the sample
alongside an oligosaccharide standard ladder which designated the
migration distance in Degree of Polymerization (DP) units.
[0333] H. Screening Methods to Identify Compounds that Modulate
sHASEGP Activity.
[0334] Several types of assays are exemplified and described
herein. It is understood that the Hyaluronidase domains can be used
in other assays. It is shown here, however, that the Hyaluronidase
domains exhibit catalytic activity.
[0335] As such they are ideal for in vitro screening assays.
[0336] They can also be used in binding assays.
[0337] The sHASEGP full length zymogens, activated enzymes, and
Hyaluronidase domains are contemplated for use in any screening
assay known to those of skill in the art, including those provided
herein. Hence the following description, if directed to
Hyaluronidase assays is intended to apply to use of a single chain
Hyaluronidase domain or a catalytically active portion thereof of
any Hyaluronidase, including a sHASEGP. Other assays, such as
binding assays are provided herein, particularly for use with a
sHASEGP, including any variants, such as splice variants
thereof.
[0338] 1. Catalytic Assays for identification of agents that
modulate the Hyaluronidase activity of a sHASEGP protein. Methods
for identifying a modulator of the catalytic activity of a sHASEGP,
particularly a single chain Hyaluronidase domain or catalytically
active portion thereof, are provided herein. The methods can be
practiced by: contacting the sHASEGP, a full-length zymogen or
activated form, and particularly a single-chain domain thereof,
with a substrate of the sHASEGP in the presence of a test
substance, and detecting the proteolysis of the substrate, whereby
the activity of the sHASEGP is assessed, and comparing the activity
to a control. For example, a control can be the activity of the
sHASEGP assessed by contacting a sHASEGP, including a full-length
zymogen or activated form, and particularly a single-chain domain
thereof, particularly a single-chain domain thereof, with a
substrate of the sHASEGP, and detecting the proteolysis of the
substrate, whereby the activity of the sHASEGP is assessed. The
results in the presence and absence of the test compounds are
compared. A difference in the activity indicates that the test
substance modulates the activity of the sHASEGP. Activators of
sHASEGP activation cleavage are also contemplated; such assays are
discussed below.
[0339] In one embodiment a plurality of the test substances are
screened simultaneously in the above screening method. In another
embodiment, the sHASEGP is isolated from a target cell as a means
for then identifying agents that are potentially specific for the
target cell.
[0340] In another embodiment, a test substance is a therapeutic
compound, and whereby a difference of the sHASEGP activity measured
in the presence and in the absence of the test substance indicates
that the target cell responds to the therapeutic compound.
[0341] One method includes the steps of (a) contacting the sHASEGP
polypeptide or Hyaluronidase domain thereof with one or a plurality
of test compounds under conditions conducive to interaction between
the ligand and the compounds; and (b) identifying one or more
compounds in the plurality that specifically binds to the
ligand.
[0342] Another method provided herein includes the steps of a)
contacting a sHASEGP polypeptide or Hyaluronidase domain thereof
with a substrate of the sHASEGP polypeptide, and detecting the
degradation of substrate, whereby the activity of the sHASEGP
polypeptide is assessed; b) contacting the sHASEGP polypeptide with
a substrate of the sHASEGP polypeptide in the presence of a test
substance, and detecting the degradation of the substrate, whereby
the activity of the sHASEGP polypeptide is assessed; and c)
comparing the activity of the sHASEGP polypeptide assessed in steps
a) and b), whereby the activity measured in step a) differs from
the activity measured in step b) indicates that the test substance
modulates the activity of the sHASEGP polypeptide.
[0343] In another embodiment, a plurality of the test substances is
screened simultaneously. In comparing the activity of a sHASEGP
polypeptide in the presence and absence of a test substance to
assess whether the test substance is a modulator of the sHASEGP
polypeptide, it is unnecessary to assay the activity in parallel,
although such parallel measurement is typical. It is possible to
measure the activity of the sHASEGP polypeptide at one time point
and compare the measured activity to a historical value of the
activity of the sHASEGP polypeptide.
[0344] For instance, one can measure the activity of the sHASEGP
polypeptide in the presence of a test substance and compare with
historical value of the activity of the sHASEGP polypeptide
measured previously in the absence of the test substance, and vice
versa. This can be accomplished, for example, by providing the
activity of the sHASEGP polypeptide on an insert or pamphlet
provided with a kit for conducting the assay.
[0345] Methods for selecting substrates for a particular sHASEGP
are described in the EXAMPLES, and particular Hyaluronidase assays
are exemplified.
[0346] Combinations and kits containing the combinations optionally
including instructions for performing the assays are provided. The
combinations include a sHASEGP polypeptide and a substrate of the
sHASEGP polypeptide to be assayed; and, optionally reagents for
detecting proteolysis of the substrate. The substrates, which can
be chromogenic or fluorogenic molecules, including
glycosaminoglycans, subject to proteolysis by a particular sHASEGP
polypeptide, can be identified empirically by testing the ability
of the sHASEGP polypeptide to cleave the test substrate. Substrates
that are cleaved most effectively i.e. at the lowest concentrations
and/or fastest rate or under desirable conditions), are
identified.
[0347] Additionally provided herein is a kit containing the
above-described combination. The kit optionally includes
instructions for identifying a modulator of the activity of a
sHASEGP polypeptide. Any sHASEGP polypeptide is contemplated as
target for identifying modulators of the activity thereof.
[0348] 2. Binding assays. Also provided herein are methods for
identification and isolation of agents, particularly compounds that
bind to sHASEGPs. The assays are designed to identify agents that
bind to the isolated Hyaluronidase domain (or a protein, other than
a sHASEGP polypeptide, that contains the Hyaluronidase domain of a
sHASEGP polypeptide), and to the activated form, including the
activated form derived from the full-length zymogen or from an
extended Hyaluronidase domain. The identified compounds are
candidates or leads for identification of compounds for treatments
of disorders and diseases involving aberrant Hyaluronidase
activity. The sHASEGP polypeptides used in the methods include any
sHASEGP polypeptide as defined herein, including the sHASEGP single
chain Hyaluronidase domain or proteolytically active portion
thereof.
[0349] A variety of methods are provided herein. These methods can
be performed in solution or in solid phase reactions in which the
sHASEGP polypeptide (s) or Hyaluronidase domain (s) thereof are
linked, either directly or indirectly via a linker, to a solid
support. Screening assays are described in the Examples, and these
assays have been used to identify candidate compounds.
[0350] For purposes herein, all binding assays described above are
provided for sHASEGP.
[0351] Methods for identifying an agent, such as a compound, that
specifically binds to a sHASEGP single chain Hyaluronidase domain,
a full-length activated sHASEGP or two chain Hyaluronidase domain
thereof are provided herein. The method can be practiced by (a)
contacting the sHASEGP with one or a plurality of test agents under
conditions conducive to binding between the sHASEGP and an agent;
and (b) identifying one or more agents within the plurality that
specifically binds to the sHASEGP.
[0352] For example, in practicing such methods the sHASEGP
polypeptide is mixed with a potential binding partner or an extract
or fraction of a cell under conditions that allow the association
of potential binding partners with the polypeptide. After mixing,
peptides, polypeptides, proteins or other molecules that have
become associated with a sHASEGP are separated from the mixture.
The binding partner that bound to the sHASEGP can then be removed
and further analyzed. To identify and isolate a binding partner,
the entire protein, for instance the entire disclosed protein of
SEQ ID No. 1 can be used. Alternatively, a fragment of the protein
can be used.
[0353] A variety of methods can be used to obtain cell extracts or
body fluids, such as blood, serum, urine, sweat, synovial fluid,
CSF and other such fluids.
[0354] For example, cells can be disrupted using either physical or
chemical disruption methods. Examples of physical disruption
methods include, but are not limited to, sonication and mechanical
shearing. Examples of chemical lysis methods include, but are not
limited to, detergent lysis and enzyme lysis. A skilled artisan can
readily adapt methods for preparing cellular extracts in order to
obtain extracts for use in the present methods.
[0355] Once an extract of a cell is prepared, the extract is mixed
with the sHASEGP under conditions in which association of the
protein with the binding partner can occur. A variety of conditions
can be used, including conditions that resemble conditions found in
the cytoplasm of a human cell or in a body fluid, such as blood.
Features, such as osmolarity pH, temperature, and the concentration
of cellular extract used, can be varied to optimize the association
of the protein with the binding partner. Similarly, methods for
isolation of molecules of interest from body fluids are known.
[0356] After mixing under appropriate conditions, the bound complex
is separated from the mixture. A variety of techniques can be used
to separate the mixture. For example, antibodies specific to a
sHASEGP can be used to immunoprecipitate the binding partner
complex. Alternatively, standard chemical separation techniques
such as chromatography and density/sediment centrifugation can be
used.
[0357] After removing the non-associated cellular constituents in
the extract, the binding partner can be dissociated from the
complex using conventional methods. For example, dissociation can
be accomplished by altering the salt concentration or pH of the
mixture.
[0358] To aid in separating associated binding partner pairs from
the mixed extract, the sHASEGP can be immobilized on a solid
support. For example, the protein can be attached to a
nitrocellulose matrix or acrylic beads. Attachment of the protein
or a fragment thereof to a solid support aids in separating
peptide/binding partner pairs from other constituents found in the
extract. The identified binding partners can be either a single
protein or a complex made up of two or more proteins.
[0359] Alternatively, the nucleic acid molecules encoding the
single chain Hyaluronidases can be used in a yeast two-hybrid
system. The yeast two-hybrid system has been used to identify other
protein partner pairs and can readily be adapted to employ the
nucleic acid molecules herein described.
[0360] Another in vitro binding assay, particularly for a sHASEGP,
uses a mixture of a polypeptide that contains at least the
catalytic domain of one of these proteins and one or more candidate
binding targets or substrates. After incubating the mixture under
appropriate conditions, the ability of the sHASEGP or a polypeptide
fragment thereof containing the catalytic domain to bind to or
interact with the candidate substrate is assessed. For cell-free
binding assays, one of the components includes or is coupled to a
detectable label. The label can provide for direct detection, such
as radioactivity, luminescence, optical or electron density, etc.,
or indirect detection such as an epitope tag, an enzyme, etc. A
variety of methods can be employed to detect the label depending on
the nature of the label and other assay components. For example,
the label can be detected bound to the solid substrate or a portion
of the bound complex containing the label can be separated from the
solid substrate, and the label thereafter detected.
[0361] 3. Detection of signal transduction sHASEGP, which is a
membrane anchored protein, can be involved directly or indirectly
in signal transduction directly as a cell surface receptor or
indirectly by activating proteins, such as pro-growth factors that
can initiate signal transduction.
[0362] In addition, secreted sHASEGP, such as the soluble domain of
sHASEGP as described in SEQ ID NO. 4, can be involved in signal
transduction either directly by binding to or interacting with a
cell surface receptor or indirectly by activating proteins, such as
pro-growth factors that can initiate signal transduction. Assays
for assessing signal transduction are well known to those of skill
in the art, and can be adapted for use with the sHASEGP
polypeptide.
[0363] Assays for identifying agents that affect or alter signal
transduction mediated directly or indirectly, such as via
activation of a pro-growth factor, by a sHASEGP, particularly the
full length or a sufficient portion to anchor the extracellular
domain or a functional portion thereof of a sHASEGP on the surface
of a cell are provided. Such assays, include, for example,
transcription based assays in which modulation of a transduced
signal is assessed by detecting an effect on an expression from a
reporter gene (see, e. g., U.S. Pat. No. 5,436,128).
[0364] 4. Methods for Identifying Agents that Modulate the
Expression a Nucleic Acid encoding a sHASEGP. Another embodiment
provides methods for identifying agents that modulate the
expression of a nucleic acid encoding a sHASEGP. Such assays use
any available means of monitoring for changes in the expression
level of the nucleic acids encoding a sHASEGP.
[0365] Assay formats can be used to monitor the ability of the
agent to modulate the expression of a nucleic acid encoding a
sHASEGP. For instance, mRNA expression can be monitored directly by
hybridization to the nucleic acids. Also enzyme assays as described
can be used to detect agents that modulate the expression of
sHASEGP.
[0366] Cell lines are exposed to the agent to be tested under
appropriate conditions and time and total RNA or mRNA is isolated
by standard procedures (see, e.g. Sambrook et al (1989) MOLECULAR
CLONING: A LABORATORY MANUAL, 2nd Ed. Cold Spring Harbor Laboratory
Press). Probes to detect differences in RNA expression levels
between cells exposed to the agent and control cells can be
prepared from the nucleic acids. It is typical, but not necessary,
to design probes which hybridize only with target nucleic acids
under conditions of high stringency. Only highly complementary
nucleic acid hybrids form under conditions of high stringency.
Accordingly, the stringency of the assay conditions determines the
amount of complementarity that should exist between two nucleic
acid strands in order to form a hybrid. Stringency should be chosen
to maximize the difference in stability between the probe: target
hybrid and potential probe: non-target hybrids.
[0367] For example, N-and C-terminal fragments of the sHASEGP can
be expressed in bacteria and used to search for proteins that bind
to these fragments. Fusion proteins, such as His-tag or GST fusion
to the N-or C-terminal regions of the sHASEGP can be prepared for
use as a substrate. These fusion proteins can be coupled to, for
example, Glutathione-Sepharose beads and then probed with cell
lysates or body fluids. Prior to lysis, the cells or body fluids
can be treated with a candidate agent that can modulate a sHASEGP
or proteins that interact with domains thereon. Lysate proteins
binding to the fusion proteins can be resolved by SDS-PAGE,
isolated and identified by protein sequencing or mass spectroscopy,
as is known in the art.
[0368] Antibody probes are prepared by immunizing suitable
mammalian hosts in appropriate immunization protocols using the
peptides, polypeptides or proteins if they are of sufficient length
(e. g., 4,5,6,7,8,9,10,11,12,13,14,15,20, 25,30,35,40 or more
consecutive amino acids the sHASEGP polypeptide or if required to
enhance immunogenicity, conjugated to suitable carriers. Methods
for preparing immunogenic conjugates with carriers, such as bovine
serum albumin (BSA), keyhole limpet hemocyanin (KLH), or other
carrier proteins are well known in the art. In some circumstances,
direct conjugation using, for example, carbodiimide reagents can be
effective; in other instances linking reagents such as those
supplied by Pierce Chemical Co., Rockford, Ill., can be desirable
to provide accessibility to the hapten. Hapten peptides can be
extended at either the amino or carboxy terminus with a Cys residue
or interspersed with cysteine residues, for example, to facilitate
linking to a carrier.
[0369] Administration of the immunogens is conducted generally by
injection over a suitable time period and with use of suitable
adjuvants, as is generally understood in the art. During the
immunization schedule, titers of antibodies are taken to determine
adequacy of antibody formation.
[0370] Anti-peptide antibodies can be generated using synthetic
peptides corresponding to, for example, the carboxy terminal amino
acids of the sHASEGP.
[0371] Synthetic peptides can be as small as 1-3 amino acids in
length, generally at least 4 or more amino acid residues long. The
peptides can be coupled to KLH using standard methods and can be
immunized into animals, such as rabbits or ungulates. Polyclonal
antibodies can then be purified, for example using Actigel beads
containing the covalently bound peptide.
[0372] While the polyclonal antisera produced in this way can be
satisfactory for some applications, for pharmaceutical
compositions, use of monoclonal preparations are generally used.
Immortalized cell lines which secrete the desired monoclonal
antibodies can be prepared using the standard method of Kohler et
al., (Nature 256: 495-7 (1975)) or modifications which effect
immortalization of lymphocytes or spleen cells, as is generally
known. The immortalized cell lines secreting the desired antibodies
are screened by immunoassay in which the antigen is the peptide
hapten, polypeptide or protein.
[0373] When the appropriate immortalized cell culture secreting the
desired antibody is identified, the cells can be cultured either in
vitro or by production in vivo via ascites fluid. Of particular
interest, are monoclonal antibodies that recognize the catalytic
domain or activation cleavage site (region) of a sHASEGP.
[0374] The antibodies or fragments can also be produced. Regions
that bind specifically to the desired regions of receptor also can
be produced in the context of chimeras with multiple species
origin.
[0375] Agents that are assayed in the above method can be randomly
selected or rationally selected or designed.
[0376] The agents can be, as examples, peptides, small molecules,
and carbohydrates. A skilled artisan can readily recognize that
there is no limit as to the structural nature of the agents.
[0377] The peptide agents can be prepared using standard solid
phase (or solution phase) peptide synthesis methods, as is known in
the art. In addition, the DNA encoding these peptides can be
synthesized using commercially available oligonucleotide synthesis
instrumentation and produced recombinantly using standard
recombinant production systems. The production using solid phase
peptide synthesis is necessitated if non-gene-encoded amino acids
are to be included.
[0378] I. Methods of Treatment
[0379] sHASEGPs identified by the methods herein are used for
treating or preventing abnormal accumulations of sHASEGP substrates
in an animal, particularly a mammal, including a human. In one
embodiment, the method includes administering to a mammal an
effective amount of a sHASEGP glycoprotein, whereby the disease or
disorder is treated or prevented.
[0380] In another embodiment, a sHASEGP inhibitor can be used in
the treatment of an excess amount of neutral hyaluronidase
activity. The mammal treated can be a human. The inhibitors
provided herein are those identified by the screening assays. In
addition, antibodies and antisense nucleic acids or double-stranded
RNA (dsRNA), such as RNAi, are contemplated.
[0381] 1. Antisense treatment: In a specific embodiment, as
described hereinabove, sHASEGP polypeptide function is reduced or
inhibited by sHASEGP polypeptide antisense nucleic acids, to treat
or prevent excessive chondroitinase activity. The therapeutic or
prophylactic use of nucleic acids of at least six nucleotides,
generally up to about 150 nucleotides, that are antisense to a gene
or cDNA encoding sHASEGP polypeptide or a portion thereof is
provided. A sHASEGP polypeptide "antisense" nucleic acid as used
herein refers to a nucleic acid capable of hybridizing to a portion
of a sHASEGP polypeptide RNA (generally mRNA) by virtue of some
sequence complementarity, and generally under high stringency
conditions. The antisense nucleic acid can be complementary to a
coding and/or noncoding region of a sHASEGP polypeptide mRNA. Such
antisense nucleic acids have utility as therapeutics that reduce or
inhibit sHASEGP polypeptide function, and can be used in the
treatment or prevention of disorders as described supra.
[0382] The sHASEGP polypeptide antisense nucleic acids are of at
least six nucleotides and are generally oligonucleotides (ranging
from 6 to about 150 nucleotides including 6 to 50 nucleotides). The
antisense molecule can be complementary to all or a portion of the
Hyaluronidase domain. For example, the oligonucleotide is at least
10 nucleotides, at least 15 nucleotides, preferably at least 20,
40, 80 to at least 100 nucleotides, or at least 125 nucleotides.
The oligonucleotides can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone. The oligonucleotide
can include other appending groups such as peptides, or agents
facilitating transport across the cell membrane (see e.g.,
Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989);
Lemaitre et al., Proc. Natl. Acad. Sci. USA 84: 648-652 (1987); PCT
Publication No. WO 88/09810, published Dec. 15, 1988) or
blood-brain barrier (see e.g., PCT Publication No. WO 89/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents
(see e.g., Krol et al., BioTechniques 6: 958-976 (1988)) or
intercalating agents (see e.g., Zon. Pharm. Res. 5: 539-549
(1988)).
[0383] The sHASEGP polypeptide antisense nucleic acid generally is
an oligo-nucleotide, typically single-stranded DNA or RNA or an
analog thereof or mixtures thereof. For example, the
oligonucleotide includes a sequence antisense to a portion of a
nucleic acid that encodes a human sHASEGP polypeptide. The
oligonucleotide can be modified at any position on its structure
with substituents generally known in the art.
[0384] The sHASEGP polypeptide antisense oligonucleotide can
include at least one modified base moiety which is selected from
the group including, but not limited to 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5-apos-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-n-2-carboxypropyl) uracil, (ACP3) w, and
2,6-diaminopurine.
[0385] In another embodiment, the oligonucleotide includes at least
one modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose. The
oligonucleotide can include at least one modified phosphate
backbone selected from a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0386] The oligonucleotide can be an a-anomeric oligonucleotide. An
a-anomeric oligonucleotide forms specific double-stranded hybrids
with complementary RNA in which the strands run parallel to each
other (Gautier et al., Nucl. Acids Res. 15: 6625-6641 (1987)).
[0387] The oligonucleotide can be conjugated to another molecule,
such as, but are not limited to, a peptide; hybridization triggered
cross-linking agent, transport agent or a hybridization-triggered
cleavage agent. The oligonucleotides can be synthesized by standard
methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides can be synthesized by the method of Stein et al.,
Nucl. Acids Res. 16: 3209 (1988)), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., Proc. Natl. Acad. Sci. USA 85:
7448-7451 (1988)), etc. In a specific embodiment, the sHASEGP
polypeptide antisense oligonucleotide includes catalytic RNA or a
ribozyme (see, e.g., PCT International Publication WO 90/11364,
published Oct. 4, 1990; Sarver et al., Science 247: 1222-1225
(1990)). In another embodiment, the oligonucleotide is a
2'-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:
6131-6148 (1987)), or a chimeric RNA-DNA analogue Inoue et al.,
FEBS Lett. 215: 327-330 (1987)).
[0388] Alternatively, the oligonucleotide can be double-stranded
RNA (dsRNA) such as RNAi.
[0389] In an alternative embodiment, the sHASEGP polypeptide
antisense nucleic acid is produced intracellularly by transcription
from an exogenous sequence.
[0390] For example, a vector can be introduced in vivo such that it
is taken up by a cell, within which cell the vector or a portion
thereof is transcribed, producing an antisense nucleic acid (RNA).
Such a vector would contain a sequence encoding the sHASEGP
polypeptide antisense nucleic acid. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such vectors can
be constructed by recombinant DNA technology methods standard in
the art. Vectors can be plasmid, viral, or others known in the art,
used for replication and expression in mammalian cells. Expression
of the sequence encoding the sHASEGP polypeptide antisense RNA can
be by any promoter known in the art to act in mammalian, including
human, cells. Such promoters can be inducible or constitutive. Such
promoters include but are not limited to: the SV40 early promoter
region (Bernoist and Chambon, Nature 290: 304-310 (1981), the
promoter contained in the 3' long terminal repeat of Rous sarcoma
virus (Yamamoto et al., Ce//22: 787-797 (1980), the herpes
thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci.
USA 78: 1441-1445 (1981), the regulatory sequences of the
metallothionein gene (Brinster et al., Nature 296: 39-42 (1982),
etc.
[0391] The antisense nucleic acids include sequence complementary
to at least a portion of an RNA transcript of a sHASEGP polypeptide
gene, including a human sHASEGP polypeptide gene. Absolute
complementarity is not required. The amount of sHASEGP polypeptide
antisense nucleic acid that is effective in the treatment or
prevention of neoplastic disease depends on the nature of the
disease, and can be determined empirically by standard clinical
techniques.
[0392] Where possible, it is desirable to determine the antisense
cytotoxicity in cells in vitro, and then in useful animal model
systems prior to testing and use in humans.
[0393] 2. RNA interference RNA interference (RNAi) (see, e.g.
Chuang et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4985) can be
employed to inhibit the expression of a gene encoding a sHASEGP.
Interfering RNA (RNAi) fragments, particularly double-stranded (ds)
RNAi, can be used to generate loss-of-sHASEGP function. Methods
relating to the use of RNAi to silence genes in organisms
including, mammals, C. elegans, Drosophila and plants, and humans
are known (see, e.g., Fire et al. (1998) Nature 391: 806-811; Fire
(1999) Trends Genet. 15: 358-363; Sharp (2001) Genes Dev. 15:
485-490; Hammond et al. (2001) Nature Rev, Genet. 2: 110-119;
Tuschl (2001) Chem. Biochem. 2: 239-245; Hamilton et al. (1999)
Science 286: 950-952; Hammond et al. (2000) Nature 404: 293-296;
Zamore et al. (2000) Cell 101: 25-33; Bernstein et al. (2001)
Nature 409: 363-366; Elbashir et al. (2001) Genes Dev. 15: 188-200;
Elbashir et al. (2001) Nature 411: 494-498; International PCT
application No. WO 01/29058; International PCT application No. WO
99/32619).
[0394] Double-stranded RNA (dsRNA)-expressing constructs are
introduced into a host, such as an animal or plant using, a
repliable vector that remains episomal or integrates into the
genome. By selecting appropriate sequences, expression of dsRNA can
interfere with accumulation of endogenous mRNA encoding a sHASEGP.
RNAi also can be used to inhibit expression in vitro.
[0395] Regions include at least about 21 (or 21) nucleotides that
are selective (i.e. unique) for sHASEGP are used to prepare the
RNAi. Smaller fragments of about 21 nucleotides can be transformed
directly (i.e., in vitro or in vivo) into cells; larger RNAi dsRNA
molecules are generally introduced using vectors that encode them.
dsRNA molecules are at least about 21 bp long or longer, such as
50, 100, 150, 200 and longer. Methods, reagents and protocols for
introducing nucleic acid molecules into cells in vitro and in vivo
are known to those of skill in the art.
[0396] 3. Gene Therapy in an exemplary embodiment, nucleic acids
that include a sequence of nucleotides encoding a sHASEGP
polypeptide or functional domains or derivative thereof, are
administered to promote sHASEGP polypeptide function, by way of
gene therapy. Gene therapy refers to therapy performed by the
administration of a nucleic acid to a subject. In this embodiment,
the nucleic acid produces its encoded protein that mediates a
therapeutic effect by promoting sHASEGP polypeptide function. Any
of the methods for gene therapy available in the art can be used
(see, Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu
and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, An. Rev. Pharmacol.
Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993);
and Morgan and Anderson, An. Rev. Biochem. 62: 191-217 (1993);
TIBTECH 11 5: 155-215 (1993). For example, one therapeutic
composition for gene therapy includes a sHASEGP
polypeptide-encoding nucleic acid that is part of an expression
vector that expresses a sHASEGP polypeptide or domain, fragment or
chimeric protein thereof in a suitable host. In particular, such a
nucleic acid has a promoter operably linked to the sHASEGP
polypeptide coding region, the promoter being inducible or
constitutive, and, optionally, tissue-specific. In another
particular embodiment, a nucleic acid molecule is used in which the
sHASEGP polypeptide coding sequences and any other desired
sequences are flanked by regions that promote homologous
recombination at a desired site in the genome, thus providing for
intrachromosomal expression of the sHASEGP protein nucleic acid
(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 8932-8935
(1989); Zijlstra et al., Nature 342: 435-438 (1989)).
[0397] Delivery of the nucleic acid into a patient can be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vector, or indirect, in which
case, cells are first transformed with the nucleic acid in vitro,
then transplanted into the patient. These two approaches are known,
respectively, as in vivo or ex vivo gene therapy.
[0398] In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any of numerous methods known
in the art, e.g., by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that it
becomes intracellular, e.g., by infection using a defective or
attenuated retroviral or other viral vector (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering it in linkage to a peptide which
is known to enter the nucleus, by administering it in linkage to a
ligand subject to receptor-mediated endocytosis (see e.g., Wu and
Wu, J. Biol. Chem, 262: 44294432 (1987)) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, a nucleic acid-ligand complex can be formed in
which the ligand is a fusogenic viral peptide to disrupt endosomes,
allowing the nucleic acid to avoid lysosomal degradation. In yet
another embodiment, the nucleic acid can be targeted in vivo for
cell specific uptake and expression, by targeting a specific
receptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16,
1992 (Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.);
WO 92/20316 dated Nov. 26, 1992 (Findeis et al.); WO 93/14188 dated
Jul. 22, 1993 (Clarke et al.), WO 93/20221 dated Oct. 14, 1993
(Young)). Alternatively, the nucleic acid can be introduced
intracellularly and incorporated within host cell DNA for
expression, by homologous recombination (Koller and Smithies, Proc.
Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijistra et al., Nature
342: 435-438 (1989)).
[0399] In a specific embodiment, a viral vector that contains the
sHASEGP polypeptide nucleic acid is used. For example, a retroviral
vector can be used (see Miller et al., Meth. Enzymol. 217: 581-599
(1993)). These retroviral vectors have been modified to delete
retroviral sequences that are not necessary for packaging of the
viral genome and integration into host cell DNA. The sHASEGP
polypeptide nucleic acid to be used in gene therapy is cloned into
the vector, which facilitates delivery of the gene into a patient.
More detail about retroviral vectors can be found in Boesen et al.,
Biotherapy 6: 291-302 (1994), which describes the use of a
retroviral vector to deliver the mdrl gene to hematopoietic stem
cells in order to make the stem cells more resistant to
chemotherapy.
[0400] Other references illustrating the use of retroviral vectors
in gene therapy are: Clowes et al., J. Clin. Invest. 93: 644-651
(1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and
Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and
Wilson, Curr. Opin. In Genetics And Devel. 3: 110-114 (1993).
[0401] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3: 499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5: 3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252: 431-434 (1991); Rosenfeld et al., Cell 68: 143-155
(1992); and Mastrangeli et al., J. Clin. Invest. 91: 225-234
(1993).
[0402] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:
289-300 (1993).
[0403] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0404] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see e.g.,
Loeffler and Behr, Meth. Enzymol. 217: 599-618 (1993); Cohen et
al., Meth. Enzymol. 217: 618-644 (1993); Cline, Pharmac. Ther. 29:
69-92 (1985)) and can be used, provided that the necessary
developmental and physiological functions of the recipient cells
are not disrupted. The technique should provide for the stable
transfer of the nucleic acid to the cell, so that the nucleic acid
is expressible by the cell and generally heritable and expressible
by its cell progeny.
[0405] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. In an embodiment,
epithelial cells are injected, e.g., subcutaneously. In another
embodiment, recombinant skin cells can be applied as a skin graft
onto the patient. Recombinant blood cells (e.g., hematopoietic stem
or progenitor cells) can be administered intravenously. The amount
of cells envisioned for use depends on the desired effect, patient
state, etc., and can be determined by one skilled in the art.
[0406] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., such as stem cells
obtained from bone marrow, umbilical cord blood, peripheral blood,
fetal liver, and other sources thereof.
[0407] For example, a cell used for gene therapy is autologous to
the patient. In an embodiment in which recombinant cells are used
in gene therapy, a sHASEGP polypeptide nucleic acid is introduced
into the cells such that it is expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells that
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment.
[0408] Such stem cells include but are not limited to hematopoietic
stem cells (HSC), stem cells of epithelial tissues such as the skin
and the lining of the gut, embryonic heart muscle cells, liver stem
cells (PCT Publication WO 94/08598, dated Apr. 28, 1994), and
neural stem cells (Stemple and Anderson, Cell 71: 973-985
(1992)).
[0409] Epithelial stem cells (ESC) or keratinocytes can be obtained
from tissues such as the skin and the lining of the gut by known
procedures (Rheinwald, Meth. Cell Bio. 21A: 229 (1980)). In
stratified epithelial tissue such as the skin, renewal occurs by
mitosis of stem cells within the germinal layer, the layer closest
to the basal lamina. Stem cells within the lining of the gut
provide for a rapid renewal rate of this tissue. ESC or
keratinocytes obtained from the skin or lining of the gut of a
patient or donor can be grown in tissue culture (Rheinwald, Meth.
Cell Bio. 21A: 229 (1980); Pittelkow and Scott, Cano. Clinic Proc.
61: 771 (1986)). If the ESC are provided by a donor, a method for
suppression of host versus graft reactivity (e.g., irradiation,
drug or antibody administration to promote moderate
immunosuppression) also can be used.
[0410] With respect to hematopoietic stem cells (HSC), any
technique which provides for the isolation, propagation, and
maintenance in vitro of HSC can be used in this embodiment.
Techniques by which this can be accomplished include (a) the
isolation and establishment of HSC cultures from bone marrow cells
isolated from the future host, or a donor, or (b) the use of
previously established long-term HSC cultures, which can be
allergenic or xenogenic.
[0411] Non-autologous HSC generally are used with a method of
suppressing transplantation immune reactions of the future
host/patient. In a particular embodiment, human bone marrow cells
can be obtained from the posterior iliac crest by needle aspiration
(see, e.g., Kodo et al., J. Clin. Invest. 73: 1377-1384 (1984)).
For example, the HSC can be made highly enriched or in
substantially pure form. This enrichment can be accomplished
before, during, or after long-term culturing, and can be done by
any techniques known in the art. Long-term cultures of bone marrow
cells can be established and maintained by using, for example,
modified Dexter cell culture techniques (Dexter et al., J. Cell
Physiol. 91: 335 (1977)) or Witlock-Witte culture techniques
(Witlock and Witte, Proc. Natl. Acad. Sci. USA 79: 3608-3612
(1982)).
[0412] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy includes an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0413] 3. Prodrugs-A method for treating tumors is provided. The
method is practiced by administering a prodrug that is cleaved at a
specific site by a HASEGP to release an active drug or precursor
that can be converted to active drug in vivo. Upon contact with a
cell that expresses sHASEGP activity, the prodrug is converted into
an active drug. The prodrug can be a conjugate that contains the
active agent, such as an anti-tumor drug, such as a cytotoxic
agent, or other therapeutic agent (TA), linked to a substrate for
the targeted sHASEGP, such that the drug or agent is inactive or
unable to enter a cell, in the conjugate, but is activated upon
cleavage. The prodrug, for example, can contain an chondroitin
sulfate molecule, typically a relatively short, less than about 20
disaccharide units, that is catalytically cleaved by the targeted
sHASEGP. Cytotoxic agents, include, but are not limited to,
alkylating agents, antiproliferative agents and tubulin binding
agents. Others include, vinca drugs, mitomycins, bleomycins and
taxanes.
[0414] J. Pharmaceutical Compositions and Modes of
Administration
[0415] 1. Components of the compositions. Pharmaceutical
compositions containing an active sHASEGP are provided herein. Also
provided are combinations of compounds that modulate the activity
of a sHASEGP polypeptide and another treatment or compound for
treatment of a hyaluronidase disorder, such as an antibody
compound.
[0416] The sHASEGP polypeptide and a second agent can be packaged
as separate compositions for administration together or
sequentially or intermittently. Alternatively, they can be provided
as a single composition for administration or as two compositions
for administration as a single composition. The combinations can be
packaged as kits.
[0417] 2. Formulations and Route of Administration
[0418] The sHASEGP polypeptides and soluble human hyaluronidase
domain thereof provided herein can be formulated as pharmaceutical
compositions, typically for single dosage administration. The
concentrations of the polypeptides in the formulations are
effective for delivery of an amount, upon administration, that is
effective for the intended treatment. Typically, the compositions
are formulated for single dosage administration. To formulate a
composition, the weight fraction of a sHASEGP polypeptide, soluble
human hyaluronidase domains thereof or mixture thereof is
dissolved, suspended, dispersed or otherwise mixed in a selected
vehicle at an effective concentration such that the treated
condition is relieved or ameliorated.
[0419] Pharmaceutical carriers or vehicles suitable for
administration of the sHASEGP or soluble human hyaluronidase
domains thereof provided herein include any such carriers known to
those skilled in the art to be suitable for the particular mode of
administration.
[0420] In addition, the polypeptides can be formulated as the sole
pharmaceutically active ingredient in the composition or can be
combined with other active ingredients. Liposomal suspensions,
including tissue-targeted liposomes, can also be suitable as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art. For
example, liposome formulations can be prepared as described in U.S.
Pat. No. 4,522,811.
[0421] The active sHASEGP or soluble human hyaluronidase domain
thereof is included in the pharmaceutically acceptable carrier in
an amount sufficient to exert a therapeutically useful effect in
the absence of undesirable side effects on the patient treated. The
therapeutically effective concentration can be determined
empirically by testing the polypeptides in known in vitro and in
vivo systems such as by using the assays provided herein or see,
e.g., Taliani et al. (1996) Anal. Biochem. 240: 60-67, Filocamo et
al. (1997) J. Virology 71: 1417-1427, Sudo et al. (1996) Antiviral
Res. 32: 9-18, Buffard et al. (1995) Virology 209: 52-59, Bianchi
et al (1996) Anal. Biochem. 237: 239-244, Hamatake et al. (1996)
Intervirology 39:249-258, Steinkuhler et al. (1998) Biochem.
37:8899-8905, D'Souza et al. (1995) J. Gen. Virol. 76:1729-1736,
Takeshita et al. (1997) Anal. Biochem. 247: 242-246; see also,
e.g., Shimizu et al. (1994) J. Virol. 68: 8406-8408; Mizutani et
al. (1996) J. Virol. 70: 7219-7223, Mizutani et al. (1996) Biochem.
Biophys. Res. Commun. 227: 822-826, Lu et al. (1996) Proc. Natl.
Acad. Sci. (USA) 93: 1412-1417, Hahm et al. (1996) Virology 226:
318-326, Ito et al. (1996) J. Gen. Virol. 77:1043-1054, Mizutani et
al. (1995) Biochem. Biophys. Res. Commun. 212: 906-911, Cho et al.
(1997) J. Virol. Meth. 65 :201-207 and then extrapolated therefrom
for dosages for humans.
[0422] Typically a therapeutically effective dosage is
contemplated. The amounts administered can be on the order of 0.001
to 1 mg/ml, including about 0.005-0.05 mg/ml and about 0.01 mg/ml,
of blood volume. Pharmaceutical dosage unit forms are prepared to
provide from about 1 mg to about 1000 mg, including from about 10
to about 500 mg, and including about 25-75 mg of the essential
active ingredient or a combination of essential ingredients per
dosage unit form. The precise dosage can be empirically
determined.
[0423] In some instances, a high Unit dose of human sHASEGP is
preferable. For example, with intravenous administration of sHASEGP
concentrations of sHASEGP from 500-100,000 Units per ml are
preferable. Lyophilized formulations of sHASEGP are also ideal for
storage of large Unit doses of sHASEGP. 200,000 Unit lyophilized
vials of sHASEGP are contemplated for intravenous delivery.
[0424] High concentration doses are also contemplated for the
delivery of small volumes of sHASEGP. Administration of 10-100 ul
of 5000 Units/ml sHASEGP is contemplated for injection in the
anterior chamber to dissolve pre administered viscoelastic
substances during cataract and phakic intraocular lens implantation
surgeries. Small volume injections of 50-200 U/ml doses are also
contemplated for intravitreal procedures such as the treatment of
vitreous hemorrhage or vitro-retinal detachment in diabetic
retinopathy.
[0425] Somewhat larger volume injections (e.g. between about one to
several ml, or more) are contemplated for somewhat larger and/or
more readily drained spaces within the body. For example, as
illustrated herein, periorbital injections of one to several ml or
more can be made into one or more spaces surrounding the eye, e.g.
by injection into the sub-Tenon's or episcleral space, in order to
promote the transcleral delivery of pharmacologic agents to tissues
at the back of the eye such as the choroid plexus and/or retina. As
will be appreciated by those of skill in the art, some spaces
within the body can readily accommodate larger volumes. By way of
illustration, the episcleral or sub-Tenon's space around the eye
can readily accommodate one to several ml or even larger volumes
(e.g. up to or greater than 5 to 10 ml, depending for example on
the characteristics of the particular tissue and the rate at which
the volume is delivered).
[0426] In addition, as described and illustrated herein, it appears
that the introduction of a sHASEGP (potentially in combination with
one or more additional pharmacologic or other agents) in a larger
volume can be used to further drive delivery of agents to desired
target sites within the body. Without being restricted to a
particular mechanism of action, it is believed that such larger
volume injections can further drive permeation or spreading of both
sHASEGP as well as other agent(s) present in the treated tissue by
a process of convective transport that is promoted by an increased
pressure differential. Again, without wishing to be restricted to a
particular mechanism of action, it is believed that such an
increased pressure differential can be brought about in the context
of the present invention by, inter alia, one or both of the
following: (i) an elevated driving pressure or elevated "pressure
head" (e.g. caused by increased hydrostatic pressure associated
with an injection that largely fills or somewhat overfills or
distends a space or site of injection); and (ii) a reduced
downstream pressure or reduced "impedance" (e.g. caused by the
opening of channels in the interstitial matrix associated with
localized hyaluronidase activity and consequent degradation of
relatively hydrated hyaluronan). Preferred hyaluronidases for use
in this and other applications of the present invention (such as
facilitating the delivery pharmacologic and other agents) are
sHASEGPs, particularly human sHASEGPs in the context of delivery of
agents to one or more tissues in the human body. However, these and
other approaches to the use and application of hyaluronidases can
also be applied using non-human sHASEGPs, such as mammalian
sHASEGPs, as well other human or mammalian hyaluronidases,
preferably soluble forms of such hyaluronidases.
[0427] The active ingredient can be administered at once, or can be
divided into a number of smaller doses to be administered at
intervals of time. It is understood that the precise dosage and
duration of treatment is a function of the disease being treated
and can be determined empirically using known testing protocols or
by extrapolation from in vivo or in vitro test data. It is to be
noted that concentrations and dosage values can also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or use of the claimed compositions
and combinations containing them.
[0428] Pharmaceutically acceptable derivatives include acids,
salts, esters, hydrates, solvates and prodrug forms. The derivative
is typically selected such that its pharmacokinetic properties are
superior to the corresponding neutral sHASEGP or soluble human
hyaluronidase domain thereof.
[0429] Thus, effective concentrations or amounts of one or more of
the polypeptides herein or pharmaceutically acceptable derivatives
thereof are mixed with a suitable pharmaceutical carrier or vehicle
for systemic, topical or local administration to form
pharmaceutical compositions. sHASEGP polypeptides or soluble human
hyaluronidase domains thereof are included in an amount effective
for ameliorating or treating the disorder for which treatment is
contemplated. The concentration of active polypeptide in the
composition depends on absorption, inactivation, excretion rates of
the active polypeptide, the dosage schedule, amount administered,
particular formulation as well as other factors known to those of
skill in the art.
[0430] The therapeutic agents for use in the methods can be
administered by any route known to those of skill in the art, such
as, but are not limited to, topically, intraarticularly,
intracisternally, intraocularly, peri-orbitally,
intraventricularly, intrathecally, intravenously, intraarterially,
intramuscularly, intraperitoneally, intradermally, subcutaneously,
intratracheally, as well as by any combination of any two or more
thereof. Dry powder pulmonary formulations can be envisioned as
well.
[0431] The most suitable route for administration will vary
depending upon the proposed use, such as, for example, use as a
delivery agent to facilitate subcutaneous delivery of fluids, use
to reduce intraocular pressure in the eyes of glaucoma patients
receiving viscoelastics or use as a "spreading agent" to enhance
the activity of chemotherapeutics, and the location of interest,
such as a particular internal organ, a tumor growth, intraocular
cavity and the epidermis. Modes of administration include, but are
not limited to, topically, locally, intraarticularly,
intracisternally, intraocularly, peri-orbitally,
intraventricularly, intrathecally, intravenously, intramuscularly,
intratracheally, intraperitoneally, intradermally, subcutaneously,
and by a combination of any two or more thereof. For example, for
treatment of various cancers, such as squamous cell carcinoma,
breast cancer, urinary bladder cancer and gastrointestinal cancer,
local administration, including administration to the site of the
tumor growth (e.g., intrathecally, intraventricularly, or
intracisternally) provides the advantage that the therapeutic agent
can be administered in a high concentration without risk of the
complications that can accompany systemic administration of a
therapeutic agent.
[0432] As an exemplary class of pharmacologic agents that can be
combined with sHASEGPs (or other glycosaminoglycanases) (e.g.
combined by co-formulation with one or more sHASEGPs or by
co-administration with one or more sHASEGPs (which may be
administered prior to, coincident with or following administration
of the agent)), a number of agents having effectiveness or
potential effectiveness in the treatment of various cancers have
been described in the art, and novel anti-cancer agents are
regularly being developed which may likewise be combined with one
or more sHASEGPs (e.g. provided by co-formulation or by
co-administration before, coincident with and/or following
administration of the agent) to improve the pharmacokinetics,
pharacodynamics or other useful properties of the agent.
[0433] By way of illustration (and without limitation) a number of
anti-cancer or chemotherapeutic agents have now been approved for
the treatment of various cancers which can be combined with
sHASEGPs (or other glycosaminoglycanases) by co-formulation or
co-administration (before, coincident with or after administration
of the anti-cancer agent). Such agents include, for example,
Aldesleukins (e.g. PROLEUKIN.TM.); Alemtuzumabs (e.g. CAMPATH.TM.);
Alitretinoins (e.g. PANRETIN.TM.); Allopurinols (e.g.
ZYLOPRIM.TM.); Altretamines (e.g. HEXALEN.TM.); Amifostines (e.g.
ETHYOL.TM.); Anastrozoles (e.g. ARIMIDEX.TM.); Arsenic Trioxides
(e.g. TRISENOX.TM.); Asparaginases (e.g. ELSPAR.TM.); BCG Live
(e.g. TICE.TM. BCG); Bexarotenes (e.g. TARGRETIN.TM.); Bleomycins
(e.g. BLENOXANE.TM.); Busulfan intravenous (e.g. BUSULFEX.TM.);
Busulfan orals (e.g. MYLERANTm); Calusterones (e.g. METHOSARB.TM.);
Capecitabines (e.g. XELODA.TM.); Carboplatins (e.g.
PARAPLATIN.TM.); Carmustines (e.g. BCNU.TM., BiCNU.TM.);
Carmustines with Polifeprosans (e.g. GLIADEL.TM. Wafer); Celecoxibs
(e.g. CELEBREX.TM.); Chlorambucils (e.g. LEUKERAN.TM.); Cisplatins
(e.g. PLATINOL.TM.); Cladribines (e.g. LEUSTATIN.TM., 2-CdA.TM.);
Cyclophosphamides (e.g. CYTOXAN.TM., NEOSAR.TM.); Cytarabines (e.g.
CYTOSAR-U.TM.); Cytarabine liposomals (e.g. DepoCyt.TM.);
Dacarbazines (e.g. DTIC-Dome.upsilon.): Dactinomycins (e.g.
COSMEGEN.TM.); Darbepoetin Alfas (e.g. ARANESP.TM.); Daunorubicin
liposomals (e.g. DANUOXOME.TM.); Daunorubicins/Daunomycins (e.g.
CERUBIDINE.TM.); Denileukin Diftitoxes (e.g. ONTAK.TM.);
Dexrazoxanes (e.g. ZINECARD.TM.); Docetaxels (e.g. TAXOTERE.TM.);
Doxorubicins (e.g. ADRIAMYCIN.TM., RUBEX.TM.); Doxorubicin
liposomals (e.g. DOXIL.TM.); Dromostanolone propionates (e.g.
DROMOSTANOLONE.TM. and MASTERONE.TM. Injection); Elliott's B
Solutions (e.g. Elliott's B Solution.TM.); Epirubicins (e.g.
ELLENCE.TM.); Epoetin alfas (e.g. EPOGEN.TM.); Estramustines (e.g.
EMCYT.TM.); Etoposide phosphates (e.g. ETOPOPHOS.TM.); Etoposide
VP-16s (e.g. VEPESID.TM.); Exemestanes (e.g. AROMASIN.TM.);
Filgrastims (e.g. NEUPOGEN.TM.); Floxuridines (e.g. FUDR.TM.);
Fludarabines (e.g. FLUDARA.TM.); Fluorouracils incl. 5-FUs (e.g.
ADRUCIL.TM.); Fulvestrants (e.g. FASLODEX.TM.); Gemcitabines (e.g.
GEMZAR.TM.); Gemtuzumabs/Ozogamicins (e.g. MYLOTARG.TM.); Goserelin
acetates (e.g. ZOLADEX.TM.); Hydroxyureas (e.g. HYDREA.TM.);
Ibritumomabs/Tiuxetans (e.g. ZEVALIN.TM.); Idarubicins (e.g.
IDAMYCIN.TM.); Ifosfamides (e.g. IFEX.TM.); Imatinib mesylates
(e.g. GLEEVEC.TM.); Interferon alfa-2as (e.g. ROFERON-A.TM.);
Interferon alfa-2bs (e.g. INTRON A.TM.); Irinotecans (e.g.
CAMPTOSAR.TM.); Letrozoles (e.g. FEMARA.TM.); Leucovorins (e.g.
WELLCOVORIN.TM., LEUCOVORIN.TM.); Levamisoles (e.g. ERGAMISOL.TM.);
Lomustines/CCNUs (e.g. CeeBU.TM.); Meclorethamines/Nitrogen
mustards (e.g. MUSTARGEN.TM.); Megestrol acetates (e.g.
