United States Patent (19) 11 Patent Number: 5,032,404 Lopez-Berestein Et Al

United States Patent (19) 11 Patent Number: 5,032,404 Lopez-Berestein et al. 45) Date of Patent: Jul. 16, 1991

(54) LIPSOME-INCORPORATION OF Panse et al., Hindustan Antibiot. Bull. 8 (1):10-14 POLYENES (1965). Panse, M. V., Indian J. Exp. Biol., 5 (2):112-114 (1966). 75) Inventors: Gabriel Lopez-Berestein; Reeta Panse et al., Hindustan Antibiot. Bull. 16 (1):25-28 Mehta, both of Houston, Tex. (1973). 73) Assignee: Board of Regents, The University of Pansy et al., Antimicrob. Agents Chemother., Texas System, Austin, Tex. 6:399-404 (1966). Parekh et al., Life Sci., 19 (11):1737-1791 (1976). 21 Appl. No.: 314,710 Shadomy et al., Antimicrob. Agents Chemother., (22) Filed: Feb. 23, 1989 8:452-460 (1968). Shadomy et al., J. Bacteriol, 97 (2):481-487 (1969). 51 Int. Cl...... A61K 37/22 Shende et al., Hindustan Antibiot. Bull., 8 (2):51-58 52 U.S. Cl...... 424/450; 424/122 (1965). 58) Field of Search ...... 424/450, 122 Shende et al., Hindustan Antibiot. Bull., 9 (4):229-232 (56) References Cited (1967). Thirumalacher et al., Sabouraudia, 4 (1):6-10 (1965). U.S. PATENT DOCUMENTS Thirumalacher et al., Hindustan Antibiot. Bull., 9 4,237,117 12/1980 Bruzzese et al...... 424/122 (4):246-251 (1967). 4,272,525 6/1981 Wright ...... Thirumalacher et al., Hindustan Antibot. Bull. 14 4,781,871 11/1988 West et al...... 424/450 (3):123-126 (1972). FOREIGN PATENT DOCUMENTS Utz et al., Antimicrob. Agents Chemother, 7:113-117 (1967). 212426 6/1972 France . Utz et al., Am. Rev. Respir. Dis., 95 (3):506-509 (1967). 2593394 7/1987 France . Williams et al., Antimicrob. Agents Chemother, 87/01933 4/1987 PCT Int'l Appl. . 5:700-705 (1965). 88/03831 4/1989 PCT Int'l Appl. . Williams et al., Proc. Soc. Exp. Biol. Med., 120 OTHER PUBLICATIONS (2):481-484 (1965). Capuozzo et al., Biochem. Biophys. Acta, 820 (1):63-73 Primary Examiner-Thurman K. Page (1985). Assistant Examiner-P. L. Prater Majuk, Dissertation Abstracts International, vol. Attorney, Agent, or Firm-Arnold, White & Durkee 41/05-B (1980). Mehta et al., Antimicrob. Agents Chemother, 31 (57) ABSTRACT (12):1901-1903 (1987). The present invention involves a liposomal agent for Mehta et al., Antimicrob. Agents Chemother., 31 treating disseminated fungal infection in an animal. This (12):1897-1900 (1987). liposomal agent comprises a variety of polymer antifun Dialog Search. gal compounds, particularly hamycin. These antifungal Divekar, et al., Antibiot. (Tokyo), 19 (1):63-64 (1966). compounds are encapsulated within a liposome decreas Maniar et al., Antimicrob. Agents Chemother, ing toxicity. This liposome is preferably a stable mul 5:349-352 (1965). tilamellar vesicle. The method of administration is pref Maniar et al., Can. J. Microbiol., 12 (2):377-384 (1965). erably parenteral, but may be oral or topical. Padhye, A. A., Mykosen, 12 (3):203-205 (1969). Padhye, A. A., Sabouraudia, 7 (3):182-185 (1969). 19 Claims, 15 Drawing Sheets AAC 72/6//Y 2a AAAA

Vf A/AOJO/744 - AA7%/AV WO X AAAA A/4/71z/AW O A/AOJO/74A. AA-714/AW 2 A)1/

of A, (5 (27 4A (29 A0 4 6 cowca w/AA77Ow (49/7/)

U.S. Patent July 16, 1991 Sheet 4 of 15 5,032,404

72/4/7Y 2a AAAA VAAS// AAAP(AAV7 A/a24/144 - Z4AAM Saa/YO//V 72 AA94 A 1/U /02

5 (2 O AAAA O A/A2 (7/14A.

