MXPA00003132A - Peptide/lipid complex formation by co-lyophilization - Google Patents

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.

Description

TRAINING OF THE PEPTIDE / LIPID COMPLEX BY CO-LIOFILIZATION 1. FIELD OF THE INVENTION The invention relates to the formation of peptide / lipid vesicles and complexes through the co-lyophilization of the peptides, preferably they can adopt an alpha-helical, antipathetic conformation and one or more lipids. It is possible to lyophilize a single solution which solubilises both the peptides and the lipids or two separate solutions. 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 composite vesicles of at least one lipid bilayer membrane containing an aqueous core. In general, phospholipids comprise the lipid bilayer, but the bilayer may be composed of other lipids. The aqueous solution within the liposome is known as the "captured volume". Liposomes have been developed as vehicles to supply medicines, cosmetics, bioactive compounds among other applications. The lipid bilayer encapsulates the medicament, cosmetic, active compound and the like within the captured volume of the liposome and the medicament is expelled from the liposome nucleus when the lipid bilayer comes into contact with a cell surface membrane. Liposomes release their contents to the cell by lipid exchange, fusion, endocytosis or adsorption. Ostro et al., 1989, Am. J. Hosp. . Pharm. 46: 1576. Otherwise, the cosmetic medicament, bioactive compound and the like may be associated with or inserted into the bilayer lipid membrane of the vesicle. In addition to the vesicles, the lipid-containing complexes have been used to deliver compounds in the form of particles. For example, many researchers have found it useful to prepare reconstituted lipoprotein-like particles or complexes that 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. Occasionally, unesterified cholesterol is also included. The most common methods of preparing these particles are: (1) co-sonification of the constituents, by sonification in a bath or with a sonic probe, (2) spontaneous interaction of the protein constituent with the 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 & Annantharamaiah, 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 replacing antipathetic helix-forming peptides for the apoprotein components. Unfortunately, each of these methods presents serious problems for the formation of large quantities of pure complexes on a reasonably cost-effective basis. Furthermore, none of these publications describe the co-lyophilization of peptides or peptide analogs that can adopt an alpha helical conformation, antipathetic, and a lipid. A range of technologies are known to produce lipid and complex vesicles. Vesicles, or liposomes, have been produced using a variety of protocols, forming different types of vesicles. Different types of liposomes include: multilamellar vesicles, small unilamellar vesicles and large unilamellar vesicles. The hydration of phospholipids (or other lipids) by aqueous solution can also cause the dispersion of lipids and the spontaneous formation of multimelar vesicles [sic] ("MLV"). An MLV is a liposome with multiple lipid bilayers surrounding the aqueous core. These types of liposomes are larger than small unilamellar vesicles (SUV) and can be 350-400 nm in diameter. The 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 rehydrated. The vesicles formed as the flask moved in a swirl. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citing Bangha, et al., 1965, J. Mol. Biol. 13: 238) Johnson et al., Subsequently reported that this method also generated individual lamellar vesicles. Johnson et al., 1971, Biochim. Biophys. Acta 233: 820. A small unilamellar vesicle (SUV) is a liposome with a single lipid bilayer enveloping an aqueous core. Depending on the method used to generate the SUVs, these can be in the size range from 25-110 nm in diameter. The first SUVs were prepared by drying the 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 clarify. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York. SUVs prepared in this way produced liposomes in the 25-50 nm diameter range. Another method for preparing the SUVs is by 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, Bi ophys, Acta 298: 1015). The SUVs produced by this method have a size range from 30-110 nm in diameter. SUVs can also be produced by passing the multilamellar vesicles through a French press four times at 20,000 psi. The SUV produced will have a size range 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 that they are individual lipid bilayers surrounding the central aqueous core, but LUVs are much larger than SUVs. Depending on their constituent parts and the method used to prepare them, the LUV can be in the size range 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: dilution in detergent, reverse phase evaporation and infusion. In the detergent dilution technique, detergent solutions such as cholate, deoxycholate, octylglucoside, heptylglucoside and Triton X-100 are used to form micelles from the lipid preparation. The solution is then dialyzed to remove the detergent and the liposomes are formed. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York. This method is delayed and the removal of the detergent is usually incomplete. The presence of detergent in the final preparation may give rise to some toxicity of the liposome preparation and / or modification of the physicochemical properties of the liposome preparation. The reverse phase evaporation technique solubilizes the lipids in non-polar aqueous solutions, forming inverted micelles. The non-polar solvent is evaporated and the micelles are added to form LUV. This method generally requires a great handling of lipids. The infusion method injects a lipid solubilized in a non-polar solution to the aqueous solution to be encapsulated. As the non-polar solution evaporates, the lipids are collected at the gas / aqueous interface. The lipid sheets form the LUV and the oligolamellar liposomes as the gas bubbles through the aqueous solution. Liposomes are sized to size 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). The infusion procedures require a very high temperature for the infusion and may have a relatively low encapsulation efficiency. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York. It has been a goal of liposome research to develop liposome preparations that can be stored for prolonged periods before use. For example, U.S. Patent No. 4,229,360 to Schneider et al., Describes a method for 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 the hydrophilic compounds are hydrophilic polymers of high molecular weight or low molecular weight compounds such as sucrose. U.S. Patent No. 4,411,894 to Shrank et al., Discloses the use of high sucrose concentrations in sonicated preparations of liposomes. Liposomes contain fat-soluble products in the captured volume, although the preparations could be lyophilized, the method can not prevent the loss of a significant amount of the captured content despite the high concentration of sucrose. Crowe et al., U.S. Patent No. 4,857,319 discloses the use of disaccharides such as sucrose, maltose, lactose and trehalose to stabilize liposomes when the 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 compared to that produced by the method described by Shrank in U.S. Patent No. 4,441,894. Janoff et al., US Patent No. 4,880,635 discloses a method for dehydrating liposomes, in which the liposomes were lyophilized in the presence of protective sugars such as trehalose and sucrose, preferably in the inner and outer leaflets of the lipid bilayer. In the method of Janoff et al., Sufficient water is retained, so that rehydration of the dehydrated liposomes produces liposomes with substantial structural integrity. However, there is a need in the art for a simple and cost-effective method for forming lyophilized peptide / lipid complexes that can then be rehydrated. The method of the present invention produces peptide / lipid mixtures in a stable, lyophilized powder that can be stored, used as a powder, or used after rehydration to form peptide / lipid complexes. 3. COMPENDIUM OF THE INVENTION The invention is a method for preparing peptides or protein- (phospho) lipid complexes or vesicles that may have the characteristics similar to high density lipoproteins (HDL). The method includes a solvent system in which at least one peptide is solubilized in a solution, and at least one lipid is solubilized in another solution. The two solutions are selected so that they are miscible with each other. The solutions are then combined and the resulting solution is lyophilized. The method can also be practiced by a second type of solvent system consisting of a solution in which the protein or peptide and the lipid can be solubilized. This solution can be a single solution, or it can be a composite solution prepared by combining two or more solutions before the addition of the peptides and lipids. The peptides and lipids are solubilized in the solution or compound solution and the peptide / lipid solution is then lyophilized. Preferably, the peptides of the present invention are peptides that can adopt an antipathetic helical conformation. In a specific embodiment of the invention, the peptide is a protein that binds to lipids. In another embodiment, analogous peptides of ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III ApoE, other apolipoprotein analogs and the like are used in place of or in combination with the peptides. ApoA-I / lipid complexes are useful in the treatment of abnormalities associated with dyslipoproteinemias including, but not limited to, hypercholesterolemia, hypertriglyceridemia, low HDL and apolipoprotein Al deficiency, septic shock for in vitro diagnostic assays as markers for HDL populations , and for use with image forming technology. The method of the invention allows the preparation of peptide / lipid complexes for parenteral administration including, but not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular and bolus injections to animals or humans. In addition, the peptide / lipid complexes can also be formulated for oral, rectal, mucosal (eg, oral cavity) or topical administration to animals or humans, or for in vitro experimentation.

