Peptide/lipid complex formation by co-lyophilization

Details Peptide/lipid complex formation by co-lyophilization Peptide/lipid complex formation by co-lyophilization

1997-10-02

2001-09-11

Kishore, Gollamudi S. (Department: 1615)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C514S002600, C514S012200, C514S013800

Reexamination Certificate

active

06287590

ABSTRACT:

1. FIELD OF THE INVENTION
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
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.”
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.
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.
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.
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.
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° 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.
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.
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).
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)
A large unilamellar vesicle (LUV) is similar to SUVs in hat 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.
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.
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.
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
It is has been a goal of liposome research to develop liposome preparations that may be stored for long periods of time before use.

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