2 /2 20 0 10 52 62 77 cawcz/v74770/w (a 9/72/) F. G. 5 U.S. Patent July 16, 1991 Sheet 5 of 15 5,032,404

% ZY / (roy/c//y)

f(70

M 2. J. f. 6.25 /2.9 25 fs (2 27 /7(2 Cowa WA470V (47/27/) A 77 (72/4 / 4A24 = 22/772A% O AWGaAv 0.47%/W + / 767 (404 O 40/CAA/50/7YC/W - 2 /767 (40/ A 40Ca WJ 0.7%W + 3/767 (40/ F. G. 6 U.S. Patent July 16, 1991 Sheet 6 of 15 5,032,404

AMPHOTERICIN B (AMB)

PERCENT SURVIVAL EL n N F-AMB (8) y -L-AMB (8) O L-AMB (2)1

O 40 2O 30 40 50 SURVIVAL TIME (DAYS) F. G. 7A

MEPARTRICN (SPA)

100 . -L-SPA (4) PERCENT SURVIVAL ---L-SPA (8) 50 F-SPA (8) . \, .;-- . . SPA (2) C.'

SURVIVAL TIME DAYS) F G. 7B U.S. Patent July 16, 1991 Sheet 7 of 15 5,032,404

NYSTATIN (NYS)

L-NYS (12) M. , . W L-NYS (8) PERCENT SURVIVAL W O 'y-L-NYS (4)'. s .

SURVIVAL TIME (DAYS) FIG.7C

CANDDIN (CAND)

OO . . . . Li-CAND (1.0) 4. N Y'.V .." V -L-CAND (8) PERCENT SURVIVAL ; . 50

SURVIVAL TIME (DAYS) F. G. 7D U.S. Patent July 16, 1991 Sheet 8 of 15 5,032,404

AMPHOTERCN B (AMB)

100

PERCENT SURVIVAL 50

O 0.4 1.O 20 O DOSE (mg/kg) F. G. 8A

MEPARTRCIN (SPA)

100 - ' ' ' '. ... L-SPA ''' (DEPC.PE:CHOL) . 6:3: V . FREE L-SPA PERCENT SURVIVAL \SPA-222 Y. (PECHOL) 50 V . 9:1 d V . L-SPA \ \(DOPPECHOL) (DMPPMPS)7.3a./. &v 63-1 O 4. 8 12 16 20 DOSE (mg/kg) F. G. 8B U.S. Patent July 16, 1991 Sheet 9 of 15 5,032,404

NYSTATIN (NYS)

L-NYS

PERCENT SURVIVAL

O 4. 8 12 16 2O 24 DOSE (mg/Kg) F. G. 8C

CANDDIN (CAND)

PERCENT SURVIVAL 50

O 0.4 0.8 1.2 1.6 DOSE (mg/Kg) FG. 8D, U.S. Patent July 16, 1991 Sheet 10 of 15 5,032,404

AMPHOTERICN B -L-AMB (0.8 x 5) ------N - . . . V N N N p N N N PERCENT SURVIVAL : L-AMB (4) ---n N. -F-AmB (0.8 x 5)

SURVIVAL TIME (DAYS) F. G. 9A

MEPARTRICN (SPA)

-"-F-SPA (8) 100 Tig':-L-SPA (2 x 5) o ... --L-SPA (8 x 5) PERCENT SURVIVAL ...L-SPA (2 x 5) 50 FSPA(8.5" L-SPA (12) na...... L-SPA (4 x 5)