The method can be used for large-scale production of amphiphatic peptide / phospholipid complexes, lipid-binding protein / phospholipid complexes and / or apoA-I peptide analog / phospholipid complexes. The lyophilized material can be prepared for bulk preparations or, otherwise, the combined peptide / lipid solution can be distributed in smaller containers (eg, individual dosage units) before lyophilization, and these smaller units can be prepared as sterile unit dosage forms. The lyophilized powder, prepared by the method of the invention, can be rehydrated in sterile, particle-free solution immediately before injection or, otherwise, the lyophilized powder can be formulated into a suitable solid dosage form and administered directly. The method may also be suitable for the storage of compounds that may otherwise be unstable or insoluble in the absence of lipids. The method can be used for the formulation of products for the treatment or prevention of human diseases, including applications such as the co-presentation of antigens in vaccines, the treatment or prevention of dyslipoproteinemias, including but not limited to hypercholesterolemia, hypertriglyceridemia, HDL low, apolipoprotein Al deficiency, cardiovascular diseases such as atherosclerosis, septic shock, or infectious diseases. The method can be used for the preparation of complexes that could be used as carriers of drugs, as vectors (to deliver drugs, DNA, genes), for example, to the liver or extrahepatic cells, or as purifiers to trap toxins (for example , pepticides, LPS, etc.). 3. 1. DEFINITIONS As used herein, a "solvent system" refers to one or more solvents that can solubilize peptides and / or lipids and, if more than one, that are miscible with each other. As used herein, "peptide / lipid complexes" refers to an aggregation of lipid portions and peptides forming particles within the size range of high density lipoproteins (HDL). As used herein, "co-freeze-dried" refers to lyophilization, freeze drying or vacuum drying of more than one compound (eg, peptide, protein, lipid, phospholipid) in solution, in the same container. For example, a lipid solution can be combined with a solution of peptides in the same container and the resulting combination of the solutions is lyophilized by lyophilizing the peptides and lipids at the same time. As used herein "unfriendly peptides" or "helical, antipathetic alpha peptides" means peptides that may adopt an unsympathetic helical or helical conformation, respectively. The antipathic alpha helix is a secondary structure motif that is often found in biologically active peptides and proteins. See, Amphipatic helix motif: classes and properties by Jere P. Segrest, Hans de Loof, Jan G. Dolhman, Christie G. Brouillette, and G. M. Anantharamaiah. PROTEINS: Structure Functions and Genetics 8: 103-117 (1990). An antipathic alpha helix is an alpha helix with opposing polar and non-polar faces oriented along the longitudinal axis of the helix. A specific distribution of the waste with charge along the polar face is evident. The antipathic helices, as defined, are complementary to the polar-non-polar interface of the mass phospholipid, hydrated; these domains of lipid association have been postulated to interact with the phospholipid by partial immersion of these at the interface between the fatty acyl chains and the polar head groups. Jere P. Segrest. Febs Letters 1976, 69 (1): 111-114. The term "peptide" and "protein" can be used interchangeably herein. In addition, the peptide analogs of the invention may be peptides, proteins or non-peptides, ie, peptide mimetics. However, all analogues are preferably bioactive molecules. The term "lipid" as used herein includes, but is not limited to, natural and synthetic phospholipids. In addition, the terms "lipid" and "phospholipid" can be used interchangeably herein. 4. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Superlose 6 chromatography of HDL prepared by density ultracentrifugation from 200 μl of human serum. Figure 2 (bottom): chromatography on Super6 complex 6 (DPPC: peptide 1) (PVLDLFRELLNELLEALKQKLK; SEQ ID NO: 1) prepared in a ratio 1: 1 (p: p) Figure 2 (upper): chromatography on Superóse 6 de complexes (DPPC: peptide 1) prepared in a 2: 1 ratio (p: p) Figure 3 (bottom): chromatography on Super 6 complexes (DPPC: peptide 1) prepared in a 3: 1 ratio (P: p) Figure 3 (upper): chromatography on Super 6 complexes (DPPC: peptide 1) prepared in a 4: 1 ratio (P: p) Figure 4 (bottom): chromatography on Super 6 complexes (DPPC: peptide 1) prepared in a 5: 1 ratio (p: p) Figure 4 (upper): chromatography on Super 6 complexes (DPPC: peptide 1) prepared in a 7.5: 1 ratio (p: p) Figure 5: Super 6 complex chromatography (DPPC: peptide 1) prepared in a 10: 1 ratio (p: p). Figure 6: Superfilter 6 chromatography of peptide 1 complexes labeled with 14C to Ri = 3.1. Figure 7: Chromatography on Super6 6 of peptide 1 complexes labeled with 14C to Ri = 4.1. Figure 8: Superfilter 6 chromatography of peptide 1 complexes labeled with 14C to Ri = 5.1.

. DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The alpha helical peptides or proteins, unfriendly, lipid binding proteins, ApoA-I agonist peptides, apoprotein analogs and the like, which are useful in the present invention, can be synthesized or manufactured using any known technique. Stable preparations of the peptides having a prolonged storage life can be prepared by lyophilizing the peptides, preparing a bulk for reformulation, or preparing individual aliquots or dosage units that can be reconstituted by rehydration with sterile water or a buffered solution , sterile, suitable before administration to an individual.

As far as the inventors are aware, this invention is the first case of a method for co-lyophilizing a helical, antipathetic or peptide analog peptide with a lipid to form a mixture that can be reconstituted into a sterile peptide / lipid complex. In certain embodiments, it may be preferable to formulate and administer the ApoA-I analog (s) included, but not limited to, the ApoA-I agonists, in a peptide / lipid complex. This approach has some advantages since the complex must have an increased half-life in the circulation, particularly when the complex has a size and density similar to the HDL class of the proteins, especially the pre-beta populations of the HDL. The HDL class of lipoproteins can be divided into some subclasses based on characteristics such as size, density and electrophoretic mobility. Some examples, in order of increasing size are HDL pre-beta micellar diameters of 50 to 60 angstroms, discoidal HDL of intermediate size, that is, with a mass of 65 kDa (approximately 70 angstroms), HDL3 or spherical HDL2 of diameter 90 to 120 angstroms. (J. Kane, 1996 in V. Fuster, R. Ross and E. Topol [eds.] Atherosclerosis and Coronary Artery Disease, p.99; A. Tall and J. Breslow, ibid., P.106; Barrans et al. ., Biochemica et Biophysica Acta 1300, pp. 75-85, and Fielding et al., 1995, J. Lipid Res 36, p, 211-228). However, peptide / lipid complexes of smaller or larger size than HDL can also be formed by the invention. The peptide / lipid complexes of the present invention can be conveniently prepared as stable preparations, having a prolonged storage life, by the co-lyophilization process 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 that can be reconstituted by rehydration with sterile water or a suitable buffered solution prior to administration to an individual . Applicants have developed a simple method for preparing peptide or protein- (phospho) lipid complexes having characteristics similar to HDL. This method can be used to prepare complex ApoA-I-lipid peptides, and has the following advantages: (1) most or all of the included ingredients are used to form the designed complexes, thus avoiding wasting of the initial material that is common in other methods. (2) Freeze-dried compounds are formed which are very stable during storage. The resulting complexes can be reconstituted immediately before use. (3) Usually, the resulting complexes do not require further purification after training or before use. (4) toxic compounds are avoided, including detergents such as cholate. In addition, the production method can easily be scaled and is suitable for manufacturing by BPM (ie, in an environment without endotoxins). According to the preferred method, the peptide and lipid are combined in a solvent system that co-solubilizes each ingredient. For this purpose, the pair of solvents must be carefully selected to guarantee the co-solubility of the antipático peptide and the hydrophobic lipid. In one embodiment, the protein (s) or peptide (s) to be incorporated into the particles can be dissolved in a solvent or mixture of solvents (solvent 1), aqueous or organic. The component (toasted) lipid is dissolved in an aqueous or organic solvent or mixture of solvents (solvent 2) that is miscible with solvent 1, and the two solutions are combined. Otherwise, the (tost) lipid component dissolves directly in the peptide (protein) solution. Otherwise, the peptide and the lipid can be incorporated into a co-solvent system, i.e., a mixture of miscible solvents. Depending on the lipid binding properties of the peptide or protein, those skilled in the art will recognize that improved solubilization or even complete and / or improved mixing may be necessary prior to lyophilization, in this manner, the solvents can be chosen accordingly. A suitable ratio 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 HDL2 or HDL3. The molar ratio of the lipid to the protein / peptide should be in the range of about 2 to about 200, and preferably 5 to 50, depending on the type of complexes desired. Examples of these size classes of the peptide / lipid or protein / lipid complexes include, but are not limited to, micellar or discoidal particles (usually smaller than HDL3 or HDL2), spherical particles of similar size to HDL2. or HDL3 and larger complexes that are larger than HDL2. The HDL used by us as a standard during chromatography (Figure 1) are mainly HDL2 mature, spherical. Pre-ßl HDL are micellar complexes of apolipoprotein and few phospholipid molecules. Pre-ß2 HDL are discoidal apolipoprotein complexes and phospholipid molecules. The more lipid triglycerides) cholesterol, phospholipids, are incorporated the larger HDL will be and their shape is modified. (HDL pre-ßl (micellar complex) = >; HDL pre-ß2 (discoidal complex) = > HDL3 (spherical complex) = > HDL2 (spherical complex). Once the solvent is chosen and the peptide and lipid are incorporated, the resulting mixture is frozen and lyophilized to dryness. Occasionally, an additional solvent is added to the mixture to facilitate lyophilization. This lyophilized product can be stored for prolonged periods and will remain stable. In the working examples described below, the peptide 1 PVLDLFRELLNELLEALKQKLK (SEQ ID NO: 1) and the (phospho) lipid were dissolved separately in methanol, combined and then mixed with xylene before lyophilization. The peptide and lipid can be added to a mixture of the two solvents. Otherwise, a solution of the peptide dissolved in methanol can be mixed with a solution of the lipid dissolved in xylene. Care must be taken to avoid saline displacement of the peptide. The resulting solution containing the peptide and lipid co-solubilized in methanol / xylene is lyophilized to form a powder. The lyophilized product can be reconstituted to obtain a solution or suspension of the peptide / lipid complex. To this end, the lyophilized powder is rehydrated with an aqueous solution at an adequate volume (frequently about 5 mg of the peptide / ml which is convenient for intravenous injection). In a preferred embodiment, the lyophilized powder is rehydrated with saline buffered with phosphate or a physiological saline solution. It is possible that the mixture has to be stirred to facilitate rehydration, and in some cases, the reconstitution step must be performed at a temperature equal to or greater than the transition temperature of the phase (Tm) of the lipid component of the complexes. In a few minutes a solution of the reconstituted lipid-protein complexes is obtained (a clear solution when the complexes are small). It is possible to characterize an aliquot of the resulting reconstituted preparation to confirm that the complexes in the preparation have the desired size distribution, for example, the size distribution of the HDL. For this purpose it is possible to use gel filtration chromatography [sic]. In the working examples described infra, gel filtration chromatography with Superóse 6 FPLC from Pharmacia was used. The eluent used contained 150 mM NaCl in deionized water. A common sample volume is 20 to 200 μl of the complexes containing 5 mg peptide / ml. The flow velocity in the column is 0.5 ml / min. A series of proteins of known molecular weight and diameter of stokes, as well as human HDL are used as standards to calibrate the column. Protein and lipoprotein complexes are monitored by absorbance or light scattering of wavelength 254 or 280 nm.

