HU209647B - Process for production of low toxicity composition containing bioactive agent and lipid - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to a process for the preparation of combined compositions (HDLCk) containing a biologically active substance and a lipid, wherein the ratio of the active ingredient to the lipid is at least 6 mol%, and which do not contain liposomes. The process involves dissolving the lipid in a medium polar organic solvent, dissolving the active ingredient in a biocompatible solvent, and mixing the two solutions. HDLC systems of the present invention have reduced toxicity. EN 209 647 B Description scope: 36 pages (including 20 sheets)

Description

FIELD OF THE INVENTION The present invention relates to a combination of low toxicity biologically active agent and lipid, high active ingredient / lipid ratio, essentially liposome free (HDLC).

These formulations generally have the same or greater potency and are less toxic in the case of formulations which otherwise contain the same active ingredient in free form. Preferably, the process of the present invention provides compositions comprising polyene antifungal antibiotics, namely amphotericin B and nystatin, as the biologically active agent.

High biologically active agent: lipid ratio combinations (HDLCs) can be prepared by methods similar to liposomes. The method of the present invention provides drug / lipid systems (HDLCs) in which the ratio of active ingredient to lipid is at least 6 mol%.

Liposomes are completely closed, bilayer lipid membranes that contain a certain volume of water. These can be unilamellar (monolayer) bubbles (surrounded by a single bilayer membrane lock) or multilamellar (multilayer, bulbous structures where each layer is separated by an aqueous layer). The bilayer consists of two single lipid layers which have a hydrophobic "tail" part and a hydrophilic "up" part. The structure of the bilayer membrane is structured in such a way that the hydrophobic (apolar) "tail" part of the single lipid layers is directed towards the center of the bilayer, while the hydrophilic "heads" are directed towards the aqueous phase.

The original liposome preparation method of Bangham et al., J.Mol. Bioi., 1965, 13: 238-252 consists of leaving the phospholipid film in the vessel by evaporation to dryness of the phospholipid in an organic solvent. In the next step, an appropriate volume of aqueous phase is added, the mixture is allowed to swell, and the resulting liposomes, which consist of multilamellar bubbles (MLV), are dispersed by mechanical means. This procedure forms the basis for the method of forming small, ultrasound-scattered unilamellar bubbles described by Papahadjopoulos et al., Biochem. Biophys. Acta, 1967, 135: 624-638.

Liposomes can be prepared using techniques for forming large unilamellar bubbles (LUVs), such as reverse-phase evaporation, infusion techniques or detergent dilution. A review of these and other methods for producing liposomes can be found in Marc Ostro's (Liposomes, ed. Marcel Dekker, Inc., New York, 1983, Chapter 1) and Szoka, Jr. et al. (1980, Ann. Rev. Biophys. Bioeng. 9: 467). A particularly preferred method of producing LUVs is described in Cullis et al., "Extrusion Technique Foal Producing Unilamellar Vesicles" (PCT Publication No. 87/00238, Jan. 16, 1986). The so-called bubbles, so-called LUVETs, are extruded under pressure through a membrane filter. Bubbles can be formed with extrusion through 200 nm filters, referred to as EA ₂ oo. Such bubbles may also be subjected to at least one freezing and thawing cycle prior to extrusion as described by Mayer et al. (1985, Biochem. et Biophys. Acta, 817: 193-196).

A number of steroids and their water-soluble salts have previously been used in the preparation of liposomes, see, for example, Janoff et al., Jan. 1988, Steroidal Liposomes. United States Patent No. 4,721,612, filed March 26,. Mayhew et al. PCT Publication No. WO 85/00968 of WO 04/00968 describes a method for reducing the toxicity of therapeutic agents, which comprises embedding these substances in liposomes containing α-tocopherol and certain derivatives thereof. In addition, a number of tocopherols or their water soluble derivatives can be used to prepare liposomes, cf. Janoff et al. 23, PCT Publication No. 87/02219, "Alpha-Tocopherols-Based Vesicles".

In a liposome / drug delivery system, the biologically active agent is administered to the patient to be treated embedded in the liposome. for example, Rahman et al., U.S. Patent No. 3,993,754; Sears, U.S. Patent 4,145,410; Paphadjopoulos et al., U.S. Patent 4,235,871; Schneider, U.S. Patent 4,224,179; Leen et al., U.S. Patent 4,522,803; and Fountain et al., U.S. Patent 4,588,578. If the drug is lipophilic, it may be linked to the lipid bilayer. As used herein, the term "embedded" refers to a therapeutic agent that is present both in the aqueous medium of the liposome and that binds to the lipid bilayer.

Most medicinal substances that are useful in the treatment of diseases also exhibit toxic effects; this may be cardiotoxicity such as doxorubicin against tumor or nephrotoxicity such as aminoglycoside or polyene antibiotics such as amphotericin B. Amphotericin B is a highly toxic, antifungal polyene antibiotic, but is uniquely reliable in the treatment of life-threatening fungal infections (Taylor et al., Am. Rev. Respir. Dis., 1982, 125: 610-611). Because amphotericin B is a hydrophobic drug, it is insoluble in water and is therefore marketed in the form of a deoxycolate medium colloidal dispersion. A detergent which is toxic in itself is used as a suspending agent. The ethological studies of acquired immune deficiency syndrome (AIDS) are simulated2

Amphotericin B and its methyl ester have been found to be effective against HTLV-III / LAV virus, a lipid-embedded retrovirus (Schaffner et al., Biochem. Pharmacol. 35: 4110-4113 (1986)). In this assay, the water soluble ascorbic acid salt of amphotericin B methyl ester was used to separate cells infected with HTLV-III / LAV virus, and the cells were tested for viral replication. The results show that amphotericin B and its methyl ester protect target cells from the cytopathic effects of the virus in a manner similar to that observed for herpes viruses (Stevens et al., Arch. Virol., 1975, 48: 391).

There are reports in the literature about the use of amphotericin B embedded in liposomes. Juliano et al. (Annals Y. Y. Acad. Sci. 1985, 446: 390-402) discuss the treatment of a general fungal infection with amphotericin B combined with liposomes. Such liposomes include, for example, phospholipids, e.g. a 7: 3 molar mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) and cholesterol. The LD ₅₀ values obtained in acute toxicity studies and in vitro studies comparing free and liposome-embedded amphotericin B have shown that liposomal formulations have less toxicity, while their anti-fungal activity remains essentially unchanged. Lopez-Berestein et al. (J. Infect. Dis., 1986, 151: 704-710) administered amphotericin B embedded in liposomes to patients with general fungal infections. The liposome consisted of a 7: 3 molar mixture of DMPC and DMPG, and the degree of drug incorporation was greater than 90%. 66% of patients responded positively to treatment with 5 mol% Amphotericin B, with partial or complete resolution of the fungal infection. Lopez-Berestein et al. (7th Infect. Dis. 1983, 147: 939-945), Ahrens et al. (S. Jour. Med. Vet. Mycol. 1984, 22: 161-166), Panosian et al. Antimicrob. Agents Chemo., 1984, 25: 655-656) and Tremblay et al. Comparative studies of the effect of free and liposome-embedded amphotericin B were performed. In the treatment of candidiasis, amphotericin B embedded in liposomes has been shown to have a higher therapeutic index. In each case, the results showed that much higher doses of amphotericin B were tolerated when applied to the liposome. In contrast to leishmaniasis, the amphotericin / liposome combinations show very little or no effect.

Proliposomes containing DMPC: DMPG blend, ergosterol and amphotericin B have also been prepared, which are granules containing the lipid and the drug as a coating on a soluble carrier. (Payne et al., 7th Pharm. Sci., 1986, 75: 330-333).

In other studies (Graybill et al., (7)

Infect. Dis., 1982, 145: 748-752) in cryptococcosis of mice with liposome-embedded amphotericin B or mice. treatment of amphotericin with B-deoxycholate was compared. Mice treated with the liposome / amphotericin B combination showed a higher survival rate and lower tissue content and acute toxicity of cryptococci. A multilamellar liposome containing ergosterol was used in the experiment. Taylor et al. (Am. Rev. Respir. Dis., 1982, 125: 610-611) treated histoplasmosis of mice with a liposome / amphotericin B combination formulation containing the liposome portion of ergosterol Is phospholipids. These formulations were less toxic than those containing free amphotericin B and were more effective in the treatment of histoplasmosis, and altered the distribution in serum and tissues by decreasing serum concentrations and increasing liver and spleen concentrations.