SURVIVAL TIME (DAYS) F G. 9B U.S. Patent July 16, 1991 Sheet 11 of 15 5,032,404

NYSTATIN (NYS)

PERCENT SURVIVAL 50 Wu-L-NYS\--- (6 a a A.N.E)s N-L-NY-L-NYS (6:5) is-L-NYS (245) O 2O 40 60 SURVIVAL TIME DAYS) F. G. 9C

CANDDIN (CAND)

-F-CAND (.4) - F - CAND (.4 x 5) -- - - -L-CAND (25) PERCENT SURVIVAL .N L-CAND(8x5. N. ... * L-CAND (4 5)

SURVIVAL TIME (DAYS) F. G. 9D U.S. Patent July 16, 1991 Sheet 12 of 15 5,032,404

AMPHOTERICN B (AMB)

PERCENT LYSS

POSOMAL

O 2O 40 6O 80 OO DOSE (pg/mL) F. G. 10 A

MEPARTRCIN (SPA)

OO SPA-222 SPA:169) SS F1 - PERCENT LYSIS

LPOSOMAL

2 4 6 810 20 50 OO 500 OOO DOSE (pg/ml ) FIG. 10B U.S. Patent July 16, 1991 Sheet 13 of 15 5,032,404

NYSTATIN (NYS)

PERCENT LYSIS 50 EL + FREENYS LPOSOMAL

O 20 4O 6O OO 250 500 DOSE (pg/mL) F. G. 10C

CAND DIN (CAND)

100

PERCENT LYSIS 50

LIPOSOMAL

1 5 10 50 100 800 DOSE (pg/mt) F. G. OD U.S. Patent July 16, 1991 Sheet 14 of 15 5,032,404

RBC TOXICITY OF SMALL POLYENES FLIPN

PERCENT LYSIS (% OF CONTROL) 50

O 20 40 60 LOG CONCENTRATION (pg/mL) F. G. 11 A RBC TOXCITY OF SMALL POLYENES LUCENSOMYCIN

PERCENT LYSIS (% OF CONTROL) LPOSOMAL

O 10 20 30 40 50 60 70 LOG CONCENTRATION (pg/mL) F.G. 11B U.S. Patent July 16, 1991 Sheet 15 of 15 5,032,404

