U.S. patent application number 10/967921 was filed with the patent office on 2005-05-12 for fusogenic properties of saposin c and related proteins and polypeptides for application to transmembrane drug delivery systems.
Invention is credited to Qi, Xiaoyang.
Application Number | 20050100591 10/967921 |
Document ID | / |
Family ID | 26877479 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100591 |
Kind Code |
A1 |
Qi, Xiaoyang |
May 12, 2005 |
Fusogenic properties of saposin C and related proteins and
polypeptides for application to transmembrane drug delivery
systems
Abstract
The present invention comprises a method for delivering
pharmaceutical agents within and/or through the dermal and mucosal
membranes, utilizing a fusogenic protein. The fusogenic protein is
associated with a phospholipid membrane, such as a liposome. The
liposome contains the pharmaceutical agent. Preferred fusogenic
proteins include saposin C and other proteins, polypeptides and
peptide analogs derived from saposin C. The active agent contained
within the liposome may comprise large biomolecules and/or small
organic molecules. This technology can be used for both cosmetic
and medicinal applications in which the objective is delivery of
the active agent within and/or beneath the biological membrane.
Inventors: |
Qi, Xiaoyang; (Cincinnati,
OH) |
Correspondence
Address: |
FROST BROWN TODD, LLC
2200 PNC CENTER
201 E. FIFTH STREET
CINCINNATI
OH
45202
US
|
Family ID: |
26877479 |
Appl. No.: |
10/967921 |
Filed: |
October 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10967921 |
Oct 18, 2004 |
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09780438 |
Feb 9, 2001 |
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6872406 |
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60181754 |
Feb 11, 2000 |
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Current U.S.
Class: |
424/450 ;
435/458 |
Current CPC
Class: |
C07K 14/475 20130101;
A61K 9/127 20130101 |
Class at
Publication: |
424/450 ;
435/458 |
International
Class: |
A61K 009/127; C12N
015/88 |
Goverment Interests
[0002] THIS WORK WAS SUPPORTED IN PART BY NIH GRANT RO1 DK57690-01.
Claims
What is claimed is:
1) A method for delivering a pharmaceutical agent through a
membrane, wherein the method comprises applying to said membrane a
composition comprising: a) anionic phospholipids; b) a safe and
effective amount of the pharmaceutical agent contained within the
aqueous interior of the phospholipids; and c) a fusogenic protein
or polypeptide derived from prosaposin in a pharmaceutically
acceptable carrier, wherein the concentration of the fusogenic
protein or polypeptide is of a sufficient amount to deliver the
pharmaceutical agent through the membrane.
2) The method of claim 2 wherein the concentration of phospholipids
are in at least a 10-fold excess, by weight, to that of the
fusogenic protein or polypeptide.
3) The method of claim 2 wherein the pH of the composition is
between about 5.5 and 2.
4) The method of claim 3 wherein the anionic phospholipid is an
anionic liposome.
5) The method of claim 4 wherein the fusogenic protein or
polypeptide is associated with the liposome through an
electrostatic and hydrophobic interaction.
6) The method of claim 5 wherein the membrane is selected from the
group consisting of dermal and mucosal membranes.
7) The method of claim 6 wherein the fusogenic protein or
polypeptide is selected from the group consisting of saposin A,
saposin C, and mixtures thereof.
8) The method of claim 6 wherein the fusogenic protein or
polypeptide is saposin C.
9) The method of claim 6 wherein the fusogenic protein or
polypeptide is SEQ. ID. NO. 1.
10) The method of claim 6 wherein the fusogenic protein or
polypeptide is SEQ. ID. NO. 2.
11. The method of claim 6 wherein the fusogenic protein or
polypeptide is selected from the group consisting of those proteins
or polypeptides given by SEQ ID Nos. 3-6.
12) The method of claim 7 wherein administration of the composition
is via a transdermal patch.
13) The method of claim 7 wherein the composition is administered
either enterally or topically.
14) A method for delivering a pharmaceutical agent through either a
dermal or mucosal membrane, wherein the method comprises the
administration to said membrane of a composition comprising: a)
anionic liposomes; b) a safe and effective amount of the
pharmaceutical agent contained within the aqueous interior of the
liposomes; and c) saposin C; in a pharmaceutically acceptable
carrier, wherein the concentration of the liposomes are of a
sufficient amount to deliver a safe and effective amount of the
pharmaceutical agent through the membrane, the pH of the
composition is between about 5.5 and 2, and the saposin C is
associated with the surface of the liposome through an
electrostatic and hydrophobic interaction.
15) The method of claim 14 wherein the concentration of the
liposomes is in at least a 10-fold excess, by weight, to that of
saposin C.
16) A therapeutic phospholipid composition comprising: a) an
anionic phospholipid; b) a safe and effective amount of the
pharmaceutical agent contained within the aqueous interior of the
phospholipids; and c) a fusogenic protein or polypeptide derived
from prosaposin; in a pharmaceutically acceptable carrier, wherein
the fusogenic protein or polypeptide is present in a sufficient
concentration to deliver the pharmaceutical agent through a
biological membrane and the fusogenic protein or polypeptide is
associated with the phospholipid through an electrostatic and
hydrophobic interaction.
17) The therapeutic phospholipid composition of claim 16 wherein
the concentration of anioinic phospholipids is in at least a
10-fold excess, by weight, to that of the fusogenic protein or
polypeptide.
18) The therapeutic phospholipid composition of claim 17 wherein
the pH of the composition is between about 5.5 and 2.
19) The therapeutic phospholipid composition of claim 18 wherein
the anionic phospholipid is an anionic liposome.
20) The therapeutic phospholipid composition of claim 19 wherein
the biological membrane is selected from the group consisting of
dermal and mucosal membranes.
21) The therapeutic phospholipid composition of claim 20 wherein
the fusogenic protein or polypeptide is selected from the group
consisting of saposin A, and saposin C, and mixtures thereof.
22) The therapeutic phospholipid composition of claim 20 wherein
the fusogenic protein or polypeptide is saposin C.
23) The therapeutic phospholipid composition of claim 20 wherein
the fusogenic protein or polypeptide is SEQ. ID. NO. 1.
24) The therapeutic phospholipid composition of claim 20 wherein
the fusogenic protein or polypeptide is SEQ. ID. NO. 2.
25) The therapeutic phospholipid composition of claim 20 wherein
the fusogenic protein or polypeptide is selected from the group
consisting of those proteins or polypeptides given by SEQ ID Nos.
3-6.
26) The therapeutic phospholipid composition of claim 21 wherein
the composition is formulated as part of a transdermal patch.
27) The therapeutic phospholipid composition of claim 21 wherein
the composition is formulated for enteral or topical
administration.
28) An anionic liposomal composition used to deliver a
pharmaceutical agent through either a dermal or mucosal membrane,
wherein the composition comprises: a) anionic liposomes; b) a safe
and effective amount of the pharmaceutical agent contained within
the aqueous interior of the liposomes; and c) saposin C; in a
pharmaceutically acceptable carrier where the pH of the composition
is between about 5.5 and 2, wherein the concentration of the
saposin C is of a sufficient amount to deliver the pharmaceutical
agent through a biological membrane and the saposin C is associated
with the surface of the liposomes through an electrostatic and
hydrophobic interaction.
29) The anionic liposomal composition of claim 28 wherein the
concentration of the anionic liposomes is in at least a 10-fold
excess, by weight, to that of saposin C.
30) A composition comprising a safe and effective amount of a
pharmaceutical agent contained in anionic liposomes, which are
associated with a prosaposin-derived fusogenic protein or
polypeptide via an electrostatic and hydrophobic interaction,
wherein the concentration of the fusogenic protein or polypeptide
is of a sufficient amount to deliver the pharmaceutical agent
through a biological membrane, the composition contained in a
pharmaceutically acceptable carrier, wherein the pH of the
composition is between about 5.5 and 2.
31) The composition of claim 30 wherein the concentration of
anionic liposomes is in at least a 10-fold excess, by weight, to
that of the fusogenic protein or polypeptide.
32) The composition of claim 31 wherein the biological membrane is
selected from the group consisting of dermal and mucosal
membranes.
33) The composition of claim 32 wherein the fusogenic protein or
polypeptide is selected from the group consisting of saposin A,
saposin C, and mixtures thereof.
34) The composition of claim 31 wherein the fusogenic protein or
polypeptide is saposin C.
35) The composition of claim 31 wherein the fusogenic protein or
polypeptide is SEQ. ID. NO. 1.
36) The composition of claim 31 wherein the fusogenic protein or
polypeptide is SEQ. ID. NO. 2.
37. The composition of claim 31 wherein the fusogenic protein or
polypeptide is selected from the group consisting of those proteins
or polypeptides given by SEQ ID Nos. 3-6.
38) A phospholipid composition used to deliver a pharmaceutical
agent through either a dermal or mucosal membrane, wherein the
composition comprises: a) anionic liposomes; b) a safe and
effective amount of the pharmaceutical agent contained within the
aqueous interior of the liposomes; and c) saposin C; in a
pharmaceutically acceptable carrier, wherein the pH of the
composition is between about 5.5 and 2, the concentration of the
saposin C is of a sufficient amount to deliver the pharmaceutical
agent through the membrane and the saposin C is associated with the
surface of the liposome through an electrostatic and hydrophobic
interaction.
39) The phospholipid composition of claim 38 wherein the
concentration of the anionic liposomes is in at least a 10-fold
excess, by weight, to that of saposin C.
40) The polypeptide consisting of SEQ. ID. NO. 1.
41) The polypeptide consisting of SEQ. ID. NO. 2.
42. A compound of the formula consisting of SEQ ID Nos. 3-6.
43) A method for treating Gauchers Disease wherein the method
comprises the administration of a composition comprising: a)
anionic liposomes; b) a safe and effective amount of acid
beta-glucosidase contained within the aqueous interior of the
liposomes; and c) saposin C; in a pharmaceutically acceptable
carrier, wherein the pH of the composition between about 5.5 and 2,
the concentration of the saposin C is of a sufficient amount to
deliver the pharmaceutical agent through the membrane and the
saposin C is associated with the surface of the liposome through an
electrostatic and hydrophobic interaction.
44) The method of claim 43 wherein the concentration of the
liposome is in at least a 10-fold excess, by weight, to that of
saposin C.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/780,438, filed Feb. 9, 2001, Xiaoyang
Qi
FIELD OF INVENTION
[0003] The present invention relates to methods of delivering
pharmaceutical agents across biological membranes, where the
pharmaceutical agent is contained within a phospholipid membrane
and delivery is facilitated by a membrane fusion protein. More
particularly, the present invention relates to methods for
enhancing the transport and delivery of pharmaceutical agents
across and/or within dermal and mucosal membranes, where the
pharmaceutical agent is contained within a liposome, and delivery
is facilitated using saposin C, which is in association with the
liposome.
