JPH02502460A - Low toxicity drugs - lipid-based - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

, -, 2 *ia, s* This application is a part of U.S. patent application Ser. No. 069,908, filed July 6, 1987. This U.S. application is a continuation application filed on March 5, 1987, in U.S. Patent Application No. 022. This is a continuation-in-part application of No. 157. BJ Shimine IJJ 1 The present invention is directed to the use of drugs with high drug:lipid ratios (elevating agent:lipid complexes, i.e., HDLCs). formulations and preparations for creating lipid complexes associated with agents; and manufacturing method. Such preparations are substantially equivalent or more potent in potency to the free form of the same drug, and are less toxic. Furthermore, this A method for manufacturing HDLCs is disclosed. More particularly, the present invention relates to toxic antifungal polyene antibiotics, specifically amphotericin B and nystatin. et al. high drug: regarding the use of lipid complexes. The high drug:lipid conjugates (HDLCs) of the present invention can be made using substantially the same techniques as those used to make liposomes. This invention relates to drugs, specifically antibiotics. Includes the use of HDLC structures associated with bioactive agents such as photoricin B and nyscutin. Another feature of the present invention is the disclosure of a novel method for producing liposomes (i.e., HDLCs) without the use of Ikf8-containing media. Incorporation or association of drugs into liposomes proceeds using ethanol or aqueous media. I can't stand it. Liposomes are completely closed lipid bilayer membranes 1 with an entrapped aqueous volume. Liposomes contain unilamellar vesicles (with a single membrane bilayer) or multilamellar vesicles (with multiple membrane bilayers). It has an onion-like structure characterized by sublayers, each layer separated from the next by an aqueous layer). This bilayer N has a hydrophobic "tail" region and a hydrophilic "tail" region. It is composed of two lipid monolayers with a "head" region. The structure of this membrane bilayer is such that the hydrophobic (nonpolar) lipid “tails” of the lipid monolayer point toward the center of the bilayer, while the hydrophilic (polar) The "head" of the aqueous layer is directed toward the aqueous layer. Early liposome production such as Bangha (Journal on More) Biological Biology (J, Mo1. Biol.), 13, 238-252.1 965] suspends phospholipids in an organic solvent, then evaporates the solvent to dryness, and forms a phospholipid film on a reaction vessel. It includes leaving behind. Subsequently, an appropriate amount of aqueous phase is added, the mixture is allowed to swell, and the resulting multilayer Liposomes consisting of ultilamellar endoplasmic reticulum (MLVs) are dispersed by mechanical means. This technique was described by Papahadj iopoulos et al. Uni provides the basis for the development of lamellar endoplasmic reticulum. Techniques for manufacturing large unilamellar vesicles (LUVs) 1 e.g. reverse phase evaporation, leaching method Infusion procedures and detergent dilutions used for the production of gum. A list of these and other methods for producing liposomes is given in Chapter 1 of the text "Liposomes J, Mare Stro, ed., Mareal Dekker+Inc, wNew York*1 983e, and is not relevant to this invention. The parts shown are for reference only. Incorporated into the specifications. Also, 5zoka, Jr. [1980, Annual See Biophysical Bioengineering (Ann, Rev. Biophys, Bioeng., 9:467), the portions related to this invention are also incorporated herein by reference. A particularly preferred method for the production of LUVs1i1 is described in Cu1lis1E+ (71 PCT Publication No. 8710 O238, January 16, 1986, entitled "Extrusion Techniques for the Production of Unilamellar Endoplasmic Reticulum"), which is incorporated by reference. Please write it here. The endoplasmic reticulum produced using this technique, called LTJVETS, is forced through a membrane filter under pressure. The endoplasmic reticulum also has 200 files. The vesicles can also be made by extrusion techniques through a VET membrane. S and It is known as These vesicles are subjected to at least one freeze-thaw process prior to extrusion techniques. Can undergo a solution process. This method is described by Mayer et al. Biochem, et. Biophys, Acta, 817, 193-196.1985) J. The relevant portions are cited here for reference. In the practice of the present invention, lamellar solutes are characterized by having a substantially uniform lamellar solute distribution. Preferred types of liposomes and methods of making the same include stable plurilamellar vesicles (SPLVs) as defined in Lenk et al., US Pat. No. 4,522,803. and contains the unilamellar vesicles described in Fountain et al., U.S. Pat. No. 4,588,578 and the freeze-thawed multilamellar vesicles (FATM LV) described above. . Various sterols and their water-soluble derivatives have been used to make liposomes. In particular, Janoff et al. See U.S. Pat. No. 4,721,612, issued January 26, 1988, entitled Ming. See Mayhew et al., PCT Publication No. WO 3510096g, 1985. Published on March 14, the paper explores the use of alpha-tocopherol as a way to reduce drug toxicity. encapsulating the drug in liposomes containing polymers and certain derivatives thereof. A method for encapsulating a file is described. Similarly, various tocopherols and their water-soluble derivatives have been used for liposome formation. Giamp et al., a development called “alpha-tocopherol-based endoplasmic reticulum” PCT Publication No. 87102219 under the name of Ming Dynasty, published on April 23, 1987, Please refer to those incorporated herein for consideration. In liposomal drug delivery systems, bioactive agents such as drugs are incorporated into liposomes. For example, Rahman et al., U.S. Pat. No. 43,993,754; Sears U.S. Pat. No. 4,145,410; Pabahadjopoulos et al. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179, Renda et al., U.S. Pat. On the other hand, if the drug is enzymatic, it can associate with the lipid bilayer. In the present invention, the term "entrap" or "encapsulata" This includes both the drug associated with the lipid bilayer and the drug in the aqueous volume of the liposome. Many drugs useful for treating disease are toxic to patients; Examples include cardiotoxicity in the case of the anticancer drug doxorubicin, and nephrotoxicity in the case of aminoglycone or polyene antibiotics such as amphotericin B. Amphotericin B is used in the treatment of life-threatening bacterial infections. Only the most reliable. Highly toxic antibacterial poly It is an antibiotic [Taylor et al., American Review of Spiral Disclosures (Am, Rev, Re5pir, Dis, 1999), 1 982, 125:610-611). Amphotericin B is a hydrophobic drug, so it is insoluble in aqueous solutions and It is commercially available as a colloidal suspension of M in suoxycholate, and the surfactant used for suspension is itself toxic. Amphotericin B methyl ester and Amphotericin B also plays a role in the pathogenesis of acquired immune deficiency syndrome (AIDS). (Schaffner et al., piochemical Biochem, Pharmacol, 1986.35:4110-4113) has been shown to be active against HT LV-m/LAV viruses, ie, lipid-enveloped retroviruses. In this study, amphotericin B methyl ester ascorbate (water soluble) and amphotericin Ricin B was added to separate cultures of HTLV-m/LAV-infected cells and cells were measured for virus reproduction. The results showed that amphotericin methyl ester and amphotericin B showed that it protected target cells from the cytopathic effects of this virus, similar to that shown for viruses [Stevens et al. Virology (Arch, Virol, ), 1975.48:391). Reports on the use of amphotericin B encapsulated in liposomes have already appeared in the literature. Juliano' et al. (Annals N, Y. Acad, Sci., 1985.446:390-402) discuss the treatment of systemic fungal infections using liposomal amphotericin B. ing. This kind of resource Posomes are phospholipids, e.g. simyristoylphosphatidyl in a molar ratio of 7:3. Phosphorus (DMPC) and Simyristoylphosphatidylcholine (DMPC) 7 :3 molar ratio and cholesterol. Free and liposomal Acute toxicity studies (LD50S) and in vitro measurements comparing loaded amphotericin B have shown that liposomal formulations have substantially unchanged antibacterial efficacy, as well as improved It showed low toxicity. L. pez-Berestein et al. Liposomal-encapsulated amphoteric acid Treatment B was administered. The liposomes contained a 7:3 molar ratio of DMPC:DMPG and the drug was encapsulated with greater than 90% efficiency. 5 mol% anhote As a result of this liposomal drug treatment with Ricin B, 66% of treated patients showed a favorable response in the form of partial or complete relief of the fungal infection. Robetz Berstein et al. (Journal on Infectious Diseases, 1983.14 7:939-945), Ahrens et al. [Antimicrob, A gente Chemotherapy], Panosian et al. ,). 19B4, 25:655-6563, and Trembley et al. (Antimicrobial Agents Chemotherapy, 1984.26:1 70-173) also describe systemic candidiasis and leishmaniasis in mice. (Badgian et al., supra) for the treatment and prevention of The effects of free amphotericin B and liposomal amphotericin B were compared and tested. They reported that liposome-capsule encapsulation in the treatment of candidiasis It has been found that mbotericin B increases the therapeutic index. In all cases, When Amphotericin B is encapsulated in liposomes, very large amounts of Amphotericin B are encapsulated in liposomes. It was found that ricin B was tolerable. Amphotericin B-liposomal preparations were either not or only slightly effective in treating leishmaniasis. DMPC: A protein containing DMPG, ergosterol and amphotericin B. Loliposomes (lipids and drugs coated on soluble carriers to create particulates) have also been created [Payne et al., Journal on Pharmacy. J. Pharm. Sci., 19g6, 75:330-333). Other studies have tested eryptococeosis in mice with lipolytic Amphotericin B-desoxycholesterol was treated intravenously with somal amphotericin B. [Graybill et al., Journal Luon Infectious Disease. 