RBC TOXCITY OF SMALL POLYENES LAGOSN

100

PERCENT LYSIS (% OF CONTROL) LPOSOMAL 50

O 5 1.O 5.0 LOG CONCENTRATION (pg/mL) F.G. 11C

RBC TOXCITY OF SMALL POLYENES NATAMYCIN

100

PERCENT LYSIS LPOSOMAL (% OF CONTROL) V 50

O 5 O 50 100 500 000 LOG CONCENTRATION (pg/mL) F.G. 11D 5,032,404 1. 2 cally important fungi activate complement. Such acti LPSOME-INCORPORATION OF POLYENES vation may be important in defense against some myco ses; a positive correlation has been demonstrated be The Government may own certain rights to the in tween animals deficient in late-acting complement com vention as the development of part of the present inven 5 ponents (C3-C9) and increased susceptibility to fungi tion was supported by contract number NIAID 72639 such as C. neoformans and C. albicans. Assuming that from the National Institutes of Health, Department of phagocytic cells are important in resistance to fungi, complement activation may play a role by provoking an Health and Human Services. acute inflammatory response on generation of comple BACKGROUND OF THE INVENTION O ment fragments C3a and C5a, and by coating the fungal The present invention relates to the treatment of elements with opsonic fragments C3b and C3d for in systemic fungal infections by administration of liposo gestion by phagocytic cells. meincorporated polyenes. The systemic mycoses of humans and other animals Clinical observations and animal experimental studies are caused by some fungi that are pathogenic and cause have added to the understanding of host-fungal interac 15 disease in the healthy host, and by other fungi (opportu tions. It is becoming recognized that host defense nistic pathogens) that are usually innocuous but cause against fungal disease is multifactorial and may vary, disease in patients whose immune defenses are impaired. depending on the etiologic agent. The mechanisms of Some of these fungi may be saprophytes in nature (soil, resistance are not well defined in most instances, but bird droppings), whereas others are a part of the normal various innate barriers and cell-mediated immune re 20 human flora (commensals). In no case are humans the sponses seem to be of primary importance. At this time, solitary or necessary host. the role of antibody in resistance is uncertain. Clearly, An example of a soil saprophyte is Histoplasma cap debilitation of innate defenses and of cell-mediated im sulatum, which commonly causes infection in endemic mune responses can increase an individual's susceptibil areas; 80%-90% of adults react positively to histoplas ity to severe fungal disease from opportunistic agents 25 min in delayed cutaneous hypersensitivity tests. An such as Cryptococcus neoformans and species of Candida example of an opportunistic pathogen is Candida albi and Aspergillus, as well as from fungal pathogens such cans, normally present in the oral cavity, gastrointesti as Histoplasma capsulatum and Coccidioides immitis. The nal tract, and probably the skin. In the patient with difficulty in gaining a complete understanding of the acute leukemia, however, C. albicans is commonly pres critical host defenses has been further complicated by 30 ent in bloo a fulminant, usually fatal, septicemia. Other many studies that show fungi may affect various host opportunistic infections are seen in patients with dia immune functions adversely. Although it is too early to betic acidosis (mucormycosis) and Hodgkin's disease evaluate the clinical importance of many of these exper (for example, cryptococcosis and histoplasmosis). The imental findings, investigators have demonstrated that pathogenesis of these mechanisms is obscure, but cell fungi impair neutrophil function, induce IgE responses, 35 mediated immunity seems to be essential for a good and cause suppression of cell-mediated immune re prognosis. sponses. Neither active vaccines nor passive immune serum Host changes likely to be associated with increased immunization has been sufficiently successful to result susceptibility may be accidentally induced, as in trau in commercially available preparations. matic injuries (such as burns or puncture wounds); self Treatment of active disease may be symptomatic (for induced, as in chronic alcoholism; naturally occurring, example, pain relief), sometimes surgical (resection of as in diabetes mellitus, various congenital immune defi irremedially damaged tissue and correction of hydro ciencies, collagen diseases, lymphoreticular neoplastic cephalus), and, most successfully, chemotherapeutic disease, and other types of tumors; or iatrogenically (Table 1). Among