BACKGROUND
[0004] Drug Delivery
[0005] The therapeutic efficacy of pharmaceutical or therapeutic
agents relies on the delivery of adequate doses of a pharmaceutical
agent to the site of action. Many modes of delivery have been
developed, including, for example, enteral (oral), parenteral
(intramuscular, intravenous, subcutaneous), and topical
administration. In most instances the administration system is
chosen for reliable dosage delivery and convenience.
[0006] Typically, parenteral administration is the most reliable
means of delivering a pharmaceutical to a patient. See, Goodman et
al., Goodman and Gilman's Pharmacological Basis of Therapeutics,
Pergamon Press, Elmsford, N.Y. (1990) and Pratt et al. Principles
of Drug Action: The Basis of Pharmacology, Churchill Livingstone,
New York, N.Y. (1990). Each parenteral mechanism insures that a
prescribed dosage of the pharmaceutical agent is inserted into the
fluid compartment of the body where it can be transported. The
disadvantage of these modes of delivery is that they require an
invasive procedure. The invasive nature of administration is
inconvenient, painful and subject to infectious contamination.
[0007] Enteral and topical administration are more convenient,
generally non-painful, and do not predispose to infection, however
both have limited utility. The gastrointestinal and dermal surfaces
present formidable barriers to transport and therefore, some
pharmaceutical agents are not absorbed across these surfaces.
Another drawback to patient directed modes of administration
(enteral, topical and subcutaneous) is compliance. Pharmaceutical
agents that have a short half-life require multiple daily doses. As
the number of doses increases, patient compliance and therapeutic
efficacy decrease. Simplified and/or less frequent administration
schedules can aid in optimizing patient compliance. Wilson et al.
(1991) Harrison's Principles of Internal Medicine, 12th Ed.,
McGraw-Hill, Inc., New York, N.Y.
[0008] The skin is an efficient barrier to the penetration of water
soluble substances, and the rate of transdermal pharmaceutical
agent absorption is primarily determined by the agent's lipid
solubility, water solubility, and polarity. Highly polar or water
soluble pharmaceutical agents are effectively blocked by the skin.
Even very lipophilic pharmaceutical agents penetrate the dermis
very slowly compared with the rate of penetration across cell
membranes. See Pratt et al. supra.
[0009] Efforts to develop more effective and convenient modes of
pharmaceutical administration have led to the development of
transdermal delivery systems. Many current transdermal
pharmaceutical agent delivery systems rely upon pharmaceutical
agents that are absorbed when admixed with inert carriers. See
Cooper et al. (1987) "Penetration Enhancers", in Transdermal
Delivery of Drugs, Vol. II, Kyodonieus et al., Eds., CRC Press,
Boca Raton, Fla. Few pharmaceutical agents fit this profile and
those which do are not always predictably absorbed. Various forms
of chemical enhancers, such as those enhancing lipophilicity, have
been developed to improve transdermal transport when physically
mixed with certain therapeutic agents and provide more predictable
absorption. See for example, U.S. Pat. Nos. 4,645,502; 4,788,062;
4,816,258; 4,900,555; 3,472,931; 4,006,218; and 5,053,227. Carriers
have also been coupled to pharmaceutical agents to enhance
intracellular transport. See Ames et al. (1973) Proc. Natl. Acad.
Sci. USA, 70:456-458 and (1988) Proc. Int. Symp. Cont. Rel. Bioact.
Mater., 15:142.
[0010] Fusogenic Proteins and Polypeptides of the Saposin
Family
[0011] Saposins, a family of small (.about.80 amino acids) heat
stable glycoproteins, are essential for the in vivo hydrolytic
activity of several lysosomal enzymes in the catabolic pathway of
glycosphingolipids (see Grabowski, G. A., Gatt, S., and Horowitz,
M. (1990) Crit. Rev. Biochem. Mol. Biol. 25, 385-414; Furst, W.,
and Sandhoff, K., (1992) Biochim. Biophys. Acta 1126, 1-16;
Kishimoto, Y., Kiraiwa, M., and O'Brien, J. S. (1992) J. Lipid.
Res. 33, 1255-1267). Four members of the saposin family, A, B, C,
and D, are proteolytically hydrolyzed from a single precursor
protein, prosaposin (see Fujibayashi, S., Kao, F. T., Hones, C.,
Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37,
741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A.,
Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101;
Rorman, E. G., and Grabowski, G. A. (1989) Genomics 5, 486-492;
Nakano, T., Sandhoff, K., Stumper, J., Christomanou, H., and
Suzuki, K. (1989) J. Biochem. (Tokyo) 105, 152-154; Reiner, O.,
Dagan, O., and Horowitz, M. (1989) J. Mol. Neurosci. 1, 225-233).
The complete amino acid sequences for saposins A, B, C and D have
been reported as well as the genomic organization and cDNA sequence
of prosaposin (see Fujibayashi, S., Kao, F. T., Jones, C., Morse,
H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37,
741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A.,
Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101;
Rorman, E. G., and Grabowski, G. A. (1989) Genomics 5, 486-492). A
complete deficiency of prosaposin with mutation in the initiation
codon causes the storage of multiple glycosphingolipid substrates
resembling a combined lysosomal hydrolase deficiency (see Schnabel,
D., Schroder, M., Furst, W., Klien, A., Hurwitz, R., Zenk, T.,
Weber, J., Harzer, K., Paton, B. C., Poulos, A., Suzuki, K., and
Sandhoff, K. (1992) J. Biol. Chem. 267, 3312-3315).
[0012] Saposins are defined as sphingolipid activator proteins or
coenzymes. Structurally, saposins A, B, C, and D have approximately
50-60% similarity including six strictly conserved cysteine
residues (see Furst, W., and Sandhoff, K., (1992) Biochim. Biophys.
Acta 1126, 1-16) that form three intradomain disulfide bridges
whose placements are identical (see Vaccaro, A. M., Salvioli, R.,
Barca, A., Tatti, M., Ciaffoni, F., Maras, B., Siciliano, R.,
Zappacosta, F., Amoresano, A., and Pucci, P. (1995) J. Biol. Chem.
270, 9953-9960). All saposins contain one glycosylation site with
conserved placement in the N-terminal sequence half, but
glycosylation is not essential to their activities (see Qi. X., and
Grabowski, G. A. (1998) Biochemistry 37, 11544-11554; Vaccaro, A.
M., Ciaffoni, F., Tatti, M., Salvioli, R., Barca, A., Tognozzi, D.,
and Scerch, C. (1995) J. Biol. Chem. 270, 30576-30580). In
addition, saposin A has a second glycosylation site in C-terminal
half.
[0013] All saposins and saposin-like proteins and domains contain a
"saposin fold" when in solution. This fold is a multiple
.alpha.-helical bundle motif, characterized by a three conserved
disulfide structure and several amphipathic polypeptides. Despite
this shared saposin-fold structure in solution, saposins and
saposin-like proteins have diverse in vivo biological functions in
the enhancement of lysosomal sphingolipid (SL) and
glycosphingolipid (GSL) degradation by specific hydrolases. Because
of these roles, the saposins occupy a central position in the
control of lysosomal sphingolipid and glycosphingolipid metabolisms
(see Kishimoto, Y., Kiraiwa, M., and O'Brien, J. S. (1992) J.
Lipid. Res. 33, 1255-1267; Fujibayashi, S., Kao, F. T., Hones, C.,
Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37,
741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A.,
Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101).
[0014] The structural characteristic of these saposins is of great
importance to the diverse mechanisms of activation. Since all of
these proteins have high sequence similarity, but different
mechanisms of action with lipid membranes, one can speculate that
the specific biological functions of saposins and saposin-like
proteins are the result of the differential interactions with the
biological membrane environments. In vitro, saposin A enhances acid
.beta.-glucosidase activity at .mu.M concentration, but saposin C
deficiency leads to glucosylceramide storage and a "Gaucher
disease-like" phenotype (see Schnable, D., Schroder, M., and
Sandhoff, K. (1991) FEBS Lett. 284, 57-59; Rafi. M. A., deGala, G.,
Zhang, X. L., and Wenger, D. A. (1993) Somat. Cell Mol. Genet. 19,
1-7). Activation of saposin B takes place through solubilizing and
presenting glycosphingolipid substrates to lysosomal enzymes (see
Furst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta 1126,
1-16).
[0015] Saposin C promotes acid .beta.-glucosidase activity by
inducing in the enzyme conformational change at acidic pH (see
Berent, S. L., and Radin, N. S. (1981) Biochim. Biophys. Acta 664,
572-582; Greenberg, P., Merrill, A. H., Liotta, D. C., and
Grabowski, G. A. (1990) Biochim. Biophys. Acta 1039, 12-20; Qi. X.,
and Grabowski, G. A. (1998) Biochemistry 37, 11544-11554). This
interaction of saposin C with the enzyme occurs on negatively
charged phospholipid surfaces. In vitro and ex vivo saposins A and
D function to enhance the degradation of galactosylceramide and
ceramide/sphingomyelin, respectively (see Harzer, K., Paton, B. C.,
Christomanou, H., Chatelut, M., Levade, T., Hiraiwa, M. and
O'Brien, J. S. (1997) FEBS Lett. 417, 270-274; Klien, A., Henseler,
M., Klein, C., Suzuki, K., Harzer, K., and Sandhoff, K. (1994)
Biochem. Biophys. Res. Commun, 200, 1440-1448). Patients lacking
the individual saposins B and C showed a variant form of
metachromatic leukodystrophy and Gaucher disease, respectively.
(see Wenger, D. A., DeGala, G., Williams, C., Taylor, H. A.,
Stevenson, R. E., Pruitt, J. R., Miller, J., Garen, P. D., and
Balentine, J. D. (1989) Am. J. Med. Genet. 33, 255-265) (see
Christomanou, H., Aignesberger, A., and Linke, R. P. (1986) Biol.
Chem. Hoppe-Seyler 367, 879-890).
[0016] Membrane fusion is a major event in biological systems
driving secretion, endocytosis, excocytosis, intracellular
transport, fertilization, and muscle development (see Christomanou,
H., Chabas, A., Pampols, T., and Guardiola, A. (1989) Klin,
Wochenschr. 67, 999-1003). Recent experimental evidence generated
by this inventor has indicated that saposin-lipid membrane
interactions play a critical role in saposin-mediated membrane
fusion of lipids thereby facilitating transport of active agents
across these biological membranes.
[0017] Accordingly, there exists a significant need for nontoxic
agents which can improve the delivery or transport of
pharmaceutical agents across or through biological membranes. The
present invention fulfills these needs.