In the aforementioned studies, lipid-containing liposomes were used to reduce the toxicity of the embedded drug and the goal was to further reduce the toxicity of the drug by increasing the lipid content of the formulations. Surprisingly, our studies have shown that low lipid concentrations greatly reduced toxicity when used in the preparation of HDLCs by any of the MLV methods of the present invention, resulting in a mixed HDLC population with MLVs. These formulations contain at least 6 mol% of the therapeutic agent (amphotericin B) and the proportion of HDLCs increases with the concentration of the therapeutic agent. Formulations containing about 25 to about 50 mole% amphotericin B are virtually pure liposome-free HDLCs. However, compositions containing about 5 mole% or less of the therapeutic agent are virtually liposomes that contain a small amount of HDLC. If necessary, HDLCs from heterogeneous populations are separated by methods known in the art, such as density gradient centrifugation.

HDLCs are prepared in a manner similar to liposomes, but the high active ingredient / lipid ratio process of the present invention essentially produces a liposome-free composition consisting essentially of HDLC, which results in an unexpected increase in toxicity compared to liposomes. reduced.

The present invention therefore relates to a process for the preparation of high active ingredient / lipid ratio complex systems (HDLCs) containing lipids and biologically active substances which are substantially liposome-free. These HDLCs may contain phospholipids, such as DMPC or DMPG, preferably in a 7: 3 molar ratio, or saturated or fatty acid phospholipids. Preferably, the biologically active agent is a therapeutic agent, such as nystatin or amphotericin B, present in the composition in an amount of at least 6 mol%, preferably 3050 mol%. The HDLCs

In combination with a pharmaceutically acceptable carrier or diluent, parenteral pharmaceutical compositions can be prepared. These compositions are used to treat various infections, such as fungal infections, when administered to a mammal such as a human. The amount of lipid present in the composition is generally no more than about 10% by weight, preferably no more than about 5% by weight, more preferably no more than about 3% by weight.

In the process of the invention, a solution of the lipid in a medium polar organic solvent is mixed with a solution of the drug in a biocompatible solvent and optionally

dehydrating the mixture, or

- evaporating the solvent under reduced pressure and adding an aqueous solution to the resulting dry film, or

adding an aqueous solution to the mixture, evaporating the solvent under reduced pressure and adding an aqueous solution to the resulting paste.

A variety of methods can be used to prepare the HDLCs in question, including methods for first solubilizing the active ingredient, namely amphotericin B, in an organic solvent such as DMSO or methanol. The lipid (preferably 7: 3 molar ratio DMPC: DMPG) is also solubilized in a solvent such as methylene chloride and the two solutions are mixed. The solvents were evaporated under reduced pressure to give a thin film of active ingredient / lipid. The grass may be hydrolyzed in an aqueous solution such as aqueous saline in PBS or glycerol buffer to form HDLC. Alternatively, the aqueous solution is added to the solvent active ingredient / lipid phase prior to the solvent evaporator. Alternatively, the resulting dry drug / lipid film is resuspended in a solvent such as methylene chloride and the solvent is again evaporated under reduced pressure before hydration of the film. A dehydration process may also be employed; this operation involves dehydrating the dry drug / lipid film to form a fluffy material which is then hydrated with an aqueous solution.

From the absorption spectrum of the composition of the present invention, the toxicity of drug (e.g., polyene, antifungal agents such as amphotericin B) lipid combinations can be determined. The absorption spectrum is specific to the particular drug; the drug may be represented by a peak or a series of peaks in the ultraviolet or visible light range. The B peak of amphotericin B (taken from the deoxycholate solution shown in Figure 12) is between 300 and 500 nm and has characteristic peaks, the most typical being 413 nm. When the drug is combined with a lipid, this peak flattens out and this phenomenon can be used to quantify HDLC toxicity. In other words, the degree of toxicity is characterized by the height of the absorption peak.

Drawings

Figure 1: Acute toxicity as measured by LD50 as a function of amphotericin B in mole% formulation

Figure 2: Differential scanning calorimetric spectrum of MLV liposome formulation containing 5 mol% amphotericin B

Figure 3: Differential scanning calorimetric spectrum of an HDLC formulation (prepared by MLV) containing 25 mol% amphotericin B.

Figure 4: Isopycnic gradient of a multilamellar liposome preparation containing 5 mol% of amphotericin B. Solid line = lipid; dashed line = amphotericin B

Figure 5: Isopycnic gradient of HDLG formulation prepared by MLV containing 50 mol% of amphotericin B. Solid line = lipid; dashed line = amphotericin B

Figure 6: 5 and 5 respectively. Containing 50 mole% amphotericin B,

MLV methods produced liposomes and liposomes. X-ray diffraction pattern of HDLC composition

Figure 7: Figure 25 Containing 50 mole% amphotericin B,

³¹ P NMR spectrum of the HDLC preparation prepared by MLV

Figure 8: 0 and. Containing 5 mole% amphotericin B,

³¹ P-NMR spectrum of a liposome preparation prepared by MLV

Figure 9: Isopycnic density gradient of acid / ethanol liposome composition containing mole% amphotericin B. Solid line = lipid; dashed line = amphotericin B

Figure 10: Absorption spectrum of amphotericin B dissolved in deoxycholate

Figure 11: Embedded in a 7: 3 DMPC: DMPG mixture of (a) 5 mol% amphotericin B, (b) 25 mol% amphotericin B, (c) 25 mol% amphotericin B after heating, and (d) 50 mol% absorption spectrum of a composition containing amphotericin B

Figure 12: Absorption spectrum of a formulation containing 25 mol% amphotericin B embedded in a 7: 3 DMPC: DMPG mixture (a) for 10 minutes before heating at 60 ° C and (b)

Figure 13: Absorption spectrum of a formulation containing 25 mole% amphotericin B embedded in a 7: 3 DMPC: DMPG mixture (a) before and after the heating cycle and (c) as a comparison of pure DMPC: DMPG mixture

Figure 14: ^3I P-NMR spectrum of a 7: 3 DMPC: DMPG mixture containing 25 mole% amphotericin B before (a) the 60 ° C heating cycle and (b)

Figure 15: Effect of ionic strength on amphotericin B

DMPC: For incorporation of DMPG into liposomes

HU 209 647 B

Figure 16: Effect of incubation temperature on incorporation of amphotericin B into DMPC: DMPG liposomes

Figure 17: Effect of liposome structure on amphotericin

B for DMPC: DMPG incorporated into MLVs or LUVs

The following describes non-liposomal high drug / lipid combinations (HDLCs) with specific properties and methods for their preparation. These HDLCs contain biologically active substances, such as a hydrophobic drug, which, when administered to an animal, may have the same or greater potency and less toxicity than the drug administered in free or liposome form. Such HDLCs have a drug / lipid ratio of greater than 6 mol%. Surprisingly, it has been found that formulations containing less lipids, such as fast dissolving HDLC systems, are less toxic than liposomal systems. HDLCs prepared by the process of the invention, for example containing antifungal polyene antibiotics such as amphotericin B or nystatin, can be used as a drug delivery system that is stable in commercial solutions, such as saline.

