U.S. patent application number 12/870030 was filed with the patent office on 2011-02-24 for conjugates comprising a biodegradable polymer and uses therefor.
Invention is credited to DAVID R. ELMALEH, MIKHAIL L. PAPISOV, SIMON C. ROBSON.
Application Number | 20110044967 12/870030 |
Document ID | / |
Family ID | 27757725 |
Filed Date | 2011-02-24 |
United States Patent
Application |
20110044967 |
Kind Code |
A1 |
ELMALEH; DAVID R. ; et
al. |
February 24, 2011 |
Conjugates Comprising a Biodegradable Polymer and Uses Therefor
Abstract
Biologically active agents covalently linked to a polymer. The
polymer is preferably a biodegradable polymer are provided. The
biologically active agent is preferably a protein, such as an
extracellular soluble protein, e.g., an extracellular enzyme. The
enzyme can be an apyrase, e.g., NTPDase. Conjugates of the
invention can be used as therapeutics in subjects. For example, a
conjugate comprising an apyrase can be used for treating and
preventing thrombosis, atherosclerotic plaque complications and
vascular disorders.
Inventors: |
ELMALEH; DAVID R.; (NEWTON,
MA) ; ROBSON; SIMON C.; (WESTON, MA) ;
PAPISOV; MIKHAIL L.; (WINCHESTER, MA) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Family ID: |
27757725 |
Appl. No.: |
12/870030 |
Filed: |
August 27, 2010 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10922378 |
Aug 20, 2004 |
7785618 |
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12870030 |
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PCT/US03/04845 |
Feb 19, 2003 |
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10922378 |
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60358303 |
Feb 20, 2002 |
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Current U.S.
Class: |
424/94.3 ;
435/188 |
Current CPC
Class: |
C12N 11/08 20130101;
C08L 59/00 20130101; A61K 47/60 20170801 |
Class at
Publication: |
424/94.3 ;
435/188 |
International
Class: |
A61K 38/46 20060101
A61K038/46; C12N 9/96 20060101 C12N009/96 |
Goverment Interests
GOVERNMENT FUNDING
[0001] This invention was made with support under Grant Number
HL57307 and HL63972, awarded by the National Institutes of Health;
the government, therefore, has certain rights in the invention.
Claims
1-49. (canceled)
50. A conjugate comprising a protein and a biodegradable polymer,
wherein the activity of the protein is higher relative to that of
the protein in the absence of the biodegradable polymer; and/or the
half-life of the protein is longer relative to that of the protein
in the absence of the biodegradable polymer; and wherein the
biodegradable polymer is a polyketal or polyacetal.
51. The conjugate of claim 50, wherein the activity of the protein
is at least 10 fold higher relative to that of the protein in the
absence of the biodegradable polymer.
52. The conjugate of claim 50, wherein the half-life of the protein
is at least 10 fold longer relative to that of the protein in the
absence of the biodegradable polymer.
53. The conjugate of claim 50, wherein the biodegradable polymer is
negatively charged.
54. The conjugate of claim 50, wherein the biodegradable polymer is
positively charged.
55. The conjugate of claim 50, wherein the biodegradable polymer
has a molecular weight from about 2 kDa to about 250 kDa.
56. The conjugate of claim 50, wherein the biodegradable polymer
has a molecular weight from about 20 kDa to about 100 kDa.
57. The conjugate of claim 50, wherein the biodegradable polymer is
a polyketal.
58. The conjugate of claim 50, wherein the biodegradable polymer is
a polyacetal.
59. The conjugate of claim 50, wherein the biodegradable polymer is
a polyacetal; and the polyacetal is poly(hydroxymethylethylene
hydroxymethylacetal).
60. The conjugate of claim 50, wherein the protein is conjugated to
the biodegradable polymer through a covalent bond.
61. The conjugate of claim 50, comprising a tether between the
protein and the biodegradable polymer.
62. The conjugate of claim 61, wherein the tether is a derivative
of a compound chosen from the group consisting of ethylene
glycol-bis-succinimidylsuccinate, succinic acid or succinic
anhydride, diaminohexane, glyoxlic acid, short chain polyethylene
glycol, and glycine.
63. The conjugate of claim 50, further comprising a targeting
agent.
64. A pharmaceutical preparation comprising a conjugate of any one
of claims 50-63 and a pharmaceutically acceptable carrier.
Description
BACKGROUND OF THE INVENTION
[0002] A major challenge in the area of the parenteral
administration of biologically active materials is the development
of a controlled delivery device that is small enough for
intravenous application and which has a long circulating half-life.
Biologically active materials administered in such a controlled
fashion into tissue or blood are expected to exhibit decreased
toxic side effects compared to when the materials are injected in
the form of a solution, and may reduce degradation of sensitive
compounds in the plasma.
[0003] A number of injectable drug delivery systems have been
investigated, including microcapsules, microparticles, liposomes
and emulsions. A significant obstacle to the use of these
injectable drug delivery materials is the rapid clearance of the
materials from the blood stream by the macrophages of the
reticuloendothelial system (RES). For example, polystyrene
particles as small as sixty nanometers in diameter are cleared from
the blood within two to three minutes. Polystyrene particles are
also not biodegradable and therefore not therapeutically
useful.
[0004] Polymers which are degraded by a physical or chemical
process in response to contact with body fluid, while implanted or
injected, are generally considered to be biodegradable.
Biodegradable polymers have been the subject of increasing interest
as materials which can be employed to form a wide variety of
pharmaceutical preparations and other biomedical products. Examples
of medical applications for biodegradable polymers include tablet
coatings, plasma substitutes, gels, contact lenses, surgical
implants, as ingredients of eyedrops, and as long-lived circulating
and targeted drugs.
[0005] However, many polymers have hydrophobic domains and,
consequently, their biocompatability is limited. Hydrophobic
polymers are vulnerable to non-specific interactions with proteins
and lipids which also may cause undesirable side effects. In
addition, synthetic polymers, such as vinyl, acrylic and
methacrylic polymers, which typically have a hydrophobic main
chain, do not degrade readily in vivo.
[0006] Hydrophilic polymers are common in nature. For example,
polysaccharides are naturally-occurring polymers which include
hydrolytically-sensitive acetals in their main chain. However,
polysaccharides can interact with cell receptors and/or plasma
opsonins, and can cause adverse reactions and other non-desirable
effects.
[0007] In addition, the activity and half-life of biological
agents, such as ecto-enzymes, which are introduced into the blood
stream is transient, which therefore limits the biological agents'
potential application. To achieve effective therapies, repeated
large enzyme dosing may be required. This may in turn result in
increased toxicity and side effects as an immune response to the
extended enzyme introduction.
[0008] Therefore, a need exists for a polymer system which
overcomes or minimizes the above-referenced problems and is
amenable to modification by a biologically active agent wherein the
agent is stabilized and released in a timed manner.
SUMMARY OF THE INVENTION
[0009] In one embodiment, the invention provides a conjugate
comprising a biologically active agent and a polymer. The polymer
is preferably a biodegradable polymer. In a preferred embodiment,
the polymer is a polyacetal. The polymer or conjugate can be
positively or negatively charged. The biologically active agent can
be conjugated to the polymer through covalent, ionic or hydrogen
bond(s). Alternatively, the biologically active agent is linked to
the polymer through a tether, e.g., a derivative of a compound
selected from the group consisting of ethylene
glycol-bis-succinimidylsuccinate, succinic acid or succinic
anhydride, diaminohexane, glyoxlic acid, short chain polyethylene
glycol, and glycine.
[0010] The polymer can be crosslinked with epibromohydrin or
epichlorhydrin to form a gel. Exemplary polymers have a molecular
weight between 0.5 and 500 kDa, preferably between 1 and 300 kDa.
In a preferred embodiment the biodegradable polyacetal is
poly(hydroxymethylethylenehydroxymethylacetal).
[0011] In a preferred embodiment, the biologically active agent is
a protein, preferably an extracellular protein, even more
preferably an enzyme, and most preferably the enzyme is nucleoside
triphosphate diphosphohydrolase (NTPDase), e.g., human CD39 or
related ectoenzymes. The NTPDase can comprise an amino acid
sequence that is at least about 90% identical to SEQ ID NO: 2 and
catalyze hydrolysis of NTPs and/or NDPs. In a further embodiment
the apyrase comprises the catalytic domain set forth in SEQ ID NO:
2.
[0012] The biodegradable polymer may also be selected from the
group consisting of polycarbonates, polyanhydrides,
polyorthoesters, polyglycolide (PGA), copolymers of glycolide,
poly(glycolide-co-caprolactone) (PGA/PCL), glycolide/L-lactide
copolymers (PGA/PLLA), lactide/trimethylene carbonate copolymers
(PLA/TMC), glycolide/trimethylene carbonate copolymers (PGA/TMC),
polylactides (PLA), stereo-copolymers of PLA, poly-L-lactide
(PLLA), poly-DL-lactide (PDLLA), L-lactide/DL-lactide copolymers,
copolymers of PLA, lactide/tetramethylglycolide copolymers,
lactide/.alpha.-valerolactone copolymers, lactide/s-caprolactone
copolymers, hyaluronic acid and its derivatives, polydepsipeptides,
PLA/polyethylene oxide copolymers, unsymmetrical 3,6-substituted
poly-1,4-dioxane-2,5-diones, carboxymethyl cellulose (CMC),
poly-.beta.-hydroxybutyrate (PHBA), PHBA/bhydroxyvalerate
copolymers (PHBA/HVA), poly-p-dioxanone (PDS), poly-a-valerlactone,
poly-.epsilon.-caprolactone, methacrylate-N-vinyl-pyrrolidone
copolymers, polyesteramides, polyesters of oxalic acid,
polydihydropyranes, polyalkyl-2-cyanoacrylates, polyurethanes,
polypeptides, poly-.beta.-malic acid (PMLA), poly-.beta.-alcanoic
acids, polybutylene oxalate, polyethylene adipate, polyethylene
carbonate, polybutylene carbonate, and other polyesters containing
silyl ethers, acetals or ketals, and alginates.
[0013] Conjugates of the invention can be used for treating
subjects. For example, conjugates comprising apyrases can be used
for treating subjects that may benefit from modulation of
circulating levels of nucleotides in the blood. In an illustrative
embodiment, the subject to be treated may be a subject suffering
from diseases relating to atherosclerotic disease, abnormal
platelet aggregation, excessive angiogenesis, and cellular
hyperproliferation, e.g., cancer. Transplant subjects, e.g., those
with rejection or preservation injury, may also benefit from
administration of conjugates comprising apyrases.
[0014] Conjugates comprising apyrases can also be used in vitro,
e.g., in stored blood, such as to prevent platelet aggregation
and/or leukocyte activation in blood; platelet preparation, or
leucopheresis product. Thus, for example, conjugates may be added
to a sample of blood or cells (e.g., leukocytes) thereof.
[0015] At least one advantage of the conjugates comprising apyrases
relative to non conjugated apyrases is that the conjugated apyrases
have an increased biological activity and extended in vivo
half-life. In addition, conjugates comprising apyrases are
potentially less toxic sytemically due to the polymeric
biodegradation and slow apyrase release.
BRIEF DESCRIPTION OF DRAWINGS
[0016] FIG. 1 depicts the amino acid sequence of human CD39 (SEQ ID
NO: 2) and the location of ACR domains.
[0017] FIG. 2 depicts the .sup.13C NMR spectrum of a polyacetal
used in the biologically active agent conjugates of the current
invention dissolved in deuterium oxide.
[0018] FIG. 3 depicts the levels of NTPDase present in the blood of
mice injected with apyrase or apyrase conjugated to a positively or
negatively charged polyacetal 5, 60 and 240 minutes after the
injection.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The features and other details of the invention, either as
steps of the invention or as combination of parts of the invention,
will now be more particularly described and pointed out in the
claims. It will be understood that the particular embodiments of
the invention are shown by way of illustration and not as
limitations of the invention. The principle features of the
invention may be employed in various embodiments without departing
from the scope of the invention.
[0020] The invention is based at least in part on the discovery
that modification of apyrase with a biodegradable polymer, such as
Fleximer, provides for stable enzyme activity by several fold. The
modification involves in part linking apyrase to the biodegradable
polymer.
DEFINITIONS
[0021] The terms used herein have their usual meaning in the art,
however, to even further clarify the present invention, for
convenience, the meaning of certain terms and phrases employed in
the specification, examples, and appended claims are provided
below.
[0022] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage (Immunology--A
Synthesis, 2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
Associates, Sunderland, Mass. (1991), which is incorporated herein
by reference). Stereoisomers (e.g., D-amino acids) of the twenty
conventional amino acids, unnatural amino acids such as,
.alpha.,.alpha.-disubstituted amino acids, N-alkyl amino acids,
lactic acid, and other unconventional amino acids may also be
suitable components for polypeptides of the present invention.
Examples of unconventional amino acids include: 4-hydroxyproline,
.gamma.-carboxyglutamate, .epsilon.-N,N,N-trimethyllysine,
.epsilon.-N-acetyllysine, O-phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
.omega.-N-methylarginine, and other similar amino acids and imino
acids (e.g., 4-hydroxyproline). In the polypeptide notation used
herein, the lefthand direction is the amino terminal direction and
the righthand direction is the carboxy-terminal direction, in
accordance with standard usage and convention.
[0023] When referring to an amino acid position as being "about
amino acid" it is meant that the amino acid could be up to 10 or
preferably 5 amino acids upstream or downstream of the enumerated
amino acid.
[0024] "Apyrase" refers to an enzyme capable of catalyzing the
sequential hydrolysis of nucleoside and deoxynucleotide
triphosphate (NTP) to nucleoside diphosphate (NDP) to nucleoside
monophosphate (NMP). Nucleoside and deoxynucleoside triphosphates
and diphosphates can be, e.g., ATP, ADP, CTP, CDP, GTP, GDP, TTP,
TDP, UTP and UDP. The enzyme is also alternately referred to as
NTPDase; ADPase; ATPDase; ATPase; ADP monophosphatase and ATP
diphosphohydrolase. Exemplary apyrases include CD39 proteins and
potato apyrase. The term "apyrase" includes naturally-occurring
apyrases, such as those further described herein, as well as
fragments and homologs thereof, provided that they have at least
one biological activity of an apyrase. For example, an apyrase can
be a protein having an amino acid sequence which is at least 70%,
preferably at least 80%, more preferably at least 90% (e.g., 95% or
greater, e.g. 99% or 100%) identical or similar to SEQ ID NO: 2 or
4 or a protein that is encoded by a nucleic acid that hybridizes to
SEQ ID NO: 1 or 3.
[0025] "Apyrase conjugate" used interchangeably herein with
"apyrase complex" or "modified apyrase" refers to a conjugate or a
complex of an apyrase with a polymer. The apyrase and polymer can
be linked directly, or indirectly, e.g., through a tether, and can
be linked together through a covalent bond or a non covalent bond,
e.g., an ionic bond or hydrogen bonds.
[0026] "Biocompatible," as that term is used herein, means
exhibition of essentially no cytotoxicity while in contact with
body fluids. "Biocompatibility" also includes essentially no
interactions with recognition proteins, e.g., naturally occurring
antibodies, cell proteins, cells and other components of biological
systems. However, substances and functional groups specifically
intended to cause the above effects, e.g., drugs and prodrugs, are
considered to be biocompatible.
[0027] "Biodegradable," as that term is used herein, means polymers
which are degraded in response to contact with body fluid while
implanted or injected in vivo. Examples of biodegradation processes
include hydrolysis, enzymatic action, oxidation and reduction.
Suitable conditions for hydrolysis, for example, include exposure
of the biodegradable polymers to water at a temperature and a pH of
circulating blood. Biodegradation of enzyme delivery systems of the
present invention can be enhanced in low pH regions of the
mammalian body, e.g. an inflamed area.
[0028] A "biologically active agent" refers to a molecule or
complex having a biological activity. A preferred biologically
active agent is a protein having one or more polypeptide chains. A
biologically active agent can also be a nucleic acid, e.g., a
ribozyme, a polysaccharide, a lipid, derivatives thereof, or a
small organic molecule.
[0029] "Biological activity of an apyrase" refers to the ability of
the apyrase to catalyze the sequential hydrolysis of NTPs or NDPs.
The biological activity can be determined, e.g., in an
ectonucleotidase or apyrase assay (e.g., ATPase or ADPase assay) or
in a biological assay that measures inhibition of platelet
aggregation. Exemplary assays are further described herein.
[0030] "Conjugated to" in the context of a biologically active
agent and a polymer refers to a covalent bond or a non covalent
bond, e.g., an ionic interaction or interaction through hydrogen
bonds.
[0031] "Graft," "transplant" or "implant" are used interchangeably
to refer to biological material derived from a donor for
transplantation into a recipient, and to the act of placing such
biological material in the recipient.
[0032] "Host" or "recipient" refers to the body of the patient in
whom donor biological material is grafted.
