U.S. patent application number 11/683784 was filed with the patent office on 2007-07-19 for peptide/lipid complex formation by co-lyphilization.
Invention is credited to Jean-Louis Dasseux.
Application Number | 20070167351 11/683784 |
Document ID | / |
Family ID | 25478329 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070167351 |
Kind Code |
A1 |
Dasseux; Jean-Louis |
July 19, 2007 |
Peptide/Lipid Complex Formation by Co-Lyphilization
Abstract
The invention relates to the formation of peptide/lipid vesicles
and complexes through the co-lyophilization of peptides, preferably
that are able to adopt an amphipathic alphahelical conformation,
and one or more lipids. A single solution which solubilizes both
the peptides and lipids or two separate solutions may be
lyophilized.
Inventors: |
Dasseux; Jean-Louis;
(Mannheim, DE) |
Correspondence
Address: |
WARNER-LAMBERT COMPANY
2800 PLYMOUTH RD
ANN ARBOR
MI
48105
US
|
Family ID: |
25478329 |
Appl. No.: |
11/683784 |
Filed: |
March 8, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10252940 |
Sep 23, 2002 |
7189411 |
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11683784 |
Mar 8, 2007 |
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09642363 |
Aug 21, 2000 |
6455088 |
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10252940 |
Sep 23, 2002 |
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08942597 |
Oct 2, 1997 |
6287590 |
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09642363 |
Aug 21, 2000 |
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Current U.S.
Class: |
424/450 ;
514/19.3 |
Current CPC
Class: |
A61K 47/62 20170801;
A61K 9/1075 20130101; C07K 14/775 20130101; A61P 3/00 20180101;
A61K 9/1275 20130101; A61K 9/1277 20130101; A61K 9/19 20130101;
A61K 38/1709 20130101 |
Class at
Publication: |
514/002 |
International
Class: |
A61K 38/16 20060101
A61K038/16 |
Claims
1. A method of preparing a lyophilized peptide/lipid product which
comprises co-lyophilizing one or more peptides, which are able to
adopt an amphipathic conformation, or peptide analogues, and one or
more lipids in a solvent system to form a peptide/lipid product,
wherein said product can be rehydrated to form peptide/lipid
complexes.
2. The method of claim 1 wherein said peptide is a lipid binding
protein.
3. The method of claim 1 wherein said peptide analogue is an
analogue of ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III,
ApoE or another apoprotein.
4. The method of claim 1 wherein said peptide is a protein.
5. The method of claim 1 wherein said lipid is a natural lipid,
synthetic lipid, saturated lipid, unsaturated lipid or mixtures
thereof.
6. The method of claim 5 wherein said lipid is selected from the
group consisting of egg phosphatidylcholine, soybean
phosphatidylcholine, ether phospholipids, small alkyl chain
phospholipids, cholesterol, cholesterol derivatives,
dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine,
distearoylphosphatidylcholine
1-myristoyl-2-palmitoylphosphatidylcholine,
1-palmitoyl-2-myristoylphosphatidylcholine,
1-palmitoyl-2-stearoylphosphatidylcholine,
1-stearoyl-2-palmitoylphosphatidylcholine,
dioleoylphosphatidylcholine dioleophosphatidylethanolamine,
dilauroylphosphatidylglycerol phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol,
sphingomyelin sphingolipids, phosphatidylglycerol,
diphosphatidylglycerol dimyristoylphosphatidylglycerol,
dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,
dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid
dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine,
dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,
dipalmitoylphosphatidylserine, brain phosphatidylserine, brain
sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin,
phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,
dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,
aminophenylglycoside, and 3-cholesteryl-6'-(glycosylthio)hexyl
ether glycolipids, and mixtures thereof.
7. The method of claim 1 which further comprises sterilizing said
product prior to, during or after lyophilization.
8. The method of claim 1 wherein said peptide/lipid complex is
sterile.
9. The method of claim 1 further comprising aliquoting a solution
of said peptide and said lipid into individual containers before
lyophilization to form a single unit dosage form.
10. A pharmaceutical unit dosage form which comprises a sterile
lyophilized peptide/lipid mixture prepared according to claim 1 or
7.
11. A method of preparing a lyophilized peptide/lipid product which
comprises (a) solubilizing at least one amphipathic peptide or
peptide analogue in a first solution, (b) solubilizing at least one
lipid in a second solution, wherein said second solution is
miscible with said first solution, (c) combining said first
solution with said second solution to form a peptide/lipid
solution, and (d) lyophilizing said peptide/lipid solution so that
lyophilized peptide/lipid product is formed which can be rehydrated
to form peptide/lipid complexes.
12. The method of claim 11 wherein said peptide is a lipid binding
protein.
13. The method of claim 1 wherein said peptide is a protein.
14. The method of claim 9 wherein said peptide analogue is an
ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoE or
another apoprotein analogue.
15. The method of claim 11 wherein said lipid is a natural lipid, a
synthetic lipid, a saturated lipid, unsaturated lipid or mixtures
thereof.
16. The method of claim 15 wherein said lipid is selected from the
group consisting of egg phosphatidylcholine, cholesterol,
cholesterol derivatives, ether phospholipidsr soybean
phosphatidycholine, small alkyl chain phospholipids,
dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine,
distearoylphosphatidylcholine
1-myristoyl-2-palmitoylphosphatidylcholine,
1-palmitoyl-2-myristoylphosphatidylcholine,
1-palmitoyl-2-stearoylphosphatidylcholine,
1-stearoyl-2-palmitoylphosphatidylcholine,
dioleoylphosphatidylcholine dioleophosphatidylethanolamine,
dilauroylphosphatidylglycerol phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol,
sphingomyelin sphingolipids, phosphatidylglycerol,
diphosphatidylglycerol dimyristoylphosphatidylglycerol,
dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,
dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid
dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine,
dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,
dipalmitoylphosphatidylserine, brain phosphatidylserine, brain
sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin,
phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,
dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,
aminophenylglycoside, and 3-cholesteryl-6'-(glycosylthio)hexyl
ether glycolipids, and mixtures thereof.
17. The method of claim 11 wherein said peptide/lipid solution is
sterile.
18. The method of claim 11 where said peptide/lipid complex is
sterile.
19. The method of claims 11, further comprising aliquoting said
peptide/lipid solution into individual containers before
lyophilization to form a single unit dosage form.
20. The method of claim 11 which further comprises a sterilization
step prior to, during or after lyophilization.