The term HDLC refers to a drug / lipid combination consisting of non-liposome particles. An example of HDLC produced by MLV is characterized by the following methods: (1) detection of freeze-fracture electron microscopy (Deamer et al., Biochim. Biophys. Acta, 1970,219: 47-60), non-liposome complexes; (2) measuring bound volume (Deamer et al., Chem. Phys. Lipids, 1986, 40: 167-188) showing that the embedded volume is virtually zero, so that the resulting complex is non-liposomal; (3) differential scanning calorimetry (DSC), (Chapman, D., in: Laposome Technology, Gregoriadis, G., ed., 1984, CRC Press, Boca Raton), which did not show the presence of a bilayer in either pre-transition or and in the main transition phase; (4) ^31- P-NMR spectra (Cullis et al., 1982, in: Membrane Fluidity in Biology, Academic Press, Inc., London & N.Y.), showing highly immobilized (bound), (broadly isotropic) lipid characteristics. and (5) X-ray diffraction measurements (Shipley et al., in: Biomembranes, 1973, Chapman, D. and Wallach, D. Eds., Vol. 2: 1, Academic Press, Inc., London & NY), which indicated the presence of gel phase lipid. Also characterized by these systems is the complete association of the drug with the lipid, as detected by density gradient centrifugation. This method is capable of increasing the strength (approx. 230,000 g) to approx. Centrifugation for 24 hours ensures that each component of the gradient reaches a steady state density. The elution (dissolution) curves of these systems overlap between drug and lipid peaks, indicating that the total amount of drug is present in the lipid-bound state.

The method of the present invention results in the formation of HDCLs having the same or higher potency but less toxicity as the biologically active agent, as demonstrated by the LD ₅₀ values in mice (see Figure 1). It has been found that at least 6, and preferably from about 15 to 50, more preferably from about 25 to 50, most preferably from about 25 to 35 mol% of the hydrophobic drug fulfills these requirements. If the drug concentration is greater than 6 mol% and approaches 25 mol%, mixed populations of liposomes and HDLCs may be formed. Within this range, as soon as the drug concentration reaches 25 mole%, the structures will have more HDLC than the liposome. At concentrations of about 5 mole% or less, up to the lower limit known as "critical micelle concentration" in the literature, there are mixed HDLC / liposome populations, which are, however, more liposome-structured.

The above-mentioned HDLCs are characterized by the presence of various drug dispersion dispersions, which can be observed by density centrifugation. Separation of the 7: 3 molar DMPC: DMPG drug / lipid combination on isopycnic sucrose density columns results in layering that is independent of drug to lipid ratio and method of preparation. MLV systems containing 5 mole% of drug exhibit two major layers of material: one composed of liposomes and the other containing lipid-bound active ingredient (HDLCs). Compositions containing 25 to 50 mol% of active ingredient exhibit a single layer in which most of the active ingredient binds to the lipid. Surprisingly, these systems, which contain low levels of lipid and higher concentrations of the drug, are less toxic. In Figure 1, measured in mice LD ₅₀ values (mg / kg), amphotericin B composition shown in mol -% - as a function of. LD ₅₀ values increase from 5 to 50 mol% drug concentration and then drop abruptly at 60 mol%. The commercially available form (Fungizone) of this drug has an LD _S0 of about 3.5 mg / kg. In vitro erythrocyte degradation studies show the same toxicity phenomenon.

The results of the fixed volume measurements (mixed solids μ kö / pmol lipid embedded solids) of the variable drug formulations produced by MLV show that HDLC formulations with a concentration of 25 mol% or higher exhibit unusual behavior: such systems they do not bind solute, so they are not liposome-like. Freeze-break electron microscopy images of these systems show the presence of liposomes at 0 to 5 mol% drug content, while non-liposomal HDLCs are present at 25 to 50 mol%.

Differential-scanning calorimetric studies on MLV-based variable drug formulations show the same phenomenon, that is, the spectra show peaks at 5 mole%, unchanged bilayer lipid (Figure 2), but not at 25 mole%. occurs in the transformation

EN 209 647 Β peak (Figure 3). At 25 mol% drug content, all lipids are present in the drug bound state, i.e., the acyl chains of the lipid are not free for a cooperative transformation. These data confirm the density centrifugation results, which at drug concentration of 5 mol% show a double peak, one corresponding to lipid bound and the other corresponding to free drug (Figure 6) but 25 and 50 mol% concentration, where all lipids are bound to the drug, only one peak appears (Figure 5).

X-ray data for 5 mol% MLV formulations show the presence of a gel at low temperature in the gel phase which is converted to a liquid crystalline phase at 23 ° C. However, at 50 mol% drug content, the lipid is gel phase at any temperature; there is no conversion point because all lipids are in close association with the drug (Figure 6). Similar data from ³¹ P-NMR studies of these higher (25 mol%) drug-containing formulations (HDLCs) also show a lack of free lipid signal; the curves do not show the flat shoulder and steep peak (Fig. 7), which can be observed on the 0 and 5 mol% drug content samples (Fig. 8).

Lipids useful in the preparation of the lipid combinations of the invention include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PG). PA), phosphatidylinositol (P1), sphingomyelin (SPM), etc. These lipids can be used individually or in combination. Saturated phospholipids such as hydrogenated PC may also be used. The phospholipids may be of synthetic origin or may be derived from natural sources, such as eggs or soybeans. Lipid formulations may also contain saturated fatty acids such as myristic acid. In preferred embodiments of the process according to the invention, dimyristol phosphatidylcholine (DMPC) and dimyristol phosphatidylglycerol (DMPG) are used in any molar ratio of 99: 1 to 1:99, preferably 7: 3. The combination of DMPC and dimyristol phosphatidylserine (DMPS) or DMPC may be used alone. Lipid combinations and liposomes may also contain a steroidal component as part of the lipid phase, such as cholesterol, polyethylene glycol, cholesterol derivatives (PEG-cholesterol), coprostanol, cholestanol, cholestane, organic acid derivatives of sterols. cholesterol hemisuccinate (CHS), etc. Organic acid derivatives of tocopherols, such as α-tocopherol hemisuccinate (THS), may also be used in combination or in combination. as a liposome-forming component. Both the CHS and THS-containing combinations and their tris salt forms can be prepared by any method known in the art for the preparation of liposomes containing such sterols, see U.S. Patent No. 4,721,614 to Janoff et al. Liposomes ”, Jan. 26, 1988) and the same authors Apr. Methods disclosed in PCT application 87/02219 of 23 September 1986, filed September 24, 1986, entitled "Alpha-Tocopherol Based Vesicles".

The compositions of the present invention employ biologically active substances, namely hydrophobic substances. Examples of such biologically active substances include, but are not limited to, polyene antibiotics such as antifungal agents such as pyramine, candicidin, filipine and preferably nystatin and amphotericin B. Other useful biologically active substances include, but are not limited to, antibacterial compounds, for example, aminoglycosides such as gentamicin, antiviral compounds such as rifampacin, anti-parasitic agents such as antimony derivatives, antineoplastic agents such as vinblastine, vincristine, mitomycin C, doxorubicin, daunorubicin, methotrexate and cisplatin, proteins such as albumin, toxins such as diphtheria toxin, enzymes such as catalase, hormones such as estrogens, neurotransmitters such as acetylcholine, lipoproteins such as α-lipoprotein, glycol proteins, such as. hyaluronic acid, immunoglobulins such as IgG, immunomodulators such as interferon or interleukins, dyes such as Arsenazo III, radiolabels elements than ^l4 C, radio-opaque compounds such as Te, fluorescent compounds such as carboxyfluorescein, polysaccharides such as glycolene, cell receptor binding molecules such as estrogen receptor proteins, nonsteroidal anti-inflammatory drugs such as indomethacin, salicylic acid acetate, ibuprofen, sulindac, piroxicam and naproxen, anti-inflammatory drugs, agents such as timolol or pilocaprin, analgesics such as dibucaine, nucleic acids such as thymine, polynucleotides such as RNA polymers, cardiovascular agents such as α-blockers, β-blockers, calcium channel blockers, ACE inhibitors, and the like. CNS reagents and prostaglandins.

Organic solvents are also used in the preparation of HDLCs, generally liposomes. Suitable solvents for this purpose are those having medium polar and dielectric properties (having a moderate degree of polarity with respect to electrical charge repulsion) which solubilize (solubilize) lipids, such as, but not limited to, chloroform, methanol, dimethylsulfoxide (DMSO), methylene chloride; and solvent mixtures such as chloroform / methanol 70:30 and benzene / methanol 70:30. This results in uniform distribution of lipids. Solvents are preferably selected for their biocompatibility, low toxicity and flame retardancy. DMSO is preferably used for the solubilization of the therapeutic agent, namely amphotericin B, since it is most soluble in DMSO. Substitution of methanol with DMSO results in an increase in solvent volume. Methylene chloride is preferably used for the solubilization of lipids because of its low toxicity to humans. Ethanol or aqueous solutions are preferably used as solvents in the novel liposome preparation process of the present invention.