MEGACE.TM.); Melphalans/L-PAMs (e.g. ALKERAN.TM.); Mercaptopurine
incl. 6-MPs (e.g. PURINETHOL.TM.); Mesnas (e.g. MESNEX.TM.);
Methotrexates; Methoxsalens (e.g. UVADEX.TM.); Mitomycin Cs (e.g.
MUTAMYCIN.TM., MITOZYTREX.TM.); Mitotanes (e.g. LYSODREN.TM.);
Mitoxantrones (e.g. NOVANTRONE.TM.); Nandrolone Phenpropionates
(e.g. DURABOLIN-50.TM.); Nofetumomabs (e.g. VERLUMA.TM.);
Oprelvekins (e.g. NEUMEGA.TM.); Oxaliplatins (e.g. ELOXATIN.TM.);
Paclitaxels (e.g. PAXENE.TM., TAXOL.TM.); Pamidronates (e.g.
AREDIA.TM.); Pegademases (e.g. ADAGEN.TM.); Pegaspargases (e.g.
ONCASPAR.TM.); Pegfilgrastims (e.g. NEULASTA.TM.); Pentostatins
(e.g. NIPENT.TM.); Pipobromans (e.g. VERCYTE.TM.);
Plicamycin/Mithramycins (e.g. MITHRACIN.TM.); Porfimer sodiums
(e.g. PHOTOFRIN.TM.); Procarbazines (e.g. MATULANE.TM.);
Quinacrines (e.g. ATABRINE.TM.); Rasburicases (e.g. ELITEK.TM.);
Rituximabs (e.g. RITUXAN.TM.); Sargramostims (e.g. PROKINE.TM.);
Streptozocins (e.g. ZANOSAR.TM.); Talcs (e.g. SCLEROSOL.TM.);
Tamoxifens (e.g. NOLVADEX.TM.); Temozolomides (e.g. TEMODAR.TM.);
Teniposides/VM-26s (e.g. VUMON.TM.); Testolactones (e.g.
TESLAC.TM.); Thioguanines incl. 6-TG; Thiotepas (e.g.
THIOPLEX.TM.); Topotecans (e.g. HYCAMTIN.TM.); Toremifenes (e.g.
FARESTON.TM.); Tositumomabs (e.g. BEXXAR.TM.); Trastuzumabs (e.g.
HERCEPTIN.TM.); Tretinoins/ATRA (e.g. VESANOID.TM.); Uracil
Mustards; Valrubicins (e.g. VALSTAR.TM.); Vinblastines (e.g.
VELBAN.TM.); Vincristines (e.g. ONCOVIN.TM.); Vinorelbines (e.g.
NAVELBINE.TM.); Zoledronates (e.g. ZOMETA.TM.); as well as other
agents described herein and in the art.
[0434] As with other classes of pharmacologic agents described
herein, a sHASEGP (or other glycosaminoglycanase) may be used in
conjunction with the administration of (or co-formulated with) an
individual pharmacologic agent (such as an antineoplastic for
purposes of illustration, or with a member of another class of
agents as described herein and/or known in the art). Alternatively,
a sHASEGP (or other glycosaminoglycanase) may be used in
conjunction with the administration of (or co-formulated with) more
than one pharmacologic agent of a given class (such as multiple
antineoplastic agents for purposes of illustration, or multiple
members of other classes of agents as described herein and/or known
in the art). In the case of combinations with two or more agents of
a given class, it is often preferred that the two or more agents
act by two or more different mechanisms of action. A sHASEGP (or
other glycosaminoglycanase) may also be used in conjunction with
the administration of (or co-formulated with) one pharmacologic
agent of a first given class (such as an antineoplastic for
purposes of illustration, or a member of one of the other classes
of agents as described herein and/or known in the art), and one or
more additional pharmacologic agents of one or more different
classes that are useful for treating any of a variety of
conditions. While certain principles are thus referred to here in
the case of anticancer agents for purposes of illustration, these
principles are also applicable to, for example, anti-infectives and
the numerous members of other classes of agents that are described
and illustrated herein, as well as additional agents that are
already known in the art or are newly developed.
[0435] In the case of any of the combinations and/or
co-formulations described herein (comprising at least one sHASEGP
(or other glycosaminoglycanase) and at least one other agent (such
as a pharmacologic agent, e.g., a member of one of the classes of
agents illustrated herein)), it is also contemplated that such
combination and/or co-formulation can further comprise one or more
agents (such as various stabilizers, excipients and the like) which
do not themselves exert substantial pharmacodynamic effects but
which promote the usefulness of the combination and/or
co-formulation (e.g. by stabilizing one or more of the components
or an entire formulation, by helping to promote or target the
activity of one or more of the agents or of the sHASEGP, and/or by
reducing toxicity or otherwise improving the safety profile of the
combination or co-formulation.
[0436] As will be appreciated by those of skill in the art, new
versions and formulations of these and other anti-cancer agents (as
well as members of other classes of agents as described herein) are
regularly being developed that can likewise be combined with
sHASEGPs (or other glycosaminoglycanases). Furthermore, since such
enzymes can be used to improve pharmacokinetic and/or
pharmacodynamic profiles of a large variety of agents, and allow
agents to be delivered by different (and potentially more targeted)
routes, for example, sHASEGPs (or other glycosaminoglycanases) can
be used to enhance the PK and/or PD profiles of a number of agents
previously considered to have sub-optimal profiles. Testing of
agents in appropriate animal models with and without concomitant
sHASEGP can be used to assess the relative effectiveness and
toxicity of particular agents.
[0437] As illustrated and exemplified herein, a number of large
molecule agents (such as antibodies and other proteins,
particularly those administered by non-IV parenteral injection),
exhibit relatively poor pharmacokinetic profiles and can be
particularly enhanced by co-formulation or co-administration with
sHASEGPs (or other glycosaminoglycanases). Without being restricted
to a particular mechanism of action, it is generally considered
that small molecule agents exhibiting relatively low hydrophobicity
are more effectively dispersable by, e.g., convective transport
within interstitial fluids as can be promoted by sHASEGPs (or other
glycosaminoglycanases). A commonly used method for predicting such
attributes is to assess the calculated and/or estimate
octanol:water partition coefficients (K.sub.ow) which have been
reported for a number of compounds. In that regard, agents
exhibiting log K.sub.ow of <3, more preferably <2 or <1,
more preferably <0, still more preferably <(-1). As will be
appreciated by those of skill in the art, many agents can also be
modified covalently and/or non-covalently to increase their
hydrophilicity, and/or to encompass such agents within vessicles or
macromolecular complexes to facilitate their permeation and/or
delivery.
[0438] For purposes of further illustration (and without
limitation), examplary anti-cancer or chemotherapeutic
pharmacologic agents that can be combined with sHASEGPs (or other
glycosaminoglycanases) are included in the following list (and
others are known in the art): Acivicins; Aclarubicins; Acodazoles;
Acronines; Adozelesins; Aldesleukins; Alitreninoins (9-Cis-Retinoic
Acids); Altretamines; Alvocidibs; Ambazones; Ambomycins;
Ametantrones; Aminoglutethimides; Amsacrines; Anastrozoles;
Anaxirones; Ancitabines; Anthramycins; Apaziquones; Argimesnas;
Asparaginases; Asperlins; Atrimustines; Azacitidines; Azetepas;
Azotomycins; Banoxantrones; Batabulins; Batimastats; Benaxibines;
Bendamustines; Benzodepas; Bexarotenes; Bicalutamides;
Bietaserpines; Biricodars; Bisantrenes; Bisantrenes; Bisnafide
Dimesylates; Bizelesins; Bleomycins; Bortezomibs; Brequinars;
Bropirimines; Budotitanes; Busulfans; Cactinomycins; Calusterones;
Canertinibs; Capecitabines; Caracemides; Carbetimers; Carboplatins;
Carboquones; Carmofurs; Carmustines; Carubicins; Carzelesins;
Cedefingols; Cemadotins; Chlorambucils; Cioteronels; Cirolemycins;
Cisplatins; Cladribines; Clanfenurs; Clofarabines; Crisnatols;
Cyclophosphamides; Cytarabines; Dacarbazines; Dactinomycins;
Daunorubicins; Decitabines; Dexniguldipines; Dexonnaplatins;
Dezaguanines; Diaziquones; Dibrospidiums; Dienogests; Dinalins;
Disermolides; Docetaxels; Dofequidars; Doxifluridines;
Doxorubicins; Droloxifenes; Dromostanolone Propionates;
Duazomycins; Ecomustines; Edatrexates; Edotecarins; Eflornithines;
Elacridars; Elinafides; Elsamitrucins; Emitefurs; Enloplatins;
Enpromates; Enzastaurins; Epipropidines; Epirubicins; Eptaloprosts;
Erbulozoles; Esorubicins; Estramustines; Etanidazoles; Etoglucids;
Etoposides; Etoprines; Exisulinds; Fadrozoles; Fazarabines;
Fenretinides; Floxuridines; Fludarabines; Fluorouracils;
Fluoxymesterones; Flurocitabines; Fosquidones; Fostriecins;
Fostriecins; Fotretamines; Galarubicins; Galocitabines;
Gemcitabines; Geroquinols; Gimatecans; Gimeracils; Gloxazones;
Glufosfamides; Hydroxyureas; Idarubicins; Ifosfamides; Ilmofosines;
Ilomastats; Imexons; Improsulfans; Indisulams; Inproquones;
Interferons; Interferon Alfas; Interferon Betas; Interferon Gammas;
Interleukin-2s and other Interleukins (including recombinant
Interleukins); Intoplicines; Iobenguanes [131-I]; Iproplatins;
Irinotecans; Irsogladines; Ixabepilones; Ketotrexates;
L-Alanosines; Lanreotides; Lapatinibs; Ledoxantrones; Letrozoles;
Leuprolides; Leuprorelins (Leuprorelides); Lexacalcitols;
Liarozoles; Lobaplatins; Lometrexols; Lomustines; Lonafarnibs;
Losoxantrones; Lurtotecans; Mafosfamides; Mannosulfans;
Marimastats; Masoprocols; Maytansines; Mechlorethamines;
Megestrols; Melengestrols; Melphalans; Menogarils; Mepitiostanes;
Mercaptopurines; Metesinds; Methotrexates; Metomidates; Metoprines;
Meturedepas; Miboplatins; Miproxifenes; Misonidazoles;
Mitindomides; Mitocarcins; Mitocromins; Mitoflaxones; Mitogillins;
Mitoguazones; Mitomalcins; Mitomycins; Mitonafides; Mitoquidones;
Mitospers; Mitotanes; Mitoxantrones; Mitozolomides; Mivobulins;
Mizoribines; Mofarotenes; Mopidamols; Mubritinibs; Mycophenolic
Acids; Nedaplatins; Nelzarabines; Nemorubicins; Nitracrines;
Nocodazoles; Nogalamycins; Nolatrexeds; Nortopixantrones;
Octreotides; Ormaplatins; Ortataxels; Oteracils; Oxisurans;
Oxophenarsines; Paclitaxels; Pamidronates; Patubilones;
Pegaspargases; Peldesines; Peliomycins; Pelitrexols; Pemetrexeds;
Pentamustines; Peplomycins; Perfosfamides; Perifosines;
Picoplatins; Pinafides; Pipobromans; Piposulfans; Pirfenidones;
Piroxantrones; Pixantrones; Plevitrexeds; Plicamycins; Plomestanes;
Plomestanes; Porfimers; Porfiromycins; Prednimustines;
Procarbazines; Propamidines; Prospidiums; Pumitepas; Puromycins;
Pyrazofurins; Ranimustines; Riboprines; Ritrosulfans; Rogletimides;
Roquinimexs; Rufocromomycins; Sabarubicins; Safingols;
Satraplatins; Sebriplatins; Semustines; Simtrazenes; Sizofirans;
Sobuzoxanes; Sorafenibs; Sparfosates; Sparfosic Acids;
Sparsomycins; Spirogermaniums; Spiromustines; Spiroplatins;
Spiroplatins; Squalamines; Streptonigrins; Streptovarycins;
Streptozocins; Sufosfamides; Sulofenurs; Tacedinalines;
Talisomycins; Tallimustines; Tariquidars; Tauromustines;
Tecogalans; Tegafurs; Teloxantrones; Temoporfins; Teniposides;
Teroxirones; Testolactones; Thiamiprines; Thioguanines; Thiotepas;
Tiamiprines; Tiazofurins; Tilomisoles; Tilorones; Timcodars;
Timonacics; Tirapazamines; Topixantrones; Toremifenes; Trabectedins
(Ecteinascidin 743); Trastuzumabs; Trestolones; Triciribines;
Triciribines; Trilostanes; Trimetrexates; Triplatin Tetranitrates;
Triptorelins; Trofosfamides; Tubulozoles; Ubenimexs; Uracil
Mustards; Uredepas; Valspodars; Vapreotides; Verteporfins;
Vinblastines; Vincristines; Vindesines; Vinepidines; Vinflunines;
Vinformides; Vinglycinates; Vinleucinols; Vinleurosines;
Vinorelbines; Vinrosidines; Vintriptols; Vinzolidines; Vorozoles;
Xanthomycin A's (Guamecyclines); Zeniplatins; Zilascorbs [2-H];
Zinostatins; Zorubicins; Zosuquidars; and the like.
[0439] Pharmaceutical and cosmetic carriers or vehicles suitable
for administration of the sHASEGP polypeptides or soluble human
hyaluronidase domain thereof provided herein include any such
carriers known to those skilled in the art to be suitable for the
particular mode of administration. In addition, the polypeptides
can be formulated as the sole pharmaceutically active ingredient in
the composition or can be combined with other active ingredients
that do not impair the desired action, or with materials that
supplement the desired action known to those of skill in the art.
For example, the sHASEGP polypeptides provided herein can be used
as a delivery or "spreading" agent in combination with a second
active compound, such as a therapeutically effective agent,
including, but not limited to a drug or a prodrug, to facilitate
delivery of or to enhance the activity of the second active
ingredient. In a particular embodiment, a sHASEGP polypeptide or a
soluble human hyaluronidase domain thereof can be co-formulated or
co-administered with an anesthetic agent, such as Lignocaine,
Bupivicaine or a mixture of the two, and, optionally, a hormonal
agent, such as epinephrine, to decrease or stop blood uptake during
ophthalmic surgery. In another particular embodiment, a sHASEGP
polypeptide or a soluble human hyaluronidase domain thereof can be
co-formulated or co-administered with a pharmacologic agent such as
an anti-inflammatory agent to enhance the delivery of the
pharmacologic to mammalian tissues in vivo (e.g. to portions of the
eye) where such agent is desired for treating a particular
condition. By co-administration is intended the administration of
sHASEGP (or other glycosaminoglycanase) together with or in close
temporal proximity to the administration of a pharmacologic agent.
Typically it is preferred that the sHASEGP (or other
glycosaminoglycanase) be administered shortly before (e.g. 5 to 60
minutes before) or, for maximal convenience, together with the
pharmacologic agent. As will be appreciated by those of skill in
the art, the desired proximity of co-administration depends in
significant part on the effective half lives of the agents in the
particular tissue setting, and the particular disease being
treated, and can be readily optimized by testing the effects of
administering the agents at varying times in suitable models, such
as in suitable animal models. In some situations, the optimal
timing of administration of the sHASEGP (or other
glycosaminoglycanase) will exceed 60 minutes. By way of
illustration and without wishing to be bound by theory, in some
tumor situations for example the maximal entry of chemotherapeutic
agent into the tumor may occur after the degradation of hyaluronan
in the tumor is substantially complete so as to reduce tumor
interstitial volume, which is followed by a hyaluronan-regenerative
phase during which fluid is drawn back into the tumor resulting in
increased adsorption of a chemotherapeutic agent from plasma. In
the latter case, optimal administration of sHASEGP (or other
glycosaminoglycanase) may be obtained between about one to twenty
hours prior to administration of the pharmacologic agent. In order
to optimize the timing of pre-administration relative to
pharmacologic agent for a particular combination of disease
condition and pharmacologic agent, one of skill can conduct a
routine time course of pre-administration, typically comparing one
or several time points of an hour or less with one or several
timepoints of greater than hour (but typically not exceeding a day)
in order to optimize the timing of administration. A sHASEGP
polypeptide or a soluble human hyaluronidase domain thereof can
also be co-formulated or co-administered with various
chemotherapeutics, such as a toxin and a tumor necrosis factor, to
enhance the activity of the chemotherapeutic and/or the
accessibility of the target tumors to the chemotherapeutic. The
active compound is included in the carrier in an amount sufficient
to exert a therapeutically useful effect in the absence of serious
toxic effects on the treated individual. The effective
concentration can be determined empirically by testing the
compounds using in vitro and in vivo systems, including the animal
models described herein.
[0440] In the case of pharmacologic agents that are delivered by
parenteral administration (i.e. by routes other than through the
digestive tract, such as by subcutaneous, intradermal,
intramuscular or intravenous routes for example), sHASEGPs (or
other glycosaminoglycanases) can be readily co-formulated with or
co-administered with any of a varety of such pharmacologic agents
to enhance their delivery to desired sites within the body. For
example, depending on the particular combination of pharmacologic
agent and route of administration, delivery can be enhanced by
increasing the extent (and/or rate) at which the agent diffuses
through a tissue at or near the site of delivery (e.g. following
its injection) and/or the extent (and/or rate) at which the agent
diffuses and/or is carried by convective transport through a target
tissue (e.g. following its release from the bloodstream, lymph,
cerebrospinal fluid or other fluid system). The extent by which
delivery is enhanced can be readily evaluated, for example, in
animal model systems in which the addition of a sHASEGP results in
a measurable increase in the bioavailability of the agent in
tissues of interest within the body.
[0441] Without wishing to be bound by theory, it is believed that
in the case of non-intravenous parenterals (such as subcutaneous,
intradermal and intramuscular injectables), the presence of sHASEGP
(or other glycosaminoglycanase) in the vicinity of an injection
site and in "downstream" sites such as those between an injection
site and a distal vascular bed, can enhance the diffusion and/or
convective transport of the agent into a delivery system such as
the vasculature. Enhanced delivery in such a situation can be
associated with an increase in the amount of the agent that
effectively passes through and out of the initial delivery site
(potentially by both increasing the volume and rate of bulk fluid
flow).
[0442] In the case of many pharmacologic agents, which are subject
to degradation, removal or other forms of inactivation in tissues,
increasing the rate of its passage through a tissue can in turn
substantially increase the amount of effective agent that transits
through the tissue, further increasing its bioavailability. For
both non-intravenously-administered parenterals that subsequently
enter the bloodstream or other conduit, as well agents delivered
directly into the conduit vessels (such as by intravenous
administration, IV), the ability of the agent to diffuse through a
tissue supplied by the conduit can also be enhanced by the presence
of sHASEGP in the tissue. Whether sHASEGP is present in the
"delivery tissue" such as in the former case, and/or in the "target
tissue" as in the latter case, the presence of sHASEGP can thus
enhance the effective extent and/or rate of delivery of the
pharmacologic to target tissues within the body. In either case,
the sHASEGP can be used to increase the effective bioavailability
of a pharmacologic agent with which it has been co-formulated or
co-administered. As a result, the sHASEGP can be used to achieve
elevated and/or more rapidly achieved concentrations of the agent
to provide, for example, a more potent and/or more rapid response
for a given dose. Alternatively, the sHASEGP can be used to allow a
given response to be achieved with a lower dose of administered
agent, for example. The ability of a sHASEGP (or other
glycosaminoglycanase) to enhance bulk fluid flow at and near a site
of injection can also improve other aspects of associated
pharmacologic delivery. For example, the increase in bulk fluid
flow can help to allow the volume of fluid injected to be more
readily dispersed from the site of injection (reducing potentially
painful or other adverse consequences of injection). Indeed, it has
long been recognized that such adverse sensations associated with
non-IV parenteral injections are a key reason that patients may
resist or avoid non-IV parenteral administration (which may
effectively require a product to be administered intravenously when
it might otherwise be given more conveniently and/or safely through
a non-IV route).
[0443] In using a sHASEGP (or other glycosaminoglycanase) of the
present invention to enhance delivery of one or more pharmacologic
or other agents in the body (a use that is sometimes referred to as
"spreading"), the sHASEGP (or other glycosaminoglycanase) itself
may be administered by the same route as is the pharmacologic agent
(in which case it may be co-formulated or otherwise co-administered
together with the pharmacologic agent or it may be administered
separately, preferably in advance of the pharmacologic agent as
discussed above. Alternatively, or in addition, the sHASEGP (or
other glycosaminoglycanase) may be administered by a different
route of administration. For example, the pharmacologic agent may
be introduced locally by non-IV parenteral injection and the
sHASEGP may be introduced intravenously. As another illustrative
and non-limiting example, the sHASEGP may be introduced locally by
non-IV parenteral injection (in a tissue that is desired to be
targeted by the pharmacologic agent) and the pharmacologic agent
itself may be introduced by another route, such as intravenous,
non-parenteral (e.g. oral) or other route. As will thus be
appreciated by those of skill in the art, sHASEGPs of the present
invention can be used to enhance the delivery and bioavailability
of a variety of pharmacologic and other agents including agents
that are typically delivered by parenteral as well as non-parental
routes.
[0444] For example, sHASEGPs (or other glycosaminoglycanases) can
be co-formulated or co-administered with a variety of pharmacologic
agents that are typically administered by parenteral delivery, e.g.
to enhance their effectiveness and/or to improve their risk/benefit
profile in treating disease.
[0445] By way of illustration of the variety of pharmacologic
agents that can be parenterally-administered and their delivery
and/or bioavailability enhanced through co-formulation and/or
co-administration with sHASEGPs (or other glycosaminoglycanases) of
the present invention, the following list is not exhaustive (and is
thus not intended to be limiting) but simply to provide exemplary
agents: Adalimumabs (e.g. HUMIRA.TM.), Agalsidase Betas (e.g.
FABRAZYME.TM.), Aldesleukins (PROLEUKIN.TM. IL-2), Alefacepts (e.g.
AMEVIVE.TM.), Ampicillins (e.g. UNASYN.TM. Injection), Anakinras
(e.g. KINERE.TM.), Antipoliomyelitic Vaccines (e.g. PEDIARIX.TM.),
Anti-Thymocytes (e.g. THYMOGLOBULIN.TM.), Azithromycins (e.g.
ZITHROMAX.TM. IV), Becaplermins (e.g. REGRANEX.TM.), Caspofungins
(e.g. CANCIDAS.TM.), Cefazolins (e.g. ANCEF.TM. and CEFAZOLIN.TM.),
Cefepimes (e.g. MAXIPIME.TM.), Cefotetans (e.g. CEFOTAN.TM.),
Ceftazidimes (e.g. FORTAZ.TM.), Ceftriaxones (e.g. ROCEFIN.TM.),
Cetuximabs (e.g. ERBITUX.TM.), Cilastatins (e.g. PRIMAXIN.TM. IV),
Clavulanic Acids (e.g. in conjunction with Amoxicillins such as in
AUGMENTIN.TM.), Clindamycins (e.g. CLEOCIN.TM.), Darbepoetin Alfas
(e.g. ARANESP.TM.), Deaclizumabs (e.g. ZENAPAX.TM.), Diphtheria
Toxoids (typically in combinations e.g. DAPTACEL.TM., INFANRIX.TM.
and PEDIARIX.TM.), Efalizumabs (e.g. RAPTVA.TM.), Epinephrines
(e.g. EPIPEN.TM.), Erythropoietin Alphas (e.g. EPOGEN.TM. And
PROCRIT.TM.), Etanercepts (e.g. ENBREL.TM.), Filgrastims (e.g.
NEUPOGEN.TM.), Fluconazoles (e.g. DIFLUCAN.TM. Injection),
Follicle-Stimulating Hormones such as Follitropin Alphas (e.g.
GONAL-F.TM.) and Follitropin Betas (e.g. FOLLISTIM.TM.),
Fosphenytoins (e.g. CEREBYX.TM.), Fluconazoles (e.g. DIFLUCAN.TM.),
Gadodiamides (e.g. OMNISCAN.TM.), Gadopentetates (e.g.
MAGNEVIS.TM.), Gatifloxacins (e.g. TEQUIN.TM.), Glatiramers (e.g.
COPAXONE.TM.), GM-CSF's (e.g. LEUKINE.TM.), Goserelins (e.g.
ZOLADEX.TM.), Granisetrons (e.g. KYTRIL.TM.), Haemophilus Influenza
B's (e.g. COMVAX.TM. and HibTITER.TM.), Haloperidols (e.g.
HALDOL.TM.), Hepatitis A Vaccines (e.g. HAVRIX.TM., TWINRIX.TM. and
VAQTA.TM.), Hepatitis B Vaccines (e.g. recombinants COMVAX.TM.,
ENGERIX.TM.-B, RECOMBIVAX.TM.-HB, TWINRIX.TM., and non-recombinants
BAYHEP.TM.-B and NABI.TM.4-HB), Ibritumomab Tiuxetans (e.g.
ZEVALIN.TM.), Immunoglobulins (including mixtures of
immunoglobulins such as GAMMAGARD.TM. and the like, as well as any
of a variety of purified immunoglobulins), Influenza Virus Vaccines
(e.g. FLUMIS.TM.), Infliximabs (e.g. REMICADE.TM.), Insulins (e.g.
HUMALOG.TM., HUMALOG.TM. MIX 75/25.TM., HUMULIN.TM. products (incl.
50/50, 70/30, Regular, NPH, Ultra and Ultralente), and
NOVOLIN.TM.), Insulin Glargines (e.g. LANTUS.TM.), Interferon
Alfa-2a's (ROFERAN.TM.-A), Interferon Alfa-2b's (e.g.
INTRON-A.TM.), Interferon Alfacon-l's (e.g. INFERGEN.TM.),
Interferon Alfa-n3s (e.g. ALFERON N.TM.), Interferon Betas (e.g.
BETASERON.TM. And BETAFERON.TM.), Interferon Beta-1a's (e.g.
AVONEX.TM. And REBIF.TM.), Interferon Gammas (e.g. ACTIMMUNE.TM.),
Iodixanols (e.g. VISIPAQUE.TM.), Iohexols (OMNIPAQUE.TM.),
Iopamidols, Ioversols (e.g. OPTIRAY.TM.), Ketorolacs (e.g.
TORADOL.TM.), Laronidases (e.g. ALDURAZYME.TM.), Levofloxacins
(e.g. LEVAQUIN.TM.), Lidocaines, Linezolids (e.g. ZYVOX.TM.),
Lorazepams (e.g. ATIVAN.TM.), Measles Vaccines (e.g.
ATTENUVAX.TM.), Measles-Mumps-Rubella Virus Vaccines (e.g.
M-M-R.TM. II), Medroxyprogesterones (e.g. DEPO-PROVERA.TM.),
Meropenems (e.g. MERREM.TM. IV), Methylprednisolones (e.g.
SOLU-MEDROL.TM.), Midazolams (e.g. VERSED.TM.), Morphines (e.g.
ASTRAMORPH/PF.TM.), Octreotides (e.g. SANDOSTATIN.TM.), Omalizumabs
(e.g. XOLAIR.TM.), Ondansetrons (e.g. ZOFRAN.TM.), Palivizumabs
(e.g. SYNAGIS.TM.), Pantoprazoles (e.g. PROTONIX.TM.),
Pegaspargases (e.g. ONCASPAR.TM.), Pegfilgrastims (e.g.
NEULASTA.TM.), Peg-Interferon Alfa-2a's (e.g. PEGASYS.TM.),
Peg-Interferon Alfa-2b's (e.g. PEG-INTRON.TM.), Pegvisomants (e.g.
SOMAVERT.TM.), Pertussis vaccines, Piperacillins (e.g. ZOSYN.TM.),
Pneumococcal Vaccines (e.g. PNEUMOVAX.TM. 23) and Pneumococcal
Conjugate Vaccines (e.g. PREVNAR.TM.), Promethazines (e.g.
PHENERGAN.TM.), Reteplases (e.g. RETAVASE.TM.), Somatropins (e.g.
GENOTROPIN.TM., HUMATROPE.TM., NORDITROPIN.TM., NUTROPIN.TM.,
SAIZEN.TM., SEROSTIM.TM., ZORBTIVE.TM.), Sulbactams, Sumatriptans
(e.g. IMITREX.TM.), Tazobactams, Tenecteplases (e.g. TNKASE.TM.),
Tetanus Purified Toxoids, Ticarcillins (e.g. TIMENTIN.TM.),
Tositumomabs (e.g. BEXXAR.TM.), Triamcinolone Acetonides,
Vancomycins (e.g. VANCOCIN.TM.), Varicella vaccines (e.g.
VARIVAX.TM.) as well as other vaccines; and the like.
[0446] As described and illustrated herein, pharmacologic agents of
a number of different classes and routes of administration can be
benefited by co-administration and/or co-formulation with sHASEGPs
(or other glycosaminoglycanases) to improve one or more of their
aspects of use. Such improvements can comprise, for example, one or
any combination of the following: (i) improving the extent and/or
the rate of absorption and thus bioavailability of an agent (for
example by causing more of it to reach the bloodstream after non-IV
parenteral administration, and/or less of it to be degraded after
such local administration by more rapid permeation), which can be
brought about for example by accelerating interstitial flow and
potentially convective transport following non-IV parenteral
administration (e.g. by applying hydrostatic pressure associated
with the volume of injection combined with a reduction in impedance
to flow associated with degradation of hyaluronan); (ii) providing
for a safer or more convenient route of administration (for example
allowing for non-IV parenteral administration in place of IV, or
for improved or more convenient non-IV parenteral administrations
such as intradermal or subcutaneous in place of intramuscular);
(iii) more favorable dosing regimens (for example less frequent
dosing, or a more frequent but lower dose non-IV route as opposed
to a less frequent but higher dose IV); (iv) reducing a side effect
or otherwise decreasing toxicity (for example an effect associated
with the initially high concentration (high Cmax) generated by IV
dosing, or from a high local dose generated by slow and/or
incomplete absorption following non-IV parenteral administration);
(v) otherwise improving the pharmacokinetics and/or
pharmacodynamics (for example by allowing more of an agent to reach
a target site (e.g. by increased interstitial fluid flow and
convective transport due to the presence of sHASEGP at or near a
delivery site such as an injection site), and/or by enhancing the
permeation within a target site (e.g. by increased interstitial
fluid flow and convective transport due to the presence of sHASEGP
within the target site), or by delivering an agent preferentially
to a target site (e.g. by local administration to a target site in
connection with local or systemic administration of a sHASEGP to
promote permeation of the target, or by using vehicles or
macromolecular complexes to preferentially target an agent to a
particular site combined with local or systemic administration of
sHASEGP to promote permeation of the target site); Agents that have
been delivered orally can also be benefited by reformulations for
parenteral delivery to, e.g., avoid loss of agent to the
gastrointestinal tract or subsequent degradation or toxicity, allow
for more effective targeting to a desired site of action and
therefore potentially greater effectiveness and/or less toxicity,
or otherwise provide for more favorable pharmacokinetic and/or
pharmacodynamic profiles. In addition, sHASEGPs can be used to
improve the pharmacodynamics of agents that are administered orally
(for example, by enhancing the permeation of an orally administered
agent within its desired target site (e.g. by increased
interstitial fluid flow and convective transport due to the
presence of sHASEGP within the target site following systemic
administration of sHASEGP and/or following local administration of
sHASEGP to the target site).
[0447] Even in situations in which the desired target site for an
agent such as pharmacologic is the bloodstream itself, such agents
can be enhanced by co-administration and/or co-formulation with
sHASEGPs (or other glycosaminoglycanases). For example, there are a
number of agents that act as blood modifiers, which may be
administered by one of several routes including oral dosing as well
as parenteral administration (such as IV injection). A number of
agents that are or could be administered orally nevertheless
exhibit relatively limited or slow uptake and/or are subject to
undesirable degradation (and there are other agents that are
generally safe and effective once introduced into the bloodstream
but are not administered orally for that reason). Parenteral
administration of such agents (e.g. by intramuscular, subcutaneous,
intradermal or transdermal routes in combination with
administration of sHASEGP, preferably at or near the site of
parenteral administration) can thus be employed to provide for
introduction into the bloodstream. In addition, there are a number
of urgent medical situations in which rapid delivery of an agent to
the bloodstream is essential but oral dosing is too slow and
intravenous dosing is either inconvenient, unsafe or unavailable.
These urgent situations include those in which the blood is the
target as well as those in which the bloodstream is simply the
route of delivery to another target tissue. In such situations, the
ability to combine administration with a sHASEGP (or apply a
ready-prepared co-formulation with a sHASEGP) can allow for rapid
delivery of the agent via non-IV parenteral administration. As will
also be appreciated by those of skill in the art, there are a
number of formulations and devices designed for non-IV parenteral
administrations that can provide for, e.g., timed or other
triggered release of agents, as well as variably controlled dosing
and the like. A large number of such pharmacologic agents (that can
be co-formulated with or used in combination with a sHASEGP) are
regularly applied in medical practice and aspects of their use well
described (see, e.g., the Physicians' Desk Reference, Thomson 2003,
2004, 2005), and many other such agents are known in the art. In
the latter regard, as will be appreciated by those of skill in the
art, sHASEGPs can be employed in combination with agents currently
used (to e.g. improve their applicability as described herein),
with agents that are not currently used (to e.g. improve their
pharacokinetics and/or pharmacodynamics and thus make them useful),
or with agents that are newly developed.
[0448] Thus for pharmacologic agents (such as anticancer agents,
blood modifers or members of other classes of agents as described
herein) that are typically introduced parenterally (e.g. by IV
injection), sHASEGPs (or other glycosaminoglycanases) can be used
for example to allow such agents to be administered by other
potentially more convenient and/or safe routes such as by non-IV
parenteral adminstration (e.g. by intramuscular, subcutaneous,
intradermal or transdermal routes). For agents that are already
deliverable by a non-IV parenteral route, sHASEGPs may be used to
enhance its extent and/or rate of uptake and delivery (e.g. by
promoting its dispersion and subsequent entry into the bloodstream,
which can likely be aided by convective transport as described
herein). Advantages of such effects include having a greater
portion of the agent reach the bloodstream, having reduced local
exposure to agent (in terms of concentration and/or time) (e.g. at
or near the injection site, which often limits dosing or results in
local toxicity issues), increasing the speed at which an agent can
be delivered into the bloodstream, other improved pharmacokinetic
and/or pharmadynamic properties, and convenience of administration.
In the last regard, non-IV parenteral administrations can allow
many agents to be administered out-of-hospital that would otherwise
require hospitalization or other medical attention typically
required for intravenous administrations.
[0449] Illustrative examples of blood modifiers include agents such
as antihemophilic agents and blood factor deficiency agents
(including, for example, antihemophilic factors (e.g. ADVATE.TM.,
HEMOFIL.TM., KOATE.TM.-DVI, KOGENATE.TM.-FS, RECOMBINATE.TM. and
REFACTO.TM.), anti-inhibitor coagulent complexes (e.g. FEIBA.TM.
VH), antithrombin IIIs (e.g. THROMBATE.TM. III), coagulations
Factor VIIs (e.g. NOVOSEVEN.TM.), coagulation Factor VIIIs (e.g.
ADVATE.TM., KOGENATE.TM.-FS and RECOMBINATE.TM.), coagulation
Factor IXs (e.g. BEBULIN.TM., BENEFIX.TM. and PROPLEX.TM. T),
plasma protein fractions (e.g. PLASMANATE.TM.), von Willebrand
factors)); antiplatelet agents (including, for example, abciximabs
(e.g. REOPRO.TM.), anagrelides (e.g. AGRYLIN.TM.), cilostazols
(e.g. PLETAL.TM.), clopidogrel bisulfates (e.g. PLAVIX.TM.),
dipyridamoles (e.g. AGGRENOX.TM. and PERSANTINE.TM.), epoprostenols
(e.g. FLOLAN.TM.), eptifibatides (e.g. INTEGRILIN.TM.), tirofibans
(e.g. AGGRASTAT.TM.)); colony stimulating factors (CSFs)
(including, for example, Granulocyte CSFs (e.g. NEULASTA.TM. and
NEUPOGEN.TM.) and Granulocyte Macrophage CSFs (e.g. LEUKINE.TM.));
erythropoiesis stimulators (including, for example, erythropoietins
such as darbepoetin alfas (e.g. ARANESP.TM.) and epoetin alfas
(e.g. EPOGEN.TM. and PROCRIT.TM.); hemostatics and albumins
(including, for example, aprotinins (e.g. TRASYLOL.TM.),
combinations of antihemophilic factors and plasma (e.g.
ADVATE.TM.), Desmopressin Acetates (e.g. DDAVP.TM.), and albumins
(e.g. BUMINATET.TM. and PLASBUMIN.TM.)); immune globulins (e.g.
BAYGAM.TM., CARIMUNE.TM. NF IV, FLEBOGAMMA.TM., GAMIMUNE.TM.,
GAMMAGARD S/D.TM., GAMUNEX.TM., IVEEGAM.TM., IVEEGAM.TM. EN IGIV,
MICRHOGAM.TM., RHOGAM.TM., PANGLOBULIN.TM. and THYMOGLOBULIN.TM.,
as well as hepatitis B immune globulins (e.g. BAYHEP.TM. B,
NABI-HB.TM., and NOVAPLUS.TM. NABI-HB); thrombin inhibitors
(including for example direct thrombin inhibitors (e.g.
ARGATROBAN.TM.) and lepirudin (e.g. REFLUDAN.TM.), and drotecogin
alfas (e.g. XIGRIS.TM.); anticoagulants (including, for example,
dalteparins, enoxaperins and other heparins (e.g. FRAGMIN.TM. and
LOVENOX.TM.), warfarins (e.g. COUMADIN.TM. and JANTOVEN.TM.)); and
the like.
[0450] By way of further illustration, examples of various types of
agents that have been administered paraneterally and which can be
co-administered and/or co-formulated with sHASEGPs (or other
glycosaminoglycanases) are included in the following list:
Acetazolamides; Acyclovirs; Adipiodones; Alatrofloxacins;
Aldesleukins; Alemtuzumabs; Alfentanils; Allergenic extracts;
Allopurinols; Alpha 1-proteinase inhibitors; Alprostadils;
Amifostines; Amikacins; Aminoacids; Aminocaproic acids;
Aminophyllines; Amitriptylines; Amobarbitals; Amrinones; Anakinras;
Analgesics; Anti-poliomyelitic vaccines; Anti-rabic serums;
Anti-tetanus immunoglobulins; Antithrombin IIIs; Antivenom serums;
Argatrobans; Arginines; Arsenics; Ascorbic acids; Asparaginases;
Atenolols; Atracuriums; Atropines; Aurothioglucoses; Azathioprines;
Azithromycins; Aztreonams; Bacillus Calmette-Guerin (BCG) vaccines;
Bacitracins; Baclofens; Basiliximabs; Benzoic acids; Benztropines;
Betamethasones; Biotins; Bivalirudins; Bleomycins; Botulism
antitoxins; Bretyliums; Bumetanides; Bupivacaines; Buprenorphines;
Busulfans; Butorphanols; Calcitonins; Calcitriols; Calciums;
Capreomycins; Carboprosts; Carmustines; Carnitines; Caspofungins;
Cefamandoles; Cefazolins; Cefoperazones; Cefotaximes; Cefotetans;
Cefoxitins; Ceftazidimes; Ceftizoximes; Cefuroximes;
Chloramphenicols; Chloroprocaines; Chloroquines; Chlorothiazides;
Chlorpromazines; Chondroitinsulfuric acids; Choriogonadotropin
alfas; Chromiums; Cidofovirs; Cimetidines; Ciprofloxacins;
Cisatracuriums; Cisplatins; Cladribines; Clavulanic acids;
Clindamycins; Clonidines; Codeines; Colchicines; Colistins;
Collagens; Corticorelin ovine triflutates; Corticotrophins;
Cosyntropins; Cyanocobalamins; Cyclophosphamides; Cyclosporines;
Cysteines; Cytarabines; Dacarbazines; Dacliximabs; Dactinomycins;
Dalfopristins; Dalteparins; Danaparoids; Dantrolenes;
Daunorubicins; Deferoxamines; Denileukin diftitoxs; Desmopressins;
Dexamethasones; Dexmedetomidines; Dexpanthenols; Dexrazoxanes;
Dextrans; Diatrizoic acids; Diazepams; Diazoxides; Dicyclomines;
Digibinds; Digoxins; Dihydroergotamines; Diltiazems;
Diphenhydramines; Diphteria, tetanus and pertussis vaccines;
Diptheria antitoxins; Dipyridamoles; Dobutamines; Dopamines;
Doxacuriums; Doxaprams; Doxercalciferols; Doxycyclines;
Droperidols; Drotrecogin alfas; Dyphyllines; Edetic acids;
Edrophoniums; Enalaprilats; Ephedrines; Epoprostenols;
Ergocalciferols; Ergonovines; Ertapenems; Erythromycins; Esmolols;
Estradiols; Estrogenics; Ethacrynic acids; Ethanolamines; Ethanols;
Ethiodized oils; Etidronic acids; Etomidates; Etoposides; Factor
VIIIs; Famotidines; Fenoldopams; Fentanyls; Floxuridines;
Fludarabines; Flumazenils; Fluoresceins; Fluorouracils;
Fluphenazines; Folic acids; Follicle-stimulating hormones;
Fomepizoles; Fomivirsens; Fondaparinuxs; Foscarnets; Fosphenytoins;
Fulvestrants; Furosemides; Gadoteridols; Gadoversetamides;
Ganciclovirs; Gatifloxacins; Gemtuzumab ozogamicins; Gentamicins;
Glucagons; Glucoses; Glycines; Glycopyrrolates; Gonadorelins;
Gonadotropin chorionics; Haemophilus B polysaccarides;
Haloperidols; Hemins; Hemophilus influenza vaccines; Hepatitis
vaccines; Herbals; Histamines; Hydralazines; Hydrocortisones;
Hydromorphones; Hydroxocobalamins; Hydroxyzines; Hyoscyamines;
Ibutilides; Idarubicins; Ifosfamides; Imiglucerases;
Immunoglobulins; Indigo carmines; Indomethacins; Interferon
alfa-con1s; Interferon alfa-2As; Interferon alfa-N3s; Interferon
beta-1As; Iodides; Iopromides; Iothalamic acids; Ioxaglic acids;
Ioxilans; Iron dextran injections; Isoniazids; Isoproterenols;
Japanese encephalitis vaccines; Kanamycins; Ketamines; Ketorolacs;
Labetalols; Lepirudins; Leucovorins; Leuprolides; Levobupivacaines;
Levothyroxines; Lidocaines; Lincomycins; Linezolids; Liothyronines;
Lorazepams; Luteinising hormones; Lyme disease vaccines;
Mangafodipirs; Mannitols; Measles virus; Mechlorethamines;
Melphalans; Meningococcal polysaccharide vaccines; Meperidines;
Mepivacaines; Meropenems; Mesnas; Mesoridazines; Metaraminols;
Methadones; Methocarbamols; Methohexitals; Methotrexates;
Methyldopates; Methylergonovines; Metoclopramides; Metoprolols;
Metronidazoles; Midazolams; Minocyclines; Mithramycins; Mitomycins;
Mitoxantrones; Mivacuriums; Morrhuic acids; Moxifloxacins; Mumps
viruses; Muromonab-CD3s; Mycophenolate mofetils; Nafcillins;
Nalbuphines; Nalmefenes; Naloxones; Nandrolones; Neostigmines;
Niacinamides; Nicardipines; Nitroglycerins; Nitroprussides;
Norepinephrines; Oprelvekins; Orphenadrines; Oxacillins;
Oxaliplatins; Oxymorphones; Oxytetracyclines; Oxytocins;
Pancuroniums; Panthenols; Pantothenic acids; Papaverines;
Pegaspargases; Peginterferon alfa 2As; Penicillin Gs; Pentamidines;
Pentazocines; Pentobarbitals; Pentostatins; Perflutrens;
Perphenazines; Pertussis vaccines; Phenobarbitals; Phentolamines;
Phenylephrines; Phenytoins; Physostigmines; Phytonadiones;
Pneumococcal vaccines; Polymyxin bs; Porfimers; Pralidoximes;
Prilocaines; Procainamides; Procaines; Prochlorperazines;
Progesterones; Promethazines; Propranolols; Pyridostigmine
hydroxides; Pyridoxines; Quinidines; Quinupristins; Rabies
immunoglobulins; Rabies vaccines; Ranitidines; Rasburicases;
Remifentanils; Riboflavins; Rifampins; Ropivacaines; Rubella
vaccines; Samariums; Scopolamines; Seleniums; Sermorelins;
Sincalides; Somatrems; Spectinomycins; Streptokinases;
Streptomycins; Streptozocins; Succinylcholines; Sufentanils;
Sulbactams; Sulfamethoxazoles; Sumatriptans; Tacrolimuss;
Teniposides; Terbutalines; Teriparatides; Testosterones; Tetanus
antitoxins; Tetracaines; Tetradecyl sulfates; Theophyllines;
Thiamines; Thiethylperazines; Thiopentals; Thyroid stimulating
hormones; Ticarcillins; Tinzaparins; Tirofibans; Tobramycins;
Tolazolines; Tolbutamides; Topotecans; Torsemides; Tranexamic
acids; Treprostinils; Triamcinolone acetonides; Triamcinolone
hexacetonides; Triamcinolones; Triethylenethiophosphoramides
(Thiotepa); Trifluoperazines; Trimethobenzamides; Trimethoprims;
Trimetrexates; Triptorelins; Tromethamines; Tuberculins; Typhoid
vaccines; Urofollitropins; Urokinases; Valproic acids; Valrubicins;
Varicella Zoster immunoglobulins; Vasopressins; Vecuroniums;
Verapamils; Vinblastines; Vincristines; Voriconazoles; Warfarins;
Yellow fever vaccines; Zidovudines; Zincs; Ziprasidone
hydrochlorides; and agents related to the foregoing or in the same
or similar classes as the foregoing.
[0451] Solutions or suspensions used for parenteral administration
(including by intramuscular, intradermal, subcutaneous, topical
application and other non-oral routes) can include any of the
following components: a sterile diluent, such as water for
injection, saline solution, fixed oil, polyethylene glycol,
glycerine, propylene glycol or other synthetic solvent;
antimicrobial agents, such as benzyl alcohol and methyl parabens;
antioxidants, such as ascorbic acid and sodium bisulfite; cheating
agents, such as ethylenediaminetetraacetic acid (EDTA); buffers,
such as acetates, citrates and phosphates; and agents for the
adjustment of tonicity, including, but not limited to sodium
chloride, calcium chloride, magnesium chloride, dextrose, glycerol
or boric acid. Parenteral preparations can be enclosed in ampoules,
disposable syringes or single or multiple dose vials made of glass,
plastic or other suitable material.
[0452] The sHASEGP polypeptides or soluble human hyaluronidase
domains thereof can be suspended in micronized or other suitable
form or can be derivatized to produce a more soluble active product
or to produce a prodrug. The form of the resulting mixture depends
upon a number of factors, including the intended mode of
administration and the solubility of the polypeptide in the
selected carrier or vehicle. The effective concentration is
sufficient for ameliorating the targeted condition and can be
empirically determined using methods known to those of skill in the
art. To formulate a composition, the weight fraction of polypeptide
is dissolved, suspended, dispersed, or otherwise mixed in a
selected vehicle at an effective concentration such that the
targeted condition is relieved or ameliorated.
[0453] In instances in which the sHASEGP polypeptides or soluble
human hyaluronidase domain thereof exhibit insufficient solubility,
methods for solubilizing polypeptides can be used. Such methods are
known to those of skill in this art, and include, but are not
limited to, using cosolvents, such as dimethylsulfoxide (DMSO),
using surfactants, such as TWEEN.TM. and Pluronic.TM. F68; or
dissolution in aqueous sodium bicarbonate. Derivatives of the
polypeptides, such as prodrugs of the polypeptides can also be used
in formulating effective pharmaceutical compositions. For
ophthalmic indications, the compositions are formulated in an
ophthalmically acceptable carrier. For the ophthalmic uses herein,
local administration, either by topical administration or by
injection is contemplated. Time-release formulations are also
desirable. Typically, the compositions are formulated for single
dosage administration, so that a single dose administers an
effective amount.