SUMMARY OF THE INVENTION
[0018] As described herein, the present invention comprises a
method for delivering a pharmaceutical agent through a biological
membrane, wherein the method comprises applying to said membrane a
composition comprising anionic phospholipids, a safe and effective
amount of the pharmaceutical agent contained within the
phospholipids, and a fusogenic protein or polypeptide derived from
prosaposin in a pharmaceutically acceptable carrier. The pH of the
composition is between about 5.5 and 2, and preferably between
about 5.5 and about 3.5. Generally, the concentration of the
fusogenic protein or polypeptide is of a sufficient amount to
deliver the pharmaceutical agent within and/or through the
membrane. The concentration of phospholipids are in at least a
10-fold exess to that of the fusogenic protein or polypeptide. The
membrane fusion protein is associated with the phospholipid
membrane, through electrostatic and hydrophobic and hydrophobic
interactions.
[0019] In accordance with the present invention, the targeted
biological membranes include, but are not limited to, dermal
membrane and mucosal membranes. The preferred membrane fusion
proteins include saposin C as well as other proteins, polypeptide
analogues or polypeptides derived from either saposin C, SEQ. ID
NO. 1, SEQ. ID. NO. 2, polypeptides of the formula:
h-u-Cys-Glu-h-Cys-Glu-h-h-h-Lys-Glu-h-u-Lys-h-h-Asp-Asn-Asn-Lys-u-Glu-Lys--
Glu-h-h-Asp-h-h-Asp-Lys-h-Cys-u-Lys-h-h,
[0020] where h=hydrophobic amino acids, including, Val, Leu, Ile,
Met, Pro, Phe, and Ala; and u=uncharged polar amino acids,
including, Thr, Ser, Tyr, Gly, Gln, and Asn., and mixtures
thereof.
[0021] The phopholipid membranes may be anionic liposomes
containing the pharmaceutical agent. The pharmaceutical agent
contained within the liposome may comprise large biomolecules
and/or small organic molecules. This technology can be used for
both cosmetic and medicinal applications in which the objective is
delivery of the active agent within and/or beneath the dermal or
mucosal membrane.
[0022] Finally, the present invention also comprises a method for
treating Gauchers Disease, wherein the method comprises the
administration of a composition comprising anionic liposomes, a
safe and effective amount of acid beta-glucosidase contained within
the liposomes; and saposin C, all contained in a pharmaceutically
acceptable carrier, wherein the pH of the composition is about 5.5
or less and the saposin C is associated with the surface of the
liposome through an electrostatic and hydrophobic interaction.
Generally, the concentration of the liposome is in at least a
10-fold excess to that of saposin C.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1: Clip-on model for saposin C induced fusion:
Liposome-bound saposin Cs clip one to another through hydrophobic
interaction, and induce liposome fusion.
[0024] FIG. 2: Saposin C and liposome vesicle association: A
conformational alteration of the saposin-fold found in lipid-bound
saposin C. Membrane Topological interaction of saposin C indicated
that amphipathic helices at amino- and carboxyl-termini were
embedded into the lipid bilayer and the middle region of saposin C
is exposed to aqueous phase.
[0025] FIG. 3: Hypothetical model of membrane-saposin C
interaction. Phospholipid bilayers contain acidic
phosphatidylserine. Cylinders are presented in the amphipathic
helices in saposin C. The membrane topological structure of saposin
C shows a conformational alteration of the saposin fold found in
lipid-bound saposin C. The amphipathic helices at amino- and
carboxyl termini of saposin C are inserted into the membrane. The
middle region of saposin C is exposed to the aqueous phase.
[0026] FIG. 4: A schematic of the functional organization of the
neuritogenic, acid .beta.-glucosidase activation and lipid-binding
properties of saposin C. Except for the box indicating the
predicated turn and the disulfide bonds, the figure is not meant to
represent known physical structure. The residues from 22-32 are of
major significance to the neurotrophic effect. The region spanning
residues 42-61 is critical to the acid .beta.-glucosidase
activation effects of saposin C, and the presence of all three
disulfide bonds is also important for this function. In addition,
higher order structure is required to have full activities of
saposin C. Lipid/lipid membrane interaction regions are located at
both NH.sub.2-- and COOH-terminal regions.
[0027] FIG. 5: Size changes of BPS (brain phosphatidylserine)
liposomes induced by Ca.sup.2+ (a) or Saposin C (b) at pH 4.7 or
7.4. Fair autocorrelation function, dust=0.0%, base line
error<1%, room temperature.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Contents
[0029] I. Definitions
[0030] II. Fusogenic Proteins or Polypeptides of the Saposin
Family
[0031] III. The Phospholipid Membrane
[0032] IV. Nature and pH Dependence of Protein-Membrane
Attachment
[0033] V. The Pharmaceutical Agent
[0034] a) General
[0035] b) Digitalis Drugs
[0036] c) Steroidal Compounds
[0037] d) Nonsteroidal Anti-inflammatories
[0038] e) Protein and Peptide Drugs
[0039] f) Nucleotide-based Drugs
[0040] g) Heterocyclic Drugs
[0041] VI. Formulation and Delivery of Pharmaceutical Agents
[0042] a) General
[0043] b) Transdermal Delivery
[0044] i. General
[0045] ii. Passive Transdermal Drug Delivery
[0046] iii. Topical Application
[0047] c) Transmucosal Delivery
[0048] i. Buccal Administration
[0049] ii. Nasal Administration
[0050] VII. Treatment of Gaucher Disease with Fusogenic Saposin
Proteins and Polypeptides
[0051] VIII. Experimental Examples
[0052] I. Definitions
[0053] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. Where "amino acid sequence" is recited
herein to refer to an amino acid sequence of a naturally occurring
protein molecule, "amino acid sequence" and like terms are not
meant to limit the amino acid sequence to the complete native amino
acid sequence associated with the recited protein molecule.
[0054] The term "amphipathic lipid" means a molecule that has a
hydrophilic "head" group and hydrophobic "tail" group and has
membrane-forming capability.
[0055] As used herein, the terms "anionic phospholipid membrane"
and "anionic liposome" refer to a phospholipid membrane or liposome
that contains lipid components and has an overall negative charge
at physiological pH.
[0056] The term "contained (with)in" refers to a pharmaceutical
agent being enveloped within a phospholipd membrane, such that the
pharmaceutical agent is protected from the outside environment.
This term may be used interchangably with "encapsulated."
[0057] A "deletion," as the term is used herein, refers to a change
in the amino acid or nucleotide sequence that results in the
absence of one or more amino acid residues or nucleotides.
[0058] The term "derivative," as used herein, refers to the
chemical modification of a polypeptide sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide
sequence can include, for example, replacement of hydrogen by an
alkyl, acyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains at least one biological function of the
natural molecule. A derivative polypeptide is one modified, for
instance by glycosylation, or any other process which retains at
least one biological function of the polypeptide from which it was
derived.
[0059] The term "fusogenic protein or polypeptide" as used herein
refers to a protein or peptide that when added to two separate
bilayer membranes can bring about their fusion into a single
membrane.
[0060] The words "insertion" or "addition," as used herein, refer
to changes in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, to the sequence found in the naturally occurring
molecule.
[0061] The terms "lipid" and "phospholipid" are used
interchangeabley and to refer to structures containing lipids,
phospholipids, or derivatives thereof comprising a variety of
different structural arrangements which lipids are known to adopt
in aqueous suspension. These structures include, but are not
limited to, lipid bilayer vesicles, micelles, liposomes, emulsions,
vesicles, lipid ribbons or sheets. In the preferred embodiment, the
lipid is an anioinic liposome. The lipids may be used alone or in
any combination which one skilled in the art would appreciate to
provide the characteristics desired for a particular application.
In addition, the technical aspects of lipid constructs and liposome
formation are well known in the art and any of the methods commonly
practiced in the field may be used for the present invention.
[0062] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to a nucleotide, oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer
to DNA or RNA of genomic or synthetic origin which may be
single-stranded or double-stranded and may represent the sense or
the antisense strand, or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which, when translated, would produce polypeptides retaining some
functional characteristic, e.g., having membrane fusogenic
properties, of the full-length polypeptide.
[0063] As used herein, the term "nucleotide-based pharmaceutical
agent" or "nucleotide-based drug" refer to a pharmaceutical agent
or drug comprising a nucleotide, an oligonucleotide or a nucleic
acid.
[0064] The phrases "percent identity" or "percent homology" refers
to the percentage of sequence similarity found in a comparison of
two or more amino acid or nucleic acid sequences.
[0065] As used herein, "pharmaceutical agent or drug" refers to any
chemical or biological material, compound, or composition capable
of inducing a desired therapeutic effect when properly administered
to a patient. Some drugs are sold in an inactive form that is
converted in vivo into a metabolite with pharmaceutical activity.
For purposes of the present invention, the terms "pharmaceutical
agent" and "drug" encompass both the inactive drug and the active
metabolite.
[0066] The phrase "pharmaceutically or therapeutically effective
dose or amount" refers to a dosage level sufficient to induce a
desired biological result. That result may be the delivery of a
pharmaceutical agent, alleviation of the signs, symptoms or causes
of a disease or any other desired alteration of a biological system
and the precise amount of the active depends on the physical
condition of the patient, progression of the illness being treated
etc.
[0067] As used herein, the term "saposin" refers to the family of
prosaposin-derived proteins and polypeptides, including but not
limited to naturally occurring saposins A, B, C and D as well as
synthetic saposin-derived proteins and peptides and peptide analogs
showing fusogenic activity. The preferred saposin is saposin C and
polypeptides derived therefrom.
[0068] As used herein, the terms "transport" and "delivery" refers
to the passage of a substance across or through the skin (i.e.,
transdermal), including the epidermis and dermis, or across a
mucosal membrane, where the substance can contact, and be absorbed
by the cells of that particular membrane.
[0069] II. Fusogenic Proteins or Polypeptides
[0070] Suitable lysosomal fusogenic proteins and polypeptides for
use in this invention include, but are not limited to, proteins of
the saposin family, preferably saposin C. Also included are
homologues of saposin C, wherein the homologue possesses at least
80% sequence homology, due to degeneracy of the genetic code which
encodes for saposin C, and polypeptides and peptide analogues
possessing similar biological activity as saposin C.
[0071] Examples of preferred peptide or peptide analogues
include:
1 (SEQ. ID. No. 1) Ser-Asp-Val-Tyr-Cys-Glu-Val-Cys-Glu-Phe--
Leu-Val- Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-
Glu-Lys-Glu-Ile-Leu-Asp-Ala-Phe-Asp-Lys-Met-Cys- Ser-Lys-Leu-Pro;
(SEQ. ID. No. 2) Val-Tyr-Cys-Glu-Val-Cys-Glu-Phe-- Leu-Val-Lys-Glu-
Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-
Glu-Ile-Leu-Asp-Ala-Phe-Asp-Lys-Met-Cys-Ser-Lys- Leu-Pro,
[0072] and derivatives, analogues, homologues, fragments and
mixtures thereof.