[0033] As used herein, the terms "label" or "labeled" refers to
incorporation of a detectable marker, e.g., by incorporation of a
radiolabeled amino acid or nucleotide or attachment to a
polypeptide or nucleic acid of biotinyl moieties that can be
detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical or colorimetric methods). Various methods of labeling
polypeptides, nucleic acids, and glycoproteins are known in the art
and may be used. Examples of labels for polypeptides include, but
are not limited to, the following: radioisotopes (e.g., .sup.3H,
.sup.14C, .sup.35S, .sup.125I, .sup.131I), fluorescent labels
(e.g., FITC, rhodamine, lanthanide, phosphors), enzymatic labels
(e.g., horseradish peroxidase, .beta.-galactosidase, luciferase,
alkaline phosphatase), biotinyl groups, predetermined polypeptide
epitopes recognized by a secondary reporter (e.g., leucine zipper
pair sequences, binding sites for secondary antibodies, metal
binding domains, epitope tags). In some embodiments, labels are
attached by spacer arms of various lengths to reduce potential
steric hindrance.
[0034] A "patient" or "subject" to be treated by the subject method
can mean either a human or non-human animal.
[0035] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0036] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject compound from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0037] "Polymeric system" is used interchangeably herein with
"polymeric complex" or "conjugate" to refer to a biological agent,
e.g., a protein, conjugated to a polymer.
[0038] The term "polypeptide having activity of an ATP
diphosphohydrolase" includes native ecto-ATP diphosphohydrolase
protein, as well as homologs thereof, e.g., oxidation resistant
peptide analogs thereof, and soluble truncated forms.
[0039] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein when referring to a gene product.
[0040] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity or more (e.g., 99 percent sequence identity). Preferably,
residue positions which are not identical differ by conservative
amino acid substitutions. Conservative amino acid substitutions
refer to the interchangeability of residues having similar side
chains. For example, a group of amino acids having aliphatic side
chains is glycine, alanine, valine, leucine, and isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is
serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, and asparagine-glutamine.
[0041] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the
composition), and preferably a substantially purified fraction is a
composition wherein the object species comprises at least about 50
percent (on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition will comprise more than
about 80 to 90 percent of all macromolecular species present in the
composition. Most preferably, the object species is purified to
essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0042] The phrase "therapeutically-effective amount" as used herein
means that amount of a compound, material, or composition
comprising a compound of the present invention which is effective
for producing some desired therapeutic effect in an animal at a
reasonable benefit/risk ratio applicable to any medical
treatment.
[0043] "Treating" a disease or condition refers to preventing,
curing, as well as ameliorating at least one symptom of the
condition or disease.
[0044] The following definitions pertain to the chemical structure
of compounds:
[0045] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
[0046] The term "electron-withdrawing group" is recognized in the
art, and denotes the tendency of a substituent to attract valence
electrons from neighboring atoms, i.e., the substituent is
electronegative with respect to neighboring atoms. A quantification
of the level of electron-withdrawing capability is given by the
Hammett sigma (.sigma.) constant. This well known constant is
described in many references, for instance, J. March, Advanced
Organic Chemistry, McGraw Hill Book Company, New York, (1977
edition) pp. 251-259. The Hammett constant values are generally
negative for electron donating groups (.sigma.[P]=-0.66 for
NH.sub.2) and positive for electron withdrawing groups
(.sigma.[P]=0.78 for a nitro group), .sigma.[P] indicating para
substitution. Exemplary electron-withdrawing groups include nitro,
acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride, and the
like. Exemplary electron-donating groups include amino, methoxy,
and the like.
[0047] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain'allcyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In preferred embodiments, a straight chain or branched
chain alkyl has 30 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.30 for straight chain, C.sub.3-C.sub.30 for branched
chain), and more preferably 20 or fewer. Likewise, preferred
cycloalkyls have from 3-10 carbon atoms in their ring structure,
and more preferably have 5, 6 or 7 carbons in the ring
structure.
[0048] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Preferred alkyl
groups are lower alkyls. In preferred embodiments, a substituent
designated herein as alkyl is a lower alkyl.
[0049] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or heteroaromatic
group).
[0050] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond respectively.
[0051] The term "aryl" as used herein includes 5-, 6- and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics." The
aromatic ring can be substituted at one or more ring positions with
such substituents as described above, for example, halogen, azide,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,
amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,
ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic
moieties, --CF.sub.3, --CN, or the like. The term "aryl" also
includes polycyclic ring systems having two or more cyclic rings in
which two or more carbons are common to two adjoining rings (the
rings are "fused rings") wherein at least one of the rings is
aromatic, e.g., the other cyclic rings can be cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
[0052] The terms ortho, meta and para apply to 1,2-, 1,3- and
1,4-disubstituted benzenes, respectively. For example, the names
1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
[0053] The terms "heterocycle" or "heterocyclic group" refer to 3-
to 10-membered ring structures, more preferably 3- to 7-membered
rings, whose ring structures include one to four heteroatoms.
Heterocycles can also be polycycles. Heterocyclyl groups include,
for example, thiophene, thianthrene, furan, pyran, isobenzofuran,
chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole,
isothiazole, thiazole, thiazolidines, thiazolidin-4-ones,
thiazolidin-5-ones, isoxazole, pyridine, pyrazine, pyrimidine,
pyridazine, indolizine, isoindole, indole, indazole, purine,
quinolizine, isoquinoline, quinoline, tetrahydroquinoline,
tetrahydroisoquinoline, phthalazine, naphthyridine, quinoxaline,
quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,
phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine,
oxolane, thiolane, oxazole, piperidine, piperazine, morpholine,
lactones, lactams such as azetidinones and pyrrolidinones, sultams,
sultones, and the like. The heterocyclic ring can be substituted at
one or more positions with such substituents as described above, as
for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
[0054] The terms "polycyclyl" or "polycyclic group" refer to two or
more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls
and/or heterocyclyls) in which two or more carbons are common to
two adjoining rings, e.g., the rings are "fused rings". Rings that
are joined through non-adjacent atoms are termed "bridged" rings.
Each of the rings of the polycycle can be substituted with such
substituents as described above, as for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,
sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,
carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde,
ester, a heterocyclyl, an aromatic or heteroaromatic moiety,
--CF.sub.3, --CN, or the like.
[0055] The term "carbocycle", as used herein, refers to an aromatic
or non-aromatic ring in which each atom of the ring is carbon.
[0056] As used herein, the term "nitro" means --NO.sub.2; the term
"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl"
means --SH; the term "hydroxyl" means --OH; and the term "sulfonyl"
means --SO.sub.2--.
[0057] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines, e.g., a moiety that
can be represented by the general formula:
##STR00001##
wherein R.sub.9, R.sub.10 and R'.sub.10 each independently
represent a hydrogen permitted by the rules of valence.
[0058] The term "acylamino" is art-recognized and refers to a
moiety that can be represented by the general formula:
##STR00002##
wherein R.sub.9 is as defined above, and R'.sub.11 represents a
hydrogen, an alkyl, an alkenyl or --(CH.sub.2).sub.m--R.sub.8,
where m and R.sub.8 are as defined above.
[0059] The term "amido" is art recognized as an amino-substituted
carbonyl and includes a moiety that can be represented by the
general formula:
##STR00003##
wherein R.sub.9, R.sub.10 are as defined above. Preferred
embodiments of the amide will not include imides which may be
unstable.
[0060] The term "alkylthio" refers to an alkyl group, as defined
above, having a sulfur radical attached thereto. In preferred
embodiments, the "alkylthio" moiety is represented by one of
--S-alkyl, --S-alkenyl, --S-alkynyl, and
--S--(CH.sub.2).sub.m--R.sub.8, wherein m and R.sub.8 are defined
above. Representative alkylthio groups include methylthio, ethyl
thio, and the like.
[0061] The term "carbonyl" is art recognized and includes such
moieties as can be represented by the general formula:
##STR00004##
wherein X is a bond or represents an oxygen or a sulfur, and
R.sub.11 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sub.8 or a pharmaceutically acceptable salt,
R.sub.11 represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8 are as defined
above. Where X is an oxygen and R.sub.11 or R'.sub.11 is not
hydrogen, the formula represents an "ester". Where X is an oxygen,
and R.sub.11 is as defined above, the moiety is referred to herein
as a carboxyl group, and particularly when R.sub.11 is a hydrogen,
the formula represents a "carboxylic acid". Where X is an oxygen,
and R'.sub.11 is hydrogen, the formula represents a "formate". In
general, where the oxygen atom of the above formula is replaced by
sulfur, the formula represents a "thiolcarbonyl" group. Where X is
a sulfur and R.sub.11 or R'.sub.11 is not hydrogen, the formula
represents a "thiolester." Where X is a sulfur and R.sub.11 is
hydrogen, the formula represents a "thiolcarboxylic acid." Where X
is a sulfur and R.sub.11' is hydrogen, the formula represents a
"thiolformate." On the other hand, where X is a bond, and R.sub.11
is not hydrogen, the above formula represents a "ketone" group.
Where X is a bond, and R.sub.11 is hydrogen, the above formula
represents an "aldehyde" group.
[0062] The terms "alkoxyl" or "alkoxy" as used herein refers to an
alkyl group, as defined above, having an oxygen attached thereto.
Representative alkoxyl groups include methoxy, ethoxy, propyloxy,
tert-butoxy and the like. An "ether" is two hydrocarbons covalently
linked by an oxygen. Accordingly, the substituent of an alkyl that
renders that alkyl an ether is or resembles an alkoxyl, such as can
be represented by one of --O-alkyl, --O-alkenyl, --O-alkynyl,
--O--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8 are described
above.
[0063] The term "sulfonate" is art recognized and includes a moiety
that can be represented by the general formula:
##STR00005##
in which R.sub.41 is an electron pair, hydrogen, alkyl, cycloalkyl,
or aryl.
[0064] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0065] The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations. The abbreviations contained in said
list, and all abbreviations utilized by organic chemists of
ordinary skill in the art are hereby incorporated by reference.
[0066] The term "sulfate" is art recognized and includes a moiety
that can be represented by the general formula:
##STR00006##
in which R.sub.41 is as defined above.
[0067] The term "sulfonylamino" is art recognized and includes a
moiety that can be represented by the general formula:
##STR00007##
[0068] The term "sulfamoyl" is art-recognized and includes a moiety
that can be represented by the general formula:
##STR00008##
[0069] The term "sulfonyl", as used herein, refers to a moiety that
can be represented by the general formula:
##STR00009##
in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,
or heteroaryl.
[0070] The term "sulfoxido" as used herein, refers to a moiety that
can be represented by the general formula:
##STR00010##
in which R.sub.45 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aralkyl, or aryl.
[0071] Analogous substitutions can be made to alkenyl and alkynyl
groups to produce, for example, aminoalkenyls, aminoalkynyls,
amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls,
thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or
alkynyls.
[0072] As used herein, the definition of each expression, e.g.
alkyl, m, n, etc., when it occurs more than once in any structure,
is intended to be independent of its definition elsewhere in the
same structure.
[0073] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc.
[0074] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
herein above. The permissible substituents can be one or more and
the same or different for appropriate organic compounds. For
purposes of this invention, the heteroatoms such as nitrogen may
have hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This invention is not intended to be limited in
any manner by the permissible substituents of organic
compounds.
[0075] The phrase "protecting group" as used herein means temporary
substituents which protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 2.sup.nd ed.; Wiley: New York, 1991).
[0076] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover. Also for purposes of this invention, the term
"hydrocarbon" is contemplated to include all permissible compounds
having at least one hydrogen and one carbon atom. In a broad
aspect, the permissible hydrocarbons include acyclic and cyclic,
branched and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic organic compounds which can be substituted or
unsubstituted.
Exemplary Polymers
[0077] A preferred polymer of the invention is preferably a
biodegradable and/or biocompatible polymer, which preferably also
contains functionality throughout the backbone chain that can be
modified to form linkages with the biologically active agents. In
an even more preferred embodiment the biodegradable polymer is a
polyacetal. The biodegradable biocompatible polyacetals of the
present invention have the following chemical structure:
##STR00011##
[0078] R.sup.1 is biocompatible and includes a carbon atom
covalently attached to C.sup.1. R.sup.x includes a carbon atom
covalently attached to C.sup.2. "n" is an integer. R.sup.2,
R.sup.3, R.sup.4 and R.sup.5 are biocompatible and are selected
from the group consisting of hydrogen and organic moieties. At
least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 is
hydrophilic. Examples of suitable organic moieties are aliphatic
groups having a chain of atoms in a range of between about one and
twelve atoms.
[0079] The term "hydrophilic" as it relates to R.sup.1, R.sup.2,
R.sup.3, R.sup.4 and R.sup.5 denotes organic moieties which contain
ionizable, polar, or polarizable atoms, or which otherwise may bind
water molecules. Examples of particular hydrophilic organic
moieties which are suitable include carbamates, amides, hydroxyls,
carboxylic acids and their salts, carboxylic acid esters, amines,
sulfonic acids and their salts, sulfonic acid esters, phosphoric
acids and their salts, phosphate esters, polyglycol ethers,
polyamines, polycarboxylates, polyesters, polythioethers, etc. In
preferred embodiments of the present invention, at least one of
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 include a carboxyl
group (COOH), an aldehyde group (CHO) or a methylol (CH.sub.2OH).
In another preferred embodiment of the present invention, R.sup.1,
R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are methylols. In still
another preferred embodiment of the present invention, R.sup.1 and
R.sup.2 are methylols and R.sup.3, R.sup.4, and R.sup.5 are
hydrogens.
[0080] In yet another embodiment of the present invention, at least
one of R.sup.1, R.sup.2, R.sup.3, R.sup.4 or R.sup.5 is a
nitrogen-containing compound. The nitrogen-containing compound can
be a drug or a cross-linking agent or a functional group which is
suitable as a modifier of biodegradable biocompatible polyacetal
behavior in vivo. Examples of such functional groups include
antibodies, their fragments, receptor ligands and other compounds
that selectively interact with biological systems.
[0081] Alternatively, the nitrogen-containing compound can have a
chemical structure of --C.sub.nH.sub.2nNR.sup.6R.sup.7, wherein "n"
is an integer. In one embodiment, "n" is one. R.sup.6 and R.sup.7
can include hydrogen, organic or inorganic substituents. Examples
of suitable organic or inorganic groups include aliphatic groups,
aromatic groups, complexes of heavy metals, etc.
[0082] The biodegradable biocompatible polyacetals of the invention
can be cross-linked. A suitable cross-linking agent has the formula
X.sup.1--(R)--X.sup.2, where R is a spacer group and X.sup.1 and
X.sup.2 are reactive groups. Examples of suitable spacer groups
include biodegradable or nonbiodegradable groups, for example,
aliphatic groups, carbon chains containing biodegradable inserts
such as disulfides, esters, etc. The term "reactive group," as it
relates to X.sup.1 and X.sup.2, means functional groups which can
be connected by a reaction within the biodegradable biocompatible
polyacetals, thereby cross-linking the biodegradable biocompatible
polyacetals. Suitable reactive groups which form cross-linked
networks with the biodegradable biocompatible polyacetals include
epoxides, halides, tosylates, mesylates, carboxylates, aziridines,
cyclopropanes, esters, N-oxysuccinimde esters, disulfides,
anhydrides etc.
[0083] In a preferred embodiment, the biodegradable biocompatible
polyacetals are cross-linked with epibromohydrin or
epichlorohydrin. More preferably, the epibromohydrin or
epichlorohydrin is present in an amount in the range of between
about one and twenty five percent by weight of the cross-linked
biodegradable biocompatible polyacetals.
[0084] Alternatively, the term "reactive" group as it relates to
X.sup.1 and x.sup.2 means a nucleophilic group that can be reacted
with an aldehyde intermediate of the biodegradable biocompatible
polyacetals, thereby cross-linking the biodegradable biocompatible
polyacetals. Suitable reactive groups for the aldehyde intermediate
include amines, thiols, polyols, alcohols, ketones, aldehydes,
diazocompounds, boron derivatives, ylides, isonitriles, hydrazines
and their derivatives and hydroxylamines and their derivatives,
etc.
[0085] In one embodiment, the biodegradable biocompatible
polyacetals of the present invention have a molecular weight of
between about 0.5 and 500 kDa. In a preferred embodiment of the
present invention, the biodegradable biocompatible polyacetals have
a molecular weight of between about 1 and 300 kDa; 2 and 250 kDa or
20 and 100 kDa.
[0086] The biodegradable biocompatible polyacetal of the present
invention can be formed by combining a suitable polysaccharide with
a molar excess of a glycol-specific oxidizing agent to form an
aldehyde intermediate. A "molar excess of a glycol-specific
oxidizing agent," as that phrase is employed herein, means an
amount of the glycol-specific oxidizing agent that provides
oxidative opening of essentially all carbohydrate rings of the
polysaccharide. The aldehyde intermediate may then combined with a
reducing agent to form the biodegradable biocompatible polyacetal.
The biodegradable biocompatible polyacetals of the present
invention can form linear or branched structures. The biodegradable
biocompatible polyacetal of the present invention can be optically
active. Optionally, the biodegradable biocompatible polyacetal of
the present invention can be racemic.
[0087] Structure, yield and molecular weight of the resultant
polyaldehyde depend on the initial polysaccharide. Polysaccharides
that do not undergo significant depolymerization in the presence of
glycol-specific oxidizing agents, for example, poly (1.fwdarw.6)
hexoses, are preferable. Examples of suitable polysaccharides
include starch, cellulose, dextran, etc. A particularly preferred
polysaccharide is dextran. Examples of suitable glycol-specific
oxidizing agents include sodium periodate, lead tetra-acetate, etc.