21. A pharmaceutical unit dosage form which comprises a sterile and
stable lyophilized peptide/lipid mixture prepared according to
claim 11 or 19.
22. A peptide/lipid complexes formed by the process comprising
lyophilizing one or more amphipathic peptides or peptide analogues
and at least one lipid in a solvent system to form a dehydrated
peptide/lipid product which can be rehydrated to form peptide/lipid
complexes.
23. The peptide/lipid complexes of claim 22 wherein said peptide is
a lipid binding protein.
24. The peptide/lipid complexes of claim 22 wherein said peptide is
an ApoA1 analog.
25. The peptide/lipid complex of claim 22 wherein said lipid is a
natural, synthetic, saturated, unsaturated lipid or mixtures
thereof.
26. The peptide/lipid complexes of claim 25 wherein said lipid is
selected from the group consisting of egg phosphatidylcholine,
soybean phosphatidycholine, cholesterol, cholesterol derivatives,
small alkyl chain phospholipids, ether phospholipids,
dipalmitoylphosphatidylcholine, dinyristoylphosphatidylcholine,
distearoylphosphatidylcholine
1-myristoyl-2-palmitoylphosphatidylcholine,
1-palmitoyl-2-myristoylphosphatidylcholine,
1-palmitoyl-2-stearoylphosphatidylcholine,
1-stearoyl-2-palmitoylphosphatidylcholine,
dioleoylphosphatidylcholine dioleophosphatidylethanolamine,
dilauroylphosphatidylglycerol phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol,
sphingomyelin sphingolipids, phosphatidylglycerol,
diphosphatidylglycerol dimyristoylphosphatidylglycerol,
dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,
dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid
dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine,
dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,
dipalmitoylphosphatidylserine, brain phosphatidylserine, brain
sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin,
phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,
dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,
aminophenylglycoside, and 3-cholesteryl-6t-(glycosylthio)hexyl
ether glycolipids, and mixtures thereof.
27. The peptide/lipid complexes of claim 22 wherein said complex is
sterile.
28. The peptide/lipid complexes of claim 22, wherein said complex
is formulated into sterile unit dosage.
29. A sterile, lyophilized composition comprising a sterile
preparation of a complex formed between a peptide which can adopt a
amphipathic alpha helical conformation, or a peptide analogue and a
lipid.
30. The composition of claim 28 wherein said preparation is
provided in a sterile unit dosage formulation.
31. A lyophilized composition which comprises a peptide/lipid
complex wherein the peptide is a lipid binding protein or an ApoA1,
ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoE or another
apoprotein analogue.
32. The lyophilized composition of claim 31 wherein the
peptide/lipid complex is a vesicle, micelle, liposome, discoidal
particle, spherical particle, or mixture thereof.
33. A lyophilized composition which comprises a peptide/lipid
complex wherein said peptide can adopt an amphipathic alpha helical
conformation.
34. The lyophilized composition of claim 33 wherein the
peptide/lipid complex is a vesicle, micelle, liposome, discoidal
particle, spherical particle, or mixture thereof.
35. The composition of claims 31 or 33 wherein said composition is
sterile.
36. The composition of claim 29 or 31 said analogue is not a
peptide or a protein.
37. A method of preparing a lyophilized peptide/lipid roduct which
comprises: co-lyophilizing one or more peptides or peptides
analogues, which are able to adopt an amphipathic conformation,
with one or more lipids in a ratio of peptide to lipid of from
about 2 to about 200 in a solvent system for an amount of time
sufficient to form a peptide/lipid product which can be rehydrated
to form peptide/lipid complexes in solution.
Description
1. FIELD OF THE INVENTION
[0001] The invention relates to the formation of peptide/lipid
vesicles and complexes through the co-lyophilization of peptides,
preferably that are able to adopt an amphipathic alpha-helical
conformation, and one or more lipids. A single solution which
solubilizes both the peptides and lipids or a two separate
solutions may be lyophilized. The methods are used to generate
stable peptide/lipid vesicles and complexes including but not
limited to micellar, spherical and discoidal complexes in bulk
preparations and in smaller units, as may be suitable for
dosages.
2. BACKGROUND OF THE INVENTION
[0002] Liposomes are vesicles composed of at least one lipid
bilayer membrane enclosing an aqueous core. Generally,
phospholipids comprise the lipid bilayer, but the bilayer may be
composed of other lipids. The aqueous solution within the liposome
is referred to as the "captured volume."
[0003] Liposomes have been developed as vehicles to deliver drugs,
cosmetics, bioactive compounds among other applications. The lipid
bilayer encapsulates the drug, cosmetic, bioactive compound, and
the like, within the captured volume of the liposome and the drug
is expelled from the liposome core when the lipid bilayer comes in
contact with a cell surface membrane. The liposome releases its
contents to the cell by lipid exchange, fusion, endocytosis, or
adsorption. Ostro et al., 1989, Am. J. Hosp. Pharm. 46:1576.
Alternatively, the drug, cosmetic, bioactive compound and the like
could be associated with or inserted into the lipid bilayer
membrane of the vesicle.
[0004] In addition to vesicles, lipid-containing complexes have
been used to deliver agents in particle form. For instance, many
researchers have found it useful to prepare reconstituted
lipoprotein-like particles or complexes which have similar size and
density as high density lipoprotein (HDL) particles. These
reconstituted complexes usually consist of purified apoproteins
(usually apoprotein A-1) and phospholipids such as
phosphatidylcholine. Sometimes unesterified cholesterol is included
as well. The most common methods of preparing these particles are
(1) co-sonication of the constituents, either by bath sonication or
with a probe sonicator, (2) spontaneous interaction of the protein
constituent with preformed lipid vesicles, (3) detergent-mediated
reconstitution followed by removal of the detergent by dialysis.
Jonas, 1986, Meth. in Enzymol. 128:553-582; Lins et al., 1993,
Biochimica et Biophysica Acta, 1151:137-142; Brouillette &
Anantharamaiah, 1995, Biochimica et Biophysica Acta, 1256:103-129;
Jonas, 1992, Structure & Function of Apoproteins, Chapter
8:217-250. Similar complexes have also been formed by substituting
amphipathic helix-forming peptides for the apoprotein components.
Unfortunately, each of these methods presents serious problems for
the formation of large amounts of pure complexes on a reasonably
cost-effective basis. Further, none of these publications disclose
the co-lyophilization of peptides/or peptides analogues which are
able to adopt an amphipathic alpha helical conformation and a
lipid.