[0073] Also included are polypeptides of the formula:
h-u-Cys-Glu-h-Cys-Glu-h-h-h-Lys-Glu-h-u-Lys-h-h-Asp-Asn-Asn-Lys-u-Glu-Lys--
Glu-h-h-Asp-h-h-Asp-Lys-h-Cys-u-Lys-h-h,
[0074] where h=hydrophobic amino acids, including, Val, Leu, Ile,
Met, Pro, Phe, and Ala; and u=uncharged polar amino acids,
including, Thr, Ser, Tyr, Gly, Gln, and Asn.
[0075] As used herein, term "peptide analog" refers to a peptide
which differs in amino acid sequence from the native peptide only
by conservative amino acid substitutions, for example, substitution
of Leu for Val, or Arg for Lys, etc., or by one or more
non-conservative amino acid substitutions, deletions, or insertions
located at positions which do not destroy the biological activity
of the peptide (in this case, the fusogenic property of the
peptide). A peptide analog, as used herein, may also include, as
part or all of its sequence, one or more amino acid analogues,
molecules which mimic the structure of amino acids, and/or natural
amino acids found in molecules other than peptide or peptide
analogues.
[0076] By "analogs" is meant substitutions or alterations in the
amino acid sequences of the peptides of the invention, which
substitutions or alterations do not adversely effect the fusogenic
properties of the peptides. Thus, an analog might comprise a
peptide having a substantially identical amino acid sequence to a
peptide provided herein as SEQ ID NO:1 and 2 and in which one or
more amino acid residues have been conservatively substituted with
chemically similar amino acids. Examples of conservative
substitutions include the substitution of a nonpolar (hydrophobic)
residue such as isoleucine, valine, leucine or methionine for
another. Likewise, the present invention contemplates the
substitution of one polar (hydrophilic) residue such as between
arginine and lysine, between glutamine and asparagine, and between
glycine and serine. Additionally, the substitution of a basic
residue such as lysine, arginine or histidine for another or the
substitution of one acidic residue such as aspartic acid or
glutamic acid for another is also contemplated.
[0077] III. Phospholipid Membrane
[0078] This invention utilizes an anioinic phospholipid membrane to
effect the saposin-mediated membrane fusion for delivery of a
particular pharmaceutical agent across either a dermal or mucosal
membrane. These anionic phospholipid membranes are generally used
for preparing liposomes. Liposomes are microscopic vesicles
consisting of concentric lipid bilayers and, as used herein, refer
to small vesicles composed of amphipathic lipids arranged in
spherical bilayers. Structurally, liposomes range in size and shape
from long tubes to spheres, with dimensions from a few hundred
angstroms to fractions of a millimeter. Regardless of the overall
shape, the bilayers are generally organized as closed concentric
lamellae, with an aqueous layer separating each lamella from its
neighbor. Vesicle size normally falls in a range of between about
20 and about 30,000 nm in diameter. The liquid film between
lamellae is usually between about 3 and 10 nm. A variety of methods
for preparing various liposome forms have been described in the
periodical and patent literature. For specific reviews and
information on liposome formulations, reference is made to reviews
by Pagano and Weinstein (see Ann. Rev. Biophysic. Bioeng., 7,
435-68 (1978) and Ann. Rev. Biophysic. Bioeng., 9, 467-508
(1980)).
[0079] In general, the liposomes utilized in the present invention
can be divided into three categories based on their overall size
and the nature of the lamellar structure (see New York Academy
Sciences Meeting, "Liposomes and Their Use in Biology and
Medicine," of December 1977). The four classifications include
multi-lamaellar vesicles (MLV's), small uni-lamellar vesicles
(SUV's), large uni-lamellar vesicles (LUV's) and giant unilamellar
vesicles (GUV's). SUVs and LUVs, by definition, have only one
bilayer, whereas MLVs contain many concentric bilayers.
[0080] Liposomes exhibit a wide variety of characteristics,
depending upon their size, composition, and charge. For example,
liposomes having a small percentage of unsaturated lipids tend to
be slightly more permeable, while liposomes incorporating
cholesterol or other sterols tend to be more rigid and less
permeable. Liposomes may be positive, negative, or neutral in
charge, depending on the hydrophilic group. For example,
choline-based lipids impart an overall neutral charge, phosphate
and sulfate based lipids contribute a negative charge,
glycerol-based lipids are generally negatively-charged, and sterols
are generally neutral in solution but have charged groups. The
lipids used in the present invention are anionic lipids.
[0081] In order for many drugs to have therapeutic potential, it is
necessary for them to be delivered to the proper location in the
body. Liposomes can form the basis for sustained drug release and
delivery to specific cell types, or parts of the body. The
therapeutic use of liposomes also includes the delivery of drugs
which are normally toxic in the free form. In the liposomal form,
the toxic drug is occluded, and may be directed away from the
tissues sensitive to that drug and targeted to selected areas.
Liposomes can also be used therapeutically to release drugs over a
prolonged period of time, reducing the frequency of administration.
In addition, liposomes can also provide a method for forming
aqueous dispersions of hydrophobic or amphiphilic drugs, which are
normally unsuitable for intravenous delivery.
[0082] The liposomes of the present invention contain
pharmaceutical agents, which have been trapped in the aqueous
interior or between bilayers, or by trapping hydrophobic molecules
within the bilayer. Several techniques can be employed to use
liposomes to target encapsulated drugs to selected host tissues,
and away from sensitive tissues. These techniques include
manipulating the size of the liposomes, their net surface charge,
and their route of administration.
[0083] The liposomes of the present invention may also be delivered
by a passive delivery route. Passive delivery of liposomes involves
the use of various routes of administration, e.g., intravenous,
subcutaneous, intramuscular and topical. Each route produces
differences in localization of the liposomes.
[0084] IV. The Nature and pH Dependence of the
Protein/Polypeptide-Membran- e Association
[0085] Without intending to be limited by theory, one possible
mechanism as to how saposin-mediated membrane fusion occurs is
through protein conformational changes. Of the pro-saposin derived
proteins, saposin A and saposin C show the highest degree of amino
acid identity/similarity. Computationally, both proteins are
predicted to fold into amphipathic helical bundle motifs. In
general, the saposin-fold is a common super secondary structure
with five amphipathic .alpha.-helices folded into a single globular
domain and is common to both proteins. The presentation of the fold
is along a centrally located helix at amino-terminal, against which
helices 2 and 3 are packed from one side and helices 4 and 5 from
the other side. This fold may provide an interface for membrane
interaction.
[0086] A mechanism for saposin-mediated membrane fusion with
anionic phospholipid membranes is thought to be a two-step process.
In the first step, electrostatic interactions between the
positively charged amino acids (Glu and Asp) of the saposins and
the negatively charged phospholipid membrane results in an
association between these two species (see FIG. 1). In the second
step, intramolecular hydrophobic interactions between the helices
of two adjacent saposin proteins brings the two membranes in close
enough proximity for fusion of the membranes to take place (see
FIG. 2).
[0087] Thus, in accordance with the present invention, the
association of saposins, and in particular saposin C, with a lipid
generally requires a pH range from about 5.5 or less since the
initial association of saposin C with the membrane arises through
an electrostatic interaction of the positively charged basic amino
acid residues of saposin C with the anionic membrane. Thus, it is
highly desirable to have the basic amino acids exist in their
protonated forms in order to achieve a high number of electrostatic
interactions.
[0088] Alternatively, related fusion proteins and peptides derived
from the saposin family of proteins may not have this lower pH
range limitation and thus the pH range of other membrane fusion
proteins and peptides can range from physiological pH (pH of about
7) to lower pH ranges.
[0089] IV. Pharmaceutical Agents
[0090] In accordance with the present invention, pharmaceutical
agents are contained within the anioinic phospholipid membrane or
liposome for saposin-mediated transport within and/or beneath the
dermal and mucosal membranes. The active agents may be large
biomolecules including, but not limited to lipids, in particular
ceramide, steroids, fatty acids, triacylglycerols, genes and
proteins. The active agent may also be comprised of small organic
molecules. As used herein, "pharmaceutical agent" means any
material or mixture of materials which provides a cosmetic or
therapeutic benefit when delivered within or through the dermal or
mucosal membranes.
[0091] a) General
[0092] Exemplary pharmaceutical agents or drugs that may be
delivered by the system of the present invention may include, but
are not limited to, analgesics, anesthetics, antifungals,
antibiotics, antiinflammatories, anthelmintics, antidotes,
antiemetics, antihistamines, antihypertensives, antimalarials,
antimicrobials, antipsychotics, antipyretics, antiseptics,
antiarthritics, antituberculotics, antitussives, antivirals,
cardioactive drugs, cathartics, chemotherapeutic agents, corticoids
(steroids), antidepressants, depressants, diagnostic aids,
diuretics, enzymes, expectorants, hormones, hypnotics, minerals,
nutritional supplements, parasympathomimetics, potassium
supplements, sedatives, sulfonamides, stimulants, sympathomimetics,
tranquilizers, urinary antiinfectives, vasoconstrictors,
vasodilators, vitamins, xanthine derivatives, and the like.
[0093] b) Digitalis Drugs
[0094] Preferred examples of pharmaceutical agents include the
digitalis drugs, such as digoxin, digitoxin, digoxigenin, and
digitoxigenin. These drugs are all primarily used as cardiac
agents.
[0095] c) Steroidal Compounds
[0096] Steroidal compounds form another preferred class of
pharmaceutical agent. An example of a steroidal pharmaceutical
agent is testosterone (17 beta-hydroxyandrost-4-en-3-one), the
principal male steroid. Its main therapeutic use is in the
treatment of deficient endocrine function of the testes. Estradiol
(estra-1,3,5(10)-triene-3,17 beta-diol) is also a preferred
steroidal pharmaceutical agent. Estradiol and its ester derivatives
are indicated for the treatment of symptoms of menopause and other
conditions that cause a deficiency of endogenous estrogen
production. Progesterone is also a preferred steroidal
pharmaceutical agent. Progesterone is used primarily to suppress or
synchronize estrus as well as to control habitual abortion and
diagnose and treat menstrual disorders. Additional preferred
steroidal pharmaceutical agents include 3-hydroxy-5
alpha-pregnan-20-one, 3-beta-hydroxy-pregn-5-ene-20-one and related
compounds.