Examples of suitable reducing agents include sodium borohydride,
sodium cyanoborohydride, etc.
[0088] In an embodiment wherein dextran is employed as a reactant
to form the biodegradable biocompatible polyacetal, the
glycol-specific oxidation can be conducted at a temperature between
about 25.degree. C. and 40.degree. C. for a period of about eight
hours at a suitable pH. Temperature, pH and reaction duration can
affect the reaction rate and polymer hydrolysis rate. The reaction
is preferably conducted in the absence of light. One skilled in the
art can optimize the reaction conditions to obtain polymers of
desired composition. The resultant aldehyde intermediate can be
isolated and combined with a solution of a reducing agent for a
period of about two hours to form the biodegradable biocompatible
polyacetal after isolation. Alternatively, aldehyde groups can be
conjugated with a variety of compounds or converted to other types
of functional groups.
[0089] It is believed that the carbohydrate rings of a suitable
polysaccharide can be oxidized by glycol-specific reagents with
cleavage of carbon bonds between carbon atoms that are connected to
hydroxyl groups. The following mechanism is an example of what is
believed to occur:
##STR00012##
This process can be complicated by the formation of intra and
interpolymer hemiacetals which can inhibit further polysaccharide
oxidation. However, oxidative opening of the polysaccharide rings
can be controlled by controlling the reaction conditions. In the
present invention, it can be demonstrated that the polysaccharide
oxidation, followed by reduction, causes synthesis of
macromolecular biodegradable biocompatible polyacetals. The
structure of the biodegradable biocompatible polyacetal obtained by
the above mentioned method is dependent upon the precursor
polysaccharide. Although it is generally not desirable, the
polyacetal can contain intermittent irregularities throughout the
polyacetal, such as incompletely oxidized additional groups or
moieties in the main chain or in the side chains, as shown
below:
##STR00013##
wherein k, m, and n are integers greater than or equal to one.
[0090] Since it is believed that oxidation does not affect
configurations at the C.sup.1 and C.sup.2 positions, the aldehyde
intermediate and the polyacetal retain the configuration of the
parent polysaccharide and are formed in stereoregular isotactic
forms.
[0091] The resultant biodegradable biocompatible polyacetal can be
chemically modified by, for example, cross-linking the polyacetals
to form a gel. The cross-link density of the biodegradable
biocompatible polyacetal is generally determined by the number of
reactive groups in the polyacetal and by the number of
cross-linking molecules, and can be controlled by varying the ratio
of polyacetal to the amount of cross-linker present.
[0092] For example, the biodegradable biocompatible polyacetal can
be combined with a suitable aqueous base, such as sodium hydroxide,
and cross-linked with epibromohydrin. Control of the amounts of
epibromohydrin can determine the degree of cross-linking within the
biodegradable biocompatible polyacetal gel. For example,
biodegradable biocompatible polyacetals can be exposed to varying
amounts of epibromohydrin for a period of about eight hours at a
temperature about 80.degree. C. to form cross-linked biodegradable
biocompatible polyacetal gels which vary in cross-link density in
relation to the amount of epibromohydrin utilized.
[0093] Treatment of the biodegradable biocompatible polyacetal with
a suitable base, such as triethylamine in dimethylsulfoxide (DMSO),
and an anhydride provides, for example, a derivatized polyacetal
solution. Control of the amount of anhydride within the
biodegradable biocompatible polyacetal can determine the degree of
derivitization of the polyacetal in the solution.
[0094] Polyacetals of this invention can have a variety of
functional groups. For example, aldehyde groups of an intermediate
product of polysaccharide oxidation can be converted not only into
alcohol groups, but also into amines, thioacetals, carboxylic
acids, amides, esters, thioesters, etc.
[0095] Terminal groups of the polymers of this invention can differ
from R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5. Terminal
groups can be created, for example, by selective modification of
each reducing and non-reducing terminal unit of the precursor
polysaccharide.
[0096] One skilled in the art can utilize known chemical reactions
to obtain desired products with varying terminal groups. For
example, a hemiacetal group at the reduced end of the polyacetal
can be readily and selectively transformed into a carboxylic acid
group and further into a variety of other functional groups. A
primary alcohol group at the non-reduced end can be selectively
transformed into an aldehyde group and further into a variety of
functional groups.
[0097] Alternatively, the biodegradable biocompatible polyacetals
of the present invention can be formed by combining a cationic
initiator with a precursor compound having the chemical
structure:
##STR00014##
which forms a polymer having the chemical structure:
##STR00015##
P.sup.1 is a protected hydrophilic group which includes a carbon
atom covalently attached to C.sup.1. P.sup.x includes a carbon atom
covalently attached to C.sup.2. "n" is an integer. At least one of
P.sup.1, P.sup.2, P.sup.3, P.sup.4 and P.sup.5 is selected from
hydrogen and protected hydrophilic groups suitable for conversion.
P.sup.1, P.sup.2, P.sup.3, P.sup.4 and P.sup.5 do not interfere
with the cationic polymerization. Furthermore, P.sup.1, P.sup.2,
P.sup.3, P.sup.4, and P.sup.5 are suitable for conversion to
hydrophilic groups as described above.
[0098] "Protected hydrophilic group," as that term is used herein,
means a chemical group which will not interfere with decyclization
of the precursor compound by the cationic initiator or subsequent
polymerization, and which, upon additional treatment by a suitable
agent, can be converted to a hydrophilic functional group. Examples
of protected hydrophilic groups include esters, ethers, thioesters,
thioethers, vinyl groups, haloalkyl groups, etc.
[0099] Other polymers that can be used in the present invention are
all biodegradable polymers. These polymers include, but are not
limited to, polycarbonates, polyanhydrides, polyorthoesters,
polyglycolide (PGA), copolymers of glycolide,
poly(glycolide-co-caprolactone) (PGA/PCL), glycolide/L-lactide
copolymers (PGA/PLLA), lactide/trimethylene carbonate copolymers
(PLA/TMC), glycolide/trimethylene carbonate copolymers (PGA/TMC),
polylactides (PLA), stereo-copolymers of PLA, poly-L-lactide
(PLLA), poly-DL-lactide (PDLLA), L-lactide/DL-lactide copolymers,
copolymers of PLA, lactide/tetramethylglycolide copolymers,
lactide/.alpha.-valerolactone copolymers,
lactide/.epsilon.-caprolactone copolymers, hyaluronic acid and its
derivatives, polydepsipeptides, PLA/polyethylene oxide copolymers,
unsymmetrical 3,6-substituted poly-1,4-dioxane-2,5-diones,
carboxymethyl cellulose (CMC), poly-.beta.-hydroxybutyrate (PHBA),
PHBA/bhydroxyvalerate copolymers (PHBA/HVA), poly-p-dioxanone
(PDS), poly-a-valerlactone, poly-.epsilon.-caprolactone,
methacrylate-N-vinyl-pyrrolidone copolymers, polyesteramides,
polyesters of oxalic acid, polydihydropyranes,
polyalkyl-2-cyanoacrylates, polyurethanes, polypeptides,
poly-.beta.-malic acid (PMLA), poly-.beta.-alcanoic acids,
polybutylene oxalate, polyethylene adipate, polyethylene carbonate,
polybutylene carbonate, and other polyesters containing silyl
ethers, acetals, or ketals, alginates, and blends or other
combinations of the aforementioned polymers. In addition to the
aforementioned aliphatic link polymers, other aliphatic polyesters
may also be appropriate for producing aromatic/aliphatic polyester
copolymers. These include aliphatic polyesters selected from the
group of oxalates, malonates, succinates, glutarates, adipates,
pimelates, suberates, azelates, sebacates, nonanedioates,
glycolates, and mixtures thereof. All of the above polymers are
degraded in the body by hydrolysis. The different polymers vary in
their structural and chemical aspects, which afford them
differences in strength, action, degradation time, and utility.
[0100] The biodegradable polymers of the present invention
preferably include one or more chemical functional groups through
which the biologically active agents can be linked. Those
biodegradable polymers which do not contain chemical functional
groups may have to be modified in order to introduce chemical
functionality into the polymer. For example, the biodegradable
polymers poly(lactic acid) and poly(glycolic acid) do not contain
any chemically functional groups along the hydrocarbon backbone of
the materials to which a biologically active species can be
covalently coupled. One strategy that has been proposed for
introducing functional groups into poly(lactic acid) is the
copolymerization of lactide with a cyclic monomer of lactic acid
and the amino acid lysine to create poly(lactic acid-co-lysine)
(see U.S. Pat. No. 5,399,665, issued to Barrera, et al.). This
copolymer provides side chains that terminate in amino (NH.sub.2)
groups. These amino groups can be used as attachment sites for the
immobilization of bioactive species. Since this method chemically
alters the polymer, many of the properties of the polymer are
subject to change. For example, the degradation rate and the
tensile strength may be effected by the alteration to the
polymer.
[0101] Certain biodegradable polymers of the present invention may
exist in particular geometric or stereoisomeric forms. The present
invention contemplates all such compounds, including cis- and
trans-isomers, R-- and S-enantiomers, diastereomers, (D)-isomers,
(L)-isomers, the racemic mixtures thereof, atactic, syndiotactic,
isotactic, and other mixtures thereof, as falling within the scope
of the invention. Additional asymmetric carbon atoms may be present
in a substituent such as an alkyl group. All such isomers, as well
as mixtures thereof, are intended to be included in this
invention.
[0102] If, for instance, a particular stereoisomeric form of a
biodegradable polymer of the present invention is desired, it may
be prepared by asymmetric synthesis. Contemplated equivalents of
the biodegradable polymeric systems described above include
polymers which otherwise correspond thereto, and which have the
same general properties thereof, wherein one or more simple
variations of substituents are made which do not adversely affect
the efficacy of the polymer in treating such disorders as mediated
by plaque build up.
[0103] The biodegradable polymers may contain a basic functional
group, such as amino or alkylamino, and are, thus, capable of
forming pharmaceutically-acceptable salts with
pharmaceutically-acceptable acids. The term
"pharmaceutically-acceptable salts" in this respect, refers to the
relatively non-toxic, inorganic and organic acid addition salts of
compounds of the present invention. These salts can be prepared in
situ during the final isolation and purification of the
biodegradable polymers of the invention, or by separately reacting
a purified polymer of the invention in its free base form with a
suitable organic or inorganic acid, and isolating the salt thus
formed. Representative salts include the hydrobromide,
hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,
valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,
phosphate, tosylate, citrate, maleate, fumarate, succinate,
tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and
laurylsulphonate salts and the like. (See, for example, Berge et
al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
[0104] Pharmaceutically acceptable salts of the subject polymers
include the conventional nontoxic salts or quaternary ammonium
salts of the compounds, e.g., from non-toxic organic or inorganic
acids. For example, such conventional nontoxic salts include those
derived from inorganic acids such as hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts
prepared from organic acids such as acetic, propionic, succinic,
glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,
palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,
salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isothionic, and the
like.
[0105] Biodegradable polymers of the present invention may contain
one or more acidic functional groups and, thus, are capable of
forming pharmaceutically-acceptable salts with
pharmaceutically-acceptable bases. These salts can be prepared in
situ during the final isolation and purification of the compounds,
or by separately reacting the purified compound in its free acid
form with a suitable base, such as the hydroxide, carbonate or
bicarbonate of a pharmaceutically-acceptable metal cation, with
ammonia, or with a pharmaceutically-acceptable organic primary,
secondary or tertiary amine. Representative alkali or alkaline
earth salts include the lithium, sodium, potassium, calcium,
magnesium, and aluminum salts and the like. Representative organic
amines useful for the formation of base addition salts include
ethylamine, diethylamine, ethylenediamine, ethanolamine,
diethanolamine, piperazine and the like. (See, for example, Berge
et al., supra).
[0106] In general, the biodegradable polymers of the present
invention may be prepared by the methods illustrated in the
Examples, or by modifications thereof, using readily available
starting materials, reagents and conventional synthesis procedures.
In these reactions, it is also possible to make use of variants
which are in themselves known, but are not mentioned here.
[0107] Biodegradability of polymers can be determined by assays
known in the art, e.g., by the incubation of the polymer in blood
or by injection of the polymer into an animal. Biocompatibility of
a polymer can also be determined according to methods known in the
art.
Exemplary Biologically Active Agents
[0108] In one embodiment, the biological agent provided by the
invention is a membraneous or soluble polypeptide or protein, e.g.,
a polypeptide or protein linked to the cellular membrane and having
an extracellular domain or a polypeptide or protein secreted from a
cell. In a preferred embodiment, the soluble or membrane-bound
polypeptide or protein is an enzyme, in particular, an
extracellular enzyme, also referred to as an "ectoenzyme."
[0109] A preferred ectoenzyme is an ectonucleotidase, i.e., an
extracellular nucleotidase. An exemplary ectonucleotidase is a
nucleoside triphosphate diphosphohydrolase (NTPDase), also referred
to as an "apyrase," "ecto-ATPase," "ecto-ADPase," "nucleotide
phosphohydrolase," and "ATP pyrophosphohydrolase" (EC 3.6.1.5).
NTPDases are Ca.sup.2+/Mg.sup.2+ dependent ectoenzymes that
sequentially hydrolyze nucleoside 5'-triphosphates (NTPs) and
nucleoside 5' diphosphates (NDPs). Thus, NTPDases catalyze the
sequential phosphorolysis (i.e., removal of phosphate groups) of
ATP to ADP to AMP. In general, proteins of this class exhibit
non-specificity toward nucleoside di- or triphosphates and towards
different nucleoside di- and tri-phosphates. For example, the same
enzyme can catalyze the conversion of ATP into ADP and ADP into
AMP. The same enzyme may also hydrolyze CTP, CDP, GTP, GDP, TTP,
TDP, UTP and/or UDP. These enzymes belong to the E-type ATPase or
ecto-ATPDase family, of which the members degrade nucleotide tri-
and/or diphosphates, but not monophosphates (Plesner L. (1995) Int.
Rev. Cytol. 158:141).
[0110] An exemplary NTPDase that can be used according to the
invention is NTPDase-1, also referred to as "CD39," which is a 78
kDa glycosylated protein. This protein was originally described as
a B-cell activation marker. Human CD39 is a protein of 510 amino
acids, encoded by a cDNA of 1704 nucleotides. The nucleotide and
amino acid sequences of human CD39 can be found under GenBank
Accession No. U87967 and S73813; in Kaczmarek et al. (1996) J.
Biol. Chem. 271: 33116; Maliszewski et al. (1994) J. Immunol.
153:3574; WO 96/30532 and in WO 00/23459. The nucleotide sequence
is set forth as SEQ DI NO: 1 and the encoded amino acid sequence
(encoded by nucleotides 31 to 1563) is set forth as SEQ ID NO: 2
and shown in FIG. 1.
[0111] CD39 contains two putative transmembrane regions located
near the N-- and C-termini, respectively. These regions are likely
to be used for anchoring of the protein to the cell membrane. The
portion of the molecule that is between these two putative
transmembrane regions forms a loop that is external to the cell and
contains five small Apyrase-Conserved Regions (ACRs; ACR1 to ACR5;
see, Schulte et al. (1999) Biochemistry 38:2248 and FIG. 1). ACR1
corresponds to amino acids 54-61 of SEQ ID NO: 2; ACR2 corresponds
to amino acids 125-135 of SEQ ID NO: 2; ACR3 corresponds to amino
acids 171-183 of SEQ ID NO: 2; ACR4 corresponds to amino acids 213
to 220 of SEQ ID NO: 2 and ACR5 corresponds to amino acids 447-454
of SEQ ID NO: 2 (Kaczmarek et al. (1996), J. Biol. Chem. 271:33116
and Schulte et al., supra). These ACRs are characteristic of
apyrases and are involved in the enzymatic activity of apyrases. In
particular, intact ACR1, ACR4 and ACR5 within CD39 are involved in
biochemical activity (Schulte et al., supra). Removal of the N-- or
C-terminal transmembrane regions did not dramatically affect
biological activity when the enzyme was coupled to a GPI-anchor
(Schulte et al., supra). A putative ATP-binding domain is located
at amino acids 52-58 of SEQ ID NO: 2.
[0112] CD39 also has six potential N-linked glycosylation sites and
11 cysteine residues that may be implicated in the formation of
oligomers. Glycosylation does not appear to affect activity of the
enzyme, but this remains to be evaluated (Schulte et al., supra).
There are also several sites that may be modified by ectoprotein
kinases and potential intracellular protein kinase C
phosphorylation sites (Schulte et al., supra). CD39 also undergoes
plamitoylation within the N-terminal intracytoplasmic region on the
cysteine at amino acid 13 (Koziak et al. (2000) J. Biol. Chem.
275:2057). CD39 undergoes multimerization (Schulte et al.,
supra).