[0005] A range of technologies is known for producing lipid
vesicles and complexes. Vesicles, or liposomes, have been produced
using a variety of protocols, forming different types of vesicles.
The various types of liposomes include: multilamellar vesicles,
small unilamellar vesicles, and large unilamellar vesicles.
[0006] Hydration of phospholipids (or other lipids) by aqueous
solution can also result in the dispersion of lipids and
spontaneous formation of multimellar vesicles ("MLVs"). An MLV is a
liposome with multiple lipid bilayers surrounding the central
aqueous core. These types of liposomes are larger than small
unilamellar vesicles (SUVS) and may be 350-400 nm in diameter. MLVs
were originally prepared by solubilizing lipids in chloroform in a
round-bottom flask and evaporating the chloroform until the lipid
formed a thin layer on the wall of the flask. The aqueous solution
was added and the lipid layer was allowed to rehydrate. Vesicles
formed as the flask is swirled or vortexed. Deamer et al., 1983, in
Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citing
Bangham et al., 1965, J. Mol. Biol. 13:238). Johnson et al.
subsequently reported that this method also generated single
lamellar vesicles. Johnson et al., 1971, Biochim. Biophys. Acta
233:820.
[0007] A small unilamellar vesicle (SUV) is a liposome with a
single lipid bilayer enclosing an aqueous core. Depending on the
method employed to generate the SUVs, they may range in size from
25-110 nm in diameter. The first SUVs were prepared by drying a
phospholipid preparation in chloroform under nitrogen, adding the
aqueous layer to produce a lipid concentration in the millimolar
range, and sonicating the solution at 45.degree. C. to clarity.
Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc.
New York. Suvs prepared in this fashion yielded liposomes in the
range of 25-50 nm in diameter.
[0008] Another method of making SUVs is rapidly injecting an
ethanol/lipid solution into the aqueous solution to be
encapsulated. Deamer et al., 1983, in Liposomes (Ostro, Ed.),
Marcel Dekker, Inc. New York (citing Batzri et al., 1973, Biochim.
Biophys. Acta 298:1015). SUVs produced by this method range in size
from 30-110 nm in diameter.
[0009] SUVs may also be produced by passing multilamellar vesicles
through a French Press four times at 20,000 psi. The SUVs produced
will range in size from 30-50 nm in diameter. Deamer et al., 1983,
in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citing
Barenholz et al., 1979, FEBS Letters 99:210).
[0010] Multilamellar and unilamellar phospholipid vesicles can also
be formed by extrusion of aqueous preparations of phospholipids at
high pressure through small-pore membranes (Hope et al., 1996,
Chemistry and Physics of Lipids, 40:89-107)
[0011] A large unilamellar vesicle (LUV) is similar to SUVs in that
they are single lipid bilayers surrounding the central aqueous
core, but LUVs are much larger that SUVs. Depending on their
constituent parts and the method used to prepare them, LUVs may
range in size from 50-1000 nm in diameter. Deamer et al., 1983, in
Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York. LUVs are
usually prepared using one of three methods: detergent dilution,
reverse-phase. evaporation, and infusion.
[0012] In the detergent dilution technique, detergent solutions
such as cholate, deoxycholate, octyl glucoside, heptyl glucoside
and Triton X-100 are used to form micelles from the lipid
preparation. The solution is then dialyzed to remove the detergent
and results in the formation of liposomes. Deamer et al., 1983, in
Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York. This method
is time consuming and removal of the detergent is generally
incomplete. The presence of detergent in the final preparation may
result in some toxicity of the liposome preparation and/or
modification of the physicochemical properties of the liposome
preparation.
[0013] The reverse-phase evaporation technique solubilizes lipid in
aqueous-nonpolar solutions, forming inverted micelles. The nonpolar
solvent is evaporated and the micelles aggregate to form LUVs. This
method generally requires a great deal of lipid.
[0014] The infusion method injects a lipid solubilized in a
non-polar solution into the aqueous solution to be encapsulated. As
the nonpolar solution evaporates, lipids collect on the gas/aqueous
interface. The lipid sheets form LUVs and oligolamellar liposomes
as the gas bubbles through the aqueous solution. Liposomes are
sized by filtration. Deamer et al., 1983, in Liposomes (Ostro,
Ed.), Marcel Dekker, Inc. New York (citing Deamer et al., 1976,
Biochim. Biophys. Acta 443:629 and Schieren et al., 1978, Biochim.
Biophys. Acta 542:137). Infusion procedures require a fairly high
temperature for infusion and may have a relatively low
encapsulation efficiency. Deamer et al., 1983, in Liposomes (Ostro,
Ed.), Marcel Dekker, Inc. New York
[0015] It is has been a goal of liposome research to develop
liposome preparations that may be stored for long periods of time
before use. For example, U.S. Pat. No. 4,229,360 to Schneider et
al., discloses a method of dehydrating liposomes by adding a
hydrophilic compound to a colloidal dispersion of liposomes in an
aqueous liquid and dehydrating the solution, preferably by
lyophilization. Examples of hydrophilic compounds are high
molecular weight hydrophilic polymers or low molecular weight
compounds such as sucrose.
[0016] U.S. Pat. No. 4,411,894 to Shrank et al., discloses the use
of high concentrations of sucrose in sonicated preparations of
liposomes. The liposomes contain fat-soluble products in the
captured volume, although the preparations could be lyophilized,
the method could not prevent the loss of a significant amount of
the captured contents despite the high concentration of
sucrose.
[0017] Crowe et al., U.S. Pat. No. 4,857,319 disclosed the use of
disaccharides such as sucrose, maltose, lactose and trehalose to
stabilize liposomes when liposomes are freeze dried. The amount of
disaccharide with respect to the lipid content of the component
(w/w) is within 0.1:1 to 4:1. Crowe achieved greater success in
preserving liposomal integrity using this method than that afforded
by the method disclosed by Shrank in U.S. Pat. No. 4,441,894.
[0018] Janoff et al, U.S. Pat. No 4,880,635 disclose a method for
dehydrating liposomes in which liposomes were lyophilized in the
presence of protective sugars such as trehalose and sucrose,
preferably on both the inner and outer leaflets of the lipid
bilayer. Sufficient water is retained in the method of Janoff et
al. so that rehydration of the dried liposomes yields liposomes
with substantial structural integrity.
[0019] However, there is a need in the art for a simple and cost
effective method of forming lyophilized peptide/lipid complexes
which may be then be rehydrated. The method of the present
invention yields peptide/lipid mixtures in a stable, lyophilized
powder which may be stored, used as a powder, or used after
rehydration to form peptide/lipid complexes.