[0097] d) Nonsteroidal Anti-Inflammatory Drugs (NSAID's)
[0098] Examples of NSAID's includes piroxicam
(4-hydroxy-2-methyl-N-2-pyri-
dinyl-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide), diclofenac,
ibuprofen, ketoprofen, meperidine, propoxyphene, nalbuphine,
pentazocine, buprenorphine, asprin, indomethacin, diflunisal,
acetominophen, naproxen, fenoprofen, piroxicam, sulindac, tolmetin,
meclofenamate, zomepirac, penicillamine, phenylbutazone,
oxyphenbutazone, chloroquine, hydroxychloroquine, azathiaprine,
cyclophosphamide, levamisole, prednisone, prednisolone,
betamethasone, triamcinolone, and methylprednisolone and
indomethacin (1-(4-chlorobenzoyl)-5-methoxy-2-meth-
yl-1H-indole-3-acetic acid).
[0099] e) Amino Acid-Based Drugs
[0100] Protein and peptide-based drugs, as well as other amino
acid-based drugs, may also be used as pharmaceutical agents
according to the present invention. The problems associated with
conventional delivery strategies for protein and peptide drugs are
widely appreciated. Oral administration of these drugs is generally
impractical due to degradation and non-absorption in the
gastrointestinal tract. Thus, the parenteral route remains the
principal delivery route.
[0101] Amino acid-based drugs, such as the cephalosporins, will
typically have molecular weight less than about 5000, and
preferably, less than about 2500, and more preferably, less than
about 1000. Protein and peptide drugs typically have a molecule
weight of at least about 100 daltons, and more typically a
molecular weight in the range of about 200 to 40,000 daltons.
Examples of peptides and proteins in this size range include, but
are not limited to Luteinizing hormone-releasing hormone,
Somatostatin, Bradykinin, Goserelin, Somatotropin, Buserelin,
Platelet-derived growth factor, Triptorelin, Gonadorelin,
Asparaginase, Nafarelin, Bleomycin sulfate, Leuprolide,
Chymopapain, Growth hormone-releasing factor, parathyroid hormone
(PTH), Cholecystokinin, Chorionic gonadotropin, Insulin,
Corticotropin (ACTH), Calcitonin Erythropoietin, Glucagon,
Hyaluronidase, Interferons, e.g., alpha, Interleukins, e.g., IL-1
Thyrotropin-releasing hormone, Menotropins, Pituitary hormones
(e.g. Urofollitropin (Follicle HGH, HMG, HCG, FSH, etc.),
Melanocyte-stimulating hormone, Gonadotropin releasing hormone,
Oxytocin, Vasopressin, Streptokinase, Tissue plasminogen activator,
Angiotensin II antagonists, Bradykinin potentiator B, Bradykinin
antagonists, Bradykinin potentiator C, Enkephalins, Insulin-like
growth factors, Prostaglandin antagonists, Tumor necrosis factor,
Epidermal growth factor (EGF), Amylin, Lipotropin, and Thyroid
stimulating hormone.
[0102] An example of a preferred peptide pharmaceutical agent is
parathyroid hormone (PTH) (see Harper et al., Eds., Review of
Physiological Chemistry, 16th Ed., Lange Medical Publications, Los
Altos, Calif. (1977) p. 468). Also, a fragment consisting of about
34 amino acid residues from the N-terminal has been isolated and
found to display the full biological activity of PTH (see Potts et
al., in Parathyroid Hormone and Thyrocalcitonin (Calcitonin), R. V.
Talmage, et al., Eds. Excerpta Medica, New York (1968)). The
sequence of the polypeptide varies slightly among mammalian
species. According to the present invention, PTH is meant to
include human parathyroid hormone, as well as the other variants
and the 34 amino acid fragment. PTH serves as a regulatory factor
in the homeostatic control of calcium and phosphate metabolism
(see, e.g., Parsons, et al. "Physiology and Chemistry of
Parathyroid Hormone" in Clinics in Endocrinology and Metabolism, I.
MacIntyre, Ed. Saunders, Philadelphia (1972) pp. 33-78). The main
therapeutic use for PTH is in the treatment of osteoporosis. PTH
has also been used as a blood calcium regulator.
[0103] Calcitonin is also a preferred peptide pharmaceutical agent.
Calcitonin is a polypeptide containing 32 amino acid residues (see
Harper et al., Eds., Review of Physiological Chemistry, 16th Ed.,
Lange Medical Publications, Los Altos, Calif. (1977), p. 469).
According to the present invention, calcitonin is meant to include
all calcitonin, including that of humans, mammals, and fish, as
well as other variants. Calcitonin is a calcium regulating hormone
and has been used in the treatment of osteoporosis, hypercalcemia,
and Paget's disease.
[0104] An additional preferred protein drug is the cytokine IL-10.
Il-10 is produced by the TH2 helper subset, B cell subsets and
LPs-activated monocytes. IL-10 inhibits several immune functions
that are relevant to the skin immune response and thus, the
development of the irritation and inflammation that is sometimes
associated with the transdermal delivery of drugs. More
specifically, the release of IFN-alpha, which initiates the cascade
of cellular activation leading to the skin's immune response, is
inhibited by IL-10. IL-10 also suppresses the synthesis of numerous
pro-inflammatory cytokines by macrophages, as well as the
proliferation of antigen-specific T cell proliferation by down
regulating class II MHC expression.
[0105] e) Nucleic Acid-Based Drugs
[0106] Generally, nucleic acid-based drugs have had limited success
as therapeutic agents, in part, because of problems associated with
their stability and delivery. Nucleotide-based pharmaceutical
agents frequently contain a phosphodiester bond which is sensitive
to degradation by nucleases. Such degradation would be a
significant impediment to the use of an oligonucleotide or nucleic
acid as a pharmaceutical agent that depends upon the integrity of
the sequence for its recognition specificity. Thus, naturally
occurring oligonucleotides and nucleic acids often must typically
be chemically modified to render them resistant to nucleases which
would degrade them in vivo, or even in vitro unless care is taken
to choose appropriate conditions. However, this is not necessary
using the drug delivery system of the present invention.
[0107] The nucleotide-based drugs of the present invention include
aptamers, antisense compounds, and triple helix drugs. The
nucleotide-based drugs typically will have a molecular weight
greater than about 350 and may range up to about 100 bases.
Examples of nucleotide-based drugs include di- and trinucleotides,
such as GS 375, a dinucleotide analog with potential therapeutic
activity against the influenza virus (Gilead Sciences, Inc., Foster
City, Calif.).
[0108] Aptamers (or nucleic acid antibody) are single- or
double-stranded DNA or single-stranded RNA molecules that bind
specific molecular targets. Generally, aptamers function by
inhibiting the actions of the molecular target, e.g., proteins, by
binding to the pool of the target circulating in the blood.
Examples of aptamers include Gilead's antithrombin inhibitor GS 522
and its derivatives (Gilead Science, Foster City, Calif.; see also
Macaya et al. (1993) Proc. Natl. Acad. Sci. USA 90:3745-9; Bock et
al. (1992) Nature (London) 355:564-566; and Wang et al. (1993)
Biochem. 32:1899-904).
[0109] For diseases that result from the inappropriate expression
of genes, specific prevention or reduction of the expression of
such genes represents an ideal therapy. In principle, production of
a particular gene product may be inhibited, reduced or shut off by
hybridization of a single-stranded deoxynucleotide or
ribodeoxynucleotide complementary to an accessible sequence in the
mRNA, or a sequence within the transcript which is essential for
pre-mRNA processing, or to a sequence within the gene itself. This
paradigm for genetic control is often referred to as antisense or
antigene inhibition.
[0110] Antisense compounds are oligonucleotides that are designed
to bind and disable or prevent the production of the mRNA
responsible for generating a particular protein. Antisense
compounds can provide a therapeutic function by inhibiting in vivo
the formation of one or more proteins that cause or are involved
with disease. Antisense compounds complementary to certain gene
messenger RNA or viral sequences have been reported to inhibit the
spread of disease related to viral and retroviral infectious agents
(see, for example, Matsukura et al. (1987) Proc. Natl. Acad. Sci.
USA 84:7706, and references cited therein). Others have reported
that oligonucleotides can bind to duplex DNA via triple helix
formation and inhibit transcription and/or DNA synthesis.
[0111] Antisense compounds include antisense RNA or DNA, single or
double stranded, oligonucleotides, or their analogs, which can
hybridize specifically to individual mRNA species and prevent
transcription and/or RNA processing of the mRNA species and/or
translation of the encoded polypeptide and thereby effect a
reduction in the amount of the respective encoded polypeptide (see
Ching et al. Proc. Natl. Acad. Sci. U.S.A. 86:10006-10010 (1989);
Broder et al. Ann. Int. Med. 113:604-618 (1990); Loreau et al. FEBS
Letters 274:53-56 (1990)).
[0112] Triple helix compounds (also referred to as triple strand
drugs) are oligonucleotides that bind to sequences of
double-stranded DNA and are intended to inhibit selectively the
transcription of disease-causing genes, such as viral genes, e.g.,
HIV and herpes simplex virus, and oncogenes, i.e., they stop
protein production at the cell nucleus. These drugs bind directly
to the double stranded DNA in the cell's genome to form a triple
helix and thus, prevents the cell from making a target protein
(see, for example U.S. Pat. No. 5,176,996, Hogan et al, Jan. 5,
1993).
[0113] The site specificity of oligonucleotides (e.g., antisense
compounds and triple helix drugs) is not significantly affected by
modification of the phosphodiester linkage or by chemical
modification of the oligonucleotide terminus. Consequently, these
oligonucleotides can be chemically modified; enhancing the overall
binding stability, increasing the stability with respect to
chemical degradation, increasing the rate at which the
oligonucleotides are transported into cells, and conferring
chemical reactivity to the molecules. The general approach to
constructing various oligonucleotides useful in antisense therapy
has been reviewed by vander Krol et al. (1988) Biotechniques
6:958-976 and Stein et al. (1988) Cancer Res. 48:2659-2668.
[0114] Accordingly, aptamers, antisense compounds and triple helix
drugs also can include nucleotide substitutions, additions,
deletions, or transpositions, so long as specific hybridization to
or association with the relevant target sequence is retained as a
functional property of the oligonucleotide. For example, some
embodiments will employ phosphorothioate analogs which are more
resistant to degradation by nucleases than their naturally
occurring phosphate diester counterparts and are thus expected to
have a higher persistence in vivo and greater potency (see,
Campbell et al. (1990) J. Biochem. Biophys. Methods 20:259-267).
Phosphoramidate derivatives of oligonucleotides also are known to
bind to complementary polynucleotides and have the additional
capability of accommodating covalently attached ligand species and
will be amenable to the methods of the present invention (see
Froehler et al. (1988) Nucleic Acids Res. 16(11): 4831).