HU 209 647 B

Aqueous solutions such as distilled water (such as USP water for injection), saline or aqueous buffer solutions may be used in the hydration step for the preparation of HDLCs. Aqueous buffers include, but are not limited to, pH 7.0 to 7.5, preferably pH 7.2, such as phosphate salt ("PBS") or tris (hydroxymethyl). aminomethane hydrochloride ("tris") buffers.

During the production of HDLCs, a sonication step (ultrasound distribution) may also be performed. This is done in a sonication bath at 25 ° C, 5060 Hz for 15 to 30 minutes.

The size of the produced HDLCs can be adjusted by filter extrusion as described by Cullis et al., PCTPubl. No. 88/00238, published January 16, 1986. Such size adjustment methods yield populations with a homogeneous particle size distribution. Filtration of HDLCs can be accomplished, for example, by extrusion through a serpentine path or through a straight membrane filter such as a polycarbonate filter.

The HDLCs produced by the novel processes of the present invention may be lyophilized or dehydrated at various stages of the process. For example, the lipid film may be lyophilized after removal of the solvent and before the drug is added, or the drug / lipid film may be lyophilized before HDLC or liposome hydration. This dehydration can be accomplished by subjecting the lipid, HDLC or liposome to reduced pressure, thereby removing any solvent for suspension. In addition, the hydrated HDLC or liposome, which has already been hydrated, may be dehydrated by freezing it in liquid nitrogen prior to the dehydration step. Pre-freezing dehydration was performed as described in Bally et al., PCT Publication No. 86/01103, Feb. 27, 1986, and Schneider et al., U.S. Patent 4,229,360, Oct. 21, 1980. or in the presence of multiple protective sugars. These methods improve the shelf life and stability of the formulations. Other methods known or to be discovered for the dehydration of the above lipid combination formulations may also be used.

One method of producing HDLCs of the present invention is an MLV method comprising dissolving a biologically active substance (e.g., a drug) in methanol in a flask and adding a 7: 3 mixture of DMPC: DMPG in chloroform. The solution is evaporated in a rotary evaporator under reduced pressure, the dried film is resuspended in PBS and sonicated in a sonication bath (about 25 ° C, about 50-60 Hz for about 30 minutes) until the solution clears. For drug-to-lipid ratios of about 6 mol% to higher (about 60 mol%), the compositions are non-liposome-like HDLC structures that are substantially free of liposomes. Toxicity of HDLC formulations depends on the drug: lipid ratio, formulations with a higher drug dipid ratio (containing about 16-50 mole% of the drug) show less acute toxicity than the lower drug dipid ratio (about 6-15 mole% of the drug) preparations containing. As mentioned above, formulations containing up to 5 mol% of drug are virtually pure liposomes.

In another modified MLV process, the drug dipid film produced in the above manner is dried in a glass bulb under reduced pressure to obtain a fluffy drug dipid composition. This fluffy material is atomized and homogeneously hydrated upon addition of the resulting aqueous solution. In this process, the drug is dissolved in an organic solvent, such as DMSO or methanol, and mixed with the lipid (preferably a 7: 3 molar ratio DMPC: DMPG) in methylene chloride. The mixture is dried under reduced pressure to give a film which is then dried, for example, in a glass bulb under reduced pressure. The resulting dry powder was hydrated with aqueous saline and then heated to form a drug dipid suspension. As mentioned above, the formation of HDLCs, liposomes, or mixtures thereof, respectively. the toxicity of the substances formed depends on the drug dipid ratio.

Other methods may be used to produce HDLCs. One such method is an SPLV process wherein the drug is dissolved in a solvent such as DMSO. A lipid (e.g., 7: 3 DMPC: DMPG) in a solvent (e.g., chloroform or methylene chloride) is dried under reduced pressure to a thin film coating and then an organic solvent, such as methylene chloride, is added. An aliquot of the drug (e.g., amphotericin B) in solution is added to the lipid suspension and the resulting mixture is sonicated until the solution is clear. A buffered aqueous solution (e.g., buffered saline) is then added and the mixture is again placed under reduced pressure to remove methylene chloride. The resulting pulp is further hydrated with PBS and sonicated until the solution clears. Similarly to the above procedure, the resulting composition will be HDLC or liposomal, depending on the molar ratio of the drug dipid used. The LD ₅₀ values of the formulations in mice showed lower toxicity at higher drug dipid ratios.

In a further modified SPLV process, the drug dissolved in an organic solvent (e.g., DMSO) is mixed with a lipid (e.g., 7: 3 molar mixture of DMPC and DMPG) dissolved in an organic solvent, followed by the addition of a buffered aqueous solution (e.g., PBS). evaporate under a nitrogen atmosphere while sonicating the material. Additional PBS is then added and the resulting solution is filtered, preferably through a 5 micron filter, and centrifuged at about 10,000 g for about 10 minutes. The mother liquor was removed and discarded, and the pellet resuspended in aqueous solvent. After a second "wash" by centrifugation, the resulting pellet was resuspended in PBS. As with the above formulations, acute toxicity is directly related to the drug dipid ratio; the higher this ratio7

(Up to about 60 mol%) the lower the toxicity of the preparation.

In a further SPLV process, the drug and lipid solution are mixed in a similar manner, but instead of saline, USP water for injection is added and the solution is heated to 37 ° C.

The formulations were filtered under sterile conditions using a 0.1 or 0.15 μm filter using LUVET. This method is an extrusion process for preparation of liposomes by filtration at a pressure of about 50 atm (Cullis et al., PCT Publ. No. 86/99238, publ. Jan. 16, 1986). These compositions may also be dehydrated by the method described by Bally et al., Supra. Again, acute toxicity studies showed that increasing the drug: lipid ratio improves drug toxicity.

The aforementioned high molar ratio of amphotericin B / lipid formulations is expected to be less toxic due to interactions between the lipid matrix and free amphotericin B. This phenomenon can be detected by absorption spectroscopy. For example, when dissolved in deoxycholate, the absorption of free amphotericin B at 413 nm is greater than that of 5 mole% amphotericin B embedded in lipid without heating, and that of 25 mole% amphotericin B is greater than lipid embedded in lipid. mole% amphotericin B (Figure 10).

The absorption spectra procedure is used to determine the toxicity of drug (e.g., amphotericin B) lipid combinations. The absorption spectrum of a drug is specific to that drug; the amphotericin B characteristic (see Figure 12, dissolved in deoxycholate) is between 300 and 500 nm, with characteristic peaks, the most characteristic being at 413 nm. The flattening of this peak in the spectrum of the drug dipid complex can be used to determine the toxicity of HDLC. Any one of the above-mentioned lipids, when complexed, is expected to give this characteristic flattened absorption curve because the spectrum is specific to the particular drug.

If samples exhibiting less than maximum toxicity reduction (e.g., 25 mol%) are heated to 25-60 ° C, the system toxicity will be reduced (see Figure 11). This decrease progresses to the maximum toxicity observed when amphotericin B is combined with 50 mol% lipid (Figure 11). One possible explanation for this phenomenon is that the decrease is caused by the separation of the lipid phase, thereby releasing amphotericin B from the heat during heating, thereby tightening the association of the amphotericin B particles, resulting in a less toxic system. Excluded lipid is indicated by the appearance on the differential scanning calorimetric curve (Figure 12) of an endotherm associated with a lipid essentially free of amphotericin B. Also, the lipid kizáródott indicates the heated systems ³¹ P-NMR signal of the taper (13), which is also very little lipid containing amphotericin B is inherent.

Finally, freeze-refraction electron microscopy images of heated systems show the presence of lipids (and liposomes) that were not previously seen. The unheated sample shows combinations that do not contain free lipid, which is typical of systems in which intramolecular interactions between lipid and amphotericin B are present.