[0454] Upon mixing or addition of the polypeptide with the vehicle,
the resulting mixture can be a solution, suspension, emulsion or
other composition and can be formulated as an aqueous mixtures, a
creams, gels, ointments, emulsions, solutions, elixirs, lotions,
suspensions, tinctures, pastes, foams, aerosols, irrigations,
sprays, suppositories, bandages, or any other formulation suitable
for systemic, topical or local administration.
[0455] The form of the resulting mixture depends upon a number of
factors, including the intended mode of administration and the
solubility of the compound in the selected carrier or vehicle. If
necessary, pharmaceutically acceptable salts or other derivatives
of the compounds are prepared. For local internal administration,
such as, intramuscular, parenteral or intra-articular
administration, the compounds are preferably formulated as a
solution suspension in an aqueous-based medium, such as
isotonically buffered saline or are combined with a biocompatible
support or bioadhesive intended for internal administration.
[0456] The sHASEGP polypeptide or soluble human hyaluronidase
domain thereof is included in the pharmaceutically acceptable
carrier in an amount sufficient to exert a therapeutically useful
effect in the absence of undesirable side effects on the patient
treated. It is understood that number and degree of side effects
depends upon the condition for which the compounds are
administered. For example, certain toxic and undesirable side
effects are tolerated when treating life-threatening illnesses that
would not be tolerated when treating disorders of lesser
consequence. Amounts effective for therapeutic use will, of course,
depend on the severity of the disease and the weight and general
state of the subject as well as the route of administration. Local
administration of the therapeutic agent will typically require a
smaller dosage than any mode of systemic administration, although
the local concentration of the therapeutic agent can, in some
cases, be higher following local administration than can be
achieved with safety upon systemic administration.
[0457] Since individual subjects can present a wide variation in
severity of symptoms and each therapeutic agent has its unique
therapeutic characteristics, it is up to the practitioner to
determine the response of a subject to treatment and vary the
dosages accordingly. Dosages used in vitro can provide useful
guidance in the amounts useful for in situ administration of the
pharmaceutical composition, and animal models can in some cases be
used to determine effective dosages for treatment of particular
disorders. In general, however, for local administration, it is
contemplated that an effective amount of the therapeutic agent will
be an amount within the range from about 0.1 picograms (pg) up to
about 1 ng per kg body weight. Various considerations in arriving
at an effective amount are known to those of skill in the art and
are described (see, e.g., Goodman And Gilman's: The Pharmacological
Bases of Therapeutics, 8th ed., Pergamon Press, 1990; Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton,
Pa., 1990; and Mantyh et al., (Science, 278: 275-79, 1997)
involving the intrathecal injection of a neuronal specific
ligand-toxin, each of which is herein incorporated by reference in
its entirety).
[0458] The formulations of the sHASEGP polypeptides or soluble
human hyaluronidase domains thereof for use herein include those
suitable for oral, rectal, topical, inhalational, buccal,
sublingual, other parenteral (e.g., subcutaneous, intramuscular,
intradermal, transdermal or intravenous), administration or any
route. The most suitable route in any given case depends on the
nature and severity of the condition being treated and on the
nature of the particular active compound that is being used. The
formulations are provided for administration to humans and animals
in unit dosage forms, such as tablets, capsules, pills, powders,
granules, sterile parenteral solutions or suspensions, and oral
solutions or suspensions, and oil-water emulsions containing
suitable quantities of the polypeptides and/or other agents or
pharmaceutically acceptable derivatives thereof. The pharmaceutical
therapeutically active polypeptides and/or other agents and
derivatives thereof are typically formulated and administered in
unit-dosage forms or multiple-dosage forms. A unit-dose form as
used herein refers to physically discrete units suitable for human
and animal subjects and packaged individually as is known in the
art.
[0459] The pharmaceutical compositions are provided for
administration to humans and animals in unit dosage forms, such as
tablets, capsules, pills, powders, granules, sterile parenteral
solutions or suspensions, and oral solutions or suspensions, and
oil-water emulsions containing suitable quantities of the sHASEGP
polypeptide or soluble human hyaluronidase domain thereof and,
optionally, another agent or pharmaceutically acceptable
derivatives thereof. The pharmaceutically therapeutically active
compounds and derivatives thereof are typically formulated and
administered in unit-dosage forms or multiple-dosage forms. A
unit-dose form as used herein refers to physically discrete units
suitable for human and animal subjects and packaged individually as
is known in the art. Each unit-dose contains a predetermined
quantity of the therapeutically active compound sufficient to
produce the desired therapeutic effect, in association with the
required pharmaceutical carrier, vehicle or diluent. Examples of
unit-dose forms include, but are not limited to, ampoules, syringes
and individually packaged tablets or capsules. For example, a small
volume formulation containing a stabilized solution with 1 to 5000
Units of sHASEGP in a small volume, such as 5 to 50 .mu.l, can be
prepackaged in a syringe for use, such as after viscoelastic
injection. Unit-dose forms can be administered in fractions or
multiples thereof. A multiple-dose form is a plurality of identical
unit-dosage forms packaged in a single container to be administered
in segregated unit-dose form. Examples of multiple-dose forms
include vials, bottles of tablets or capsules or bottles of pints
or gallons. Hence, multiple dose form is a multiple of unit-doses
that are not segregated in packaging.
[0460] The composition can contain along with the active
ingredient, such as a sHASEGP polypeptide: a diluent, such as
lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a
lubricant, such as magnesium stearate, calcium stearate and talc;
and a binder such as starch, natural gums, such as gum
acaciagelatin, glucose, molasses, polvinylpyrrolidine, celluloses
and derivatives thereof, povidone, crospovidones and other such
binders known to those of skill in the art. Liquid pharmaceutically
administrable compositions can, for example, be prepared by
dissolving, dispersing, or otherwise mixing an active compound as
defined above and optional pharmaceutical adjuvants in a carrier,
such as, for example, water, saline, aqueous dextrose, glycerol,
glycols, ethanol, and the like, to thereby form a solution or
suspension. If desired, the pharmaceutical composition to be
administered can also contain minor amounts of nontoxic auxiliary
substances such as wetting agents, emulsifying agents, or
solubilizing agents, pH buffering agents and the like, for example,
acetate, sodium citrate, cyclodextrine derivatives, sorbitan
monolaurate, triethanolamine sodium acetate, triethanolamine
oleate, and other such agents. Methods of preparing such dosage
forms are known, or will be apparent, to those skilled in this art
(see e.g., Remington's Pharmaceutical Sciences, Mack Publishing
Company, Easton, Pa., 15th Edition, 1975). The composition or
formulation to be administered contains a quantity of the active
compound in an amount sufficient to alleviate the symptoms of the
treated subject. For example, a standard stabilized formulation of
sHASEGP or a soluble human hyaluronidase domain thereof as provided
herein includes 150 Units/ml of the soluble glycoprotein formulated
in EDTA, NaCl and CaCl.sub.2. Additionally, an anti-bacterial or
anti-fungal agent, including, but not limited to thiomersal, can be
present in the formulation. Another formulation provided herein is
a stabilized solution or lyophilized form of sHASEGP or a soluble
human hyaluronidase domain thereof in EDTA, NaCl and CaCl.sub.2,
containing an effective active amount of the soluble glycoprotein,
such as 150 Unit/ml, with the addition of lactose, such as 13
mg/ml. Also provided herein is a formulation containing a
stabilized solution or lyophilized form of sHASEGP or a soluble
human hyaluronidase domain thereof in EDTA, NaCl and CaCl.sub.2
containing an effective active amount of the soluble glycoprotein,
such as 150 Unit/ml, with the addition of lactose, such as 13
mg/ml, and Albumin, Pluronic.TM. F68, TWEEN.TM. and/or other
detergent. Another formulation provided herein, either lyophilized
or as a stabilized solution, contains an effect amount of sHASEGP
or a soluble human hyaluronidase domain thereof, such as 1 to 300
Units/ml, in EDTA, NaCl and CaCl.sub.2.
[0461] Dosage forms or compositions containing active ingredient in
the range of 0.005% to 100% with the balance made up from non-toxic
carrier can be prepared. For oral administration, the
pharmaceutical compositions can take the form of, for example,
tablets or capsules prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinized maize starch, polyvinyl pyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods well known in the art.
[0462] The sHASEGPs or a soluble human hyaluronidase domain thereof
or pharmaceutically acceptable derivatives can be prepared with
carriers that protect the soluble glycoprotein against rapid
elimination from the body, such as time release formulations or
coatings. The compositions can include other pharmaceutically
effective agents known in the general art to be of value in
treating one or more of the diseases or medical conditions,
including, but not limited to, a chemotherapeutic agent, an
analgesic agent, an anti-inflammatory agent, an antimicrobial
agent, an amoebicidal agent, a trichomonocidal agent, an
anti-parkinson agent, an anti-malarial agent, an anticonvulsant
agent, an anti-depressant agent, and antiarthritics agent, an
anti-fungal agent, an antihypertensive agent, antipyretic agent, an
anti-parasite agent, an antihistamine agent, an alpha-adrenargic
agonist agent, an alpha blocker agent, an anesthetic agent, a
bronchi dilator agent, a biocide agent, a bactericide agent, a
bacteriostat agent, a betadrenergic blocker agent, a calcium
channel blocker agent, a cardiovascular drug agent, a contraceptive
agent, a decongestant agent, a diuretic agent, a depressant agent,
a diagnostic agent, a electrolyte agent, a hypnotic agent, a
hormone agent, a hyperglycemic agent, a muscle relaxant agent, a
muscle contractant agent, an ophthalmic agent, a
parasympathomimetic agent, a psychic energizer agent, ophthalmic
agent, a parasympathomimetic agent, a psychic energizer agent, a
sedative agent, a sympathomimetic agent, a tranquilizer agent, an
urinary agent, a vaginal agent, a viricide agent, a vitamin agent,
a non-steroidal anti-inflammatory agent, an angiotensin converting
enzyme inhibitor agent, a polypeptide, a protein, a nucleic acid, a
drug, a prodrug, a organic molecule and a sleep inducer, to obtain
desired combinations of properties. It is to be understood that
such combination therapy constitutes a further aspect of the
compositions and methods of treatment provided herein.
[0463] 1. Compositions for Oral Administration
[0464] Oral pharmaceutical dosage forms are solid, gel or liquid.
The solid dosage forms are tablets, capsules, granules, and bulk
powders. Types of oral tablets include compressed, chewable
lozenges and tablets, which can be enteric-coated, sugarcoated or
film-coated. Capsules can be hard or soft gelatin capsules, while
granules and powders can be provided in non-effervescent or
effervescent form with the combination of other ingredients known
to those skilled in the art.
[0465] The pharmaceutical compositions containing a sHASEGP or a
soluble human hyaluronidase domain thereof can be in liquid form,
for example, solutions, syrups or suspensions, or can be presented
as a drug product for reconstitution with water or other suitable
vehicle before use. Such liquid preparations can be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid).
[0466] In certain embodiments, the formulations are solid dosage
forms, preferably capsules or tablets. The tablets, pills,
capsules, troches and the like can contain any of the following
ingredients, or compounds of a similar nature: a binder; a diluent;
a disintegrating agent; a lubricant; a glidant; a sweetening agent;
and a flavoring agent.
[0467] Examples of binders include microcrystalline cellulose, gum
tragacanth, glucose solution, acacia mucilage, gelatin solution,
sucrose and starch paste. Lubricants include talc, starch,
magnesium or calcium stearate, lycopodium and stearic acid.
Diluents include, for example, lactose, sucrose, starch, kaolin,
salt, mannitol and dicalcium phosphate. Glidants include, but are
not limited to, colloidal silicon dioxide. Disintegrating agents
include crosscarmellose sodium, sodium starch glycolate, alginic
acid, corn starch, potato starch, bentonite, methylcellulose, agar
and carboxymethylcellulose. Coloring agents include, for example,
any of the approved certified water soluble FD and C dyes, mixtures
thereof; and water insoluble FD and C dyes suspended on alumina
hydrate. Sweetening agents include sucrose, lactose, mannitol and
artificial sweetening agents such as saccharin, and any number of
spray dried flavors. Flavoring agents include natural flavors
extracted from plants such as fruits and synthetic blends of
compounds which produce a pleasant sensation, such as, but not
limited to peppermint and methyl salicylate. Wetting agents include
propylene glycol monostearate, sorbitan monooleate, diethylene
glycol monolaurate and polyoxyethylene laural ether.
Emetic-coatings include fatty acids, fats, waxes, shellac,
ammoniated shellac and cellulose acetate phthalates. Film coatings
include hydroxyethylcellulose, sodium carboxymethylcellulose,
polyethylene glycol 4000 and cellulose acetate phthalate.
[0468] If oral administration is desired, the sHASEGP or a soluble
human hyaluronidase domain thereof could be provided in a
composition that protects it from the acidic environment of the
stomach. For example, the composition can be formulated in an
enteric coating that maintains its integrity in the stomach and
releases the active compound in the intestine. The composition can
also be formulated in combination with an antacid or other such
ingredient.
[0469] When the dosage unit form is a capsule, it can contain, in
addition to material of the above type, a liquid carrier such as a
fatty oil. In addition, dosage unit forms can contain various other
materials which modify the physical form of the dosage unit, for
example, coatings of sugar and other enteric agents. The compounds
can also be administered as a component of an elixir, suspension,
syrup, wafer, sprinkle, chewing gum or the like. A syrup can
contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
[0470] The sHASEGP or a soluble human hyaluronidase domain thereof
can also be mixed with other active materials which do not impair
the desired action, or with materials that supplement the desired
action, such as antacids, H2 blockers, and diuretics. The active
ingredient is a compound or pharmaceutically acceptable derivative
thereof as described herein. Higher concentrations, up to about 98%
by weight of the active ingredient can be included.
[0471] Pharmaceutically acceptable carriers included in tablets are
binders, lubricants, diluents, disintegrating agents, coloring
agents, flavoring agents, and wetting agents. Enteric-coated
tablets, because of the enteric-coating, resist the action of
stomach acid and dissolve or disintegrate in the neutral or
alkaline intestines. Sugar-coated tablets are compressed tablets to
which different layers of pharmaceutically acceptable substances
are applied. Film-coated tablets are compressed tablets which have
been coated with a polymer or other suitable coating. Multiple
compressed tablets are compressed tablets made by more than one
compression cycle utilizing the pharmaceutically acceptable
substances previously mentioned. Coloring agents can also be used
in the above dosage forms. Flavoring and sweetening agents are used
in compressed tablets, sugar-coated, multiple compressed and
chewable tablets. Flavoring and sweetening agents are especially
useful in the formation of chewable tablets and lozenges.
[0472] Liquid oral dosage forms include aqueous solutions,
emulsions, suspensions, solutions and/or suspensions reconstituted
from non-effervescent granules and effervescent preparations
reconstituted from effervescent granules. Aqueous solutions
include, for example, elixirs and syrups. Emulsions are either
oil-in-water or water-in-oil.
[0473] Elixirs are clear, sweetened, hydroalcoholic preparations.
Pharmaceutically acceptable carriers used in elixirs include
solvents. Syrups are concentrated aqueous solutions of a sugar, for
example, sucrose, and can contain a preservative. An emulsion is a
two-phase system in which one liquid is dispersed in the form of
small globules throughout another liquid. Pharmaceutically
acceptable carriers used in emulsions are non-aqueous liquids,
emulsifying agents and preservatives. Suspensions use
pharmaceutically acceptable suspending agents and preservatives.
Pharmaceutically acceptable substances used in non-effervescent
granules, to be reconstituted into a liquid oral dosage form,
include diluents, sweeteners and wetting agents. Pharmaceutically
acceptable substances used in effervescent granules, to be
reconstituted into a liquid oral dosage form, include organic acids
and a source of carbon dioxide. Coloring and flavoring agents are
used in all of the above dosage forms.
[0474] Solvents include glycerin, sorbitol, ethyl alcohol and
syrup. Examples of preservatives include glycerin, methyl and
propylparaben, benzoic add, sodium benzoate and alcohol. Examples
of non-aqueous liquids utilized in emulsions include mineral oil
and cottonseed oil. Examples of emulsifying agents include gelatin,
acacia, tragacanth, bentonite, and surfactants such as
polyoxyethylene sorbitan monooleate. Suspending agents include
sodium carboxymethylcellulose, pectin, tragacanth, Veegum and
acacia. Diluents include lactose and sucrose. Sweetening agents
include sucrose, syrups, glycerin and artificial sweetening agents
such as saccharin. Wetting agents include propylene glycol
monostearate, sorbitan monooleate, diethylene glycol monolaurate
and polyoxyethylene lauryl ether. Organic additives include citric
and tartaric acid. Sources of carbon dioxide include sodium
bicarbonate and sodium carbonate. Coloring agents include any of
the approved certified water soluble FD and C dyes, and mixtures
thereof. Flavoring agents include natural flavors extracted from
plants such fruits, and synthetic blends of compounds that produce
a pleasant taste sensation.
[0475] For a solid dosage form, the solution or suspension, in for
example propylene carbonate, vegetable oils or triglycerides, is
encapsulated in a gelatin capsule. Such solutions, and the
preparation and encapsulation thereof, are disclosed in U.S. Pat.
Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form,
the solution, e.g., for example, in a polyethylene glycol, can be
diluted with a sufficient quantity of a pharmaceutically acceptable
liquid carrier, e.g., water, to be easily measured for
administration.
[0476] Alternatively, liquid or semi-solid oral formulations can be
prepared by dissolving or dispersing the sHASEGP or a soluble human
hyaluronidase domain thereof in vegetable oils, glycols,
triglycerides, propylene glycol esters (e.g., propylene carbonate)
and other such carriers, and encapsulating these solutions or
suspensions in hard or soft gelatin capsule shells. Other useful
formulations include those set forth in U.S. Pat. Nos. Re 28,819
and 4,358,603.
[0477] Formulations suitable for buccal (sublingual) administration
include, for example, lozenges containing the sHASEGP or a soluble
human hyaluronidase domain thereof in a flavored base, usually
sucrose and acacia or tragacanth; and pastilles containing the
compound in an inert base such as gelatin and glycerin or sucrose
and acacia.
[0478] In all embodiments, tablets and capsules formulations can be
coated as known by those of skill in the art in order to modify or
sustain dissolution of the active ingredient. Thus, for example,
they can be coated with a conventional enterically digestible
coating, such as phenylsalicylate, waxes and cellulose acetate
phthalate.
[0479] 2. Injectables, Solutions and Emulsions
[0480] Parenteral administration of the sHASEGP or a soluble human
hyaluronidase domain thereof, generally characterized by injection,
either subcutaneously, intramuscularly or intravenously is also
contemplated herein. Injectables can be prepared in conventional
forms, either as liquid solutions or suspensions; solid forms
suitable for solution or suspension in liquid prior to injection,
or as emulsions. Suitable excipients are, for example, water,
saline, dextrose, glycerol or ethanol. In addition, if desired, the
pharmaceutical compositions to be administered can also contain
minor amounts of non-toxic auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, stabilizers, solubility
enhancers, and other such agents, such as, for example, sodium
acetate, sorbitan monolaurate, triethanolamine oleate and
cyclodextrins. Implantation of a slow-release or sustained-release
system, such that a constant level of dosage is maintained (see,
e.g., U.S. Pat. No. 3,710,795) is also contemplated herein. The
percentage of the sHASEGP or a soluble human hyaluronidase domain
thereof contained in such parenteral compositions is dependent on
the specific nature thereof, as well as the activity of the
compound and the needs of the subject.
[0481] Parenteral administration of the compositions includes
intravenous, subcutaneous and intramuscular administrations.
Preparations for parenteral administration include sterile
solutions ready for injection, sterile dry soluble products, such
as lyophilized powders, ready to be combined with a solvent or
sterile solution just prior to use, including hypodermic tablets,
sterile suspensions ready for injection, sterile dry insoluble
products ready to be combined with a vehicle just prior to use and
sterile emulsions. The solutions can be either aqueous or
nonaqueous.
[0482] If administered intravenously, suitable carriers include
physiological saline or phosphate buffered saline (PBS), and
solutions containing thickening and solubilizing agents, such as
glucose, polyethylene glycol, and polypropylene glycol and mixtures
thereof.
[0483] Pharmaceutically acceptable carriers used in parenteral
preparations include aqueous vehicles, nonaqueous vehicles,
antimicrobial agents, isotonic agents, buffers, antioxidants, local
anesthetics, suspending and dispersing agents, emulsifying agents,
sequestering or chelating agents and other pharmaceutically
acceptable substances.
[0484] Examples of aqueous vehicles include Sodium Chloride
Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile
Water Injection, Dextrose and Lactated Ringers Injection.
Nonaqueous parenteral vehicles include fixed oils of vegetable
origin, cottonseed oil, corn oil, sesame oil and peanut oil.
Antimicrobial agents in bacteriostatic or fungistatic
concentrations must be added to parenteral preparations packaged in
multiple-dose containers which include phenols or cresols,
mercurials, benzyl alcohol, chlorobutanol, methyl and propyl
p-hydroxybenzoic acid esters, thiomersal, benzalkonium chloride and
benzethonium chloride. Isotonic agents include sodium chloride and
dextrose. Buffers include phosphate and citrate. Antioxidants
include sodium bisulfate. Local anesthetics include procaine
hydrochloride. Suspending and dispersing agents include sodium
carboxymethylcelluose, hydroxypropyl methylcellulose and
polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80
(TWEEN.TM. 80). A sequestering or chelating agent of metal ions
includes EDTA. Pharmaceutical carriers also include ethyl alcohol,
polyethylene glycol and propylene glycol for water miscible
vehicles and sodium hydroxide, hydrochloric acid, citric acid or
lactic acid for pH adjustment.
[0485] The concentration of the pharmaceutically active compound is
adjusted so that an injection provides an effective amount to
produce the desired pharmacological effect. The exact dose depends
on the age, weight and condition of the patient or animal as is
known in the art.
[0486] The unit-dose parenteral preparations are packaged in an
ampoule, a vial or a syringe with a needle. All preparations for
parenteral administration must be sterile, as is known and
practiced in the art.
[0487] Illustratively, intravenous or intraarterial infusion of a
sterile aqueous solution containing an active compound is an
effective mode of administration. Another embodiment is a sterile
aqueous or oily solution or suspension containing an active
material injected as necessary to produce the desired
pharmacological effect.
[0488] Injectables are designed for local and systemic
administration. Typically a therapeutically effective dosage is
formulated to contain a concentration of at least about 0.1% w/w up
to about 90% w/w or more, preferably more than 1% w/w of the active
compound to the treated tissue(s). The active ingredient, such as a
sHASEGP or a soluble human hyaluronidase domain thereof, can be
administered at once, or can be divided into a number of smaller
doses to be administered at intervals of time. It is understood
that the precise dosage and duration of treatment is a function of
the tissue being treated and can be determined empirically using
known testing protocols or by extrapolation from in vivo or in
vitro test data. It is to be noted that concentrations and dosage
values can also vary with the age of the individual treated. It is
to be further understood that for any particular subject, specific
dosage regimens should be adjusted over time according to the
individual need and the professional judgment of the person
administering or supervising the administration of the
formulations, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed formulations.
[0489] The compounds provided herein can be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection can be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions can be suspensions,
solutions or emulsions in oily or aqueous vehicles, and can contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient can be in
powder form for reconstitution with a suitable vehicle, e.g.,
sterile pyrogen-free water or other solvents, before use. For
example, provided herein are parenteral formulations containing an
effective amount of sHASEGP or a soluble human hyaluronidase domain
thereof, such as 500 to 500,000 Units, in a stabilized solution or
a lyophilized from.
[0490] The compound can be suspended in micronized or other
suitable form or can be derivatized to produce a more soluble
active product or to produce a prodrug. The form of the resulting
mixture depends upon a number of factors, including the intended
mode of administration and the solubility of the compound in the
selected carrier or vehicle. The effective concentration is
sufficient for ameliorating the symptoms of the condition and can
be empirically determined.
[0491] 3. Lyophilized Powders
[0492] Also provided herein are lyophilized powders containing
sHASEGP or a soluble human hyaluronidase domain thereof, which can
be reconstituted for administration as solutions, emulsions and
other mixtures. These formulations can also be reconstituted and
formulated as solids or gels.
[0493] The sterile, lyophilized powder is prepared by dissolving a
solid portion of or mixing an aliquot of a solution containing a
sHASEGP or a soluble human hyaluronidase domain thereof in a
suitable solvent. The solvent can contain an excipient that
improves the stability or other pharmacological component of the
powder or reconstituted solution, prepared from the powder.
Excipients that can be used include, but are not limited to,
dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin,
glucose, sucrose, lactose or other suitable agent. The solvent can
also contain a buffer, such as citrate, sodium or potassium
phosphate or other such buffer known to those of skill in the art
at, typically, about neutral pH. Subsequent sterile filtration of
the solution followed by lyophilization under standard conditions
known to those of skill in the art provides the lyophilized
formulation. Generally, the solution resulting from the sterile
filtration is apportioned into vials for lyophilization. Each vial
can contain a single dosage, such as 10-1000 mg or 100-500 mg, or
multiple dosages of the compound.
[0494] Briefly, the lyophilized powder is prepared by dissolving
dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin,
glucose, sucrose, lactose or other suitable agent, about 1-20%, in
a suitable buffer, such as citrate, sodium or potassium phosphate
or other such buffer known to those of skill in the art at about
neutral pH. Then, a selected salt, such as, for example, the sodium
salt of the sHASEGP (about 1 gm of the salt per 10-100 gms of the
buffer solution, typically about 1 gm/30 gms), is added to the
resulting mixture above room temperature, such as at about
30-35.degree. C., and stirred until it dissolves. The resulting
mixture is diluted by adding more buffer, to decrease the resulting
concentration of the salt by about 10-50%, typically about 15-25%).
The resulting mixture is sterile filtered or treated to remove
particulates and to insure sterility, and apportioned into vials
for lyophilization. The lyophilized powder can be stored under
appropriate conditions, such as at about 4 C to room
temperature.
[0495] Reconstitution of this lyophilized powder with water for
injection provides a formulation for use in parenteral
administration. For reconstitution, a therapeutically effective
amount of the lyophilized powder containing a sHASEGP or a soluble
human hyaluronidase domain thereof is added per milliliter of
sterile water or other suitable carrier. The precise amount depends
upon the selected compound and can be empirically determined by
methods known to those of skill in the art.
[0496] 4. Topical administration
[0497] Topical mixtures are prepared as described for the local and
systemic administration. Topical administration, which is sometimes
referred to in the art as percutaneous administration, typically
involves applications designed for absorption across skin, mucous
membranes or other surfaces of the body (e.g., applications applied
directly onto the skin, under the tongue (sublingual), against the
cheek (buccal), to the eyes, nose or ears, or to the lungs
(inhalation)). The resulting mixture can be a solution, suspension,
emulsions or the like and are formulated as creams, gels,
ointments, emulsions, solutions, elixirs, lotions, suspensions,
tinctures, pastes, foams, aerosols, irrigations, sprays,
suppositories, bandages, dermal patches or any other formulations
suitable for topical administration.
[0498] The compositions of sHASEGP or a soluble human hyaluronidase
domain thereof or pharmaceutically acceptable derivatives thereof
can be formulated as aerosols for topical application, such as by
inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and
4,364,923, which describe aerosols for delivery of a steroid useful
for treatment inflammatory diseases, particularly asthma). These
formulations for administration to the respiratory tract can be in
the form of an aerosol or solution for a nebulizer, or as a
microfine powder for insufflation, alone or in combination with an
inert carrier such as lactose. In such a case, the particles of the
formulation will typically have diameters of less than 50 microns,
such as less than 10 microns.
[0499] For administration by inhalation, the compositions for use
herein can be delivered in the form of an aerosol spray
presentation from pressurized packs or a nebulizer, with the use of
a suitable propellant, including, but not limited to,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide and other suitable gases.
In the case of a pressurized aerosol, the dosage unit can be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges of, e.g., gelatin, for use in an inhaler or
insufflator can be formulated containing a powder mix of the
compound and a suitable powder base such as lactose or starch.
[0500] The compositions can be formulated for local or topical
application, such as for topical application to the skin and mucous
membranes, such as in the eye, in the form of gels, creams, and
lotions and for application to the eye or for intracisternal or
intraspinal application. Topical administration is contemplated for
transdermal delivery and also for administration to the eyes or
mucosa, or for inhalation therapies. Nasal solutions of the active
compound alone or in combination with other pharmaceutically
acceptable excipients can also be administered.
[0501] For example, formulations suitable for topical application
to the skin or to the eye generally are formulated as an ointment,
cream, lotion, paste, gel, spray, aerosol and oil. Carriers that
can be used include vaseline, lanoline, polyethylene glycols,
alcohols, and combinations of two or more thereof. The topical
formulations can further advantageously contain 0.05 to 15 percent
by weight of thickeners, including, but not limited to,
hydroxypropylmethylcellulose, methylcellulose,
polyvinylpyrrolidone, polyvinyl alcohol, poly (alkylene glycols),
poly/hydroxyalkyl, (meth) acrylates or poly (meth) acrylamides. A
topical formulation is often applied by instillation or as an
ointment into the conjunctival sac. It also can be used for
irrigation or lubrication of the eye, facial sinuses, and external
auditory meatus. The topical formulations in the liquid state can
be also present in a hydrophilic three-dimensional polymer matrix
in the form of a strip, contact lens, and the like from which the
active components are released. It can also be injected into the
anterior eye chamber, into the episcleral space, and other places.
For example, provided herein is a formulation for intraocular use
after viscoelastic injecting containing a stabilized solution of an
effective amount of a sHASEGP or a soluble human hyaluronidase
domain thereof, such as 1 to 5000 Units of the soluble glycoprotein
with 30 to 150,000 Units/mg of specific activity, in a small
volume, such as 5 to 50 .mu.l. As another example, provided herein
is a formulation for intraocular use to enhance delivery of one or
more pharmacologic agents to tissues within the eye following
injection by routes of administration such as a Sub-Tenon's
injection into the episcleral space surrounding the sclera,
comprising a stabilized solution of an effective amount of a
sHASEGP or a soluble human hyaluronidase domain thereof, such as 50
to 5,000 Units of the soluble glycoprotein with 30 to 150,000
Units/mg of specific activity, in a volume such as 0.05 to 10
ml.
[0502] These solutions, particularly those intended for ophthalmic
use, can be formulated as 0.01% -10% isotonic solutions, pH about
5-7, with appropriate salts.
[0503] Co-Applications with Iontophoresis
[0504] In the case of topical administrations and other
applications to tissues that are susceptible to iontophoresis, the
use of one or more sHASEGPs (potentially co-formulted or
co-administered with another pharmacologic or other agent) can be
combined with iontophoretic technqiues in order to further drive
permeation of a tissue. In these regards, iontophoresis and
sHASEGPs can function cooperatively in that iontophoresis can
promote delivery of sHASEGPs into a tissue or other location within
the body, and the sHASEGP so delivered can further drive permeation
of the enzyme, as well as co-formulated or co-administered or other
molecules present in the tissue, i.e. by enzymatically opening
channels that facilitate diffusion as well as promoting bulk fluid
flow and thus convective transport of solutes such as
pharmacologics.
[0505] 5. Compositions for Other Routes of Administration
[0506] Other routes of administration, such as topical application,
transdermal patches, and rectal administration are also
contemplated herein.
[0507] For example, pharmaceutical dosage forms for rectal
administration are rectal suppositories, capsules and tablets for
systemic effect. Rectal suppositories are used herein mean solid
bodies for insertion into the rectum that melt or soften at body
temperature releasing one or more pharmacologically or
therapeutically active ingredients. Pharmaceutically acceptable
substances utilized in rectal suppositories are bases or vehicles
and agents to raise the melting point. Examples of bases include
cocoa butter (theobroma oil), glycerin-gelatin, carbowax
(polyoxyethylene glycol) and appropriate mixtures of mono-, di- and
triglycerides of fatty acids. Combinations of the various bases can
be used. Agents to raise the melting point of suppositories include
spermaceti and wax. Rectal suppositories can be prepared either by
the compressed method or by molding. The typical weight of a rectal
suppository is about 2 to 3 gm.
[0508] Tablets and capsules for rectal administration are
manufactured using the same pharmaceutically acceptable substance
and by the same methods as for formulations for oral
administration.
[0509] Formulations suitable for transdermal administration can be
presented as discrete patches adapted to remain in intimate contact
with the epidermis of the recipient for a prolonged period of time.
Such patches suitably contain the active compound as an optionally
buffered aqueous solution of, for example, 0.1 to 0.2 M
concentration with respect to the active compound. Formulations
suitable for transdermal administration can also be delivered by
iontophoresis (see e.g., Pharmaceutical Research 3 (6): 318 (1986))
and typically take the form of an optionally buffered aqueous
solution of the active compound.
[0510] The pharmaceutical compositions can also be administered by
controlled release means and/or delivery devices (see e.g., in U.S.
Pat. Nos. 3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770;
3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543;
5,120,548; 5,354,566; 5,591,767; 5,639,476; 5,674,533 and
5,733,566). The active compounds or pharmaceutically acceptable
derivatives can be prepared with carriers that protect the compound
against rapid elimination from the body, such as time-release
formulations or coatings.
[0511] In one embodiment of the compositions and methods provided
herein, the therapeutic agent is administered locally in a slow
release delivery vehicle, for example, encapsulated in a colloidal
dispersion system or in polymer stabilized crystals. Useful
colloidal dispersion systems include nanocapsules, microspheres,
beads, and lipid-based systems, including oil-in-water emulsions,
micelles, mixed micelles, and liposomes. For example, the colloidal
dispersion system can be a liposome or microsphere. Liposomes are
artificial membrane vesicles that are useful as slow release
delivery vehicles when injected or implanted. Some examples of
lipid-polymer conjugates and liposomes are disclosed in U.S. Pat.
No. 5,631,018, which are incorporated herein by reference in its
entirety. Other examples of slow release delivery vehicles are
biodegradable hydrogel matrices (U.S. Pat. No. 5,041,292),
dendritic polymer conjugates (U.S. Pat. No. 5,714,166), and
multivesicular liposomes (Depofoam.TM.' Depotech, San Diego,
Calif.) (U.S. Pat. Nos. 5,723,147 and 5,766,627). One type of
microspheres suitable for encapsulating therapeutic agents for
local injection (e.g., into subdermal tissue) is poly(D,L)lactide
microspheres, as described in D. Fletcher, Anesth. Analg. 84:90-94,
(1997). For example, a slow release formulation containing an
effective amount of sHASEGP or a soluble human hyaluronidase domain
thereof, such as 1 to 5000 Units/ml, can be employed for various
uses or to treat various conditions, including, but not limited to,
cosmetic formulations, treatment of spinal cord injuries and other
nervous system disorders, certain opthalmic disorders, inflammatory
or autoimmune conditions, cancers, and other chronic disease
conditions, in which prolonged delivery of the sHASEGP is
beneficial.
[0512] Desirable blood levels can be maintained by a continuous
infusion of the active agent as ascertained by plasma levels. It
should be noted that the attending physician would know how to and
when to terminate, interrupt or adjust therapy to lower dosage due
to toxicity, or bone marrow, liver or kidney dysfunctions.
Conversely, the attending physician would also know how to and when
to adjust treatment to higher levels if the clinical response is
not adequate (precluding toxic side effects).
[0513] The efficacy and/or toxicity of the sHASEGP polypeptide
and/or its inhibitor (s), alone or in combination with other
agents, such as therapeutically effective agents, also can be
assessed by the methods known in the art (see, e.g., O & Apos;
Reilly, Investigational New Drugs 15: 5-13 (1997)).
[0514] 6. Articles of Manufacture
[0515] The sHASEGP polypeptides or soluble human hyaluronidase
domains thereof or compositions containing any of the preceding
agents can be packaged as articles of manufacture containing
packaging material, a compound or suitable derivative thereof
provided herein, which is effective for treatment of a diseases or
disorders contemplated herein, within the packaging material, and a
label that indicates that the compound or a suitable derivative
thereof is for treating the diseases or disorders contemplated
herein. The label can optionally include the disorders for which
the therapy is warranted.
[0516] The articles of manufacture provided herein contain
packaging materials. Packaging materials for use in packaging
pharmaceutical products are well known to those of skill in the art
(see, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,352).
Examples of pharmaceutical packaging materials include, but are not
limited to, blister packs, bottles, tubes, inhalers, pumps, bags,
vials, containers, syringes, bottles, and any packaging material
suitable for a selected formulation and intended mode of
administration and treatment. A wide array of formulations of the
compounds and compositions provided herein are contemplated, as are
variety treatments for any disorder in which HCV infection is
implicated as a mediator or contributor to the symptoms or
cause.
[0517] Kits containing the compositions and/or the combinations
with instructions for administration thereof are also provided
herein. The kit can further include a needle or syringe, typically
packaged in sterile form, for injecting the complex, and/or a
packaged alcohol pad. Instructions are optionally included for
administration of the active agent by a clinician or by the
patient. For example, provided herein is a kit containing a small
volume syringe with an effective amount of sHASEGP or a soluble
human hyaluronidase domain thereof, such as 1 to 5000 Units of the
soluble glycoprotein, in a 5 to 50 .mu.l volume, optionally
containing a second syringe containing a viscoelastic. Also
provided herein is a kit containing a small volume syringe
containing an effective amount of sHASEGP or a soluble human
hyaluronidase domain thereof, such as 1 to 500 Units of the soluble
glycoprotein, and a therapeutic amount of a second active
ingredient, such as a drug, a small molecule, a protein or a
nucleic acid.
[0518] K. Animal Models
[0519] Transgenic animal models and animals, such as rodents,
including mice and rats, cows, chickens, pigs, goats, sheep,
monkeys, including gorillas, and other primates, are provided
herein. In particular, transgenic non-human animals that contain
heterologous nucleic acid encoding a sHASEGP polypeptide or a
transgenic animal in which expression of the polypeptide has been
altered, such as by replacing or modifying the promoter region or
other regulatory region of the endogenous gene are provided. Such
an animal can by produced by promoting recombination between
endogenous nucleic acid and an exogenous sHASEGP gene that could be
over-expressed or mis-expressed, such as by expression under a
strong promoter, via homologous or other recombination event.
[0520] Transgenic animals can be produced by introducing the
nucleic acid using any know method of delivery, including, but not
limited to, microinjection, lipofection and other modes of gene
delivery into a germline cell or somatic cells, such as an
embryonic stem cell. Typically the nucleic acid is introduced into
a cell, such as an embryonic stem cell (ES), followed by injecting
the ES cells into a blastocyst, and implanting the blastocyst into
a foster mother, which is followed by the birth of a transgenic
animal. Generally introduction of a heterologous nucleic acid
molecule into a chromosome of the animal occurs by a recombination
between the heterologous sHASEGP-encoding nucleic acid and
endogenous nucleic acid. The heterologous nucleic acid can be
targeted to a specific chromosome. In some instances, knockout
animals can be produced. Such an animal can be initially produced
by promoting homologous recombination between a sHASEGP polypeptide
gene in its chromosome and an exogenous sHASEGP polypeptide gene
that has been rendered biologically inactive (typically by
insertion of a heterologous sequence, e.g., an antibiotic
resistance gene). In one embodiment, this homologous recombination
is performed by transforming embryo-derived stem (ES) cells with a
vector containing the insertionally inactivated sHASEGP polypeptide
gene, such that homologous recombination occurs, followed by
injecting the ES cells into a blastocyst, and implanting the
blastocyst into a foster mother, followed by the birth of the
chimeric animal ("knockout animal") in which a sHASEGP polypeptide
gene has been inactivated (see Capecchi, Science 244: 1288-1292
(1989)). The chimeric animal can be bred to produce homozygous
knockout animals, which can then be used to produce additional
knockout animals. Knockout animals include, but are not limited to,
mice, hamsters, sheep, pigs, cattle, and other non-human mammals.
For example, a knockout mouse is produced. The resulting animals
can serve as models of specific diseases, such as cancers, that
exhibit under-expression of a sHASEGP polypeptide. Such knockout
animals can be used as animal models of such diseases e.g., to
screen for or test molecules for the ability to treat or prevent
such diseases or disorders.
[0521] Other types of transgenic animals also can be produced,
including those that over-express the sHASEGP polypeptide. Such
animals include "knock-in" animals that are animals in which the
normal gene is replaced by a variant, such as a mutant, an
over-expressed form, or other form. For example, one species', such
as a rodent's endogenous gene can be replaced by the gene from
another species, such as from a human. Animals also can be produced
by non-homologous recombination into other sites in a chromosome;
including animals that have a plurality of integration events.
[0522] After production of the first generation transgenic animal,
a chimeric animal can be bred to produce additional animals with
over-expressed or mis-expressed sHASEGP polypeptides. Such animals
include, but are not limited to, mice, hamsters, sheep, pigs,
cattle and other non-human mammals. The resulting animals can serve
as models of specific diseases, such as cancers, that are exhibit
over-expression or mis-expression of a sHASEGP polypeptide. Such
animals can be used as animal models of such diseases e.g., to
screen for or test molecules for the ability to treat or prevent
such diseases or disorders. In a specific embodiment, a mouse with
over-expressed or mis-expressed sHASEGP polypeptide is
produced.
[0523] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
[0524] L. Therapeutic Uses of sHASEGP
[0525] Naturally occurring hyaluronidase enzymes from
slaughterhouses have been the principle source of clinical enzyme
preparations for over forty years. Bovine and Ovine testicles are
the principle source of this material. These clinical enzyme
preparations however are very crude, sold in preparations ranging
from 0.5-5% purity based upon known specific activities between
30-100,000 Units/mg. Thus their lack of purity combined with their
slaughterhouse origin, leave them as both immunogenic to humans and
as potential source of Jacob Creutzfeld disease and other bovine
and ovine pathogens. Anaphylactic reactions to bovine and ovine
hyaluronidase preparations are known to occur.
[0526] Cattle or bacterially derived hyaluronidase have been used
in the treatment of diseases associated with excess hyaluronic acid
and to enhance the circulation of physiological fluids and/or
therapeutic agents. For example, bovine hyaluronidase can be co
injected with anesthesia in peribulbar, retrobulbar and sub-Tenon's
blocks for ophthalmic surgical procedures. Moreover, increased
surgical complications occur in its absence (Brown SM et al. J
Cataract Refract Surg. September 1999; 25(9): 1245-9.). Bovine
hyaluronidase is also used as an antidote to local necrosis from
paravenous injection of necrotic substances such as vinka alkaloids
(Few, B. J. (1987) Amer. J. Matern. Child Nurs. 12, 23-26). Bovine
testes hyaluronidase is also useful for the treatment of ganglion
cysts (Paul et al. J Hand Surg April 1997; 22 (2): 219-21).
Hyaluronidase can also be used to facilitate subcutaneous delivery
of fluids in hypodermoclysis (Berger E Y, Am Geriatr Soc March
1984; 32(3): 199-203). Hyaluronidase has also been utilized to
reduce intraocular pressure in the eyes of glaucoma patients and
cataract patients receiving viscoelastics (U.S. Pat. No. 4,820,516
issued Apr. 11, 1989).
[0527] Cattle or bacterially derived hyaluronidases have also been
used as a "spreading agent" to enhance the activity of
chemotherapeutics and/or the accessibility of tumors to
chemotherapeutics (Schuller et al., 1991, Proc. Amer. Assoc. Cancer
Res. 32:173, abstract no. 1034; Czejka et al., 1990, Pharmazie
45:H.9). Combination chemotherapy with hyaluronidase is effective
in the treatment of a variety of cancers including urinary bladder
cancer (Horn et al., 1985, J. Surg. Oncol. 28:304-307), squamous
cell carcinoma (Kohno et al., 94, J. Cancer Res. Oncol.
120:293-297), breast cancer (Beckenlehner et al., 1992, J. Cancer
Res. Oncol. 118:591-596), and gastrointestinal cancer (Scheithauer
et al., 1988, Anticancer Res. 8:391-396). Hyaluronidase is
effective as the sole therapeutic agent in the treatment of brain
cancer (gliomas) (PCT published application no. WO88/02261,
published Apr. 7, 1988). Administration of hyaluronidase also
induces responsiveness of previously chemotherapy-resistant tumors
of the pancreas, stomach, colon, ovaries, and breast (Baumgartner
et al., 1988, Reg. Cancer Treat. 1:55-58; Zanker et al., 1986,
Proc. Amer. Assoc. Cancer Res. 27:390). Unfortunately, the
contaminants and non human nature of such hyaluronidases result in
anaphylactic reactions.
[0528] In addition to its indirect anticancer effects, cattle
derived hyaluronidase has direct anticarcinogenic effects.
Hyaluronidase prevents growth of tumors transplanted into mice (De
Maeyer et al., 1992, Int. J. Cancer 51:657-660) and inhibits tumor
formation upon exposure to carcinogens (Pawlowski et al., 1979,
Int. J. Cancer 23:105-109; Haberman et al., 1981, Proceedings of
the 17th Annual Meeting of the American Society of Clinical
Oncology, Washington, D.C., 22:105, abstract no. 415).
[0529] Given the value of cattle-derived hyaluronidases as a
therapeutic, particularly in chemotherapy in conjunction with
conventional chemotherapeutics or as a chemotherapeutic in and of
itself, there is a need in the field for substantially pure
preparations of hyaluronidase of human origin. There is also a need
for efficient, cost-effective methods of making hyaluronidase to
provide commercially significant quantities of the enzyme. The
present invention addresses these problems.
[0530] Hyaluronic acid is an essential component of the
extracellular matrix. Hyaluronic acid is found in the connective
tissue of mammals and is the main constituent of the vitreous of
the eye. In connective tissue, the water of hydration associated
with hyaluronic acid creates spaces between tissues, thus creating
an environment conducive to cell movement and proliferation.
Hyaluronic acid plays a key role in biological phenomena associated
with cell motility including rapid development, regeneration,
repair, embryogenesis, embryological development, wound healing,
angiogenesis, and tumorigenesis (Toole, 1991, Cell Biol. Extracell.
Matrix, Hay (ed), Plenum Press, New York, 1384-1386; Bertrand et
al., 1992, Int. J. Cancer 52:1-6; Knudson et al., 1993, FASEB J.
7:1233-1241). In addition, hyaluronic acid levels correlate with
tumor aggressiveness (Ozello et al., 1960, Cancer Res. 20:600-604;
Takeuchi et al., 1976, Cancer Res. 36:2133-2139; Kimata et al.,
1983, Cancer Res. 43:1347-1354).
[0531] Following spinal cord injury, glial scars are produced by
astrocytes and contain chondroitin sulfate proteoglycans (CSPGs).
CSPGs play a crucial role in the inhibition of axon growth (Levine,
1994; Powell et al., 1997). For example, during fetal development,
CSPGs repel axons and inhibit neural cell adhesion. CSPG's also
play an important role in boundary formation (Snow et al., 1990,
1992; Powell and Geller, 1999). In addition the expression of CSPG
increases following injury of CNS (Mckeon et al., 1991; Davies et
al., 1997).
[0532] Studies indicate that the inhibitory effects of CSPGs are
principally due to the chondroitin sulfate (CS) glycosaminoglycan
(GAG) sugar chain (Snow et al., 1990; Cole and McCable, 1991;
Geisert and Bidanset, 1993). This is supported by the finding that
administration of bacterial chondroitinase in fact promote axon
regeneration when administered intrathecally. Moreover,
electrophysiological experiments determined that regenerated CST
axons established functional connections (Bradbury, et al 2002). In
addition to their direct inhibitory effects, CSPGs could also
interact with cell adhesion molecules or neurotrophic factors to
influence neurite outgrowth (Roberts et al., 1988; Ruoslahti and
Yamaguchi, 1991; Milev et al., 1994). Recombinant mammalian
Hyaluronidases are thus useful to reverse the inhibition of CSPG's
in the glial scar and to promote axon regeneration following
injury.
[0533] The amount of sHASEGP required to sufficiently degrade
CSPG's in the glial scar will vary. In some cases repeated
administration of 10-5000 Units of sHASEGP by intrathecal delivery
will be required to remove the CSPG's in the scar. In other cases,
sustained release of sHASEGP through use of a slow release
formulation may be preferred. Alternatively, administration of gene
therapy vectors encoding sHASEGP may be effective to enhance
clearance of CSPG's.