19g2,145nia4g-752), treated with liposomal amphotericin B The mice showed higher survival times, lower tissue counts of mycobacterium, and reduced acute toxicity. The nine multilamellar liposomes used in this study are ergosteroid Contains Tiller et al. [American Review, Rev, Re5p, Dis, 1982.125=610-610) J*? Us (7) k-stoff5,! ! ” hist. plas+5osis was treated with liposomal amphotericin B. Somes contained ergosterol and phospholipids. This liposomal formulation is effective in treating histoplasmosis with low toxicity and improves serum and tissue distribution with lower serum levels compared to free amphotericin B. It was changed so that the degree of A in the pancreas was high. In the above studies, lipid-containing liposomes were used to improve the toxicity of the entrapped drug and tended to increase the lipid content in the formulation to buffer drug toxicity. Applicants have surprisingly found that a low lipid composition is in fact the most efficient at reducing this toxicity. When producing the HDLCs of the present invention by the MLV method, a mixed population of HDLCs and MLVs can result, and these formulations contain increasing proportions of HDLCs as the mole percent of drug increases. About 5 to about 25 mole percent of the drug (amphotericin B) is used. Formulations using 25 mol% to about 50 mol% of the drug are substantially lipolytic with HDLCs. There are no somesomes, and on the other hand, formulations containing 5 mol% and less hydrophobic drugs are essentially liposomes with some HDLCs. Mother collection of different species If it is necessary to separate the HDLCs from the group, this can be done by known separation techniques, such as density gradient centrifugation. The methods used to make these HDLCs can be substantially the same as those used to make liposomes, but with essentially no differences. In the present invention, using a high drug:lipid ratio, toxicity was unexpectedly greatly reduced compared to liposomal formulations. More HDLCs are formed than liposomes. And 1st time

The present invention discloses an HDLC (i-drug-to-IM ratio complex) system containing bioactive agents, including lipids and drugs. The HDLC5 may contain phospholipids such as DMPC and DMPG, preferably in a molar ratio of 7:3, and may be saturated. It can contain Japanese phospholipids or fatty acid phospholipids. The bioactive agent is preferably a drug, such as an antimicrobial agent such as nystatin or amphotericin. The mole percent of drug present is preferably from about 6 to about 50 mole percent, and preferably from about 30 to about 50 mole percent. Pharmaceutical compositions of HDLCs, preferably containing a drug such as amphotericin B, particularly preferably 50 mol %, are prepared containing a pharmaceutically acceptable carrier or diluent, and these compositions are The substance can be administered orally. Such compositions are used to treat infectious diseases such as fungal infections, and they are carried out by administration to mammals such as humans. Compositions include compositions that are substantially free of liposomes and liposomes that incorporate drug. The composition contains a composition that is substantially free of gum. Substantially usually less than 10% by weight of lipids, preferably or less than 5% by weight, more preferably less than 3% by weight. Various methods have been disclosed for producing the HDLCs of the present invention, including the technique of dissolving the drug, particularly amphotericin B, in a solvent such as DMSO or methanol. The lipid (preferably DMPC:DMPG in a 7:3 molar ratio) is dissolved in a solvent such as methylene chloride, and the lipid and drug solution are mixed. The solvent is distilled under reduced pressure, resulting in a thin lipid-drug film. This film is Hydration in an aqueous solution such as saline, PBS or glucine buffer produces HDLCs. Alternatively, the aqueous solution can be added to the solvent containing the drug and lipid phase prior to evaporation of the solvent. In addition, as a further alternative, the obtained dried Resuspend the lipid-drug film in a solvent such as methylene chloride to hydrate the film. It can also be re-evaporated under reduced pressure before being removed. Dehydration methods can also be used. child In this method, the dried lipid-drug film is dehydrated to form flakes, which are then hydrated with an aqueous solution. Another method of forming the HDLCs of the present invention is to use a A bioactive agent containing 50 mol% amphotericin B (drug 1 e.g. amphotericin B) Lipid particles (or liposomes) containing a polyene antimicrobial agent such as tericin B are formed and the particles (or liposomes) are then subjected to a heating cycle from about 25°C to about 60°C, most preferably at about 60°C. . Such cycles are more highly ordered and less toxic. Forms a ricin B/lipid complex. Another aspect of the present invention is to use drugs such as polyene antiseptics such as embotericin B. to determine the toxicity of fungal agents)-lipid complexes. Absorption spectral techniques are used. The absorption spectrum of a drug is specific to that drug. It is. The drug is characterized by a peak or peak in the ultraviolet or visible region. It can be a series of blocks. The peak characteristics of amphotericin B (which is represented in Figure 12 as dissolved in dexoxylate) are between 300 and 500 μm, with several characteristic peaks, and the The most typical one is the one that appears at 413 μm. The decrease in this peak due to the complexing of this drug with lipid can be used quantitatively to measure the toxicity of this HDLC. In other words. The degree of toxicity can be measured by the intensity of the absorption peak height. A liposomal drug loading process is also disclosed, but the drug, particularly polyene antibiotic antibiotics, Hotelicin B is dispersed by sonication into a solvent such as ethanol to which an acid such as salt is added. Lipid membranes, particularly membranes containing about a 7:3 molar ratio of DMPC:DMPG, are hydrated with an aqueous solution. In particular, an aqueous buffer such as DBS is used and a certain amount of the acidified ethanol solution containing the drug is added to the liposome. It is loaded into liposomes by adding it to a drug. eta in the resulting suspension The nol is removed and the solution is resuspended with aqueous solution. Due to the molar ratio of drugs co-mixed with lipids, this process produces more HDLCs than liposomes. For example, when the mole percent of drug is about 16 or more, more HDLCs are formed than liposomes. On the other hand, for 0-15 mol% drug, more liposomes are formed with this method. The liposomes or HDLCs produced by this acidified ethanol pharmaceutical process can be prepared for use as a pharmaceutical composition by the addition of a pharmaceutically acceptable carrier or diluent. It can be used to treat fungal infections by administering it to mammals such as humans. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. LD as a function of mol% of amphotericin B in the formulation. This is a graph depicting acute toxicity measured in . FIG. 2 is a differential scanning calorimetry spectrum of a liposomal (MLV) formulation containing 5 mol% amphotericin B. FIG. 3 is a differential scanning calorimetry spectrum of HDLC Ju agent containing 25 mol% amphotericin B (MLV method). Figure 4 shows the condensation of multilamellar liposomes containing 5 mol% amphotericin B. It is a graph of the slope of degree. Lipid (solid line); amphotericin B (dashed line). FIG. 5 is a graph of the isodensity slope of HDLCs made with 50 mol% amphotericin B (MLV method). N (solid line); amphotericin B (dashed line). Figure 6 shows liposomes containing 5 and 50 mol% amphotericin B, respectively. and a graph of X-ray diffraction data of HDLCm agent (MLV method). Figure 7 shows the 11P-NMRX pect/L of the DLC formulation (MI, V method) containing 25 and 50 mol% amphotericin B. Figure 9 shows the "P-NMR spectrum of the liposome (ML V) la agent containing 5 mol% amphotericin B. FIG. lipids (solid line), amphotericin B (dashed line). Figure 10 shows the absorption spectra of free amphotericin B dissolved in deoxycholate. It is Kutle. FIG. 11 shows 5 mol% amphotericin B (a), 25 mol% amphotericin B (b) in 7:3 DMPC:DMPG. 25 mol% amphotericin B (c) and 50 mol% amphotericin after heating It is an absorption spectrum of SynB (d). Figure 12 shows the absorption spectra of 25 mol% amphotericin B in 7:3 DMPC:DMPG before heating (a) and after heating at 60''C for 10 minutes (b). 