When examining samples containing 50 mole% amphotericin B, little difference was found between measurements before and after heating. At a concentration of 50 mol%, the interaction between the drug and the lipid is presumed to be such that no displaceable lipid is present in the structure. Thus, these samples show essentially the same signals in DSC and NMR before and after heating.

The theory that the isolated lipid phase leaves amphotericin B in an environment that promotes greater interaction between amphotericin B and amphotericin B (and thus reduces toxicity) can be supported by absorption spectroscopy. The spectra of heated systems are much more intense (indicating a decrease in toxicity) than those of untreated systems. In one case, heating resulted in an LD _{50 of} greater than 30 mg / kg, while without heating, the formulation had an LD ₅₀ of 15 mg / kg. In this case, a decrease in absorbance was observed at 413 nm after heating.

The above heating process involves the use of any type of lipid particles or solids. lipid preparation process, such as any of the above, but in this example the MLV process was used. Similarly, any of the lipids or phospholipids listed above, as well as any of the solvents and aqueous buffers mentioned above, may be used. For example, the lipid is suspended in an organic solvent, such as chloroform, and then dried on a wall of a round-bottomed flask to form a thin film. Amphotericin B (or any other drug as described above) is dissolved in a suitable organic solvent (a solvent which dissolves the drug, for example, amphotericin B). In the process of the invention, the drug (amphotericin B) is used at a concentration of about 6 to about 50 mol%, and at a drug concentration of 25 mol% and above, the population is essentially HDLC.

In this heating embodiment of the process of the invention, 10 mg of amphotericin B is dissolved in 100 ml of methanol, i.e. the concentration of amphotericin B is 0.1 mg / l. A solution containing amphotericin B (100 mL) was added to the lipid film and resuspended. The resulting slurry is then concentrated in a rotary evaporator under reduced pressure to a thin film. The resulting lipid / amphotericin B film is resuspended in a known amount (e.g. 4 ml) of an aqueous solution sufficient to form liposomes or lipid particles. The suspension is then stirred and sonicated for a few minutes to about 1 hour and then on a water bath at about 25 to about 60 ° C.

EN 209 647 Β approx. 10 minutes to approx. Heat for 400 minutes.

In another method of preparing the HDLCs of the present invention, a homogenization step is used instead of sonication. For example, HDLCs can be prepared by the SPLV process, whereby the drug (e.g., amphotericin B) is mixed with a suitable solvent (e.g., DMSO) with a mechanical stirrer until completely dissolved. If necessary, the solution is filtered to remove any remaining drug particles. A 7: 3 molar mixture of DMPC and DMPG is dissolved in a suitable solvent (e.g., methylene chloride) and the lipid is mixed with the drug solution in DMSO. An aqueous solution, such as brine, is added to the mixture, and the organic solvents are removed in a rotary evaporator at about 35-45 ° C. The resulting drug-lipid mixture is diluted with an aqueous solution, such as brine, and the HDLC suspension is ground in a homogenizer. Any homogenizing apparatus or colloidal mill which comminutes the particles may be used, but preferably Gifford-Wood colloidal mill. The milling is carried out until a suitable particle size is obtained (for example, 90% of the particles have a diameter of less than 10 μ, preferably between about 4 and about 10 μ), i.e. about 10 μm. Continue for 15-30 minutes. Depending on the desired size and homogeneity, the particles are passed through the grinding apparatus once or more. The particle size distribution of HDLCs can be analyzed using the Maivem classifier. If necessary, larger and smaller particles are removed by any method known in the art for separating particles, such as by filtration. With this method, approx. 0.2 to approx. Particles with a diameter of 10.0 μιη are obtained.

Other methods known to those skilled in the art for the preparation of liposomes may be used in the practice of the invention; the preparation of HDLCs according to the invention is not limited to the aforementioned methods.

The HDLC compositions of the present invention are therapeutically useful in animals (including man) for the treatment of a variety of infections and other conditions which include (1) repeated administration, (2) delayed release of a biologically active drug, or (3) preservation of the efficacy of a free drug. or increase in toxicity as they increase. Such conditions include, but are not limited to, local or general fungal infections that can be treated with antifungal agents such as amphotericin B or nystatin, viral diseases that cause immune deficiency syndrome (AIDS), and herpes. The compositions of the present invention are stable in aqueous solution.

The route of administration may determine the sites and cells of the body to which the active ingredient is delivered. The HDLCs and liposomes produced by the process of the invention may be administered alone but will generally be administered with a pharmaceutical carrier selected according to the intended route of administration and general pharmacological practice. For example, transportation to a specific location is easier with topical administration (if the infection is external to areas such as eyelids, skin, ears, or injuries such as wounds or burns). Such topical application can be effected, for example, by applying the compositions directly in the form of creams, ointments, gels, emulsions or pastes to the affected surfaces. In other cases, the compositions are administered parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be used, for example, in the form of a sterile aqueous solution which may contain other solutes, such as salt and glucose, sufficient to render the solution isotonic. One of ordinary skill in the art will recognize that other routes of administration may be employed, depending upon the properties of the formulations.

The compositions of the present invention may be used to treat asthma by inhalation of an aerosolized aqueous suspension of liposome or HDLC into the lungs. For example, the liposomes or HDLCs are suspended in a suitable solvent and the suspension is aerosolized by pneumatic or ultrasonic nebulizer or, more simply, by a single nebulizer that operates on gas pressure from a fluorocarbon pellet. Other inhalation systems may be used, such as those in which the liposomes and HDLCs are in the form of particles suspended in dry powder or in a suitable carrier system. After conversion to the aerosol, most of the solvent is instantaneously evaporated and replaced by moisture in the respiratory tract, resulting in the precipitation of the particles.

When administered to humans for therapeutic or prophylactic use, the appropriate dosage for the individual patient will ultimately be determined by the attending physician and will vary with the patient's age, weight and response, and the nature and severity of the symptoms. The dose of the HDLC preparation is approximately the same as that of the drug in free form, but in some cases doses outside these limits may be required.

The following examples are intended to illustrate the invention in more detail and are not intended to limit its scope.

Example 1

In a 500 mL round bottom flask, 10 mg of amphotericin B (total drug content 5 mol%) was added to 100 mL of methanol and the mixture was treated until clarified. This sonication step was carried out in a sonication bath for 15 minutes at 25 ° C for approx. 50-60 Hz. Dissiristoyl phosphatidylcholine (DMPC) (100 mg) and dimyristoyl phosphatidylglycerol (0.42 ml) were then added to the solution in 1.0 ml of chloroform. The resulting dispersion is evaporated to dryness under reduced pressure at 60 ° C in a rotary evaporator to form a thin film on the wall of the flask. This was resuspended in 4.0 mL of PBS, filled into a test tube and sonicated until clear.

The above procedure was repeated with 16.7, 20, 25, 33, 50 and 60 mol% of amphotericin B. Acute toxicity

Results (LD ₅₀ ) - that is, doses of the drug that cause 50% mortality in mice - increase with 50% increase in amphotericin B ratio.

Example 2

To 1.5 ml of dimethyl sulfoxide (DMSO) is added 140 mg of amphotericin B (total drug content 5 mol%). DMPC (1400 mg) and DMPG (600 mg) were dissolved in methylene chloride (50 mL), placed in a flask and stirred until clear, then evaporated to dryness under reduced pressure on a rotary evaporator. The film formed on the wall of the flask was dried under a glass bulb for 1-4 days. The film formed into dry flakes is pulverized and mixed with 100 or 50 ml of 0.9% solution or 50 ml of USP water. The resulting suspension was heated at 37 ° C for 1 hour.

The above example was repeated using DMSO instead of methanol to suspend amphotericin B and 10, 16.7 and 10% of amphotericin B, respectively. 33 mol%. Instead of a 7: 3 DMPCDMPG blend, 1 and 2 respectively. 2 g of egg phosphatidylcholine are used.