[0113] Another exemplary apyrase is CD39L1 (or NTPDase-2). Other
apyrases that can be used include CD39L2 (or NTPDase-6); CD39L3;
and CD39L4 (or NTPDase-5) (see, e.g., Chadwick and Frischauff
(1998) Genome 50:357). The nucleotide sequence of CD39-L4 is set
forth in Chadwick and Frischauff (1998) Genome 50:357. Similarly to
CD39, CD39-L1 and CD39-L3 have hydrophobic domains at their N-- and
C-termini. CD39-L2 and CD39-L4, however, have a hydrophobic region
only at their N-terminus and are potentially soluble ecto-enzymes
postcleavage.
[0114] Whereas CD39 (NTPDase 1) hydrolyzes ATPs and ADPs at about
the same rate, CD39-L1 (NTPDase2) hydrolyzes preferentially ATP
over ADP. In particular, whereas recombinant NTPDase 1 hydrolyzes
ATP to AMP with the formation of only minor amounts of free ADP,
ADP appears as the major free product when ATP is hydrolyzed by
NTPDase2 (Heine et al. (2001) Eur J Biochem 268:364. Experiments
with chimeric enzymes comprising portions of each of the NTPDases
showed that amino-acid residues between ACR3 and ACR5 and in
particular the cysteine-rich region between ACR4 and ACR5 conferred
a phenotype to the chimeric enzymes that corresponded to the
respective wild-type enzyme. These results indicated that protein
structure rather than the conserved ACRs may be of major relevance
for determining differences in the catalytic properties between the
related wild-type enzymes (Heine et al., supra).
[0115] CD39-L4 hydrolyzes NDP preferably over NTP. For example, it
has been described that CD39-L4 hydrolyze ADP at a rate of about 20
fold higher than that of ATP (Mulero et al. (1999) J. Biol. Chem.
274:20064).
[0116] In some embodiments, the apyrase is a non-mammalian or
non-animal apyrase, e.g., apyrase from potato (Handa and Guidotti
(1996) Biochem. Biophys. Res. Comm. 218:916 and GenBank Accession
number U58597). The nucleotide and amino acid sequences of potato
apyrase are set forth as SEQ ID NOs: 3 and 4, respectively.
[0117] GenBank Accession numbers for nucleic acids encoding
exemplary apyrases are set forth below: [0118] U87967: Human ATP
diphosphohydrolase; [0119] S73813: CD39=lymphoid cell activation
antigen [human, B lymhpoblastoid cell line, MP-1]; [0120] XM
055699, XM 055698, XM 005712 and XM 051047: Homo sapiens
ectonucleoside triphosphate diphosphohydrolase 1; [0121] NM 020354
and AF269255: Homo sapiens lysosomal apyrase-like protein 1
(LALP1); [0122] AF 034840: Homo sapiens E-type ATPase (HB6); [0123]
NM 001249: Homo sapiens ectonucleoside triphosphate
diphosphohydrolase 5 (ENTPD5): [0124] NM 001248: Homo sapiens
ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3): [0125]
NM 001247: Homo sapiens ectonucleoside triphosphate
diphosphohydrolase 6 (putative function) (ENTPD6); [0126] NM
001246: Homo sapiens ectonucleoside triphosphate diphosphohydrolase
2 (ENTPD2); [0127] NM 001776: Homo sapiens ectonucleoside
triphosphate diphosphohydrolase 1 (ENTPD 1); [0128] NM 053103: Mus
musculus lysosomal apyrase-like 2 (Lysal2); [0129] AV254553 RIKEN
full-length enriched, adult male testis (DH10B) Mus musculus cDNA;
[0130] AF 288221: Mus musculus LALP1; [0131] NM 009849: Mus
musculus ectonucleoside triphosphate diphosphohydrolase 2 (Entpd2);
[0132] NM 009848: Mus musculus ectonucleoside triphosphate
diphosphohydrolase 1 (Entpd1); [0133] NM 007647: Mus musculus
ectonucleoside triphosphate diphosphohydrolase 5 (Entpd5); [0134]
NM 022587: Rattus norvegicus ecto-apyrase (U81295); [0135] AF
041048: Drosophila melanogaster CD39-like NTPase gene; [0136] AF
005940: Bos taurus ecto-apyrase CD39; [0137] U58597 and P80595:
Solanum tuberosum (potato) ATP-diphosphohydrolase (RROP1); [0138]
P32621 Saccharomyces cerevisiae DGA1 guanosine diphosphatase;
[0139] S48859: pea NTP-ase, P. sativum (garden pea) nucleoside
triphosphatase; and [0140] gi1049394: C. elegans cosmid. Yet other
apyrases, such as Shistosoma mansoni, are described in Vasconcelos
et al. (1996) J. Biol. Chem. 271:22139.
[0141] Other apyrase and apyrase-type genes that can be used
according to the invention can be found in GenBank.
[0142] The amino acid sequences of potato apyrase and that of other
NTPDases have a high degree of similarity, particularly within
several small Apyrase-Conserved Regions (ACRs).
[0143] Another sequence that is conserved among apyrases is
(I/V)(V/M/I)(I/UF/C)DAGS(S/T) (SEQ ID NO: 5), which is located near
the amio-terminal of the apyrases (see Vasconcelos et al., supra).
Sequences of strong homology between potato apyrase and pea NTPase
include PGLSSYA (SEQ ID NO: 6) and LYVHSYL (SEQ ID NO: 7) (see
Vasconcelos et al., supra).
[0144] Fragments of apyrases can also be used according to the
invention. Preferred fragments retain at least some of the
biological activity of the full-length apyrase. Exemplary fragments
consists of at least about 50, 100, 200, 300, or 500 amino acids.
In one embodiment, an apyrase lacking one or both of the
hydrophobic regions is used, such that the apyrase is soluble and
not membrane bound. Accordingly, for example, a polypeptide having
about amino acids 37 to about 510 of SEQ ID NO: 2 (lacking the
N-terminal transmembrane region); amino acids 1 to about 477 of SEQ
ID NO: 2 (lacking the C-terminal transmembrane region); about amino
acids 37 to about 477 of SEQ ID NO: 2 (lacking both N-- and
C-terminal transmembrane region) can be used (Schulte et al.,
supra). Fragments of apyrases may contain only the portion that is
external to the cell and contains one or more of the ACRs.
Preferred fragments include ACR-1, ACR4 and/or ACRS. NTPDase
proteins lacking ACR-2 and/or ACR3 can be used according to the
invention. Mutants wherein one of the transmembrane regions or ACRs
is mutated, rather than deleted can also be used according to the
invention. The biological activity of fragments can be tested as
further described herein.
[0145] Preferred apyrases (or apyrases conjugated to a polymer) of
the invention have a greater affinity for ADP than for ATP. For
example, preferred apyrases or fragments of homologs may have an
affinity constant (Kd) for ADP of at least about 10.sup.-6 M,
10.sup.-7 M, 10.sup.-8 M, or 10.sup.-9 M, and an affinity constant
for ATP of at most about 10.sup.-6 M or 10.sup.-7 M. Apyrases may
have an affinity for ADP that is at least 2 fold, 5 fold, 10 fold,
20 fold, 50 fold or 100 fold higher than that for ATP. In other
embodiments, an apyrase will have a greater affinity for ATP than
for ADP.
[0146] Apyrases may have an ADPase or ATPase activity of at least
about 50, preferably at least about 100, 500, 1,000, 2,000, 3,000,
5,000, or 10,000 nmol Pi min.sup.-1 mg.sup.-1 as measured in an
ADPase assay, e.g., as described herein.
[0147] Another ectonucleotidase is ecto-5'nucleotidase (CD73; EC
3.1.3.5), which catalyzes the hydrolysis of nucleoside
5'-monophosphates, such as AMP, UMP and GMP, and generates the
respective nucleosides that may be taken up by specific membrane
transporters (Zimmerman H. (1992) Biochem. J. 285:345). Other
ectonucleosidases that can be used according to the invention
include members of the family of ecto-phosphdiesterase/nucleotide
pyrophosphatase or PDNP family (Goding et al. (1998) Immunol. Rev.
161:11). One member of the PDNP family is is the glycoprotein PC-1,
which has two enzymatic activities, i.e., a 5' nucleotide
phosphodiesterase activity (EC 3.1.4.1) and a nucleotide
pyrophosphatase activity (EC 3.6.1.9), and is capable of
hydrolyzing 3', 5'-cAMP to AMP, or ATP to AMP and pyrophosphate
(Belli and Goding (1994) Eur. J. Biochem. 226:443). Another
ectoenzyme involved in regulating local extracellular
concentrations of nucleosides is adenosine deaminase (ADA; EC
3.5.4.4), which degrades adenosine to inosine.
[0148] In other embodiments, the biological agent is a secreted
polypeptide or protein which is not necessarily an enzyme. For
example, the biological agent can be an interleukin, a lymphokine,
a hormone, a growth factor, a differentiation factor, a complement
factor, or a soluble form of a membrane protein, e.g., a cell
surface receptor. The biological agent can be, e.g., an interferon,
such as interferon-.alpha., -.beta., or -.gamma.. Portions of
secreted polypeptides or proteins can also be used.
[0149] Biological agents that can be used include polypeptides
encoded by any form of a gene, i.e., by any allele of the gene or
mutated versions of the gene, provided that the desired biological
activity is present.
[0150] The biological agents can originate from any species,
including plants and animals. In some embodiments, the biological
agent is from a mammal, e.g., a human, a non-human primate, an
ovine, a bovine, a porcine, an equine, a feline, a canine or an
avian. Nucleotide and amino acid sequences of such homologs may be
known in the art or can be determined by isolating the
corresponding homolog and determining the nucleotide sequence.
Isolating orthologs or a gene can be performed by hybridization
under non stringent conditions as further described herein, or by
PCT using degenerate primers. Such methods are known in the art.
Considering that apyrases are present in distant evolutionary
species, isolation of additional apyrases, is within the skill in
the art.
[0151] Other biological agents that can be used include homologs of
wild-type polypeptide, e.g., apyrases, e.g., those described above.
"Homologs" of apyrases refers to polypeptides having a significant
degree of homology (identity or similarity) in amino acid sequence
and biological activity to wild-type apyrases. Homologs include
polypeptides differing from wild-type polypeptides in one or more
amino acid substitution, deletion or addition. For example, an
homolog of a wild-type apyrase can have from 1 to 50 amino acid
substitutions, deletions or additions, preferably from 1 to 25,
even more preferably from 1 to 15, from 1 to 10 or from 1 to 5
amino acid substitutions, deletions or additions.
[0152] Accordingly, the biological agent can be a polypeptide which
is a least about 70%, at least about 80%, at least about 90%, at
least about 95%, at least about 97% or at least about 99% identical
or homologous to that of the wild-type counterpart polypeptide or
portion thereof, provided that the polypeptide has the desired
biological activity. Based on the description of wild-type
polypeptides provided herein and further described in publications,
a person of skill in the art would know where amino acid
substitutions can be made without significantly affecting the
biological activity of the polypeptide. The biological activity of
such homologues can also be tested as further described herein, or
according to methods known in the art.
[0153] Homologs of naturally occurring polypeptides within the
scope of the invention are those that are encoded by nucleic acids
which hybridize under stringent conditions to nucleic acids of
wild-type polypeptides, e.g., the nucleic acid encoding human CD39,
e.g., set forth as SEQ ID Nos: 1. Appropriate stringency conditions
which promote DNA hybridization, for example, 6.0.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by a
wash of 2.0.times.SSC at 50.degree. C., are known to those skilled
in the art or can be found in Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For
example, the salt concentration in the wash step can be selected
from a low stringency of about 2.0.times.SSC at 50.degree. C. to a
high stringency of about 0.2.times.SSC at 50.degree. C. In
addition, the temperature in the wash step can be increased from
low stringency conditions at room temperature, about 22.degree. C.,
to high stringency conditions at about 65.degree. C. Both
temperature and salt may be varied, or temperature of salt
concentration may be held constant while the other variable is
changed. In a preferred embodiment, a nucleic acid encoding a
mutated PDGFR of the present invention will hybridize to SEQ ID
NOs: 1 or GenBank Accession numbers set forth herein or complement
thereof under moderately stringent conditions, for example at about
2.0.times.SSC and about 40.degree. C.
[0154] Other homologs, e.g., homologs of an apyrase, include those
which differ from the wild-type polypeptide by conservative amino
acid substitutions. Preferred homologs contain from 1 to 20, amino
acid substitutions, preferably from 1 to 10 and even more
preferably from 1 to 5 amino acid substitutions. It is reasonable
to expect that an isolated replacement of a leucine with an
isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid (i.e. isosteric and/or isoelectric
mutations) will not have a major effect on the biological activity
of the resulting molecule. Conservative replacements are those that
take place within a family of amino acids that are related in their
side chains. Genetically encoded amino acids can be divided into
four families: (1) acidic=aspartate, glutamate; (2) basic=lysine,
arginine, histidine; (3) nonpolar=alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan; and (4)
uncharged polar=glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine. In similar fashion, the amino acid repertoire
can be grouped as (1) acidic=aspartate, glutamate; (2)
basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine,
valine, leucine, isoleucine, serine, threonine, with serine and
threonine optionally be grouped separately as aliphatic-hydroxyl;
(4) aromatic=phenylalanine, tyrosine, tryptophan; (5)
amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and
methionine. (see, for example, Biochemistry, 2.sup.nd ed., Ed. by
L. Stryer, WH Freeman and Co.: 1981).
[0155] In yet other embodiments, a biological agent is fused to a
heterologous polypeptide. For example, a polypeptide may be fused
in frame to a marker sequence, also referred to herein as "Tag
sequence" encoding a "Tag peptide", which allows for marking and/or
purification of the polypeptide of the present invention. In a
preferred embodiment, the marker sequence is a hexahistidine tag,
e.g., supplied by a PQE-9 vector. Numerous other Tag peptides are
available commercially. Other frequently used Tags include
myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem
266:21150-21157), which includes a 10-residue sequence from c-myc,
the pFLAG system (International Biotechnologies, Inc.), the
pEZZ-protein A system (Pharmacia, N.J.), and a 16 amino acid
portion of the Haemophilus influenza hemagglutinin protein.
Polypeptides may also be fused to a maltose binding protein (MBP),
glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for
expression and purification of such fusion proteins are
commercially available from New England BioLab (Beverly, Mass.),
Pharmacia (Piscataway, N.J.) and InVitrogen, respectively.
Furthermore, any polypeptide can be used as a Tag so long as a
reagent, e.g., an antibody interacting specifically with the Tag
polypeptide is available or can be prepared or identified.
[0156] In another embodiment, a fusion polypeptide, e.g., a
polypeptide having a poly-(His) Tag further comprises a cleavage
site located between the polypeptide and the Tag, thereby allowing
the Tag to be removed following the purification step using the
Tag. For example, the cleavage site can be a consensus sequence
that is recognized by proteases. The site can be, e.g., an
enterokinase cleavage site which is cleavable with enterokinase
(e.g., see Hochuli et al. (1987) J. Chromatography 411:177; and
Janknecht et al. PNAS 88:8972).
[0157] Alternatively, a heterologous polypeptide may be added for
stabilizing the protein; or for facilitating the folding of the
polypeptide or for prolonging its half-life. For example, a
polypeptide may be fused to an immunoglobulin constant region (see,
e.g., U.S. Pat. No. 5,434,131).
[0158] Polypeptides may also be chemically modified to create
derivatives by forming covalent or aggregate conjugates with other
chemical moieties, such as glycosyl groups, lipids, phosphate,
acetyl groups and the like. Covalent derivatives of proteins can be
prepared by linking the chemical moieties to functional groups on
amino acid sidechains of the protein or at the N-terminus or at the
C-terminus of the polypeptide.
[0159] Biologically active agents may also be labeled, which help
in the detection of the conjugate. A label can be radioactive or
non-radioactive. When the conjugate is administered to a subject,
any label suitable for administration to a subject can be used.
Numerous labels and methods for attaching them to molecules are
known in the art.
[0160] In another embodiment, an enzyme may be subjected to limited
proteolysis prior to linking to a polymer. For example, apyrase can
be incubated at 37.degree. C. with about 3.3 .mu.g/ml trypsin (Life
Technologies, Grand Island, N.Y.) for about 5 minutes or in 0.3%
Triton X-100 with 0.25 units of N-glycosidase F (Life Technologies,
Grand Island, N.Y.) for 12 hours. The reaction can be stopped with
PMSF at a final concentration of about 1 mM. This is further
described in Schulte et al., supra.
Methods of Making Biological Agents
[0161] Biological agents can be prepared according to methods known
in the art. For example, polypeptides or proteins can be isolated
from their natural environment; they can be synthesized de novo, or
they can be produced by recombinant technology.
[0162] In a preferred embodiment, a polypeptide is produced
recombinantly. In an illustrative embodiment, a nucleic acid
encoding the polypeptide and operably linked to at least one
transcriptional regulatory sequence, such as a promoter, is
introduced into a host cell, the polypeptide is expressed in the
cell or is secreted from the cell, and the polypeptide is isolated.
The host cell can be a eukaryotic (e.g., yeast, avian, insect,
mammalian or plant) or a prokaryotic cell (e.g., bacterial).
Alternatively, the polypeptide can be synthesized in lysates. For
example, RNA can be synthesized in vitro, and the RNA can be
translated into polypeptide in a reticulocyte lysate or wheat germ
extract, according to methods known in the art.