25 mol% amphoteric acid before circulation (a) and after circulation (b) The DSC spectrum of 7:3 DMPC/DMPG containing Contain B is compared with the spectrum of pure MPC/DMPG (c). Figure 14 before (a) and after (b) heating cycling at 60°C. P-NMR spectrum of a 7:3 molar ratio DMPC/DMPG complex containing 25 mol% amphotericin B. Figure 16 shows the effect of culture temperature on the rate of incorporation of amphotericin B into DMPC:DMPG liposomes. Figure 3 shows the influence of liposome structure on uptake of protein B. Supervised by IK Gotachi Bifu High quality non-liposomal drugs with unique properties: H quality complexes (HDLCs), and a method for its manufacture are described. These HDLCs contain bioactive agents such as hydrophobic drugs. When this HDLC is administered to animals, it is present in free or liposomal form. The drug may have substantially equal or greater efficiency and lower toxicity compared to drugs administered in the same manner. Such HDLCs consist of a high drug:H mole ratio (approximately 5 mol% greater than drug).The present invention surprisingly shows that compared to liposomal systems, less lipid The present invention provides HDLC II agents for use as drug carrier systems in association with drugs such as the antibacterial polyene antibiotics amphotericin B and nystatin. contains. These preparations, unlike commercially available preparations, are stable in aqueous solutions such as saline. Ru. Additionally, new liposome pharmaceutical methods have been described in which liposomes are loaded by incorporating the drug via a solvent intermediate. This solvent is amphoteric It has the characteristics of dissolving bioactive agents such as N-B, which does not disrupt the liposome membrane structure, and which is compatible with aqueous solutions such as water. With such medium Then there is ethanol. This method proceeds without the use of organic solvents. child depending on the mole percent of the drug used in the formulation. HDLCs rather than liposomes are produced. If formation of HDLCs is preferred, the method loads the HDLCs with the drug. HDLCs are associated with drug-associated lipids in a special form, and such particulate matter is non-liposomal in nature. When formed using the MLV method, such HDLCs have, for example, the following characteristics: (1) freeze-fractured electron micrographs; [Deamer et al., Biochim, Biphys, Acta, 1970.219: 47-603] (2) Capture capacity measurement (Deamer et al., Chemist (3) Differential scanning calorimetry (Chew, Phys, Lipids, 1986, 40:167-188) shows virtually zero uptake capacity and is therefore non-liposomal. (Chapman, D., Liposome Technology) Molecular membrane pre-transition phase or no main transition; (4) "P-NMR spectrum (Cullis et al., 1982. Fluidity in Biol Ogy + Academic Press Incorporated, London and New York ] suggests the character of a highly immobile lipid (broadly isotropic); and (5) Xi! Diffraction data (5hipley et al., Biomembranes t 197L Chapman, O. and Vallach, Dli, 2: 1 volume, Academic Press Inc. Do... :/Door and New York] indicates gel layer lipids. A feature of these systems is also the complete association of the drug with lipids, as evidenced by density gradient centrifugation. In this technique, the gradient is centrifuged at elevated pressure (about 230,000 sg) for about 24 hours. This ensures that all components in this gradient reach their equilibrium density positions. child The elution status of these systems shows that the drug and lipid peaks overlap, indicating that all drugs are associated with this lipid. One feature of the invention is the drug:lipid ratio that forms HDLCs, which in mice are LD. (Figure 1), while drug The effect of the agent is to form HDLCs that produce substantially the same or greater effect. Applicants believe that between about 6 and 50 mole percent of the hydrophobic agent will meet such requirements, with preferred ratios between about 15 and 50, more preferably between about 25 and 50, and most preferably about It was found to be between 25 and 35 mol%. medicine When the drug concentration exceeds about 6 mol% and reaches 25 mol%, a mixed population of liposomes and HDLCs is formed. Within this range, as the mole % of drug approaches 2 degrees, a greater proportion of structures become present in HDLC than in liposomes. At drug concentrations of about 5 mol % and lower, and up to a lower lipid limit known to those skilled in the art as the "critical micelle concentration," mixed HDLC/lipo Although it is a group of somes, there is essentially a liposomal structure. such lipo Somes can be formed by the new ethanol intermediate method of the present invention. A feature of the HDLCs mentioned above is the various drug-lipid dispersions found in density centrifugation. This drug-lipid (DMPC:DMPG in a 7:3 molar ratio) *combination is When separated on a sucrose density column, a system containing 5 mol% drug made by the MLV method shows bands depending on drug:lipid ratio and method of preparation. In this case, two major bands of material are observed: one of the liposomes and one where the drug is associated with lipids (HDLCs). Formulations containing 25 and 50 mol% drug showed a single band with most of the drug associated with lipids. Surprisingly, these low lipid/high mole % drug systems have low toxicity. Figure 1 shows the molpercent of the drug amphotericin B in the formulation. LD so S (mg/kg) in mice is shown as a relationship between the two. child LD,. increases between 5 and 50 mol% drug and then tapers off at 60 mol%, while the LD of the approximately 3.5 mg/kg commercial form of the drug fungizon is plotted. . In vitro, hemolysis of red blood cells exhibits the same toxic phenomenon. Studies on incorporated capacity [uptake of medium (in ul) relative to U moles of lipids A study was carried out on drug-lipid associated MLV formulations with various mole percentages of drug, which showed that preparations of 25 mole percent and larger (HDLC) exhibited different properties than normal ones. these systems do not take up any medium and are therefore not liposomal; cryo-fracture electron micrographs of these same systems show liposomes at 0 and 5 mol% drug, but at 25 and 50 mol%. % medicine The drug is non-liposomal HDLCs. Differential scanning calorimetry follow-up performed on MLV formulations containing various mole percents of drug. The traces show the same phenomenon; i.e., the spectrum changes to 5 mole percent drug. The 25 mol% drug shows a transition peak characteristic of unperturbed bilayer lipids (Figure 2), but the 25 mol% drug does not show any transition peak (Figure 3). -cent drugs, this lipid is completely associated with the drug, i.e. Since the quality acyl chains are not free, no cooperative transfer occurs; this data shows that the two components of lipid-associated drug and free lipid at a 5 mole percent drug concentration (Figure 6). This confirms density centrifugation data showing that drug and all lipids are associated in a single band at 25 and 50 mole percent drug concentrations (Figure 5). Ru. The X-ray data at 5 mole percent (MLV method) is for the liquid at a specific temperature of 23°C. However, at 50 mole percent drug, this lipid is in the gel phase at all temperatures; at all the lipids are tightly associated with the drug. Similar data for 11P-NMR studies show that these high (25 and 50) mole percent drug preparations do not occur because metastases do not occur (Figure 6). This type of The peak characteristics of the low shoulder/high lipid region of and 5 mole percent drug samples (Figure 8). The lipid complex system with its associated high drug-lipid ratio is a feature of the present invention. However, liposome-forming means can be used to form this lipid complex. Wear. In particular, these methods include forming liposomes known as multilamellar vesicles (MLVs). Other methods used include creating stable plurilamellar vesicles (SPLV) - large unilamellar vesicles (LUVETS) made by extrusion or other liposome formation methods known in the art. A method for forming 5PLVS is described in Lenk et al., US Pat. No. 4,522,803. The LUVET method is disclosed in PCT Application Publication No. WO36100238 of Cullis et al., published on June 11, 1985. Published on January 16, 1986; each relevant part is included in the present invention for reference, and in the new liposome formation of the present invention, Posomes are formed in an aqueous solution into which the drug to be taken up is placed in an acidified ether. In other liposome-forming embodiments of the present invention, the Hotelicin B is introduced into liposomes via an aqueous intermediate. In this technique, amphotericin B is suspended in an aqueous solution, for example in distilled water, by sonication. Ru. This sulfuric agent is then dissolved in an aqueous solution such as distilled water or sodium chloride solution. mixed with a lipid suspension in liquid. This mixture should be adjusted to the transition temperature of the lipids used or is further incubated to form MLVs. The N substances that can be used to form this lipid complex (1) and used in the novel posome formation technique of the present invention (2) include phosphatidylcholine (PC), atidylethanolamine (PE), phosphatidylserine (ps), phospha Tidylglycerol (PG), phosphatidyl! i* (FA), phospha Rises such as tidylinositol (PI), sphingomyelin ('SPM), etc. lipids may be used alone or in combination. Saturated phospholipids such as hydrogenated soybean PC Quality can also be used. This phospholipid may be synthetic or egg-based. can also be obtained from natural sources such as soybeans. Other lipid compositions include Milis Contains saturated fatty acids such as cic acid. In a preferred embodiment, phospholipid similis Toylphosphatidylcholine (DMPC) and simiristoylphosphatidyl glycerol (DMPG) are used in combination in any molar ratio, such as from about 99:1 to about 1=99 DMPC:DMPC ratio, but preferably about 7 :3 molar ratio. DMPC and simyristoyl phosphatidylserine (DMPS) can also be used in combination. However, DMPC alone can also be used. These lipid complexes and liposomes are part of this lipid phase. It can also contain steroid ingredients. This steroid is cholesterol, polyethylene glycoside of cholesterol. alcohol derivatives (PEG-cholesterol), cobrostal, cholestanol, cholesterol The presence of sterols such as restane, cholesterol