Example 3

In 2.0 ml of DMSO, 100 mg of amphotericin B (total drug content 5 mol%) was dissolved. In a flask was dissolved 100 mg of DMPC in 1.0 ml of solution and 42 mg of DMPG in 0.42 ml of solution was dissolved in chloroform and the chloroform was removed in a rotary evaporator under reduced pressure. Then, 20 ml of methylene chloride are added to the flask, followed by 0.2 ml of amphotericin B stock solution. This suspension is cleared until clear (about 1 minute)

After sonication under the conditions described in Example 1, 0.3 mL of pH 7.2 PBS was added and methylene chloride was removed by sonication under a stream of nitrogen. The resulting paste is resuspended in 10.0 ml of PBS and centrifuged at 10,000 g for 10 minutes, followed by sonication in a sonication bath for approximately 20 minutes.

The above procedure was repeated with 16.7, 20, 25, 33, 50 and 60 mol% of amphotericin B.

Example 4

Amphotericin B 140 mg (total drug content 5 mol%) was dissolved in 1.5 ml methylene chloride. The solution is stirred until clear, filled into a flask and mixed with 8.0 ml PBS. The methylene chloride is removed in a sonication bath under a stream of nitrogen. Sonication was performed as described in Example 1. To the solution was added 32 mL of PBS and then filtered through a 5 μm polytetrafluoroethylene (PTFE) Teflon filter, centrifuged twice, and resuspended in 100 mL of PBS.

The above procedure was repeated with 4.0 and 4.0 µg of lipid hydration, respectively. 12.0 ml PBS was used.

Example 5

Amphotericin B 140 mg (total drug content 5 mol%) was dissolved in 1.5 ml DMSO. DMPC (1400 mg) and DMPG (600 mg) were dissolved in methylene chloride (50 ml) and the two solutions were stirred until they were clear, then transferred to a flask and evaporated to a thin film under reduced pressure on a rotary evaporator. The film was then resuspended in 50 mL of methylene chloride and 8.0 mL of PBS was added. The methylene chloride is evaporated in a stream of nitrogen during sonication as described in Example 1. The resulting paste was hydrolyzed with 32 mL of PBS, washed twice by centrifugation, resuspended in 100 mL of PBS, and passed through a 5 μm Teflon (PTFE) filter. The above example is repeated with 5.0, 10.0, 15.0 and 5% as the moisturizing buffer. 20.0 ml PBS was used.

Example 6

Amphotericin B 140 mg (total drug content 5 mol%) was dissolved in 1.5 ml DMSO. 2.0 g of egg-derived phosphodylcholine (EPC) is dissolved in 50 g of methylene chloride and the solutions are mixed in a flask. PBS (8.0 ml) was then added and the solvent was evaporated under nitrogen flow as in Example 1 during sonication. Finally, 200 ml of PBS was added.

The above example is illustrated in Figs. This is repeated using 20 mole% amphotericin B.

Example 7

Amphotericin B 140 mg (total drug content 5 mol%) was dissolved in 1.5 ml DMSO. 1400 mg of DMPC and 600 mg of DMPG are dissolved in 50 g of methylene chloride in a 500 ml round-bottomed flask and mixed with 10 ml of USP (water for injections) at 37 ° C. The solvent was evaporated in a stream of nitrogen during sonication as described in Example 1. 50 ml of Usp water are then added and the solution is heated for 30 minutes at 37 ° C.

The above example is illustrated in Figs. It is repeated using 16.7 mol% of amphotericin B and the solution is passed through a 3 m linear pass filter made by Nucleopore.

Example 8

Amphotericin B (140 mg total drug content, 1.67 mol%) was mixed with 50 ml of methanol, sonicated for 15 minutes and the resulting solution was filtered through a Millex 0.22 μ twisted-pass filter (mixed cellulose). and acetate nitrate esters). 350 mg of DMPC and 150 mg of DMPG are dissolved in 50 g of methylene chloride and mixed with the filtered solution. The solvents were evaporated in a stream of nitrogen as described in Example 1 during sonication and 50 ml of PBS was added. Half of the preparation (25 ml) was lyophilized using standard techniques.

Example 9

To 2.0 ml of anhydrous ethanol was added 10 μΐ IN hydrochloric acid. To the acidified ethanol was added 20 mg of amphotericin B (total drug content 5 mol%) and the mixture was sonicated until clarification as described in Example 1.

EN 209 647 Β, except that the operation is performed for 30 seconds instead of 60. To a solution of 200 mg of DMPC and 42 mg of DMPG was added a 70:30 mixture of benzene / methanol in a tube and lyophilized as described in Example 8. The dried lipid was dispersed in turbulent stirring with 2.0 ml PBS. 0.2 ml of amphotericin B solution was added to the lipid and the mixture was stirred overnight. The resulting DMPC: DMPG MLVs (200 μΐ) were layered on a sucrose density gradient solution and centrifuged at 230,000 g for 24 hours at 22 ° C. The results are all. all amphotericin B is bound to the lipid, most of the lipid on the wide distribution curve appears at the peak, and the amphotericin B peak appears at the bottom of the gradient. Alternatively, the resulting DMPC: DMPG MLVs are lyophilized and rehydrated with 2.0 mL of distilled water.

Examples 7, 9, 16,7, 17, 20, 25, 33, 50 and 7, respectively. This is repeated using 60 mole% amphotericin B. At 16.7 mol% drug content, HDLCs are mainly formed. Such high drug: lipid ratio compositions have a density-centrifugation gradient similar to that of Figure 5, where all drugs and lipids are combined in a single peak.

Example 10 DMPC: DMPG samples containing mole% amphotericin B were prepared for X-ray diffraction, DSC, freeze-fracture electron microscopy and ³¹ P-MNR as follows: DMPC: DMPG 7: 3 (total lipid: 44 mg) and Amphotericin Bt (20 mg) was suspended in a 70:30 mixture of chloroform and methanol and concentrated under reduced pressure at 55 ° C to a thin film deposited on the wall of the flask. The film was hydrated in 20 ml of pH 7.2, 20 mM Hepes / 250 mM sodium chloride, in a glass bead, with turbulent stirring. The preparation was centrifuged at 10,000 g for 10 minutes, the mother liquor was discarded, and the pellet was resuspended in 1.0 ml Hepes / sodium chloride buffer. The composition is then sonicated in a sonication bath at 25-50 Hz for 20 minutes.

The above example was repeated using 0.5 and 20 mole% amphotericin B.

Example 11

The volume of amphotericin B embedded in liposomes and HDLCs is determined as follows:

100 mg of DMPC and 42 mg of DMPG (both dissolved in chloroform) are mixed in a flask with 10 mg (25 mol%) of amphotericin B (0.1 mg / ml solution). The solvent was removed under reduced pressure at 37 ° C. To the dry lipid film was added 3.7 mL of PBS containing 0.3 mL of dilute aqueous ³ H-inulin solution with stirring. An aliquot (10.2 ml) of the resulting solution was placed in a scintillation mixture and counted for radioactivity on a β-synthesis counter. Another aliquot was assayed for phosphate by the method of Artlett B (7th Biol. Chem., 1959, 234: 466-468). The lipid / drug suspension was centrifuged at 10,000 g for 10 minutes, the mother liquor was discarded, and the pellet was resuspended in PBS. The centrifugation and resuspension of the pellet was repeated two more times; take a sample of 100 μ utolsó from the last resuspension and measure with a β-synthesis counter. Another aliquot of the last resuspension is tested for phosphate content as described above. Percentage of inulin incorporation is calculated by multiplying the final radioactivity measure by the starting number multiplied by 100. The incorporation volume (μΐ embedded inulin / pmol lipid) is also calculated.

The above example was repeated using 0.5 and 20 mole% amphotericin B.

Example 72

Amphotericin B particles were prepared by drying 100 mg of DMPC and 42 mg of DMPG (7: 3 molar ratio) in about 10 ml of chloroform solution onto a 100 ml round bottom flask, and adding 10 mg of amphotericin B suspended in 100 ml of methanol, and a slurry of the lipid film was prepared containing 25 mole% amphotericin B. The mixture was dried in a rotary evaporator at 37 ° C to a film deposited on the wall of the flask. This film was then hydrated in a glass bead bed with 10 mM Hepes / 150 mM sodium chloride pH 7.2 and sonicated for 30 minutes. A 10 ml sample of the final suspension thus obtained was taken and heated in a water bath for 10 minutes at 60 ° C. The resulting suspension is subjected to DSC, NMR and freeze-fracture electron microscopy.