the agents commonly used are hy induced by instrumentation (such as catheterization), 45 droxystilbamidine isethionate, amphotericin B, 5 surgical procedures (such as open heart surgery), or by fluorocytosine (Flucytosine), miconazole, and use of cytotoxic drugs (as in an attempt to prevent graft ketoconazole. Response to these drugs varies according rejection and to treat neoplastic disease), corticosteroid to the fungus, type of disease, and course of illness. For therapy, and long-term use of broad-spectrum antibod example, response is good in most B. dermatitidis infec S. 50 tions, but is poor in most diseases caused by A. fumiga Chemical factors that aid resistance to fungal diseases tus. Response is better for skin lesions caused by B. are poorly defined. Knowledge of these substances is dermatitidis than for meningitis due to C. inmitis; re based primarily on circumstantial evidence at the clini sponse is better in chronic cryptococcosis than in fulmi cal level and in vitro observations at the experimental nant candidiasis. Table 1 shows a listing of some sys level. Hormonally associated increases in lipid and fatty 55 temic mycoses and generally accepted chemotherapeu acid content on the skin occurring at puberty have been correlated with increased resistance to tinea capitis tic agents. caused by the dermatophyte Microsporum audouini, TABLE 1. although pubescent changes are not the sole factors in CHEMOTHERAPEUTICAGENTS resistance. Substances in serum, cerebrospinal fluid, and FORSYSTEMIC MYCOSES saliva may limit growth of Cryptococcus neoformans, and Disease First Choice Second Choice basic peptides in body fluids have been shown to inhibit Aspergillosis Amphotericin B Ketoconazole Candida albicans. Blastomycosis Amphotericin B Hydroxystilbanidine isethionate Results of clinical and experimental studies indicate Candidiasis Amphotericin B Flucytosine or that C. albicans, C. neoformans, Aspergillus fumigatus, 65 ketoconazole and C. immitis activate the alternative pathway of the Coccidioidomycosis Amphotericin B Ketoconazole complement cascade. Because of the polysaccharide Cryptococcosis Amphotericin B Either drug alone" nature of fungal cell walls, it is expected that all medi Flucytosine 5,032,404 3 4. TABLE 1-continued TABLE 2-continued CHEMOTHERAPEUTICAGENTS SOME USEFUL ANTIFUNGALAGENTS, FORSYSTEMIC MYCOSES THEIR CHEMICAL CLASSIFICATION, Disease First Choice Second Choice AND THER MECHANISMS OF ACTION 5 Class Compounds Mechanism Histoplasmosis Amphotericin B Ketoconazole' Mucornycosis Amphotericin B Miconazole" ribosome Paracoccidioidomycosis Amphotericin B Sulfonamides, Ketoconazole' The polyene macrollide antibiotics are secondary "Depending on minimal inhibitory concentration necessary for the fungus. 10 metabolites produced by various species of Streptomy ces. Several common features of these compounds are Infection is the cause of death in 51% of patients with useful in classifying the more than 80 different polyenes lymphoma and 75% of patients with leukemia. Al that have been isolated. All are characterized by a mac though bacteria are the causative organisms of many rolide ring, composed of 26-38 carbon atoms and con such infections, fungi account for 13% of the fatal infec 15 taining a series of unsaturated carbon atoms and hy tions in patients with lymphoma and for more than 20% droxyl groups. These features of the molecule contrib of patients with leukemia. The fungus Candida albicans ute to the polyenes, amphipathic properties (those relat causes more than 80% of these infections, and Aspergil ing to molecules containing groups with different prop lus spp. is also a frequent cause of such infections. In erties, for example, hydrophilic and hydrophobic). The addition, fungal infection is a major cause of morbidity 20 ring structure is closed by the formation of an internal and mortality in patients with congenital and acquired ester or lactone bond (FIG. 1). The number of conju deficiencies of the immune system. Much concerted gated double bonds vary with each polyene, and the effort has been expended in search of agents useful in compounds are generally classified according to the treating fungal infections of humans. As a result, many degree of unsaturation. compounds have been isolated and shown to have anti 25 Toxic effects of polyene macrollides appear to be fungal activity, but problems associated with solubility, dependent on binding to cell membrane sterols. Thus, stability, absorption, and toxicity have limited the thera they bind to membranes of fungus cells as well as to peutic value of most of them in human infections. The those of other eukaryotic cells (human, plant, and pro most useful antifungal antibiotics fall into one of two tozoa), but not to bacterial cell membranes, which do categories: those that affect fungal cell membranes and 30 not contain membrane sterols. The interaction of poly those that are taken up by the cell and interrupt vital ene macrollides with mammalian and fungal membrane cellular processes such as RNA, DNA, or protein syn sterols results in transmembrane channels that allow the thesis. Table 2 lists some useful antifungal agents and leakage of intracellular components leading to cell their mechanisms of action, deaths. 