3. SUMMARY OF THE INVENTION
[0020] The invention is a method for preparing peptide or
protein-(phospho)lipid complexes or vesicles which may have
characteristics similar to high density lipoprotein (HDL). The
method utilizes a solvent system in which at least one peptide is
solubilized in one solution, and at least one lipid is solubilized
in another solution. The two solutions are selected such that they
are miscible with one another. The solutions are then combined, and
the resulting solution is lyophilized.
[0021] The method also may be practiced by a second type of solvent
system comprising a solution into which both the protein or peptide
and the lipid may be solubilized. This solution may be a single
solution, or may be a composite solution made by combining two or
more solutions before the addition of peptides and lipids. Peptides
and lipids are solubilized in the solution or composite solution
and the peptide/lipid solution is then lyophilized.
[0022] Preferably, the peptides of the present invention are
peptides which are able to adopt an amphipathic helical
conformation. In one specific embodiment of the invention, the
peptide is a lipid binding protein. In other embodiment, peptide
analogues of ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, Apoc-III,
ApoE, other apolipoprotein analogues and the like are utilized in
place of or in combination with the peptides. In another specific
embodiment, the method is used to prepare ApoA1
analogue/(phospho)lipid complexes similar to HDL. The ApoA1/lipid
complexes are useful in treating disorders associated with
dyslipoproteinemias including but not limited to
hypercholesterolemia, hypertriglyceridemia, low HDL, and
apolipoprotein A-1 deficiency, septic shock, for in vitro
diagnostic assays as markers for HDL populations, and for use with
imaging technology.
[0023] The method of the invention enables the preparation of
peptidellipid complexes for parenteral administration including but
not limited to intravenous, intraperitoneal, subcutaneous,
intramuscular, and bolus injections to animals or humans. Further,
the peptide/lipid complexes can also be formulated for oral,
rectal, mucosal (e.g. oral cavity) or topical administration to
animals or humans, or for in vitro experimentation.
[0024] The method may be used for large scale production of
amphipathic peptide/phospholipid complexes, lipid binding
protein/phospholipid complexes, and/or ApoA1 peptide
analogue/phospholipid complexes. The lyophilized material may be
prepared for bulk preparations, or alternatively, the mixed
peptide/lipid solution may be apportioned in smaller containers
(for example, single dose units) prior to lyophilization, and such
smaller units may be prepared as sterile unit dosage forms.
[0025] The lyophilized powder prepared by the method of the
invention can be rehydrated into a particulate-free sterile
solution immediately before injection, or alternatively, the
lyophilized powder can be formulated into an appropriate solid
dosage form and administered directly.
[0026] The method may also be suitable for storage of compounds
which may be otherwise unstable or insoluble in the absence of
lipids.
[0027] The method may be used for the formulation of products for
the treatment or prevention of human diseases, including such
applications as co-presentation of antigens in vaccines, treatment
or prevention of dyslipoproteinemias, including but not limited to
hypercholesterolemia, hypertriglyercidemia, low HDL, and
apolipoprotein A-1 deficiency, cardiovascular disease such as
atherosclerosis, septic shock, or infectious diseases.
[0028] The method may be used for the preparation of complexes that
could be used as carriers for drugs, as vectors (to deliver drugs,
DNA, genes), for example, to the liver or to extrahepatic cells, or
as scavengers to trap toxin (e.g. pesticides, LPS, etc.).
3.1. Definitions
[0029] As used herein, a "solvent system" refers to one or more
solvents which are capable of solubilizing peptides and/or lipids
and, if more than one, which are miscible with one another.
[0030] As used herein, "peptide/lipid complexes" refers to an
aggregation of lipid moieties and peptides forming particles within
the size range of high density lipoproteins (HDLs).
[0031] As used herein, "co-lyophilized" refers to the
lyophilization, freeze-drying, or vacuum drying of more than one
compound (e.g., peptide, protein, lipid, phospholipid) in solution
in the same vessel. For example, a lipid solution may be combined
with a peptide solution in the same vessel and the resulting
combination of solutions is lyophilized together, thereby
lyophilizing the peptides and lipids simultaneously.
[0032] As used herein "amphipathic peptide" or "amphipathic alpha
helical peptides" means peptides which are able to adopt an
amphipathic or amphipathic helical conformation, respectively. The
amphipathic alpha helix is an often encountered secondary
structural motif in biologically active peptides and proteins. See
Amphipathic helix motif: classes and properties by Jere P. Segrest,
Hans de Loof, Jan G. Dohlman, Christie G. Brouillette, and G. M.
Anantharamaiah. PROTEINS: Structure Functions and Genetics
8:103-117 (1990). An amphipathic alpha helix is an alpha helix with
opposing polar and nonpolar faces oriented along the long axis of
the helix. A specific distribution of charged residues is evident
along the polar face. Amphipathic helices, as defined, are
complementary for the polar-nonpolar interface of hydrated bulk
phospholipid; these lipid-associating domains have been postulated
to interact with the phospholipid by partially immersing themselves
at the interface between the fatty acyl chains and the polar head
groups, Jere P. Segrest. Febs letters 1976, 69 (1): 111-114.
[0033] The term "peptide" and "protein" may be used interchangeably
herein. Further, the peptide analogues of the invention can be
peptides, proteins or non-peptides i.e., peptidomimetics. However,
all the analogues are preferably bioactive molecules.
[0034] The term "lipid" as used herein includes but is not limited
to natural and synthetic phospholipids. Further, the terms, "Lipid"
and "phospholipid" may be used interchangeably herein.
4. BRIEF DESCRIPTION OF THE FIGURES
[0035] FIG. 1: Superose 6 chromatography of HDL prepared by density
ultracentrifugation from 200 .mu.l human serum.
[0036] FIG. 2 (bottom): Superose 6 chromatography of (DPPC:peptide
1) (PVLDLFRELLNELLEALKQKLK; SEQ ID NO:1) complexes prepared at a
ratio of 1:1 (w:w).
[0037] FIG. 2 (top): Superose 6 chromatography of (DPPC:peptide 1)
complexes prepared at a ratio of 2:1 (W:W).
[0038] FIG. 3 (bottom): Superose 6 chromatography of (DPPC:peptide
1) complexes prepared at a ratio of 3:1 (w:w).
[0039] FIG. 3 (top): Superose 6 chromatography of (DPPC:peptide 1)
complexes prepared at a ratio of 4:1 (w:w).