The above procedure is repeated without heating the sample. Maintain the sample at 22 ° C and perform the above tests.

Example 13

Using the materials and methods described in Example 12 and using 50 and 5 mole% amphotericin B, the procedure is repeated. The systems thus obtained were characterized by DSC, NMR and freeze-fracture electron microscopy.

Example 14

DSC measurements were performed on a Micro Cal MC-1 Unit of Micro Cal, Inc., Amherst, MA. The sample cuvettes were injected with 0.7 ml of a suspension containing 5-9 mg of substance, and the control cuvettes were injected with an equal volume of buffer solution. The samples were exposed to approximately 26 and 1 hour, respectively. we kept about 37 'Con. Samples with the same heat history were tested twice, making the set and actual temperatures reproducible with an accuracy of 0.2 ° C. To ensure the same thermal history, samples containing amphotericin B were generally heated to a maximum of 60 ° C and then cooled to 7-4 ° C for at least 2 hours.

Example 75

NMR Spectra at 145.7 MHz, Brucker AM360 Wide Spectrometer11

It was manufactured at 209,647 Β using an 8K interrogation data point with a scanning frequency of 50,000 Hz and a pulse width of 20 psec corresponding to a 45 ° pulse. The spectra were summed from 10,000 transients.

Example 16

An aliquot of 0.1-0.3 mL of sample was placed between two Balzer (Nashua, NH) copper support plates at 23 ° C and suddenly pressed into liquid propane. The samples were then fractured, followed by freeze-crushing unit Balzer, 2 x 10 ⁶ mbar or even greater vacuum -115 ° C were prepared copies of them a double replicating device. The copies were pelleted in 3N nitric acid and washed with a series of sodium hypochlorite solutions. Finally, purified with distilled water, it was placed on a 300 Hex mesh copper screen (Polysciences, PA). Copies were examined with a Philips 300 electron microscope at 3,000 to 22,000 × magnification.

Example 17

The absorption spectra (shown in Figures 12 and 13) were made by diluting Amphotericin B to 25 pmol / L with Hepes / sodium chloride buffer. A sample was placed in a Beckman spectrophotometer cuvette and read between 300 and 500 nm, and the reading was repeated with a clear buffer cuvette.

Example 18

For ESR studies, the samples were labeled with doxyl stearic acid with a series of positional isomers in which the doxyl label moiety appeared at different sites on the fatty acid chain. Labeling was performed by vortexing the sample with ethanol solution and sonication, and then drying the solution as a thin film on the tube wall. Each sample is labeled 1 mol%.

ESR spectra were recorded on an IBM Instruments ER100D ESR spectrometer with nitrogen gas flow temperature control. The sample temperature was measured with an external calibrated thermistor probe (Omega Engineering, Inc., Stamford, CA). ESR spectra were recorded at 10 MW microwave power, 9.11 GHz microwave frequency at 100 G and 100 kHz, with a modulation amplitude of 0.32. The row parameter was calculated from the maximum hyperfine division (A max).

Example 19

To 3375 ml of DMSO was added 337.5 g of amphotericin B and the resulting 100 mg / ml solution of amphotericin B was stirred until clear (about 3 hours with mechanical stirrer). The mixture was poured into a 5 liter stainless steel pressure vessel and Filter through a Sartoflour filter with a 22 μπι aperture and a thick popolpropylene filter (Pali Profile, Pali, Inc., GlenCove, NY) in a liter of pressurized container of 50.8 kg methylene chloride with 225.0 g DMPC and 99.0 g DMPG 3, After stirring for 5 hours, dissolve, the resulting lipid solution is passed through a 0.2 M ² Sartofluor 0.22 µm Teflon filter into a stainless steel processing vessel, the 40 L pressure vessel is washed with an additional 55.2 kg of methylene chloride and the wash solution The lipid solution is mixed with a rotary mixer at 195 rpm and a solution of amphotericin B in DMSO is introduced. Finally, 16.2 liters of 0.9% sodium chloride in USP was introduced through a Millipak 100 polycarbonate filter.

Using a processing vessel connected in series to a 35 ° C heat exchanger, a stream of preheated liquid nitrogen was passed through the vessel and the solvent removed within 12 hours. The resulting drug dipid complex is diluted with 7000 mL of 0.9% USP sodium chloride solution introduced through a Millipak 100 polycarbonate filter.

The resulting HDLCs were homogenized with a Gifford-Wood colloid mill and a rotary lobed pump. The HDLCs were passed through a gap of 13 µm at the head of the colloid mill at 0.7 atm head pressure for 3.0 hours to give particles smaller than 20 µm. The particle size of the HDLCs was analyzed with a Maivem particle counter.

Example 20

A total of 102.6 pmol of chloroform-dissolved lipid (DMPC: DMPG molar ratio 70:30) was pipetted into a 500 ml round bottom flask. Dissolve 500 mg of Nystatin in 1.0 L of methanol using a Branson sonication bath to make a 0.5 mg / ml solution. Add 5.0 ml of the Nystatin solution to the lipid in the round-bottom flask and mix by removing the solvent in a rotary evaporator at 45 ° C for 15 minutes and rehydrate with 5.0 ml of saline in a glass bead bed with turbulent stirring. . The liposomes formed are visible under a light microscope.

The above was repeated using 25.50, 75 and 100 mol% Nystatin. Freeze-fracture electron microscopy showed that the composition containing 25 mole% Nystatin was non-liposome.

Example 27

14.8 pmol / ml lipid (DMPC: DMPG molar ratio 70: 30) was hydrated with distilled water and the solution was incubated at 4 ° C. The resulting MLVs are ten times extruded on two superimposed polycarbonate filters using the LUVET procedure.

Amphotericin B is dispersed in distilled water at a concentration of 10.8 pmol / L in a sonication bath. Amphotericin B dispersant is added to the lipid suspension to a final concentration of 13.5 and. 0.98 pmol / ml. To remove unmoveted amphotericin B, a 20 ml sample was centrifuged in a Beckman L8-60 ultracentrifuge at 15,000 g, Ti60 or SW 27 (Beckman) rotor at 22 ° C for 30 minutes.

HU 209 647 B. The mother liquor containing free amphotericin B was removed, taking care not to mix the liposome pellet. The resulting liposomes have a diameter greater than 1.0 µm as measured by quasi-elastic light scattering.

The above procedure was repeated by incubation at 23 ° C using a solution of 150 mM sodium chloride, 10 mM sodium phosphate (pH 7.4) to hydrate the lipids. The resulting liposomes have a diameter greater than 1.0 μιη, measured by quasi-elastic light scattering. The incorporation of amphotericin B into liposomes in a lower ionic strength medium (using distilled water instead of 150 mM sodium chloride) was greater (Fig. 17).

Figure 16 shows the incorporation of amphotericin B into DMPC: DMPG liposomes under various incubation conditions. The incorporation at 23 ° C is similar to that at 45 ° C. The drug also accumulates when the lipid is present in the gel phase, i.e. at 15 ° C.

Example 22

The materials and procedures described in Example 20 were used, but the lipid suspended in distilled water was incubated at 22 ° C. The liposomes thus obtained are unilamellar with a mean diameter of 0.1-0.2 μιη, measured by quasi-elastic light scattering. Figure 19 shows that during the first two hours, uptake of amphotericin B in these LUVs was faster than in MLVs.

Example 23

Preparation of Nystatin-Lipid Blends

Nystatin was dissolved in methanol to make a 0.1 mg / ml solution and poured into a round-bottomed flask containing a sufficient amount of lipid dried from chloroform to a thin film. The chloroform was previously removed in a rotary evaporator and the resulting polyene lipid film was resuspended in 45 'Con in 20 mM HEPES and 150 mM sodium chloride, pH 7.4. (This buffer solution is used everywhere). This suspension is then sonicated for 30 minutes at a temperature between 5 and 20 ° C for the LD ₅₀ assay.