Solvents that can be used according to the method of the present invention include, but are not limited to, non-polar, polar, aprotic and organic protic solvents, and the like such as ethanol, methanol, cyclohexane, 1-butanol, isopropyl alcohol, xylene, THF, ether, methylene chloride, benzene and chloroform. The invention also includes the use of solvent mixtures as well as individual solvents. In addition, before use within the present methods, organic solvents can be dried to remove water; however, it is possible to use hydrated solvents or water with certain lipids, peptides or proteins. In other words, water can be a suitable solvent, or hydrated solvents or organic solvent / water mixtures can be used, however, if water is used it must be free of detergent. As already mentioned, the solvents are preferably of the purest quality (to avoid concentration of impurities after lyophilization), and the solvents must be free of salts and free of particulates. However, it is not necessary for the solvents to be sterile since the resulting product can be sterilized before, during or after lyophilization, according to known pharmaceutical techniques, such as those described in Remington Pharmaceutical Sciences, issues 16 and 18 , Mack Publishing Co., Easton, Pennsylvania (1980 and 1990), which is incorporated herein by reference in its entirety, and in the United States / National Formulary Pharmacopeia (USP / NF) XVII, incorporated herein by reference in their strengths. Lipids which can be used according to the method of the present composition [sic] include but are not limited to: natural and synthesized (synthetic) lipids and phospholipids including phospholipids with small alkyl chains, phosphatidylcholine, egg, soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoyl phosphatidylcholine, l-myristoyl-2-palmitoylphosphatidylcholine, l-palmitoyl-2-myristoylphosphatidylcholine, l-palmitoyl-2-stearoylphosphatidylcholine, l-stearoyl-2-palmitoylphosphatidylcholine , dioleofosfatidiletanolamina, dilauroilfosfatidilglicerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, sphingolipids, phosphatidylglycerol, diphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimiristoilfosfatídico acid, palmitoilfosfatídico acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimiristoilfosfatidilserina, dipalmitoylphosphatidylserine, phosphatidylserine, brain sphingomyelin brain , dipalmitolesfingo ielina, distearoilesfingomielina, fosfatídico acid, galactocerebrósido, gangliósi two, cerebrosides, dilaurylphosphatidylcholine, (1,3) -D-mannosyl- (1,3) diglyceride, aminophenylglucoside, 3-cholesteryl-6 '- (glucosylthio) hexyl ether glycolipids, and cholesterol and its derivatives. Peptides that are suitable for use with the present invention include, but are not limited to, those that are described in the three co-pending applications [Series Nos., Identified by the attorney-in-fact's dossiers.