[0040] FIG. 4 (bottom): Superose 6 chromatography of (DPPC:peptide
1) complexes prepared at a ratio of 5:1 (w:w).
[0041] FIG. 4 (top): Superose 6 chromatography of (DPPC:peptide 1)
complexes prepared at a ratio of 7.5:1 (w:w).
[0042] FIG. 5: Superose 6 chromatography of (DPPC:peptide 1)
complexes prepared at a ratio of 10:1 (w:w).
[0043] FIG. 6: Superose 6 chromatography of .sup.14C-labeled
peptide 1 complexes at Ri=3:1.
[0044] FIG. 7: Superose 6 chromatography of .sup.14C-labeled
peptide 1 complexes at Ri=4:1.
[0045] FIG. 8: Superose 6 chromatography of .sup.14C-labeled
peptide 1 complexes at Ri=5:1.
10 5. DETAILED DESCRIPTION OF THE PREFERRED EMODIMENTS
[0046] The amphipathic alpha helical peptides or proteins, lipid
binding proteins, ApoA-I agonist peptides, apoprotein analogues,
and the like, which are useful in the present invention, can be
synthesized or manufactured using any technique known in the art.
Stable preparations of peptides which have a long shelf life may be
made by lyophilizing the peptides--either to prepare bulk for
reformulation, or to prepare individual aliquots or dosage units
which can be reconstituted by rehydration with sterile water or an
appropriate sterile buffered solution prior to administration to a
subject.
[0047] To the inventor's knowledge, this invention is the first
instance of a method for co-lyophilizing an amphipathic alpha
helical peptide or peptide analogue with a lipid to form a mixture
that can be reconstituted into a sterile peptide/lipid complex.
[0048] In certain embodiments, it may be preferred to formulate and
administer the ApoA-I analog(s) including but not limited to ApoA-I
agonists, in a peptide-lipid complex. This approach has several
advantages since the complex should have an increased half-life in
the circulation, particularly when the complex has a similar size
and density to the HDL class of proteins, especially the pre-beta
HDL populations. The HDL class of lipoproteins can be divided into
a number of subclasses based on such characteristics as size,
density and electrophoretic mobility. Some examples, in order of
increasing size are micellar pre-beta HDL of diameter 50 to 60
Angstroms, discoidal HDL of intermediate size i.e., with a mass of
65 kDa (about 70 Angstroms), spherical HDL.sub.3 or HDL.sub.2 of
diameter 90 to 120 Angstroms. (J. Kane, 1996 in V. Fuster, R. Ross
and E. Topol [eds.] Atberosclerosis and Coronary Artery Disease, p.
99; A. Tall and J. Breslow, ibid., p. 106; Barrans et al.,
Blochemica et Biophysica Acta 1300, p. 73-85; and Fielding et al.,
1995, J. Lipid Res 36, p. 211-228). However, peptide/lipid
complexes of smaller or larger size than RDL may also be formed by
the invention.
[0049] The peptide-lipid complexes of the present invention can
conveniently be prepared as stable preparations, having a long
shelf life, by the co-lyophilization procedure described below. The
lyophilized peptide-lipid complexes can be used to prepare bulk
drug material for pharmaceutical reformulation, or to prepare
individual aliquots or dosage units which can be reconstituted by
rehydration with sterile water or an appropriate buffered solution
prior to administration to a subject.
[0050] The applicants have developed a simple method for preparing
peptide or protein-(phospho)lipid complexes which have
characteristics similar to HDL. This method can be used to prepare
the ApoA-I peptide-lipid complexes, and has the following
advantages: (1) Most or all of the included ingredients are used to
form the designed complexes, thus avoiding waste of starting
material which is common to the other methods. (2) Lyophilized
compounds are formed which are very stable during storage. The
resulting complexes may be reconstituted immediately before use.
(3) The resulting complexes usually do not require further
purification after formation or before use. (4) Toxic compounds,
including detergents such as cholate, are avoided. Moreover, the
production method can be easily scaled up and is suitable for GMP
manufacture (i.e., in an endotoxin-free environment).
[0051] In accordance with the preferred method, the peptide and
lipid are combined in a solvent system which co-solubilizes each
ingredient. To this end, solvent pairs must be carefully selected
to ensure co-solubility of both the amphipathic peptide and the
hydrophobic lipid. In one embodiment, the protein(s) or peptide(s)
to be incorporated into the particles can be dissolved in an
aqueous or organic solvent or mixture of solvents (solvent 1). The
(phospho) lipid component is dissolved in an aqueous or organic
solvent or mixture of solvents (solvent 2) which is miscible with
solvent 1, and the two solutions are combined. Alternatively, the
(phospho)lipid component is dissolved directly in the peptide
(protein) solution. Alternatively, the peptide and lipid can be
incorporated into a co-solvent system, i.e., a mixture of the
miscible solvents. Depending on the lipid binding properties of the
peptide or protein, those skilled in the art will recognize that
enhanced or even complete solubilization (and/or enhanced mixing)
may be necessary prior to lyophilization; thus, the solvents can be
chosen accordingly.
[0052] A suitable proportion of peptide (protein) to lipids is
first determined empirically so that the resulting complexes
possess the appropriate physical and chemical properties, usually
but not always meaning similar in size to HDL.sub.2 or HDL.sub.3.
The lipid to protein/peptide molar ratio should be in the range of
about 2 to about 200, and preferably 5 to 50 depending on the
desired type of complexes. Examples of such size classes of
peptide/lipid or protein/lipid complexes include, but are not
limited to, micellar or discoidal particles (usually smaller than
HDL.sub.3 or HDL.sub.2), spherical particles of similar size to
HDL.sub.2 or HDL.sub.3 and larger complexes which are larger than
HDL.sub.2. The HDLs used by us as a standard during chromatography
(FIG. 1) are mainly spherical mature HDL.sub.2. Pre-.beta.1 HDL are
micellar complexes of apolipoprotein and few molecules of
phospholipids. Pre-.beta.2 HDL are discoidal complexes of
apolipoprotein and molecules of phospholipids. The more lipids
(triglycerides, cholesterol, phospholipids) are incorporated the
bigger will become the HDL and its shape is modified. (Pre-.beta.1
HDL (micellar complex) Pre-.beta.2 HDL (discoidal complex)) HDL3
(spherical complex) HDL2 (spherical complex).
[0053] Once the solvent is chosen and the peptide and lipid have
been incorporated, the resulting mixture is frozen and lyophilized
to dryness. Sometimes an additional solvent is added to the mixture
to facilitate lyophilization. This lyophilized product can be
stored for long periods and will remain stable.