[0163] Preferred mammalian expression vectors contain both
prokaryotic sequences, to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine
papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived
and p205) can be used for transient expression of proteins in
eukaryotic cells. The various methods employed in the preparation
of the plasmids and transformation of host organisms are well known
in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells, as well as general recombinant
procedures, see Molecular Cloning A Laboratory Manual, 2.sup.nd
Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor
Laboratory Press: 1989) Chapters 16 and 17.
[0164] A number of types of cells may act as suitable host cells
for expression of a protein. Mammalian host cells include, for
example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human
kidney 293 cells, human epidermal A431 cells, human Colo205 cells,
3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, primary explants, HeLa cells, mouse L cells, BHK,
HL-60, U937, HaK or Jurkat cells.
[0165] Suitable vectors for the expression of a polypeptide in
prokaryotes, e.g., E. coli, include plasmids of the types:
pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived
plasmids, pBTac-derived plasmids and pUC-derived plasmids.
Potentially suitable bacterial strains include Escherichia coli,
Bacillus subtilis, Salmonella typhimurium, or any bacterial strain
capable of expressing heterologous proteins.
[0166] A number of vectors exist for the expression of recombinant
proteins in yeast. For instance, YEP24, YIPS, YEP51, YEP52, pYES2,
and YRP17 are cloning and expression vehicles useful in the
introduction of genetic constructs into S. cerevisiae (see, for
example, Broach et al. (1983) in Experimental Manipulation of Gene
Expression, ed. M. Inouye Academic Press, p. 83, incorporated by
reference herein). These vectors can replicate in E. coli
[0167] due the presence of the pBR322 ori, and in S. cerevisiae due
to the replication determinant of the yeast 2 micron plasmid. In
addition, drug resistance markers such as ampicillin can be used.
Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any yeast strain capable of expressing heterologous
proteins.
[0168] In some instances, it may be desirable to express the
recombinant polypeptide by the use of a baculovirus expression
system. Examples of such baculovirus expression systems include
pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),
pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived
vectors (such as the .beta.-gal containing pBlueBac III).
[0169] When it is desirable to express only a portion of a protein,
such as a form lacking a portion of the N-- or C-terminus, i.e. a
truncation mutant which lacks the signal peptide, it may be
necessary to add a start codon (ATG) to the oligonucleotide
fragment containing the desired sequence to be expressed. It is
well known in the art that a methionine at the N-terminal position
can be enzymatically cleaved by the use of the enzyme methionine
aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat
et al. (1987) J. Bacteriol. 169:751-757) and Salmonella typhimurium
and its in vitro activity has been demonstrated on recombinant
proteins (Miller et al. (1987) PNAS 84:2718-1722). Therefore,
removal of an N-terminal methionine, if desired, can be achieved
either in vivo by expressing the derived polypeptides in a host
which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in
vitro by use of purified MAP (e.g., procedure of Miller et al.,
supra).
[0170] The protein of the invention may be prepared by culturing
transformed host cells under culture conditions suitable to express
the recombinant protein. The resulting expressed protein may then
be purified from such culture (i.e., from culture medium or cell
extracts) using known purification processes, such as gel
filtration and ion exchange chromatography. The purification of the
protein may also include an affinity column containing agents which
will bind to the protein; one or more column steps over such
affinity resins as concanavalin A-agarose, heparin-toyopearl.TM. or
Cibacrom blue 3GA Sepharose.TM.; one or more steps involving
hydrophobic interaction chromatography using such resins as phenyl
ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
[0171] One or more reverse-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify the protein. Some or all of the
foregoing purification steps, in various combinations, can also be
employed to provide a substantially homogeneous isolated
recombinant protein. Purification of proteins is further described,
e.g., in Robert K Scopes "Protein Purification: Principles and
Practice" Third Ed. Springer-Verlag, N.Y. 1994. The protein thus
purified is substantially free of other mammalian proteins and is
defined in accordance with the present invention as an "isolated
protein."
[0172] For example, soluble human CD39 or portions thereof can be
prepared, e.g., by expression of a construct expressing the CD39 or
portions thereof in a host cell, e.g., COS-1 and CHO cells, as
described, e.g., in Gayle et al. (1998) J. Clin. Invest. 101:1851
and in Kacmarek et al. (1996), supra. Preferred portions are
fragments that do not include one or both of the transmembrane
domains. The CD39 or portion thereof can then be isolated from the
supernatant of the culture by methods including, e.g., affinity
chromatrography using an antibody binding specifically to the CD39
or portion thereof.
[0173] The polypeptides can also be produced in an in vitro system,
e.g., in an in vitro transcription and translation system. Many
vectors for in vitro transcription are available commercially.
These may contain one or more of the promoters SP6, T3 and T7 and
may additionally contain a polyA sequence at the 3' end of the
polylinker in which the DNA of interest is inserted. A "polylinker"
refers to a nucleotide sequence containing several restriction
enzyme recognition sites. Examples of vectors include the series of
SP6 vectors, e.g,. SP64 (Krieg and Melton, infra), BlueScript, and
pCS2+. Vectors that can be used for in vitro transcription are also
described, e.g., in U.S. Pat. No. 4,766,072. In vitro transcription
can be conducted with a nucleic acid that is not per se a vector,
but merely contains the elements necessary for in vitro
transcription. For example, such a template nucleic acid may
comprise an RNA polymerase promoter located upstream of the
sequence to transcribe. Such template nucleic acid can be obtained,
e.g., by polymerase chain reaction (PCR) amplification of a
sequence of interest using a primer that contains an RNA polymerase
promoter. PCR amplification methods are well known in the art.
[0174] An in vitro transcription reaction can be carried out
according to methods well known in the art. Kits for performing in
vitro transcription kits are also commercially available from
several manufacturers. In an illustrative embodiment, an in vitro
transcription reaction is carried out as follows. A vector
containing an RNA Polymerase promoter and an insert of interest is
preferably first linearized downstream of the insert, by e.g.,
restriction digest with an appropriate restriction enzyme. The
linearized DNA is then incubated for about 1 hour at 37 or
40.degree. C. (depending on the RNA polymerase) in the presence of
ribonucleotides, an RNAase inhibitor, an RNA polymerase recognizing
the promoter that is operably linked upstream of the insert to be
transcribed, and an appropriate buffer containing Tris.Cl,
MgCl.sub.2, spermidine and NaCl. Following the transcription
reaction, RNAase free DNAse can be added to remove the DNA template
and the RNA can be purified by, e.g., a phenol-chlorophorm
extraction. Usually about 5-10 .mu.g of RNA can be obtained per
microgram of template DNA. Further details regarding this protocol
are set forth, e.g., in Molecular Cloning A Laboratory Manual, 2nd
Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor
Laboratory Press: 1989).
[0175] In another embodiment, the RNA is "capped" prior to
contacting it with an in vitro translation system. In certain
situations, efficient translation of eukaryotic RNA requires that
the 5' end of an RNA molecule is "capped", i.e., that the 5'
nucleotide at the 5' end of the RNA has a 5'-5' linkage with a
7-methylguanylate ("7-methyl G") residue. The presence of a
7-methyl G on an RNA molecule in a 5'-5' linkage is referred to as
a "cap." It has been proposed that recognition of the translational
start site in mRNA by the eukaryotic ribosomes involves recognition
of the cap, followed by binding to specific sequences surrounding
the initiation codon on the RNA. Accordingly, it is possible that
in certain embodiments of the invention, capping of the RNA
synthesized in vitro prior to contacting the RNA with an in vitro
translation system improves the translation efficiency of the RNA.
Thus, in one embodiment, the RNA is contacted with methyl-7
(5')PPP(5')guanylate (available, e.g., from Boehringer Mannheim
Biochemicals) in the presence of an in vitro transcription reaction
mixture, to obtain capped RNA. In the case of in vitro transcribed
RNA, capping is preferably carried out during in vitro
transcription, but can also be carried out during in vitro
translation by, e.g., addition of a cap analog (GpppG or a
methylated derivative thereof). Cap analogs and protocols
pertaining to their use are commercially available, e.g, in in
vitro transcription and/or translation kits.
[0176] In vitro synthesized RNA can be in vitro translated using an
in vitro translation system. The term "in vitro translation
system," which is used herein interchangeably with the term
"cell-free translation system" refers to a translation system which
is a cell-free extract containing at least the minimum elements
necessary for translation of an RNA molecule into a protein. An in
vitro translation system typically comprises at least ribosomes,
.sup.tRNAs, initiator methionyl-.sup.tRNA.sup.Met, proteins or
complexes involved in translation, e.g., eIF.sub.2, elF.sub.3, the
cap-binding (CB) complex, comprising the cap-binding protein (CBP)
and eukaryotic initiation factor 4F (eIF.sub.4F). A variety of in
vitro translation systems are well known in the art and include
commercially available kits. Examples of in vitro translation
systems include eukaryotic lysates, such as rabbit reticulocyte
lysates, rabbit oocyte lysates, human cell lysates, insect cell
lysates and wheat germ extracts. Lysates are commercially available
from manufacturers such as Promega Corp., Madison, Wis.;
Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, Ill.;
and GIBCO/BRL, Grand Island, N.Y. In vitro translation systems
typically comprise macromolecules, such as enzymes, translation,
initiation and elongation factors, chemical reagents, and
ribosomes.
[0177] In cases where plant expression vectors are used, the
expression of a polypeptide of the invention may be driven by any
of a number of promoters. For example, viral promoters such as the
35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984,
Nature, 310:511-514), or the coat protein promoter of TMV
(Takamatsu et al., 1987, EMBO J., 6:307-311) may be used;
alternatively, plant promoters such as the small subunit of RUBISCO
(Coruzzi et al., 1994, EMBO J., 3:1671-1680; Broglie et al., 1984,
Science, 224:838-843); or heat shock promoters, e.g., soybean hsp
17.5-E or hsp 17.3-B (Gurley et al., 1986, Mol. Cell. Biol.,
6:559-565) may be used. These constructs can be introduced into
plant cells using Ti plasmids, Ri plasmids, plant virus vectors;
direct DNA transformation; microinjection, electroporation, etc.
For reviews of such techniques see, for example, Weissbach &
Weissbach, 1988, Methods for Plant Molecular Biology, Academic
Press, New York, Section VIII, pp. 421-463; and Grierson &
Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch.
7-9.
[0178] An alternative expression system which can be used to
express a polypeptide of the invention is an insect system. In one
such system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus
grows in Spodoptera frugiperda cells. The PGHS-2 sequence may be
cloned into non-essential regions (for example the polyhedrin gene)
of the virus and placed under control of an AcNPV promoter (for
example the polyhedrin promoter). Successful insertion of the
coding sequence will result in inactivation of the polyhedrin gene
and production of non-occluded recombinant virus (i.e., virus
lacking the proteinaceous coat coded for by the polyhedrin gene).
These recombinant viruses are then used to infect Spodoptera
frugiperda cells in which the inserted gene is expressed. (e.g.,
see Smith et al., 1983, J. Virol., 46:584, Smith, U.S. Pat. No.
4,215,051).
[0179] In a specific embodiment of an insect system, the DNA
encoding the subject polypeptide is cloned into the pBlueBacill
recombinant transfer vector (Invitrogen, San Diego, Calif.)
downstream of the polyhedrin promoter and transfected into Sf9
insect cells (derived from Spodoptera frugiperda ovarian cells,
available from Invitrogen, San Diego, Calif.) to generate
recombinant virus. After plaque purification of the recombinant
virus high-titer viral stocks are prepared. that in turn would be
used to infect Sf9 or High Five.TM. (BTI-TN-5B 1-4 cells derived
from Trichoplusia ni egg cell homogenates; available from
Invitrogen, San Diego, Calif.) insect cells, to produce large
quantities of appropriately post-translationally modified subject
polypeptide. Although it is possible that these cells themselves
could be directly useful for drug assays, the subject polypeptides
prepared by this method can be used for in vitro assays.
[0180] In another embodiment, the subject polypeptides are
expressed in transgenic animals, such that in certain embodiments,
the polypeptide is secreted, e.g., in the milk of a female
animal.
[0181] Alternatively, apyrases can be isolated from tissue. For
example, CD39 can be isolated from vascular endothelial cells or
placenta, which are known to constitutively express a cell-surface
ADPDase or NTPDase. Apyrases can be isolated from plant material
(e.g., potato) as described, e.g., in Handa and Guidotti (1996)
Biochem. Biophys. Res. Commun. 218:916.
[0182] Nucleic acids for use in recombinant technology are known in
the art, and can be found, e.g., in GenBank. Purification methods
for each of these biological agents are described in the art, or
they can be derived from purification methods of other, e.g.,
similar, biological agents.
[0183] Homologs and fragments of wild-type polypeptides, e.g.,
homologs and fragments having one or more amino acid deletions,
substitutions or additions, which retain at least a significant
amount of the biological activity of the wild-type polypeptide can
be obtained by creating combinatorial libraries and screening of
these libraries for the desired activity. In one embodiment, the
variegated library of variants is generated by combinatorial
mutagenesis at the nucleic acid level, and is encoded by a
variegated gene library. For instance, a mixture of synthetic
oligonucleotides can be enzymatically ligated into gene sequences
such that the degenerate set of potential sequences are expressible
as individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display) containing the set of
sequences therein.
[0184] There are many ways by which such libraries of potential
homologs can be generated from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
carried out in an automatic DNA synthesizer, and the synthetic
genes then ligated into an appropriate expression vector. The
purpose of a degenerate set of genes is to provide, in one mixture,
all of the sequences encoding the desired set of potential homolog
sequences. The synthesis of degenerate oligonucleotides is well
known in the art (see for example, Narang, S A (1983) Tetrahedron
39:3; Itakura et al. (1981) Recombinant DNA, Proc 3.sup.rd
Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam:
Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem.
53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)
Nucleic Acid Res. 11:477. Such techniques have been employed in the
directed evolution of other proteins (see, for example, Scott et
al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS
89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et
al. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
[0185] Likewise, a library of coding sequence fragments can be
provided for a clone in order to generate a variegated population
of fragments of the gene of interest for screening and subsequent
selection of bioactive fragments. A variety of techniques are known
in the art for generating such libraries, including chemical
synthesis. In one embodiment, a library of coding sequence
fragments can be generated by (i) treating a double stranded PCR
fragment of a coding sequence of interest with a nuclease under
conditions wherein nicking occurs only about once per molecule;
(ii) denaturing the double stranded DNA; (iii) renaturing the DNA
to form double stranded DNA which can include sense/antisense pairs
from different nicked products; (iv) removing single stranded
portions from reformed duplexes by treatment with Si nuclease; and
(v) ligating the resulting fragment library into an expression
vector. By this exemplary method, an expression library can be
derived which codes for N-terminal, C-terminal and internal
fragments of various sizes.
[0186] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations or truncation, and for screening cDNA libraries for gene
products having a certain property. Such techniques will be
generally adaptable for rapid screening of the gene libraries
generated by the combinatorial mutagenesis of homologs. The most
widely used techniques for screening large gene libraries typically
comprises cloning the gene library into replicable expression
vectors, transforming appropriate cells with the resulting library
of vectors, and expressing the combinatorial genes under conditions
in which detection of a desired activity facilitates relatively
easy isolation of the vector encoding the gene whose product was
detected. Each of the illustrative assays described below are
amenable to high through-put analysis as necessary to screen large
numbers of degenerate sequences created by combinatorial
mutagenesis techniques.
[0187] Combinatorial mutagenesis has a potential to generate very
large libraries of mutant proteins, e.g., in the order of 10.sup.26
molecules. Recrusive ensemble mutagenesis (REM) can be used to
allow one to avoid the very high proportion of non-functional
proteins in a random library and simply enhances the frequency of
functional proteins, thus decreasing the complexity required to
achieve a useful sampling of sequence space. REM is an algorithm
which enhances the frequency of functional mutants in a library
when an appropriate selection or screening method is employed
(Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan et al.,
1992, Parallel Problem Solving from Nature, 2., In Maenner and
Manderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401-410;
Delgrave et al., 1993, Protein Engineering 6(3):327-331).
[0188] The invention also provides for reduction of proteins to
generate mimetics, e.g., peptide or non-pepide agents, such as
small molecules. Thus, such mutagenic techniques as described above
are also useful to map the determinants of the proteins which
participate in biological activity. To illustrate, the critical
residues of a subject apyrase which are involved in enzymatic
activity can be determined and used to generate peptidomimetics or
small molecules which have the same biological activity or which
act as competitive inhibitors. By employing, for example, scanning
mutagenesis to map the amino acid residues of the subject proteins
which are involved in biological activity, e.g., enzymatic
activity, peptidomimetic compounds can be generated which mimic
those residues of the protein. For instance, non-hydrolyzable
peptide analogs of such residues can be generated using
benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry
and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), azepine (e.g., see Huffinan et al. in Peptides:
Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), substituted gamma lactam rings (Garvey et al.
in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM
Publisher: Leiden, Netherlands, 1988), keto-methylene
pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and
Ewenson et al. in Peptides: Structure and Function (Proceedings of
the 9.sup.th American Peptide Symposium) Pierce Chemical Co.
Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al. (1985)
Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin
Trans 1:1231), and b-aminoalcohols (Gordon et al. (1985) Biochem
Biophys Res Commun126:419; and Dann et al. (1986) Biochem Biophys
Res Commun 134:71).
[0189] Peptides can also be isolated by phage display, as known in
the art.
[0190] The amount of a protein, e.g., synthesized or purified as
described herein, can be determined by the Coomassie protein assay
according to the manufacturer's instructions (Bio-Rad). The amount
and purity of proteins can also be determined by subjecting the
protein mixtures to SDS-PAGE, optionally followed by Western blot
analysis. SDS-PAGE gels can be stained, e.g., silver or Coomassie
blue stained, for visualizing polypeptides. Western blots can also
be incubated with a reagent binding specifically to the subject
polylpeptide, e.g., an antibody. The inclusion of known amounts of
reference proteins permit, by comparison, to estimate the quantity
of a particular protein on the Western blot. Protocols for Western
blot analysis are known in the art:
[0191] Those skilled in the art will appreciate that the purity of
the polypeptide preparation of the invention can be determined by
various methods. A preferred method for determining the amount of
contaminating proteins in a polypeptide preparation comprises
subjecting the polypeptide preparation to gel electrophoresis,
e.g., polyacrylamide electrophoresis, in the presence of specific
amounts of molecular markers, and staining the gel after the
electrophoresis with a protein dye. A comparison of the intensity
of the band of the subject polypeptide with the molecular markers
indicates the purity of the subject polypeptide preparation. Other
methods for determining the amount of contaminating proteins
include mass spectrometry, gel filtration and peptide sequencing
according to methods known in the art.
[0192] A preferred method for determining the amount of
contaminating cellular material in a polypeptide preparation
comprises gel electrophoresis and silver staining of the gel. Other
methods for determining the purity of a polypeptide preparation
include mass spectrometry according to methods known in the art.
Yet other measurements of the purity of a polypeptide preparation
include a measure of the activity of the polypeptide, as further
described herein.
[0193] Protein concentrations can be determined according to the
following methods: Lowry-Folin-Ciocalteau reagent; UV absorption at
280 nm (aromatic band) or 205-220 nm (peptide band); dye binding
(e.g., Coomassie Blue G-250); or bis-cinchonic acid (BCA; Pierce
Chemicals (Rockford, Ill.)) reagent. All of these methods are
described in, e.g., Robert K. Scopes, Protein Purification,
Principles and Practice, Third Ed., Springer Verlag New York, 1993,
and references cited therein. Briefly, the well-known Lowry method
is a relatively sensitive method giving a good color with 0.1 mg/ml
or protein or less. The method using Coomassie Blue G-250 is very
sensitive, fast and at least as accurate as the Lowry method. The
procedure consists in mixing a polypeptide sample with the reagent
and measure the blue color at 595 nm.
[0194] Those skilled in the art will understand that a preferred
method for determining exact protein amounts is by dry weight
determination, since it provides a suitably accurate measurement of
protein amount. Thus, in a preferred embodiment for determining the
amount of the purified polypeptide of the invention, the dry weight
of a highly pure preparation of the polypeptide of the invention is
determined, and this preparation is then used as a standard for
determining the protein concentration of other preparations of
polypeptides of the invention.
[0195] As those skilled in the art will understand, the percent
recovery and degree of purity of a preparation of polypeptide of
the invention can be calculated from the total amount of protein
recovered after purification and the amount and/or activity of the
polypeptide of interest.
Attachment of the Biologically Active Agent to a Biodegradable
Polymer
[0196] The conjugates of the invention comprise a biologically
active agent and a polymer. These two elements can be linked
covalently. Alternatively, these two elements can form a
non-covalently linked complex, and maintained together, e.g.,
through ionic interactions or hydrogen bonding.
[0197] The attachment of a biologically active material is either
done directly to the biodegradable polymeric backbone, or through a
pendant chain off of the polymeric backbone. The term "attachment"
and its derivatives refer to covalent bonding, hydrogen bonding, or
ionic bonding of a bioactive species to a biodegradable
polymer.
[0198] Ionic attachment may be employed in cases where the
biodegradable polymer is either positively or negatively charged
and the biologically active agent carries the opposite charge or
wherein at least a portion of the biologically active agent carries
the opposite charge. The oppositely charged components are held
together through electrostatic forces. The charge on the
biodegradable polymer may exist naturally, as is the case with the
anionic alginate or CMC, or through modification. For example,
carbonyl groups can be rendered positively charged through the
reaction with secondary amines to form cationic imine complexes.
Quartenary nitrogens as in the previous example are commonly used
as a source of positive charges in polymers. Any of the previously
discussed biodegradable polymers modified with a quartenary
nitrogen may be used in ionically attached biologically active
agent complexes. Charges on the biologically active compound may
originate, for example, from the amino acids of a peptide chain.
Deprotonated acid groups or protonated amines are a source of
anionic and cationic charges respectively. Attachment is carried
out by admixing the two oppositely charged components together in a
suitable solvent or more preferably a buffer solution wherein the
two species maintain their charges.
[0199] For hydrogen atoms bonded to electronegative elements
dipole-dipole interaction is uniquely important and forms the basis
of hydrogen bonding. Although hydrogen bonds are weak (about 5 kcal
mole.sup.-1 per hydrogen bond) they play an important role in
proteins and enzymes where a polypeptide chain contains many
C.dbd.O and N--H groups. The total amount of bonding that results
from many small interactions is substantial and plays an important
role in the actual shape or conformation of the protein. Reciprical
hydrogen bonding may occur between the C.dbd.O and N--H groups of
different chains and thus bind them together. In this same fashion,
binding may occur with the C.dbd.O and N--H groups of the
biodegradable polymer. As with the ionic complexes discussed above,
preparation of biologically active agents hydrogen bonded to
biodegradable polymers can be prepared by admixing the two in a
suitable solvent. Due to the weaker bonding interactions of the
hydrogen bonds, these complexes may be desirable when increased
rates of release of the biologically active agent are wanted.
[0200] Preferably, the biologically active agents in the present
invention are covalently conjugated to the biodegradable polymer.
Biologically active species can be attached to a biodegradable
polymer using mild bioconjugation techniques known to those skilled
in the art (See K. Mosbach, Immobilized Enzymes and Cells, Part B,
Academic Press (Orlando, Fla.), (1987); G. T. Hermanson, A. K.
Mallia, P. K. Smith, "Immobilized Affinity Ligand Techniques,"
Academic Press, San Diego, (1992); and S. F. Karel, S. B. Libicki,
C. R. Robertson, "The Immobilization of Whole Cells: Engineering
Principles," Chemical Eng. Sci., 40: 1321 (1985), for example).
Mild bioconjugation schemes are preferred for attachment of
bioactive species in order to eliminate or minimize damage to the
structure of the biodegradable polymer.
[0201] In some circumstances, the interaction of a polymer with the
attached bioactive species may be suboptimal. For example, steric
hindrances between the biodegradable polymer and the attached
bioactive species may limit the approach of the solution phase
reactant to the bioactive species. In addition, physical bulk,
electrostatic repulsion, or inappropriate positioning of the
bioactive species may also contribute to reduced efficiency of the
immobilized bioactive species. Accordingly, it may be desirable to
place one or more additional compounds as a "spacer" or "tether"
between the chemical functional groups of the biodegradable polymer
and the bioactive species to increase the space between the polymer
and the bioactive species. The covalent attachment of bioactive
species onto the biodegradable polymer according to the present
invention is generally reversible, i.e., the bioactive species are
released from the biodegradable polymer in a controlled, or
predictable, manner over time. In addition, spacers, or tethers,
capable of selectively releasing immobilized bioactive species
provide another degree of controlled release of bioactive species.
Suitable compounds for use as cleavable tethers, or cleavable
spacers, include, but are not limited to, polyhydroxyacids,
polyanhydrides, polyamino acids, tartarates, cysteine-linkers such
as Lomant's Reagent, for example, derivatives of ethylene
glycol-bis-succinimidylsuccinate, succinic acid or succinic
anhydride, diaminohexane, glyoxylic acid, short chain polyethylene
glycol, and glycine, for example. Other means are crosslinking via
imidocarbonates, carbonates, oxiranes, aziridine and active double
bonds and halogens. The reactive functionalities which are
available on the biologically active agent for covalently bonding
to the chemically reactive group of the biodegradable polymer are
primarily amino groups, carboxyl groups and thio groups. While any
of these may be used as the target of the chemically reactive group
on the biodegradable polymer, for the most part, bonds to amino
groups will be employed, particularly with the formation of amide
bonds. To form amide bonds, one may use as a chemically reactive
group wide-variety of active carboxyl groups, particularly esters,
where the hydroxyl moiety is physiologically acceptable at the
levels required. While a number of different hydroxyl groups may be
employed, the most convenient are N-hydroxysuccinimide (NHS), and
N-hydroxy sulfosuccinimide (sulfo-NHS), although other alcohols may
also be employed. In some cases, special reagents may be used, such
as azido, diazo, carbodiimide anhydride, hydrazine, dialdehydes,
thiol groups, or amnines to form amides, esters, imines,
thioethers, disulfides, substituted amines, or the like.
[0202] Polymers may be attached to the N-- and/or C-terminus of a
polypeptide. If the biological agent is a multimer (e.g., a dimer,
trimer, tetramer), polymers may be attached to one or both termini
of one or more polypeptide chains. Polymers may also be inserted in
a polypeptide, if such insertion does not significantly affect the
activity of the polypeptide. In yet other embodiments, one or more
polymers are attached to sidechains of amino acids within the
polypeptide. For example, the polymer can be an oligosaccharide
that is attached to an amino acid of an apyrase.
[0203] Conjugates comprising a biological agent and a biodegradable
polymer can further be conjugated to one or more other moieties.
For example, they may be linked to a label, e.g., to permit
detection of the conjugates in vivo. The conjugates may also be
linked to a targeting agent that will target the conjugate to the
appropriate site within a subject. A targeting agent may be, e.g.,
an antibody. For example, when the biological agent is an apyrase,
it may be desirable to target a conjugate comprising the apyrase to
endothelial cells. This can be accomplished, e.g., by linking to
the apyrase or to the polymer an antibody binding specifically to
membrane antigens of endoplasmic cells. Other targeting agents are
known in the art for targeting various sites. Targeting agents can
be linked to either the biological agent or the polymer, as
described herein or according to methods known in the art.
Determination of Biological Activity of Biological Agents and
Conjugates Thereof
[0204] The biological activity of biological agents, whether or not
linked to a polymer, can be determined according to methods known
in the art. Exemplary assays are set forth below.
[0205] Where the biological agent is an apyrase or derived from
(e.g., a fragment), or homologous to, an apyrase, several assays
can be used to determine the rate of catalysis of substrates by the
apyrase. For example, nucleotidase activity can be determined by
measuring the amount or inorganic phosphate released from
nucleotide substrates using, e.g., the technique of Daly and
Ertingshausen (Clin. Chem. 18:263 (1972)). In this method, the
complex of inorganic phosphate with phosphor reagent (ammonium
molybdate in the presence of sulfuric acid) produces an unreduced
phosphomolybdate compound. The absorbance of this complex at 340 nm
is directly proportional to the inorganic phosphorus concentration.
The nucleotide can be added to a final concentration of 1 mM and
incubated at 37.degree. C. for 30 minutes. The reagent can then be
stopped with 100 volumes of phosphor reagent, and the amount of
phosphate released from the reaction can be quantitated using a
calcium/phosphorus combined standard (Sigma). This assay is further
described in Mulero et al. (1999) J. Biol. Chem. 274:20064.
[0206] Alternatively, hydrolysis of ADP or ATP can be measured by
incubating a sample of apyrase, e.g., cell lysates of cells
expressing an apyrase, with either 200 .mu.M ADP or 200 .mu.M ATP,
and Ca.sup.2+ or Mg.sup.2+ dependent release of free phosphate is
determined. Malachite green can be used to stop the reaction, and
absorbance at 610 nm can be used to determine levels of phosphate
against the standard curve of KH.sub.2PO.sub.4 as described, e.g.,
in Geladopoulos et al. (1991) Anal. Biochem. 192: 112 and in
Kaczmarek et al. (1996) J. Biol. Chem. 271:33116.
[0207] ATPDase activity of intact cells can also be determined by
measuring the hydrolysis of [.sup.14C]ADP to AMP, as described,
e.g., in Kaczmarek et al. (1996) J. Biol. Chem. 271:33116. Briefly,
a sample of apyrase or monolayers of cells expressing an apyrase,
e.g., cells transfected with an expression construct encoding an
apyrase, and appropriate control cells are incubated with
[.sup.14C]ADP (50 .mu.Ci/reaction; DuPont NEN) and the products
analyzed on thin layer chromatography (TLC) plates (Whatman
Labroatory Division, Clifton, N.J.). The solvent system may
comprise isobutyl alcohol: 1-pentanol: ethylene glycol monoethyl
ether: NH4OH water at ratios 90:60:180:90:120. The seperated
compounds can be scanned for radioactivity with a PhosphorImager
(Molecular Dynamics, Sunnyvale, Calif.) and degradation of the
[.sup.14C]ADP can be determined by ImageQuant software according to
the manufacturer's instructions (Kaczmarek et al. (1996) J. Biol.
Chem. 271:33116).
[0208] Platelet aggregration assays can be conducted as follows.
Blood is obtained from an apparently healthy human volunteer and
anticoagulated with 0.1 volume 3.2% sodium citrate. Platelet rich
plasma is then prepared by centrifugation of whole blood at
280.times.g for 15 minutes at 22.degree. C. Platelet-rich plasma is
the preincubated with cell membrane or a cell lysate containing
apyrase or with soluble apyrase for 10 minutes at 37.degree. C. in
a siliconized glass cuvette containing a stirring bar, followed by
stimulation with either ADP (5 .mu.g/ml), or thrombin (0.1
unit/nil) (Chronolog Corp., Havertown, Pa.). Platelet aggregation
can be recorded for at least 10 minutes with Blood
Lumi-Aggregometer, e.g., model 560 (Chronolog Corp., Havertown,
Pa.), which detects platelet shape change. Data can be expressed as
the percentage of light transmission with platelet-poor plasma
equal to 100%. Further details are provided in Kaczmarek et al.
(1996) J. Biol. Chem. 271:33116.
[0209] Cell lysates or membrane preparations, e.g., from cells
expressing an apyrase, can be prepared, e.g., as described in
Kaczmarek et al. (1996) J. Biol. Chem. 271:33116.
[0210] The angiostatic (angiogenesis inhibiting) activity of a
protein can be determined, e.g., in a HUVEC-endothelial cell
proliferation assay. The assay can be performed in a 96 well plate.
Primary human umbilical cells (HUVECs) are seeded to
3.times.10.sup.3 cells per well in EGM medium (Clonetics)/20% FCS
(fetal bovein serum) and incubated at 37.degree. C. for 24 hr. The
cells can then be starved in M199 medium (0113C0 BRL) containing
0.5% FCS (M199-0.5% FCS) for 48 hr at 37.degree. C. 100 ng/ml FGF
is added to the solution containing the protein to be tested, e.g.,
conditioned media or membrane fractions derived from transfected
host cells. The medium is added to starved cells and incubated for
72 hr at 37.degree. C. The cells are then radiolabeled by
.sup.3H-thymidine for 6 hr. Radiolabeled cells are washed with PBS
and trypsinized for liquid scintillation counting. Results can be
plotted using Kaleidograph software (Abelbeck Software). The extent
of angiogenesis in a tissue, can be evaluated by a variety of
methods, such as are described in U.S. Pat. No. 6,248,327, for
detecting immature and nascent vessel structures by
immunohistochemistry.
[0211] The efficacy of the modified apyrases can also be tested in
vivo, e.g., in animal models. One model is the CD39 null mouse,
that displays prolonged bleeding times and yet, paradoxically has a
predisposition to thrombogenesis in vivo (Enjyoji et al. (Nature
Med. 5:1010 (1999) and WO 00/23459). It has been shown that i.v.
injection of apyrases correct the abnormal bleeding times of these
mice (Enjoyji et al., supra). Accordingly, modified apyrases can be
administered to these mice, and their bleeding time and/or clot
production monitored relative to CD39 null mice that have not
received modified apyrase.
[0212] Graft viability can be determined according to methods known
in the art, e.g., using animals, e.g., nude mice.
[0213] Where the biological agent is not an apyrase, biological
activity tests of the particular biological agent can be conducted
as known in the art. Exemplary tests are set forth in U.S. Pat. No.
6,312,921.
Diseases That Can be Treated with the Compounds of the
Invention
[0214] The soluble apyrase conjugates provided by the invention can
be used for treating any disease that can benefit from an increase
in enzymatic activity. Exemplary diseases include those that can
benefit from a modulation of circulating levels of nucleotides in
the blood. Such diseases or conditions include those associated
with abnormal platelet aggregation, cardiac function, immune
responses (inflammation).
[0215] Briefly, platelets are anuceleate cell fragments that are
released from large hematopoietic precursors named megakaryocytes.