[0534] sHASEGPs can also be utilized for the treatment of herniated
disks in a process known as chemonucleolysis. Chondroitinase ABC,
and enzyme cleaving similar substrates as sHASEGP can induce the
reduction of intradiscal pressure in the lumbar spine. (Sasaki et
al., 2001, Ishikawa et al., 1999). There are three types of disk
injuries. A protruded disk is one that is intact but bulging. In an
extruded disk, the fibrous wrapper has torn and the NP has oozed
out, but is still connected to the disk. In a sequestered disk, a
fragment of the NP has broken loose from the disk and is free in
the spinal canal. Chemonucleolysis is effective on protruded and
extruded disks, but not on sequestered disk injuries. In the United
States, chemonucleolysis is approved only for use in the lumbar
(lower) spine. In other countries, it has also been used
successfully to treat cervical (upper spine) hernias.
Chemonucleolysis is thus a conservative alternative to disk surgery
when it is preferable to reduce disk pressure.
[0535] The precise composition and structure of the carbohydrate
chain(s) on a glycoprotein can directly influence its serum
lifetime, since cells in the liver and reticulo-endothelial system
can bind and internalize circulating glycoproteins with specific
carbohydrates. Hepatocytes have receptors on their surfaces that
recognize oligosaccharide chains with terminal (i.e., at the
outermost end(s) of glycans relative to the polypeptide) Gal
residues, macrophages contain receptors for terminal Man or GlcNAc
residues, and hepatocytes and lymphocytes have receptors for
exposed fucose residues. No sialic acid-specific receptors have
been found, however. Although somewhat dependent on the spatial
arrangement of the oligosaccharides, as a general rule, the greater
the number of exposed sugar residues recognized by cell surface
receptors in the liver and reticulo-endothelial system, the more
rapidly a glycoprotein will be cleared from the serum. Because of
the absence of sialic acid-specific receptors, however,
oligosaccharides with all branches terminated, or "capped," with
sialic acid will not promote the clearance of the protein to which
they are attached.
[0536] The presence and nature of the oligosaccharide chain(s) on a
glycoprotein can also affect important biochemical properties in
addition to its recognition by sugar-specific receptors on liver
and reticulo-endothelial cells. Removal of the carbohydrate from a
glycoprotein will usually decrease its solubility, and it may also
increase its susceptibility to proteolytic degradation by
destabilizing the correct polypeptide folding pattern and/or
unmasking protease-sensitive sites. For similar reasons, the
glycosylation status of a protein can affect its recognition by the
immune system.
[0537] sHASEGPs can be used to remove the cumulus cells surrounding
an egg prior to cryopreservation and other In Vitro fertilization
techniques such an intracytoplasmic sperm injection (ICSI).
Hyaluronidase can be added to harvested oocytes between 10-200 U/ml
in buffered salt solutions. Oocytes are separated from the released
cumulus cells through aspiration and washed through several washes
with media lacking hyaluronidase. The eggs can then be processed
for cryopreservation or IVF techniques.
[0538] sHASEGPs are also useful for the more effective penetration
of chemotherapeutic agents into solid tumors. sHASEGPs can be
injected intratumorally with anti-cancer agents or intravenously
for disseminated cancers or hard to reach tumors. The anticancer
agent can be a chemotherapeutic, an antibody, a peptide, or a gene
therapy vector, virus or DNA. Additionally, sHASEGPs can be used to
recruit tumor cells into the cycling pool for sensitization in
previously chemorefractory tumors that have acquired multicultural
drug resistance St Croix et al Cancer Lett September 1998 11;
131(1): 35-44). sHASEGPs are also useful to enhance delivery of
biologics such as monoclonal antibodies, cytokines and other drugs
to tumors that accumulate glycosaminoglycans. Many tumors delete
genes involved with the catabolism of glycosaminoglycans such that
localized accumulation can prevent antineoplastic agents and the
immune system from reaching the tumor mass.
[0539] sHASEGP can also be used to increase the sensitivity of
tumors that are resistant to conventional chemotherapy. In one
embodiment, sHASEGP is administered to a patient having a tumor
associated with a LuCa-1 defect in an amount effective to increase
diffusion around the tumor site (e.g., to increase circulation of
chemotherapeutic factors (e.g., to facilitate circulation and/or
concentrations of chemotherapeutic agents in and around the tumor
site), inhibit tumor cell motility (e.g., by HA degradation) and/or
to lower the tumor cell(s) threshold of apoptosis (i.e., bring the
tumor cell(s) to a state of anoikis), a state that renders the
tumor cell(s) more susceptible to the action of chemotherapeutic
agents or other agents that may facilitate cell death, preferably
preferentially facilitate programmed cell death of cells in
anoikis. Chemotherapeutic agents as used herein is meant to
encompass all molecules, synthetic (e.g., cisplatin) as well as
naturally occurring (e.g., tumor necrosis factor IF)), that
facilitate inhibition of tumor cell growth, and preferably
facilitate, more preferably preferentially facilitate tumor cell
death.
[0540] Of particular interest is the use of sHASEGP for the
treatment of metastatic and non-metastatic cancers, particularly
metastatic cancers, having decreased to undetectable hyaluronidase
activity relative to non-cancerous (normal) cells. sHASEGP can be
used as a chemotherapeutic agent (alone or in combination with
other chemotherapeutics) in the treatment of any of a variety of
cancers, particularly invasive tumors. For example, sHASEGP can be
used in the treatment of small lung cell carcinoma, squamous lung
cell carcinoma, as well as cancers of the breast, ovaries, head and
neck, or any other cancer associated with depressed levels of
hyaluronidase or with a defective LuCa-1 (hpHAse) gene (e.g., a
LuCa-1 gene that does not provide for expression of adequate hpHAse
levels or encodes a defective hpHAse that does not provide for an
adequate level of hyaluronidase activity) or other defect
associated with decreased hyaluronan catabolism. sHASEGP is
preferable for the treatment of malignancies associated with
deficient HA catabolism as it does not require cellular
participation for degradation to occur.
[0541] The specific dosage appropriate for administration can be
readily determined by one of ordinary skill in the art according to
the factors discussed above (see, for example, Harrison's
Principles of Internal Medicine, 11th Ed., 1987). In addition, the
estimates for appropriate dosages in humans may be extrapolated
from determinations of the level of enzymatic activity of sHASEGP
in vitro and/or dosages effective in animal studies. For example,
70-300 TRU hyaluronidase is effective in reducing the tumor load in
a scid mouse. Given this information, the corresponding dosages in
the average 70 kg human would range from about 250,000-1,200,000
TRU hyaluronidase. The amount of sHASEGP administered to a human
patient is generally in the range of 1 TRU to 5,000,000 TRU of
enzymatic activity, preferably between about 1,000 TRU to 2,500,000
TRU, more preferably between about 100,000 TRU to 1,500,000 TRU,
normally between about 250,000 TRU and 1,200,000 TRU, with about
725,000 TRU representing average prescribed doses.
[0542] In one embodiment, a sHASEGP is formulated in a 0.15 M
saline solution containing sHASEGP at a concentration of about
150,000 TRU/cc. The formulation is then injected intravenously at
15,000 TRU/kg body weight of the patient. Alternatively, the enzyme
formulation may also be injected subcutaneously to allow the
hyaluronidase to perfuse around the tumor site. In a preferred
embodiment, sHASEGP is injected peritumorally or into the tumor
mass. In another preferred embodiment, sHASEGP is formulated as a
liposome and is delivered by injection either intravenously or at
or near the site of cancerous cells associated with a defect in the
LuCa-1 (hpHAse) gene. Injection of sHASEGP intravenously results in
sHASEGP in the tumor site. Moreover, Super Sialated sHASEGP is
preferably for parenteral administration in that the terminal
sialic acids on sHASEGP prevent the clearance of the enzyme from
circulation by the reticuloendothelial system. Comparisons of super
sialated sHASEGP to non-sialated bovine and ovine hyaluronidases
reveal that substantially more favorable pharmacokinetics is
achieved.
[0543] Facilitation of Gene Therapy
[0544] The efficacy of most gene delivery vehicles in vivo does not
correspond to the efficacy found in vitro. Glycosaminoglycans can
hinder the transfer and diffusion of DNA and viral vectors into
many cell types. The levels such extracellular matrix material can
hinder the process considerably. Dubensky et al., (Proc Natl Acad
Sci USA December 1984;81(23):7529-33) demonstrated that
hyaluronidase when combined with collagenase could facilitate
transduction of DNA in vivo. It has been demonstrated that adeno
associated virus is also amenable to hyaluronidase mediated gene
therapy Favre et al, (Gene Ther August 2000;7(16):1417-20).
[0545] We have determined herein that channels of defined size in
the extracellular matrix are opened with sHASEGP. These pores
generally do not enhance the diffusion of substances greater than
about 200-500 nm in diameter. However, smaller molecules such as
retroviruses, adenoviruses, adeno-associated viruses and DNA
complexes are amenable to sHASEGP mediated diffusion. The fact that
sHASEGPs generally act to open channels that are substantially
large enough to permit the movement of most therapeutic and other
pharmacological agents of interest (including macromolecules such
as proteins and nucleic acids as well as macromolecular complexes
such as gene therapy vectors) and yet are generally not so large as
to promote the dislocation and movement of cells, provides
particularly advantageous applications in treating, preventing
and/or diagnosing a variety of diseases for example.
[0546] Alternatively, viruses can be armed with the sHASEGP gene to
facilitate their replication and spread within a target tissue for
example. The target tissue can be a cancerous tissue whereby the
virus is capable of selective replication within the tumor. The
virus can also be a non-lytic virus wherein the virus selectively
replicates under a tissue specific promoter. As the viruses
replicate, the coexpression of sHASEGP with viral genes will
facilitate the spread of the virus in vivo.
[0547] Alternatively the nucleic acid of interest and a sHASEGP,
can be used simultaneously or consecutively or so as to be
staggered over time. Simultaneously refers to a coadministration.
In this case, these two essential components can be mixed to form a
composition prior to being administered, or can be administered at
the same time to the cell or the host organism. It is also possible
to administer them consecutively, that is to say one after the
other, irrespective of which component of the combination product
according to the invention is administered first. Finally, it is
possible to use a mode of administration which is staggered over
time or is intermittent and which stops and restarts at intervals
which may or may not be regular. It is pointed out that the routes
and sites of administration of the two components can be different.
According to one particularly preferred embodiment, the sHASEGP is
administered before the nucleic acid, with the route of
administration of the two components preferably being similar. The
time interval between the injections is not critical and can be
defined by the skilled person. It is possible to recommend an
interval of from 10 min to 72 h, advantageously of from 30 min to
48 h, preferably of from 1 to 24 h and, very preferably, of from 1
to 6 h.
[0548] In addition, the combination product according to the
invention can also be combined with one or more molecule(s) which
is/are intended to improve the nucleic acid administration. The
molecules can be molecules which have a protective effect on the
nucleic acid (protection with regard to degradation in the cell),
which improve its penetration or its expression in the host cell
(fusogenic peptide, nuclear localization signal, etc.), which
enable one particular cell type to be targeted (ligand or antibody
which recognizes a cell surface protein, etc.), or which prolong
the therapeutic effect (immunosuppressive agent, etc.). The
combination product can also be combined with agents that
facilitate transfection (proteins, etc.).
[0549] The combination product according to the invention can be
prepared with a view to local or parenteral administration or to
administration by the digestive route. Routes which may in
particular be mentioned are the intragastric, subcutaneous,
intracardiac, intravenous, intraperitoneal, intrasynovial,
intratumor, intrapulmonary, intranasal and intratracheal routes,
and, very particularly, the intramuscular route. The administration
can be effected by means of any technique of the art (injection,
oral route, aerosol, instillation, etc.), as a single dose or as a
dose that is repeated once or several times after a particular time
interval. The route of administration can be adjusted to suit the
gene of interest to be transferred and the disease to be treated.
The formulation can include pharmaceutically acceptable vehicles
(excipients, adjuvants, etc.). The substance leading to
disorganization of the extracellular matrix and the nucleic acid of
interest are preferably dissolved in a buffer which is suitable for
pharmaceutical use and which can be hypertonic, hypotonic or
isotonic. Various buffers can be envisaged. Those which may be
mentioned by way of illustration are a physiological saline
solution (0.9% NaCl), a nonphysiological saline solution (1.8%
NaCl), a Hepes-Ringer solution, a Lactate-Ringer solution, a buffer
which is based on Tris-HCl (10 mM Tris-HCl, pH 7.5 to 8, 1 mM EDTA;
10 mM Tris-HCl, pH 7.5 to 8, 1 mM MgCl.sub.2), a phosphate buffer
(Krebs phosphate H.sub.2O buffer), a sugar (glucose, sucrose,
trehalose, etc.) solution, or simply water.
[0550] Hypodermoclysis
[0551] Hypodermoclysis, the subcutaneous infusion of fluids, is a
useful and easy hydration technique suitable for mildly to
moderately dehydrated adult patients, especially the elderly. The
method is considered safe and does not pose any serious
complications. The most frequent adverse effect is mild
subcutaneous edema that can be treated by local massage or systemic
diuretics. Approximately 3 L can be given in a 24-hour period at
two separate sites. Common infusion sites are the chest, abdomen,
thighs and upper arms. The preferred solution is normal saline, but
other solutions, such as half-normal saline, glucose with saline or
5 percent glucose, can also be used. Potassium chloride can be
added to the solution bag if needed. Additionally, other drugs can
be delivered through similar routes. Human sHASEGP can be added to
enhance fluid absorption and increase the overall rate of
administration. Human sHASEGP is preferable for repeated
Hypodermoclysis over slaughter house-derived enzymes in that it not
likely to be immunogenic as the bovine enzyme is known to be. It
may be administered at home by family members or a nurse; the
technique should be familiar to every family physician.
[0552] In ambulatory patients, hypodermoclysis sites include the
abdomen, upper chest, above the breast, over an intercostal space
and the scapular area. In bedridden patients, preferred sites are
the thighs, the abdomen and the outer aspect of the upper arm.
After one to four days, the needle and tubing should be changed,
although infusion sets have been left in place for much longer
periods without complications. Administration of 500-mL boluses
over one or two hours three times a day can also be given, with 150
U of sHASEGP given at the subcutaneous site before the first
morning infusion
[0553] Facilitation of Therapeutic Injections.
[0554] Many molecules injected percutaneously (e.g. by using a
needle or other device that penetrates the skin and is used to
deposit the molecules into a sub-dermal layer) reach circulation
slowly or with very low efficiency. Several factors regulate the
pharmacokinetics and pharmacodynamics of molecules injected
subcutaneously (SC) or intramuscularly (IM). Generally, larger
molecules reach circulation more slowly and less efficiently
without active transport into circulation. Subcutaneous
bioavailability is determined by calculating the ratio of area
under the curves for SC verses intravenous administration
(AUC.sub.SC/AUC.sub.intravenous). A second factor is charge and
affinity for matrix molecules that may play a role in sequestration
of molecules subcutaneously. If these materials are degraded
locally they may never reach their desired targets and thus
demonstrate a decreased overall systemic bioavailability to the
target organs.
[0555] Large proteins are normally given intravenously so the
medicament directly available in the blood stream. It would however
be advantageous if a medicament could be given subcutaneously,
intramuscularly or intradermally (ID) as these administration forms
are much easier to handle for the patient. Especially, if the
medicament must be taken regularly during the whole life and
treatment is to start early, already when the patient is a child.
However, a medicament with a very large and labile molecule, such
as coagulation factor VIII of 170 to 300 kDa, have normally a very
low bioavailability if given subcutaneously, intramuscularly or
intradermally, since the uptake is not enough and degradation is
severe.
[0556] In addition to the need to increase bioavailability of many
subcutaneously administered biologics, more rapid pharmacokinetics
is also critically important in instances of emergency medicine.
The time required to reach intravenous access in many patients can
prevent an otherwise rapid acting drug when administered
systemically from being utilized. In some cases failure to reach
intravenous access is then followed by subcutaneous injection,
which leads to additional delay in reaching the target organs.
Thus, the more rapid availability of subcutaneous drugs would be of
benefit as a first line of treatment rather than to risk the time
required to achieve intravenous access. Examples of molecules that
can be delivered subcutaneously as well as intravenously include
epinephrine, atropine, narcan, lignocaine, and dextrose.
[0557] An additional benefit of the invention lies in the ability
to deliver equivalent or larger volumes of solutions ID, SC or IM
without the pain and morbidity associated with the pressure and
volume of the solution at the site of injection.
[0558] By way of illustration (and without limitation), there are a
number of biologics that can be enhanced by combination with one or
more sHASEGPs (e.g. by co-formulation or co-administration of
sHASEGP before, coincident with or after administration of the
biologic), including for example: Abciximabs (e.g. REOPRO.TM.),
Adalimumabs (e.g. HUMIRA.TM.), Agalsidase betas (e.g.
FABRAZYME.TM.), Aldesleukins (e.g. PROLEUKIN.TM.), Alefacepts (e.g.
AMEVIVE.TM.), Alemtuzumabs (e.g. CAMPATH.TM.), Alteplases (e.g.
ACTIVASE.TM.), Alteplases (e.g. CATHFLO ACTIVASE.TM.), Anakinras
(e.g. KINERET.TM.), Arcitumomabs (e.g. CEA-Scan.TM.), Asparaginases
(e.g. ELSPAR.TM.), Basiliximabs (e.g. SIMULECT.TM.), Becaplermins
(e.g. REGRANEX.TM.), bevacizumabs (e.g. AVASTIN.TM.), Botulinum
Toxin Type A's (e.g. BOTOX.TM.), Botulinum Toxin Type B's (e.g.
MYOBLOC.TM.), Capromab Pendetides (e.g. PROSTASCINT.TM.),
Cetuximabs (e.g. ERBITUX.TM.), Daclizumabs (e.g. ZENAPAX.TM.),
Darbepoetin alfas (e.g. ARANESP.TM.), Denileukin diftitoxs (e.g.
ONTAK.TM.), Dornase alfas (e.g. PULMOZYME.TM.), Drotrecogin alfas
(e.g. XIGRIS.TM.), Efalizumabs (e.g. RAPTIVA.TM.), Epoetin alfas
(e.g. EPOGEN.TM.), Etanercepts (e.g. ENBREL.TM.), Filgrastims (e.g.
NEUPOGEN.TM.), Ibritumomabs/Tiuxetans (e.g. ZEVALIN.TM.), Imciromab
Pentetates (e.g. MYOSCINT.TM.), Infliximabs (e.g. REMICADE.TM.),
Interferon alfa-2as (e.g. ROFERON-A.TM.), Interferon alfacon-1s
(e.g. INFERGEN.TM.), Interferon alfa-n3s (e.g. ALFERON N.TM.),
Interferon beta-1as (e.g. AVONEX.TM., BETASERON.TM.), Interferon
beta-1as (e.g. REBIF.TM.), Interferon gamma-1bs (e.g.
ACTIMMUNE.TM.), Laronidases (e.g. ALDURAZYME.TM.), Nofetumomabs
(e.g. VERLUMA.TM.), Omalizumabs (e.g. XOLAIR.TM.), Oprelvekins
(e.g. NEUMEGA.TM.), Palivizumabs (e.g. SYNAGIS.TM.), Pegfilgrastims
(e.g. NEULASTA.TM.), Peg-interferon alfa-2as (e.g. PEGASYS.TM.),
Peginterferon alfa-2bs (e.g. PEG-INTRON.TM.), Rasburicases (e.g.
ELITEK.TM.), Reteplases (e.g. RETAVASE.TM.), Rituximabs (e.g.
RITUXAN.TM.), Tenecteplases (e.g. TNKase.TM.), Tositumomab and
Iodine I-13 Is (e.g. BEXXAR.TM.), Trastuzumabs (e.g.
HERCEPTIN.TM.), Urokinases (e.g. ABBOKINASE.TM.).
[0559] Vitreous Hemorhhage
[0560] In an effort to minimize the potential for causing further
detachment or tearing of the retina during performance of
vitrectomy, it has previously been proposed in U.S. Pat. No.
5,292,509 (Hageman), to inject certain protease-free
glycosaminoglycanase enzymes into the vitreous body, to cause the
vitreous body to become uncoupled or "disinserted" from the retina,
prior to removal of the vitreous body. Such disinsertion or
uncoupling of the vitreous body is purported to minimize the
likelihood that further tearing or detachment of the retina will
occur as the vitreous body is removed. Examples of specific
protease-free glycosaminoglycanase enzymes which may be used to
bring about this vitreal disinsertion purportedly include;
chondroitinase ABC, chondroitinase AC, chondroitinase B,
chondroitin 4-sulfatase, chondroitin 6-sulfatase, hyaluronidase and
beta-glucuronidase.
[0561] Although hyaluronidase enzyme has been known to be usable
for various ophthalmic applications, including the vitrectomy
adjunct application described in U.S. Pat. No. 5,292,509 (Hageman),
published studies have indicated that the hyaluronidase enzyme may
itself be toxic to the retina and/or other anatomical structures of
the eye. See, The Safety of Intravitreal Hyaluronidase; Gottleib,
J. L.; Antoszyk, A. N., Hatchell, D. L. and Soloupis, P., Invest
Ophthalmol Vis Sci 31:11, 2345-52 (1990). Moreover, the used of
impure slaughterhouse preparations of hyaluronidase can cause
uveitis or inflammation of the eye. The use of human sHASEGP is
thus preferable in both its increased potency, purity and lack of
animal origin that can give rise to immunogenic reactions and
antibody mediated neutralization following repeated administration.
In another embodiment, a pegylated form of a sHASEGP can be
injected into the eye. Such a pegylated sHASEGP is not cleared from
the vitreous in such a rapid fashion and maintains its activity in
the vitreous for a longer period of time.
[0562] The ophthalmic toxicity of some hyaluronidase preparations
has been confirmed by other investigators, who have proposed that
such hyaluronidase preparations be used as a toxic irritant for
causing experimentally induced neovascularization of the eye, in
animal toxicity models, (see An Experimental Model of Preretinal
Neovascularization in the Rabbit; Antoszyk, A. N., Gottleib, J. L.,
Casey, R. C., Hatchell, D. L. and Machemer, R., Invest Ophthalmol
Vis Sci 32:1, 46-51 (1991). The use of a highly purified sHASEGP
devoid of mercury-based and cattle or bacterially derived
contaminants is preferable for intraocular procedures. Moreover, a
recombinant human sHASEGP is preferable over slaughterhouse derived
preparations in both purity lack of bovine pathogens and reduced
risk of immunogenicity. Most preferably a pegylated sHASEGP is
envisioned.
[0563] An enzymatic method using a human sHASEGP is thus provided
for treating ophthalmic disorders of the mammalian eye. In one
embodiment of the invention, said sHASEGP is PEGylated to prolong
its residence within the vitreous and prevent localized uptake.
Prevention of neovascularization, and the increased rate of
clearance from the vitreous of materials toxic to retina, are
accomplished by administering an amount of hyaluronidase effective
to liquefy the vitreous humor of the treated eye without causing
toxic damage to the eye. Liquefaction of the vitreous humor
increases the rate of liquid exchange from the vitreal chamber.
This increase in exchange removes those materials and conditions
whose presence causes ophthalmologic and retinal damage.
[0564] Cosmetic Uses of sHASEGP
[0565] It is known that hyaluronidase has the effect of
depolymerizing the long mucopolysaccharide chains of the
fundamental substance, responsible for the retention of bound water
and of the slowing, by capillary compression, of the diffusion of
organic liquids, which eliminate metabolic wastes. Such retention
of water and wastes associated with fat overloading of the
lipocytes, constitutes classical "pigskin" edema or "orange peel"
edema. This depolymerization will therefore cut the long chains of
mucopolysaccharides into shorter chains, whence the elimination of
the bound water, of wastes, restoration of the venous and lymphatic
circulation and disappearance of local edema.
[0566] Use of sHASEGP by way of subcutaneous administration is thus
preferred for the removal of glycosaminoglycans involved in the
accumulation of so-called cellulite and to promote lymphatic flow.
Human sHASEGP is preferred for the treatment of cellulite in that
it is capable of removal of said glycosaminoglycans without the
inflammatory components of slaughter house derived proteins and is
of high purity and is not likely to be immunogenic. The sHASEGP can
be administered through repeated subcutaneous injections, through
transdermal delivery in the form of ointments or creams or through
the use of injectable slow release formulations to promote the
continual degradation of glycosaminoglycans and prevent their
return.
[0567] Organ Transplantation
[0568] Hyaluronan has several biological effects, that are in part
related to its molecular size (West, D. C., Kumar, S. Exp. Cell.
Res. 183, 179-196, 1989). The content of hyaluronan in an organ
increases in different conditions of inflammation of that organ.
Thus, an increased concentration of hyaluronan has been shown in
tissue from different organs characterized by
inflammatory-immunological injury such as alveolitis (Nettelbladt 0
et al, Am Rev Resp Dis 1989; 139: 759-762) and myocardial
infarction (Waldenstrom et al, J Clin Invest 1991; 88(5):
1622-1628). Other examples are allograft rejection after a renal
(Ha'llgren et al, J Exp Med 1990a; 171: 2063-2076; Wells et al,
Transplantation 1990; 50: 240-243), small bowel (Wallander et al,
Transplant Int 1993; 6: 133-137) or cardiac (Hallgren et al, J Clin
Invest 1990b;85:668-673) transplantation; or a myocardial
inflammation of viral origin (Waldenstrdm et al, Eur J Clin Invest
1993; 23: 277-282).
[0569] The occurrence of interstitial edemas in connection with the
grafting of an organ constitutes a severe problem in the field of
transplantation surgery. As much as 25% of the grafts, will swell
to such a degree that the function will temporarily be lost.
Moreover, in 2-3% of the cases, the swelling causes disruption of
the kidney, resulting in a massive haemorrhage.
[0570] SHASEGP may be used to degrade accumulated
glycosaminoglycans in an organ transplant. Removal of such
glycosaminoglycans promotes removal of water from the graft and
thus organ function. Dose ranging from 500-10,000 Units/kg may be
administered to reduce interstitial pressure as such.
[0571] Pathologic Accumulations of Glycosaminoglycans in the
Brain
[0572] Hyaluronan levels are elevated in a number of cerebrospinal
pathologic conditions. Levels of cerebrospinal hyaluronan are
normally less than 200 ug/L in adults (Laurent et al, Acta Neurol
Scand September 1996;94(3):194-206). These levels can elevate to
over 8,000 ug/L in diseases such as meningitis, spinal stenosis,
head injury and cerebral infarction. Thus administration of sHASEGP
(for example by intrathecal delivery or systemic injection of a
sHASEGP, PEGylated sHASEGP or super-sialated sHASEGP) can be
utilized to degrade critically elevated levels of substrate.
[0573] The lack of effective lymphatics in the brain can also lead
to life threatening edema following head trauma. Hyaluronan
accumulation is a result of increased synthesis by HA synthases,
and decreased degradation. Accumulation of hyaluronan can initially
serve the beneficial purpose of increasing water content in the
damaged tissue, to facilitate leukocyte extravasation, but
continued accumulation can be lethal. Administration of human
sHASEGP to a patient suffering from head trauma can thus removal
tissue hyaluronan accumulation and the water associated with it.
Human sHASEGP can be administered intrathecally through a shunt or
alternatively, sHASEGP, PEGylated sHASEGP or Super-Sialated sHASEGP
(preferably human versions thereof) can be administered
intravenously to reach the brain tissue.
[0574] Following ischemia and reperfusion of the brain as occurs in
stroke, the hyaluronan content typically increases dramatically due
to increased expression of HA synthases and decreased catabolism.
Failure of ion pumps and leakage of plasma into the interstitium
results in fluid retention that if not properly cleared by the
lymphatics, results in tissue necrosis. Some groups have attempted
to prevent interstitial fluid accumulation following ischemia
reperfusion by blocking vascular permeability. However, once the
fluid has extravasated, preventing vascular permeability can
prevent resolution of edema and exacerbate conditions. As noted
above, human sHASEGP can be administered intrathecally (e.g.
through a shunt), or sHASEGP, PEGylated sHASEGP or Super-Sialated
sHASEGP (preferably human versions thereof) can be administered
intravenously to reach the brain tissue.
[0575] Human sHASEGP can also be used in the treatment of edema
associated with brain tumors, particularly that associated with
glioblastoma multiform. The edema associated with brain tumors
results from the accumulation of hyaluronan in the non-cancerous
portions of the brain adjacent the tumor. Administration of
hyaluronidase to the sites of hyaluronan accumulation (e.g., by
intravenous injection or via a shunt) can relieve the edema
associated with such malignancies by degrading the excess
hyaluronan at these sites. Thus, hyaluronidase is successful in the
treatment of brain tumors not only in the reduction of the tumor
mass and inhibition of tumor growth and/or metastasis, but it also
is useful in relieving edema associated with the malignancy. Human
sHASEGP can be administered for treatment of edema in a manner
similar to that for administration of bovine testicular
hyaluronidase to treat edema (see, e.g., Sa Earp Arq. Braz. Med.
44:217-20).
[0576] Without being bound to a particular application or mechanism
of action, sHASEGPs can potentially ameliorate ischemic or
hemorrhagic stroke and/or other CNS damaging conditions by one or
more of the following: (i) increasing oxygenation of damaged tissue
by reducing intracranial pressure; (ii) facilitating the
dissolution of clots and resorption of toxic metabolites; (iii)
suppressing leukocyte extravasation into infarcted or hemorrhaged
regions (e.g. by removal of hyaluronan which is a receptor for
CD44); (iv) promotion of neural regeneration of damaged tissue; or
(v) increasing cerebral vasculogenesis.
[0577] Treatment of Glycosaminoglycan Accumulation in
Cardiovascular Disease
[0578] It has been shown that the administration of hyaluronidase
in animal models following experimental myocardial infarct can
reduce infarct size (Maclean, et. al Science October 1976
8;194(4261):199-200). The proposed mechanism by which bovine
hyaluronidase reduces infarct size in animals is by reducing
hyaluronan accumulation that occurs following ischemia reperfusion.
Reduction of infarct size is believed to occur from increased lymph
drainage and increased tissue oxygenation and reduction of
myocardial water content. While reduced infarct size could be
obtained in animal models, the benefits were not realized in larger
clinical studies in humans. Bovine testes hyaluronidase possesses a
remarkably short serum half life of approximately 3 minutes in
animals-and man Wolf, et. al., J Pharmacol Exp Ther August
1982;222(2):331-7. This short half-life is due to the terminal
mannose residues that are readily recognized by the scavenger
receptors of the reticuloendothelial system. While small animals
may benefit from hyaluronidase due to a smaller vascular bed, an
enzyme with increased half-life is needed. Super sialated sHASEGP
possesses more favorable pharmacokinetics due to sialation for
which there is no scavenger receptor. Super sialated sHASEGP in
doses ranging from 100-200,000 Units/kg may be utilized to
facilitate resolution of excess hyaluronan following ischemia
reperfusion and to reduce infarct size.
[0579] Super sialated sHASEGP may also be used to limit coronary
plaques from arteriosclerosis. Such plaques accumulate
glycosaminoglycans and mediate macrophage and foam cell adhesion
Kolodgie et al, Arterioscler Thromb Vasc Biol. October 2002
1;22(10):1642-8. Administration of Super Sialated sHASEGP can be
used to reduce plaque formation. As repeated administration of
hyaluronidase is contemplated at doses from 100-100,000 U/kg, the
need to utilize a human recombinant protein with low risk of
immunogenicity and increased half-life will result in superior
reduction of plaques.
[0580] Treatment of Peripheral Tissues Necrosis
[0581] Tissue necrosis occurs in many diseases due to venous
insufficiency. The lack of sufficient oxygenation is one of the
main obstacles for regrowth of the tissue. It has been demonstrated
that intra-arterial hyaluronidase treatment significantly improves
the clinical picture in patients with peripheral arterial occlusive
disease (Elder et. al, Lancet (1980) 648-649). sHASEGP can be
injected intra-arterially 3-5 times a week at doses from 10-200,000
Units.
[0582] Enhanced Delivery of Anesthetics and Other Pharmacologic
Agents to Mammalian Tissues
[0583] Slaughterhouse-derived hyaluronidase is commonly used for
peribulbar block in local anesthesia prior ophthalmic surgery. The
presence of the enzyme prevents the need for additional blocks and
speeds the time to the onset of akinesia (loss of eye movement).
Peribulbar and sub-Tenon's block are the most common applications
of hyaluronidase for ophthalmic procedures. Since the
discontinuation of Wydase.TM., reports of increased diplopia and
ptosis have been reported with peribulbar block (Brown et al J
Cataract Refract Surg 1999; 25:1245-9).
[0584] With Wyeth's discontinuance of Wydase.TM., bovine
testes-derived hyaluronidase material is now supplied by
compounding pharmacies. However, there are several concerns with
using an extemporaneously compounded sterile
product.http://www.ashp.org/shortage/hyaluronidase.cfm?cfid=11944667&CFTo-
ken=942 6953-ref#ref Compounded preparations are not FDA-approved
products. As such, the FDA has no control over the quality or
consistency of the manufacturing process.
[0585] SHASEGP from 10-500 Units can be mixed directly with 5 ml 2%
lidocaine (Xylocaine), 5 ml 0.5% bupivacaine (Marcaine) and
optionally with epinephrine 1:200,000. sHASEGP can be used to
increase the onset of akinesia and to remove the need for
additional blocks. sHASEGP is also ideal for akinesia for cosmetic
surgery in blepharoplasties and face lifts. sHASEGP can also be
utilized following such surgical procedures to diffuse anti
inflammatories and to reduce tissue swelling.
[0586] SHASEGP may also be mixed with a buffering solution such as
bicarbonate to prevent discomfort during the injection procedure.
SHASEGP can also be mixed with anesthesia for lacerations to both
reduce the total volume of material required for injection and to
reduce pain from swelling of tissue.
[0587] Use of sHASEGPs to Promote Dispersion of Pharmacologic and
Other Agents
[0588] As described herein, the compositions and methods of the
present invention can be used to promote or enhance the delivery of
anesthetics and other pharmacologic agents (such as members of the
various classes of pharmaceutically effective agents referred to
herein) to any of a variety of mammalian tissues in vivo. In that
regard, sHASEGP or a soluble human hyaluronidase domain thereof can
be used to facilitate the diffusion and therefore promote the
delivery of small molecule pharmacologic agents, such as
anesthetics, anti-infectives and anti-inflammatories for example,
as well as larger molecule pharmacologic agents, such as proteins
(including, e.g., enzymes, monoclonal antibodies, and the like),
nucleic acids (including deoxyribonucleic acids (e.g. genes and
antisense molecules) and ribonucleic acids (including molecules for
RNA interference (RNAi) and the like), and macromolecular
compositions which may comprise a combination of such components
(including combinations of nucleic acids, proteins, carbohydrates,
lipids and lipid-based molecules, and the like). By way of
illustration and without limitation, molecules and macromolecular
complexes ranging from less than about 10 nm to around 500 nm in
diameter, particularly from about 20 to 200 nm, are likely to
exhibit dramatic improvements in delivery through interstitial
spaces when the interstitial space has been previously, or is
coincidently, exposed to a sHASEGP as described and illustrated
herein.
[0589] Without being restricted to a particular theory of mechanism
of action, it is believed that sHASEGP of the present invention
functions principally by hydrolyzing glycosaminoglycans within the
extracellular matrix surrounding mammalian cells to create
pore-like passages or channels that facilitate diffusion or other
movement of molecules within the exposed extracellular matrix; and,
furthermore, that permeabilizing such portions of the extracellular
matrix has the greatest effect on the diffusion or "spreading" of
molecules that are not so large as to be also hindered by other
macromolecular components of the extracellular matrix, such as an
overarching network of collagen. The extent of the spreading effect
obtainable with a given sHASEGP or a soluble human hyaluronidase
domain thereof in a particular tissue setting can be readily
ascertained as illustrated herein by assessing the effect of the
sHASEGP or domain thereof on the diffusion of detectable test
molecules of various sizes (such as fluoresceinated or otherwise
labeled beads) within the test tissue, either with or without
pretreatment or concomitant treatment with sHASEGP or domain
thereof at a range of times and concentrations. By way of
illustration, in the case of recombinant rHuPH20 injected
subcutaneously into the lateral skin of mice, fluoresceinated bead
diffusion tests (as exemplified herein) indicated that the effects
on diffusion were greatest with particles of less than about 500 nm
in diameter.
[0590] It is further believed that larger macromolecules
(exemplified by beads of 500 nm or larger) may be hindered in
diffusion not only by their larger size but by their potential
interaction with other relatively more open networks within the
extracellular matrix, such as a network formed from strands of
interstitial collagen. In addition, and unlike described
animal-derived hyaluronidases, presently preferred sHASEGPs of the
present invention do not contain agents that promote extravasation
or contaminating agents such as collagenases (that may open larger
channels) or other contaminants that may provoke immune reactions.
In particular applications in which larger pores or channels are
desired, sHASEGP or a soluble human hyaluronidase domain thereof
can be combined with preferably relativelty pure forms of other
matrix-degrading enzymes such as collagenases to facilitate further
spreading of larger size macromolecular complexes or cells. For
most applications, the use of an agent that promotes the
permeability of macromolecules having a size of less than about 500
nm, and more preferably less than about 200 nm, is very
advantageous in that the agent does not substantially promote the
movement of cells within a treated tissue but promotes the passage
of smaller complexes and agents.
[0591] As described and illustrated herein, sHASEGPs can be used to
enhance the delivery of a variety of other pharmacologic and/or
other agents to tissues within the human body (including tissues
such as those at the back of the eye that are typically relatively
difficult to reach). Without wishing to be bound by theory, it is
believed that sHASEGPs can enhance the pharmacokinetic and/or
pharmacodynamic profiles of a number of such agents by potentially
enhancing their diffusion or permeation within a tissue, and/or by
enhancing their convective transport by increasing bulk fluid flow
within the interstitial space of a tissue. For purposes of
illustration (and without limitation), examples of pharmacologic
agents that can be combined with sHASEGPs of the present invention
(e.g. combined by co-formulation with one or more sHASEGPs or by
co-administration with one or more sHASEGPs (which may be
administered prior to, coincident with or following administration
of the agent) for treatment of various conditions affecting the eye
include, for example, Angiogenesis inhibitors (such as
angiostatins, anecortave acetates (e.g. RETAANE.TM.), endostatins,
thrombospondins (e.g. thrombospondin-1 and thrombospondin-2),
anti-angiogenic antibodies and other anti-angiogenic agents or
vascular targeting agents (VTAs) (e.g. anti-VEGF antibodies such as
bevacizumabs (e.g. AVASTIN.TM.), carboxyamido triazole (CAI),
Interleukin-12 (IL-12), OLTIPRAZ.TM.
(5-[2-pyrazinyl]-4-methyl-1,2-3-thione), SU-5416, TNP-470,
thalidomide), and the like); Anti-cataract and anti-diabetic
retinopathy substances (such as aldose reductase inhibitors (ARIs)
for example tolrestats (e.g. ALREDASE.TM.) and zopolrestats (e.g.
ALOND.TM.), lisinoprils, enalaprils, statils, and thiol
cross-linking substances and the like); Anti-inflammatory agents
(including steroidal and non-steroidal anti-inflammatory agents as
described herein and in the art); Anti-infectives (including
compounds as described below, as well as a number of opthalmic
preparations thereof such as BLEPHAMIDE.TM., CILOXAN.TM.,
POLYTRIM.TM., QUIXIN.TM., TOBRADEX.TM., VIGAMOX.TM., ZYMAR.TM.);
Beta-adrenergic blocking agents (such as betaxolols (e.g. BETOPTIC
S.TM.), timolols (e.g. BETIMOL.TM.and TIMOPTIC.TM.) and
combinations thereof (e.g. timolols and a carbonic anhydrase
inhibitor such as a dorzolamide (e.g. COSOP.TM.); Carbonic
anhydrase inhibitors (such as acetazolamides, brinzolamides (e.g.
AZOPT.TM.), dichlorophenamides, dorzolamides (e.g. TRUSOP.TM., and
in combinations with other agents as in e.g. COSOPT.TM.),
methazolamides and the like); Mydriatics (such as atropine
sulfates, cyclopentolates, homatropines, scopolamines,
tropicamides, eucatropines, and hydroxyamphetamines and the like);
Photodynamic therapy agents (such as verteporfin (e.g.
VISUDYNE.TM.); Prostaglandin analogs (such as bimatoprosts (e.g.
LUMIGAN.TM.), latanoprosts (e.g. XALATAN.TM.), and travoprosts
(e.g. TRAVATAN.TM.); Sympathomimetics (such as brimonidines (e.g.
ALPHAGAN.TM. P); as well as combinations thereof (such as
combinations of a beta-adrenergic blocking agent and a carbonic
anhydrase inhibitor as in COSOP.TM.); and numerous other classes of
agents such as anti-infectives and various other physiological
modulators as described below or elsewhere herein, and/or as known
in the art or newly developed.
[0592] A number of other pharmacologic agents that can be combined
with sHASEGPs of the present invention (e.g. combined by
co-formulation with one or more sHASEGPs or by co-administration
with one or more sHASEGPs (which may be administered prior to,
coincident with or following administration of the agent) can be
used for treatment of various conditions affecting the eye or
affecting various other tissues of the body. For purposes of
illustration (and without limitation), such agents include, for
example: Anesthetics; Anti-metbolites, anti-neoplastics and other
anti-cancer agents; Anti-virals; Anti-infectives, including
Anti-bacterials and other antibiotics, Anti-fungals and other
anti-infectives; Immunomodulatory agents; Steroidal and
non-steroidal anti-inflammtories; Beta blockers; Sympathomimetics;
Ducosanoids, prostaglandins and prostaglandin analogs; Miotics,
cholinergics and anti-cholinesterases; Anti-allergenics and
Decongestants; Hormonal agents; Growth factors; other synthetic and
biologically derived agents; and combinations thereof. Some
illustrative examples of agents having such properties that can be
combined with sHASEGPs in the formulation of novel pharmacologics
and/or reformulations of existing pharmacologics include the
various categories of agents described above and below (as well as
other known and novel agents that are regularly being identified
for the diagnosis, prevention or treatment of various disease):
[0593] Anesthetics, particularly non-inhalation local anesthetics
(such as ambucaines; amoxecaines; amylocaines; aptocaines;
articaines; benoxinates; benzyl alcohols; benzocaines;
betoxycaines; biphenamines; bucricaines; bumecaines; bupivacaines;
butacaines; butambens; butanilicaines; carbizocaines;
chloroprocaine; clibucaines; clodacaines; cocaines; dexivacaines;
diamocaines; dibucaines; dyclonines; elucaines; etidocaines;
euprocins; fexicaines; fomocaines; heptacaines; hexylcaines;
hydroxyprocaines; hydroxytetracaines; isobutambens; ketocaines;
leucinocaines; lidocaines; mepivacaines; meprylcaines; octocaines;
orthocaines; oxethacaines; oxybuprocaines; phenacaines;
pinolcaines; piperocaines; piridocaines; polidocanols; pramocaines;
prilocaines; procaines; propanocaines; propipocaines;
propoxycaines; proxymetacaines; pyrrocaines; quatacaines;
quinisocaines; risocaines; rodocaines; ropivacaines; salicyl
alcohols; suicaines; tetracaines; trapencaines; trimecaines) and
the like; as well as various other non-inhalation anesthetics (such
as alfaxalones; amolanones; etoxadrols; fentanyls; ketamines;
levoxadrols; methiturals; methohexitals; midazolams; minaxolones;
propanidids; propoxates; pramoxines; propofols; remifentanyls;
sufentanyls; tiletamines; zolamine and the like);
[0594] Anti-metabolites (such as folic acid analogs (e.g.,
denopterins, edatrexates, methotrexates, piritrexims, pteropterins,
Tomudex, trimetrexates), purine analogs (e.g., cladribines,
fludarabines, 6-mercaptopurines, thiamiprines, thiaguanines), and
pyrimidine analogs (e.g., ancitabines, azacitidines, 6-azauridines,
carmofurs, cytarabines, doxifluridines, emitefurs, enocitabines,
floxuridines, fluorouracils, gemcitabines, tegafurs) and the
like;
[0595] Anti-neoplastics (such as aclacinomycins, actinomycins,
adriamycins, ancitabines, anthramycins, azacitidines, azaserines,
6-azauridines, bisantrenes, bleomycins, cactinomycins, carmofurs,
carmustines, carubicins, carzinophilins, chromomycins, cisplatins,
cladribines, cytarabines, dactinomycins, daunorubicins,
denopterins, 6-diazo-5-oxo-L-norleucines, doxifluridines,
doxorubicins, edatrexates, emitefurs, enocitabines, fepirubicins,
fludarabines, fluorouracils, gemcitabines, idarubicins,
loxuridines, menogarils, 6-mercaptopurines, methotrexates,
mithramycins, mitomycins, mycophenolic acids, nogalamycins,
olivomycines, peplomycins, pirarubicins, piritrexims, plicamycins,
porfiromycins, pteropterins, puromycins, retinoic acids,
streptonigrins, streptozocins, tagafurs, tamoxifens, thiamiprines,
thioguanines, triamcinolones, trimetrexates, tubercidins,
vinblastines, vincristines, zinostatins, zorubicins and the like)
(see also the illustrative anticancer agents described herein);
[0596] Anti-virals (such as abacavirs (e.g. ZIAGEN.TM.), acyclovirs
(e.g. ZOVIRAX.TM.), amantadines and rimantadines (e.g.
SYMMETREL.TM.), amprenavirs (e.g. AGENERASE.TM.), cidofovirs (e.g.
VISTIDE.TM.), delavirdines (e.g. RESCRIPTOR.TM.), didanosines (e.g.
VIDEX.TM.), efavirenz (e.g. SUSTIVA.TM.), famciclovirs (e.g.
FAMVIR.TM.), fomivirsens (e.g. VITRAVENE.TM.), foscarnets (e.g.
FOSCAVIR.TM.), ganciclovirs (e.g. CYTOVENE.TM.) and valganciclovirs
(e.g. VALCYTE.TM.), idoxuridines (e.g. HERPLEX.TM.), indinavirs
(e.g. CRIXIVAN.TM.), interferons (e.g. ALFERON N.TM., INTRON.TM. A,
PEGASYS.TM., PEG-INTRON.TM., ROFERON-A.TM., and combinations of
interferons and other antivirals such as ROBETRON.TM. and
PEGASYS/COPEGUS.TM.), lamivudines (e.g. 3TC.TM. and EPIVIR.TM.) and
combinations of lamivudines and zidovudines (e.g. COMBIVIR.TM.),
monoclonal antibodies such as palivizumabs (e.g. SYNAGIS.TM.),
nelfinavirs (e.g. VIRACEP.TM.), nevirapines (e.g. VIRAMUNE.TM.),
oseltamivirs (TAMIFLU.TM.), penciclovirs (e.g. DENAVIR.TM.),
pleconarils, ribavirins (e.g. COPEGUS.TM. and REBETOL.TM.),
rimantadines (e.g. FLUMADINE.TM.), ritonavirs (NORVIR.TM.),
saquinavirs (e.g. INVERASE.TM.), stavudines (d4T, e.g., ZERIT.TM.),
valacyclovirs (e.g. VALTREX.TM.), vidarabines (e.g. VIRA-A.TM.),
zalcitabines (e.g. HIVID.TM.), zanamivirs (RELENZA.TM.),
zidovudines (e.g. AZT, RETROVIR.TM.), and the like);
[0597] Anti-bacterials and other antibiotics, including, for
example, Aminoglycosides; Amphenicols; Ansamycins; Carbacephems;
Carbapenems; Cephalosporins or Cephems; Cephamycins; Clavams;
Cyclic lipopeptides; Diaminopyrimidines; Ketolides; Lincosamides;
Macrolides; Monobactams; Nitrofurans; Oxacephems; Oxazolidinones;
Penems, thienamycins and miscellaneous beta-lactams; Penicillins;
Polypeptides antibiotics; Quinolones; Sulfonamides; Sulfones;
Tetracyclines; and other antibiotics, illustrative members of which
are provided below (and others known in the art or newly
developed): [0598] Aminoglycosides (e.g., amikacins, apramycins,
arbekacins, bambermycins, butirosins, dibekacins,
dihydrostreptomycins, fortimicins, gentamicins, isepamicins,
kanamycins, micronomicins, neomycins, neomycin undecylenates,
netilmicins, paromomycins, ribostamycins, sisomicins,
spectinomycins, streptomycins, tobramycins, trospectomycins and the
like); [0599] Amphenicols (e.g., azidamfenicols, chloramphenicols,
florfenicols, thiamphenicols and the like); [0600] Ansamycins
(e.g., rifamides, rifampins or rifampicins (e.g. RIFADIN.TM.,
RIFADIN IV.TM., as well as combinations comprising rifampins such
as rifampin/isoniazid (e.g. RIFAMATE.TM.) and
rifampin/isoniazid/pyrazinamide (e.g. RIFATER.TM.), rifamycins,
rifapentines, rifaximins (e.g. XIFAXAM.TM.), and the like); [0601]
Carbacephems (e.g. loracarbefs, 3-chloro-1-carbacephems, 3-thio
substituted carbacephems, carbacephems described in U.S. Published
Patent Application No. 20050004095, and the like); [0602]
Carbapenems (e.g., biapenems, imipenems, meropenems, panipenems and
the like); [0603] Cephalosporins or cephems (e.g., cefaclors (e.g.