hemisuccinate (CHS) Examples include organic acid derivatives and the like. Organic acid derivatives of tocopherols can also be used as complex or liposome-forming components, e.g. Both CHS and TH5-containing complexes, such as phytocopherol hemisuccinate (TH5) and their Tris salt forms, are commonly used in these compounds. Any of the methods known in the art for producing liposomes containing terols It can also be made using this. In particular, Janoff et al., US Pat. No. 4,721,614, issued January 26, 1988, "Steroidal Liposomes" and Janoff et al., PCT Publication No. 87102219, published April 23, 1987, filed September 24, 1986, "Endoplasmic Reticulum-Based Alpha-Tocophenol," pertinent portions of which are incorporated herein by reference. has been incorporated into. Bioactive agents such as drugs can be used in the present invention. For HDLCs, for The bioactive agent used is hydrophobic, and in the case of liposomes, the liposome The bioactive agent can be either hydrophobic or hydrophilic, since it incorporates a type of agent. These bioactive agents include viramycin, candidine, and filiform. pins and preferably polyene antiseptics such as nystatin and amphotericin B. Other bioactive agents include, but are not limited to, biological agents, such as antibacterial compounds such as aminoglyconds, such as gentamicin; Antiviral compounds such as Funpacin, antiparasitic agents such as antimony derivatives, vinblastin, vincristin, mitomycin C, doquinol. Anti-tumor agents such as bicine, daunorubicin, methotrexate and cisplatin, among others proteins such as albumin, toxins such as diphtheria toxin, -ions such as catalase, hormones such as estrogen, and neurotransmitters such as acetylcholine. Masterpiece. lipoproteins such as alpha lipoprotein, glycoproteins such as hyaluronic acid, immunoglobulins such as IgG, interferons or interleukin immunomodulators such as arsenazo, dyes such as arsenazo, radiolabels such as '4C, radiation shielding substances such as 99Te, fluorescent compounds such as carboxyfluorescein, polysaccharides such as glycogen, estrogen receptors. Cell receptor binding molecules such as proteins, indomethacin, salicylic acid acetate, and ibuprof non-steroidal anti-inflammatory agents such as fenugreek, sulindac, piroxicam and naproxen, anti-inflammatory agents such as dexamethacin, anti-glaucoma agents such as timolol or pirocarbin, opiate agents such as dibucaine, and nucleic acids such as thymine. Polynucleotides such as RNA polymers, alpha shields, beta shields, and calcium sium pathway blockers, cardiovascular agents such as ACE inhibitors, and prostaglandins. These include, but are not limited to, CNS agents. During the production of HDLCs, organic solvents can be used as in the general production process of liposomes. Suitable organic solvents also have intermediate polarity and dielectric properties. (having an intermediate polarity opposite to the charge), which dissolves lipids. solution, chloroform, methanol, dimethyl sulfoxide (DMSO), methylene and solvent mixtures such as chloroform:methanol (70:30) + benzene:methanol (70:30); As a result, a lipid-containing solution is formed, which is defined as a uniformly distributed mixture of components. The solvent is preferably selected on the basis of its biocompatibility, low toxicity and non-flammability. When dissolving drugs, especially amphotericin B, it is most soluble in DMSO. In which DMSO is preferred, methanol can be substituted for DMSO with increasing coexistence in solvent volume. To dissolve lipids, methylene chloride is preferably used due to its low toxicity in humans.9 In the novel liposome formation method of the present invention, ethanol or aqueous solutions are preferred solvents. In the hydration step of HDLC formation, aqueous solutions such as distilled water (eg, USP water for injection), saline, or aqueous buffers may be used. The aqueous buffer is about 7.0 Ishi 7.5. Preferably, in 7.2, phosphorus buffered saline (PBS), tris-(hydroxymethyl)-aminomethane salt buffer (Tris), or glycine salt buffer is added. buffer solutions and the like are used, but are not limited to these. During the formation process of HDLCs or new liposomes, a sonication step is performed. It can be done. Such methods are carried out in a sonic bath at 25 DEG C. and about 50-60 Hz for about 15 minutes to about 30 minutes. The formed HDLCs or liposomes are described in PCT Publication No. 8 of Cullis et al. 810 O238, published January 16, 1986, by extrusion through a filter. The relevant parts are for reference only. It is introduced here for this purpose. In this way, the lath process allows for uniformity according to the size. For example, in the filtration of HDLCs, polycarbonate A tortuous or straight path through a membrane filter such as a bonate filter. This can be done through a wire path. HDLCs or liposomes formed by the novel method of the present invention are subjected to lyophilization or dehydration steps at various stages of formation. For example, the lipid membrane is lyophilized after removal of the solvent and before addition of the drug. Otherwise, the lipid-drug film can be lyophilized before the HDLC or liposomes are hydrated; dehydration This is done by exposing the room to vacuum and removing all suspending solvent. If (or, additionally, finally hydrated) (DLC or liposome) The formulation can also be dehydrated by placing it in a surrounding medium in liquid nitrogen and freezing it before the dehydration step. Ru. First, dehydration accompanied by freezing is carried out in the presence of 1, 1 or more protective sugars + according to the technique of Baly et al. It is included here for this purpose. It is also carried out by the technique of 5chneider et al., US Pat. No. 4,229.360, issued October 21, 1980. This technology is suitable for long-term storage and and enhance the stability of formulations. Any other suitable method, now known or later discovered, may be used to Quality complex preparations can be used in dehydration. One method for forming HDLCs in the present invention is the MLV method. In this method, a bioactive agent (eg, drug) is dissolved in methanol in a flask to which a lipid such as DMPC and DMPG is added in chloroform at an approximately 7:3 molar ratio of DMPC:DMPG. This solution was rotary evaporated under reduced pressure and then dried. Re-incubate the dried membrane in PBS! ! about 6 and above (about 6 o mole percent With a molar ratio of drug to drug:II. The toxicity of this HDLC formulation is drug:lipid ratio dependent, with formulations with higher drug:lipid ratios (approximately 16-50 mole percent drug) compared to formulations with lower drug:lipid ratios (approximately 6-15 mole percent drug). low acute toxicity As discussed above, formulations containing up to about 5 mole percent drug are substantially liposomal. Another method is the modified MLV method, in which drug-lipid films, such as those created above, are dried in a Pelger under reduced pressure to form drug-lipid flakes. Ru. The pieces are ground to a powder and an aqueous solution is added to homogeneously hydrate. This method involves a solution of the drug in an organic solvent such as DMSO or methanol and Mix with lipids in tylene chloride (most preferably DMPC:DMPG in a 7:3 molar ratio). The mixture is subsequently dried under reduced pressure to form a membrane, which is then dried, for example, in a Perger under reduced pressure. The resulting dry powder is hydrated with an aqueous physiological saline solution and then heated to produce a drug-lipid suspension. As previously discussed, HDLCs, liposomes and their The formation of these mixtures and the degree of toxicity depend on the drug:lipid ratio. Other methods can be used to form HDLCs. One such method is the 5PLV method, in which the drug is dissolved in a solvent such as DMSO. The lipid (eg DMPC:DMPG in a 7:3 molar ratio) in a solvent (eg chloroform or methylene chloride) is dried under reduced pressure into a thin film and then an organic solvent such as methylene chloride is added to the lipid. An aliquat of the drug (eg, amphotericin B) in a solvent is added to the lipid suspension 1 and the resulting mixture is sonicated until clear. Buffered aqueous solution (e.g. mild saline solution) is added, then the mixture is again placed under vacuum and methylene chloride is added. Remove ride. The resulting paste is further hydrated with PBS and sonicated until transparent. Regarding the above method, the resulting formulation will be HDLC or liposomal depending on the drug:lipid molar ratio used; the LD g in mice with this formulation will be higher. The drug:lipid ratio showed lower toxicity. Yet another method is the modified 5PLV method, in which a drug in an organic solvent (e.g. DMSO) is mixed with a lipid in solution (e.g. DMPC: DMPG) and the primary Add a buffered aqueous solution (e.g. PBS) and then dissolve under nitrogen while sonicating. The medium was evaporated. Additional PBS is added and the resulting solution is filtered, preferably through a 5 micron filter, and then centrifuged at about 10,000 x g for about 10 minutes. The synovial fluid is removed and discarded, and the pellet is then resuspended in an aqueous solvent (PBS). After the second “wash”, centrifugation is performed and the resulting pellets are Resuspend the cells in PBS. For the above formulations, the acute toxicity of the formulation is directly related to the drug:lipid ratio; the higher the ratio (up to about 60 mole percent drug), the less toxic the formulation. Yet another 5PLV method is used for formulation, in which the drug-solvent and lipid solutions are mixed as before, but water for