35 The usefulness of an antibiotic is usually measured by TABLE 2 the differential sensitivity of the pathogen and host. The SOME USEFUL ANTIFUNGAL AGENTS, polyene macrolide compounds, for example, hamycin THEIR CHEMICAL CLASSIFICATION, and lucensomycin, are relatively specific for fungi and AND THEIR MECHANISMS OF ACTION are potentially useful in humans. The relative specificity Class Compounds Mechanism of these two polyene macrollides is based on their Polyene Amphotericin B Interacts with sterols Nystatin (ergosterol) in fungal greater avidity for ergosterol, the principal sterol of Hamycin cell membrane, rendering fungal membranes, compared to cholesterol, the princi Lucensomycin cells selectively pal sterol of human cell membranes. However, it is the permeable to the outflow binding to cholesterol which causes the toxicities typi of vital constituents, e.g. potassium cally associated with the polyene macrollide com Imidazole Miconazole Inhibits demethylation of pounds. Because the polyene macrollides are so poten Ciotrinazole lanosterol thus tially useful, researchers are actively investigating ways Ketoconazole preventing formation of to reduce the toxic effects of these compounds. ergosterol, a vital It has recently been shown that the encapsulation of component of fungal cell 50 certain drugs in liposomes before administration to the membrane; also has a direct cidal effect on patient can markedly alter the pharmacokinetics, tissue fungal cells distribution, metabolism and therapeutic efficacy of Pyrimidine 5-Fluorocytosine Is taken up and deaminated these compounds. Liposomes may be defined as lipid by susceptible cell to vesicles which are formed spontaneously on addition of form 5-fluorouracil, 55 which in turn inhibits an aqueous solution to a dry lipid film. Further, the RNA synthesis; also distribution and pharmacokinetics of these drugs can be thought to inhibit modified by altering the lipid composition, size, charge thymidylate synthetase and membrane fluidity of the liposome in which they and DNA synthesis are encapsulated. Grisan Griseofulvin Binds to tubulin and inhibits microtubule Recently, liposomes have been used as carriers of assembly Amphotericin B for treatment of murine leishmaniasis 3-Arylpyrrole Pyrrolnitrin Appears to inhibit terminal (New, R. R. C., et al., “Antileishmanial Activity of electron transport Amphotericin and Other Antifungal Agents Entrapped between succinate or in Liposomes.' J. Antimicrob. Chemother, Vol. 8 (1981), NADH and coenzyme Q 65 Glutaramide Cycloheximide Inhibits protein synthesis pp. 371-381), histoplasmosis (Taylor, R. L., et al., "Am at 80S ribosomal level, photericin B in Liposomes: A Novel Therapy for histo preventing transfer of plasmosis.' Am. Rey. Respir. Dis., Vol. 125 (1982), pp. aminoacyl tRNA to the 610-611), cryptococosis (Graybill, J. R., et al., "Treat 5,032,404 5 6 ment of Murine Cryptococosis with Liposome of sterols drastically reduces the toxicity typically asso Associated Amphotericin B." J. Infect. Dis, Vol. 145 ciated with polyene macrollide compounds. (1982), pp. 748-752), and candidiasis (Tremblay, C., et An important aspect of the present invention involves al., "Comparative Efficacy of Amphotericin B (AMB) a method for treating disseminated fungal infection in and Liposomal AMB (lip-AMB) in Systemic Candidia 5 an animal. This method comprises administering to an sis in Mice." Abstr. 