[0115] In addition, nucleotide analogs, for example where the sugar
or base is chemically modified, can be employed in the present
invention. Analogous forms of purines and pyrimidines are those
generally known in the art, many of which are used as
chemotherapeutic agents.
[0116] Terminal modification also provides a useful procedure to
modify cell type specificity, pharmacokinetics, nuclear
permeability, and absolute cell uptake rate for oligonucleotide
pharmaceutical agents. For example, substitutions at the 5' and 3'
ends include reactive groups which allow covalent crosslinking of
the nucleotide-based pharmaceutical agent to other species and
bulky groups which improve cellular uptake (see
Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression,
(1989) Cohen, Ed., CRC Press; Prospects for Antisense Nucleic Acid
Therapeutics for Cancer and ADS, (1991), Wickstrom, Ed.,
Wiley-Liss; Gene Regulation: Biology of Antisense RNA and DNA,
(1992) Erickson and Izant, Eds., Raven Press; and Antisense RNA and
DNA, (1992), Murray, Ed., Wiley-Liss. For general methods relating
to antisense compounds, see Antisense RNA and DNA, (1988), D. A.
Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.).
[0117] f) Heterocyclic Drugs
[0118] Heterocyclic drugs, and particularly those containing at
least one nitrogen heterocyclic ring can be employed as
pharmaceutical agents in the methods described herein. For example,
yohimbine is an indole alkaloid that blocks alpha-2-adrenergic
receptors. Its peripheral effects are to increase cholinergic
activity at the same time that it decreases adrenergic activity.
This combination has led to the use of yohimbine in the treatment
and diagnostic classification of certain types of male erectile
impotence.
[0119] Other examples of heterocyclic drugs includes, but is not
limited to morphine, methotrexate (formerly Amethopterin,
N-[4-[[(2,4-diamino-6-p-
teridinyl)-methyl]methylamino]benzoyl]-L-glutamic acid), Lorazepam
(7-chloro-5-(o-chloro-phenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin--
2-one), 6-Mercaptopurine, (1,7-dihydro-6H-purine-6-thione
monohydrate), 5-fluorouracil, nicotine, nicotinic acid and
niacin.
[0120] VI. Formulations and Delivery of Pharmaceutical Agents
[0121] a. General
[0122] The compositions of the present invention generally comprise
a fusogenic saposin protein or polypeptide, which is associated
with an anioinc liposome containing a pharmaceutical agent in a
safe and effective amount for the desired effect, all contained in
a pharmaceutically acceptable carrier with an appropriate pH. A
safe and effective amount of the active agent is defined as an
amount which would cause the desired cosmetic or therapeutic effect
in a patient. An experienced practioner, skilled in this invention
would have knowledge of the appropriate dosing ratios.
[0123] The appropriate dosage administered in any given case will,
of course, vary depending upon known factors, such as the
pharmacodynamic characteristics of the particular pharmaceutical
agent, and its mode and route of administration; the age, general
health, metabolism, weight of the recipient and other factors which
influence response to the compound; the kind of concurrent
treatment, the frequency of treatment, and the effect desired.
[0124] A preferred embodiment comprises a desired pharmaceutical
agent, in a safe and effective amount, which is incorporated into
anioinic liposomes, in a buffered aqueous solution of a pH of about
5.5 or less. The preferred fusogenic protein or polypeptide is
saposin C, in concentrations from about 20 nM to about 100 nM
(nanomolar), preferably about 40 to about 50 nM, which is then
introduced to the liposome-pharmacuetical agent mixture. The
concentration of the liposomes is in excess to that of the
fusogenic protein or polypeptide and is preferably at least in a
10-fold excess, by weight, to that of saposin C (i.e. at least a
1:10 by weight ratio of saposin C:liposome). An electrostatic
association occurs between the fusogenic protein or polypeptide and
the liposme. Such a composition could then be applied topically to
the skin. Other examples of preparing such liposome-fusion protein
complexes, in which an active agent is contained within the
liposome, are given in U.S. Pat. No. 6,099,857, Gross, Aug. 8, 2000
and U.S. Pat. No. 5,766,626, Gross, Jun. 16, 1998, which are herein
incorporated by reference.
[0125] b) Transdermal Delivery
[0126] The pharmaceutical agent-chemical modifier complexes
described herein can be administered transdermally. Transdermal
administration typically involves the delivery of a pharmaceutical
agent for percutaneous passage of the drug into thee systemic
circulation of the patient. The skin sites include anatomic regions
for transdermally administering the drug and include the forearm,
abdomen, chest, back, buttock, mastoidal area, and the like.
[0127] Transdermal delivery is accomplished by exposing a source of
the complex to a patient's skin for an extended period of time.
Transdermal patches have the added advantage of providing
controlled delivery of a pharmaceutical agent to the body (see
Transdermal Drug Delivery: Developmental Issues and Research
Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989);
Controlled Drug Delivery: Fundamentals and Applications, Robinson
and Lee (eds.), Marcel Dekker Inc., (1987); and Transdermal
Delivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds.), CRC
Press, (1987)). Such dosage forms can be made by dissolving,
dispersing, or otherwise incorporating the pharmaceutical agent,
saposin C and anioinic liposomes in a proper medium, such as an
elastomeric matrix material. Absorption enhancers can also be used
to increase the flux of the compound across the skin. The rate of
such flux can be controlled by either providing a rate-controlling
membrane or dispersing the compound in a polymer matrix or gel.
[0128] i. Passive Transdermal Drug Delivery
[0129] A variety of types of transdermal patches will find use in
the methods described herein. For example, a simple adhesive patch
can be prepared from a backing material and an acrylate adhesive.
The pharmaceutical agent-chemical modifier complex and any enhancer
are formulated into the adhesive casting solution and allowed to
mix thoroughly. The solution is cast directly onto the backing
material and the casting solvent is evaporated in an oven, leaving
an adhesive film. The release liner can be attached to complete the
system.
[0130] Alternatively, a polyurethane matrix patch can be employed
to deliver the pharmaceutical agent-chemical modifier complex. The
layers of this patch comprise a backing, a polyurethane
drug/enhancer matrix, a membrane, an adhesive, and a release liner.
The polyurethane matrix is prepared using a room temperature curing
polyurethane prepolymer. Addition of water, alcohol, and complex to
the prepolymer results in the formation of a tacky firm elastomer
that can be directly cast only the backing material.
[0131] A further embodiment of this invention will utilize a
hydrogel matrix patch. Typically, the hydrogel matrix will comprise
alcohol, water, drug, and several hydrophilic polymers. This
hydrogel matrix can be incorporated into a transdermal patch
between the backing and the adhesive layer.
[0132] For passive delivery systems, the rate of release is
typically controlled by a membrane placed between the reservoir and
the skin, by diffusion from a monolithic device, or by the skin
itself serving as a rate-controlling barrier in the delivery system
(see U.S. Pat. Nos. 4,816,258; 4,927,408; 4,904,475; 4,588,580,
4,788,062). The rate of drug delivery will be dependent, in part,
upon the nature of the membrane. For example, the rate of drug
delivery across membranes within the body is generally higher than
across dermal barriers. The rate at which the complex is delivered
from the device to the membrane is most advantageously controlled
by the use of rate-limiting membranes which are placed between the
reservoir and the skin. Assuming that the skin is sufficiently
permeable to the complex (i.e., absorption through the skin is
greater than the rate of passage through the membrane), the
membrane will serve to control the dosage rate experienced by the
patient.
[0133] Suitable permeable membrane materials may be selected based
on the desired degree of permeability, the nature of the complex,
and the mechanical considerations related to constructing the
device. Exemplary permeable membrane materials include a wide
variety of natural and synthetic polymers, such as
polydimethylsiloxanes (silicone rubbers), ethylenevinylacetate
copolymer (EVA), polyurethanes, polyurethane-polyether copolymers,
polyethylenes, polyamides, polyvinylchlorides (PVC),
polypropylenes, polycarbonates, polytetrafluoroethylenes (PTFE),
cellulosic materials, e.g., cellulose triacetate and cellulose
nitrate/acetate, and hydrogels, e.g., 2-hydroxyethylmethacrylate
(HEMA).
[0134] Other items may be contained in the device, such as other
conventional components of therapeutic products, depending upon the
desired device characteristics. For example, the compositions
according to this invention may also include one or more
preservatives or bacteriostatic agents, e.g., methyl
hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium
chlorides, and the like. These pharmaceutical compositions also can
contain other active ingredients such as antimicrobial agents,
particularly antibiotics, anesthetics, analgesics, and antipruritic
agents.
[0135] ii) Topical Treatments
[0136] Another aspect of this invention provides for the topical
delivery of pharmaceutical compositions. This treatment regimen is
suitable either for the systemic administration of the
pharmaceutical agent or for localized therapy, i.e., directly to
pathological or diseased tissue.
[0137] Typically, the topical formulations will comprise a
preparation for delivering the pharmaceutical agent-chemical
modifier complex directly to the affected skin comprising the
complex, typically in concentrations in the range of from about
0.001% to 10%; preferably, from about 0.01 to about 10%; more
preferably, from about 0.1 to about 5%; and most preferably, from
about 1 to about 5%, together with a non-toxic, pharmaceutically
acceptable topical carrier (see Dermatological Formulations:
Percutaneous Absorption, Barry (ed.), Marcel Dekker Inc., (1983);
for standard dosages of conventional pharmaceutical agents, see,
e.g., Physicians Desk Reference (1992 Edition); and American
Medical Association (1992) Drug Evaluations Subscriptions).
[0138] Topical preparations can be prepared by combining the
pharmaceutical agent-chemical modifier complex with conventional
pharmaceutical diluents and carriers commonly used in topical dry,
liquid, cream and aerosol formulations. Ointment and creams may,
for example, be formulated with an aqueous or oily base with the
addition of suitable thickening and/or gelling agents. Such bases
may include water and/or an oil such as liquid paraffin or a
vegetable oil such as peanut oil or castor oil. Thickening agents
which may be used according to the nature of the base include soft
paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol,
polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, and
the like. Lotions may be formulated with an aqueous or oily base
and will, in general, also include one or more of the following:
stabilizing agents, emulsifying agents, dispersing agents,
suspending agents, thickening agents, coloring agents, perfumes,
and the like. Powders may be formed with the aid of any suitable
powder base, e.g., talc, lactose, starch, and the like. Drops may
be formulated with an aqueous base or non-aqueous base also
comprising one or more dispersing agents, suspending agents,
solubilizing agents, and the like.