injection, USP, rather than saline is added. and the suspension is warmed to 37°C. This solvent is again treated with sonication. The suspension is evaporated under nitrogen, more water for injection is added and the suspension is kept at 37°C for 10 minutes. This preparation was prepared using the LUVET method to obtain a It is filtered and sterilized through a Chron filter. This method is an extrusion method in which liposomes are produced by filtration at a pressure of about 700 psi and is the subject of Culis et al., PCT Publication 86/99238, published January 16, 1986, the relevant parts of which are It is introduced here for reference. Such preparations also. It can be dehydrated by the method of Bally et al. mentioned above. Acute toxicity studies have again shown that lower drug:lipid ratios have an improved effect on drug toxicity. Further includes dissolution of the drug by sonication into imaged absolute ethanol. Lipids (eg DMPC:DMPG in a 7:3 molar ratio) dissolved in a solvent (eg benzene:methanol) are lyophilized. Next, it is stirred and mixed with an aqueous solvent (for example, PBS), and then this chromatized alcohol-drug solution is added and stirred at room temperature. This formulation is then lyophilized and rehydrated with an aqueous buffer. Otherwise, this solvent is evaporated and the lipid-drug A drug film can also be left behind, which film can be hydrated with an aqueous solution. Again, lower acute toxicity is coupled with higher drug 2rN quality in this formulation. Approximately 5 mole percent This method produces exclusively liposomes when using drugs of up to 6 mole percent and lower, compared to concentrations of about 6 mole percent and greater than that. It mainly forms HDLCs. Centrifuge this formulation on a density gradient This confirms that all lipids are associated with this drug. This formulation is then lyophilized, which removes the ethanol, and then Rehydrated in liquid. In other liposome formation embodiments of the invention, amphotericin B is introduced into the liposomes via an aqueous intermediate. In this technique, amphotericin B is suspended in an aqueous solution, for example distilled water, by sonication. This suspended drug is then mixed with distilled water. or mixed with a lipid S suspension in an aqueous solution such as a saline solution. This mixture is incubated at or above the transition temperature of the lipid used, resulting in the formation of MLVs. Discussed above. High molar ratio amphotericin B-lipid formulations. It is believed that it exhibits low toxicity due to the enhanced amphotericin B-amphotericin B interaction in this lipid matrix. This can be shown by the absorption spectrum. For example, at an absorption f of 413 nm, the rise of free amphotericin B in deoxycholate is greater than that of unheated 5 mol % amphotericin B in lipids. Furthermore, in lipids, the absorbance of 25 mol% amphotericin B is higher than that of 50 mol% amphotericin B. (See Figure 10). This absorbance spectral technique is used to analyze the toxicity of drug (e.g. amphotericin B)-lipid complexes. Used to determine sex. The absorbance spectrum of a drug is unique to a single drug; amphotericin B (dissolved in deoxycholate is represented in Figure 12) has a distinctive peak between 300 and 500 nm; The location of these peaks is also unique, with one at 413 nm. The reduction of this peak by forming a complex of this drug with a lipid is used to quantitatively measure the toxicity of HDLC. Since the spectra are drug-specific, when complexed in this way, any of the above-named lipids will have the characteristic absorbance discussed here, even though it may be reduced. It is expected that If an amphotericin B-lipid system exhibiting less than the maximum toxicity buffer (e.g. a 25 mol% sample) is heated to 25-60°C, the toxicity of the resulting system is reduced (see Figure 11); this reduction , proceeding to the maximum toxicity seen when amphotericin B is complexed with 50 mol % lipids (Figure 11). One possible explanation is that this decrease is due to the fact that the lipids are in the separated phase and that amphotericin B is complexed with 50 mol% lipid. kicked out of B to allow amphotericin B to bind to itself, resulting in better This means that it produces a system with low toxicity. This expelled lipid is detected by differential scanning thermal imaging. This is indicated by the reappearance (after heating) of the endotherm seen in the mass spectrum (see Figure 12), consistent with a lipid that is substantially free of amphotericin B. Furthermore, this displaced lipid can be indicated by the narrowing of the "P-NMR signal resulting from this heated system (see Figure 13); again, this narrowing results in a substantial decrease in amphotericin B concentration. matched lipids are doing. Finally, cryo-fractured electron micrographs of the heated system showed that the lipids (and lipoproteins) after heating were (somes), which were not observed before heating. This heating Samples that do not show complexes with no free lipids, indicating features of the system indicating intermolecular interactions between lipids and amphotericin B. When this above study was carried out on samples containing 50 mol% amphotericin B, small differences were observed between before and after heating of the studied samples. Perhaps this structure There are no more lipids to be expelled. These samples were then subjected to DSC and and NMR analysis showed virtually the same signal before and after heating. did. The theory that the lipid-separated phase leaves amphotericin B in an environment more conducive to amphotericin B-amphotericin B interactions (and thus less toxic) is dictated by the absorbance spectrum. The spectrum resulting from the heated system has a much lower intensity (meaning less toxicity) compared to the untreated system. In one case, this heating is LD of the unheated formulation. produced an LD of greater than 30 mg/kg compared to 15 mg/kg. It was. In this case, a decrease in absorbance at 413 nm was noted after heating. The heating process described above can be applied to any type of liposome or lipid particle, and This is shown in any liposome or lipid particle formation method described above. The MLV method was used in the examples. Similarly, the lipids or lipids mentioned so far For example, lipids (such as DMPC:DMPG in a 7:3 molar ratio) can be used, and any solvent or aqueous buffer mentioned so far can be used. (in any amount known to form liposomes) are suspended in an organic solvent such as chloroform and placed in a round bottom flask. Dry until a thin film forms on the sides of the lask. In the present invention, amphotericin B (or any of the other drugs previously described) is dissolved in any suitable organic solvent (a suitable solvent is one in which the drug, particularly one such as amphotericin B) is dissolved. is a drug that forms liposomes or lipid particles (amphoteric acid). B) is used in any mole %. In particular, in the present invention, from about 5 to about 50 moles % amphotericin B is used. When liposomes are formed, The molar ratio of the drug used is 5 mol% drug or less. About 6 to about 50 mole percent of drug is used in forming HD LCs. At 25 mol% drug and above, the population is essentially HDLC. In the heating aspect of the invention, amphotericin B was dissolved in methanol at a concentration of 10 mg amphotericin B per 100 ml methanol, or 0.1 mg/+sl amphotericin B. This methanol containing dissolved amphotericin B (100.0 ml) was added to the lipid membrane to resuspend the membrane. The resulting resuspension is then evaporated once under reduced pressure to obtain a thin film. The lipid-amphotericin B membrane is resuspended in a volume of aqueous solution. The volume of this solution is The amount is sufficient to form somesomes or lipid particles, such as about 4.0 ml. This suspension was then stirred. Sonicate for a few minutes to about 1 hour, then heat from about 25℃ to about 60℃ for about 1o. Heat in a water bath for approximately 400 minutes. Other methods for forming HDLCcs of the present invention employ a homogenization process rather than sonication. For example, HDLCs can be produced by the 5PLV method, in which drugs (e.g. For example, amphotericin B) is added to a suitable solvent (e.g. MDSO) and mixed mechanically. Mix in a mixer to dissolve all drugs. If necessary, the solution can be filtered to remove insoluble particles of drug. Then, a 7:3 molar ratio of DMPC:DMPG is dissolved in a suitable solvent (e.g., methylene chloride) to remove the lipids. Mix with previous drug-DMSO solution. An aqueous solution such as saline is then added to the mixture and the organic solvent is removed by rotary evaporation at about 35-45°C. melt Following vehicle removal, the resulting drug-lipid mixture is diluted with an aqueous solution such as saline. Interpretation. This suspension of HDLCs is pulverized using a homogenizer. Any homogenizer or colloid mill that attempts to pulverize zero particles can be used in this method, but it is preferable that a particle size that can tolerate grind to However, for example, 90% of the particles are smaller than 10 μm in diameter, and about 4 to about 10 μm in diameter. A range of grain size is preferred and milled for about 15-30 minutes. The particles are milled one to many times depending on the desired size and uniformity. The HDLC particles were measured using a Halvarn particle counter. The size distribution is then analyzed. For single particle dispersion such as filtration if necessary Particles that are too large or too small can be removed by any method known in the art. Preferably, such a