Nos. 9196-0004-999, 9196-0005-999 and 9196-0006-999, each of which was filed on September 29, 1997] each of which is incorporated herein by reference in its entirety. It is preferred, although not necessary in each case, that the precipitates must be solubilized or removed before agitation mixing of the lipid and peptide solutions or before lyophilization. The method can be used for large-scale production of peptide / lipid complexes, peptide / antiphatic (phospho) lipid complexes, lipid / (phospho) lipid binding protein complexes, and / or peptide analog complexes. ApoA-I / (phospho) lipid. The lyophilized material can be prepared for bulk preparations, or else, the mixed peptide / lipid solution can be distributed in smaller containers (eg, single dose units) before lyophilization, and these smaller units can be prepared as sterile unit dosage forms. The vacuum-dried compositions of the present invention can be provided in container forms for single dose or multiple doses by aseptic filling of the suitable containers with the sterile, previously vacuum-dried solution at a prescribed content; preparing the desired vacuum-dried compositions; and then sealing the container with the single dose or multiple doses. It is intended that these filled containers will allow rapid dissolution of the dehydrated composition upon reconstitution with suitable sterile diluents in situ producing a suitable 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 small bottle, capable of delivering a vacuum dried product, hermetically sealed by a capping means. In addition, suitable containers imply adequacy in size, considering the volume of the solution to be maintained with the reconstitution of the composition dried in vacuum; and adequacy of the material of the container, generally type I glass. The capping means used, for example, sterile rubber closures or equivalent, should be understood as those that provide the aforementioned seal, but also allow entry for the purpose of introduction of a diluent, for example, sterile water for injection, USP, normal USP saline or 5% dextrose in water, USP, for the reconstitution of the desired solution. These and other aspects of the suitability of the containers for pharmaceutical products such as those of the present invention are well known to those skilled in the pharmaceutical art practice. In specific embodiments, the sizes of unit dosages of the product 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 a concentration after reconstitution of about 1 to 50. mg / ml, preferably approximately 2 to 25 mg / ml. The method of the invention allows 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 administration (e.g., oral cavity) ) or topical to animals or humans, or for in vitro experimentation. The lyophilized powder prepared by the method of the invention can be rehydrated immediately prior to injection, or otherwise, the lyophilized powder can be administered directly. The lyophilized powder includes, but is not limited to, lipid and peptides that can form complexes in the form of vesicles, liposomes, particles including spherical or discoidal particles, micelles and the like. To reconstitute or rehydrate the lyophilized powder a solution is chosen depending on the desired end use. For pharmaceutical use it is possible to use any sterile solution. further, buffered solutions are preferred for certain uses and these include, but are not limited to, phosphate, citrate, tris, barbital, acetate, glycine-HCl, succinate, cacodylate, boric acid-borax, amediol and carbonate. The lyophilized powder of the present invention can be formed using any lyophilization method known in the art, including, but not limited to, freeze dehydration in which the solution containing the peptide / lipid is subjected to freezing followed by pressure evaporation reduced. The method may also be suitable for storing compounds that may otherwise be unstable or insoluble in the absence of lipids. The method can be used for the formulation of products for the treatment or prevention of diseases in humans, including applications such as co-presentation of antigens in vaccines, treatment or prevention of dyslipoproteinemias including, but not limited to, hypercholesterolemia, hypertriglyceridemia, low HDL and apolipoprotein Al deficiency, cardiovascular diseases such as atherosclerosis, septic shock, or infectious diseases. The method can be used for the preparation of complexes which can be used as carriers for drugs, as vectors (for supplying drugs, DNA, genes) for example, to the liver or extrahepatic cells, or as purifiers for trapping toxins (for example, pesticides, LPS, etc.). Otherwise, the method can be used to prepare complexes for in vitro study systems, or to use in imaging technology. In specific embodiments, the method can be used for the preparation of the ApoA-I analogue (which includes, but is not limited to agonists) complexes that can be used in in vitro diagnostic assays and as markers for populations and sub-populations. HDL populations. In other specific embodiments, complexes of ApoA-I agonists can be used for immunoassays or for imaging technology (e.g., CAT scans, MRI scans). The following examples are proposed as illustrative of the present invention and should not be considered, in any sense, as a limitation thereof. 6 EXAMPLE: PREPARATION OF THE PEPTIDE-LIPID COMPLEX BY THE CO-LIOFILIZATION METHOD The following protocol was used to prepare the peptide-lipid complex. Peptide 1 (PVLDLFRELLNELLEALKQKLK; SEQ ID NO: 1) (22.4 mg) was dissolved in methanol in a concentration of 3.5 mg / ml by incubation for a few minutes and mixing by vortex intermittently. To this solution was added dipalmitoyl phosphatidylcholine (DPPC) in methanol (100 mg / ml of standard solution) so that the final ratio of DPPC / peptide was 2.5: 1 (w / w). This solution was mixed producing vortex. Xylene was added to the solution for a final concentration of 36%. Aliquots of the resulting solution were separated for further analysis by gel filtration chromatography. The solutions were frozen in liquid nitrogen and lyophilized to dryness under vacuum. An aliquot containing 20 mg of peptide 1 (SEQ ID NO: 1) and 50 mg of DPPC was rehydrated in sterile saline (0.9% NaCl), mixed and heated at 41 ° C for a few minutes until a clear complex solution resulted peptide / phospholipid reconstituted. 6. 