CECLOR.TM.), cefadroxils, cefamandoles, cefatrizines, cefazedones,
cefazolins (e.g. ANCEF.TM.), cefcapene pivoxils, cefclidins,
cefdinirs, cefditorens, cefepimes (e.g. MAXIPIME.TM.), cefetamets,
cefiximes, cefinenoximes, cefmetazoles, cefodizimes, cefonicids,
cefoperazones, ceforanides, cefotaximes, cefotiams, cefotetans,
cefoxitins, cefozoprans, cefpimizoles, cefpiramides, cefpiromes,
cefpodoxime proxetils, cefprozils, cefroxadines, cefsulodins,
ceftazidimes (e.g. FORTAZ.TM.), cefterams, ceftezoles, ceftibutens,
ceftizoximes, ceftriaxones (e.g. ROCEPHIN.TM.), cefuroximes (e.g.
ZINACEF.TM.), cefuzonams, cephacetriles, cephalexins,
cephaloglycins, cephaloridines, cephalosporins, cephalothins,
cephapirins, cephradines, pivcefalexins and the like); [0604]
Cephamycins (e.g., cefbuperazones, cefinetazoles, cefininoxes,
cefotetans, cefoxitins (e.g. MEFOXIN.TM.), and the like); [0605]
Clavams (e.g., clavulinic acid or clavulanates, 2- and 3-phenyl
clavams, and combinations comprising such agents such as
amoxicillin/clavulanates (e.g. AUGMENTIN.TM.) and
ticarcillin/clavulanates (e.g. TIMENTIN.TM.), and the like); [0606]
Cyclic lipopeptides (e.g. daptomycins (e.g. CUBICIN.TM.); [0607]
Diaminopyrimidines such as 2,4-diaminopyrimidines (e.g.,
brodimoprims, tetroxoprims, trimethoprims and the like); [0608]
Ketolides (e.g. telithromycins (e.g. KETEK.TM.), and bridged
bicyclic ketolides (e.g. Enanta EP-013420 and 6-11 bycyclic
ketolides as described in U.S. Published patent application
20040157787), and the like); [0609] Lincosamides (e.g.,
clindamycins, lincomycins and the like); [0610] Macrolides (e.g.,
azithromycins (e.g. ZITHROMAX.TM.), carbomycins, clarithromycins,
diritthromycins, erythromycins, erythromycin acistrates,
erythromycin estolates, erythromycin glucoheptonates, erythromycin
lactobionates, erythromycin propionates, erythromycin stearates,
josamycins, leucomycins, midecamycins, miokamycins, oleandomycins,
primycins, rokitamycins, rosaramicins, roxithromycins, spiramycins,
troleandomycins and the like); [0611] Monobactams (e.g., aztreonams
(e.g. AZACTAM.TM.), carumonams, tigemonams and the like); [0612]
Nitrofurans (e.g., furaltadones, furazolium chlorides, nifuradenes,
nifuratels, nifurfolines, nifurpirinols, nifurprazines,
nifurtoinols, nitrofuirantoins and the like); [0613] Oxacephems
(e.g. flomoxefs, latamoxefs or moxalactams (e.g. MOXAM.TM.), and
the like); [0614] Oxazolidinones (e.g. linezolids (e.g. ZYVOX.TM.),
eperezolids, and the like); [0615] Penems, thienamycins and
miscellaneous beta-lactams (e.g. ertapenems (e.g. INVANZ.TM.),
imipenems (e.g. with Cilastatin as in PRIMAXIN.TM.), meropenems
(e.g. MERREM.TM.), and the like); [0616] Penicillins (e.g.,
amdinocillins, amdinocillin pivoxils, amoxicillins, ampicillins
(including combinations thereof (e.g. with sublactams as in
UNASYN.TM.)), apalcillins, aspoxicillins, azidocillins,
azlocillins, bacampicillins, benzylpenicillinic acids,
benzylpenicillin sodiums, carbenicillins, carindacillins,
clometocillins, cloxacillins, coamoxiclavs, cyclacillins,
dicloxacillins, epicillins, fenbenicillins, floxacillins,
hetacillins, lenampicillins, metampicillins, methicillin sodiums,
mezlocillins, nafcillins, oxacillins, penamecillins, penethamate
hydriodides, penicillin G benethamines, penicillin G benzathines
(e.g. BICILLIN.TM. L-A), penicillin G benzhydrylamines, penicillin
G calciums, penicillin G hydrabamines, penicillin G potassiums,
penicillin G procaines, penicillin N, penicillin O, penicillin V,
penicillin V benzathines, penicillin V hydrabamines,
penimepicyclines, phenethicillin potassiums, piperacillins
(including combinations thereof (e.g. with beta-lactamase
inhibitors such as tazobactam, as in ZOSYN.TM.)), pivampicillins,
propicillins, quinacillins, sulbenicillins, sultamicillins,
talampicillins, tazocillins, temocillins, ticarcillins (e.g. with
clavulanates as in TIMENTIN.TM.), and the like); [0617]
Polypeptides antibiotics (e.g., amphomycins, bacitracins,
capreomycins, colistimethates (e.g. COLI-MYCIN.TM. M), colistins,
enduracidins, enviomycins, fusafungines, gramicidins, mikamycins,
polymyxins, pristinamycins (and pristinamycin derivatives such as
quinupristins and dalfopristins (e.g. SYNERCID.TM.)), ristocetins,
teicoplanins, thiostreptons, tuberactinomycins, tyrocidines,
tyrothricins, vancomycins, viomycins, virginiamycins, zinc
bacitracins and the like); [0618] Quinolones (incl.
fluoroquinolones and analogs) (e.g., alatrofloxacin mesylates,
cinoxacins, ciprofloxacins (e.g. CIPRO.TM.), clinafloxacins,
difloxacins, enoxacins, fleroxacins, flumequines, gatifloxacins
(e.g. TEQUIN.TM.), grepafloxacins, levofloxacins (e.g.
LEVAQUIN.TM.), lomefloxacins, miloxacins, moxyfloxacins (e.g.
AVELOX.TM.), nadifloxacins, nalidixic acids, norfloxacins,
ofloxacins, oxolinic acids, pazufloxacins, pefloxacins, pipemidic
acids, piromidic acids, rosoxacins, rufloxacins, sparfloxacins,
temafloxacins, tosufloxacins, trovafloxacin mesylates and the
like); [0619] Sulfonamides (e.g., acetyl sulfamethoxypyrazines,
benzylsulfamides, chloramine-B, chloramine-T, dichloramine T,
formylsulfisomidines, beta-glucosylsulfanilamides, mafenides,
4'-(methylsulfamoyl)sulfanilanilides, noprylsulfamides,
phthalylsulfacetamides, phthalylsulfathiazoles,
salazosulfadimidines, succinylsulfathiazoles, sulfabenzamides,
sulfacetamides, sulfachlorpyridazines, sulfachrysoidines,
sulfacytines, sulfadiazines, sulfadicramides, sulfadimethoxines,
sulfadoxines, sulfaethidoles, sulfaguanidines, sulfaguanols,
sulfalenes, sulfaloxic acids, sulfamerazines, sulfameters,
sulfamethazines, sulfamethizoles, sulfamethomidines,
sulfamethoxazoles, sulfamethoxypyridazines, sulfametroles,
sulfamidochrysoidines, sulfamoxoles, sulfanilamides,
4-sulfanilamidosalicylic acids, sulfanilylsulfanilamides,
sulfanilylureas, n-sulfanilyl-3,4-xylamides, sulfanitrans,
sulfaperines, sulfaphenazoles, sulfaproxylines, sulfapyrazines,
sulfapyridines, sulfasomizoles, sulfasymazines, sulfathiazoles,
sulfathioureas, sulfatolamides, sulfisomidines, sulfisoxazoles and
the like); [0620] Sulfones (e.g., acedapsones, acediasulfones,
acetosulfone sodiums, dapsones, diathymosulfones, glucosulfone
sodiums, solasulfones, sublactams, succisulfones, sulfanilic acids,
p-sulfanilylbenzylamines, sulfoxone sodiums, thiazolsulfones and
the like); [0621] Tetracyclines (e.g., apicyclines,
chlortetracyclines, clomocyclines, demeclocyclines, doxycyclines,
guamecyclines, lymecyclines, meclocyclines, methacyclines,
minocyclines, oxytetracyclines, penimepicyclines, pipacyclines,
rolitetracyclines, sancyclines, tetracycline), and others (e.g.,
cycloserines, mupirocins, tuberins and the like); [0622] and other
antibiotics (such as clofoctols, fusidic acids, hexedines,
methenamines (including e.g., methenamine, methenamine
anhydromethylene-citrates, methenamine hippurates, methenamine
mandelates, methenamine sulfosalicylates), nitrofurantoins (e.g.
FURADANTN.TM.), nitroxolines, ritipenems, taurolidines, xibomols
and the like);
[0623] Anti-fungals, including polyenes such as amphotericin Bs
(e.g. ABELCE.TM. and AMBISOME.TM.), candicidins, dermostatins,
filipins, fungichromins, hachimycins, hamycins, lucensomycins,
mepartricins, natamycins (e.g. Natacyn), nystatins, pecilocins,
perimycins, and others (e.g., azaserines, griseofulvins,
oligomycins, neomycin undecylenates, pyrrolnitrins, siccanins,
tubercidins, viridins); as well as synthetic anti-fungals such as
allylamines (e.g., butenafines, naffifines, terbinafines);
imidazoles (e.g., bifonazoles, butoconazoles, chlordantoins,
chlormidazoles, cloconazoles, clotrimazoles, econazoles,
enilconazoles, fenticonazoles, flutrimazoles, isoconazoles,
itraconazole, ketoconazoles, lanoconazoles, miconazoles,
omoconazoles, oxiconazole nitrates, sertaconazoles, sulconazoles,
tioconazoles, voriconazoles (e.g. VFEND.TM.)); thiocarbamates
(e.g., tolciclates, tolindates, tolnaftates); triazoles (e.g.,
fluconazoles (e.g. DIFLUCAN.TM.), itraconazoles (e.g.
SPORANOX.TM.), saperconazoles, terconazoles); and other synthetics
(e.g., acrisorcins, amorolfines, biphenamines,
bromosalicylchloranilides, buclosamides, calcium propionates,
chlorphenesins, ciclopiroxes, cloxyquins, coparaffinates,
diamthazole dihydrochlorides, exalamides, flucytosines,
halethazoles, hexetidines, lipopeptides such as echinocandin (as in
caspofungin CANCIDAS.TM.), loflucarbans, nifuratels, potassium
iodides, propionic acids, pyrithiones, salicylanilides, sodium
propionates, sulbentines, tenonitrozoles, triacetins, ujothions,
undecylenic acids, zinc propionate) and the like;
[0624] Amebicides and anti-protozoals (including, e.g., atovaquone,
chloroquine hydrochloride, chloroquine phosphate, metronidazole,
metronidazole hydrochloride, pentamidine isethionate and the like);
Anti-helmenthics and anti-parasitics (including, e.g., mebendazole,
pyrantel pamoate, thiabendazole and the like); Anti-malarials
(including, e.g., chloroquine hydrochloride, chloroquine phosphate,
doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride,
primaquine phosphate, pyrimethamine, pyrimethamine with
sulfadoxine; Anti-tuberculotics and anti-leprotics (including,
e.g., clofazimine, cycloserine, dapsone, ethambutol hydrochloride,
isoniazid, pyrazinamide, rifabutin, rifampin , rifapentine,
streptomycin sulfate and the like, as well as combinations thereof
such as rifampin/isoniazid (e.g. RIFAMATE.TM.) and
rifampin/isoniazid/pyrazinamide (e.g. RIFATER.TM.).
[0625] For purposes of further illustration (and without
limitation), examples of immuno-modulatory agents that can be
combined with sHASEGPs of the present invention (e.g. combined by
co-formulation with one or more sHASEGPs or by co-administration
with one or more sHASEGPs (which may be administered prior to,
coincident with or following administration of the agent) include,
for example, members of the following classes of agents: [0626]
Immunosuppressants (including, e.g., azathioprine, basiliximab,
cyclosporine, daclizumab, lymphocyte immune globulin,
muromonab-CD3, mycophenolate mofetil, mycophenolate mofetil
hydrochloride, sirolimus, tacrolimus and the like); [0627] Vaccines
and toxoids (including, e.g., BCG vaccine, cholera vaccine,
diphtheria and tetanus toxoids (adsorbed), diphtheria and tetanus
toxoids and acellular pertussis vaccine adsorbed, diphtheria and
tetanus toxoids and whole-cell pertussis vaccine, Haemophilius b
conjugate vaccines, hepatitis A vaccine (inactivated), hepatisis B
vaccine (recombinant), influenza virus vaccines (based on purified
surface antigens), influenza virus vaccines (based on subvirions or
purified subvirions), influenza virus vaccines (based on whole
virions), Japanese encephalitis virus vaccine (inactivated), Lyme
disease vaccine (recombinant OspA), measles and mumps and rubella
virus vaccine (live), measles and mumps and rubella virus vaccine
(live attenuated), measles virus vaccine (live attenuated),
meningococcal polysaccharide vaccine, mumps virus vaccine (live),
plague vaccine, pneumococcal vaccine (polyvalent), poliovirus
vaccine (inactivated), poliovirus vaccine (live, oral, trivalent),
rabies vaccine (adsorbed), rabies vaccine (human diploid cell),
rubella and mumps virus vaccine (live), rubella virus vaccine
(live, attenuated), tetanus toxoid (adsorbed), tetanus toxoid
(fluid), typhoid vaccine (oral), typhoid vaccine (parenteral),
typhoid VI polysaccharide vaccine, varicella virus vaccine, yellow
fever vaccine and the like); [0628] Antitoxins and antivenins
(including, e.g., black widow and other spider antivenins,
Crotalidae antivenom (polyvalent), diphtheria antitoxin (equine),
Micrurus fulvius antivenin and the like); [0629] Immune sera
(including, e.g., cytomegalovirus immune globulin (intraveneous),
hepatitis B immune globulin (human), immune globulin intramuscular,
immune globulin intravenous, rabies immune globulin (human),
respiratory syncytial virus immune globulin intravenous (human),
immune globulins (human), tetanus immune globulin (human),
varicella-zoster immune globulin and the like); [0630] and other
immunomodulators (including, e.g., aldesleukin, epoetin alfa,
filgrastim, glatiramer acetate for injection, interferon alfacon-1,
interferon alfa-2a (recombinant), interferon alfa-2b (recombinant),
interferon alfa-n3, interferon beta-1a, interferon beta-1b
(recombinant), interferon gamma-1b, levamisole hydrochloride,
oprelvekin, sargramostim and the like).
[0631] Other agents that can be combined with sHASEGPs of the
present invention (e.g. combined by co-formulation with one or more
sHASEGPs or by co-administration with one or more sHASEGPs (which
may be administered prior to, coincident with or following
administration of the agent) include, e.g., the following: [0632]
Steroidal anti-inflammatories (such as alclomethasones, algestones,
beclomethasones, betamethasones, budesonides, clobetasols,
clobetasones, clocortolones, cloprednols, corticosterones,
cortisones and hydrocortisones (e.g. hydrocortisone acetate),
cortivazols, deflazacorts, desonides, desoximetasones,
dexamethasones (e.g. dexamethasone 21-phosphate), difluorosones,
diflucortolones, difluprednates, enoxolones, fluazacorts,
flucloronides, flumethasones, flunisolides, fluocinolones,
fluocinonides, fluocortins, fluocortolones, fluorometholones,
fluperolones, fluprednidenes, fluprednisolones, flurandrenolides,
fluticasones, formocortals, halcinonides, halobetasols,
halometasones, halopredones, hydrocortamates, hydrocortisones,
loteprednol etabonate, mazipredones, medrysones, meprednisones,
methylprednisolones, mometasone furoate, paramethasones,
prednicarbates, prednisolones (e.g. prednisolone
25-diethylamino-acetate, prednisolone sodium phosphate),
prednisones, prednivals, prednylidenes, rimexolones, tixocortols,
triamcinolones (e.g. triamcinolone acetonide, triamcinolone
benetonide, and triamcinolone hexacetonide) and the like); [0633]
Non-steroidal anti-inflammatories (such as cromoglicates,
diclofenacs, flurbiprofens, indomethacins, ibuprofens, ketorolacs
(e.g. ketorolac tromethamine), lodoxamides, nedocromils,
piroxicams, salicylates, and the like); [0634] Beta blockers (such
as betaxolols (e.g. betaxolol hydrochloride), carteolols, esmolols,
levobunolols, metipranalols, timolols (e.g. timolol hemihydrate and
various forms of timolol maleate) and the like); [0635]
Sympathomimetics (such as adrenaline, brimonidines, dipivefrins,
epinephrines and the like); [0636] Ducosanoids, prostaglandins and
prostaglandin analogs (such as bimatoprosts, latanoprosts,
travoprosts, unoprostones, and the like) as well as
antiprostaglandins and prostaglandin precursors; [0637] Miotics,
cholinergics and anti-cholinesterases (such as acetylcholine
chlorides, carbachols, demecarium bromides, diisopropyl
fluorophosphates, eserines, physostigmines, pilocarpines,
phospholine iodines, salicylates, and the like); [0638]
Anti-allergenics (such as sodium antazolines, cetirizines,
chlorpheniramines, cromoglycates, cromolyn sodium (Crolom),
emedastine difumarate, ketotifins (e.g. ketotifen fumarate),
levocabastines, methapyrilines, nedocromil sodium, olopatadines,
pyrilamines, prophenpyridamines, and the like); [0639]
Decongestants (such as phenylephrines, naphazolines,
tetrahydrozolines and the like); [0640] Hormonal agents (such as
estrogens, estradiols, progestationals, progesterones, insulins,
calcitonins, parathyroid hormone and peptide and vasopressin
hypothalamus releasing factor and the like); [0641] Growth factors
(such as epidermal growth factor, fibroblast growth factor,
platelet derived growth factor, transforming growth factor beta,
somatotropin and fibronectin and the like); [0642] Analgesics (such
as acetaminophen; alfentanil; aminobenzoate; aminobenzoate;
anidoxime; anileridine; anileridine; anilopam; anirolac;
antipyrine; aspirin; benoxaprofen; benzydamine; bicifadine
hydrochloride; brifentanil; brom adoline; bromfenac; buprenorphine;
butacetn; butixirate; butorphanol; butorphanol; carbamazepine;
carbaspirin calcium; carbiphene; carfentanil; ciprefadol succinate;
ciramadol; ciramadol; clonixeril; clonixin; codeine; codeine
phosphate; codeine sulfate; conorphone; cyclazocine; dexoxadrol;
dexpemedolac; dezocine; diflunisal; dihydrocodeine; dimefadane;
dipyrone; doxpicomine; drinidene; enadoline; epirizole; ergo tamine
tartrate; ethoxazene; etofenamate; eugenol; fenoprofen; fenoprofen
calcium; fentanyl citrate; floctafenine; flufenisal; flunixin;
flunixin meglumine; flupirtine; fluproquazone; fluradoline;
flurbiprofen; hydromorphone; ibufenac; indoprofen; ketazocine;
ketorfanol; ketorolac; letimide; levomethadyl acetate; levomethadyl
acetate hydrochloride; levonantradol; levorphanol; lofemizole;
lofentanil oxalate; lorcinadol; lomoxicam; magnesium salicylate;
mefenamic add; menabitan; meperidine; meptazinol; methadone;
methadyl acetate; methopholine; methotrimeprazine; metkephamid
acetate; mimbane; mirfentanil; molinazone; morphine sulfate;
moxazocine; nabitan; nalbuphine; nalmexone; namoxyrate; nantradol;
naproxen; naproxen; naproxol; nefopam; nexeridine; noracymethadol;
ocfentanil; octazamide; olvanil; oxetorone; oxycodone; oxycodone;
oxycodone terephthalate; oxymorphone; pemedolac; pentamorphone;
pentazocine; pentazocine; phenazopyridine; phenyramidol; picenadol;
pinadoline; pirfenidone; piroxicam olamine; pravadoline;
prodilidine; profadol; propiram; propoxyphene; propoxyphene
napsilate; proxazole; proxorphan; pyrroliphene; remifentanil;
salcolex; salethamide maleate; salicylamide; salicylate meglumine;
salsalate; salicylate; spiradoline; sufentanil; sufentanil;
sucapsaicin; talmetacin; talniflumate; talosalate; tazadolene;
tebufelone; tetrydamine; tifurac; tilidine; tiopinac; tonazocine;
tramadol; trefentanil; trolamine; veradoline; verilopam;
volazocine; xorphanol; xylazine; zenazocine mesilate; zomepirac;
and the like); [0643] Other macromolecular pharmacologic agents
such as other proteins, nucleic acids, and the like; as well as
complexes of macromolecules such as various drug delivery, protein
delivery and/or gene delivery vehicles (including
synthetically-prepared vehicles such as liposomes and the like, as
well as biologically-generated vehicles such as viral vectors and
the like); and [0644] Other agents used for a variety of conditions
(and additional information regarding usage, dosages,
contraindications etc. of various agents including those identified
herein), as known in the art, including for example those
additional agents identified in references such as the Physicians'
Desk Reference (PDR) (Thomson 2003, 2004, 2005); the American
College of Physicians Complete Home Medical Guide (DK Publishing
2003); and additional agents indentified in patent references
including, for example, U.S. Published applications 20040224023,
20040224012 and 20050002865 (which lists of agents are hereby
incorporated by reference herein).
[0645] Combinations of agents (e.g. two or more agents selected
from one or more of the preceding groups of agents and/or from
other groups or classes of pharmacologic and other agents specified
herein or known in the art).
[0646] Use of sHASEGPs for Reduction of Intraocular Pressure and
Other Opthalmic Indications
[0647] A common side effect occurring in postoperative cataract
patients is a significant early, and occasionally prolonged, rise
in intraocular pressure. Such a condition is sometimes serious,
especially in patients with glaucomatous optic disc changes.
Although the pressure increase tends to be more severe when
visco-elastic agents such as hyaluronic acid are injected into the
eye during surgery, the intraocular pressure can become elevated
postoperatively even when such agents are not utilized.
Furthermore, such a pressure increase can occur even when no
additional medications are used during the surgical procedure. In
some cases, it is advantageous to leave a viscoelastic agent in the
eye, which often necessitates giving patients large doses of
carbonic anhydrase inhibitors. These inhibitors lower the
intraocular pressure by decreasing the formation of aqueous humor,
a fluid that is normally secreted in the eye, by the ciliary body.
Current methods for relieving postoperative pressure increases in
the eye include various types of eye drops such as beta-adrenergic
blocking agents, sympathomimetic agents, miotics, alpha II
selective agents, carbonic anhydrase inhibitors and prostaglandin
agents.
[0648] A preferred method of removing the viscoelastic such as
hyaluronic acid is by injection of sHASEGP during or immediately
following anterior segment or posterior segment surgical
procedures, although other methods of administration known in the
art are possible as well. It is preferred if the hyaluronic acid
and the sHASEGP are administered by injection into the anterior
chamber during anterior segment ocular surgical procedures to allow
the hyaluronic acid to act as a spacer during the start of the
surgical procedure. In some cases of corneal transplantation, the
hyaluronic acid and sHASEGP combination may be placed on the
surface of the intraocular structures prior to suturing the corneal
transplant in place. This combination may also be used in posterior
segment surgery, such as retina or vitreous surgery.
[0649] In some cases, it may be advisable to leave a visco-elastic
agent such as Healon.TM., Viscoat.TM., or other space-occupying
substances in the anterior chamber of the eye at the conclusion of
surgery. This is especially true in positive pressure rise when the
intraocular contents tend to come forward and press against the
posterior surface of the cornea. If this occurs in an eye with a
synthetic intraocular lens in place, pressure on the corneal
endothelium can cause significant damage to the cells and
subsequent corneal swelling and opacification can occur, which are
associated with decreased vision. Typically, if a patient's
intraocular pressure is significantly elevated at the conclusion of
the operative procedure, it is necessary to give such a patient
large doses of carbonic anhydrase inhibitors, as well as topical
eye drops such as beta-blockers and alpha II agonists in order to
decrease aqueous formation and/or to increase aqueous outflow.
These agents all have significant side effects and, in some
instances, are contraindicated in patients with various types of
medical conditions such as breathing problems, heart disease or
high blood pressure. However, the use of sHASEGP in these
situations will eliminate the necessity of giving these patients
large doses of such drugs.
[0650] Furthermore, there is a significant amount of hyaluronic
acid in the trabecular meshwork. The sHASEGP will break this down
and therefore improve the outflow of the aqueous through the
trabecular meshwork. The patient's intraocular pressure will
therefore decrease. The combination of sHASEGP with other anterior
chamber agents, such as a methylcellulose (Ocucoat.TM. for example,
commercially available from Storz Instrument Co.), used as spacers
and/or protective agents in cataract surgery, will also be
efficacious in preventing significant pressure rises because it
will in effect open the trabecular meshwork and allow more aqueous
humor drainage by breaking down a significant amount of the
hyaluronic acid present in the trabecular meshwork.
[0651] Removal of glycosaminoglycans from the trabecular meshwork
is also useful for the reduction of intraocular pressure in
individuals suffering form open angle glaucoma. Human sHASEGP can
be administered by subconjunctuval injection or injection directly
in the anterior chamber. Since the sHASEGP acts enzymatically, even
relatively limited amounts of sHASEGP can effectively function as a
viscoelastic antidote or for other situations that are aided or
improved by application of a glycosaminoglycanase. As with other
uses, the units of hyaluronidase activity provided by the sHASEGP
for optimum application will depend in part on the particular
situation being treated--especially the amount of glycosaminoglycan
degradation that is desired--but can be readily assessed using
appropriate models and then optimized as appropriate for particular
uses. By way of illustration, the units of sHASEGP to be applied as
an antidote will be influenced, inter alia, by the particular
procedure employed and the amount of viscoelastic applied, and also
on whether any of the applied viscoelastic is to be removed using
other procedures such as irrigation and aspiration. Where sHASEGP
is used following irrigation and aspiration, generally amounts
providing in the range of 10 to 12,000 units, typically 100 to
5,000 units, more typically 500 to 2,000 units may be applied,
although this will again depend on the particular application.
Where sHASEGP is used to obviate the need for irrigation and
aspiration, larger amounts will be applied in order to effectively
digest a larger volume of applied viscoelastic, with amounts
generally providing in the range of 50 to 20,000 units, typically
400 to 10,000, more typically 1,500 to 5,000 units may be applied,
although this will again depend on the particular application.
[0652] In the case of sHASEGPs being used to reduce intraocular
pressure associated with glaucoma, application of sHASEGPs can also
be used as a primary treatment to reduce IOP associated with
glaucoma and thereby potentially avoid the need for surgical
procedures such as glaucomal filtration, glaucomal drainage
implants (tube shunts), laser trabeculoplasty,
cyclophotocoagulation or iridotomy. Thus sHASEGPs of the present
invention can be introduced, for example, by injection into the
anterior chamber of the eye to provide a minimally invasive means
for reducing intraocular pressure associated with glaucoma. Without
wishing to be bound by theory, it is believed that the
administration of sHASEGPs in such settings can enhance fluid
outflow via the trabecular meshwork and thus lower IOP by reducing
hydrostatic and/or oncotic pressures within the anterior
chamber.
[0653] In the case of sHASEGPs being used to used to dissolve
vitreous aggregates or "floaters", which are generally macroscopic
opaque aggregates of vitreous fibers, the administration of sHASEGP
to the vitreous body can be used to promote disaggregation.
[0654] Similarly, sHASEGPs as described herein can be applied to
the vitreous to promote the resolution of vitreous hemorrhages
(i.e. extravasation of blood into the vitreous) which can occur in
connection with conditions such as diabetic retinopathy, retinal
neovascularization, retinal vein occlusion, posterior vitreous
detachment, retinal tears, ocular traumas and the like. The
presence of vitreous hemorrhages, which are typically slow to
resolve, can delay, complicate or prevent procedures that require
the retina to be visualized through the vitreous for diagnosis
and/or for treatment procedures such as laser photocoagulation and
the like which are often primary treatments for conditions such as
proliferative diabetic retinopathy. Use of sHASEGPs to promote
resolution of such vitreous hemorrhages can thus be used to
facilitate diagnosis of retinal conditions and/or to obviate the
need for more radical procedures such as vitrectomy.
[0655] In the case of sHASEGPs being used to promote vitreo-retinal
detachment in treating conditions such as diabetic retinopathy, the
administration of sHASEGP into the vitreous can be used to promote
detachment or uncoupling of the vitreous body from the retina. In
this context, sHASEGP may not only promote the detachment but help
it to occur in a manner that reduces the potential for retinal
distortion or tearing.
[0656] sHASEGPs of the present invention can also be used for
treating corneal opacities. In that regard, a variety of
distortions or deformities can affect the normally organized
structure of the cornea and lead to disruptions in the passage of
light through the cornea and concomitant impairments in vision.
Thus, a number of conditions and causes lead to corneal distortions
that may appear as scars, hazing or opacification. In applying
sHASEGPs to the improvement or treatment of such corneal
conditions, the sHASEGP (in a pharmaceutically acceptable solution
which may comprise one or more additional pharmacologic agents) can
be introduced to the cornea by any of several routes of
administration, depending in part on the extent and location of the
corneal injury. By way of illustration, sHASEGPs can be introduced
onto the outer surface of the eye (using drops or other topical
application for example), potentially encapsulated within carriers
such as liposomes and the like that can be used to facilitate
transfer across the corneal epithelium, by injection or
microinjection onto or into the cornea itself, or by other
approaches to the corneal tissue. In addition to biochemical or
mechanical aids for promoting delivery, other means for promoting
permeation such as iontophoresis can likewise be applied.
[0657] sHASEGPs of the present invention can also be used, for
example, to enhance the permeability of agents employed in
photodynamic therapy (PDT). PDT is a treatment for choroidal
neovascular membranes (CNVM), which are generally leaky vascular
structures under the retina in the "wet" form of age-related
macular degeneration (AMD). In PDT, a photosensitive dye such as
VISUDYNE.TM. (verteporfin) is typically administered intravenously
(IV) and allowed to perfuse the CNVM, as well as the remainder of
the body. An ophthalmologist then generally treats the CNVM with a
red laser of a specific wavelength (e.g. 689 nm) for about 90
seconds. The non-thermal laser light activates the dye (such as
VISUDYNE.TM.) producing an active form of oxygen that both
coagulates and reduces the growth of abnormal blood vessels, which
in turn inhibits the leakage of fluid from the CNVM. sHASEGPs of
the present invention can be used, for example, to promote more
localized administration of a dye (such as VISUDYNE.TM.) directly
to the eye; and/or to facilitate the delivery of other agents that
are usefully administered to the eye in connection with procedures
such as PDT.
[0658] Ganglion Cysts
[0659] The ganglion cyst (also known as a wrist cyst, Bible cyst,
or dorsal tendon cyst) is the most common soft tissue mass of the
hand. It is a fluid filled sac that can be felt below the skin. It
is usually attached to a tendon sheath (lining which lubricates the
tendon) in the hand or wrist or connected with an underlying joint;
however, some have no obvious connection to any structures. These
may also occur in the foot. It often occurs when there is a tear in
the ligaments overlying the lining of tendons or joints and the
lining herniates out of the ligamentous defect causing a bump under
the skin. Because there is often inflammation associated, the
inflamed tissue produces a jelly-like fluid that fills the
protruding sac. They may be rock hard due to a high pressure of the
mucous like fluid contained within the cyst, and are often mistaken
for a bony prominence.
[0660] sHASEGP can be used to ameliorate ganglion cysts.
Intralesional injection of sHASEGP from 5-1000 Units followed by
fine needle aspiration will remove the cyst without the need for
surgery. Corticosteroids may be optionally injected as well with
the sHASEGP. Additional injection may be required for some
patients.
[0661] Myxedema
[0662] Glycosaminoglycan (GAG) infiltration of the skin is a
feature of hyperthyroidism, hypothyroidism, pretibial myxedema,
scleromyxedema, and scleredema. Hyaluronic acid is the main GAG in
all the conditions and in normal skin. There is minimal histologic
variability of GAG dermal distribution. The acquired cutaneous
mucinoses exhibit similar skin GAG distribution and biochemical
composition. The morphologic differences in fibroblastic activity
suggest that the mucinoses of scleredema and scleromyxedema
represent a local process, whereas the GAG infiltration of thyroid
diseases may have a systemic origin. These disorders may be
ameliorated with a sHASEGP from both a local and systemic route of
administration. For chronic therapy, a PEGylated sHASEGP may be
envisioned.
[0663] Pulmonary Uses of sHASEGP
[0664] Levels of Hyaluronan in broncheoalveolar lavages (BAL) from
normal individuals are generally below 15 ng/ml. However, BAL
levels rise dramatically in conditions of respiratory distress
(Bjermer Br Med J (Clin Res Ed) Oct. 3, 1987;295(6602):803-6). In
ARDS for example, hyaluronan levels can increase to 500 ng/ml
whereas in farmers lung, BAL levels can surpass 1000 ng/ml
(Hallgren et al Am Rev Respir Dis. March 1989;139(3):682-7),
(Larrson et al Chest. January 1992;101(1):109-14). The increased
hyaluronan in the lung can prevent oxygen diffusion and gas
exchange as well as activating neutrophil and macrophage
responses.
[0665] Bovine preparations of hyaluronidase are no preferable for
the treatment of such conditions for a number of reasons. First,
slaughterhouse testes-derived preparations of hyaluronidase are
known to be contaminated with serine proteases such as acrosin.
Secondly, the foreign nature of the bovine enzymes increase the
probability of an anaphylactic reaction, which could result in
death of the patient. Thus a highly purified preparation of
recombinant human sHASEGP can be delivered by either pulmonary or
intravenous delivery. Human sHASEGP can also be administered to
patients suffering from other pulmonary complications that are
associated with elevated glycosaminoglycans or to enhance the
delivery of other co delivered molecules to the lung.
[0666] The invention will now be described in greater detail by
reference to the following non-limiting examples
EXAMPLE 1
Microtiter Based Hyaluronidase Assays
[0667] The following example provides for a rapid assay for
measurement of the hyaluronidase activity of sHASEGP. This assay
can be related to the TRU, the IU or NFU through use of a W.H.O.
standard preparation of hyaluronidase.
[0668] Biotinylated Hyaluromam Microtiter Assay
[0669] The free carboxyl groups on glucuronic acid residues of
Hyaluronan are biotinylated in a one step reaction using
biotin-hydrazide (Pierce), Sulfo NHS (Pierce) and 1-Ethyl
dimethylaminopropyl-carbodiimide (Sigma). This biotinylated HA
substrate is covalently coupled to a 96 well microtiter plate in a
second reaction. At the completion of the enzyme reaction, residual
substrate is detected with an avidin-peroxidase reaction that can
be read in a standard ELISA plate reader. As the substrate is
covalently bound to the microtiter plate, artifacts such as
pH-dependent displacement of the biotinylated substrate does not
occur. The sensitivity permits rapid measurement of Hyaluronidase
activity from cultured cells and biological samples with an
inter-assay variation of less than 10%.
[0670] a. Protocol
[0671] Preparation of Biotinylated HA Substrate
[0672] One hundred mg of HA (Sigma Chemicals) was dissolved in 0.1
M MES, pH 5.0, to a final concentration of 1 mg/ml and allowed to
dissolve for at least 24 hr at 4.degree. C. prior to coupling of
biotin. Sulfo-NHS (Pierce; Rockford Ill.) was added to the CS04 MES
solution to a final concentration of 0.184 mg/ml. Biotin hydrazide
(Pierce) was dissolved in DMSO as a stock solution of 100 mM and
added to the CS04 solution to a final concentration of 1 mM. A
stock solution of 1-ethyl -3-(3-dimethylaminopropyl)carbidodiimide
(EDAC) was prepared as a 100 mM stock solution in distilled water
and added to the HA biotin solution at a final concentration of 30
mM. This solution was left stirring overnight at 4.degree. C.
Unlinked biotin and EDAC were removed by dialysis against water
with 3 changes of 1000.times. volume of water. The dialyzed,
biotinylated HA (bHA) was aliquoted and stored at -20.degree. C.
for up to several months.
[0673] Sulfo-NHS was diluted to 0.184 mg/ml in water with the bHA
at a concentration of 0.2 mg/ml and pipetted into 96 well
COVALINK-NH plates (NUNC; Placerville N.J.) at 50 .mu.l per well.
EDAC was diluted to 0.123 mg/ml in water and pipetted into the
COVALINK-NH plates with the bHA solution resulting in a final
concentration of 10 .mu.g/well bHA and 6.15 .mu.g/well EDAC. The
plates were incubated overnight at 4.degree. C. or for 2 hr at
23.degree. C., which gave comparable results. After covalent
immobilization of bCS04 on the microtiter plates, the coupling
solution was removed by shaking and the plates were washed 3 times
in PBS containing 2M NaCl and 50 mM MgSO4 (Buffer A). The plates
could be stored at 4.degree. C. for up to one week.
[0674] The COVALINK-NH plates with immobilized bHA were
equilibrated with 100 .mu.l/well assay buffer--either 0.1 M
formate, pH 3.7, 0.1 M NaCl, 1% TRITON X-100 detergent, 5 mM
saccharolactone for lysosomal Hyaluronidase; or 10 mM Hepes PH 7.4
with 1 mM CaCl2 and 1 mg/ml Human Serum Albumin (ICN) for
neutral-active enzymes. A set of standards for the calibration of
enzyme activity against "relative Turbidity Reducing Units"
(rTRU's) was generated by diluting bovine testicular hyaluronidase
(Sigma Type VI-S) in neutral enzyme buffer from 1.0 to
1.times.10.sup.-6 rTRU/well and assaying 100 .mu.l/well in
triplicate. Samples of acid-active Hyaluronidase were diluted in
lysosomal assay buffer from 1:10 to 1:130,000 were pipetted in
triplicate at 100 .mu.l/well. For most assays of tissue extracts
and human plasma, a 30 min incubation at 37.degree. C. was
sufficient. Positive and negative control wells (no enzyme or no
ABC (see below), respectively) were included in triplicate.
[0675] The reaction was terminated by the addition of 200
.mu.l/well of 6M Guanidine HCl followed by three washes of 300
.mu.l/well with PBS, 2 M NaCl, 50 mM MgSO.sub.4, 0.05% TWEEN 20
detergent (Buffer B). An avidin biotin complex (ABC) kit (Vector
Labs; Burlingame Calif.) was prepared in 10 ml of PBS containing
0.1% TWEEN 20 detergent, which was preincubated for 30 min at room
temperature during the incubation. The ABC solution was added (100
.mu.l/well) and incubated for 30 min at room temperature. The plate
was washed five times with Buffer B, then an o-phenylenediamine
(OPD) substrate was added at 100 .mu.l/well by dissolving one 10 mg
tablet of OPD in 10 ml of 0.1 M citrate-PO.sub.4 buffer, pH 5.3 and
adding 7.5 .mu.l of 30% H.sub.2O.sub.2. The plate was incubated in
the dark for 10-15 min, then read using a 492 nm filter in an ELISA
plate reader (Titertek Multiskan PLUS; ICN) monitored by computer
using the Delta Soft II plate reader software from Biometallics
(Princeton N.J.). A standard curve using the bovine testicular
hyaluronidase was generated by a four parameter curve fit of the
commercial hyaluronidase preparation and unknown samples were
interpolated through their absorbance at 492 nm.
[0676] To analyze pH dependence of Hyaluronidases, purified
recombinant sHASEGP and bovine testicular hyaluronidase are used.
The pH dependence of enzyme activity is measured by diluting
purified sHASEGP or partially purified bovine testicular
hyaluronidase to 0.1 rTRU in the following buffers: 50 mM formate,
pH 3-4.5; 50 mM acetate, pH 5-6; 50 mM MES, pH 6-7; or 50 mM HEPES,
pH 7-8. Samples are assayed for 30 min at 37.degree. C. and
activity was expressed as a percent of maximal activity. NaCl was
not used in buffers, as it can alter the pH optima of testicular
hyaluronidase preparations (Gold, Biochem. J. 205:69-74, 1982;
Gacesa et al. Biochem. Soc. Trans. 7:1287-1289, 1979);
physiological salt concentrations (0.15 M) decreased the apparent
pH optimum, an effect that was more pronounced in purified
preparations of the testicular enzyme than in the original crude
sample.
[0677] b. Results
[0678] Hyaluronan was biotinylated in a one step reaction using
biotin-hydrazide and EDAC. By limiting the EDAC, which couples the
free carboxyl groups on HA with biotin hydrazide, only a small
fraction of the total glucuronic acid residues on HA were labeled.
This amount of EDAC (3.times.10.sup.-5 M) added to HA
(2.8.times.10.sup.-3 M) results in a maximum of one molecule of
biotin hydrazide coupled per 93 disaccharide units of HA.
[0679] A four-parameter curve fit of bovine testicular
hyaluronidase standard reactions measured at pH 3.7, and diluted
from 1.0 to 1.times.10.sup.-6 TRU/well, was prepared. Four
parameter curve fits were established from the equation
y=((A-D)/(1+(conc/C) B))+D) where log.sub.it y=ln (y'/1-y'),
y'=(y-D)/(A-D), B=-b/ln 10 and C=EXP (a/B). The four parameters
(A,B,C,D) were calculated with a software program that utilized the
2+2 algorithm with linear regression (Rodbard et al., Clin. Chem.
22:350, 1976). This curve fit incorporates the sigmoidal aspects
the standard curve. Optimal accuracy for measurement of a sample
typically occurs from 0.001 to 0.01 TRU/well for a 30 min
incubation. During a 60 min incubation, 1/1000th of a TRU is
detectable. A standard logarithmic curve also can be utilized over
a shorter range of values to establish a standard curve fit.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited-to such specific
embodiments.
EXAMPLE 2
Cloning of sHASEGP cDNA
[0680] Nucleic acid encoding Human sHASEGP may be obtained by one
skilled in the art through a number of procedures including, but
not limited to, artificial gene synthesis, RT-PCR, and cDNA library
hybridization (for example see, Gmachl et al FEBS 336(3) 1993,
Kimmel et al., Proc. Natl. Acad. Sci. USA 90 1993 10071-10075).
Alternatively, clones encoding human sHASEGP may be obtained from
IMAGE, or other suppliers of human gene sequences (Invitrogen Clone
ID IOH10647).
[0681] The full length human PH20 cDNA was calculated to be 2009
nucleotides in length and contained an open reading frame of 1530
nucleotides. The 5' UTR is unusually large, which can indicate a
retained intron and can inhibit translation by preventing the
ribosome from binding to the correct initiating methionine codon
due to 9 non coding start codons in the 5'UTR. The protein (Genbank
Accession number NP.sub.--003108) is predicted to comprise 509
amino acids SEQ ID No. 1 with a calculated molecular mass of 58
kDa.
[0682] For sequencing of clones, PCR amplified bands were excised,
and eluted with the Gel Extraction Kit (Qiagen) and cloned into the
appropriate vectors with compatible ends after restriction
digestion. All sequencing reactions were performed on double
stranded DNA with the Taq dye deoxy terminator cycle sequencing kit
(Applied Biosystems) according to the manufacturer's instructions,
and run on an ABI Prism.TM. automated sequencer (Applied
Biosystems).
[0683] The human PH-20 open reading frame was obtained by
amplifying a human testis cDNA library (Clontech, Palo Alto Calif.)
by Polymerase Chain Reaction using primers SEQ ID NO 14 and SEQ ID
NO 47. PCR products were digested with NheI and BamHI and cloned
into the NheI and BamHI sites of the vector IRESpuro2
(Clontech).
EXAMPLE-4
Isolation of SHASEGP From Human PH20 cDNA
[0684] A catalytically active secreted recombinant human sHASEGP
expression vector capable of effective glycosylation in mammalian
cells was generated as described below. Other expression constructs
with promoters and selection genes for different species such as
yeast and insect cells that are also capable of generating sHASEGP
are contemplated. Positive selection genes such as Glutamine
Synthase or Dihydrofolate Reductase (DHFR) may also be used. The
examples given below is not intended to restrict but is rather
provided as an example of several plasmid expression systems that
may be used.
[0685] In order to construct secreted forms of sHASEGP, truncation
mutants that lack the hydrophobic C terminal end were constructed.
Using a GPI cleavage prediction program the GPI anchor cleavage
site was located around amino acid position N 483 in the
full-length GPI-anchored protein. A set of seven nested 3' primers
were used to construct a set of seven truncated deletion mutants
lacking predicted GPI anchor starting at position Y 482 and deleted
progressively by one amino acid. These primers were designed to
have compatible NheI (5') and BamHI (3') sites to clone the
truncation mutants in vector Irespuro2 either untagged with a stop
codon in the 3' primer, or as a C terminus His tagged protein for
ease of purification and detection. For example reverse primers SEQ
ID No. 8, SEQ ID No. 9, and SEQ ID No. 10 were used to generate
deletion mutants ending at position Y 482, F 481 and I 480 without
a 6 His tag. Other mutant primers were generated with the same base
design with the appropriate modifications to include and exclude
the particular amino acids. For generating His-tagged variants the
same set of primers are used as for non tagged variants except that
primers lack the stop codon in the respective reverse primers, the
forward primer remaining the same (for His tagged construction
refer to primers with SEQ ID No 19, 20, 21, 22, 23,24 and 25 which
are the reverse primers without stop codon corresponding to non
tagged reverse primers for their respective constructs).
Overlapping primers were used to construct a six amino acid spacer
followed by hexahistidine within BamH1 and Not1 sites in Irespuro2
vector such that His-tagged mutants were generated by ligation of
the PCR amplified and restriction digested products within the Nhe1
and BamH1 sites in the his tag containing Irespuro2 vector.
[0686] To identify whether human sHASEGP could be modified at its
carboxy terminus to generate a secreted and neutral active enzyme,
a series of truncations were made from the GPI anchor attachment
site to the predicted "catalytic domain" based upon homology with
the bee venom enzyme.
[0687] DNA encoding the human sHASEGP full length GPI anchored
clone in IRESPuro2 was used as a template to generate the various
truncated deletion mutants. Software modeling programs gave several
predicted cleavage sites for the full length polypeptide. One of
such predicted sites was at amino acid position N 483 (SEQ ID No.
1). PCR primers were designed to successively truncate the protein
from N483 to generate six deletion mutants starting at Y 482
(lacking N) and ending at E 477(lacking P).
[0688] a. Protocol
[0689] Generating truncation mutant lacking N483:
[0690] The full length GPI anchored sHASEGP clone between Nhe1 and
BanH1 site in pIRESPuro2 was used as a template. This template was
amplified with 5' primer containing NheI site that starts at
starting Methionine of the native signal peptide at M 1(SEQ ID No.
14), and a 3' primer containing BamHI site that ends at Y 482 (SEQ
ID No. 8). The PCR product was ran on a 1% agarose gel to resolve
and confirm the correct sized amplified band, gel purified,
restriction digested with NheI and BamHI and cloned into vector
pIRESPuro2 (Clontech) between NheI and BamHI sites generating an
expression vector for expressing this truncation mutant of SHASEGP
ending at amino acid position N482 and lacking the GPI anchor with
amino acid sequence (SEQ ID No. 5 for the sequence of the resulting
polypeptide of sHASEGP up to Y 482) and nucleotide sequence (SEQ ID
No. 48--coding nucleotides for polypeptide in SEQ ID No. 5 ) as
indicated.
[0691] Generation of the other truncation mutants lacking Y 482, F
481, I 480, Q 479, and P 478 respectively.