[0054] In the working examples describe infra, the peptide 1
PVLDLFRELLNELLEALKQKLK (SEQ ID NO:1) and (phospho)lipid were
dissolved separately in methanol, combined, then mixed with xylene
before lyophilization. The peptide and lipid can both be added to a
mixture of the two solvents. Alternatively, a solution of the
peptide dissolved in methanol can be mixed with a solution of lipid
dissolved in xylene. Care should be taken to avoid salting out the
peptide. The resulting solution containing the peptide and lipid
co-solubilized in methanol/xylene is lyophilized to form a
powder.
[0055] The lyophilized product can be reconstituted in order to
obtain a solution or suspension of the peptide-lipid complex. To
this end, the lyophilized powder is rehydrated with an aqueous
solution to a suitable volume (often about 5 mg peptide/ml which is
convenient for intravenous injection). In a preferred embodiment
the lyophilized powder is rehydrated with phosphate buffered saline
or a physiological saline solution. The mixture may have to be
agitated or vortexed to facilitate rehydration, and in most cases,
the reconstitution step should be conducted at a temperature equal
to or greater than the phase transition temperature (TM) of the
lipid component of the complexes. Within minutes, a solution of
reconstituted lipid-protein complexes (a clear solution when
complexes are small) results.
[0056] An aliquot of the resulting reconstituted preparation can be
characterized to confirm that the complexes in the preparation have
the desired size distribution, e.g., the size distribution of HDL.
Gel filtration chromatography can be used to this end. In the
working examples described infra, a Pharmacia Superose 6 FPLC gel
filtration chromatography system was used. The eluant used contains
150 mM NaCl in deionized water. A typical sample volume is 20 to
200 microliters of complexes containing 5 mg peptide/ml. The column
flow rate is 0.5 ml/min. A series of proteins of known molecular
weight and Stokes' diameter as well as human HDL are used as
standards to calibrate the column. The proteins and lipoprotein
complexes are monitored by absorbance or scattering of light of
wavelength 254 or 280 nm.
[0057] The solvents that may be used according to the method of the
present invention include but are not limited to nonpolar, polar,
aprotic, and protic organic solvents and the like such as ethanol,
methanol, cyclohexane, 1-butanol, isopropyl alcohol, xylene, THP,
ether, methylene chloride benzene and chloroform. The invention
also includes the use of solvent mixtures as well as single
solvents. Further, prior to use within the present methods the
organic solvents maybe dried to remove water; however, hydrated
solvents or water may be used with certain lipids, peptides or
proteins. In other words, water may be a suitable solvent, or
hydrated solvents or organic solvent/water mixtures may be used,
however, if water is used it must be detergent free. As mentioned
above, the solvents are preferably of the purest quality (in order
to avoid concentrating impurities after lyophilization), and the
solvents should be salt free and free of particulates. However, the
solvents need not be sterile as the resulting product can be
sterilized before, during or after lyophilization, in accordance
with known techniques in the pharmaceutical art, such as those
described in Remington's Pharmaceutical Sciences, 16th and 18th
Eds., Mack Publishing Co., Easton, Pa. (1980 and 1990), herein
incorporated by reference in its entirety, and in the United States
Pharmacopeia/National Formulary (USP/NF) XVII, herein incorporated
by reference in its entirety.
[0058] The lipids which may be used according to the method of the
present composition include but are not limited to natural and
synthesized (synthetic) lipids and phospholipids including small
alkyl chain phospholipids, egg phosphatidylcholine, soybean
phosphatidylcholine, dipalmitoylphosphatidylcholine,
dimyristoylphosphatidylcholine, distearoylphosphatidylcholine
1-myristoyl-2-palmitoylphosphatidylcholine,
1-palmitoyl-2-myristoylphosphatidylcholine,
1palmitoyl-2-stearoylphosphatidylcholine,
1-stearoyl-2-palmitoylphosphatidylcholine,
dioleoylphosphatidylcholine dioleophosphatidylethanolamine,
dilauroylphosphatidylglycerol phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol,
sphingomyelin sphingolipids, phosphatidylglycerol,
diphosphatidylglycerol dimyristoylphosphatidylglycerol,
dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,
dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid
dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine,
dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,
dipalmitoylphosphatidylserine, brain phosphatidylserine, brain
sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin,
phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,
dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,
aminophenylglycoside, 3-cholesteryl-6'-(glycosylthio)hexyl ether
glycolipids, and cholesterol and its derivatives.
[0059] The peptides that are suitable for use with the present
invention include, but are not limited to, those described in the
three co-pending applications [Ser. Nos. ______ identified by
attorney docket nos. 9196-0004-999, 9196-0005-999 and
9196-0006-999, each of which was filed on Sep. 29, 1997] each of
which is herein incorporated by reference in its entirety.
[0060] It is preferred, although not necessary in every case, that
precipitates should be solubilized or removed prior to mixing or
stirring the lipid and peptide solutions or prior to
lyophilization.
[0061] The method may be used for large scale production of
peptide/lipid complexes, amphipathic peptide/(phospho)lipid
complexes, lipid binding protein/(phospho)lipid complexes, and/or
ApoA1 peptide analogue/(phospho)lipid complexes. The lyophilized
material may be prepared for bulk preparations, or alternatively,
the mixed peptide/lipid solution may be apportioned in smaller
containers (for example, single dose units) prior to
lyophilization, and such smaller units may be prepared as sterile
single dosage forms.
[0062] The vacuum dried compositions of the present invention may
be provided in single dose or multiple dose container forms by
aseptically filling suitable containers with the sterile pre-vacuum
dried solution to a prescribed content; preparing the desired
vacuum dried compositions; and then hermetically sealing the single
dose or multiple dose container. It is intended that these filled
containers will allow rapid dissolution of the dried composition
upon reconstitution with appropriate sterile diluents in situ
giving an appropriate sterile solution of desired concentration for
administration. As used herein, the term "suitable containers"
means a container capable of maintaining a sterile environment,
such as a vial, capable of delivering a vacuum dried product
hermetically sealed by a stopper means. Additionally, suitable
containers implies appropriateness of size, considering the volume
of solution to be held upon reconstitution of the vacuum dried
composition; and appropriateness of container material, generally
Type I glass. The stopper means employed, e.g., sterile rubber
closures or the equivalent, should be understood to be that which
provides the aforementioned seal, but which also allows entry for
the purpose of the introduction of a diluent, e.g., sterile Water
for Injection, USP, Normal Saline, USP, or 5% Dextrose in Water,
USP, for the reconstitution of the desired solution. These and
other aspects of the suitability of containers for pharmaceutical
products such as those of the instant invention are well known to
those skilled in the practice of pharmaceutical arts. In specific
embodiments, sizes of product unit dosages may be in a range of
about 10 mg to 2 g of peptide preferably in the range of about 100
mg to 1 g and at a concentration after reconstitution of about 1 to
50 mg/ml, preferably about 2 to 25 mg/ml.