1 EXAMPLE: GEL FILTRATION AND USE OF PHOSPHOLIPID 6.1.1 MATERIALS AND METHODS For the purpose of testing the conditions for the preparation of complexes it is often convenient to prepare small amounts of complexes for characterization. These preparations contained 1 mg of the peptide and were prepared as follows: 1 mg of peptide 1 (SEQ ID NO: 1) was dissolved in 250 μl of HPLC grade methanol (Perkin Elmer) in a small 1.0 ml clear glass bottle with lid (Waters # WAT025054). Peptide dissolution was aided by occasional vortex shaking for a period of 10 minutes at room temperature. After this time, a small amount of particulate, undissolved material could still be observed, but this did not adversely affect the results. To this mixture was added an aliquot containing 1, 2, 3, 4, 5, 7.5, 10 or 15 mg DPPC (Avanti Polar Lipids, 99% purity, product # 850355) of a standard solution of 100 mg / ml in methanol . The volume of the mixture was brought to 400 μl by the addition of methanol and the mixture was again stirred vortexing intermittently for a period of 10 minutes at room temperature. At this time, very little undissolved material could be observed in the tubes. To each tube, 200 μl of xylene (Sigma-Aldrich, 99% pure, HPLC grade) were added and the tubes were shaken in a vortex for 10 seconds each. In the plugs of each tube, two small holes were drilled with a 20 gauge syringe needle, the tubes were frozen for 15 seconds each in liquid nitrogen, and lyophilized overnight under vacuum. To each tube were added 200 ml of 0.9% NaCl solution. The tubes were shaken in a vortex 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 ° C. The solutions in all the tubes became clear (ie, similar to water in appearance) minus the tube containing 15 mg of PPC, which remained milky. To determine if all the phospholipids that were used in the complex preparations actually appeared in the fractions of the column corresponding to the absorbance peaks of the chromatogram, the eluate of the column, of the reconstituted peptide / lipid complexes was collected in fractions of 1 or 2 ml and the fractions were assayed enzymatically for the phospholipid content BioMerieux Phospholipides Enzymatique PAP 150 Kit (# 61491) according to the supplier's instructions. The preparations of the complexes can also be made on a large scale. An example of such a preparation is reported before. These complexes were used for in vivo experiments. 6. 2. RESULTS OF THE CHARACTERIZATION OF THE COMPLEX Figure 1: chromatography on Super6se 6 of mature HDL2 prepared by ultra centrifugation by density from 200 μl of human serum. Chromatography shows absorbance at 254 nm. The volume of the elution = 14.8 ml, corresponding to the Stokes diameter of 108 angstroms (see Table 1). Figure 2 (bottom): Super6se chromatography of DPPC complexes: peptide 1 prepared in an incubation ratio (Ri, defined as the ratio of the total phospholipid to the total peptide in the initial mixture) of 1: 1 (p: p) as described above (small scale preparation). The volumes of the elution of the absorbance peaks = 16.2 ml and 18.1 ml correspond to the particles of Stokes diameters of 74 and 82 angstroms, which are smaller than HDL. 86% of the phospholipid applied to the column was recovered in the fractions containing the absorbance peaks (see Table 1). Figure 2 (upper): Super6se chromatography of 6 complexes of PPC: peptide 1 prepared in a Ri of 2: 1 (p: p) as described above. The elution volume of the 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). Figure 3 (bottom): Super6se chromatography of DPPC complexes: peptide 1 prepared in a Ri of 3: 1 (p: p) as already described. Absorbance peak elusion volume = 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). Figure 3 (upper): Super6se 6 chromatography of DPPC complexes: peptide 1 prepared in a Ri of 4: 1 (p: p) as already described. Volume of elution 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). Figure 4 (bottom): Super6se 6 chromatography of DPPC complexes: peptide 1 prepared in a Ri of 5: 1 (p: p) as already described. Absorbance peak elusion volume = 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). Figure 4 (upper): Super6se 6 chromatography of DPPC: peptide 1 complexes prepared in a Ri of 7.5: 1 (p: p) as already described. Absorbance peak elusion volume = 13.6 ml, (134 angstroms) corresponding to particles smaller than HDL. 92% of the phospholipid applied to the column was recovered in the fractions containing the absorbance peak (see Table 1). Figure 5: Super6se 6 chromatography of DPPC: peptide 1 complexes prepared in a ratio of 10: 1 (p: p) as described above. Absorbance peak elution volume = 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). The sample containing the complexes with 15: 1 DPPC: peptide 1 (p: p) was not subjected to chromatography on Super6se 6 because it was turbid, suggesting the presence of large particles. For each of the above experiments no significant phospholipid was observed in any fraction other than those containing the material eluting with the absorbance peaks (see Figures 2-8). This suggests that almost all phospholipids (within the experimental error of the assay) were incorporated into the complexes. The experiment shows that by varying the initial ratio of the phospholipids to the peptides, it is possible to form homogeneous complexes of different sizes (smaller or larger than HDL). 6. 3. CHARACTERIZATION OF COMPLEXES USING PEPTIDE 1 MARCHED WITH 14C Peptide / phospholipid complexes containing peptide 1 labeled with 1C (specific activity 159,000 DPM / mg of peptide by weight, assuming 50% peptide content) were prepared by co-lyophilization as already described . The preparations each contained 1 mg of peptide and 3, 4 or 5 mg of DPPC by weight. After reconstituting the complexes in 200 ml of 0.9% NaCl, 20 μl (100 μg) of the complexes were applied to a Pharmacia Super 6 column using 0.9% NaCl as the liquid phase at a flow rate of 0.5 ml / min. After a delay of 5 ml (empty volume of the column = 7.7 ml) fractions of 1 ml were collected. Aliquots containing 20 μl of the fractions were assayed for the phospholipid content using BioMerieux enzymatic assay. The remainder of each fraction was counted for three minutes in a Wallach 1410 liquid scintillation counter (Pharmacia) using the Easy Count program. The results of these analyzes are shown in Figures 6-8. It can be seen that the vast majority of the phospholipid and the peptide are recovered together in a few fractions with peaks in approximately 16, 16 and 15 ml for complexes prepared in 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 in volumes 15: 1, 14.7, and 14.4 ml for complexes prepared in 3: 1, 4: 1, and 5: 1 DPPC: peptide, respectively , (the dead volume of the tube between the fraction connector and the UV flow cell is 1.3 ml, which explains a slight discrepancy between the volumes of the elution when measured by radioactivity / phospholipid assay and UV absorbance). The volumes of the elution correspond to the Stoke diameters of 106, 114, and 120 angstroms for the complexes with Ri 3: 1, 4: 1 and 5: 1, respectively. * in relation to the size of the HDL ** NR particles, it was not carried out The present invention should not be limited in scope by the specific embodiments described which are proposed as simple illustrations of the individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the aforementioned description and the accompanying drawings. These modifications are proposed to fall within the scope of the appended claims.