Lipid and Nystatin assays

Phospholipid content was determined according to the method of Chen et al. (1956, Anal. Chem. 28, 1756-1758). Aliquots of the nystatin-lipid preparation were diluted with methanol to a concentration of 5-8 μΜ (monomer) and weighed at 318 mm and recorded for light absorption using a Shimadzu UV-160 spectrophotometer (Shimadzu Corporation Kyoto, Japan). The concentration is determined by comparison with standard curves.

Carrier toxicity

LD ₅₀ measurements were performed according to the method of Reed and Muench (1938, Am, J. Hyg., 493-497). For each dose, 6 female Swiss Webster mice were injected intravenously with a total volume of 0.5 ml. The animals were observed for 10 days.

Freeze-fracture microscopic examination

For preparation of freeze-fracture replicates, aliquots of 0, ΙΟ, 3 μΐ (without the addition of antifreeze) were placed between two Blazer copper trays (Nahua, New Hampsire) from 21 ° C to -190 ° C liquid propane. Samples were chopped and replicated in a Blazer BAF 400 freeze-fracture unit at 4 x 10 ^{7 bar} at -115 ° C. The replicates were rinsed from the trays with 3M nitric acid solution, washed with distilled water and then purified overnight with 5% sodium hypochlorite solution (commercial bleach).

Results obtained with Nystatin lipid formulations

The Π. Table contains nystatin lipid mixtures obtained _LD50 values. From these, it can be seen that the toxicity of the HDLC-nystatin complex slightly decreases at 25 mol% nystatin.

In the DMPC / DMPG blend, only liposomes are seen in freeze-frost replicates containing 5 mole% nystatin (Fig. 11). In contrast, samples made with 25 mole% nystatin contain "ribbon" structures similar to those of amphotericin B (Figure 11 B, showing the 25 mole% nystatin complex). In formulations with higher nystatin content, only spherical structures were observed (Fig. 11 C).

II. Red blood cell hemolysis

This procedure is used to determine the in vitro toxicity of various polyene formulations. A 0.5 mL sample was mixed with 0.5 mL of a washed red blood cell suspension in 4% (v / v) phosphate buffered saline and incubated for 20 hours at 37 ° C with constant stirring. It is then centrifuged at low speed (2000 rpm) for 10 minutes, 200 μΐ of the resulting red blood cell pellet is removed and diluted with 1 ml of buffer. The percentage of hemolysis is determined from the absorbance at 550 nm by assuming 100% the absorbance obtained with a 4% erythrocyte solution diluted 1: 1 with distilled water.

III. In vitro efficacy

To determine the minimum inhibitory concentration (MIC), Fungizone and drug-lipid complex were diluted directly with Sabaroud dextrose vapor. The MIC values of each formulation after incubation for 18 hours at 30 ° C against Candida albicans and Saccharomyces cerevisiae were determined by McGinnis and Rinaldi (1986), Antibiotics in Laboratory Medicine, 2nd ed., Lorian, V., ed. 223-281, William & Wilkens, Baltimore) on microtiter plates.

ARC. Carrier toxicity

LD ₅₀ measurements were performed according to the method of Reed and Muech (1938, Am. J. Hyg., 493-497). All doses13

6 out of 206,647 HU female Swiss Webster mice are injected intravenously with a total volume of 0.5 ml. The animals were observed for 10 days.

Table I: Minimum Inhibitory Concentrations (MICs) of mixtures of Atnphotericin B with DMPC / DMPG and Deoxycholate P

MIC (μ _β / πύ) C. albicans S. cerevisiae 5 mol% in DMPC / DMPG (separated) high density material low density material 0.16 to 0.32 0.16 to 0.32 1.25 1.25 25 mol% in DMPC / DMPG 0.16 to 0.32 0.63 deoxycholate 0.08 to 0.16 0.16 to 0.32

^a In DMPC / DMPG, 5 mole% (7: 3) amphotericin B contained 33 mole% high density fractions and low density 1.5 mole% amphotericin B. The deoxycholate formulation was prepared from commercially available Fungizone.

II. Amphotericin B and Nystatin table made of DMPC / DMPG with a 7: 3 mixture of the LD ₅₀ value of ⁰

LD ₅₀ (mg / kg) ^b Free Nystatin 5.3 ± 1.4 Nystatin in DMPC / DMPG 5 mol% 6.7 + 4.7 25 mol% 9.4 + 3.2 50 mol% 7.1 ± 0.7 40 Mole% Amphotericin B in DMPC / DMPG > 30

^the LD ₅₀ values were determined in Swiss Webster mice at 10 day measurement.

^b = mean ± standard deviation of 4 measurements for free Nystatin, 2 for Dystoc mixed with DMPC / DMPG

Claims (23)

PATENT CLAIMS CLAIMS 1. A process for the preparation of a combination of low toxicity, biologically active agent and lipid (HDLC), characterized in that it has a high active ingredient / lipid ratio, wherein the active ingredient has a lipid content of at least 6 mol% and is essentially liposome-free. ways to a) i) dissolving at least one lipid in a medium polar solvent, ii) dissolving the biologically active agent in a biocompatible solvent, iii) mixing the solution prepared in step i) with the solution prepared in step ii); obsession b) i) dissolving at least one lipid in a medium polar organic solvent, ii) dissolving the biologically active agent in a solvent, iii) mixing the solution prepared in step i) with the solution prepared in step ii); or iv) dehydrating the mixture obtained in step iii); obsession c) i) dissolving at least one lipid in a medium polar solvent, ii) dissolving the biologically active agent in a biocompatible solvent and iii) mixing the solution obtained in step i) with the solution obtained in step ii) iv) mixing the mixture obtained in step iii) evaporating the solvent under reduced pressure, and v) adding an aqueous solution to the dry lipid film prepared in step iv); obsession d) i) dissolving the biologically active agent in a biocompatible organic solvent, ii) dissolving at least one lipid in a medium polar organic solvent and mixing the solution with the solution prepared in step i), iii) the above mixture of the solutions obtained in steps i) and ii) adding an aqueous solution and then iv) evaporating the solvent under reduced pressure and adding an aqueous solution to the resulting paste. 2. The process of claim 1 wherein the lipid is a phospholipid. The process according to claim 2, wherein the phospholipid is a saturated phospholipid or a saturated fatty acid. 4. A process according to claim 2 wherein the phospholipid is phosphatidylcholine and phosphatidylglycerol. 5. A process according to claim 4 wherein the phosphatidylcholine is dimyristoylphosphatidylcholine and the phosphatidylglycerol is dimyristoylphosphatidylglycerol. 6. The process of claim 4 wherein the ratio is 7: 3 molar phosphatidylcholine and phosphatidylglycerol. The process according to claim 1, wherein the biologically active agent is present in an amount of from 6 to 50 mol%. 8. A process according to claim 1 wherein the biologically active agent is present in an amount of 25-50 mol%. 9. The method of claim 1 wherein the biologically active agent is a medicament. 10. The method of claim 1 wherein the biologically active agent is an antifungal agent. HU 209 647 B 11. The method of claim 10 wherein the antifungal agent is a polyene antibiotic. 12. The method of claim 11, wherein the antifungal agent is amphotericin B. 13. The method of claim 11 wherein the antifungal agent is nystatin. 14. The process of claim 12 wherein the composition comprises 6-50 mol% of amphotericin B. 15. The process of claim 12 wherein the composition comprises 25 to 50 mol% of amphotericin B. 16. A process for preparing a pharmaceutical composition, wherein the HDLC produced according to claim 1 is mixed with a pharmaceutically acceptable carrier or diluent to form a pharmaceutical composition. 17. The method of claim 16, wherein the composition is for parenteral administration. 18. The method of claim 17, wherein the composition comprises an antifungal agent. 19. The method of claim 18, wherein the antifungal agent is amphotericin B. 20. The method of claim 19, wherein the HDLC contains 25 to 50 mol% of amphotericin B. 21. The method of claim 20, wherein the pharmaceutical composition comprises a HDLC having a particle size of 0.2 to 10 µ. 22. The method of claim 18, wherein the antifungal agent is nystatin. Method b) according to claim 1, characterized in that the medicament is suspended in an aqueous solution by sonication (sonication).