Platelets adhere to sites of vascular injury, are activated and
release ADP and other signaling molecules. As a result, the ADP
that was released causes the platelets to undergo a shape change
from smooth discs to spiculated spheres, the fibrinogen receptor is
activated and the platelets are caused to aggregate at the site of
injury. Interactions between activated platelet surfaces and
coagulation proteins result in thrombin generation, further
platelet activation, and formation of an insoluble plug, also
referred to as plaque. These effects are mediated by a platelet ADP
receptor termed P2-receptor, that is antagonized by ATP.
[0216] Although platelet aggregation is a necessary physiological
response to vascular injury by fulfilling essential roles in
mediating effective and immediate control of bleeding, abnormal
platelet reactivity can also cause undesirable conditions, such as
thrombotic disorders, occlusive vascular disease, unstable angina,
myocardial infarction, post-angioplasty stenosis, cerebral
ischaemia, thrombotic stroke and a variety of inflammatory vascular
disorders associated with organ and cell transplantation. Any
condition resulting from excessive or inappropriate platelet
aggregation or reactivity can be treated by administering a
modified apyrase of the invention to a subject.
[0217] As further described herein, the modified apyrases of the
invention degrade preferably ADP as opposed to ATP, and have a long
half-life in blood. Accordingly, an effective dose of a modified
apyrase can be administered to a subject, e.g., at the site of or
in the vicinity of the site of vascular injury.
[0218] Examples of therapeutic uses for modified apyrases of the
invention include the treatment of individuals who suffer from
coronary artery disease or injury following myocardial infarction;
unstable angina; atherosclerosis; preeclampsia; embolism;
platelet-associated ischemic disorders including lung, coronary and
cerebral ischemia; and the prevention of reocclusion following
thrombosis, thrombotic disorders icluding coronary artery
thrombosis, cerebral artery thrombosis, intracardiac thrombosis,
peripheral artery thrombosis, venous thrombosis, cerebral artery
thrombosis, intracardiac thrombosis, venous thrombosis and
thrombosis and coagulophathies associated with exposure to a
foreign or injured tissue surface, in combination with angioplasty,
carotid endarterectomy, anastomosis of vascular grafts, and chronic
cardiovascular devices such as in-dwelling catheters or shunts. The
modified apyrases of the invention can also be used to treat
individuals at high risk for thrombus and plaque formation or
reformation (severe arteriosclerosis), and be used for inhibition
of occlusion, reocclusion, stenosis and/or restenosis of blood
vessels. Yet other individuals that benefit from a reduction of
platelet aggregation include those at risk for advanced coronary
artery disease; individuals that are or will be undergoing
angioplasty procedures (e.g., balloon angioplasty, laser
angioplasty, coronary angioplasty, coronary atherectomy and similar
techniques); individuals undergoing surgery that has a high risk of
thrombus formation (e.g., coronary bypass surgery, insertion of a
prosthetic valve or vessel and the like); and individuals having,
or at risk of having, deep venous thrombosis (DVT), pulmonary
embolism (PE), transient ischemic attacks (TIAs) or other related
conditions where arterial occlusion is the common underlying
feature.
[0219] The modified apyrases of the invention can be used to
prevent stroke and for treating patients experiencing stroke due to
vascular occlusion. Other applications include the inhibition of
microvascular thrombosis, postischemic cerebral blood flow
improvement, redution of cerebral infarction volumes and
neurological deficit without inducing intracerebral hemorrhage, in
stroke.
[0220] In vivo experiments support a role of modified apyrases for
preventing vascular occlusion and the consequences thereof. For
example, a recombinant adenovirus encoding CD39 was used to infect
rat aortae and rabbit femoral vessels following balloon-mediated
denudation and injury. It was found that the injured vasculature in
the rat aortae displayed dramatically less intimal hyperplasia and
smooth muscle proliferation responses post-CD39 adenoviral
infection relative to the negative control adenoviral exposed
vessels (Robson et al. (2000) Emerging Therapeutic Targets 4:155).
In a contrary manner, CD39 knock-out mice have a predisposition to
thrombogenesis.
[0221] The modified apyrases of the invention can also be used to
modulate angiogenesis. A role for NTPDase1 and phosphohydrolysis of
extracellular nucleotides in the regulation of cellular
infiltration and new vessel growth in a model of angiogenesis has
been shown (Goepfert et al. (2001) Circulation 104:3109).
Accordingly, angiogenesis is promoted with NTPDase1. NTPDase 1 may
be useful for treating, e.g., ischemic peripheral vascular
diseases.
[0222] Abnormal platelet reactivity has also been linked to a
variety of inflammatory vascular disorders, e.g., associated with
transplantation. Quiescent EC express ATPDase which exerts an
important thromboregulatory function by hydrolyzing both ATP and
ADP. It has been shown that ATPDase activity is rapidly lost from
the surface of the EC following ischemia-reperfusion injury and
during graft rejection. Providing a mechanism for restoration or
augmentation of ATPDase activity would benefit patients with such
injuries or surgeries. Accordingly, modified apyrases can be used
to prolong graft survival, at least in part by preventing platelet
thrombi and vascular inflammation. For example, infusion of soluble
potato apyrase has been shown to abrogate platelet sequestration in
cardiac grafts where NTPDase activity has been lost (Koyamada et
al. Transplantation 62:1739 (1996)). It has also been shown that
cardiac xenografts of CD39 null mice undergo rejection with more
rapid vascular occlusion than the matched wild type organs when
grafted to rats with normal circulating platelets (Imai et al.
(1999) Mol. Med. 5:743). Overexpression of CD39 also significantly
prolonged graft survival when compared with a negative control in a
system involving vascularised guinea-pig (to rat) xenografts
overexpressing CD39. Moreover, in this model, platelet
sequestration was also markedly decreased and vascular integrity
better preserved (Imai et al. (2000) Transplantation 70:864).
Accordingly, the modified apyrases of the invention can be
administered to a subject receiving a graft, e.g., an autologous,
allogeneic, syngeneic or xenogeneic graft.
[0223] The invention also permits treatment of thrombotic
complications associated with myocardial infarction, deep vein
thrombosis following orthopedic surgery, transient ischemic attack,
coronary artery bypass graft, percutaneous transluminal coronary
angioplasty, acute promyelocytic leukemia, diabetes, multiple
myelomas, septic shock, purpura fulminanas, adult respiratory
distress syndrome, angina, or aortic valve or vascular prosthesis.
The methods of the invention can also be used to treat inflammatory
diseases, e.g., inflammatory bowel disease. For example, a subject
developing thrombotic complications as a result of sepsis can be
treated by administration of a conjugate comprising CD39
(NTPDase1).
[0224] Also within the invention are modified apyrases which
stimulate platelet aggregation and augment hemostasis; modulate
angiogenesis, and/or modulate immune responses. For example, the
wild-type CD39L1 stimulates platelet aggregation by converting the
competitive antagonist (ATP) of ADP receptors into the specific
agonist. Accordingly, CD39L1 conjugates can be used in such
embodiments. Such apyrases can also be used to repair blood vessels
by promoting thrombus formation. Apyrases can regulate angiogenesis
by sealing off blood vessels.
[0225] In situations in which the biological agent is not an
apyrase, use of such conjugates are based on the identity of the
biological agent. For example, if the biological agent is an
interleukin, the conjugate containing an interleukin can be used,
e.g., for stimulating lymphocyte activation and/or proliferation.
If the biological agent is an interferon, a conjugate containing
interferon can be used for treating multiple sclerosis.
Other Uses for the Agents of the Invention
[0226] Modified apyrases of the invention can also be used ex vivo,
e.g., in blood or platelet stocks, where one desires to prevent
platelet aggregation (see, e.g., U.S. Pat. No. 5,378,601). Apyrases
have been reported as protecting platelets during storage (Mirowiec
et al. (Transfusion 36:5 (1996)). Prevention of platelet
aggregation may also be desirable when obtaining blood samples from
individuals. Accordingly, modified apyrases could be added to blood
samples. In certain embodiments, recipients of blood samples, or
containers for storing blood or platelets may contain modified
apyrases prior to receiving blood or platelets. A syringe
containing modified apyrase is also contemplated.
[0227] Other ex vivo or in vitro uses for the modified apyrases
include their use in pyrophosphate-based DNA sequencing
methodologies such as those described by Ronaghi et al. (Science
281:336 (1998)).
[0228] Modified apyrases can also be used for screening for
modulators, e.g., inhibitors of apyrases.
Methods of Administrating the Compounds of the Invention
[0229] The therapeutic methods of the invention generally comprise
administering to a subject in need thereof, a pharmaceutically
effective amount of a pharmaceutical complex comprising a
biological agent, e.g., a modified apyrase. The conjugates of this
invention may be administered to mammals, preferably humans, either
alone or, preferably, in combination with pharmaceutically
acceptable carriers, excipients or diluents, in a pharmaceutical
composition, according to standard pharmaceutical practice. The
conjugates can be administered orally or parenterally, including
the intravenous, intramuscular, intraperitoneal, subcutaneous,
rectal and topical routes of administration.
[0230] Toxicity and therapeutic efficacy of the conjugates can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Conjugates
which exhibit large therapeutic indices are preferred. While
conjugates that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such reagents to
the site of affected tissue in order to minimize potential damage
to normal cells and, thereby, reduce side effects.
[0231] Data obtained from cell culture assays and animal studies
can be used in formulating a range of dosage for use in humans. The
dosage of such reagents lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage may vary within this range depending
upon the dosage form employed and the route of administration
utilized. For any reagent used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (i.e., the concentration of the conjugate which achieves
a half-maximal inhibition of symptoms) as determined in cell
culture. Levels of conjugates in plasma may be measured, for
example, by high performance liquid chromatography.
[0232] A conjugate of the invention is preferably administered
parenterally, e.g., by injection, e.g., intravenous (i.v.),
subcutaneous, or intramuscular injection. Accordingly,
pharmaceutical compositions may be in the form of a sterile
injectable aqueous solution. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution.
[0233] Sterile injectable preparation may also be a sterile
injectable oil-in-water microemulsion where the compound of the
invention is dissolved in the oily phase. For example, the active
ingredient may be first dissolved in a mixture of soybean oil and
lecithin. The oil solution then introduced into a water and
glycerol mixture and processed to form a microemulsion.
[0234] The conjugates of the invention may be delivered in a single
dose or delivered in repeat doses. The preferred time for
administration of the second and later doses will depend on the
half-life of the conjugate. For example, a second dose could be
administered 3 days, one week or one month after the first
dose.
[0235] The injectable solutions or microemulsions may be introduced
into a patient's blood-stream by local bolus injection.
Alternatively, it may be advantageous to administer the solution or
microemulsion in such a way as to maintain a constant circulating
concentration of the instant compound. In order to maintain such a
constant concentration, a continuous intravenous delivery device
may be utilized. An example of such a device is the Deltec
CADD-PLUS.TM. model 5400 intravenous pump.
[0236] The conjugates of the invention can also be administered in
a softgel, e.g., silica gel and solgel, which holds the conjugates
inside and releases them over time.
[0237] In other embodiments, modified apyrases or other conjugates
are administered by local delivery systems. Non-limiting examples
of local delivery systems for use in the present invention include
intravascular drug delivery catheters, wires, pharmacological
stents and endoluminal paving.
[0238] In a preferred embodiment the compounds for use in the
present invention are administered to a desired site, e.g., a site
of vascular injury or plaque formation, by direct intravascular
deposition using intravascular catheters. Catheter systems for use
in the present invention, include, for example, pressure-driven
catheters, diffusion catheters and mechanical catheters. See, e.g.
Wolinsky & Thung, J. Am. Coll. Cardiol., 15:475-81 (1990);
Goldman et al., Atherosclerosis, 65:215-25 (1987); Nabel et al.,
Science, 294, 1285-8 (1990); Fram et al., J Am. Coll. Cardiol.,
23:1570-71 (1994); Riessen et al., Human Gene Ther., 4, 749-58
(1993); Fernandez-Ortiz et al., Circulation, 89:1518-22 (1994).
[0239] In one embodiment, the administration of conjugates may be
by pressure-driven catheter systems, including for example, porous
catheters; microporous catheters, for example, those made by Cordis
Corporation; macroporous catheters; transport catheters, for
example, those made by Cardiovascular Dynamics/Boston Scientific;
channeled balloon catheters, for example, those made by Boston
Scientific; and infusion sleeve catheters, for example, those made
by LocalMed.
[0240] In a preferred embodiment the methods of the invention
utilize a pressure driven based catheter that is an infusion sleeve
catheter, an example of which is described by Moura et al,
Circulation, 92: 2229-2305 (1995) and further described in U.S.
Pat. No. 5,279,565. The infusion sleeve, an example of which is
that produced by LocalMed, is designed to allow independent control
of both the apposition of conjugates against the arterial wall and
the drug delivery of the conjugates into the wall. The efficacy of
drug delivery by an infusion sleeve on the arterial architecture of
a vessel is a function of proximal delivery pressure. In one
embodiment, the effect of proximal pressure on delivery of
compounds used in the methods of the present invention by an
infusion sleeve catheter can be determined in vitro by histological
evaluation of the treated artery by known methods. In one
non-limiting example, a proximal pressure of between about 50 to
200 psi may be used to deliver, by an infusion sleeve catheter, the
compounds for use in the methods of the present invention,
preferably, between about 100 to about 150 psi, and most
preferably, between about 50 to 100 psi.
[0241] In another embodiment, the compounds of the invention may be
administered locally by diffusion-based catheter systems, including
for example, double balloon, dispatch, hydrogel and coated stent
catheters. Such methods are described in, e.g., U.S. Pat. No.
6,179,817. The methods of the invention also include local
administration of the conjugates by mechanical device-based
catheter systems, including for example, iontophoretic balloon
catheters.
[0242] In another embodiment, conjugates are delivered to a site in
an individual using coated medical devices, e.g., a coated
stent.
[0243] The ability to locally deliver the compounds used in the
present invention may be evaluated in vivo using known animal
models, including for example, acute canine coronary models. For
example, a compound for use in the methods of the present invention
is administered by local delivery to a canine at a site of injury.
The canine is sacrificed and then examined by known methods,
including, for example, fluorescence microscopy.
[0244] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous or oleagenous suspension for
intramuscular and subcutaneous administration. This suspension may
be formulated according to the known art using those suitable
dispersing or wetting agents and suspending agents which have been
mentioned above. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butane diol. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic
mono- or diglycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables.
[0245] In other embodiments, pharmaceutical compositions are
administered orally or topically according to methods known in the
art.
[0246] The conjugates of the invention may also be co-administered
with other well known therapeutic agents that are selected for
their particular usefulness against the condition that is being
treated. The conjugates may be administered simultaneously or
sequentially.
[0247] For example, modified apyrases can also be administered
together with one or more other agents, e.g., antiplatelet agents,
such as aspirin or GPIIb/IIIa-antagonists), direct antithrombin
modalities (e.g., heparin) or certain fibrinolytic interventions.
The agents of the invention can also be coadministered with an
angiogenesis inhibitors, in which case the angiogenesis inhibitor
is typically administered during or after chemotherapy, although it
is preferably to inhibit angiogenesis after a regimen of
chemotherapy at times where the tumor tissue will be responding to
the toxic assault by inducing angiogenesis to recover by the
provision of a blood supply and nutrients to the tumor tissue. In
addition, it is preferred to administer the angiogenesis inhibition
methods after surgery where solid tumors have been removed as a
prophylaxis against metastases.
[0248] In addition, since adenosine is the ultimate product of the
degradation of ATP and ADP, and it is a major factor in mediating
vasodilation by ATP, it may be preferable, in certain embodiments,
to administer to a subject a modified apyrase and an agent that
blocks adenosine receptors.
[0249] When a composition according to this invention is
administered into a human subject, the daily dosage will normally
be determined by the prescribing physician with the dosage
generally varying according to the age, weight, and response of the
individual patient, as well as the severity of the patient's
symptoms.
Kits of the Invention
[0250] In one embodiment, a conjugate of the invention, and
materials and/or reagents required for administering the complexes
of the invention may be assembled together in a kit. When the
components of the kit are provided in one or more liquid solutions,
the liquid solution preferably is an aqueous solution, with a
sterile aqueous solution being particularly preferred.
[0251] The kit may further comprise one or more other conjugates of
the invention or other drug, e.g., an anti-platelet aggregating
agent. These normally will be a separate formulation, but may be
formulated into a single pharmaceutically acceptable composition.
The container means may itself be geared for administration, such
as a syringe, pipette, eye dropper, or other such like
apparatus.
[0252] The compositions of these kits also may be provided in dried
or lyophilized forms. When reagents or components are provided as a
dried form, reconstitution generally is by the addition of a
suitable solvent. It is envisioned that the solvent also may be
provided in another container means. The kits of the invention may
also include an instruction sheet defining administration of the
agent.
[0253] The kits of the present invention may include a means for
containing the vials in close confinement for commercial sale such
as, e.g., injection or blow-molded plastic containers into which
the desired vials are retained. Irrespective of the number or type
of containers, the kits of the invention also may comprise, or be
packaged with a separate instrument for assisting with the
injection/administration or placement of the conjugate within the
body of an animal. Such an instrument may be a syringe, an
inhalant, pipette, forceps, measured spoon, eye dropper or any such
medically approved delivery vehicle. Other instrumentation includes
devices that permit the reading or monitoring of reactions, e.g.,
by the conjugates, or for determining the amounts of
conjugates.