[0063] The method of the invention enables the preparation of
protein or peptide/lipid complexes for parenteral administration
including intravenous, intraperitoneal, subcutaneous, intramuscular
and bolus injections to animals or humans, or for oral, rectal,
mucosal (e.g. oral cavity) or topical administration to animals or
humans, or for in vitro experimentation.
[0064] The lyophilized powder prepared by the method of the
invention can be rehydrated immediately before injection, or
alternatively, the lyophilized powder can be administered directly.
The lyophilized powder includes, but is not limited to lipid and
peptides that are able to form complexes in the form of vesicles,
liposomes, particles including spherical or discoidal particles,
micelles and the like. In order to reconstitute or rehydrate the
lyophilized powder a solution is chosen depending upon the desired
end use. For pharmaceutical use any sterile solution may be used.
Further, buffered solutions are preferred for certain uses and
these include but are not limited to phosphate, citrate, tris,
baribital, acetate, glycine-HCl, succinate, cacodylate, boric
acid-borax, ammediol and carbonate.
[0065] The lyophilized powder of the present invention may be
formed using any method of lyophilization known in the art,
including, but not limited to, freeze-drying in which the
peptide/lipid-containing solution is subjected to freezing followed
by reduced pressure evaporation.
[0066] The method may also be suitable for storage of compounds
which may be otherwise unstable or insoluble in the absence of
lipids.
[0067] The method may be used for the formulation of products for
the treatment or prevention of human diseases, including such
applications as co-presentation of antigens in vaccines treatment
or prevention of dyslipoproteinemias including but not limited to
hypercholesterolemia, hypertriglyceridemia, low HDL, and
apolipoprotein A-1 deficiency, cardiovascular disease such as
atherosclerosis, septic shock, or infectious diseases.
[0068] The method may be used for the preparation of complexes that
could be used as carriers for drugs, as vectors (to deliver drugs,
DNA, genes), for example, to the liver or to extrahepatic cells, or
as scavengers to trap toxin (e.g. pesticides, LPS, etc.).
Alternatively, the method may be used to prepare complexes for in
vitro assay systems, or for use in imaging technology.
[0069] In specific embodiments, the method may be used for the
preparation of ApoA-I analogue (including but not limited to
agonists) complexes which may be used in in vitro diagnostic assays
and as markers for HDL populations and subpopulations. In other
specific embodiments, ApoA-I agonist complexes may be used for
immunoassays or for imaging technology (e.g., CAT scans, MRI
scans).
[0070] The following examples are intended to be illustrative of
the present invention and should not be construed, in any way, to
be a limitation thereof.
6. EXAMPLE
Preparation of Peptide-Lipid Complex by Co-Lyophilization
Approach
[0071] The following protocol was utilized to prepare peptide-lipid
complexes.
[0072] Peptide 1 (PVLDLFRELLNELLEALKQKLK; SEQ ID NO:1) (22.4 mg)
was dissolved in methanol at a concentration of 3.5 mg/ml by
incubation for several minutes and mixing by vortex intermittently.
To this solution was added dipalmitoylphosphatidylcholine (DPPC) in
methanol (100 mg/ml stock solution) such that the final ratio of
DPPC/peptide was 2.5:1 (weight/weight). This solution was mixed by
vortexing. Xylene was added to the solution to a final
concentration of 36%. Aliquots of the resulting solution were
removed for later analysis by gel filtration chromatography. The
solutions were frozen in liquid nitrogen and lyophilized to dryness
by vacuum. An aliquot containing 20 mg peptide 1 (SEQ ID NO:1) and
50 mg DPPC was rehydrated in sterile saline solution (0.9% NaCl),
mixed, and heated to 41.degree. C. for several minutes until a
clear solution of reconstituted peptide/phospholipid complexes
resulted.
6.1. EXAMPLE
Gel Filtration and Phospholipid Utilization
6.1.1. Materials and Methods
[0073] For the purpose of testing conditions for the preparation of
complexes it is often convenient to prepare small amounts of
complexes for characterization. These preparations contained one mg
of peptide and were prepared as follows: One mg of peptide 1 (SEQ
ID NO: 1) was dissolved in 250 .mu.l HPLC grade methanol (Perkin
Elmer) in a 1.0 ml clear glass vial with cap (Waters #WAT025054).
Dissolving of the peptide was aided by occasional vortexing over a
period of 10 minutes at room temperature. After this time a small
amount of undissolved particulate matter could still be seen but
this did not adversely affect the results, To this mixture an
aliquot containing either 1, 2, 3, 4, 5, 7.5, 10 or 15 mg DPPC
(Avanti Polar Lipids, 99% Purity, product #850355) from a 100 mg/ml
stock solution in methanol was added. The volume of the mixture was
brought to 400 .mu.l by addition of methanol and the mixture was
further vortexed intermittently for a period of 10 minutes at room
temperature. At this time, very little undissolved material could
be seen in the tubes. To each tube 200 .mu.l of xylene
(Sigma-Aldrich 99% pure, HPLC-grade) was added and the tubes were
vortexed for 10 seconds each. Two small holes were punched into the
tops of each tube with a 20 gauge syringe needle, the tubes were
frozen for 15 seconds each in liquid nitrogen, and the tubes were
lyophilized overnight under vacuum. To each tube 200 ml of 0.9%
NaCl solution was added. The tubes were vortexed for 20 seconds
each. At this time the solutions in the tubes were milky in
appearance. The tubes were then incubated in a water bath for 30
minutes at 41.degree. C. The solutions in all of the tubes became
clear (i.e., similar to water in appearance) except for the tube
containing 15 mg DPPC, which remained milky.
[0074] In order to determine if all of the phospholipids that were
used in the complex preparations actually appeared in the column
fractions corresponding to the chromatogram absorbance peaks, the
column eluate from reconstituted peptide/lipid complexes was
collected in one or two ml fractions and the fractions were assayed
enzymatically for phospholipid content with the BioMerieux
Phospholipides Enzymatique PAP 150 kit (#61491) according to the
instructions supplied by the manufacturer.