[0139] Dosage forms for the topical administration of a complex of
this invention include powders, sprays, ointments, pastes, creams,
lotions, gels, solutions, patches and inhalants. The active
compound may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0140] The ointments, pastes, creams and gels also may contain
excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, talc and zinc oxide, or mixtures
thereof. Powders and sprays also can contain excipients such as
lactose, talc, aluminum hydroxide, calcium silicates and polyamide
powder, or mixtures of these substances. Sprays can additionally
contain customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0141] c. Transmucosal Delivery
[0142] Although much of the discussion herein has centered on
techniques for transdermal delivery, the methods of the present
invention are also applicable to the enhanced transport and
delivery of pharmaceutical agents through mucosal membranes, such
as gastrointestinal, sublingual, buccal, nasal, pulmonary, vaginal,
corneal, and ocular membranes (see Mackay et al. (1991) Adv. Drug
Del. Rev, 7:313-338). Specifically, there are many similarities
between skin and mucosal membranes. For example, the membrane of
the buccal cavity is non-keratinized. However, the buccal membrane
is similar to the skin because both are stratified with the former
consisting of polygonal cells at the basal membrane leading to
squamous cells at the surface.
[0143] Transmucosal (i.e., sublingual, buccal and vaginal) drug
delivery provides for an efficient entry of active substances to
systemic circulation and reduce immediate metabolism by the liver
and intestinal wall flora. Transmucosal drug dosage forms (e.g.,
tablet, suppository, ointment, gel, pessary, membrane, and powder)
are typically held in contact with the mucosal membrane and
disintegrate and/or dissolve rapidly to allow immediate systemic
absorption.
[0144] i. Buccal Administration
[0145] For delivery to the buccal or sublingual membranes,
typically an oral formulation, such as a lozenge, tablet, or
capsule will be used. The method of manufacture of these
formulations are known in the art, including but not limited to,
the addition of the pharmaceutical agent-chemical modifier complex
to a pre-manufactured tablet; cold compression of an inert filler,
a binder, and either a pharmaceutical agent-chemical modifier
complex or a substance containing the complex (as described in U.S.
Pat. No. 4,806,356 incorporated by reference) and
encapsulation.
[0146] Another oral formulation is one that can be applied with an
adhesive, such as the cellulose derivative, hydroxypropyl
cellulose, to the oral mucosa, for example as described in U.S.
Pat. No. 4,940,587, incorporated by reference. This buccal adhesive
formulation, when applied to the buccal mucosa, allows for
controlled release of the pharmaceutical agent-chemical modifier
complex into the mouth and through the buccal mucosa.
[0147] ii. Nasal/Pulmonary Administration
[0148] For delivery to the nasal and/or pulmonary membranes,
typically an aerosol formulation will be employed. The term
"aerosol" includes any gas-borne suspended phase of the
pharmaceutical agent-chemical modifier complex which is capable of
being inhaled into the bronchioles or nasal passages. Specifically,
aerosol includes a gas-borne suspension of droplets of the
compounds of the instant invention, as may be produced in a metered
dose inhaler or nebulizer, or in a mist sprayer. Aerosol also
includes a dry powder composition of the pharmaceutical
agent-chemical modifier complex suspended in air or other carrier
gas, which may be delivered by inhalation from an inhaler
device.
[0149] VII. Treatment of Gaucher Disease with Fusogenic Saposin
Proteins and Polypeptides
[0150] Additionally, saposin C is essential for hydrolysis of
glucosylceramides to ceramide in vivo. A deficiency in epidermal
glucocerebrosidase results in an altered glucosylceramide to
ceramide ratio and this altered ratio is associated with skin
barrier abnormalities characterized by Gaucher Disease. It is
thought that saposin C is critical to the formation of the
epidermal permeability barrier by maintaining physiologic
concentrations of glucosylceramide and ceramide in the stratum
corneum. According to this model, the role of saposin C in
stimulating glucocerebrosidase is mediated by its destabilizing
effect on the membranes. Thus, in patients with epidermal
glucocerebrosidase deficiency, a topical application of a saposin
C-liposome complex, wherein the lipsome contains acid beta
glucosidase, the mixture contained in a pharmaceutically acceptable
carrier may be used to fuse cell membranes in order to facilitate
the hydrolysis of glucosylceramide to ceramide to aid in regulation
of skin barrier formation and function. These compositions can, for
example, be formulated as creams, lotions, solutions or gels. The
carrier may include, for example, pharmaceutically acceptable
emollients, emulsifiers, thickening agents, solvents,
preservatives, coloring agents and fragrances.
EXPERIMENTAL EXAMPLES
[0151] To elucidate the temporal and spatial interaction of
saposins with liposomal membranes, the Inventor has focused efforts
on the development of intrinsic (Trp) and/or extrinsic (NBD,
pyrene, etc.) fluorescence determination methods. These approaches
include maximal emission spectrum shift, fluorescence resonance
energy transfer, fluorescence stopped-flow analysis, flow-analysis
of fluorescent bead-saposin-liposome complexes, and fluorescence
microscopy. In addition, circular dichroism (CD) is used to
evaluate relative secondary structure changes from lipid-free to
lipid-bound saposins. Analyses of the initial results evolved into
the proposed hypothesis.
[0152] Summarized below are the studies related to the expression,
purification, functional analysis, mutagenesis, as well as
fluorescence analyses of saposin-phospholipid interaction and
membrane fusion.
[0153] 1. Purification and Characterization of Natural and
Recombinant Saposins
[0154] a. Expression of Saposins from Prokaryotic Systems
[0155] Although natural saposins have been isolated and
characterized, it is important to establish a recombinant
expression system to provide an accessible source of large amounts
of normal, mutated and Trp-labeled saposins for the proposed
investigations. A prokaryotic system was developed, based on the
following: 1) Saposins have at least one occupied N-glycosylation
site, but, for saposins B and C, occupancy of these sites are not
needed for function. 2) Expression of proteins in eukaryotic
systems is labor and resource intensive, and slow. In comparison,
prokaryotic systems are rapid and give high yields of wild-type and
mutant proteins. And 3) The proteins can be labeled with Trp
residues as intrinsic fluorescence probes, since wild-type A is the
only saposin that contains a natural Trp(37W).
[0156] b. Production of Active Saposins in E. Coli
[0157] Functional saposins were overexpressed in BL21(DE3) cells
using a pET 21a series vector. Following IPTG induction at
37.degree. C. or 30.degree. C., large amounts of saposins
containing His.cndot.Tag were found in the soluble fraction of the
disrupted cells. These were conveniently purified to
electrophoretic homogeneity on nickel-loaded columns.
Alternatively, saposins without His.cndot.Tag were generated by
introducing a stop codon after protein coding region, and then
purified using immuno-affinity columns with T7-taq monoclonal
antibody. The purified recombinant saposin C shows excellent
activation of acid .beta.-glucosidase and other biologic
properties. Circular dichroism spectra, light scattering, and ES-MS
analyses were used to evaluate the physical properties of the
purified saposins, such as aggregation status and molecular weight.
Trp-saposins without His.cndot.Tag were also generated for
necessary control experiments. The functional integrity of
recombinant saposin C was determined using delipidated and
homogenous acid .beta.-glucosidase in a liposomal reconstitution
system and in neuritogenic assays. Recombinant saposin B function
was determined using a sulfatide binding assay. The in vitro
function of recombinant saposins B and C are similar to the natural
or deglycosylated saposins.
[0158] 2. Functional Conformations of Saposins Induced by
Phospholipids
[0159] To determine the specificity of saposin C-phospholipid
interaction, a liposomal system was developed using CD,
fluorescence emission shifts, and fluorescence quenching methods.
Mutated saposins C's, produced to contain individual Trp (W), were
termed saposin C (0W), (S37W), and (81W). These Trp-labeled saposin
C's were as follows: saposin C (0W) has a Trp preceding the first
NH.sub.2-terminal amino acid of mature saposin C, saposin C (S37W)
has a Trp at residue 37 (i.e., in the middle), and saposin C(81W)
has a Trp after the last COOH-terminal amino acid. These
substitutions had no effect on the activation properties or CD
spectra of saposin C.
[0160] a. CD Spectra
[0161] Using CD spectroscopy, relative secondary structural changes
of recombinant saposins were induced by membrane binding. The
relative secondary structural changes of saposins obtained from the
acidic, unsaturated phosphatidylserine (PS)/saposin C complexes and
the neutral phosphatidylcholine (PC)/saposin B complexes are
similar and result in a decrease the .beta.-strand and an increase
the .alpha.-helix content (Table 1).
2TABLE 1 Circular Dichroism (195-250 nm) Analyses of Saposins with
Various Phospholipids Saposin % .alpha. % .beta. % T % R C Only
29.9 41.7 0.0 28.4 C + Phosphatidylserine (18: 0, 0) 30.1 40.4 1.4
28.1 C + Phosphatidylcholine (18: 1, 1) 30.6 41.0 0.0 28.4 C +
Phosphatidylserine (18: 1, 1) 49.8 3.9 14.0 32.4 B only 43.7 36.6
0.0 19.7 B + Phosphatidylserine (18: 1, 1) 43.8 38.8 0.0 17.9 B +
Phosphatidylcholine (18: 1, 1) 68.2 24.2 5.3 2.3 A only 44.0 31.9
0.0 24.1 A + Phosphatidylserine (18: 1, 1) 39.3 34.9 0.5 25.4
[0162] No changes were observed with saposin A and B in the PS
(18:1, 1) complexes. These results indicate that saposin A and B
have a different membrane interaction from that of saposins C. The
CD data were collected on a Jasco 710 instrument, and deconvoluted
using Yang's method (see Chang, C. T., Wu, C. S., and Yang, J. T.
Anal. Biochem (1978) 91, 13-31).
[0163] b. Fluorescence Emission Spectra
[0164] Emission spectra of proteins shift when the tryptophanyl
environments change polarity. The fluorescence spectra of saposins
A(0W), A(37W), A(81W), C(0W), and C(81W) obtained upon addition of
brain phosphatidylserine (BPS) liposomes, showed blue-shifts (Table
2).
3TABLE 2 Fluorescence Emission Maxima of Trp-saposins in the
Absence and Presence of Brain phosphatidylserine (BPS) Emission
Maxima (EM, nm) Saposins -BPS +BPS EM Shifts C(0W) 339 333 Blue
C(S37W) 351 351 No C(S37W, Q48N) 345 339 Blue C(S37W, Q48A/E49A)
338 329 Blue C(81W) 339 323 Blue A(0W) 345 333 Blue A(37W) 351 338
Blue A(37W, G64E) 344 358 Red A(37W, 339 350 Red K63L/G64E/M65V)
A(81W) 345 336 Blue Experiments conditions: pH 4.7, protein:lipid =
1:20 to 40. No differences were observed at 22 or 37.degree. C.