[0692] The same strategy was used with the only difference being
using the appropriate 3' primer for each mutant. The respective 3'
primers are as follows: [0693] 3' primer for sHASEGP mutant that
lacks the Y 482--SEQ ID No. 9 [0694] 3' primer for mutant that
lacks the F 481--SEQ ID No. 10 [0695] 3' primer for mutant that
lacks the I 480--SEQ ID No. 11 [0696] 3' primer for mutant that
lacks the Q 479--SEQ ID No. 12 [0697] 3' primer for mutant that
lacks the P 478--SEQ ID No. 13
[0698] Generating further deletion mutants to determine the
minimally active domain of sHASEGP:
[0699] Further deletions, in blocks of ten to twenty amino acids
were made from the 3' end of innermost neutral pH active truncation
mutant of sHASEGP, which is sHASEGP up to E 477. The NheI forward
primer SEQ ID No. 14) was used with an appropriately positioned 3'
primer to PCR amplify a deletion mutant of sHASEGP of the desired
length from the carboxy terminal end. For example PCR with primers
described in SEQ ID No. 14 and SEQ ID No. 26 as the 5' and 3'
primers respectively was used to generate the polypeptide in SEQ ID
No. 49 when expressed from an expression construct in IresPuro2
vector. Similarly, PCR with reverse3' primers described in SEQ ID
No 27,28,29,30,31 and 32 were used to generate deletion mutants
ending at amino acid positions A 447, S 430, G 413, A 372, and S
347 respectively of the mature sHASEGP. The PCR products in each
case were digested with NheI and BamHI enzymes and the digested
products cloned into pIresPuro2 vector between NheI and BamHI
sites. A few independent clones in the final expression construct
from each group were tested for secreted neutral active sHASEGP
activity by transient transfection in CHO cells in CD-CHO serum
free media (Invitrogen, Calif.) and samples drawn at indicated time
points for assay. Miniprep DNA prepared from overnight cultures was
transfected with Genejuice (Novagen, Calif.) transfection reagent
following manufacturer recommended protocols. Hyaluronidase
activity was measured by microtiter assay as described above.
[0700] b. Results
[0701] Hyaluronidase activity was measured in sHASEGP truncation
mutants to identify the minimally active domain for secreted
neutral active hyaluronidase activity. TABLE-US-00004 AMINO ACID 1
TO: U/ML/24 HRS PH7.4 347 0.000 372 0.000 394 0.000 413 0.000 430
0.000 447 0.000 467 0.089 477 0.567 478 0.692 479 0.750 480 0.575
481 0.740 482 0.329 483 0.800 509 0.044
[0702] The results showed that all six one amino acid deletion
mutants ending at indicated amino acids form Y to E 477 gave higher
secreted activity than GPI anchored sHASEGP.
[0703] The results showed that deletions beyond A 467 eliminated
any secreted activity. Secreted neutral activity from the A 467
clones decreased to approximately 10% of that found P478 or N 483
clones. It was therefore concluded that more of the carboxy
terminal domain of human sHASEGP was required to create the neutral
active hyaluronidase domain than previously assumed from the bee
venom enzyme. The cysteines in the carboxy terminal domain are thus
necessary for neutral activity. A very narrow range spanning
approximately 10 amino acids before the GPI cleavage site at N 483
thus defined the minimally active domain.
EXAMPLE-5
Effects of Signal Peptide Modification on SHASEGP Secretory
Activity
[0704] Hyman sHASEGP possesses an unusually long predicted native
leader peptide. Additionally, the existence of two adjacent
cysteine residues in the leader peptide may lead to aggregation of
polypeptide multimers within the endoplasmic reticulum during high
level expression and therefore prevent high level expression of a
sHASEGP. A series of more efficient secretory leader peptides were
therefore tested to examine for their ability to enhance the
targeting of sHASEGP for secretion.
[0705] a. Protocol
[0706] The Kappa leader peptide was constructed by overlapping
primer annealing and extension PCR with primers corresponding to
sequences in SEQ ID No 37, 38, 39 and 40. The resulting PCR
amplified kappa sequence was amplified with flanking primers
containing NheI site in the 5' end (as described in SEQ ID No. 41)
and EcoR1 site at the 3' end (as described in SEQ ID No. 42). This
allowed cloning the Kappa leader peptide (the polypeptide sequence
is as described in SEQ ID No. 43) in the Litmus 39 (NEB) vector
between NheI and EcoRI sites. sHASEGP has an internal EcoRI site;
therefore this kappa construct between NheI site and EcoRI site was
further amplified with a 5' SpeI primer (as described in SEQ ID No.
44) and a 3' MluI primer (as described in SEQ ID No. 45). sHASEGP
without GPI anchor ending at P 478 was cut out from pIresPuro2 with
NheI and BamHI and cloned into a Litmus 39(NEB) vector within the
NheI and BamHI sites of the Litmus39 vector. This resulting
sHASEGP-containing Litmus vector was digested with SpeI and MluI
restriction enzymes and the kappa leader construct amplified with
SpeI and MluI was cloned into it. Site directed mutagenesis was
performed on this Litmus 39 vector containing both Kappa and
sHASEGP sequences to generate the in frame fusion of Kappa leader
sequence to the mature polypeptide of sHASEGP. Primer pairs
corresponding to SEQ ID No. 34 and 35 were used to generate the
kappa leader with the native Asp as the terminal amino acid fused
to the F 38 of sHASEGP (up to P 478) (as described in SEQ ID No. 46
for the polypeptide sequence of the fusion protein). Other primer
pair combinations such as embodied by SEQ ID No. 33 with SEQ ID No.
35 were used to generate Kappa leader ending at the terminal Asp
(D) fused to L 36 of SHASEGP, SEQ ID No. 33 with SEQ ID No. 36 were
used to generate Kappa leader ending at the Gly (G) (before the
terminal Asp (D)) fused to L 36 of SHASEGP, and SEQ ID No. 34 with
SEQ ID No. 36 were used to generate Kappa ending at the Gly (G)
(before the terminal Asp (D)) fused to F 38 of SHASEGP. The
Kappa--sHASEGP fusions obtained by site directed mutagenesis were
gel purified, digested with enzyme DpnI to digest any carryover
parental DNA, and then digested with NheI and Bam HI and cloned in
to the NheI/BamHI digested.HisIresPuro2 backbone which has the his
tag (six amino acid spacer followed by six histidines) cloned in
between BamH1 and Not1 sites in pIRESPuro2 vector. Therefore upon
ligation we obtain a construct that is NheI-kappa-SHASEGP-BamHI-His
in pIresPuro2. Four sets of such construct were obtained that would
correspond to the combinations of G or D at the Kappa leader end
and L 36 or F 38 at the beginning of mature sHASEGP. A few
independent clones from each type of construct were transfected
into CHO cells in CD-CHO medium (Invitrogen, Calif.) to test
whether the presence of kappa secretion leader would promote
increased levels of secreted protein as compared to native
secretion leader. Miniprep DNA prepared from overnight cultures
were transfected with Genejuice (Novagen, Calif.) transfection
reagent following manufacturer recommended protocols and samples
were drawn for testing by microtiter assay at indicated time
points. Hyaluronidase activity was measured by microtiter assay as
described above.
[0707] Mouse IgG Kappa chain leader peptide sHASEGP fusion
constructs were tested to test for higher levels of secreted
neutral active sHASEGP activity.
[0708] b. Results TABLE-US-00005 HUMAN sHASEGP GENE CONSTRUCT
U/ML/24HOURS PH 7.4 IgG Kappa Leader sHASEGP AA 38-478 3.0257 HIS6
Native Leader sHASEGP AA 1-478 HIS6 0.4857
[0709] The enzyme assay results indicated that the IgG Kappa leader
was capable of enhancing secretion of sHASEGP approximately 7 to 8
fold higher than the native secretion leader when compared with
clones P478, Y 482 or N 483 that lacked such a leader. Other kappa
leader constructs with variations of the leader fusion site from
the Asp or the Gly of the Kappa leader to L36 or F38 of sHASEGP
yielded increased levels of secreted neutral active hyaluronidase
activity as well. These examples are intended to expand rather than
limit the scope of the invention, as other efficient secretory
leader sequences may be utilized with the same technology.
EXAMPLE 6
Generation of a Human sHASEGP Expression Vector
[0710] A sHASEGP without an epitope tag was generated by cloning
into a bicistronic expression cassette, HZ24 (SEQ ID NO 47). The
HZ24 plasmid vector for expression of sHASEGP comprises a pCI
vector backbone (Promega), DNA sequence encoding amino acids 1-482
of human PH20 hyaluronidase, an internal ribosomal entry site
(IRES) from the ECMV virus (Clontech), and the mouse dihydrofolate
reductase (DHFR) gene. The pCI vector backbone also includes DNA
encoding the Beta-lactamase resistance gene (AmpR), an fl origin of
replication, a Cytomegalovirus immediate-early enhancer/promoter
region (CMV), a chimeric intron, and an SV40 late polyadenylation
signal (SV40). The DNA encoding the sHASEGP construct contained a
Kozak consensus sequence in the Methionine of the native signal
leader and a stop codon at Tyrosine 482. The resultant construct
pCI-PH20-IRES-DHFR-SV40pa (HZ-24) results in a single mRNA species
driven by the CMV promoter that encodes amino acids 1-482 of PH20
and amino acids 1-187 of the dihydrofolate reductase separated by
the internal ribosomal entry site.
[0711] The human PH20 open reading frame was amplified from an
Invitrogen ORF clone (IOH10647, Invitrogen, Carlsbad Calif.) with a
5' Primer that introduced an NheI site and Kozack consensus
sequence before the Methionine of PH20 and a reverse primer that
introduced a stop codon following Tyrosine 482 and introduced a
BamH1 restriction site. The resultant PCR product was ligated into
the plasmid pIRESpuro2 (Clontech, Palo Alto, Calif.) following
digest of the PH20 PCR fragment with NheI and BamH1.
EXAMPLE-7
Generation of a sHASEGP Expressing Cell Line
[0712] Non-transfected DG44 CHO cells growing in GIBCO Modified
CD-CHO media for DHFR(-) cells, supplemented with 4 mM Glutamine
and 18 ml Plurionic F68/L (Gibco), were seeded at
0.5.times.10.sup.6 cells/ml in a shaker flask in preparation for
transfection. Cells were grown at 37.degree. C. in 5% CO.sub.2
humidified incubator with 120 rpm for shaking. Exponentially
growing non-transfected DG44 CHO cells were tested for viability
prior to transfection.
[0713] 60,000,000 viable cells of the non-transfected DG44 CHO cell
culture was pelleted and resuspended to a density of 20,000,000
cells in 0.7 mL of 2.times. transfection buffer (2.times.HeBS=40 mM
Hepes, pH 7.0, 274 mM NaCl, 10 mM KCl, 1.4 mM Na.sub.2HPO.sub.4, 12
mM dextrose). To each aliquot of resuspended cells, 0.09 mL of the
linear HZ24 plasmid (250 .mu.g) was added, and the cell/DNA
solutions were transferred into 0.4 cm gap BTX (Gentronics)
electroporation cuvettes at room temperature. A negative control
electroporation was performed with no plasmid DNA mixed with the
cells. The cell/plasmid mixes were electroporated with a capacitor
discharge of 330 V and 960 .mu.F or at 350 V and 960 .mu.F.
[0714] The cells were removed from the cuvettes after
electroporation and transferred into 5 mL of Modified CD-CHO media
for DHFR(-) cells, supplemented with 4 mM Glutamine and 18 ml
Plurionic F68/L (Gibco), and allowed to grow in a well of a 6-well
tissue culture plate without selection for 2 days at 37.degree. C.
in 5% CO.sub.2 humidified incubator.
[0715] Two days post electroporation, 0.5 mL of tissue culture
media was removed from each well and tested for presence of
hyaluronidase activity.
[0716] Initial Hyaluronidase Activity of HZ24 Transfected DG44 CHO
Cells At 40 Hours Post Transfection TABLE-US-00006 Activity
Dilution Units/ml Transfection 1 1 to 10 0.25 330 V Transfection 2
1 to 10 0.52 350 V Negative 1 to 10 0.015 Control
[0717] Cells from transfection 2 (350V), were collected from the
tissue culture well, counted and diluted to 10,000 to 20,000 viable
cells per mL. A 0.1 mL aliquot of the cell suspension was
transferred to each well of five, 96 well round bottom tissue
culture plates. 0.1 mL of CD-CHO media (GIBCO) containing 4 mM
Glutamax-1, and without hypoxanthine and thymidine supplements were
added to the wells containing cells (final volume 0.2 mL).
[0718] Ten clones were identified from the 5 plates grown without
methotrexate. TABLE-US-00007 Relative Plate/Well Hyaluronidase ID
Activity 1C3 261 2C2 261 3D3 261 3E5 243 3C6 174 2G8 103 1B9 304
2D9 273 4D10 302 1E11 242 A1 (+) 333 control H12 (-) 0 control
[0719] Six HZ24 clones were expanded in culture and transferred
into shaker flasks as single cell suspensions. Clones 3D3, 3E5,
2G8, 2D9, 1E11, and 4D10 were plated into 96-we round bottom tissue
culture plates using a two-dimensional infinite dilution strategy.
Diluted clones were grown in a background of 500 non-transfected
DG44 CHO cells per well, to provide necessary growth factors for
the initial days in culture. Ten plates were made per subclone.
[0720] Clone 3D3 produced 24 visual subclones. Significant
hyaluronidase activity was measured in the supernatants from 8 of
the 24 subclones (>50 Units/mL), and these 8 subclones were
expanded into T-25 tissue culture flasks in the presence of 50 nM
methotrexate. Clone 3D3 50 nM was further expanded in 500 nM
methotrexate giving rise to clones producing in excess of 1,000
Units/ml in shaker flasks (clone 3D3 5M).
EXAMPLE 8
Production of sHASEGP
[0721] A vial of 3D3 5M was thawed and expanded from T flasks
through 1 L spinner flasks in CHO CDM (Invitrogen, Carlsbad Calif.)
supplemented with 100 nM Methotrexate and Glutamax (Invitrogen).
Cells were transferred from spinner flasks to a 5 L bioreactor
(Braun) at an inoculation density of 4.0.times.10E5 viable cells
per ml. Parameters were temperature setpoint, 37C , pH 7.2
(starting Setpoint), with Dissolved Oxygen Setpoint 25% and an air
overlay of 0-100 cc/min. At 168 hrs, 250 ml of Feed #1 Medium (CD
CHO+50 g/L Glucose) was added. At 216 hours, 250 ml of Feed #2
Medium (CD CHO+50 g/L Glucose+10 mM Sodium Butyrate) was added, and
at 264 hours 250 ml of Feed #2 Medium was added. This process
resulted in a final productivity of 1600 Units per ml with a
maximal cell density of 6 million cells/ml. The addition of sodium
butyrate was found to dramatically enhance the production of
sHASEGP in the final stages of production. TABLE-US-00008 3D3-5M
Growth & sHASEGP Production, 5 L Bioreactor Viable Run Cells %
Hours .times.10E5 Viable Units/ml Vol (mL) [Glucose] Feed 0 4.4 100
0 4500 547 24 5.7 100 0 4500 536 48 10.1 100 37 4500 501 72 17.1 99
62 4500 421 96 28.6 99 118 4500 325 120 28.8 99 240 4500 274 144
60.2 100 423 4500 161 168 55 100 478 4500 92 250 ml Feed #1 192
66.6 98 512 4750 370 216 55.2 92 610 4750 573 250 ml Feed#2 240 53
88 710 5000 573 264 49.8 84 852 5000 474 250 ml Feed #2 288 40 70
985 5250 770 312 31 61 1467 5250 773 336 25.4 52 1676 5250 690
EXAMPLE 9
Purification of sHASEGP
[0722] Conditioned media from the 3D3 clone was clarified by depth
filtration and tengenial flow diafiltration into 10 mM Hepes pH
7.0. Soluble HASEGP was then purified by sequential chromatography
on Q Sepharose (Pharmacia) ion exhange, Phenyl Sepharose
(Pharmacia) hydrophobic interaction chromatography, phenyl boronate
(Prometics) and Hydroxapatite Chromatography (Biorad, Richmond,
Calif.).
[0723] SHASEGP bound to Q Sepharose and eluted at 400 mM NaCl in
the same buffer. The eluate was diluted with 2M Ammonium sulfate to
a final concentration of 500 mM ASO4 and passed through a Phenyl
Sepharose (low sub) column, followed by binding under the same
conditions to a phenyl boronate resin. The sHASEGP was eluted from
the phenyl sepharose resin in Hepes pH 6.9 after washing at pH 9.0
in 50 mM bicine without AS04. The eluate was loaded onto a ceramic
hydroxyapatite resin at pH 6.9 in 5 mM PO4 1 mM CaCl2 and eluted
with 80 mM PO4 pH 7.4 with 0.1 mM CaCl.sub.2.
[0724] The resultant purified sHASEGP possessed a specific activity
in excess of 65,000 USP Units/mg protein by way of the
microturbidity assay using the USP reference standard. Purified
sHASEGP eluted as a single peak from 24 to 26 minutes from a
Pharmacia 5RPC styrene divinylbenzene column with a gradient
between 0.1% TFA/H.sub.2O and 0.1% TFA/90% acetonitrile/10%
H.sub.2O and resolved as a single broad 61 kDa band by SDS
electrophoresis that reduced to a sharp 51 kDa band upon treatment
with PNGASE-F. N-terminal amino acid sequencing revealed that the
leader peptide had been efficiently removed.
[0725] N-terminal Amino Acid Sequence biochemically purified
sHASEGP. TABLE-US-00009 Position 1 2 3 4 5 6 7 8 9 10 11
Theoretical Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Observed --
Asn Phe Arg Ala Pro Pro Val Ile Pro Asn
EXAMPLE 10
Analysis of DG44 CHO-Derived sHASEGP Glycosylation
[0726] Conflicting data exists as to whether sHASEGPs from
different species require glycosylation for their catalytic
activity. For example, it is reported that enzymatically active bee
venom hyaluronidase can be synthesized in cells that lack
glycosylation machinery, i.e. such as E. coli. Moreover, treatment
of purified bovine testes hyaluronidase with PNGase did not
inactivate enzyme activity (Yamagata et al 1997). Other studies
report loss of activity following deglycosylation and that
disulfide bonds are additionally required.
[0727] As all such previous tests were made using either crude or
partially purified preparations however, it was not apparent
whether the loss of activity was a result of exposure of
deglycosylated enzyme to contaminating proteases in the crude
preparations or a direct functional relationship between
glycosylation and catalytic activity.
[0728] a. Protocol
[0729] To determine if functional N-linked glycosylation could be
introduced into human sHASEGP using a CHO based expression system
under protein free conditions, a cDNA encoding human sHASEGP-HIS6
was expressed in CHO cells using an IRESpuro bicistronic cassette
in chemically defined media. Cells were grown for 72 hours in CHO
CDM (Invitrogen/Gibco) followed by concentration and tangential
flow diafiltration on a Pellicon TFF unit (Millipore) with 30 kDa
cutoff membranes. The concentrate was exchanged with 10 mM Hepes PH
7.4 50 mM NaCl. The diafiltrate was then loaded on a DEAE
streamline sepharose resin and eluted with a NaCl gradient from 0-1
M NaCl on a Pharmacia FPLC resin. Human sHASEGP eluted between
10-30% NaCl. Levels of sHASEGP in column fractions determined that
the majority of enzyme was recovered in the 10-30% NaCl gradient.
The enzyme from the 10-30% NaCl gradient was then further purified
through affinity chromatography on an IMAC resin charged with Ni.
Human sHASEGP was eluted from the IMAC resin after washing with 10
mM Imidizole with 50 mM Acetate PH 5.0. The protein was
concentrated and dialyzed against 10 mM Hepes PH 7.4. The highly
purified enzyme was determined to possess a specific activity of
97,000 Units/mg protein in the presence of 1 mM Calcium and 1 mg/ml
HSA in the ELISA-based biotinylated substrate microtiter assay.
[0730] To detect changes in protein relative molecular mass,
purified human sHASEGP was treated with PNGASE or Neuraminidase
overnight followed by gel electrophoresis, electotransfer and
western blot analysis with an HRP linked anti His6 monoclonal
antibody (Qiagen) and ECL detection.
[0731] b. Results
[0732] Western blot analysis determined that the human sHASEGP
produced in CHO cells was sensitive to PNGASE treatment. The
relative molecule mass of human sHASEGP revealed that the protein
was highly glycosylated. Upon complete overnight digestion with
PNGASE, human sHASEGP reduced to a single species confirming that
mild heterogeneity of the undigested band could be attributed to
N-linked sugar residues. PNGaseF partial digestion showed a series
of intermediates shifting from untreated and progressive shift with
longer treatment. Although bands were somewhat diffuse on a 7% Gel,
at least 6 different intermediate isoforms could be visualized.
[0733] Treatment of sHASEGP with Neuraminidase revealed that CHO
cells were in fact capable of synthesizing sialated human sHASEGP.
Upon treatment with neuraminidase and Western Blot analysis of
sHASEGP on 7% Gels, CHO derived Human recombinant sHASEGP revealed
an approximately 1-3 kDa shift in mobility compared to untreated
sHASEGP. This is thus the first report of the generation of a
substantially sialated human sHASEGP. This is very valuable for
both stability and to enhance serum half-life of a human sHASEGP as
native sperm sHASEGP from many species lacks sialation and does not
react with sialic acid specific lectins.
[0734] FACE Analysis of sHASEGP
[0735] Analysis of active sHASEGP oligosaccharides by FACE analysis
permits rapid determination of profiles of catalytically active
sHASEGPs.
[0736] Protocol
[0737] Purified Hyaluronidase from the 3D3 5M clone was evaluated
using FACE.TM. N-Linked Oligosaccharide Profiling (Prozyme).
Oligosaccharides were cleaved from 128.7 .mu.g of glycoproteins by
enzymatic digestion with N-Glycanase (a.k.a. PNGase), labeled using
the fluorophore ANTS, and separated by electrophoresis. The
relative positions of the oligosaccharide bands were determined by
running the sample and dilutions of the sample alongside an
oligosaccharide standard ladder which designated the migration
distance in Degree of Polymerization (DP) units.
[0738] Results
[0739] The N-Profile for the Hyaluronidase sample consists of ten
bands of which six (running concomitant with the oligosaccharide
standard bands G5-G12) had intensities greater than 9%.
Furthermore, the band running alongside the G9 standard was the
most intense with intensities of 35% -46%.
[0740] sHASEGP Oligosaccharide Analysis TABLE-US-00010 sHASEGP
Degree of Oligosaccharide Polymerization Percent of Total 1 15.64
1.2 2 13.68 3.4 3 11.61 10.0 4 10.04 10.4 5 8.37 35.4 6 7.32 9.7 7
6.14 9.0 8 5.57 12.4 9 3.84 2.3 10 3.26 0.5
EXAMPLE 11
Dependence of SHASEGP N-Linked Glycosylation for Enzyme
Activity
[0741] a. Protocol
[0742] Samples of purified HIS6 sHASEGP were mixed with buffer
containing Neuraminidase and PNGASE with and without 50 mm
Octylglucoside overnight at 37 C. Oligosaccharides were verified to
have been removed by gel shift from Western Blot analysis.
[0743] b. Results TABLE-US-00011 SAMPLE U/ML No Rx 22.01
Neuraminidase O/N 50 mM OG 23.57 PNGaseF w/ 50 mM OG 0.0 PNGaseF
without 50 mM OG o/n 10.74
EXAMPLE-12
Activity of SHASEGP Towards Sulfated and Non-Sulfated
Glycosaminoglycans
[0744] In addition to the microtiter-based assay using HA, the
substrate specificity of sHASEGP towards other glycosaminoglycans
or proteoglycans can be tested using a gel shift assay with
purified substrates to determine the activity of sHASEGP towards
other glycosaminoglycans. Many Hyaluronidase assays have been based
upon the measurement of the generation of new reducing
N-acetylamino groups (Bonner and Cantey, Clin. Chim. Acta
13:746-752, 1966), or loss of viscosity (De Salegui et al., Arch.
Biochem. Biophys. 121:548-554, 1967) or turbidity (Dorfman and Ott,
J. Biol. Chem. 172:367, 1948). With purified substrates all of
these methods suffice for determination of the presence or absence
of endoglucosamidic activity.
[0745] a. Protocol
[0746] GEL SHIFT ASSAY--Purified substrates are mixed with
recombinant sHASEGP to test for endoglucosidase activity that give
rise to increased mobility in substrate within the gel. Chondroitin
Sulfate A, Aggrecan and D were from Calbiochem. Hyaluronan (Human
Umbilical Cord) Chondroitin Sulfate C, Dermatan sulfate, and
Heparan-sulfate are obtained from Calbiochem. Human umbilical cord
Hyaluronan was obtained from ICN. Each test substrate is diluted to
0.1 mg/ml. 10 .mu.l samples of purified sHASEGP or conditioned
media from sHASEGP expressing cells as well as are mixed with 90 ul
of test substrate in desired buffer and incubated for 3 hours at 37
C. Following incubation samples are neutralized with sample buffer
(Tris EDTA PH 8.0, Bromophenol Blue and glycerol) followed by
electrophoresis on 15% polyacrylamide gels. Glycosaminoglycans are
detected by staining the gels in 0.5% Alcian Blue in 3% Glacial
Acetic Acid overnight followed by destaining in 7% Glacial Acetic
Acid. Degradation is determined by comparison substrate mobility in
the presence and absence of enzyme.
[0747] b. Results
[0748] 100 Units of sHASEGP.sub.HIS6 in 10 ul was incubated with 90
ul 10 mM Hepes Buffer with 50 ug/ml Human Serum Albumin for 2 hours
at 37 C containing boug of various glycosaminoglycans and
proteoglycans. Electrophoretic analysis followed by Alcian blue
staining revealed increased mobility shifts to a single species in
Chondroitin Sulfate A, C and D, Aggrecan and Hyaluronan but not
Heparan Sulfate nor Chondroitin Sulfate B. Whereas the undigested
glycosaminoglycans ran as a smear in the middle of the gel, the
digested products showed the majority of alcian blue stain running
at the dye front with a small amount of material running as an
incremental ladder.
EXAMPLE-13
Effects of Metal Ions on sHASEGP Activation
[0749] In addition to the requirement of glycosylation for optimal
enzyme activity, human sHASEGP was found to be activated with
cations for optimal enzyme activity. In the process of
purification, sHASEGP was found to have a low specific acitivity
following successive chromatography steps. The HIS6tagged sHASEGP
was found to have a very low specific activity when purified to
homogeneity from DEAE followed by successive Ni-IMAC purifications.
As IMAC resins can chelate metal ions, various metals were added
back to sHASEGP to determine the relative enzyme activity.
[0750] a. Protocol
[0751] Purified sHASEGP was tested following incubation with 0.1 mM
nickel (Ni), Cobalt (Co) Zinc (Zn) Calcium (Ca) and Magnesium (Mg)
for 2 hours at room temperature followed by determination of
hyaluronidase activity in microtiter based assay.
[0752] b. Results TABLE-US-00012 Metal Salt Additive Neutral
Activity U/ml NO ADDITIVES 11.909 100 uM Ni 6.0306 100 uM Co 8.972
100 uM Zn 3.7476 100 uM Ca 101.9892
[0753] A significant increase in hyaluronidase activity was found
following incubation of sHASEGP with 0.1 mM Calcium or 0.1 mM
Magnesium. No such activation was found following incubation with
other metals. The addition of Calcium to sHASEGP increased the
specific activity of the enzyme to approximately 97,000 units per
mg protein based upon A280 measurement. A dose response curve of
Calcium and Magnesium metals was then tested to determine the
optimal concentration of metal ions to enzyme. TABLE-US-00013 mM
Divalent Metal [Ca++] [Mg++] 100 1 1.3 10 108 104 1 169 164 0.1 123
78 0.01 59 18 0.001 47 13 0.0001 39 13 0.00001 55 15
[0754] Activation of sHASEGP was found to occur in the micromolar
range. Concentrations above 10 mM were inhibitory for both Calcium
and Magnesium. To rule out nonspecific activation of substrate
rather than enzyme, Calcium Chloride in 10 mM Hepes buffer was
incubated with the immobilized biotinylated substrate on the
microtiter plate followed by washing. No activation was found when
the enzyme was added to the Calcium preincubated plate that had
been washed. The activation was also tested on phospholipase C
released native sHASEGP which revealed a similar activation with
Calcium ruling out an artifact of the carboxy terminus HIS6 epitope
tag.
EXAMPLE 14
Effects of Albumin on the Activity of sHASEGP
[0755] It was found that the dilution of recombinant rHUPH20 and
other preparations of slaughterhouse testes-derived hyaluronidases
required albumin in addition to Calcium for optimal activity.
[0756] a. Protocol
[0757] Human Serum Albumin (ICN) was diluted into 10 mM Hepes
buffer with Calcium to determine the effects of albumin protein on
enzyme activity. Enzyme assays with sHASEGP and commercial
preparations were examined using both 1 mM CaCl.sub.2 and 1 mg/ml
Human Serum Albumin.
[0758] b. Results
[0759] Activation of hyaluronidase activity was found at high
dilutions in the presence of albumin. It was not clear whether this
activation was a result of preventing denaturation or if the
albumin affected the availability of the substrate. A preferable
formulation of human sHASEGP could therefore include Albumin and a
metal salt consisting of either Calcium or Magnesium.
EXAMPLE-15
Spreading Activity of Purified sHASEGP in vivo
[0760] a. Protocol
[0761] Purified sHASEGP in 10 mM Hepes PH 7.4, 150 mM NaCl 0.1%
Pluronic was diluted to 0.5 U/ul in pyrogen free water with 0.15 M
NaCl. A series of dilutions in 20 ul final of Saline were made to
give a total of 0.01, 0.05, 0.1 Units per injection. 20 ul of
Trypan Blue solution was added to a final volume of 40 ul and
injected subcutaneously into the lateral skin on each side of
balb.sup.Nu/Nu mice that had been previously anesthetized i.p. by
ketamine/xylazine administration. Dye areas were measured in 2
dimensions with a microcaliper from t=0 to t=45 min. Area was
represented as mm.sup.2. As a control recombinant Human HYAL1 that
lacks neutral activity but is secreted was included.
[0762] b. Results TABLE-US-00014 TEST ARTICLE DYE AREA @ 45 MIN A.
Saline Control 51.5 mm.sup.2 B. sHASEGP 0.01U 76.8 mm.sup.2 C.
sHASEGP 0.05U 98.22 mm.sup.2 D. sHASEGP 0.10U 180.4 mm.sup.2 E.
HYAL1 100U 67.48 mm.sup.2
EXAMPLE-16
Kinetics of sHASEGP Diffusion Activity
[0763] a. Protocol
[0764] Recombinant purified sHASEGP.sub.His6 was separated into 2
aliquots. One was heated to 95 C for 15minutes in a thermocycler
with a heated lid. The other remained at room temperature. Thermal
inactivation of enzyme activity was verified in the microtiter
based enzyme assay. For kinetic analysis heat inactivated verses
native material was tested. 4 Units of purified sHASEGP or
equivalent heat inactivated material was injected subcutaneously
with trypan blue dye. Areas were tested at various time points up
to 15 minutes.
[0765] b. Results TABLE-US-00015 4 UNITS 4 UNITS HEAT INACTIVATED
t.sub.minute post injection t.sub.minute post injection t.sub.0 =
52.38 t.sub.0 = 50.58 t.sub.3 = 116.51 t.sub.3 = 65.48 t.sub.6.5 =
181.93 T.sub.6.5 = 63.87 t.sub.10 = 216.96 T.sub.10 = 65.80
t.sub.16 = 279.99 T.sub.16 = 74.3
EXAMPLE-17
Restoration of the Dermal Barrier Broken Down by sHASEGP
[0766] a. Protocol
[0767] To establish the regeneration time of the pores opened with
sHASEGP following subcutaneous administration, 2 Units of purified
sHASEGP or saline control was injected into two opposing lateral
sites subcutaneously in animals at t=0 followed by injection with
trypan blue at the same site at 30 min 60 min and 24 hours. Area of
the dye diffusion at t=15 minutes post injection was recorded for
each time point compared to the control.
[0768] b. Results TABLE-US-00016 2 UNITS SALINE CONTROL T.sub.hour
post injection sHASEGP t.sub.hour post injection sHASEGP t.sub.0.5h
= 183 t.sub.0.5h = 54 t.sub.1hr = 167 t.sub.1hr = 50 t.sub.22hr =
61 t.sub.22hr = 48
[0769] The results demonstrate that the dermal barrier
reconstitutes within 24 hours of administration of 2 Units of
enzyme.
EXAMPLE-18
Determination of the Size of Channels Opened by sHASEGP
[0770] It was shown that human sHASEGP opened channels in the
interstitial space sufficient to permit the diffusion of a small
molecule, i.e. trypan blue dye. However, it was unknown what the
upper limits were on the size of particles that could diff-use in
the presence of sHASEGP.
[0771] a. Protocol
[0772] Florescent molecules of varying sizes were used to determine
the size of the channels opened by human sHASEGP, Fluoresceinated
Dextrans of 4,400 and 2 million Da Average Molecular Weight (Sigma)
as well as fluorescein labeled beads of defined diameters from 20
nanometers to 500 nanometers (Molecular Probes), were administered
subcutaneously in a volume of 40 ul with following injection of
sHASEGP or saline control in the same sites. Area of the dye front
was then measured in two dimensions at 15 minutes post
injection.
[0773] b. Results TABLE-US-00017 Diffusion Test Diffusion Agent
Particle Size Area at 15 min Stand Dev sHASEGP 4400 Da 84.2 25.7
Control 4400 Da 38.0 5.8 sHASEGP 2 .times. 10E6 Da 141.2 4.5
Control 2 .times. 10E6 Da 51.7 8.1 sHASEGP 20 nm Diameter 92.3 20.6
Control 20 nm Diameter 51.6 3.0 sHASEGP 100 nm 61.0 5.7 Diameter
Control 100 nm 40.0 7.0 Diameter sHASEGP 200 nm 35.5 1.6 Diameter
Control 200 nm 27.9 8.2 Diameter sHASEGP 500 nm 44.8 13.6 Diameter
Control 500 nm 41.2 9.8 Diameter
[0774] The results demonstrated that molecules from approximately 1
kDa (Trypan Blue) to 50 nm in diameter (Latex Beads) showed
enhanced diffusion following administration of sHASEGP. While
bovine serum albumin (66 kDA) showed similar kinetics of diffusion
to trypan blue, the 50 nm latex beads required significantly more
time to diffuse. 500 nm beads showed no diffusion up to 480
minutes.
EXAMPLE-19
Serum Pharmacokinetics Profiles of Biotinylated Antibodies
Following Subcutaneous Co-Injection of Human SHASEGP
[0775] a. Protocol
[0776] Female Balb/c mice were anesthetized with a mixture of
ketamine /xylazine. The mice were then injected subcutaneously with
20 ul of 0.5 mg/ml solution of biotinylated mouse IgG mixed with 20
ul of either saline or 20 ul sHASEGP containing 4 Units of
activity.
[0777] b. Results TABLE-US-00018 TIME POST INJECTION CONTROL
sHASEGP (4U) Serum IgG t = 0 hrs 0 ng/ml 0 ng/ml Serum IgG t = 2
hrs 0 ng/ml 360 ng/ml Serum IgG t = 51 hrs 4152 ng/ml 4176
ng/ml
[0778] The results demonstrate that sHASEGP increases the kinetics
of serum distribution of large molecules in circulation. Where no
biotinylated IgG could be detected in the control group at 2 hours,
360ng/ml was apparent by 2 hours in the sHASEGP group.
EXAMPLE-20
Spreading Activity of Subcutaneously Injected Molecules Following
Intravenous Injection of Human sHASEGP
[0779] a. Protocol
[0780] Four sites for dye injection were utilized per dose of each
Test Article and carrier control. Dye injection was 45 minutes
after i.v. injection. Each dose of test or control article was
injected i.v. into 2 animals. Measurement of the dye front area
post 45 minute enzyme administration was calculated at 2.5, 5, 10
and 15 minutes for each dose or carrier control.
[0781] b. Results
[0782] Results demonstrated that highly purified sHASEGP was
systemically available to distal tissues upon intravenous
administration. The spreading activity of systemically administered
sHASEGP was dose dependent, with a 10 unit injection being
indistinguishable from carrier control. TABLE-US-00019 Dose Time
Mean Type IV Minutes Area (mm.sup.2 SD PH20 1000 2.5 86.417
2.834193 PH20 1000 5 102.17 2.221146 PH20 1000 10 124.53 6.304944
PH20 1000 15 129.81 1.434319 PH20 300 2.5 59.137 7.218615 PH20 300
5 73.638 7.51197 PH20 300 10 87.092 8.686008 PH20 300 15 92.337
10.66466 PH20 100 2.5 56.308 7.741934 PH20 100 5 63.156 11.42052
PH20 100 10 76.519 16.18449 PH20 100 15 77.432 17.32264 PH20 30 2.5
50.534 10.64287 PH20 30 5 59.493 5.163971 PH20 30 10 68.102
11.00071 PH20 30 15 71.118 9.934212 PH20 10 2.5 36.4 3.807072 PH20
10 5 39.859 6.680932 PH20 10 10 45.649 4.44936 PH20 10 15 48.41
6.546835 Control 0 2.5 34.652 5.935037 Control 0 5 36.279 3.614544
Control 0 10 44.687 5.821216 Control 0 15 53.002 2.812439
EXAMPLE 21
Illustrative Use of Pegylation to Generate Modified sHASEGPs
Exhibiting Improved Pharmacokinetics
[0783] As an illustrative example of the use of pegylation to
generate modified forms of sHASEGPs having improved
pharmacokinetics, such as increased serum half life, we attached
either linear or branched polyethylene glycol (PEG) moieties to an
exemplary sHASEGP, recombinant human PH20 (rHuPH20) as described
above.
[0784] A number of different functionally activated groups can be
used for attaching PEG moieties to sHASEGPs such as rHuPH20
(including, by way of illustration, aldehydes, succinimides,
maleimides and others). A number of different pegylation reagents
are commercially available from various sources including Nektar
Therapeutics (www.nektar.com) as well as a number of other
suppliers (www.pharma.dow.com, www.nof.co.jp, www.sunbio.com,
www.celares.com). For purposes of illustration, rHuPH20 has been
effectively pegylated as described herein using a number of
illustrative PEG reagents to generate a number of different
PEGylated rHuPH20 enzymes. Analogous methodologies have also been
applied to other hyaluronidase enzymes, including an illustrative
animal-derived hyaluronidase (using a preparation of ovine
testicular hyaluronidase available as Vitrase.TM. from ISTA
Pharmaceuticals), an illustrative bacterial-derived hyaluronidase
(using a preparation of bacterial hyaluronidase available as
Hyaluronate Lyase from Sigma), and an illustrative
bacterial-derived chondroitinase having hyaluronidase activity
(using a preparation of bacterial chondroitinase ABC available as
Chrondroitinase ABC from Associates of Cape Cod, Inc./Seikagaku
Corporation).
[0785] In addition to varying the functional group, PEGs of a
variety of different lengths and structures are available which can
be incorporated using a particular functional group. For purposes
of illustration, PEG lengths ranging from 5 to 60 KDa (e.g. 5, 10,
20, 30, 40 and 60 KDa) have been conjugated to rHuPH20. Again a
variety of different PEGs having different lengths and structures
are readily available from sources including Nektar, the Dow
Chemical Corporation (Dowpharma), NOF Corporation, Sunbio, Celares
and others. A large number of references are known in the art
related to PEG reagents and methods of PEGylating proteins,
including a number of reviews and articles cited therein; see,
e.g., Roberts, M J et al. Advanced Drug Delivery Review 2002, 54:
459-476; Harris J M and Zalipsky, S (eds) of "Poly(ethylene
glycol), Chemistry and Biological Applications" ACS Symposium
Series 680 (1997); Mehvar, R et al. J. Pharm. Pharmaceut. Sci.,
3(1): 125-136 (2000); Harris, J M, Nature Reviews 2: 215 et seq.
(2003); Tsubery, H., J Biol. Chem 279(37): 38118-24 (2004); and are
further described in a number of references describing PEGylation
and various reagents and methodologies (see, e.g., Monfardini, C et
al, Bioconjugate Chem. 6: 62-69 (1995); Veronese F M et al., J.
Bioactive Compatible Polymers 12: 197-207 (1997); and a variety of
U.S. and other published patents and applications including U.S.
Pat. No. 5,672,662; U.S. Pat. No. 5,932,462; U.S. Pat. No.
6,495,659; and U.S. Pat. No. 6,737,505; as well as U.S. Pat. Nos.
4,002,531; 4,179,337; 5,122,614; 5,183,550; 5,324,844; 5,446,090;
5,612,460; 5,643,575; 5,766,581; 5,795,569; 5,808,096; 5,900,461;
5,919,455; 5,985,263; 5,990,237; 6,113,906; 6,214,966; 6,258,351;
6,340,742; 6,413,507; 6,420,339; 6,437,025; 6,448,369; 6,461,802;
6,828,401; 6,858,736; US Appl Publ 20010021763; 20010044526;
20010046481; 20020052430; 20020072573; 20020156047; 20030114647;
20030143596; 20030158333; 20030220447; 20040013637; 2005000360;
20050114037; 20050171328 and 20050209416; and European publications
EP 01064951; and EP0822199; and PCT publications WO 00176640; WO
0002017; WO 0249673; WO 9428024; and WO 0187925 (each of which
publications is incorporated herein by reference).
EXAMPLE 21-A
Illustrative Pegylated Versions of Human Recombinant
Hyaluronidases
[0786] As an exemplary illustration of the pegylation of an
illustrative sHASEGP, PEG aldehydes, succinimides and carbonates
have each been applied to conjugate PEG moieties to rHuPH20. Of
these three chemistries, succinimidyl PEGs have typically been the
most convenient to use in the case of rHuPH20. For example, rHuPH20
has been conjugated with exemplary succinimidyl monoPEG (mPEG)
reagents including mPEG-Succinimidyl Propionates (mPEG-SPA),
mPEG-Succinimidyl Butanoates (mPEG-SBA), and (for attaching
"branched" PEGs) mPEG2-N-Hydroxylsuccinimide. These pegylated
succinimidyl esters contain different length carbon backbones
between the PEG group and the activated cross-linker, and either a
single or branched PEG group. These differences can be used, for
example, to provide for different reaction kinetics and to
potentially restrict sites available for PEG attachment to rHuPH20
during the conjugation process.
[0787] Succinimidyl PEGs (as above) comprising either linear or
branched PEGs have been conjugated to rHuPH20. Using compositions
and techniques as described in the art, succinimidyl PEGs have been
used to generate rHuPH20s reproducibly comprising a combination of
molecules having between about three to six PEG molecules per
sHASEGP. Such pegylated rHuPH20 compositions were readily purified
to yield compositions having specific activities of approximately
25,000 Unit/mg protein hyaluronidase activity, and being
substantially free of non-PEGylated rHuPH20 (less than 5%
non-PEGylated). As is known in the art, and described for example
in PEGylation references including those cited above, a variety of
reagents and techniques are also available for achieving
mono-disperse as opposed to poly-disperse PEG products (see, e.g.,
the reviews and references cited in the detailed description above
related to site-specific monoPEGylation and site-directed
PEGylation).
[0788] Improvements in both pharmacokinetics and pharmacodynamics
have been observed in animal models following administration of a
PEGylated sHASEGP as compared to the unmodified version of the
sHASEGP. For example, following intravenous administration to mice
of either 1,000 Units of an unmodified rHuPH20 or 1,000 Units of
rHuPH20 conjugated to PEGs having a molecular weight of
approximately 40 kDa (rHuPH20-PEG40), it was observed that the
serum half-life of neutral active hyaluronidase activity (PH20) in
serum of mice treated with rHuPH20-PEG40 was significantly longer
(16-20 fold) than that of mice treated with unmodified rHuPH20. In
addition, the effectiveness of rHuPH20-PEG40 has been compared to
non-PEGylated rHuPH20 in a rat stroke model. Initial results from
these studies demonstrate greater effectiveness of rHuPH20-PEG40
(greater than 75-85% survival, versus less than 50% in controls) in
rat survival 7 days post stroke-inducement.
[0789] Using various PEG reagents as available from Nektar, we have
prepared exemplary versions of human recombinant hyaluronidases
(particularly preferred enzymes based on rHuPH20) using mPEG-SBA
(30 kD), mPEG-SMB (30 kD), and branched versions based on mPEG2-NHS
(40 kD), mPEG2-NHS (60 kD). These reagents and numerous others are
available in various molecular weights (see, e.g., the 2005-06
Nektar Product Catalog which is available on-line at
http://www.nektar.com or
http://www.nektar.com/pdf/nektar_catalog.pdf, references therein,
which catalog and references are incorporated herein by reference).
A number of other PEG reagents and methodologies are available from
other suppliers and/or are described in the art. By way of
illustration, PEGylated versions of rHuPH20 have been generated
using NHS chemistries, as well as carbonates, and aldehydes, using
each of the following reagents available from Nektar: mPEG2-NHS-40K
branched (Nektar #2z3y0t01); mPEG-NHS-10K branched (Nektar
#2z3x0Lo1-10); mPEG-NHS-20K branched (Nektar #2z3x0Lo1-20);
mPEG-NHS-40K branched (Nektar #2z3x0Lo1-40); mPEG2-NHS-60K branched
(Nektar #2z3y0v01); mPEG-SBA-5K (Nektar #2m450h01); mPEG-SBA-20K
(Nektar #2M450p01); mPEG-SBA-30K (Nektar #2M450r01); mPEG-SMB-20K
(Nektar #2m4k0p01); mPEG-SMB-30K (Nektar #2m4k0r01);
mPEG-butyrldehyde-30K (Nektar #072M0R01); mPEG-SPA-20K (Nektar
#2m4m0p01); mPEG-SPA-30K (Nektar #2m4m0r01); and PEG-NHS-5K-biotin
(Nektar #0H4M0H02).
[0790] We have also generated PEGylated versions of hyaluronidases
(e.g. rHuPH20) using PEG reagents available from Dowpharma, a
division of Dow Chemical Corporation; including hyaluronidases
PEGylated with Dowpharma's p-nitrophenyl-carbonate PEG (30 kDa) and
with propionaldehyde PEG (30 kDa). Nektar and Dowpharma reagents
and their resulting products were used and tested essentially as
described below to generate a large variety of PEGylated
hyaluronidase compositions. Other reagents, and other
hyaluronidases, can be similarly reacted using analogous
techniques.
[0791] For generating PEGylated versions of rHuPH20, purified
enzyme (at a concentration of between about 1 to 14 mg/ml) was
mixed with a molar excess of each PEG reagent (generally at about a
10:1 molar excess of PEG:enzyme), at pH and other conditions as
recommended for each of the PEG reagent or chemistries (generally
aldehydes reactions were performed at about pH4.5, NHS reactions at
neutral pH and carbonate reactions at about pH9.5). The reaction
components were reacted with mixing for approximately 16 hours at 4
degrees C.
[0792] SDS-PAGE was used to initially examine reaction products.
Generally unmodified rHuPH20 was observed to migrate as a single
band of approximately 63 kDa molecular weight. The various
PEGylated versions of rHuPH20 generally comprised a band in the
range of about 150-200 kDa (typically about 180 kDa) as well as
several bands of greater than 200 kDa.
[0793] We then performed gel filtration of the modified enzyme
products followed by enzyme activity of fractionated proteins.
Briefly, following techniques as known in the art, separations of
PEG-modified hyaluronidases from their respective native unmodified
forms, were performed by gel filtration chromatography, using a
Superose 6 column (GE/Pharmacia). Phosphate buffered saline was
used as mobile phase for the separations, with a flow rate of 0.2
mL/minute. The column was calibrated using Gel Filtration
Calibration Standards from GE/Pharmacia. Fractions containing
separated hyaluronan degrading enzymes were assayed for relative
hyaluronidase activity by a modified USP Turbidity assay, performed
in a microtiter format. Peak retention volume (time) for rHuPH20
hyaluronidase enzyme activity fractionating through a Superose 6
gel filtration column, shifted from about 16 mL (approximately 60
to 80 KD apparent molecular weight) for the unmodified
hyaluronidase, to about 10 mL (greater than 100,000 KD to
approximately 670,000 KD apparent molecular weight) for the
Peg-modified hyaluronidase. Peg modification can apparently,
completely shift all measurable enzyme activity to the higher
molecular weight modified forms, when molar excess peg is reacted
with the enzyme.