[0075] The preparations of complexes may also be done on a larger
scale. An example of one such preparation is reported above. These
complexes were used for in vivo experiments.
6.2. Results of Complex C Characterization
[0076] FIG. 1: superose 6 chromatography of mature HDL.sub.2
prepared by density ultracentrifugation from 200 .mu.l human serum.
Chromatograph shows absorbance at 254 nm. Elution volume=14.8 ml,
corresponding to a Stokes' diameter of 108 Angstroms (See Table
1).
[0077] FIG. 2 (bottom): Superose 6 chromatography of DPPC:peptide 1
complexes prepared at a ratio of incubation (Ri, defined as the
ratio of total phospholipid to total peptide in starting mixture)
of 1:1 (w:w) as described above (small scale preparation). Elution
volumes of absorbance peaks=16.2 mls and 18.1 ml corresponding to
particles of Stokes' diameters 74 and 82 Angstroms, which are
smaller than HDL. 87% of the phospholipid applied to the column was
recovered in the fractions containing the absorbance peaks (See
Table 1).
[0078] FIG. 2 (top): Superose 6 chromatography of DPPC:peptide 1
complexes prepared at an Ri of 2:1 (w:w) as described above.
Elution volume of absorbance peak=16.4 ml, (77 Angstroms),
corresponding to particles smaller than HDL. 70% of the
phospholipid applied to the column was recovered in the fractions
containing the absorbance peak (See Table 1).
[0079] FIG. 3 (bottom): Superose 6 chromatography of DPPC:peptide 1
complexes prepared at an Ri of 3:1 (w:w) as described above.
Elution volume of absorbance peak=16.0 ml, (80 Angstroms)
corresponding to particles smaller than HDL. 79% of the
phospholipid applied to the column was recovered in the fractions
containing the absorbance peak (See Table 1).
[0080] FIG. 3 (top): Superose 6 chromatography of DPPC:peptide 1
complexes prepared at an Ri of 4:1 (w:w) as described above.
Elution volume of the absorbance peak=15.7 ml, (90 Angstroms),
corresponding to particles smaller than HDL. 106% of the
phospholipid applied to the column was recovered in the fractions
containing the absorbance peak (See Table 1).
[0081] FIG. 4 (bottom): Superose 6 chromatography of DPPC:peptide 1
complexes prepared at an Ri of 5:1 (w:w) as described above.
Elution volume of the absorbance peak=15.1 ml, (104 Angstroms),
corresponding to particles smaller than HDL. 103% of the
phospholipid applied to the column was recovered in the fractions
containing the absorbance peak (See Table 1).
[0082] FIG. 4 (top): Superose 6 chromatography of DPPC:peptide 1
complexes prepared at an Ri of 7.5:1 (w:w) as described above.
Elution volume of the absorbance peak=13.6 ml, (134 Angstroms)
corresponding to particles larger than HDL. 92% of the phospholipid
applied to the column was recovered in the fractions containing the
absorbance peaks (See Table 1).
[0083] FIG. 5: Superose 6 chromatography of DPPC:peptide 1
complexes prepared at a ratio of 10:1 (w:w) as described above.
Elution volume of absorbance peak=13.4 ml, (138 Angstroms), again
corresponding to particles larger than HDL. 103% of the
phospholipid applied to the column was recovered in the fractions
containing the absorbance peaks (See Table 1).
[0084] The sample containing complexes with 15:1 DPPC:peptide 1
(w:w) was not subjected to Superose 6 chromatography because it was
turbid, suggesting the presence of large particles.
[0085] For each of the above experiments, no significant
phospholipid was observed in any fraction other than those
containing material eluting with the absorbance peaks (See FIGS.
2-8). This suggests that virtually all of the phospholipids (within
experimental error of the assay) were incorporated into the
complexes. The experiment demonstrates that by varying the initial
ratio of phospholipids to peptides, homogeneous complexes of
various sizes (smaller or larger than HDL) can be formed.
6.3. Characterization of Complexes Using .sup.14C-Labeled Peptide
1
[0086] Peptide-phospholipid complexes containing .sup.14C-labeled
peptide 1 (specific activity 159,000 DPM/mg peptide by weight,
assuming 50% peptide content) were prepared by co-lyophilization as
described above. The preparations each contained 1 mg peptide and
3, 4 or 5 mg DPPC by weight. After reconstituting the complexes in
200 Al 0.9% NaCl, 20 .mu.l (100 .mu.g) of the complexes were
applied to a Pharmacia Superose 6 column using 0.9% NaCl as the
liquid phase at a flow rate of 0.5 ml/min. After a 5 ml delay
(column void volume=7.7 ml) 1 ml fractions were collected. Aliquots
containing 20 .mu.l of the fractions were assayed for phospholipid
content using the BioMerieux enzymatic assay. The remainder of each
fraction was counted for 3 minutes in a Wallach 1410 liquid
scintillation counter (Pharmacia) using the Easy Count program. The
results of these analyses are shown in FIGS. 6-8. It can be seen
that the vast majority of both phospholipid and peptide are
recovered together in a few fractions with peaks at approximately
16, 16, and 15 ml for complexes prepared at 3:1, 4:1 and 5:1
DPPC:peptide ratios, respectively. The UV absorbance profiles for
these samples indicate that the complexes elute from the column at
volumes 15.1, 14.7 and 14.4 ml for complexes prepared at 3:1, 4:1
and 5:1 DPPC:peptide ratios, respectively (the dead volume of
tubing between the fraction collector and UV flow cell is 1.3 ml,
which explains a slight discrepancy between the elution volumes as
measured by radioactivity/phospholipid assay and UV absorbance).
The elution volumes correspond to Stoke's diameters of 106, 114,
and 120 Angstroms for the 3:1, 4:1 and 5:1 Ri complexes,
respectively. TABLE-US-00001 TABLE 1 % of Applied DPPC:Peptide 1
Elution Relative Size of Phosphalipid in ratio Volume Particles*
Absorbance Peak HDL 14.8 -- -- 1:1 16.2 and 18.1 Smaller 87% 2:1
16.4 Smaller 70% 3:1 16.0 Smaller 79% 4:1 15.7 Smaller 106% 5:1
15.1 Smaller 103% 7.5:1 13.6 Larger 92% 10:1 13.4 Larger 103% 15:1
ND** ND ND *Relative to size of HDL particles **ND, not done
[0087] The present invention is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
Sequence CWU 1
1
* * * * *