[0165] The blue-shifts suggest interaction of saposins with lipids
during complex formation. However, saposin C(S37W) showed no shift
in the presence of BPS. This implies that the NH.sub.2-(0W) and
COOH-(81W) termini of saposin C enter the membrane whereas the
middle of the sequence does not. With saposin A, the reverse is
true with the middle of the sequence (37W) in the membrane. This
means that saposin A-membrane associations are quite different from
those of the saposins C. These results are consistent with the CD
analysis. Maximal emission wavelength changes were not observed
with saposin As or Cs in the presence of neutral EPC nor with PS
containing saturated fatty acid chains.
[0166] 3. Temporal and Spatial Interaction of Saposins and
Phospholipid Membranes
[0167] To investigate temporal and spatial interactions of saposins
and liposomal membrane, fluorescence stopped-flow and quenching
approaches were used with Trp as the intrinsic fluorescence probe
of the saposins. These experiments allowed identification of
regional interactions between saposins and lipid bilayers, and also
the kinetics of their binding.
[0168] a. Temporal Interactions
[0169] Fluorescence intensity increased significantly upon saposin
C(0W) binding to synthetic phosphatidylserine [PS(18:1, 1)]
vesicles at acidic pH. This binding induced change is
lipid-concentration dependent and requires at least one unsaturated
fatty acid chain. To evaluate the kinetics of this interaction,
stopped-flow experiments were conducted the change in fluorescence
during saposin C/liposome complex formation was monitored. When
saposin C(0W) was mixed with PS(18:1, 1) or BPS vesicles,
fluorescence of Trp was increased, but the time course of this
change was undetectable due to limitation of the machine's
capability. Apparently, the interaction of saposin C and
unsaturated PS containing membranes occurs within at least 10
ms.
[0170] From CD and emission spectra data, saposin C binds
negatively charged, unsaturated phospholipids. This suggests there
is an electrostatic interaction between positively charged residues
in saposin C and the negatively charged membrane surface. This
initial interaction is followed by the protein embedding into
membrane through a hydrophobic interaction (see FIG. 3). No shift
in emission or change in intensity of Trp fluorescence was observed
with the saposin C (0W) and PS(18:0, 0) mixture.
[0171] b. Spatial Interactions
[0172] To determine the depth of saposin insertion into BPS
liposomes, spin-labeled phosphatidylcholines (SLPCs) were
incorporated into BPS liposomes with increasing mole percentages
(0-50%). SLPCs, hydrophobic fluorescence quenchers, contain doxyl
groups which are located at different carbons (n) in the acyl
chain: SLPC5 (n=5), SLPC10 (n=10), and SLPC16 (n=16). After
addition of Trp-saposins, the protein-liposome mixture
(protein:lipid=1:20) was incubated at room temperature for 30
minutes, and then, the fluorescence intensity changes were
recorded. For the Trp-saposins that show the blue-shifts in Table
2, significant quenching effects (30-60%) were observed with
BPS/SLPC5 liposomes. The quenching efficiency was dependent upon
the location in the acyl chain of the doxyl groups on SLPC. The
deeper the doxyl group was in the membrane, the lower quenching
efficiency. With BPS/SLPC10, the tryptophanyl fluorescence of
saposin C (0W) is quenched by 30%.
[0173] 4. Saposin C-Induced Membrane Fusion
[0174] Saposin C is a multifunctional molecule having lysosomal
enzyme activation and neuritogenic activities. Detailed
function/structure organization of saposin C is shown in FIG.
4.
[0175] The amino acid residues 51-67 are necessary, but not
sufficient, for its optimal enzymatic activation function. The
disulfide structure and conformational alteration of saposin C upon
lipid binding are also required for this activity. Three approaches
were used for this study: (1) stopped-flow monitoring reduction of
self-quenching resulting from fusion of fluorescence
probe-containing vesicle with non-fluorescent vesicle induced by
saposin C; (2) monitoring the lipid vesicle size changes upon
addition of saposin C to vesicles, the size distribution as
determined using N4 plus submicron particle sizer (Coulter Co.);
(3) monitoring intrinsic fluorescence of Trp-saposin C change
during liposomal fusion. These results defined the fusogenic
activity regions at the .alpha.-helical domain at amino- and
carboxyl-terminus in saposin C, and kinetics of saposin C induced
liposomal fusion (see below).
[0176] a) Saposin C Induced Liposomal Fusion
[0177] Fluorescence probes have been widely used to determine
membrane fusion, such as fluorescence dequenching, and fluorescence
resonance energy transfer (FET), and can be used for quantitative
and kinetics analyses. The dequenching approach was used to
investigate saposin C's fusogenic activity. Octadecyl rhodamine B
(R18) was selected as fluorescence probe and was entrapped in
internal aqueous compartment of liposomal vesicles by co-sonication
with BPS or PS(18:1, 1). R18 shows self-quenching at high
concentrations. Fluorescence increase (dequenching) of R18 occurs
upon R18 concentration decreases. After non-labeled and labeled
vesicles fuse, the R18 concentration is diluted, resulting in an
increase in intensity of fluorescence. R18-labeled vesicles
(lipid:R18=96:4, mol:mol) were mixed with the same lipid vesicles
without fluorescence probe. Stopped-flow assays were conducted to
quickly mix these vesicles with saposin C or Ca.sup.2+ ion.
Time-trace curves were generated for kinetic analysis. Induction of
unsaturated PS(18:1, 1) membrane fusion by saposin C showed the
same kinetics as those with Ca.sup.2+. Fusion occurs extensively
when reaction temperature is above the phase Transition temperature
(T.sub.c) of phospholipids. The T.sub.c of synthetic PS(18:1) is
about -11.degree. C., while the T.sub.c of PS(18:0) is very high
(68.degree. C.). Thus, the lipid bilayer phase of PS(18:1, 1) is
different to that of BPS(18:0 and 18:1) at 24.degree. C. The
results indicated that kinetics of saposin C-induced membrane
fusion is determined by the physical state of the bilayer
lipids.
[0178] 5. Size Change Determination
[0179] Electron microscopy (EM) was used for vesicle fusion
analysis, since the size of fused vesicles is bigger than those of
non-fused. N4 plus submicron particle size was used to estimate
particle sizes in the range of 3 nm to 3 .mu.m since most liposomes
fit in this range. Sonication conditions with a cup sonicator gave
mono-dispersed BPS-liposomes with .about.200 nm in size. Upon
addition of saposin C, these vesicles changed to a larger size up
to 2-3 .mu.m. The size increase is related to vesicles fusion as
shown by the above dequenching experiments. Saposin C enlarged
vesicle size at pH 4.7, but not at pH 7.4 over a 10 min period (see
FIG. 5).
[0180] These data suggest a pH-sensitive fusogenic activity of
saposin C. Saposin C promotes the size changes at .about.50 nM
concentration. To define the regions responsible for this fusion
property, peptides containing only 50% of the NH.sub.2-terminal or
50% of the COOH-terminal halves in saposin C were tested. Both
peptides showed fusion activity. These data suggested linear
sequence(s) mediated fusion located on both saposin C ends.
[0181] 6. Mechanism of Fusion
[0182] Protein conformational changes are thought to play a role in
protein-mediated membrane fusion. This fusion mechanism was
evaluated using saposin C-dependent membrane fusion. First, saposin
C-PS(18:1, 1) liposome complexes were formed. In this saposin
C-anchored membrane, protein conformation is altered. This complex
is stable from pH 3 to 10, and in low concentrations of SDS
solution. This indicated that dissociated rate of saposin C from PS
vesicles is very slow.
[0183] Since the Trp in saposin C(0W) is embedded inside of lipid
bilayer, the change of its signal is indicative of that the
surrounding environment of Trp has been changed. After about 20 to
30 ms, Trp fluorescence signal decreased to the starting level.
This indicates that saposin C in the complexes interacted with
additional PS-vesicles. Shortly after this, the signal dropped back
to starting level signaling on end of the fusion process. These
data indicate that saposin C retains the fusogenic activity even
when it bound to lipid membrane. Therefore, a conformational change
of saposin C upon lipid binding is not required for its fusogenic
activity. This result is consistent with the conclusion that a
linear sequence(s) is sufficient to induce membrane fusion
Sequence CWU 1
1
4 1 38 PRT Homo sapiens MISC_FEATURE (1)..(1) Where the amino acid
located at 1 is a hydrophobic amino acid, including Val, Leu, Ile,
Met, Pro, Phe, and Ala 1 Xaa Xaa Cys Glu Xaa Cys Glu Xaa Xaa Xaa
Lys Glu Xaa Xaa Lys Xaa 1 5 10 15 Xaa Asp Asn Asn Lys Xaa Glu Lys
Glu Xaa Xaa Asp Xaa Xaa Asp Lys 20 25 30 Xaa Cys Xaa Lys Xaa Xaa 35
2 39 PRT Homo sapiens MISC_FEATURE (1)..(2) Where the amino acids
located at 1 and 2 are hydrophobic amino acids, including Val, Leu,
Ile, Met, Pro, Phe, and Ala 2 Xaa Xaa Xaa Cys Glu Xaa Cys Glu Xaa
Xaa Xaa Lys Glu Xaa Xaa Lys 1 5 10 15 Xaa Xaa Asp Asn Asn Lys Xaa
Glu Lys Glu Xaa Xaa Asp Xaa Xaa Asp 20 25 30 Lys Xaa Cys Xaa Lys
Xaa Xaa 35 3 38 PRT Homo sapiens MISC_FEATURE (1)..(1) Where the
amino acid located at 1 is a hydrophobic amino acid, including Val,
Leu, Ile, Met, Pro, Phe, and Ala 3 Xaa Xaa Cys Glu Xaa Cys Glu Xaa
Xaa Xaa Lys Glu Xaa Xaa Lys Xaa 1 5 10 15 Xaa Asp Asn Asn Lys Xaa
Glu Lys Glu Xaa Xaa Asp Xaa Xaa Asp Lys 20 25 30 Xaa Cys Xaa Lys
Xaa Xaa 35 4 38 PRT Homo sapiens MISC_FEATURE (1)..(1) Where the
amino acid located at 1 is a hydrophobic amino acid, including Val,
Leu, Ile, Met, Pro, Phe, and Ala 4 Xaa Xaa Cys Glu Xaa Cys Glu Xaa
Xaa Xaa Lys Glu Xaa Xaa Lys Xaa 1 5 10 15 Xaa Asp Asn Asn Lys Xaa
Glu Lys Glu Xaa Xaa Asp Xaa Xaa Asp Lys 20 25 30 Xaa Cys Xaa Lys
Xaa Xaa 35
* * * * *