[0254] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way. The contents of all cited references (including literature
references, issued patents, published patent applications as cited
throughout this application) are hereby expressly incorporated by
reference.
[0255] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No.: 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Exemplification
Example 1
Formation of Aldehyde-Containing Polymer by Polysaccharide
Oxidation
[0256] Dextran (MW=485 kDa), 22.5 g was dissolved in 500 mL water.
Sodium periodate, 57 g, was dissolved in 200 mL of water and mixed
with the dextran solution at 25.degree. C. After 8 hours of
incubation, the high-molecular components were extracted from the
reaction mixture by flow dialysis, using a hollow fiber Amicon.TM.
cartridge with a 10 kDa cutoff. The reaction mixture was
concentrated to 200 mL, then a 10 fold volume of water (2 liters)
was passed through. A forty mL aliquot of the reaction mixture was
lyophilized to yield 1.81 g of product. The resultant polymer was
slowly soluble in water at neutral and low pH and readily dissolved
at pH>7 and remained soluble after pH adjustment to pH<7. Ten
milligrams of the polymer were dissolved in deuterium oxide and a
proton NMR was obtained.
Example 2
Formation of Polyalcohol by Reduction of Aldehyde-Containing
Polymer
[0257] Sodium borohydride, 20 g, was dissolved in 20 mL water and
mixed with 160 mL of 4.5% solution of the aldehyde containing
polymer from Example 1. After 2 hours of incubation, the pH was
adjusted to 6. Twenty minutes later, high molecular components were
extracted by flow dialysis (as described in Example 1) and
separated into two fractions using an Amicon cartridge with a 100
kDa cutoff. Both fractions were lyophilized. Yields: low molecular
weight fractions: 2.4 g; high molecular weight fraction: 3.1 g. Ten
milligrams of low molecular weight polymer were dissolved in 1 mL
of deutero DMSO and proton NMR were obtained. FIG. 2 is a .sup.13C
NMR of the polyacetal, dissolved in deuterium oxide which
demonstrates carbons functionalized by alcohol functionality in the
biodegradable biocompatible polyacetal.
Example 3
Preparation of Apyrase
[0258] The apyrase used in the examples was a purified form of
potato apyrase. The apyrase was purchased from Aldrich-Sigma
(apyrase grade VII, cat. #A6535). The apyrase has an activity over
200 units/mg. One unit refers to the amount of apyrase that
liberates 1.0 mole of inorganic phosphate (Pi) from ATP or ADP per
minute at pH 6.5 at 30.degree. C. This preparation has a low
ATPase/ADPase ratio. The apyrase was further purified as
follows.
[0259] Purified apyrase was purified in the native conformation by
the Cibacron Blue column procedure. Potato apyrase eluted as a
single peak of active enzyme (950 units/ml of ADPase activity) in
the last step of Cibacron Blue affinity chromatography, as
described in Kettlun et al. (1982) Phytochemistry 21:551. Protein
from the active peak was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis followed by
electroblotting onto PVDF membranes (Millipore). The membrane was
stained with Ponceau S, and a single band of 49 kDa was
observed.
Example 4
Conjugation of Poly(hydroxymethylethylene hydroxymethylacetal)
(PHF) to Apyrase
[0260] PHF prepared in Example 2 was succinylated with succinic
anhydride in dimethylformamide. The succinilation was performed
under argon, at 21.degree. C. for 12 hours, at 5% PHF (w/w).
Succinic anyhdride was added at 1:5 to PHF monomer (mole/mole). The
reaction mixture was dried in vacuum. Then, the product was
dissolved in water (5% w/w), desalted on Sephadex G-25, and
lyophilized. Succinylation degree, as determined by titration, was
20.+-.1% (0.2 carboxyl groups per monomer unit). The ayrase enzyme,
obtained as described in Example 3, was dissolved in 50 mM PBS,
pH=8, at 5 mg/ml, modified with FITC (1% w/w to protein), and
purified on Sephadex G-25. The FITC labeled enzyme was conjugated
with succinyl-PHF in the presence of
ethyl-(dimethylaminopropyl)-carbodiimide (EDC) in 50 mM PBS, at
protein concentration of 1 mg/ml and succinyl-PHF concentration 50
mg/ml. The reaction mixture was monitored by SEC HPLC. When
conjugation degree reached about 95%, half of the reaction mixture
was transferred to a separate vessel and further reacted with
ethylenediamine (10:1 excess, EDC 1.5:1 to carboxyl groups) to
obtain a positively charged conjugate. Both conjugates were
purified on Sephadex G-25. SEC HPLC showed >95% conjugation with
formation of conjugates nearly equal in hydrodynamic size to the
original succinyl-PHF.
Example 5
Conjugated Apyrase Exhibits a Stronger Enzymatic Activity In Vitro
Relative to Unconjugated Apyrase
[0261] The in vitro rate of hydrolysis of ATP and ADP by apyrase,
unlinked to any polymer or linked to a positively charged PHF or a
negatively charged PHF, was determined as follows. Aliquots of
apyrase were incubated with either 200 .mu.M ADP or 200 .mu.M ATP,
and Ca.sup.2+-or Mg.sup.2+ dependent release of free phosphate was
determined. Malachite green was added to stop the reaction, and
absorbance was measured at 610 nm to determine levels of phosphate
generation against the standard curve of KH.sub.2PO.sub.4, as
described in Kaczmarek et al. (1996) J. Biol. Chem. 271:33116 and
Geladopoulos et al. (1991) Anal. Biochem. 192:112.
[0262] The results are set forth in Table 1.
TABLE-US-00001 TABLE 1 Rate of ATP and ADP breakdown of apyrase III
or VII vs. positively and negatively charged apyrase conjugates
ATPase ADPase (nanomol (nanomol mg/ml Pi/min/mg) Pi/min/mg) ATP/ADP
Unconjugated* 2 82466 13932 5.9 Conjugate + 0.5 9,074 11,621 0.8
Conjugate - 0.5 15,350 23,205 0.7 *These data were obtained in a
separate experiment relative to the data listed in the next two
lines of the table.
[0263] The results indicate that the apyrase linked to a negatively
or positively charged polymer is much more effective in degrading
ADP than the non-conjugated apyrase is. Furthermore, the conjugates
are much less effective in hydrolyzing ATP than the non-conjugated
apyrase is. Thus, whereas the non-conjugated apyrase is 5.9 times
more efficient in hydrolyzing ATP than ADP, the apyrase conjugated
to a negatively or positively charged polymer hydrolyzes ADP more
efficiently than ATP (ATP/ADP breakdown rate of more than 1 versus
less than 1, respectively). As it is the abundance of ADP that is
thought to lead to abnormal platelet aggregation and the diseases
associated with that condition, the higher consumption rate of ADP
relative to ATP of the charged conjugated apyrase will keep the
level of ADP down more effectively than the free apyrase.
Example 6
Conjugated Apyrase Exhibits a Stronger Enzymatic Activity and
Longer Half-Life In Vivo Relative to Unconjugated Apyrase
[0264] In this experiment, the pharmacokinetic effects of
conjugating an apyrase to a polymer were analyzed in an animal. The
in vivo rate of hydrolysis of ATP and ADP by apyrase, unlinked to
any polymer or linked to a positively charged PHF or a negatively
charged PHF, was determined as follows. Mice were injected with 5
units unconjugated apyrase and approximately 1 unit of conjugated
apyrase, mice were sacrificed after 5, 60 or 240 minutes, and the
activity of apyrase in the mice was determined as described in
Example 5 on samples of the animals' blood. The results, in Pi
.mu.mol/mg/min, are shown in Table 2 and in FIG. 3.
TABLE-US-00002 TABLE 2 Free and modified apyrase activities in
nanomol/minute/ml blood (normalized) 5 minutes 60 minutes 240
minutes unconjugated 0 1 1 apyrase Conjugate + 4,449 3,857 1,102
Conjugate - 73 191 23
[0265] The results show the activities of free apyrase (i.e., not
conjugated to a polymer) versus negatively and positively charged
conjugated apyrase and the marked increase at different time
intervals in activity of the conjugated apyrase over the free
apyrase.
[0266] The results also indicate that the positively charged
conjugate is more active than the negatively charged conjugate in
the in vivo assay, which is the opposite from that which was
observed in the in vitro assays. Without wanting to be limited to a
particular mechanism of action, it is believed that positively
charged conjugates are more active in vivo, since they are
sequestered in vivo to the negatively charged endothelium.
[0267] Thus, the results indicate that apyrase conjugated to a
polymer is more active in degrading ADP relative to ATP, and has a
much longer half-life in vivo.
Equivalents
[0268] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 7 <210> SEQ ID NO 1 <400> SEQUENCE: 1 000
<210> SEQ ID NO 2 <211> LENGTH: 510 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met
Glu Asp Thr Lys Glu Ser Asn Val Lys Thr Phe Cys Ser Lys Asn 1 5 10
15 Ile Leu Ala Ile Leu Gly Phe Ser Ser Ile Ile Ala Val Ile Ala Leu
20 25 30 Leu Ala Val Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu Asn
Val Lys 35 40 45 Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr
Ser Leu Tyr Ile 50 55 60 Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp
Thr Gly Val Val His Gln 65 70 75 80 Val Glu Glu Cys Arg Val Lys Gly
Pro Gly Ile Ser Lys Phe Val Gln 85 90 95 Lys Val Asn Glu Ile Gly
Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala 100 105 110 Arg Glu Val Ile
Pro Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu 115 120 125 Gly Ala
Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser Glu Glu Leu 130 135 140
Ala Asp Arg Val Leu Asp Val Val Glu Arg Ser Leu Ser Asn Tyr Pro 145
150 155 160 Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu Glu
Gly Ala 165 170 175 Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Lys
Phe Ser Gln Lys 180 185 190 Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu
Thr Asn Asn Gln Glu Thr 195 200 205 Phe Gly Ala Leu Asp Leu Gly Gly
Ala Ser Thr Gln Val Thr Phe Val 210 215 220 Pro Gln Asn Gln Thr Ile
Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg 225 230 235 240 Leu Tyr Gly
Lys Asp Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr 245 250 255 Gly
Lys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val 260 265
270 Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys
275 280 285 Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr
Lys Arg 290 295 300 Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu Ile
Gln Gly Ile Gly 305 310 315 320 Asn Tyr Gln Gln Cys His Gln Ser Ile
Leu Glu Leu Phe Asn Thr Ser 325 330 335 Tyr Cys Pro Tyr Ser Gln Cys
Ala Phe Asn Gly Ile Phe Leu Pro Pro 340 345 350 Leu Gln Gly Asp Phe
Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys 355 360 365 Phe Leu Asn
Leu Thr Ser Glu Lys Val Ser Gln Glu Lys Val Thr Glu 370 375 380 Met
Met Lys Lys Phe Cys Ala Gln Pro Trp Glu Glu Ile Lys Thr Ser 385 390
395 400 Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser Glu Tyr Cys Phe Ser
Gly 405 410 415 Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly Tyr His Phe
Thr Ala Asp 420 425 430 Ser Trp Glu His Ile His Phe Ile Gly Lys Ile
Gln Gly Ser Asp Ala 435 440 445 Gly Trp Thr Leu Gly Tyr Met Leu Asn
Leu Thr Asn Met Ile Pro Ala 450 455 460 Glu Gln Pro Leu Ser Thr Pro
Leu Ser His Ser Thr Tyr Val Phe Leu 465 470 475 480 Met Val Leu Phe
Ser Leu Val Leu Phe Thr Val Ala Ile Ile Gly Leu 485 490 495 Leu Ile
Phe His Lys Pro Ser Tyr Phe Trp Lys Asp Met Val 500 505 510
<210> SEQ ID NO 3 <400> SEQUENCE: 3 000 <210> SEQ
ID NO 4 <400> SEQUENCE: 4 000 <210> SEQ ID NO 5
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Unknown Organism <220> FEATURE: <223> OTHER
INFORMATION: Description of Unknown Organism: Amino acid sequence
which is conserved among various apyrases <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223>
OTHER INFORMATION: Ile or Val <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER
INFORMATION: Val, Met, or Ile <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHER
INFORMATION: Ile, Leu, Phe, or Cys <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (8) <223> OTHER
INFORMATION: Ser or Thr <400> SEQUENCE: 5 Xaa Xaa Xaa Asp Ala
Gly Ser Xaa 1 5 <210> SEQ ID NO 6 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Unknown Organism
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown Organism: Amino acid sequence which shares strong homology
between potato apyrase and pea NTPase <400> SEQUENCE: 6 Pro
Gly Leu Ser Ser Tyr Ala 1 5 <210> SEQ ID NO 7 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Unknown
Organism <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism: Amino acid sequence which shares
strong homology between potato apyrase and pea NTPase <400>
SEQUENCE: 7 Leu Tyr Val His Ser Tyr Leu 1 5
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 7 <210>
SEQ ID NO 1 <400> SEQUENCE: 1 000 <210> SEQ ID NO 2
<211> LENGTH: 510 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 2 Met Glu Asp Thr Lys Glu Ser
Asn Val Lys Thr Phe Cys Ser Lys Asn 1 5 10 15 Ile Leu Ala Ile Leu
Gly Phe Ser Ser Ile Ile Ala Val Ile Ala Leu 20 25 30 Leu Ala Val
Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu Asn Val Lys 35 40 45 Tyr
Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile 50 55
60 Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val His Gln
65 70 75 80 Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe
Val Gln 85 90 95 Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys
Met Glu Arg Ala 100 105 110 Arg Glu Val Ile Pro Arg Ser Gln His Gln
Glu Thr Pro Val Tyr Leu 115 120 125 Gly Ala Thr Ala Gly Met Arg Leu
Leu Arg Met Glu Ser Glu Glu Leu 130 135 140 Ala Asp Arg Val Leu Asp
Val Val Glu Arg Ser Leu Ser Asn Tyr Pro 145 150 155 160 Phe Asp Phe
Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu Glu Gly Ala 165 170 175 Tyr
Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Lys Phe Ser Gln Lys 180 185
190 Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr
195 200 205 Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln Val Thr
Phe Val 210 215 220 Pro Gln Asn Gln Thr Ile Glu Ser Pro Asp Asn Ala
Leu Gln Phe Arg 225 230 235 240 Leu Tyr Gly Lys Asp Tyr Asn Val Tyr
Thr His Ser Phe Leu Cys Tyr 245 250 255 Gly Lys Asp Gln Ala Leu Trp
Gln Lys Leu Ala Lys Asp Ile Gln Val 260 265 270 Ala Ser Asn Glu Ile
Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys 275 280 285 Lys Val Val
Asn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg 290 295 300 Phe
Glu Met Thr Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly 305 310
315 320 Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn Thr
Ser 325 330 335 Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe
Leu Pro Pro 340 345 350 Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe
Tyr Phe Val Met Lys 355 360 365 Phe Leu Asn Leu Thr Ser Glu Lys Val
Ser Gln Glu Lys Val Thr Glu 370 375 380 Met Met Lys Lys Phe Cys Ala
Gln Pro Trp Glu Glu Ile Lys Thr Ser 385 390 395 400 Tyr Ala Gly Val
Lys Glu Lys Tyr Leu Ser Glu Tyr Cys Phe Ser Gly 405 410 415 Thr Tyr
Ile Leu Ser Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp 420 425 430
Ser Trp Glu His Ile His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala 435
440 445 Gly Trp Thr Leu Gly Tyr Met Leu Asn Leu Thr Asn Met Ile Pro
Ala 450 455 460 Glu Gln Pro Leu Ser Thr Pro Leu Ser His Ser Thr Tyr
Val Phe Leu 465 470 475 480 Met Val Leu Phe Ser Leu Val Leu Phe Thr
Val Ala Ile Ile Gly Leu 485 490 495 Leu Ile Phe His Lys Pro Ser Tyr
Phe Trp Lys Asp Met Val 500 505 510 <210> SEQ ID NO 3
<400> SEQUENCE: 3 000 <210> SEQ ID NO 4 <400>
SEQUENCE: 4 000 <210> SEQ ID NO 5 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Unknown Organism
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown Organism: Amino acid sequence which is conserved among
various apyrases <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (1) <223> OTHER INFORMATION: Ile or Val
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (2) <223> OTHER INFORMATION: Val, Met, or Ile
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (3) <223> OTHER INFORMATION: Ile, Leu, Phe, or Cys
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (8) <223> OTHER INFORMATION: Ser or Thr <400>
SEQUENCE: 5 Xaa Xaa Xaa Asp Ala Gly Ser Xaa 1 5 <210> SEQ ID
NO 6 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Unknown Organism <220> FEATURE: <223> OTHER
INFORMATION: Description of Unknown Organism: Amino acid sequence
which shares strong homology between potato apyrase and pea NTPase
<400> SEQUENCE: 6 Pro Gly Leu Ser Ser Tyr Ala 1 5 <210>
SEQ ID NO 7 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Unknown Organism <220> FEATURE: <223> OTHER
INFORMATION: Description of Unknown Organism: Amino acid sequence
which shares strong homology between potato apyrase and pea NTPase
<400> SEQUENCE: 7 Leu Tyr Val His Ser Tyr Leu 1 5
* * * * *