Claims (37)

1. A method for preparing a lyophilized peptide / lipid product which consists of co-lyophilizing one or more peptides, which are capable of adopting an antipathetic conformation, or peptide analogs, and one or more lipids in a solvent system to form a peptide / lipid product , wherein the product can be rehydrated to form peptide / lipid complexes.

2. The method of claim 1, wherein the peptide is a protein that binds lipids.

3. The method of claim 1, wherein the peptide analog is an analogue of ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoE or other apoprotein.

4. The method of claim 1, wherein the peptide is a protein.

The method of claim 1, wherein the lipid is a natural lipid, synthetic lipid, saturated lipid, unsaturated lipid or mixtures thereof.

The method of claim 5, wherein the lipid is selected from the group consisting of phosphatidylcholine, egg, soybean phosphatidylcholine, ether phospholipids, small alkyl chain phospholipids, cholesterol, cholesterol derivatives, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, 1- 2-palmitoylphosphatidylcholine iristoil-, l-palmitoyl-2-miristoilfosfatidilcolina, l-palmitoyl-2-stearoyl, l-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleofosfatidiletanolamina, dilauroilfosfatidilglicerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, sphingolipids , phosphatidylglycerol, diphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolam ina, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, dipalmitolesphingomyelin, distearoylphingomyelin, phosphatidic acid, galactocerebrosides, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3) -D-mannosyl- (1,3) -diglyceride, aminophenyl glucoside and -cholesterol-6 '- (glucosylthio) hexyl ether glycolipids and mixtures thereof.

The method of claim 1, which further comprises sterilizing the product before, during or after lyophilization.

The method of claim 1, wherein the peptide / lipid complex is sterile.

9. The method of claim 1 further comprises taking aliquots of a solution of the peptide and the lipid in individual containers prior to lyophilization to prepare a single unit dosage form.

A pharmaceutical unit unit dosage form containing a lyophilized, sterile peptide / lipid mixture prepared according to claim 1 to 7.