process results in particles having a diameter between about 0.2 times m and No, Oum. The HDLCs invention incorporates the above-described method for its production, as other methods known to those skilled in the art for liposome formation may be used in the practice of the present invention. It is not limited. HDLC1 obtained from the novel method of the present invention! I-drugs and liposomes are used in animals (humans). used to treat infections or conditions that require the following: (1) repeated administration; (2) the drug in its biologically active form; or (3) substantially equivalent to the free delivery of the drug in question; has greater efficacy and reduced toxicity; these symptoms can be caused by fungal infections, naive Topical and systemic treatments that can be treated with antibacterial agents such as istatin and amphotericin B both physical symptoms and infections such as acquired immune deficiency syndrome (AIDS) and herpes. In addition, the formulations of the present invention are stable in aqueous solutions, including but not limited to carp virus diseases. The dosage form of this formulation determines the site and cells of the ffi tissue to which the compound is delivered. Although the HDLCs and liposomes of the present invention can be administered alone, the route of administration and Il! Administered in admixture with a drug carrier selected according to standard pharmaceutical practice. For example, delivery to a specific site is most easily achieved by local administration (infections are an example). In external cases, such as in the eyes, skin, or ears, or in cases of wounds or burns), such topical administration may be applied to the vulcanizate in the form of creams, ointments, gels, emulsions, or pastes. Administer directly to the affected area. On the other hand, this preparation can be administered parenterally, e.g. intravenously; intramuscularly. Injected into the flesh or subcutaneously. For parenteral administration, it can be administered, for example, in the form of a sterile aqueous solution containing other solutes 1, eg, sufficient salt or glucose to make the solution isotonic. Other uses are also conceivable by those skilled in the art due to the special properties of the formulation. The compositions of the present invention can be used in the treatment of asthma by instilling liposomes or a nebulized aqueous suspension of HDLCs into the lungs. Posomes or HDLCs are self-contained sprays extruded by air or ultrasonic atomizers, or conveniently by gas pressure from fluorocarbon pellets. The 1tFi is suspended in a suitable aerosolizable solvent by means of a nebulizer. Other inhalation systems, such as liposomes or those in which HDLCs are delivered in particulate form, dry powders and Delivery as a suspension or as a suspension in a suitable carrier system is acceptable. air Following losolization, most of the propellant is lost by flash evaporation and converted to N by the moisture in the breathing tract, leading to the precipitation of one particle. For administration to humans in the treatment or prophylaxis of bacterial or viral diseases, the prescribing physician will ultimately determine the appropriate human dose to be administered, but this will depend on the individual, as well as the nature and severity of the patient's symptoms. Varies depending on age, weight, and sensitivity. It is expected that the The dosage of drug in HDLC or liposomal form is generally about the same as that used for free drug. However, I will continue In some cases, it may be necessary to administer drugs in excess of these limits. , @<The examples are merely for illustrating the invention. It is not intended to limit the scope of the invention. 11. Amphotericin B (10 mg total drug 5 mol percent). Add to lOml methanol in a 500ml round bottom flask. This mixture was sonicated until clear. This sonication step was carried out in a sonicator at about 5 O-60 Hz and 25° C. for about 15 minutes. Similistoyl phosphatidylcoli (DMPC) was added together with 42 mg of simyristoylphosphatidylglycerol (DM PG) in 0.42 ml of chloroform (0.1 ml of chloroform). The resulting dispersion was dried by rotary evaporation at 60"C under reduced pressure. Dry to produce a thin film in a flask. The membrane was resuspended in 4.0 ml of PBS, and the solution was transferred to a glass tube and sonicated until clear. The above example was repeated using 16.7, 20, 25, 33, 50 and 60 mole percent amphotericin B. According to an acute toxicity test (LD, S) that measures the dose of a drug that causes 50% mortality in mice, LD, Amphotericin B increased as the mole percent of the agent increased up to 50 mole percent. 11 ml Amphotericin B (140 mg: 5 mole percent of total drug) in 1.5 ml diluted Added to methyl sulfoxide (DMSO). DMPC (1400 mg) and 600 mg DMPG were dissolved in 50 ml methylene chloride and the two solutions were transferred to a flask. The solution was mixed until clear, then dried by five rotary distillations under reduced pressure to form a film on the flask, and then dried in a Perger for 1 to 4 days. After this drying, the film forming the dried pieces was ground into powder and mixed with 100 ml or 50 ml of 0.9% physiological saline, or 50 ml of tJsP water to make an injection. The resulting suspension was heated to 37°C for 1 hour. The above example was modified to include methane instead of DMSO as the suspending solvent for amphotericin B. and repeated using amphotericin B mole percentages of 10, 16.7, and 33. 7:3 molar ratio of DMPC: 1g or more in place of DMPG and 2 g of egg phosphatidylcholine were used. Amphotericin B (100 mg; 5 mole percent total drug) was dissolved in 2.0 ml of DMSO. DMPC (100 mg in 1.0 ml) and DMPG (42 mg in 0.42 ml) were co-dissolved in chloroform in a flask and the chloroform was removed by rotary evaporation under reduced pressure. Methylene chloride (20 ml) was added to the flask followed by 0.2 ml of the stock amphotericin B solution. This WA? ! Solution ii was sonicated under the conditions described in Example 1 until clear (approximately 1 minute). PBS (0.3 ml), pH 7.2 was added, then sound was added. Methylene chloride was removed under a stream of nitrogen with wave treatment. The resulting paste was resuspended in 10.0 mL PBS. Ultracentrifugation was performed at 10.0 OOX g for 10 minutes, followed by sonic bath treatment for about 20 minutes. Repeated using rubercent amphotericin B. 1. Amphotericin B (140 mg: 5 mol percent of total drug) was added in a 1.5 ml volume. Dissolved in ethylene chloride. The two solutions were mixed until clear, transferred to a flask, and mixed with 8.0 ml of PBS. Methylene chloride, nitrogen flow in a sonic bath The bottom evaporated. Sonication was performed in the same manner as in Example 1. Add PBS (32 mg) and transfer the solution to a 5 micron pore polytetrafluoroethylene (PTFE) teflon tube. The mixture was filtered through a Ron filter, washed by centrifugation twice, and finally suspended in PBS at 100 m. The above example was applied to PBS 4. Lipid hydration was performed repeatedly using Omfi and 12.0 mM. Amphotericin B (140 mg; 5 mole percent total drug) was dissolved in 1.5 ml of DM SO. DMPC (1400 mg) and 600 mg D M P G were dissolved in 50 ml methylene chloride. The two solutions were mixed until clear, transferred to a flask, and dried to a thin film by rotary evaporation under reduced pressure. The membrane was subsequently resuspended in 50 ml of methylene chloride and 8.0 ml of PBS was added to this solution. Methylene chloride was removed by evaporation under nitrogen with sonication according to the procedure of Example 1. The resulting paste was further hydrated using 32 ml of PBS, the suspension was washed by centrifugation twice, and finally resuspended in 100 ml of PBS and 5 micron pore size Teflon (PTFE). ) fi Passed Ruta. The above example was repeated using glycine buffer at PH 3, 6 and 9 instead of PBS. The above examples were also modified to include hydration buffer volumes of 5.0, 10.0°, 15.0, 20. It was repeated using 0 ml (7) PBS. xLL12Mphotericin B (140 rng; 5 mole percent total drug) was dissolved in 1.5 ml of DMSO. Egg phosphatidylcholine (EPC) (2.0 g) was dissolved in 50 g of methylene chloride. The two solutions were mixed in a flask. Add PBS (8.0ml) and Then, the acid solvent was removed under a stream of nitrogen with sonication according to the procedure of Example 1. PBS (200ml) was added. The above example was repeated using 10 and 20 mole percent amphotericin B. did. z】1 ship Amphotericin B (140 mg; 5 mole percent total drug) was dissolved in 1.5 ml DMSO. DMPC (1400 mg) and 600 mg DMPG were dissolved in 50 g methylene chloride in a 50 ml round bottom flask. These two The solutions were mixed in a flask and then 10 ml of water for injection was added at 37°C. According to the method of Example 1, nitrogen treatment was carried out while sonication was performed. The solvent was evaporated using a simple stream, then 50 ml of water for injection, USP was added and the solution was warmed to 37° C. for 30 minutes. The above example was repeated using 10 and 16.7 mole percent amphotericin B. This solution was then passed through a 3 micron straight path polycarbonate filter sold by Nucleop. Amphotericin B (140 mg 16.7 mole percent of Li) was mixed with 50 ml of methanol and the mixture was sonicated for 15 minutes to form a solution. The sample was passed through a 0.22 micron tortuous path filter (cellulose and acetate mixed nitrate ester) sold by Millex. DMPC (350 mg) and 150 mg DMPG in 50 g Co-dissolved in Loride and mixed with the above liquid. this solvent. Evaporated-PBS (50 mg) under a stream of nitrogen with sonication according to the procedure of Example 1 was added. Half of the product (25 mg) was lyophilized using standard lyophilization methods. 