[0794] We then examined enzymatic activity of various native and
modified enzymes using zymography (analyses of
glycosaminoglycan-degrading activity by substrate gel
electrophoresis). Briefly, hyaluronan-containing SDS-PAGE zymogram
gels were cast and analyses performed essentially in accordance
with the procedure previously described by Miura R O, et. al.
Analysis of glycosaminoglycan-degrading enzymes by substrate gel
electrophoresis (zymography). Anal Biochem. 1995 Mar
1;225(2):333-40.
[0795] Native rHuPH20 hyaluronidase demonstrated
hyaluronan-degrading activity in zymogram gels as a single band
with an apparent molecular weight of approximately 60 kDa. Peg
modifications of rHuPH20 hyaluronidase resulted in a shift in
hyaluronan degrading activity in zymogram gels with enzymatically
active signals at approximately 180 kDa, and with at least 2
additional bands having enzyme activity with molecular masses
greater than 200 kDa.
[0796] These results and others described herein demonstrate that
glycosaminoglycanases can be effectively modified by PEGylation
(which can be used to alter their pharmacokinetic and/or other
profiles) using any of a wide variety of PEG reagents, and that the
resulting enzymes can retain substantial enzymatic activity making
them useful in any of variety of different
applications--particularly in vivo or other applications in which
modifying the enzyme to extend its half life can be useful.
EXAMPLE 21-B
Illustrative Pegylated Versions of Non-Human Hyaluronidases
[0797] Although human recombinant hyaluronidases are particularly
preferred for applications in which the enzyme is being used in
association with human cells, and even more particularly when it is
being applied to or within the human body, the techniques
illustrated herein can also be employed to generate
pharmacokinetically-improved versions of other hyaluronidase
enzymes that may have suitable application in certain contexts or
environments. Thus, by applying methodologies as illustrated herein
and described in the art to other hyaluronidase enzymes, we have
generated a number of modified enzymes that are PEGylated (which
can be used to improve their pharmacokinetics as illustrated above)
and yet retain substantial enzymatic activity. For purposes of
illustration, methodologies as described and illustrated herein
have been applied to a series of different exemplary "non-human"
hyaluronidase enzymes, including an illustrative animal-derived
hyaluronidase, an illustrative bacterial-derived hyaluronidase, and
an illustrative bacterial-derived chondroitinase having
hyaluronidase activity. PEGylated versions of such non-human
hyaluronidases, as described herein, be used to effectively provide
extended half lives (such as following intravenous or other in vivo
administration).
[0798] As an illustrative example of a non-human animal-derived
hyaluronidase, we have generated PEGylated versions of ovine
testicular hyaluronidase (available as Vitrase.TM. from ISTA
Pharmaceuticals). Vitrase (hyaluronidase for injection,
Lyophilized, Ovine NDC 67425-001-01, 6200 USP Units/vial),
purchased from ISTA pharmaceuticals, was a gift from an
ophthalmologist. Vitrase solutions were prepared by reconstituting
the lyophilized Vitrase with 0.9% Sodium Chloride Injection USP
(Baxter).
[0799] As an illustrative example of a non-human bacterial-derived
hyaluronidase, we have generated PEGylated versions of bacterial
hyaluronidase available as Hyaluronate Lyase from Sigma.
Hyaluronate Lyase from Streptomyces hyalurolyticus or other
species, CAS Number 9001-54-1, EC Number 4.2.2.1) (674 units /vial)
was purchased from Sigma. Hyaluronate lyase was reconstituted with
sterile filtered phosphate buffered saline. See, e.g., Ohya, T.,
and Kaneko, Y. Biochim. Biophys. Acta 198, 607, (1970).
[0800] As an illustrative example of a non-human chondroitinase
having hyaluronidase activity, we have generated PEGylated versions
of bacterial chondroitinase ABC (available as Chondroitinase ABC
from Associates of Cape Cod, Inc. Seikagaku Corporation).
Chondroitinase ABC from Proteus vulgaris (Chondroitin ABC lyase,
Chondroitin ABC eliminase, EC Number: 4.2.2.4, CAS Number:
9024-13-9, cat. # 100330 or 10033) was purchased from Associates of
Cape Cod Inc. Chondroitinase ABC solutions were prepared in 0.1%
BSA solution. References include: (1) Yamagata, T., et al., J.
Biol. Chem., 243, 1523 (1968); (2) Saito, H., et al., J. Biol.
Chem., 243, 153.6 (1968); (3) Suzuki, S., et al., J. Biol. Chem.,
243, 1543 (1968); and (4) Oike, Y., et al., J. Biol. Chem., 257,
9751 (1982).
[0801] As an illustrative PEG reagent, we used an mPEG-Succinimidyl
Butanoate (available as mPEG-SBA (30 kD) from Nektar). While
mPeg-SBA-30K (N-hydroxysuccinimidyl ester of methoxy poly(ethylene
glycol) butanoic acid, average M.W. 30,000, Cat # 2M450R01), was
used for this example, other N-hydroxysuccinimidyl ester of methoxy
poly(ethylene glycol) of any molecular weight or
N-hydroxysuccinimidyl esters of poly(ethylene glycol) of any
molecular weight, or peg-aldehydes of any size, or peg-carbonates
of any size, or other Nektar PEG reagents, or other pegylation
reagents can also be used. See, e.g., Zalipsky, S and C Lee,
"Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical
Applications," J. M. Harris, ed., Plenum, N.Y., 1992, cf. chapter
21.
[0802] For the generation of exemplary PEGylated versions of
animal-derived hyaluronidases, a vial of ovine testicular
hyaluronidase (Vitrase) was reconstituted with 0.62 mL of 0.9%
saline (10 units/microliter), and 0.3 mL of the reconstituted
enzyme solution was transferred to a sterile tube. The exemplary
PEG reagent, mPeg-SBA-30K (9.9 mg), was added to the tube
containing the Vitrase solution, the tube was mixed until all the
mPeg appeared to enter into solution, and then the mixture was
reacted for approximately 16 hours at 4 degrees C., with
mixing.
[0803] For the generation of exemplary PEGylated versions of
bacterial-derived hyaluronidases, one ampoule of Sigma hyaluronate
lyase was reconstituted with 0.7 mL sterile filtered phosphate
buffered saline (.about.1 unit/microliter), and 0.3 mL of the
reconstituted enzyme solution was transferred to a sterile tube.
The exemplary PEG reagent, mPeg-SBA-30K (9.8 mg), was added to the
tube containing the hyaluronate lyase solution, the tube was then
mixed until all the mPeg appeared to be in solution, and the
mixture was reacted for approximately 16 hours at 4 degrees C.,
with mixing.
[0804] For the generation of exemplary PEGylated versions of
bacterial-derived chondroitinase having hyaluronidase activity, one
vial of Chondroitinase ABC (10 Units) was reconstituted with 0.2 mL
of 0.9% saline, and the exemplary PEG reagent, mPeg-SBA-30K (12.4
mg), was added to the vial containing Chondroitinase ABC solution,
and mixed until all mPeg appeared to be in solution, and the
mixture was reacted for approximately 11 hours at 4 degrees C.,
with mixing.
[0805] Initial analyses included the application of SDS-PAGE to
examine the modified enzymes compared to unmodified controls.
Briefly, total protein was visualized with either Coomassie Blue or
silver staining after separation through 3% to 8% NuPage
Tris/Acetate gels (Invitrogen). Molecular weights of stained bands
were determined by comparison to standard proteins of known mass
run on the same gel.
[0806] Results with Vitrase were as follows: (A) Vitrase
(unmodified), 5 bands of approximately the following molecular
weights (kDa): 90, 80. 63, 50 and 40; (B) PEG-Vitrase, 7 bands of
approximately the following molecular weights (kDa): 150, 180 and
five bands of greater than 200.
[0807] Results with Chondroitinase ABC were as follows: (A)
Chondroitinase ABC (unmodified), 6 bands of approximately the
following molecular weights (kDa): 100, 80, 50, 38, 33 and 30; (B)
PEG-Chondroitinase ABC, 8 or more bands of approximately the
following molecular weights (kDa): one to two bands between 150-200
and six or more bands of greater than 200.
[0808] Results with Hyaluronate Lyase were as follows: (A)
Hyaluronate Lyase (unmodified), one band of approximately the
following molecular weight (kDa): 35; (B) PEG-Hyaluronate Lyase,
four or more bands of approximately the following molecular weights
(kDa): greater than 200.
[0809] As demonstrated, activated polyethylene glycol (e.g.:
mPeg-SBA-30K (N-hydroxysuccinimidyl ester of methoxy poly(ethylene
glycol) butanoic acid, average M.W. 30,000), can be applied to
ovine hyaluronidase (e.g. Vitrase), bacterial Chondroitinase ABCs
(e.g. from Proteus vulgaris), or bacterial hyaluronate lyases (e.g.
from Streptomyces hyalurolyticus), to induce chemical changes in
the enzyme preparations which produces new molecules of increased
average molecular mass, as would be associated with the generation
of PEGylated versions of these enzymes. Further characterization of
these products provided additional evidence that the enzymes can
not only be effectively PEGylated but that resulting enzyme
products can exhibit substantial enzymatic activity in the
degradation of hyaluronan--demonstrating that PEGylation can be
applied to these various types of enzymes (which can be used to
alter their pharmacokinetic profiles while at the same time
retaining substantial enzymatic activity).
[0810] We then performed gel filtration of the modified enzyme
products followed by enzyme activity of fractionated proteins.
Briefly, following techniques as known in the art, separations of
PEG-modified hyaluronidases from their respective native unmodified
forms, were performed by gel filtration chromatography, using a
Superose 6 column (GE/Pharmacia). Phosphate buffered saline was
used as mobile phase for the separations, with a flow rate of 0.2
mL/minute. The column was calibrated using Gel Filtration
Calibration Standards from GE/Pharmacia. Fractions containing
separated hyaluronan degrading enzymes were assayed for relative
hyaluronidase activity by a modified USP Turbidity assay, performed
in a microtiter format. The pH and buffer used to assay the
activity of each of the hyaluronan degrading enzymes tested, was as
described in the literature, for each specific enzyme.
[0811] Peak retention volume (time) for ovine hyaluronidase enzyme
activity (e.g.: Vitrase) fractionating through a Superose 6 gel
filtration column, shifted from 15.5 mL (approximately 60 to 80 KD
apparent molecular weight) for the native ovine hyaluronidase, to
9.5 mL (greater than 100,000 KD to approximately 670,000 KD
apparent molecular weight) for the Peg-modified ovine
hyaluronidase. Peg modification can apparently, completely shift
all measurable enzyme activity to the higher molecular weight
modified forms, when molar excess peg is reacted with the ovine
enzyme.
[0812] Peak retention volume (time) for native bacterial
Hyaluronate Lyase enzyme activity was approximately 16 mL
(approximately 50 to 70 KD apparent molecular weight) after
injection. Peg-modified Hyaluronate Lyase fractionated by gel
filtration demonstrated no enzyme activity in any fractions. Lack
of measurable enzyme activity included fractions where native
Hyaluronate Lyase activity is normally measurable, suggesting that
peg modification negatively effected enzyme function.
[0813] Peg-modified Chondroitinase ABC demonstrated peak enzyme
activity at approximately 11 mL and 13 mL after injection
(approximately between 200 and 600 KD apparent molecular weight),
and again at approximately 16 mL after injection (approximately 50
to 70 KD apparent molecular weight). Native Chondroitinase ABC has
enzyme activity at only approximately 16 mL after injection. This
data suggests that the Chondroitinase ABC was peg-modified, with
maintenance of enzymatic activity as higher than native, molecular
mass molecules.
[0814] Gel filtration can be used to estimate the extent of
Peg-modification on hyaluronan degrading enzymes; and analysis of
enzyme activity of fractionated preparations can be used to
evaluate the effect the Peg modification has on the enzymatic
activity of the hyaluronan degrading enzymes, with respect to total
activity and the molecular mass of constituents demonstrating
enzyme activity.
[0815] We then examined enzymatic activity of various native and
modified enzymes using zymography (analyses of
glycosaminoglycan-degrading activity by substrate gel
electrophoresis). Briefly, hyaluronan-containing SDS-PAGE zymogram
gels were cast and analyses performed essentially in accordance
with the procedure previously described by Miura R O, et. al.
Analysis of glycosaminoglycan-degrading enzymes by substrate gel
electrophoresis (zymography). Anal Biochem. Mar. 1,
1995;225(2):333-40.
[0816] Native ovine hyaluronidase demonstrates hyaluronan-degrading
activity in zymogram gels identifying primarily 2 bands of activity
at approximately 60 KD and 90 KD with some smearing of enzyme
activity between those molecular weights. No enzyme activity is
demonstrated greater than 100 KD. Peg modification of ovine
hyaluronidase demonstrates a shift in hyaluronan degrading activity
in zymogram gels with enzymatically active signals at approximately
150 KD, and with at least 2 additional bands having enzyme activity
with molecular masses greater than 200 KD.
[0817] Native Chondroitinase ABC demonstrated a single band of
hyaluronan degrading activity in zymogram gels, demonstrating a
molecular mass of approximately 80 to 90 KD. Peg modification of
Chondroitinase ABC demonstrates a shift in hyaluronan degrading
activity in zymogram gels with enzymatically active signals at
approximately 180 KD, and with at least one additional band of
activity with a molecular mass greater than 200 KD.
[0818] The zymography analysis thus confirmed that an exemplary
animal-derived hyaluronidase (Vitrase) and an exemplary
bacterial-derived chondroitinase having hyaluronidase activity
(Chrondroitinase ABC) can be modified by PEGylation (which can be
used to alter their pharmacokinetic profile as illustrated above
with respect to recombinant human hyaluronidase) and that the
resulting modified enzymes can still exhibit substantial enzymatic
activity.
[0819] As will be appreciated by those of skill in the art in view
of the foregoing examples, other hyaluronidases (both human and
other recombinant versions as described herein, as well as other
non-human animal or bacterial, prokaryotic or other hyaluronidases,
as well as chondroitinases) can likewise be modified using any of a
variety of PEG reagents as are known in the art or newly
discovered, to generate modified enzymes having altered
pharmacokinetic and/or other profiles that make them useful for
particular applications.
EXAMPLE 22
Illustrative Use of a sHASEGP to Promote Spreading of a Small
Molecule Pharmacologic Agent in vivo
[0820] As an illustrative example of the use of sHASEGP
compositions of the present invention to promote spreading or
diffusion of pharmacologic agents into and within organs and
tissues in a mammal, we assessed the use of rHUPH20 to promote
penetration of dexamethasone ("dex", an anti-inflammatory agent
used as an exemplary small molecule pharmacologic) into tissues of
the mammalian eye in vivo
[0821] For injection of agents such as anesthetics to the mammalian
eye, commonly used periorbital routes for achieving anesthetic
blocks include peribulbar, retrobulbar and sub-Tenon's injections.
For this illustrative example, we compared sub-Tenon's injections
of dex, with or without concomitant administration of sHASEGP, into
the episcleral space, and then examined the concentrations of dex
within tissues of the posterior of the eye, particularly the
choroid and retinal layers, which are important drug targets but
are often difficult to efficiently reach with pharmacologic
agents.
[0822] New Zealand red male rabbits (approximately 2.1 to 3.2 kg)
were employed and were placed under general anesthesia using
intramuscular injections of ketamine hydrochloride (21 mg/kg) and
xylazine hydrochloride (5.25 mg/kg). Topical 0.5% proparacaine was
used for anesthetizing the ocular surface. Pupils were dilated with
1% tropicamide and 2.5% phenylephrine. After opening the
conjunctiva of the right eye of each rabbit using Vannas scissors,
the tip of a Medez tri-port anesthetic cannula (Eagle Laboratories,
Rancho Cucamonga, Calif.) was inserted into the episcleral space
and 1.5 ml of either dex alone (American Pharmaceutical Partners,
n=18) or dex plus sHASEGP (3000 units of rHUPH20, n=18) was
injected adjacent to the posterior pole of the eye. A cotton-tipped
applicator was placed at the site of conjunctival entry to prevent
egress of fluid.
[0823] At 1, 2, 3 or 6 hours after the injection, rabbits were
anesthetized as described above and sacrificed by intracardiac
injection of pentobarbital sodium (120 mg/kg). Following
euthanasia, 1 ml of blood sample was taken and both eyes were
enucleated from each animal. Immediately after the enucleation, a
2-ml sample of aqueous humor was collected via anterior chamber
paracentesis with tuberculin syringe from each eye. After removal
of anterior segment from enucleated eyes, 1 ml of vitreous sample
was withdrawn and placed into labeled Eppendorf tubes. The
remainder of the eye was dissected and 6-mm trephine (Fine Science
Tools, Foster City, Calif.) was used to punch the tissue sample
inferior to the optic nerve where drug was injected on the
extrascleral side. From the punched tissue the retina was scraped
off with a blunt blade and placed into labeled tube as was the
choroid.
[0824] Mass spectrometry was used to assess dex levels in tissues
with the minimum quantifiable limits being on the order of 5 ng/ml.
For the choroid and retinal samples, a buffer containing 50 mM Tris
base, 1 mM MgCl.sub.2, pH 9.0, was added to the tissue so the final
concentration was 20 mg/mL. The tissue was disrupted using an
ultrasonic homogenizer (BioLogics, Inc., model 150 V/T) for 5-15
seconds. The samples of tissue homogenate, the vitreous fluid,
aqueous fluid, and plasma were all analyzed by the following
procedure. Samples containing dexamethasone and prednisolone as the
internal standard (I.S.) were extracted with methyl t-butyl ether
by vortex mixing for five minutes. The organic and aqueous layers
were separated by centrifugation at 1,300 g for five minutes, the
aqueous layer was frozen at -70.degree. C. for fifteen minutes, and
the organic layer was poured into new tubes and evaporated under a
stream of nitrogen at 40.degree. C. The residue was reconstituted
with water:acetonitrile (50:50 v/v). The processed samples were
analyzed by high-performance liquid chromatography using a
Phenomenex Synery Polar RP column maintained at 45.degree. C. The
mobile phase was nebulized using heated nitrogen in a Z-spray
source/interface and the ionized compounds were detected using a
tandem quadrupole mass spectrometer. Peak heights of dexamethasone
and prednisolone were acquired and integrated using MassLynx.TM.
software, Micromass, Beverly, Mass. The calibration curves were
obtained by fitting the peak height ratios of the standards to the
internal standard a power equation in MassLynx.TM.. The equation of
the calibration curve was then used to interpolate the
concentration of dexamethasone in the samples using their peak
height ratios.
[0825] The mass spec results revealed that the choroidal, retinal
and vitreous dexamethasone levels of rabbits treated with dex alone
reached peak concentrations at the first time point (i.e. 1 hour
after injection); whereas the choroidal and retinal dex
concentrations of rabbits that received concomitant sHASEGP were
higher and continued to increase to the second time point (i.e. 2
hours after injection). Peak choroidal, retinal and vitreous
concentrations were approximately 10-fold, 5-fold and 8-fold
higher, respectively, in tissues that had been treated with
sHASEGP, and the areas under the curve (AUC) were also at least 50%
higher.
[0826] These results indicate that the administration of sHASEGP
can be used to substantially enhance the delivery of pharmacologic
agents such as dex, even to relatively difficult to reach tissues
in vivo such as segments at the posterior of the mammalian eye.
Furthermore, since the relatively thick scleral layer forms a
tissue barrier between the sub-Tenon's injection space and the
targeted choroid and retinal layers, the high levels of
pharmacologic agent delivered to the latter layers indicate that
sHASEGP can be used to effectively promote delivery of
pharmacologics across tissue layers within the body.
[0827] In the case of promoting transcleral delivery, sHASEGP can
thus be applied in a minimally-invasive procedure to promote the
delivery of agents used to treat conditions affecting the back of
the eye which are of particular interest in a number of ocular
disease situations. This can provide, for example, a less invasive
delivery route for the treatment of choroidal neovascularization
(e.g. by administering steroids), macular degeneration and other
diseases affecting these tissues which are typically treated via
intravitreal injection. There are a wide variety of agents
currently used for these conditions, such as, but not limited to,
dexamethasone, triamcinolone, Macugen.TM., and VEGF
antagonists.
[0828] In addition to the enzymatic activities of sHASEGP that
contribute to spreading in the context of delivery routes such as
via sub-Tenon's injection, it is also believed that the
introduction of the sHASEGP in a volume of fluid that effectively
fills or distends the tissue space into which it is injected
generates positive hydrostatic pressures that serve to further
drive delivery of fluids, and the agents comprised therein, into
the surrounding tissues (e.g. by promoting convective
transport).
EXAMPLE 23
Illustrative Use of a sHASEGP to Reduce Tumor Interstitial Fluid
Pressure Thereby Promoting the Susceptibility of a Tumor to
Anti-Tumor Pharmacologic Agents
[0829] As described and illustrated herein, sHASEGPs can be used to
generate interstitial channels that can increase fluid flow and
facilitate the delivery of pharmacologic and other agents to
desired tissue sites in vivo. Tumors present a particularly
important target issue for a number of known and regularly
developed pharmacologic agents, especially antineoplastics,
antimetabolites, antiangiogenics and various cytotoxic and
cytostatic agents. The challenges associated with targeting such
pharmacologics to tumor sites in vivo are exacerbated by the fact
that many tumors exhibit elevated interstitial pressures that tend
to impede infusion of administered pharmacologics, particularly to
sites deep within a tumor mass. It is believed that sHASEGPs can
reduce this impedance, and therefore faciliate delivery of any of a
variety of known and newly developed pharmacologic agents into
tumors, thereby rendering them more sensitive to and more treatable
by a given dose of a pharmacologic. In these regards, sHASEGPs of
the present invention have been shown to be capable of promoting
the formation of interstitial channels and to enhance the
dispersion of markers.of various sizes as described above. In
addition, we have observed that sHASEGPs can be used to increase
hydraulic conductivity of interstitial tissues over ten fold.
[0830] As confirmation of these principles in the context of tumor
cells, application of an exemplary sHASEGP (rHuPH20, 50 Units) to
tumor cells in vitro resulted in substantial removal of
pericellular matrices, which are believed to be a major contributor
to elevated tumor interstitial fluid pressure.
[0831] In addition, administration of rHuPH20 in vivo, in a
xenograft human tumor model, resulted in a significant reduction in
tumor interstitial fluid pressure within one hour of
administration.
[0832] As described in the detailed description of the invention
above, sHASEGPs of the present invention can be applied in
combination with any of a number of known or newly developed
antitumor agents for use in the treatment of various cancers.
EXAMPLE 24
Illustrative Use of a sHASEGP to Enhance the Pharmacokinetics of a
Macromolecule Delivered by Non-IV Parenteral Injection
[0833] As discussed above, sHASEGPs can be used to modulate the
pharmacokinetics of known or newly developed pharmacologic and
other agents by, example, facilitating their absorption and/or
distribution with the body. Such improved pharmacokinetic
parameters can in turn be used to improve the pharmacodynamics of a
particular agent (e.g. reducing local toxicity by lowereing the
amount of an agent left near a site of injection and/or raising
effectiveness by increasing the amount of an agent delivered to
(and/or through) a desired target). Alternatively, since sHASEGPs
can be used to effectively drive dispersion of a
locally-administered pharmacologic agent (e.g., through a site of
injection and into the bloodstream), it is also possible to use the
sHASEGP to essentially improve the pharmacokinetics of absorption
and achieve similar pharmacodynamics with a reduced amount of
applied agent.
[0834] As an illustrative example of the use of sHASEGPs to enhance
the pharmacokinetics of a macromolecular drug that is normally
administered by non-intravenous parenteral injection, we combined
recombinant human PH20 (rHuPH20) with a pegylated version of
interferon alpha 2b (PEG-INTRON.TM. available from
Schering-Plough). PEG-INTRON is a macromolecule of approximately 31
kD that is typically administered by subcutaneous injection for the
treatment of Hepatitis C virus (HCV) (generally in combination with
the small molecule drug ribavirin). Like many macromolecules
administered subcutaneously, PEG-INTRON is relatively slowly
absorbed and a substantial portion of the agent never makes it to
the bloodstream, with bioavailability on the order of 50-60%. In
addition, a substantial fraction of patients (typically more than
half) develop various injection-site reactions, presumably as a
result of or exacerbated by the retained local concentrations of
the pharmacologic. As appreciated by those of skill in the art, the
consequences of such pharmacokinetics are several-fold; and include
not only the "wasting" of agent trapped near the injection site but
also reactions to the locally metabolized compound.
[0835] We administered Iodine-125 labelled PEG-INTRON by different
routes of administration, with or without concomitant adminisration
of rHuPH20, to examine the effects of rHuPH20 on the
pharmacokinetic parameters of the PEG-INTRON. To determine the
absolute bioavailability of rHuPH20, we also measured the
pharmacokinetic parameters of PEG-INTRON administered by
intravenous administration.
[0836] Briefly, eighteen Sprague-Dawley rats (each weighing
approximately 225-250 grams) were divided into three groups of six
animals each. A first group was administered test article (i.e.
I-125 labeled PEG-INTRON) by intravenous administration (1.5
micrograms/kg in a volume of 100 microliters via tail vein
injection). Specific activity of the I-125 labeled interferon was
55 uCi/microgram. Blood samples (100 microliters) were collected
pre-dose and at 0.5, 1, 2, 4, 8, 24, 48, 72, 96 and 120 hours after
dosing and processed for quantification of labeled drug by gamma
counting (expressed as Counts Per Minute (CPM) per gram plasma). A
second group of six animals was administered an equivalent dose and
volume of test article by intradermal injection into a shaved and
prepared spot over the back of the neck, and blood samples were
processed as for the first group. A third group of six animals was
administered an equivalent dose of test article mixed with rHuPH20
(100 I.U.) in a combined volume of 100 microliters by intradermal
injection as in the preceding group, and blood samples were then
processed as for groups one and two. The results of this study are
shown below in Table 24-1. TABLE-US-00020 TABLE 24-1 Effects of
sHASEGP on the pharmacokinetic profile of Interferon PEG-
PEG-INTRON PEG- INTRON (1.5 ug/kg) + sHASEGP INTRON (1.5 ug/kg) 100
U (1.5 ug/kg) Time (Hours) Intradermal SD Intradermal SD
Intravenous 0.5 30,289 9,989 54,808 12,995 281,781 1 43,137 9,434
76,672 17,570 185,630 2 54,655 11,128 102,159 17,626 155,591 3
55,261 11,055 115,329 24,910 128,656 6 61,214 15,528 118,364 23,817
93,065 8 59,277 14,704 110,443 21,735 79,275 12 46,696 12,752
85,710 28,078 58,457 24 16,233 6,595 26,047 9,006 16,966 36 7,954
1,777 12,498 1,762 9,277 48 3,862 499 5,517 1,030 5,847 AUC
1,222,952 2,212,583 1,910,723 Cmax 61,214 118,364 -- Tmax 6 6 --
Absolute 64% 116% 100% Bioavailability
[0837] As can be seen in Table 24-1, the bioavailability of the
exemplary pharmacologic PEG-INTRON (as assessed by AUC) was
dramatically improved when the intradermal dosing was combined with
a dose of sHASEGP. In fact, the absolute bioavailability achieved
with an intradermal dosing in combination with sHASEGP were
statistically indistinguishable from the direct intravenous
administration, where all of the material is delivered directly
into the bloodstreams. Thus, the use of sHASEGP in combination with
a pharmacologic that is introduced by non-IV parenteral
administration can provide levels of delivery comparable to
administration directly into the bloodstream. Although the
T.sub.max is necessarily longer following ID administration (since
some amount of time is necessary for the agent to reach the
bloodstream), within several hours after ID dosing with sHASEGP,
the concentrations of agent in the bloodstream essentially reached
those achieved by IV dosing, and thereafter apparently exceeded
them.
EXAMPLE 25
Illustrative Use of a sHASEGP to Facilitate Intradermal Delivery of
a Macromolecule Normally Delivered by Intravenous Injection
[0838] As discussed above, sHASEGPs can be used to facilitate the
delivery of known or newly developed pharmacologic and other agents
by different routes of administration that may provide for greater
convenience, safety, reduced costs and/or other benefits. As an
illustrative example of such a reformulation, we used a sHASEGP to
facilitate intradermal delivery of a macromolecular drug that is
normally administered by intravenous injection. For this particular
example, we combined recombinant human PH20 (rHuPH20) with a
chimeric human-murine anti-TNFalpha monoclonal antibody
(Infliximab, available as REMICADE.TM. from Centocor). REMICADE is
a macromolecule of approximately 150 kD that is typically
administered by intravenous injection for the treatment of
arthritis or Crohn's disease. Administration of REMICADE thus
generally requires the involvement of intrevenous access and
concomitant professional care and risks, and also requires on the
order of two hours for each dosing.
[0839] REMICADE (20 mg/ml) was labeled with .sup.125I to a specific
activity of 10 .mu.Ci/mg. Eighteen rats were divided into three
groups of six animals each and were administered a mixture of
radioactive and cold REMICADE (combined 10 mg/kg) in a final volume
of 100 .mu.l (i) by intravenous administration, (ii) by intradermal
administration or (iii) or by intradermal administration in
combination with 100 Unit sHASEGP (from a 100,000 U/ml stock); and
then blood and tissues processed by gamma counting at various
points after dosing, essentially as described for the preceding
example. The results are summarized in Table: TABLE-US-00021 TABLE
25-1 Effects of sHASEGP on the Pharmacokinetic Parameters of
REMICADE. CPM CPM REMICADE REMICAD (10 mg/kg) + sHASEGP CPM E (10
mg/kg) 100 U REMICADE (10 mg/kg) Time (Hours) Intradermal SD
Intradermal SD Intravenous 0.5 27,451 2,221 94,888 31,318 1,657,849
1 37,213 10,775 146,793 42,014 1,645,571 2 66,001 6,078 412,047
61,240 1,576,145 4 120,704 21,744 691,626 89,002 1,260,646 8
256,987 32,459 833,826 133,887 1,157,268 24 493,004 75,694 853,423
65,777 790,357 48 515,439 53,431 728,302 74,848 684,599 72 454,717
54,388 640,303 81,492 558,307 96 375,993 39,744 598,413 59,533
496,658 120 336,067 66,964 518,861 53,643 434,008 AUC 53,350,555
118,424,609 82,718,550 C.sub.max 515,439 853,423 1,657,849
T.sub.max 48 24 0 Absolute 62% 121% 100% Bioavailability
[0840] As can be seen in Table 5-1, the bioavailability of the
exemplary pharmacologic REMICADE (as assessed by AUC) was
dramatically improved when the intradermal dosing was combined with
a dose of sHASEGP. As was observed with enhanced delivery of
PEG-INTRON, the overall levels of REMICADE bioavailability achieved
with an intradermal dosing in combination with sHASEGP were
statistically similar to those of the intravenous administration
when all material was delivered directly into the bloodstream.
Although the REMICADE T.sub.max is necessarily longer following ID
versus IV administration, within twenty four hours after ID dosing
with sHASEGP, the concentrations of agent in the bloodstream
exceeded that observed with IV dosing, and thereafter approached or
exceeded the IV concentrations. Compared with ID dosing without
sHASEGP, the observed T.sub.max was reached about twice as quickly
with sHASEGP (24 hours versus 48 hours); and the Cmax and
bioavailability (AUC) both substantially increased (by about 65%
and 95%, respectively).
EXAMPLE 26
Illustrative Use of a sHASEGP to Facilitate Intradermal Delivery of
a Larger Macromolecular Complex Normally Delivered by Non-IV
Parenteral Injection
[0841] As another illustrative example of the use of sHASEGPs to
enhance the pharmacokinetics of a macromolecular drug that is
normally administered by non-intravenous parenteral injection, we
combined recombinant human PH20 (rHuPH20) with recombinant human
Factor VIII (RECOMBINATE.TM. available from Baxter). Factor VIII is
a large glycoprotein complex comprising multiple polypeptides, and
has a combined molecular of approximately 280 kD. Factor VIII is
typically only administered by intravenous injection for the
treatment of hemophilia. Like many macromolecules administered
subcutaneously, Factor VIII is relatively slowly absorbed and a
substantial portion of the agent never makes it to the bloodstream,
with bioavailability on the order of 0.5-1%.
[0842] Eighteen rats were divided into three groups of six animals
each and were administered 125-I labeled Factor VIII (i) by
intravenous administration, (ii) by intradermal administration or
(iii) by intradermal administration in combination with sHASEGP;
and then blood and tissues processed at various points after dosing
to measure counts per minute (CPM) per gram plasma, essentially as
described for the preceding example. TABLE-US-00022 TABLE 26-1
Effects of rHuPH20 on the pharmacokinetics of Factor VIII CPM CPM
FACTOR FACTOR VIII CPM VIII (50 IU/kg) + sHASEGP FACTOR VIII (50
IU/kg) 100 U (50 IU/kg) Time (Hours) Intradermal SD Intradermal SD
Intravenous 0.5 29,748 12,002 46,376 5,503 670,743 1 43,767 9,415
70,249 14,614 379,558 2 49,507 14,592 72,736 14,985 247,397 3
56,087 17,621 76,631 14,454 200,602 4 55,494 12,721 84,066 8,646
177,846 5 55,277 14,181 79,735 9,363 165,914 6 50,195 13,970 72,176
6,848 121,198 8 52,290 13,707 66,419 10,495 114,579 12 48,336
10,765 58,817 11,760 75,912 24 23,173 4,700 24,596 4,426 29,610 AUC
1,014,521 1,303,078 2,554,607 C.sub.max 56,087 84,066 -- T.sub.max
4 4 -- Absolute 39.7% 51% 100% Bioavailability
[0843] As shown in Table 26-1, co-adminsitration of Factor VIII
with rHuPH20 significantly increased the systemic bioavailability
relative to Factor VIII alone (P<0.01). The use of TCA
precipitation was necessary to eliminate the contribution of
completely degraded I-125 labeled Factor VIII protein entering the
blood stream.
EXAMPLE 27
Illustrative Use of a sHASEGP to Improve Survival and/or Behavioral
Outcomes Following Stroke
[0844] In a first set of studies, sHASEGPs were evaluated in a rat
model of stroke. Briefly, stroke was surgically induced in
Sprague-Dawley rats by middle cerebral artery occlusion, followed
at various times by intravenous administration of either a control
solution (saline) or a test solution (saline comprising a sHASEGP).
Animals were allowed to recover and were followed over a period of
time to assess rates of survival in control versus test groups. In
addition, survivors were assessed over a range of timepoints using
multiple analyses of behavioral conditions typically associated
with stroke. Only rats that performed normally in an initial
qualifying forelimb flexion test were included in the study, and
were then randomly assigned to a control or test group of
animals.
[0845] Aseptic surgical procedures were performed by Zivic
Laboratories essentially as follows. Rats were anesthetized in a
chamber supplied with 2.5% halothane. A ventral midline incision
was made and the musculature was separated to expose the right
external carotid artery, which was then isolated and double
ligated. An untied but constrictive 5/0 polyamide ligature was then
placed around the external carotid stump. A small hole was cut into
the lumen of the stump and an 18-20 mm length of 4/0 microfilament
suture material was inserted into the lumen and advanced into the
internal carotid artery. When completely advanced, the internalized
tip of the monofilament should block flow into the middle cerebral
artery. The constrictive polyamide ligature was then tied to secure
the monofilament. Retraction was removed and the skin incision
closed with a wound clip.
[0846] Intravenous injection procedures were performed essentially
as follows. Rats were anesthetized in a chamber supplied with 2.5%
halothane. A ventral skin incision was made to expose the jugular
vein. A sterile 1 ml disposable syringe was fitted with a 30-gauge
needle and loaded with injection material. Using the clavicle for
support, the syringe was inserted into the jugular vein (using
slight negative pressure on the syringe to draw bloodflow into the
syringe and thereby confirm proper placement for IV injection) and
then the injection material was slowly injected into the jugular
vein. After injection, the needle was slowly withdrawn (and any
bleeding controlled with a sterile cotton applicator), and then the
skin incision was closed with a wound clip.
[0847] Survival of animals in the various groups (approximately 40
animals in each group) was monitored following induction of stroke,
and the number of survivors at each day were then plotted against
days following stroke to generate a Kaplan-Meier stroke curve.
Substantial differences between the groups were apparent by the
3.sup.rd day following stroke. By one week following the induction
of stroke, more than half of the control animals had died, whereas
nearly three quarters of the sHASEGP-treated animals had survived,
which was a statistically significant difference (p=0.03).
[0848] In addition to measuring rates of survival following stroke,
survivors were also tested (at 1, 2, 3 and 4 weeks following stoke)
using other analyses designed to assess the incidence of certain
behavioral problems associated with stroke. Behavioral conditions
assessed included forelimb flexion, torso twist, hind limb, gait
disturbances and wall walk, which were used to generate a PNEGS
behavorial score (higher scores reflecting an increase in
behavioral problems associated with the stroke). By the second week
following induction of stroke, the sHASEGP-treated animals
exhibited significantly higher behavioral scores (p=0.02), and the
relative differences between the treated group and the control were
even greater over subsequent weeks (p=0.007 and p=0.003 for weeks
three and four respectively).