11. A method of preparing a lyophilized peptide / lipid product, consisting of: a) solubilizing at least one antipyretic peptide or peptide analogue in a first solution, (b) solubilizing at least one lipid in a second solution, wherein the second solution is miscible with the first solution, (c) combining the first solution with the second solution to form a peptide / lipid solution, and (d) freeze-drying the peptide / lipid solution so that the lyophilized peptide / lipid product is formed, which can be rehydrated to form peptide / lipid complexes.

The method of claim 11, wherein the peptide is a protein that binds lipids.

The method of claim 1, wherein the peptide is a protein.

The method of claim 9, wherein the 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 the lipid is a natural lipid, a synthetic lipid, a saturated lipid, an unsaturated lipid or mixtures thereof.

16. The method of claim 15, Wherein the lipid is selected from the group consisting of egg phosphatidylcholine, cholesterol, cholesterol derivatives, ether phospholipids, phosphatidylcholine soybean phospholipids chain small alkyl, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, l-myristoyl-2 -palmitoilfosfatidilcolina, 1-palmitoi1-2-miristoilfosfatidilcolina, l-palmitoyl-2-stearoyl, l-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleofosfatidiletanolamina, dilauroilfosfatidilglicerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, sphingolipids, phosphatidylglycerol, diphosphatidylglycerol, dimyristoylphosphatidylglycerol , dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimiristoilfosfatídico acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimiristoilfosfatidilserina, di palmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, dipalmitolesphingomyelin, distearoylphingomyelin, phosphatidic acid, galactocerebrosides, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3) -D-mannosyl- (1,3) -diglyceride, aminophenylglucoside and 3-cholesteryl-6 '- (glucosylthio) hexyl ether glycolipids, and mixtures thereof.

The method of claim 11, wherein the peptide / lipid solution is sterile.

18. The method of claim 11, wherein the peptide / lipid complex is sterile.

19. The method of claim 11, further comprising aliquoting of the peptide / lipid solution into individual containers before lyophilization to produce a single unit dosage form.

20. The method of claim 11, which further comprises a sterilization step before, during or after lyophilization.

21. A unit dosage form, pharmaceutical comprising a peptide / lipid, lyophilized, sterile, stable mixture prepared according to claim 11 or 19.

22. Peptide / lipid complexes formed by the process consisting of freeze-drying one or more peptide peptides or analogs and at least one lipid in a solvent system to form a dehydrated peptide / lipid product that can be rehydrated to form peptide / lipid complexes.

23. The peptide / lipid complexes of claim 22, wherein the peptide is a protein that binds lipids.

24. The peptide / lipid complexes of claim 22, wherein the peptide is an analogue of ApoA-I.

25. The peptide / lipid complexes of claim 22, wherein the lipid is a natural, synthetic, saturated, unsaturated lipid or mixtures thereof.

The peptide / lipid complexes of claim 25, wherein the lipid is selected from the group consisting of egg phosphatidylcholine, soybean phosphatidylcholine, cholesterol, cholesterol derivatives, small alkyl chain phospholipids, ether phospholipids, dipalmitoyl phosphatidylcholine , dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, l-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-miristoilfosfatidilcolina, l-palmitoyl-2-stearoyl, l-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleofosfatidiletanolamina, dilauroilfosfatidilglicerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol , sphingomyelin, sphingolipids, phosphatidylglycerol, diphosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalm itoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, dipalmitoylphingomyelin, distearoylphingomyelin, phosphatidic acid, galactocerebrosides, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3) -D-mannosyl- (1,3) -diglyceride, aminophenylglycoside and -cholesterol-6 '- (glucosylthio) hexyl ether glycolipids, and mixtures thereof.

27. The peptide / lipid complexes of claim 22, wherein the complex is sterile.

28. The peptide / lipid complexes of claim 22, wherein the complex is formulated in sterile unit dosage.

29. A sterile, lyophilized composition comprising a sterile preparation of a complex formed between a peptide that can adopt an alphahelical, antipathetic conformation, or a peptide analog and a lipid.

30. The composition of claim 28, wherein the preparation is provided in a sterile unit dosage formulation.

31. A lyophilized composition containing a peptide / lipid complex, wherein the peptide is a lipid-binding protein or an ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III , ApoE or other apoprotein analog

32. The lyophilized composition of claim 31, wherein the peptide / lipid complex is a vesicle, micelle, liposome, discoidal particle, spherical particle or mixtures thereof.

33. A lyophilized composition containing a peptide / lipid complex, wherein the peptide can adopt an alphahelicoidal, antipathetic conformation.

34. The lyophilized composition of claim 33, wherein the peptide / lipid complex is a vesicle, micelle, liposome, discoidal particle, spherical particle or mixtures thereof.

35. The composition of claim 31 or 33, wherein the composition is sterile.

36. The composition of claim 29 or 31, said analog [sic] is not a peptide or a protein.

37. A method of preparing a lyophilized peptide / lipid product consisting of: co-lyophilizing one or more peptides or peptide analogs, which can adopt an antipathetic conformation, with one or more lipids at a peptide to lipid ratio of about 2 to about 200 in a solvent system for a sufficient amount of time to form a peptide / lipid product that can be rehydrated to form peptide / lipid complexes in solution.