1. Lou l of IN hydrochloric acid was added to 2.0 ml of anhydrous ethanol. Amphotericin B (20 mg; 5 mole percent total drug) was added to this acidic ethanol. The mixture was prepared in a similar manner except that the sonication time was between 30 seconds and 60 seconds. Sonicated under the conditions described in Example 1v until clear. DMPC (200 mg) and DMPG (42 mg) were placed in a test tube, to which was added a benzene:methanol (70:30) solution, and this solution was lyophilized as in Example 8. Ta. The dry lipid was mixed with 2.0 ml of PBS and the mixture was dispersed by vortex mixing. This amphotericin Bm solution (0°2 ml) was added to the lipid, and the mixture was The mixture was shaken overnight. The obtained DMPC: DMPG MLVs (200 u 1) was layered on an LJ refined sugar density gradient, and centrifuged at 230,0 OOX g; 22° C. for 24 hours. The results are shown in Figure 11; Amphotericin B is associated with lipids and is widely distributed at the top of the gradient, which contains most of the lipids, and most amphotericin B peaks are at the bottom of the gradient. On the other hand, the obtained DMPC:DMPG MLVs were freeze-dried and rehydrated with distilled water (2.0 ml). The above embodiments were applied to 7, 9, 16.7, 17, 20, 25, 33°5o and 60 Repeat with rubercent amphotericin B. At mole percent of drug of 16.7 and above, almost no HDLCs were formed. The density gradient 2 for such high drug:lipid ratio formulations was similar to that shown in Figure 5, where all drug and lipid were co-associated in a single peak. If! For X-ray diffraction = DSC, cryo-fracture electron micrograph and 1''P-NMR examination, 25 molvar centamphotericin B-DMPC: A sample of DMPG was prepared as follows. A 7:3 molar ratio of DMPC:DMPG (44 mg total lipids) and 20 mg amphotericin B (25 molar cent amphotericin B) was suspended in chloroform:methanol (70:30 V/V). It becomes cloudy and decreases at 55℃. Pressure evaporation was performed to form a thin film on the sides of the flask. The membrane was hydrated by mixing with 8.0 ml of 20 mM Hepes, 250 mM NaCl at pH 7, 2, and 22° C. by stirring with a glass bead. The product was centrifuged at 10,000 x g for 10 minutes, the supernatant was decanted and the pellet was resuspended in 1.0 ml of Hepes/NaCQ buffer. This product was then sonicated in a sonic bath for 20 minutes at 25° C. and 50-60 Hz. Repeat the above method using 0.5 and 50 mole percent amphotericin B. did. To determine the incorporated volume of amphotericin B containing liposomes and HDLCs, the following method was followed; DMPC (100 mg) and 42 mg DMPG in oo-form solution were mixed with 10 mg amphotericin B in methanol solution ( 0.1 mg/ml amphotericin B) and mixed in a flask. The solvent was removed under reduced pressure at 37°C. A solution of 0.3 ml of 1H-inulin in dilute distilled water in PBS (3.7 ml) was added to the dried lipid film with stirring. This melt Add a certain amount of liquid (10.2ml) to a scintillation cocktail and add beta scintillation to the scintillation cocktail. Radioactivity was measured using an application counter. The second aliquot was treated with a hologram according to the method of Barttett, Journal of Biological Chemistry (J. Biol. Ches.) 1959.234:466-468. The sphate was measured. This lipid-drug 1! The suspension was centrifuged for 10 minutes at 10,000 x g, the supernatant was decanted, and the pellet was resuspended in PBS. This centrifugation and residue resuspension step was performed two more times; the final resuspended suspension was sampled (100 ul) and measured in a beta scintillation counter. I made a decision. A second aliquot of the final residue resuspension was measured for phosphate as above. I made a decision. The percent uptake of inulin was calculated by dividing the last radioactive count by the starting count and multiplying by 10o. The uptake volume (inulin ul incorporated/U mole lipid) was also calculated. The above method was repeated using 0.5 and 50 mole percent amphotericin B. Amphotericin B particles were prepared by drying 15.5 mg of DMPC and 6.5 mg of DMPG (7:3 molar ratio) from approximately 10 ml of chloroform onto the side of a 100 ml round bottom flask. Amphotericin B (10 mg) was suspended in 100 ml of methanol, and 100.0 ml of this methanol solution was added to the flask to suspend the lipid film to produce 25 mole percent amphotericin B. The mixture was dried on a rotary evaporator at 37°C. A thin film was formed on the sides of the flask. The membrane was hydrated with 4.0 ml of 10mM Hepes, 150mM NaCl (pH 7,2) using glass beads and sonicated for 30 minutes to produce the final suspension. A sample of this suspension was then immersed in a water bath and heated to 60°C for 10 minutes. The obtained 2i solution was analyzed by DSC, NMR and freezing. Characterized by crystallization electron** mirror photography. The above method was repeated without heating the sample. This sample was kept at 22°C and characterized using the techniques listed above. The raw materials and method of Example 12 were combined with 50 mole percent and 5 mole percent Repeated with amphotericin B. These systems were characterized by DSC, ESR, and freeze-pulverization electron micrographs. [! I am located in Angeles, Massachusetts. The test was carried out on a Micro Cal, Inc. (Amherst, Mass.) MC-1 Unit. 5-9 mg of J! ! Place 0.70 ml of sample containing the suspension into the sample cell and the same amount of buffer into the control cell. It was. These samples were heated at either about 6°C/hour or about 7°C/hour. When the same sample with the same history is subjected to the same operation, the starting and ending temperatures are reproducible to 0.2°C. Typically, samples containing amphotericin B were heated to 60°C (no higher) and then cooled to 7-4°C for at least 2 hours to ensure a consistent sample history. . 8 to 8 data points for acquisition, on a Bruker AM360 wide-width NMR spectrometer using a sweep width of 50,0 OOHz and a pulse width of 20 usec corresponding to a 45° pulse. An NMR spectrum at 145.7MH2 was obtained. The spectrum is 10,000 Even transients were collected. After 11 days, a 0.1-0.3 ujl aliquot of the sample was sandwiched between a pair of Balzer (Nashua, NH) copper support plates and quickly placed into liquid propane at 23°C. Crush the sample and apply a vacuum of 2 x 10-'m bar or higher at -115°C. and replicated in a Balzer freeze-grinding unit medium replicator. The replicates were floated in 3N HNO3 and then washed in a gradient series of sodium hypochlorite solutions. These were finally cleaned with distilled water and taken up on a 3008ex mesh copper grid (Polyseiences, PA). Reproductions were examined using a Ph1lips 300 electron microscope at magnifications of 3,000 to 22,000. ! I guessed it. Example 17 Amphotericin B was diluted to 25 uM liter amphotericin in Hepes/Na Cρ buffer. The absorbance spectra were prepared by dilution to telycin B (as in Figures 12 and 13), the sample was placed in the sample cuvette of a Beckman spectrophotometer, and read at 300 to 500 nm. did. This sample cuvette The samples were read using buffer as a control. Example 18 Samples for ESR were prepared using a series of regioisomers of doxylsterine w1 (doxyl 5taric acid) in which doxyl reporting groups are present at different positions along the fatty acid chain. I understood. Labeling was performed by introducing the probe from an ethanol solution by stirring and sonication, which was dried to a thin film on the side wall of the test tube. All samples were swL'd to 1 mole percent. ESR spectra were recorded on an IBM Instrument ER100D ESR spectrometer with nitrogen gas flow temperature control. External measurement thermistor The temperature of the sample was determined using a tar probe (0+mega Engineering, Inc., Stamford, Calif.). The ESR spectra were analyzed using IOMW microwave power, microwave number 9.0 with a field sweep of 100 G and a 100 KH2 field amplitude modulation of 0.32 G. ] To IG HZ I recorded it. The parameters of this dimension were measured from the maximum hyperfine splitting (maximum). Amphotericin B (337.5 g) was added to 3375 m ft of DMSO. This amphotericin B (100 wag/mΩ) was dissolved using a mechanical mixer. Stir until dissolved (approximately 3 hours). The mixture was transferred to a stainless steel compressor (5 fl capacity) - 0.22 um 5 artofluor filter and a polypropylene filter. The mixture was filtered through a deep filter (Pall Profile, Pal, Inc., GlenCove, N.Y.). + 50.8 kg of methylene chloride in a 40Q compressor with 225.0 g of DM PC and 99.8 kg of methylene chloride. Of DMPG and mixed for 3.5 hours to completely dissolve. The obtained lipid solution was diluted with 0.2M” 5artofluor or 0.22 micron Tefluor. The mixture was transferred through a filter to a stainless steel reaction tank. The 40Q compressor was washed with an additional 55.2 kg of methylene chloride and filtered into the reaction tank in the same manner. The reactor was set to rotate and mix the lipid solution at 195rP-. The amphotericin B DMSO solution was then transferred to this tank. salt Sodium solution (16,210,9% USP) was added to the tank through a 0.22um Millipak 100 polycarbonate filter. connected to this reaction tank. Heated using a heat exchanger set at 35°C. A stream of liquid nitrogen was passed through this tank and the solvent was removed over a 12 hour period. Obtained The lipid-drug complex was diluted with sodium chloride 0.9% USP (7000 mQ) and transferred to this tank through a 0.22 um Millipak 100 polycarbonate filter. The resulting HDLCs were passed through a Gifford Wood colloid mill head for 3.0 hours with a 0.5 ml gap set at a back pressure of 10 psi. Particles smaller than 20 microns were obtained. The particle size of the HDLCs was analyzed using a Malvern particle counter. Lipids (102.6 umol total, 70:30 molar ratio DMPC:DMPG dissolved in chloroform) were pipetted into a 500 mn round bottom flask. Na Istatin (500) was dissolved in a 1.0Ω methacrylate by sonication in a Branson sound bath. Dissolved nystatin (5.0 mΩ) dissolved in alcohol (0.5 I!g/mΩ) was added to the round bottom flask containing the lipids and mixed; the solvent was rotary evaporated for 15 min at 45 °C. and the resulting formulation was hydrated in physiological saline (5.0 m Q ) with rotational mixing using glass beads. Liposomes were visible under light microscopy. +25.50.75. and repeated using 100 mol,% nystatin. I went back. A cryo-pulverization electron microscopy test revealed that this 25 mol% The formulation containing tin was non-liposomal HDLC5. 