[0849] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
53 1 509 PRT Homo sapiens CARBOHYD 82, 166, 235, 254, 368, 393, 490
1 Met Gly Val Leu Lys Phe Lys His Ile Phe Phe Arg Ser Phe Val Lys 1
5 10 15 Ser Ser Gly Val Ser Gln Ile Val Phe Thr Phe Leu Leu Ile Pro
Cys 20 25 30 Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val Ile Pro
Asn Val Pro 35 40 45 Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe
Cys Leu Gly Lys Phe 50 55 60 Asp Glu Pro Leu Asp Met Ser Leu Phe
Ser Phe Ile Gly Ser Pro Arg 65 70 75 80 Ile Asn Ala Thr Gly Gln Gly
Val Thr Ile Phe Tyr Val Asp Arg Leu 85 90 95 Gly Tyr Tyr Pro Tyr
Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly 100 105 110 Gly Ile Pro
Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys 115 120 125 Lys
Asp Ile Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135
140 Ile Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro
145 150 155 160 Lys Asp Val Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln
Gln Gln Asn 165 170 175 Val Gln Leu Ser Leu Thr Glu Ala Thr Glu Lys
Ala Lys Gln Glu Phe 180 185 190 Glu Lys Ala Gly Lys Asp Phe Leu Val
Glu Thr Ile Lys Leu Gly Lys 195 200 205 Leu Leu Arg Pro Asn His Leu
Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220 Tyr Asn His His Tyr
Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240 Val Glu
Ile Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255
Thr Ala Leu Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val 260
265 270 Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg
Val 275 280 285 Ser Lys Ile Pro Asp Ala Lys Ser Pro Leu Pro Val Phe
Ala Tyr Thr 290 295 300 Arg Ile Val Phe Thr Asp Gln Val Leu Lys Phe
Leu Ser Gln Asp Glu 305 310 315 320 Leu Val Tyr Thr Phe Gly Glu Thr
Val Ala Leu Gly Ala Ser Gly Ile 325 330 335 Val Ile Trp Gly Thr Leu
Ser Ile Met Arg Ser Met Lys Ser Cys Leu 340 345 350 Leu Leu Asp Asn
Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn 355 360 365 Val Thr
Leu Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln 370 375 380
Gly Val Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385
390 395 400 Asn Pro Asp Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys
Phe Thr 405 410 415 Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gln
Phe Ser Glu Lys 420 425 430 Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser
Cys Lys Glu Lys Ala Asp 435 440 445 Val Lys Asp Thr Asp Ala Val Asp
Val Cys Ile Ala Asp Gly Val Cys 450 455 460 Ile Asp Ala Phe Leu Lys
Pro Pro Met Glu Thr Glu Glu Pro Gln Ile 465 470 475 480 Phe Tyr Asn
Ala Ser Pro Ser Thr Leu Ser Ala Thr Met Phe Ile Val 485 490 495 Ser
Ile Leu Phe Leu Ile Ile Ser Ser Val Ala Ser Leu 500 505 2 35 PRT
Homo sapiens 2 Met Gly Val Leu Lys Phe Lys His Ile Phe Phe Arg Ser
Phe Val Lys 1 5 10 15 Ser Ser Gly Val Ser Gln Ile Val Phe Thr Phe
Leu Leu Ile Pro Cys 20 25 30 Cys Leu Thr 35 3 474 PRT Homo sapiens
3 Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp 1
5 10 15 Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu
Pro 20 25 30 Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg
Ile Asn Ala 35 40 45 Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp
Arg Leu Gly Tyr Tyr 50 55 60 Pro Tyr Ile Asp Ser Ile Thr Gly Val
Thr Val Asn Gly Gly Ile Pro 65 70 75 80 Gln Lys Ile Ser Leu Gln Asp
His Leu Asp Lys Ala Lys Lys Asp Ile 85 90 95 Thr Phe Tyr Met Pro
Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp 100 105 110 Glu Glu Trp
Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125 Tyr
Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu 130 135
140 Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala
145 150 155 160 Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys
Leu Leu Arg 165 170 175 Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro
Asp Cys Tyr Asn His 180 185 190 His Tyr Lys Lys Pro Gly Tyr Asn Gly
Ser Cys Phe Asn Val Glu Ile 195 200 205 Lys Arg Asn Asp Asp Leu Ser
Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220 Tyr Pro Ser Ile Tyr
Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr 225 230 235 240 Leu Tyr
Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile 245 250 255
Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val 260
265 270 Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val
Tyr 275 280 285 Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile
Val Ile Trp 290 295 300 Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser
Cys Leu Leu Leu Asp 305 310 315 320 Asn Tyr Met Glu Thr Ile Leu Asn
Pro Tyr Ile Ile Asn Val Thr Leu 325 330 335 Ala Ala Lys Met Cys Ser
Gln Val Leu Cys Gln Glu Gln Gly Val Cys 340 345 350 Ile Arg Lys Asn
Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365 Asn Phe
Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380
Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys 385
390 395 400 Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val
Lys Asp 405 410 415 Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val
Cys Ile Asp Ala 420 425 430 Phe Leu Lys Pro Pro Met Glu Thr Glu Glu
Pro Gln Ile Phe Tyr Asn 435 440 445 Ala Ser Pro Ser Thr Leu Ser Ala
Thr Met Phe Ile Val Ser Ile Leu 450 455 460 Phe Leu Ile Ile Ser Ser
Val Ala Ser Leu 465 470 4 448 PRT Homo sapiens 4 Leu Asn Phe Arg
Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp 1 5 10 15 Ala Trp
Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30
Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35
40 45 Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr
Tyr 50 55 60 Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly
Gly Ile Pro 65 70 75 80 Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys
Ala Lys Lys Asp Ile 85 90 95 Thr Phe Tyr Met Pro Val Asp Asn Leu
Gly Met Ala Val Ile Asp Trp 100 105 110 Glu Glu Trp Arg Pro Thr Trp
Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125 Tyr Lys Asn Arg Ser
Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu 130 135 140 Ser Leu Thr
Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala 145 150 155 160
Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg 165
170 175 Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn
His 180 185 190 His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn
Val Glu Ile 195 200 205 Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn
Glu Ser Thr Ala Leu 210 215 220 Tyr Pro Ser Ile Tyr Leu Asn Thr Gln
Gln Ser Pro Val Ala Ala Thr 225 230 235 240 Leu Tyr Val Arg Asn Arg
Val Arg Glu Ala Ile Arg Val Ser Lys Ile 245 250 255 Pro Asp Ala Lys
Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val 260 265 270 Phe Thr
Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr 275 280 285
Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp 290
295 300 Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu Leu Leu
Asp 305 310 315 320 Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile
Asn Val Thr Leu 325 330 335 Ala Ala Lys Met Cys Ser Gln Val Leu Cys
Gln Glu Gln Gly Val Cys 340 345 350 Ile Arg Lys Asn Trp Asn Ser Ser
Asp Tyr Leu His Leu Asn Pro Asp 355 360 365 Asn Phe Ala Ile Gln Leu
Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380 Lys Pro Thr Leu
Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400 Ser
Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410
415 Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala
420 425 430 Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe
Tyr Asn 435 440 445 5 482 PRT Homo sapiens 5 Met Gly Val Leu Lys
Phe Lys His Ile Phe Phe Arg Ser Phe Val Lys 1 5 10 15 Ser Ser Gly
Val Ser Gln Ile Val Phe Thr Phe Leu Leu Ile Pro Cys 20 25 30 Cys
Leu Thr Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro 35 40
45 Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe
50 55 60 Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser
Pro Arg 65 70 75 80 Ile Asn Ala Thr Gly Gln Gly Val Thr Ile Phe Tyr
Val Asp Arg Leu 85 90 95 Gly Tyr Tyr Pro Tyr Ile Asp Ser Ile Thr
Gly Val Thr Val Asn Gly 100 105 110 Gly Ile Pro Gln Lys Ile Ser Leu
Gln Asp His Leu Asp Lys Ala Lys 115 120 125 Lys Asp Ile Thr Phe Tyr
Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 Ile Asp Trp Glu
Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160 Lys
Asp Val Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn 165 170
175 Val Gln Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe
180 185 190 Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu
Gly Lys 195 200 205 Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu
Phe Pro Asp Cys 210 215 220 Tyr Asn His His Tyr Lys Lys Pro Gly Tyr
Asn Gly Ser Cys Phe Asn 225 230 235 240 Val Glu Ile Lys Arg Asn Asp
Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255 Thr Ala Leu Tyr Pro
Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val 260 265 270 Ala Ala Thr
Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val 275 280 285 Ser
Lys Ile Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295
300 Arg Ile Val Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu
305 310 315 320 Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala
Ser Gly Ile 325 330 335 Val Ile Trp Gly Thr Leu Ser Ile Met Arg Ser
Met Lys Ser Cys Leu 340 345 350 Leu Leu Asp Asn Tyr Met Glu Thr Ile
Leu Asn Pro Tyr Ile Ile Asn 355 360 365 Val Thr Leu Ala Ala Lys Met
Cys Ser Gln Val Leu Cys Gln Glu Gln 370 375 380 Gly Val Cys Ile Arg
Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400 Asn Pro
Asp Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415
Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys 420
425 430 Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala
Asp 435 440 445 Val Lys Asp Thr Asp Ala Val Asp Val Cys Ile Ala Asp
Gly Val Cys 450 455 460 Ile Asp Ala Phe Leu Lys Pro Pro Met Glu Thr
Glu Glu Pro Gln Ile 465 470 475 480 Phe Tyr 6 1530 DNA Homo sapiens
CDS (1)...(1530) PH-20 GPI Anchored Hyaluronidase Glycoprotein 6
atg gga gtg cta aaa ttc aag cac atc ttt ttc aga agc ttt gtt aaa 48
Met Gly Val Leu Lys Phe Lys His Ile Phe Phe Arg Ser Phe Val Lys 1 5
10 15 tca agt gga gta tcc cag ata gtt ttc acc ttc ctt ctg att cca
tgt 96 Ser Ser Gly Val Ser Gln Ile Val Phe Thr Phe Leu Leu Ile Pro
Cys 20 25 30 tgc ttg act ctg aat ttc aga gca cct cct gtt att cca
aat gtg cct 144 Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val Ile Pro
Asn Val Pro 35 40 45 ttc ctc tgg gcc tgg aat gcc cca agt gaa ttt
tgt ctt gga aaa ttt 192 Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe
Cys Leu Gly Lys Phe 50 55 60 gat gag cca cta gat atg agc ctc ttc
tct ttc ata gga agc ccc cga 240 Asp Glu Pro Leu Asp Met Ser Leu Phe
Ser Phe Ile Gly Ser Pro Arg 65 70 75 80 ata aac gcc acc ggg caa ggt
gtt aca ata ttt tat gtt gat aga ctt 288 Ile Asn Ala Thr Gly Gln Gly
Val Thr Ile Phe Tyr Val Asp Arg Leu 85 90 95 ggc tac tat cct tac
ata gat tca atc aca gga gta act gtg aat gga 336 Gly Tyr Tyr Pro Tyr
Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly 100 105 110 gga atc ccc
cag aag att tcc tta caa gac cat ctg gac aaa gct aag 384 Gly Ile Pro
Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys 115 120 125 aaa
gac att aca ttt tat atg cca gta gac aat ttg gga atg gct gtt 432 Lys
Asp Ile Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135
140 att gac tgg gaa gaa tgg aga ccc act tgg gca aga aac tgg aaa cct
480 Ile Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro
145 150 155 160 aaa gat gtt tac aag aat agg tct att gaa ttg gtt cag
caa caa aat 528 Lys Asp Val Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln
Gln Gln Asn 165 170 175 gta caa ctt agt ctc aca gag gcc act gag aaa
gca aaa caa gaa ttt 576 Val Gln Leu Ser Leu Thr Glu Ala Thr Glu Lys
Ala Lys Gln Glu Phe 180 185 190 gaa aag gca ggg aag gat ttc ctg gta
gag act ata aaa ttg gga aaa 624 Glu Lys Ala Gly Lys Asp Phe Leu Val
Glu Thr Ile Lys Leu Gly Lys 195 200 205 tta ctt cgg cca aat cac ttg
tgg ggt tat tat ctt ttt ccg gat tgt 672 Leu Leu Arg Pro Asn His Leu
Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220 tac aac cat cac tat
aag aaa ccc ggt tac aat gga agt tgc ttc aat 720 Tyr Asn His His Tyr
Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240 gta gaa
ata aaa aga aat gat gat ctc agc tgg ttg tgg aat gaa agc 768 Val Glu
Ile Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255
act gct ctt tac cca tcc att tat ttg aac act cag cag tct cct gta 816
Thr Ala Leu Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val 260
265 270 gct gct aca ctc tat gtg cgc aat cga gtt cgg gaa gcc atc aga
gtt 864 Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg
Val 275 280 285 tcc aaa ata cct gat gca aaa agt cca ctt ccg gtt ttt
gca tat acc 912 Ser Lys Ile
Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300 cgc
ata gtt ttt act gat caa gtt ttg aaa ttc ctt tct caa gat gaa 960 Arg
Ile Val Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu 305 310
315 320 ctt gtg tat aca ttt ggc gaa act gtt gct ctg ggt gct tct gga
att 1008 Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser
Gly Ile 325 330 335 gta ata tgg gga acc ctc agt ata atg cga agt atg
aaa tct tgc ttg 1056 Val Ile Trp Gly Thr Leu Ser Ile Met Arg Ser
Met Lys Ser Cys Leu 340 345 350 ctc cta gac aat tac atg gag act ata
ctg aat cct tac ata atc aac 1104 Leu Leu Asp Asn Tyr Met Glu Thr
Ile Leu Asn Pro Tyr Ile Ile Asn 355 360 365 gtc aca cta gca gcc aaa
atg tgt agc caa gtg ctt tgc cag gag caa 1152 Val Thr Leu Ala Ala
Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln 370 375 380 gga gtg tgt
ata agg aaa aac tgg aat tca agt gac tat ctt cac ctc 1200 Gly Val
Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395
400 aac cca gat aat ttt gct att caa ctt gag aaa ggt gga aag ttc aca
1248 Asn Pro Asp Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe
Thr 405 410 415 gta cgt gga aaa ccg aca ctt gaa gac ctg gag caa ttt
tct gaa aaa 1296 Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gln
Phe Ser Glu Lys 420 425 430 ttt tat tgc agc tgt tat agc acc ttg agt
tgt aag gag aaa gct gat 1344 Phe Tyr Cys Ser Cys Tyr Ser Thr Leu
Ser Cys Lys Glu Lys Ala Asp 435 440 445 gta aaa gac act gat gct gtt
gat gtg tgt att gct gat ggt gtc tgt 1392 Val Lys Asp Thr Asp Ala
Val Asp Val Cys Ile Ala Asp Gly Val Cys 450 455 460 ata gat gct ttt
cta aaa cct ccc atg gag aca gaa gaa cct caa att 1440 Ile Asp Ala
Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile 465 470 475 480
ttc tac aat gct tca ccc tcc aca cta tct gcc aca atg ttc att gtt
1488 Phe Tyr Asn Ala Ser Pro Ser Thr Leu Ser Ala Thr Met Phe Ile
Val 485 490 495 agt att ttg ttt ctt atc att tct tct gta gcg agt ttg
taa 1530 Ser Ile Leu Phe Leu Ile Ile Ser Ser Val Ala Ser Leu * 500
505 7 509 PRT Homo sapiens CARBOHYD 82, 166, 235, 254, 368, 393,
490 7 Met Gly Val Leu Lys Phe Lys His Ile Phe Phe Arg Ser Phe Val
Lys 1 5 10 15 Ser Ser Gly Val Ser Gln Ile Val Phe Thr Phe Leu Leu
Ile Pro Cys 20 25 30 Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val
Ile Pro Asn Val Pro 35 40 45 Phe Leu Trp Ala Trp Asn Ala Pro Ser
Glu Phe Cys Leu Gly Lys Phe 50 55 60 Asp Glu Pro Leu Asp Met Ser
Leu Phe Ser Phe Ile Gly Ser Pro Arg 65 70 75 80 Ile Asn Ala Thr Gly
Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu 85 90 95 Gly Tyr Tyr
Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly 100 105 110 Gly
Ile Pro Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys 115 120
125 Lys Asp Ile Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val
130 135 140 Ile Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp
Lys Pro 145 150 155 160 Lys Asp Val Tyr Lys Asn Arg Ser Ile Glu Leu
Val Gln Gln Gln Asn 165 170 175 Val Gln Leu Ser Leu Thr Glu Ala Thr
Glu Lys Ala Lys Gln Glu Phe 180 185 190 Glu Lys Ala Gly Lys Asp Phe
Leu Val Glu Thr Ile Lys Leu Gly Lys 195 200 205 Leu Leu Arg Pro Asn
His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220 Tyr Asn His
His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240
Val Glu Ile Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245
250 255 Thr Ala Leu Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro
Val 260 265 270 Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala
Ile Arg Val 275 280 285 Ser Lys Ile Pro Asp Ala Lys Ser Pro Leu Pro
Val Phe Ala Tyr Thr 290 295 300 Arg Ile Val Phe Thr Asp Gln Val Leu
Lys Phe Leu Ser Gln Asp Glu 305 310 315 320 Leu Val Tyr Thr Phe Gly
Glu Thr Val Ala Leu Gly Ala Ser Gly Ile 325 330 335 Val Ile Trp Gly
Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu 340 345 350 Leu Leu
Asp Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn 355 360 365
Val Thr Leu Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln 370
375 380 Gly Val Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His
Leu 385 390 395 400 Asn Pro Asp Asn Phe Ala Ile Gln Leu Glu Lys Gly
Gly Lys Phe Thr 405 410 415 Val Arg Gly Lys Pro Thr Leu Glu Asp Leu
Glu Gln Phe Ser Glu Lys 420 425 430 Phe Tyr Cys Ser Cys Tyr Ser Thr
Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445 Val Lys Asp Thr Asp Ala
Val Asp Val Cys Ile Ala Asp Gly Val Cys 450 455 460 Ile Asp Ala Phe
Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile 465 470 475 480 Phe
Tyr Asn Ala Ser Pro Ser Thr Leu Ser Ala Thr Met Phe Ile Val 485 490
495 Ser Ile Leu Phe Leu Ile Ile Ser Ser Val Ala Ser Leu 500 505 8
34 DNA Artificial Sequence Reverse Primer to generate GPI anchor
lacking N483 and terminating at Y482 with BamHI site in the 5' end
8 aattggatcc tcagtagaaa atttgaggtt cttc 34 9 33 DNA Artificial
Sequence Reverse Primer to generate GPI anchor lacking Y482 and
terminating at F481 with BamHI site in the 5' end 9 aattggatcc
tcagaaaatt tgaggttctt ctg 33 10 34 DNA Artificial Sequence Reverse
Primer to generate GPI anchor lacking F481 and terminating at I480
with BamHI site in the 5' end 10 aattggatcc tcaaatttga ggttcttctg
tctc 34 11 32 DNA Artificial Sequence Reverse Primer to generate
GPI anchor lacking I480 and terminating at Q479 with BamHI site in
the 5' end 11 aattggatcc tcattgaggt tcttctgtct cc 32 12 32 DNA
Artificial Sequence Reverse Primer to generate GPI anchor lacking
Q479 and terminating at P478 with BamHI site in the 5' end 12
aattggatcc tcaaggttct tctgtctcca tg 32 13 31 DNA Artificial
Sequence Reverse Primer to generate GPI anchor lacking P478 and
terminating at E477 with BamHI site in the 5' end 13 aattggatcc
tcattcttct gtctccatgg g 31 14 32 DNA Artificial Sequence Forward
Primer with NheI restriction site at 5' end 14 aattgctagc
atgggagtgc taaaattcaa gc 32 15 1473 DNA Homo sapiens CDS
(1)...(1473) sHASEGPup to P478 and His Tagged 15 atg gga gtg cta
aaa ttc aag cac atc ttt ttc aga agc ttt gtt aaa 48 Met Gly Val Leu
Lys Phe Lys His Ile Phe Phe Arg Ser Phe Val Lys 1 5 10 15 tca agt
gga gta tcc cag ata gtt ttc acc ttc ctt ctg att cca tgt 96 Ser Ser
Gly Val Ser Gln Ile Val Phe Thr Phe Leu Leu Ile Pro Cys 20 25 30
tgc ttg act ctg aat ttc aga gca cct cct gtt att cca aat gtg cct 144
Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro 35
40 45 ttc ctc tgg gcc tgg aat gcc cca agt gaa ttt tgt ctt gga aaa
ttt 192 Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys
Phe 50 55 60 gat gag cca cta gat atg agc ctc ttc tct ttc ata gga
agc ccc cga 240 Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe Ile Gly
Ser Pro Arg 65 70 75 80 ata aac gcc acc ggg caa ggt gtt aca ata ttt
tat gtt gat aga ctt 288 Ile Asn Ala Thr Gly Gln Gly Val Thr Ile Phe
Tyr Val Asp Arg Leu 85 90 95 ggc tac tat cct tac ata gat tca atc
aca gga gta act gtg aat gga 336 Gly Tyr Tyr Pro Tyr Ile Asp Ser Ile
Thr Gly Val Thr Val Asn Gly 100 105 110 gga atc ccc cag aag att tcc
tta caa gac cat ctg gac aaa gct aag 384 Gly Ile Pro Gln Lys Ile Ser
Leu Gln Asp His Leu Asp Lys Ala Lys 115 120 125 aaa gac att aca ttt
tat atg cca gta gac aat ttg gga atg gct gtt 432 Lys Asp Ile Thr Phe
Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 att gac tgg
gaa gaa tgg aga ccc act tgg gca aga aac tgg aaa cct 480 Ile Asp Trp
Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160
aaa gat gtt tac aag aat agg tct att gaa ttg gtt cag caa caa aat 528
Lys Asp Val Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn 165
170 175 gta caa ctt agt ctc aca gag gcc act gag aaa gca aaa caa gaa
ttt 576 Val Gln Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu
Phe 180 185 190 gaa aag gca ggg aag gat ttc ctg gta gag act ata aaa
ttg gga aaa 624 Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr Ile Lys
Leu Gly Lys 195 200 205 tta ctt cgg cca aat cac ttg tgg ggt tat tat
ctt ttt ccg gat tgt 672 Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr
Leu Phe Pro Asp Cys 210 215 220 tac aac cat cac tat aag aaa ccc ggt
tac aat gga agt tgc ttc aat 720 Tyr Asn His His Tyr Lys Lys Pro Gly
Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240 gta gaa ata aaa aga aat
gat gat ctc agc tgg ttg tgg aat gaa agc 768 Val Glu Ile Lys Arg Asn
Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255 act gct ctt tac
cca tcc att tat ttg aac act cag cag tct cct gta 816 Thr Ala Leu Tyr
Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val 260 265 270 gct gct
aca ctc tat gtg cgc aat cga gtt cgg gaa gcc atc aga gtt 864 Ala Ala
Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val 275 280 285
tcc aaa ata cct gat gca aaa agt cca ctt ccg gtt ttt gca tat acc 912
Ser Lys Ile Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290
295 300 cgc ata gtt ttt act gat caa gtt ttg aaa ttc ctt tct caa gat
gaa 960 Arg Ile Val Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp
Glu 305 310 315 320 ctt gtg tat aca ttt ggc gaa act gtt gct ctg ggt
gct tct gga att 1008 Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu
Gly Ala Ser Gly Ile 325 330 335 gta ata tgg gga acc ctc agt ata atg
cga agt atg aaa tct tgc ttg 1056 Val Ile Trp Gly Thr Leu Ser Ile
Met Arg Ser Met Lys Ser Cys Leu 340 345 350 ctc cta gac aat tac atg
gag act ata ctg aat cct tac ata atc aac 1104 Leu Leu Asp Asn Tyr
Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn 355 360 365 gtc aca cta
gca gcc aaa atg tgt agc caa gtg ctt tgc cag gag caa 1152 Val Thr
Leu Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln 370 375 380
gga gtg tgt ata agg aaa aac tgg aat tca agt gac tat ctt cac ctc
1200 Gly Val Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His
Leu 385 390 395 400 aac cca gat aat ttt gct att caa ctt gag aaa ggt
gga aag ttc aca 1248 Asn Pro Asp Asn Phe Ala Ile Gln Leu Glu Lys
Gly Gly Lys Phe Thr 405 410 415 gta cgt gga aaa ccg aca ctt gaa gac
ctg gag caa ttt tct gaa aaa 1296 Val Arg Gly Lys Pro Thr Leu Glu
Asp Leu Glu Gln Phe Ser Glu Lys 420 425 430 ttt tat tgc agc tgt tat
agc acc ttg agt tgt aag gag aaa gct gat 1344 Phe Tyr Cys Ser Cys
Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445 gta aaa gac
act gat gct gtt gat gtg tgt att gct gat ggt gtc tgt 1392 Val Lys
Asp Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys 450 455 460
ata gat gct ttt cta aaa cct ccc atg gag aca gaa gaa cct gga tcc
1440 Ile Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gly
Ser 465 470 475 480 ggt tct ggt gct cac cat cac cat cac cat taa
1473 Gly Ser Gly Ala His His His His His His * 485 490 16 490 PRT
Homo sapiens 16 Met Gly Val Leu Lys Phe Lys His Ile Phe Phe Arg Ser
Phe Val Lys 1 5 10 15 Ser Ser Gly Val Ser Gln Ile Val Phe Thr Phe
Leu Leu Ile Pro Cys 20 25 30 Cys Leu Thr Leu Asn Phe Arg Ala Pro
Pro Val Ile Pro Asn Val Pro 35 40 45 Phe Leu Trp Ala Trp Asn Ala
Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60 Asp Glu Pro Leu Asp
Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg 65 70 75 80 Ile Asn Ala
Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu 85 90 95 Gly
Tyr Tyr Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly 100 105
110 Gly Ile Pro Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys
115 120 125 Lys Asp Ile Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met
Ala Val 130 135 140 Ile Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg
Asn Trp Lys Pro 145 150 155 160 Lys Asp Val Tyr Lys Asn Arg Ser Ile
Glu Leu Val Gln Gln Gln Asn 165 170 175 Val Gln Leu Ser Leu Thr Glu
Ala Thr Glu Lys Ala Lys Gln Glu Phe 180 185 190 Glu Lys Ala Gly Lys
Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys 195 200 205 Leu Leu Arg
Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220 Tyr
Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230
235 240 Val Glu Ile Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu
Ser 245 250 255 Thr Ala Leu Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln
Ser Pro Val 260 265 270 Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg
Glu Ala Ile Arg Val 275 280 285 Ser Lys Ile Pro Asp Ala Lys Ser Pro
Leu Pro Val Phe Ala Tyr Thr 290 295 300 Arg Ile Val Phe Thr Asp Gln
Val Leu Lys Phe Leu Ser Gln Asp Glu 305 310 315 320 Leu Val Tyr Thr
Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile 325 330 335 Val Ile
Trp Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu 340 345 350
Leu Leu Asp Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn 355
360 365 Val Thr Leu Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu
Gln 370 375 380 Gly Val Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr
Leu His Leu 385 390 395 400 Asn Pro Asp Asn Phe Ala Ile Gln Leu Glu
Lys Gly Gly Lys Phe Thr 405 410 415 Val Arg Gly Lys Pro Thr Leu Glu
Asp Leu Glu Gln Phe Ser Glu Lys 420 425 430 Phe Tyr Cys Ser Cys Tyr
Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445 Val Lys Asp Thr
Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys 450 455 460 Ile Asp
Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gly Ser 465 470 475
480 Gly Ser Gly Ala His His His His His His 485 490 17 39 DNA
Artificial Sequence SpacerHisFor Primer 17 ataattggat ccggttctgg
tgctcaccat caccatcac 39 18 38 DNA Artificial Sequence SpacerHisRev
Primer 18 tataattgcg gccgcctaat ggtgatggtg atggtgag 38 19 30 DNA
Artificial Sequence 3' REVERSE PRIMER WITHOUT STOP CODON FOR
GENERATING truncation product HIS-sHASEGP lacking GPI anchor and
ending at N 483 19 aatggatcca ttgtagaaaa tttgaggttc 30 20 30 DNA
Artificial Sequence 3' REVERSE PRIMER WITHOUT STOP CODON FOR
GENERATING truncation product HIS-sHASEGP lacking GPI anchor and
ending at Y
482 20 aatggatccg tagaaaattt gaggttcttc 30 21 30 DNA Artificial
Sequence 3' REVERSE PRIMER WITHOUT STOP CODON FOR GENERATING
truncation product HIS-sHASEGP lacking GPI anchor and ending at F
481 21 aattggatcc gaaaatttga ggttcttctg 30 22 30 DNA Artificial
Sequence 3' REVERSE PRIMER WITHOUT STOP CODON FOR GENERATING
truncation product HIS-sHASEGP lacking GPI anchor and ending at I
480 22 attggatcca atttgaggtt cttctgtctc 30 23 29 DNA Artificial
Sequence 3' REVERSE PRIMER WITHOUT STOP CODON FOR GENERATING
truncation product HIS-sHASEGP lacking GPI anchor and ending at Q
479 23 aattggatcc ttgaggttct tctgtctcc 29 24 29 DNA Artificial
Sequence 3' REVERSE PRIMER WITHOUT STOP CODON FOR GENERATING
truncation product HIS-sHASEGP lacking GPI anchor and ending at P
478 24 aattggatcc aggttcttct gtctccatg 29 25 28 DNA Artificial
Sequence 3' REVERSE PRIMER WITHOUT STOP CODON FOR GENERATING
truncation product HIS-sHASEGP lacking GPI anchor and ending at E
477 25 aattggatcc ttcttctgtc tccatggg 28 26 36 DNA Artificial
Sequence Reverse primer to amplify sHASEGP deletion mutant ending
at A 467 26 aattggatcc ctaagcatct atacagacac catcag 36 27 35 DNA
Artificial Sequence Reverse primer to amplify sHASEGP deletion
mutant ending at A 447 27 aattggatcc ctaagctttc tccttacaac tcaag 35
28 34 DNA Artificial Sequence Reverse primer to amplify sHASEGP
deletion mutant ending at S 430 28 aattggatcc ctaagaaaat tgctccaggt
cttc 34 29 36 DNA Artificial Sequence Reverse primer to amplify
sHASEGP deletion mutant ending at G 413 29 aattggatcc ctatccacct
ttctcaagtt gaatag 36 30 36 DNA Artificial Sequence Reverse primer
to amplify sHASEGP deletion mutant ending at S 394 30 aattggatcc
ctatgaattc cagtttttcc ttatac 36 31 35 DNA Artificial Sequence
Reverse primer to amplify sHASEGP deletion mutant ending at A 372
31 aattggatcc ctatgctagt gtgacgttga ttatg 35 32 33 DNA Artificial
Sequence Reverse primer to amplify sHASEGP deletion mutant ending
at S 347 32 aattggatcc ctaacttcgc attatactga ggg 33 33 29 DNA
Artificial Sequence LN forward primer for site directed mutagenesis
to generate sHASEGP fusion with kappa leader with L 36 as the first
sHASEGP amino acid after the kappa leader 33 ctgaatttca gagcacctcc
tgttattcc 29 34 28 DNA Artificial Sequence FR forward primer for
site directed mutagenesis to generate sHASEGP fusion with kappa
leader with F 38 as the first sHASEGP amino acid after the kappa
leader 34 ttcagagcac ctcctgttat tccaaatg 28 35 23 DNA Artificial
Sequence Asp reverse primer for site directed mutagenesis to
generate sHASEGP fusion with kappa leader with Asp as the last
Kappa leader amino acid before L 36 or F 38 of PH-20 35 gtcaccagtg
gaacctggaa ccc 23 36 24 DNA Artificial Sequence Gly reverse primer
for site directed mutagenesis to generate sHASEGP fusion with kappa
leader with Gly as the last Kappa leader amino acid before L 36 or
F 38 of PH-20 36 accagtggaa cctggaaccc agag 24 37 30 DNA Artificial
Sequence Forward primer for first fragment of kappa leader 37
gagacagaca cactcctgct atgggtactg 30 38 30 DNA Artificial Sequence
Reverse primer for first fragment of kappa leader 38 cccagagcag
cagtacccat agcaggagtg 30 39 30 DNA Artificial Sequence Forward
primer for second fragment of kappa leader 39 ggtactgctg ctctgggttc
caggttccac 30 40 27 DNA Artificial Sequence Reverse primer for
second fragment of kappa leader 40 gcgtcaccag tggaacctgg aacccag 27
41 30 DNA Artificial Sequence Nhe Forward primer for kappa leader
41 attgctagca tggagacaga cacactcctg 30 42 28 DNA Artificial
Sequence EcoR1 reverse primer for kappa leader 42 aattgaattc
gtcaccagtg gaacctgg 28 43 21 PRT Artificial Sequence Mouse
IgK-chain leader sequence 43 Met Glu Thr Asp Thr Leu Leu Leu Trp
Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp 20 44 30
DNA Artificial Sequence K-leader SPE 1 FORWARD Primer 44 actcactagt
gctagcatgg agacagacac 30 45 30 DNA Artificial Sequence K-leader
MLU1 REV primer 45 aattacgcgt gaattcgtca ccagtggaac 30 46 462 PRT
Artificial Sequence Kappa leader fusion protein with sHASEGP with F
38 as the first amino acid of the putative mature secreted sHASEGP
(up to P478) 46 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu
Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Phe Arg Ala Pro Pro Val
Ile Pro Asn Val Pro 20 25 30 Phe Leu Trp Ala Trp Asn Ala Pro Ser
Glu Phe Cys Leu Gly Lys Phe 35 40 45 Asp Glu Pro Leu Asp Met Ser
Leu Phe Ser Phe Ile Gly Ser Pro Arg 50 55 60 Ile Asn Ala Thr Gly
Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu 65 70 75 80 Gly Tyr Tyr
Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly 85 90 95 Gly
Ile Pro Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys 100 105
110 Lys Asp Ile Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val
115 120 125 Ile Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp
Lys Pro 130 135 140 Lys Asp Val Tyr Lys Asn Arg Ser Ile Glu Leu Val
Gln Gln Gln Asn 145 150 155 160 Val Gln Leu Ser Leu Thr Glu Ala Thr
Glu Lys Ala Lys Gln Glu Phe 165 170 175 Glu Lys Ala Gly Lys Asp Phe
Leu Val Glu Thr Ile Lys Leu Gly Lys 180 185 190 Leu Leu Arg Pro Asn
His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 195 200 205 Tyr Asn His
His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 210 215 220 Val
Glu Ile Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 225 230
235 240 Thr Ala Leu Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro
Val 245 250 255 Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala
Ile Arg Val 260 265 270 Ser Lys Ile Pro Asp Ala Lys Ser Pro Leu Pro
Val Phe Ala Tyr Thr 275 280 285 Arg Ile Val Phe Thr Asp Gln Val Leu
Lys Phe Leu Ser Gln Asp Glu 290 295 300 Leu Val Tyr Thr Phe Gly Glu
Thr Val Ala Leu Gly Ala Ser Gly Ile 305 310 315 320 Val Ile Trp Gly
Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu 325 330 335 Leu Leu
Asp Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn 340 345 350
Val Thr Leu Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln 355
360 365 Gly Val Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His
Leu 370 375 380 Asn Pro Asp Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly
Lys Phe Thr 385 390 395 400 Val Arg Gly Lys Pro Thr Leu Glu Asp Leu
Glu Gln Phe Ser Glu Lys 405 410 415 Phe Tyr Cys Ser Cys Tyr Ser Thr
Leu Ser Cys Lys Glu Lys Ala Asp 420 425 430 Val Lys Asp Thr Asp Ala
Val Asp Val Cys Ile Ala Asp Gly Val Cys 435 440 445 Ile Asp Ala Phe
Leu Lys Pro Pro Met Glu Thr Glu Glu Pro 450 455 460 47 40 DNA
Artificial Sequence 3' Primer BAM REV sHASEGP with GPI anchor up to
L 509 including STOP 47 aattggatcc ctacagaaga aatgataaga aacaaaatac
40 48 1449 DNA Homo sapiens 48 atgggagtgc taaaattcaa gcacatcttt
ttcagaagct ttgttaaatc aagtggagta 60 tcccagatag ttttcacctt
ccttctgatt ccatgttgct tgactctgaa tttcagagca 120 cctcctgtta
ttccaaatgt gcctttcctc tgggcctgga atgccccaag tgaattttgt 180
cttggaaaat ttgatgagcc actagatatg agcctcttct ctttcatagg aagcccccga
240 ataaacgcca ccgggcaagg tgttacaata ttttatgttg atagacttgg
ctactatcct 300 tacatagatt caatcacagg agtaactgtg aatggaggaa
tcccccagaa gatttcctta 360 caagaccatc tggacaaagc taagaaagac
attacatttt atatgccagt agacaatttg 420 ggaatggctg ttattgactg
ggaagaatgg agacccactt gggcaagaaa ctggaaacct 480 aaagatgttt
acaagaatag gtctattgaa ttggttcagc aacaaaatgt acaacttagt 540
ctcacagagg ccactgagaa agcaaaacaa gaatttgaaa aggcagggaa ggatttcctg
600 gtagagacta taaaattggg aaaattactt cggccaaatc acttgtgggg
ttattatctt 660 tttccggatt gttacaacca tcactataag aaacccggtt
acaatggaag ttgcttcaat 720 gtagaaataa aaagaaatga tgatctcagc
tggttgtgga atgaaagcac tgctctttac 780 ccatccattt atttgaacac
tcagcagtct cctgtagctg ctacactcta tgtgcgcaat 840 cgagttcggg
aagccatcag agtttccaaa atacctgatg caaaaagtcc acttccggtt 900
tttgcatata cccgcatagt ttttactgat caagttttga aattcctttc tcaagatgaa
960 cttgtgtata catttggcga aactgttgct ctgggtgctt ctggaattgt
aatatgggga 1020 accctcagta taatgcgaag tatgaaatct tgcttgctcc
tagacaatta catggagact 1080 atactgaatc cttacataat caacgtcaca
ctagcagcca aaatgtgtag ccaagtgctt 1140 tgccaggagc aaggagtgtg
tataaggaaa aactggaatt caagtgacta tcttcacctc 1200 aacccagata
attttgctat tcaacttgag aaaggtggaa agttcacagt acgtggaaaa 1260
ccgacacttg aagacctgga gcaattttct gaaaaatttt attgcagctg ttatagcacc
1320 ttgagttgta aggagaaagc tgatgtaaaa gacactgatg ctgttgatgt
gtgtattgct 1380 gatggtgtct gtatagatgc ttttctaaaa cctcccatgg
agacagaaga acctcaaatt 1440 ttctactaa 1449 49 467 PRT Homo sapiens
49 Met Gly Val Leu Lys Phe Lys His Ile Phe Phe Arg Ser Phe Val Lys
1 5 10 15 Ser Ser Gly Val Ser Gln Ile Val Phe Thr Phe Leu Leu Ile
Pro Cys 20 25 30 Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val Ile
Pro Asn Val Pro 35 40 45 Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu
Phe Cys Leu Gly Lys Phe 50 55 60 Asp Glu Pro Leu Asp Met Ser Leu
Phe Ser Phe Ile Gly Ser Pro Arg 65 70 75 80 Ile Asn Ala Thr Gly Gln
Gly Val Thr Ile Phe Tyr Val Asp Arg Leu 85 90 95 Gly Tyr Tyr Pro
Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly 100 105 110 Gly Ile
Pro Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys 115 120 125
Lys Asp Ile Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130
135 140 Ile Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys
Pro 145 150 155 160 Lys Asp Val Tyr Lys Asn Arg Ser Ile Glu Leu Val
Gln Gln Gln Asn 165 170 175 Val Gln Leu Ser Leu Thr Glu Ala Thr Glu
Lys Ala Lys Gln Glu Phe 180 185 190 Glu Lys Ala Gly Lys Asp Phe Leu
Val Glu Thr Ile Lys Leu Gly Lys 195 200 205 Leu Leu Arg Pro Asn His
Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220 Tyr Asn His His
Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240 Val
Glu Ile Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250
255 Thr Ala Leu Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val
260 265 270 Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile
Arg Val 275 280 285 Ser Lys Ile Pro Asp Ala Lys Ser Pro Leu Pro Val
Phe Ala Tyr Thr 290 295 300 Arg Ile Val Phe Thr Asp Gln Val Leu Lys
Phe Leu Ser Gln Asp Glu 305 310 315 320 Leu Val Tyr Thr Phe Gly Glu
Thr Val Ala Leu Gly Ala Ser Gly Ile 325 330 335 Val Ile Trp Gly Thr
Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu 340 345 350 Leu Leu Asp
Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn 355 360 365 Val
Thr Leu Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln 370 375
380 Gly Val Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu
385 390 395 400 Asn Pro Asp Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly
Lys Phe Thr 405 410 415 Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu
Gln Phe Ser Glu Lys 420 425 430 Phe Tyr Cys Ser Cys Tyr Ser Thr Leu
Ser Cys Lys Glu Lys Ala Asp 435 440 445 Val Lys Asp Thr Asp Ala Val
Asp Val Cys Ile Ala Asp Gly Val Cys 450 455 460 Ile Asp Ala 465 50
1536 DNA Homo sapiens 50 atgggagtgc taaaattcaa gcacatcttt
ttcagaagct ttgttaaatc aagtggagta 60 tcccagatag ttttcacctt
ccttctgatt ccatgttgct tgactctgaa tttcagagca 120 cctcctgtta
ttccaaatgt gcctttcctc tgggcctgga atgccccaag tgaattttgt 180
cttggaaaat ttgatgagcc actagatatg agcctcttct ctttcatagg aagcccccga
240 ataaacgcca ccgggcaagg tgttacaata ttttatgttg atagacttgg
ctactatcct 300 tacatagatt caatcacagg agtaactgtg aatggaggaa
tcccccagaa gatttcctta 360 caagaccatc tggacaaagc taagaaagac
attacatttt atatgccagt agacaatttg 420 ggaatggctg ttattgactg
ggaagaatgg agacccactt gggcaagaaa ctggaaacct 480 aaagatgttt
acaagaatag gtctattgaa ttggttcagc aacaaaatgt acaacttagt 540
ctcacagagg ccactgagaa agcaaaacaa gaatttgaaa aggcagggaa ggatttcctg
600 gtagagacta taaaattggg aaaattactt cggccaaatc acttgtgggg
ttattatctt 660 tttccggatt gttacaacca tcactataag aaacccggtt
acaatggaag ttgcttcaat 720 gtagaaataa aaagaaatga tgatctcagc
tggttgtgga atgaaagcac tgctctttac 780 ccatccattt atttgaacac
tcagcagtct cctgtagctg ctacactcta tgtgcgcaat 840 cgagttcggg
aagccatcag agtttccaaa atacctgatg caaaaagtcc acttccggtt 900
tttgcatata cccgcatagt ttttactgat caagttttga aattcctttc tcaagatgaa
960 cttgtgtata catttggcga aactgttgct ctgggtgctt ctggaattgt
aatatgggga 1020 accctcagta taatgcgaag tatgaaatct tgcttgctcc
tagacaatta catggagact 1080 atactgaatc cttacataat caacgtcaca
ctagcagcca aaatgtgtag ccaagtgctt 1140 tgccaggagc aaggagtgtg
tataaggaaa aactggaatt caagtgacta tcttcacctc 1200 aacccagata
attttgctat tcaacttgag aaaggtggaa agttcacagt acgtggaaaa 1260
ccgacacttg aagacctgga gcaattttct gaaaaatttt attgcagctg ttatagcacc
1320 ttgagttgta aggagaaagc tgatgtaaaa gacactgatg ctgttgatgt
gtgtattgct 1380 gatggtgtct gtatagatgc ttttctaaaa cctcccatgg
agacagaaga acctcaaatt 1440 ttctacaatg cttcaccctc cacactatct
gccacaatgt tcatttggag gctggaagtc 1500 tgggatcaag gtattagcag
aattggtttc ttctga 1536 51 6630 DNA Artificial Sequence HZ24 plasmid
vector 51 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca
atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac
atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt
gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat
atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga
ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg
acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc
ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat
taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt
ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt
ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660
cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
720 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc
acagttaaat 780 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac
ttaagctgca gaagttggtc 840 gtgaggcact gggcaggtaa gtatcaaggt
tacaagacag gtttaaggag accaatagaa 900 actgggcttg tcgagacaga
gaagactctt gcgtttctga taggcaccta ttggtcttac 960 tgacatccac
tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta 1020
aggctagagt acttaatacg actcactata ggctagcatg ggagtgctaa aattcaagca
1080 catctttttc agaagctttg ttaaatcaag tggagtatcc cagatagttt
tcaccttcct 1140 tctgattcca tgttgcttga ctctgaattt cagagcacct
cctgttattc caaatgtgcc 1200 tttcctctgg gcctggaatg ccccaagtga
attttgtctt ggaaaatttg atgagccact 1260 agatatgagc ctcttctctt
tcataggaag cccccgaata aacgccaccg ggcaaggtgt 1320 tacaatattt
tatgttgata gacttggcta ctatccttac atagattcaa tcacaggagt 1380
aactgtgaat ggaggaatcc cccagaagat ttccttacaa gaccatctgg acaaagctaa
1440 gaaagacatt acattttata tgccagtaga caatttggga atggctgtta
ttgactggga 1500 agaatggaga cccacttggg caagaaactg gaaacctaaa
gatgtttaca agaataggtc 1560 tattgaattg gttcagcaac
aaaatgtaca acttagtctc acagaggcca ctgagaaagc 1620 aaaacaagaa
tttgaaaagg cagggaagga tttcctggta gagactataa aattgggaaa 1680
attacttcgg ccaaatcact tgtggggtta ttatcttttt ccggattgtt acaaccatca
1740 ctataagaaa cccggttaca atggaagttg cttcaatgta gaaataaaaa
gaaatgatga 1800 tctcagctgg ttgtggaatg aaagcactgc tctttaccca
tccatttatt tgaacactca 1860 gcagtctcct gtagctgcta cactctatgt
gcgcaatcga gttcgggaag ccatcagagt 1920 ttccaaaata cctgatgcaa
aaagtccact tccggttttt gcatataccc gcatagtttt 1980 tactgatcaa
gttttgaaat tcctttctca agatgaactt gtgtatacat ttggcgaaac 2040
tgttgctctg ggtgcttctg gaattgtaat atggggaacc ctcagtataa tgcgaagtat
2100 gaaatcttgc ttgctcctag acaattacat ggagactata ctgaatcctt
acataatcaa 2160 cgtcacacta gcagccaaaa tgtgtagcca agtgctttgc
caggagcaag gagtgtgtat 2220 aaggaaaaac tggaattcaa gtgactatct
tcacctcaac ccagataatt ttgctattca 2280 acttgagaaa ggtggaaagt
tcacagtacg tggaaaaccg acacttgaag acctggagca 2340 attttctgaa
aaattttatt gcagctgtta tagcaccttg agttgtaagg agaaagctga 2400
tgtaaaagac actgatgctg ttgatgtgtg tattgctgat ggtgtctgta tagatgcttt
2460 tctaaaacct cccatggaga cagaagaacc tcaaattttc tactgaggat
ccatagctaa 2520 cgcccctctc cctccccccc ccctaacgtt actggccgaa
gccgcttgga ataaggccgg 2580 tgtgcgtttg tctatatgtt attttccacc
atattgccgt cttttggcaa tgtgagggcc 2640 cggaaacctg gccctgtctt
cttgacgagc attcctaggg gtctttcccc tctcgccaaa 2700 ggaatgcaag
gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 2760
caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
2820 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
accccagtgc 2880 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
tctcctcaag cgtattcaac 2940 aaggggctga aggatgccca gaaggtaccc
cattgtatgg gatctgatct ggggcctcgg 3000 tgcacatgct ttacatgtgt
ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac 3060 ggggacgtgg
ttttcctttg aaaaacacga tgataagctt gccacaaccc acagcggccg 3120
ctgccatcat ggttcgacca ttgaactgca tcgtcgccgt gtcccaaaat atggggattg
3180 gcaagaacgg agacctaccc tggcctccgc tcaggaacga gttcaagtac
ttccaaagaa 3240 tgaccacaac ctcttcagtg gaaggtaaac agaatctggt
gattatgggt aggaaaacct 3300 ggttctccat tcctgagaag aatcgacctt
taaaggacag aattaatata gttctcagta 3360 gagaactcaa agaaccacca
cgaggagctc attttcttgc caaaagtttg gatgatgcct 3420 taagacttat
tgaacaaccg gaattggcaa gtaaagtaga catggtttgg atagtcggag 3480
gcagttctgt ttaccaggaa gccatgaatc aaccaggcca cctcagactc tttgtgacaa
3540 ggatcatgca ggaatttgaa agtgacacgt ttttcccaga aattgatttg
gggaaatata 3600 aacttctccc agaataccca ggcgtcctct ctgaggtcca
ggaggaaaaa ggcatcaagt 3660 ataagtttga agtctacgag aagaaagact
aaacgcgtgg tacctctaga gtcgacccgg 3720 gcggccgctt cgagcagaca
tgataagata cattgatgag tttggacaaa ccacaactag 3780 aatgcagtga
aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 3840
cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
3900 tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat
gtggtaaaat 3960 cgataaggat ccgggctggc gtaatagcga agaggcccgc
accgatcgcc cttcccaaca 4020 gttgcgcagc ctgaatggcg aatggacgcg
ccctgtagcg gcgcattaag cgcggcgggt 4080 gtggtggtta cgcgcagcgt
gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 4140 gctttcttcc
cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 4200
gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
4260 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
ccctttgacg 4320 ttggagtcca cgttctttaa tagtggactc ttgttccaaa
ctggaacaac actcaaccct 4380 atctcggtct attcttttga tttataaggg
attttgccga tttcggccta ttggttaaaa 4440 aatgagctga tttaacaaaa
atttaacgcg aattttaaca aaatattaac gcttacaatt 4500 tcctgatgcg
gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 4560
ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
4620 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
caagctgtga 4680 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
atcaccgaaa cgcgcgagac 4740 gaaagggcct cgtgatacgc ctatttttat
aggttaatgt catgataata atggtttctt 4800 agacgtcagg tggcactttt
cggggaaatg tgcgcggaac ccctatttgt ttatttttct 4860 aaatacattc
aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 4920
attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
4980 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
aaagatgctg 5040 aagatcagtt gggtgcacga gtgggttaca tcgaactgga
tctcaacagc ggtaagatcc 5100 ttgagagttt tcgccccgaa gaacgttttc
caatgatgag cacttttaaa gttctgctat 5160 gtggcgcggt attatcccgt
attgacgccg ggcaagagca actcggtcgc cgcatacact 5220 attctcagaa
tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 5280
tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
5340 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
aacatggggg 5400 atcatgtaac tcgccttgat cgttgggaac cggagctgaa
tgaagccata ccaaacgacg 5460 agcgtgacac cacgatgcct gtagcaatgg
caacaacgtt gcgcaaacta ttaactggcg 5520 aactacttac tctagcttcc
cggcaacaat taatagactg gatggaggcg gataaagttg 5580 caggaccact
tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 5640
ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
5700 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
aatagacaga 5760 tcgctgagat aggtgcctca ctgattaagc attggtaact
gtcagaccaa gtttactcat 5820 atatacttta gattgattta aaacttcatt
tttaatttaa aaggatctag gtgaagatcc 5880 tttttgataa tctcatgacc
aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 5940 accccgtaga
aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 6000
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
6060 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
actgttcttc 6120 tagtgtagcc gtagttaggc caccacttca agaactctgt
agcaccgcct acatacctcg 6180 ctctgctaat cctgttacca gtggctgctg
ccagtggcga taagtcgtgt cttaccgggt 6240 tggactcaag acgatagtta
ccggataagg cgcagcggtc gggctgaacg gggggttcgt 6300 gcacacagcc
cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 6360
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
6420 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
tatctttata 6480 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
tttgtgatgc tcgtcagggg 6540 ggcggagcct atggaaaaac gccagcaacg
cggccttttt acggttcctg gccttttgct 6600 ggccttttgc tcacatggct
cgacagatct 6630 52 2009 DNA Homo sapiens 52 atgtggctca cataaattca
gaaagtatga tagcagtgta ggtggttagc agcacctcat 60 aaggtccttc
ctagcaaggc aaagggatgc taatgactag ccaatgctct aggaagacat 120
tgagaccagc caacttcttg ccttgataac tactgaagag acattgggtg gctggatttt
180 gaaagcagac ttctggttat aggtgatgca acttgaaaaa caatcctgaa
acatgaaaca 240 agaataataa tatttaaatg taacttaatc attatacctc
tttatccatc aaagtgaatt 300 cattccattc cctttcatct gtgctcatac
tttgcatcag atattgggta aaccaaagtg 360 tgtaggaaga aataaatgtt
ttcatagtca ttactcttta caatgggagt gctaaaattc 420 aagcacatct
ttttcagaag ctttgttaaa tcaagtggag tatcccagat agttttcacc 480
ttccttctga ttccatgttg cttgactctg aatttcagag cacctcctgt tattccaaat
540 gtgcctttcc tctgggcctg gaatgcccca agtgaatttt gtcttggaaa
atttgatgag 600 ccactagata tgagcctctt ctctttcata ggaagccccc
gaataaacgc caccgggcaa 660 ggtgttacaa tattttatgt tgatagactt
ggctactatc cttacataga ttcaatcaca 720 ggagtaactg tgaatggagg
aatcccccag aagatttcct tacaagacca tctggacaaa 780 gctaagaaag
acattacatt ttatatgcca gtagacaatt tgggaatggc tgttattgac 840
tgggaagaat ggagacccac ttgggcaaga aactggaaac ctaaagatgt ttacaagaat
900 aggtctattg aattggttca gcaacaaaat gtacaactta gtctcacaga
ggccactgag 960 aaagcaaaac aagaatttga aaaggcaggg aaggatttcc
tggtagagac tataaaattg 1020 ggaaaattac ttcggccaaa tcacttgtgg
ggttattatc tttttccgga ttgttacaac 1080 catcactata agaaacccgg
ttacaatgga agttgcttca atgtagaaat aaaaagaaat 1140 gatgatctca
gctggttgtg gaatgaaagc actgctcttt acccatccat ttatttgaac 1200
actcagcagt ctcctgtagc tgctacactc tatgtgcgca atcgagttcg ggaagccatc
1260 agagtttcca aaatacctga tgcaaaaagt ccacttccgg tttttgcata
tacccgcata 1320 gtttttactg atcaagtttt gaaattcctt tctcaagatg
aacttgtgta tacatttggc 1380 gaaactgttg ctctgggtgc ttctggaatt
gtaatatggg gaaccctcag tataatgcga 1440 agtatgaaat cttgcttgct
cctagacaat tacatggaga ctatactgaa tccttacata 1500 atcaacgtca
cactagcagc caaaatgtgt agccaagtgc tttgccagga gcaaggagtg 1560
tgtataagga aaaactggaa ttcaagtgac tatcttcacc tcaacccaga taattttgct
1620 attcaacttg agaaaggtgg aaagttcaca gtacgtggaa aaccgacact
tgaagacctg 1680 gagcaatttt ctgaaaaatt ttattgcagc tgttatagca
ccttgagttg taaggagaaa 1740 gctgatgtaa aagacactga tgctgttgat
gtgtgtattg ctgatggtgt ctgtatagat 1800 gcttttctaa aacctcccat
ggagacagaa gaacctcaaa ttttctacaa tgcttcaccc 1860 tccacactat
ctgccacaat gttcattgtt agtattttgt ttcttatcat ttcttctgta 1920
gcgagtttgt aattgcgcag gttagctgaa atgaacaata tgtccatctt aaagtgtgct
1980 ttttcgacta attaaatctt tgaaaagaa 2009 53 2395 DNA Homo sapiens
53 atgtggctca cataaattca gaaagtatga tagcagtgta ggtggttagc
agcacctcat 60 aaggtccttc ctagcaaggg atgctaatga ctagccaatg
ctctaggaag acattgagac 120 cagccaactt cttgccttga taactactga
agagacattg ggtggctgga ttttgaaagc 180 agacttctgg ttataggtga
tgcaacttga aaaacaatcc tgaaacatga aacaagaata 240 ataatattta
aatgtaactt aatcattata cctctttatc catcaaagtg aattcattcc 300
attccctttc atctgtgctc atactttgca tcagatattg ggtaaaccaa agtgtgtagg
360 aagaaataaa tgttttcata gtcattactc tttacaatgg gagtgctaaa
attcaagcac 420 atctttttca gaagctttgt taaatcaagt ggagtatccc
agatagtttt caccttcctt 480 ctgattccat gttgcttgac tctgaatttc
agagcacctc ctgttattcc aaatgtgcct 540 ttcctctggg cctggaatgc
cccaagtgaa ttttgtcttg gaaaatttga tgagccacta 600 gatatgagcc
tcttctcttt cataggaagc ccccgaataa acgccaccgg gcaaggtgtt 660
acaatatttt atgttgatag acttggctac tatccttaca tagattcaat cacaggagta
720 actgtgaatg gaggaatccc ccagaagatt tccttacaag accatctgga
caaagctaag 780 aaagacatta cattttatat gccagtagac aatttgggaa
tggctgttat tgactgggaa 840 gaatggagac ccacttgggc aagaaactgg
aaacctaaag atgtttacaa gaataggtct 900 attgaattgg ttcagcaaca
aaatgtacaa cttagtctca cagaggccac tgagaaagca 960 aaacaagaat
ttgaaaaggc agggaaggat ttcctggtag agactataaa attgggaaaa 1020
ttacttcggc caaatcactt gtggggttat tatctttttc cggattgtta caaccatcac
1080 tataagaaac ccggttacaa tggaagttgc ttcaatgtag aaataaaaag
aaatgatgat 1140 ctcagctggt tgtggaatga aagcactgct ctttacccat
ccatttattt gaacactcag 1200 cagtctcctg tagctgctac actctatgtg
cgcaatcgag ttcgggaagc catcagagtt 1260 tccaaaatac ctgatgcaaa
aagtccactt ccggtttttg catatacccg catagttttt 1320 actgatcaag
ttttgaaatt cctttctcaa gatgaacttg tgtatacatt tggcgaaact 1380
gttgctctgg gtgcttctgg aattgtaata tggggaaccc tcagtataat gcgaagtatg
1440 aaatcttgct tgctcctaga caattacatg gagactatac tgaatcctta
cataatcaac 1500 gtcacactag cagccaaaat gtgtagccaa gtgctttgcc
aggagcaagg agtgtgtata 1560 aggaaaaact ggaattcaag tgactatctt
cacctcaacc cagataattt tgctattcaa 1620 cttgagaaag gtggaaagtt
cacagtacgt ggaaaaccga cacttgaaga cctggagcaa 1680 ttttctgaaa
aattttattg cagctgttat agcaccttga gttgtaagga gaaagctgat 1740
gtaaaagaca ctgatgctgt tgatgtgtgt attgctgatg gtgtctgtat agatgctttt
1800 ctaaaacctc ccatggagac agaagaacct caaattttct acaatgcttc
accctccaca 1860 ctatctgcca caatgttcat ttggaggctg gaagtctggg
atcaaggtat tagcagaatt 1920 ggtttcttct gagagtcatg agggaaaaat
gtgtttcagg cctcttccct tggcttacag 1980 gaaatgaaaa aaccatgact
atcatcacca acatccttgg gtattaagtg cagtcactct 2040 cctagatgct
gtggggagaa ggcaagttac aaagatagac cttccctcaa gataatcaga 2100
ttttcatggt attatcctta acctttttga catcatggag gctttgggaa tctgatgaag
2160 cctatcaatt ttcttccaga agatatttat ataagattat aagaaaaatt
atgtacacag 2220 cttattttat tgcattggat caaaatgcca tttataaaga
attatgcctt ttccatcaat 2280 tttagcatgg aaaaataatt tcaggcaata
tgcttaaaaa ttgggggaag acaaaagaaa 2340 tccatatcgt gtaaataaaa
ataaattttg gttttgctca aaaaaaaaaa aaaaa 2395
* * * * *
References