1'' LL was hydrated in distilled water and incubated at 4°C. The resulting MLVs were extruded 10 times through two stacked polycarbonate filters using the LUVE T method. Amphotericin B was added to distilled water at a concentration of 10.8 umol/m Q using a sonic bath. dispersed inside. This amphotericin B dispersion was added to the above lipid suspension to give a final concentration of lipid and amphotericin B of 13°5u+ool/mD and 0.98umol/mQ, respectively. Excluding unincorporated amphotericin B The 20 mΩ samples were centrifuged for 30 min at 15,000 x g in a Ti 60 or 5W 270-tar (Beckman) at 22°C in a Beckman L8-60 ultracentrifuge. The supernatant without amphotericin B was removed without disturbing the liposome precipitate. When the obtained liposome was measured by 4:1 elastic light dispersion, the diameter was 1. It was bigger than ou m. The above method was repeated employing incubation conditions of 23° C. and 150 mM NaC Q, 10 mM Na, PO4+ pH 7.4 to hydrate the lipids. When the obtained liposome was measured by quasi-elastic light dispersion, the diameter was 1. It was bigger than um. The percentage of amphotericin B taken up by liposomes was highest when the ionic power of the solvent was low (distilled water vs. 150 m M Na CQ) (Figure 17). Figure 16 shows the DMPC:DMPG reaction under various incubation temperature conditions. Indicating the uptake of amphotericin B into the posomes, the rate of uptake was similar at 23°C and 45°C. This drug is. A similar accumulation occurs when the lipid is in the gel phase, for example at 15°C. The lipids suspended in distilled water are incubated with amphotericin B at 22°C. The process was repeated using the raw material of Example 20 under these conditions. The resulting liposo The membrane was unilamellar, and the average diameter was measured to be approximately 0.1-0.2 um by quasi-elastic light dispersion. Figure 19 shows that in the first two hours, the L U Vs than in the MLVs This shows that uptake of amphotericin B is fast. Figure 1 Mol% Amphotericin B 6F Lipid (in m) Amphotericin B (μM) Figure 10 nm Figure 11 Figure 12 Figure 13 Temperature °C Figure 14 Figure 15 Figure 16 Figure 17 International Investigation report Al? +ItAb11Ml a*s+° (11°”” two/+<9+J/nM t)

Claims (43)

[Claims] 1. Contains a bioactive agent-lipid complex (“HDLC”) and has biological activity on oils. A composition in which the proportion of the agent is at least about 6 mole percent. 2. 2. The composition of claim 1, which is esthetically free of ribosomes. 3. 2. The composition of claim 1, wherein the lipid comprises a phospholipid. 4. The composition according to claim 3, wherein the phospholipid contains a saturated phospholipid or a saturated fatty acid. thing. 5. Phospholipids contain phosphatidylcholine and phosphatidylglycerol The composition according to claim 3. 6. Phosphatidylcholine is simiristoylphosphatidylcholine, and and phosphatidylglycerol are dimyristoyl phosphatidylglycerol. 6. The composition of claim 5. 7. Approximately 7:3 molar ratio of phosphatidylcholine and phosphatidylglycerol 6. The composition according to claim 5, which is a ratio of 8. The mole percent of bioactive agent to phospholipid is approximately 6 and 50 mole percent. 2. The composition of claim 1, wherein the composition is between . 9. the mole percent of bioactive agent is between about 25 and 50 mole percent The composition according to claim 2. 10. The composition of claim 1, wherein the bioactive agent is a drug. 11. The composition of claim 1, wherein the bioactive agent is an antimicrobial agent. 12. 12. The composition of claim 11, wherein the antimicrobial agent is a polyene antibiotic. 13. 13. The composition of claim 12, wherein the antimicrobial agent is amphotericin B. 14. 13. The composition of claim 12, wherein the antimicrobial agent is nystatin. 15. Claims wherein amphotericin B is present in amounts of about 6 and 50 mole percent. 14. The composition according to 13. 16. Claims that amphotericin B is present between about 25 and 50 mole percent The composition according to item 13. 17. comprising the HDLC of claim 1 and a pharmaceutically acceptable carrier or diluent. A pharmaceutical composition comprising: 18. 18. The pharmaceutical composition of claim 17, which is administered for parenteral administration. 19. 19. The pharmaceutical composition of claim 18, wherein the composition contains an antimicrobial agent. 20. 20. The pharmaceutical composition according to claim 19, wherein the antibacterial agent is amphotericin B. 21. Claims that amphotericin B is present between about 25 and 50 mole percent Item 21. Pharmaceutical composition according to item 20. 22. 22. The HDLC has a size between about 0.2 and 10 microns. Pharmaceutical compositions. 23. 20. The composition of claim 19, wherein the antimicrobial agent is nystatin. 24. An infectious disease-effective amount of the pharmaceutical composition of claim 17 is administered to a mammal in need of treatment. A method of treating infectious diseases. 25. 25. The method according to claim 24, wherein the infectious disease is a fungal infection. 26. 25. The method according to claim 24, wherein the infectious disease is an infection by a virus. 27. The set of claim 1, which is substantially free of ribosomes containing bioactive agents. A product. 28. 6. The HDLC has a size between about 0.2 and 10 microns. Composition of. 29. (a) forming a ribosome containing at least one lipid; (b ) suspending the drug in acidified ethanol; and (c) suspending the drug of step (b). A drug and a lipid comprising the step of mixing the suspension and the ribosomes of step (a). A method of forming HDLC or ribosomes containing. 30. 30. The method of claim 29, wherein the drug is present at about 5 mole percent. 31. 31. A ribosome formed by the method of claim 30. 32. 31. The method of claim 30, wherein the drug is present at about 25 mole percent. 33. (a) dissolving at least one lipid in an organic solvent of intermediate polarity; (b) dissolving the drug in a biologically acceptable organic solvent; and (c) ( A HDLC containing step of mixing the solution of a) and the solution of step (b) A method for producing a composition. 34. 34. The drug of claim 33, wherein the drug is present between about 25 and 50 mole percent. Method. 35. (a) dissolving at least one lipid in an organic solvent of intermediate polarity; (b) dissolving the drug in a biologically acceptable organic solvent; (c) step (a); and (d) mixing the solution obtained from step (c) with the solution of step (b); A method for producing a composition containing HDLC, the method comprising the step of dehydrating a mixture containing HDLC. 36. (a) dissolving at least one lipid in an organic solvent of intermediate polarity; (b) dissolving the drug in a biologically acceptable organic solvent; (c) step (a); (d) mixing the solution of step (c) with the solution of step (b); (e) producing a dry lipid membrane by evaporating the solvent under reduced pressure; and (e) the membrane of step (d). A method for producing a composition containing HDLC, comprising the step of adding an aqueous solution to. 37. (a) dissolving the drug in a biocompatible organic solvent; (b) at least one Dissolve two lipids in an organic solvent of intermediate polarity and mix this with the solution in step (8). (c) Adding an aqueous solution to the mixed solution of (a) and (b) above and reducing the pressure (d) evaporating the solvent to form a paste; and (d) the step of step (c) above. Production of a composition containing HDLC, comprising the step of adding an aqueous solution to a yeast Method. 38. Amphotericin B and DMPC:DMPG at a molar ratio of 7:3 were prepared by the MLV method. forming a formulation containing the HDL; and heating the formulation. Method of forming C. 39. Claims that amphotericin B is present between about 25 and 50 mole percent The method according to item 38. 40. 40. The method of claim 39, wherein the formulation is heated to about 60<0>C for about 10 minutes. 41. Deciphering the absorption spectrum of HDLC preparations and measuring the intensity of its peaks A method for determining the toxicity of HDLC. 42. (a) suspending the drug in an aqueous solution; (b) suspending a lipid in an aqueous solution; (c) mixing the products of steps (a) and (b); and ( d) incubating the product of step (c) at or above the transition temperature of the lipid; HDLC liquid containing drug and lipid at a high drug molar ratio, including a step of A method of manufacturing a bosome-containing composition. 43. 43. The method of claim 42, wherein the drug is suspended in the aqueous solution by sonication.