1983 ICAAC, No. 755 (1983), p. 222). animal subject to disseminated fungal infection, a fungi Liposome-encapsulated Amphotericin B has also been cidally effective amount of a polyene macrollide antifun used for treatment of coccidioidomycosis in the Japa gal compound encapsulated within a liposome. The nese macaque (Graybill, J. R., et al., "Treatment of liposome is composed as described above. The method Coccidioidomydosis (cocci) in Primates Using Lipo 10 of administration is preferably parenteral in most in some Associated Amphotericin B (Lipo-AMB)." Abstr. stances, but may be oral or topical if specific colonies of 1982 ICCAC, No. 492 (1982), p. 152). fungus are thereby more directly reached. Parenteral The present inventors have recently demonstrated treatment is most useful when the animal is a human that liposome encapsulated amphotericin B (Ampb) suffering from disseminated fungal infection. The may be used to treat experimental murine candidiasis 5 method of treatment involves administering a fungicid (Lopez-Berestein et al., J. Infect. Dis., Vol. 120, pp ally effective amount of liposome-incorporated polyene 278-283 (1984) and in the treatment of fungal infections compound of between about 0.1 mg/kg body weight in patients with leukemia and lymphoma (Lopez-Bere and about 80 mg/kg body weight. stein et al., J. Infect. Dis., Vol. 151, pp 704–71- (1985). An additional aspect of the present invention in Liposome encapsulation has markedly reduced the 20 volves a method of treating various other diseases such toxicity and enhanced the therapeutic index of polyene as benign prostate hyperplasia and hypercholsterolemia macrolide compounds. However, liposome formula and inflammation. Moreover, the present invention may tions presently available do not sufficiently reduce tox be used in conjunction with other drugs to enhance icity of several polyene macrollide compounds, for ex their efficiency and produce synergistic effects. ample, hamycin, lucensomycin, and mepartricin. Ac 25 Yet another important aspect of the present invention cordingly, these drugs are not widely available as thera is to provide an optimal liposomal formation which peutic agents. Recently, the present inventors have significantly buffers the toxicity of mepartricin, hamy discovered that by increasing the percentage of choles cin and lucensomycin, as well as other small polyenes terol in the liposome formulation, the toxicity typically (i.e., filipin, lagosin and natamycin). associated with polyene macrolide compounds is 30 greatly reduced. Thus, the present inventors have dem BRIEF DESCRIPTION OF THE DRAWINGS onstrated that liposome formulations having cholesterol FIG. 1 shows the structures of the polyenes studied. concentrations as high as 60% by weight increased the FIG. 2 shows the in vitro toxicity of free-hamycin rate of survival in mice treated with polyene macrollide versus liposomal-hamycin to human RBCs. The human compounds threefold. In these studies, the present in 35 RBCs reincubated at 37 C. for 45 minutes with (X) and ventors demonstrated that liposomes containing a large ( ) liposomal-hamycin. percentage by weight of cholesterol, reduced the toxic FIG. 3 shows the toxicity of various lipidcomposi ity of polyene macrollide compounds so that they may tions of liposomal hamycin having varying concentra now be used relatively safely. tions of cholesterol. Mice were dosed with various amounts of hamycin incorporated into liposomes hav SUMMARY OF THE INVENTION ing increasing amounts of cholesterol. Specifically, the The present invention involves a liposomal agent for liposomal compositions were ( )PL (DMPC-DMPG), treating disseminated fungal infection in an animal. This ( )PL:CHOL (cholesterol) (5%), ( ) PL:CHOL liposomal agent comprises lipids, a polyene macrollide (10%), ( ) PL:CHOL (20%), (A) PL:CHOL (30%), antifungal compound, and cholesterol. The cholesterol 45 (1) PL:CHOL (40%), and ( ) PL:CHOL (60%). is included in concentrations of from 10 to 75% by FIG. 4 shows the antifungal affect of free versus weight. It has been determined that as the concentration liposomal-hamycin in mice. Various doses of hamycin of cholesterol in the liposome increases, the toxicity of were given to mice and their survival was measured and the incorporated polyene macrollide compound de recorded. Specifically, the doses administered were creases. The polyene macrollide antifungal compound is 50 ( ) controls, ( ) free-hamycin 5 mg/mouse, ( ) free incorporated in or encapsulated within a liposome for hamycin 10 mg/mouse, (O) free-hamycin 15 effective therapy of systemic fungal infection. mg/mouse, (A) liposomal-hamycin 15 mg/mouse, (O) The liposome in which the polyene macrollide com liposomal-hamycin 30 mg/mouse, ( ) 45 mg/mouse. pound is incorporated is preferably a multilamellar vesi FIG. 5 shows the in vitro toxicity of free-lucensomy cle. The liposome includes one or more lipids, prefera 55 cin versus liposomal-lucensomycin to human RBCs. bly phospholipids, selected from the group consisting of The human RBCs were incubated at 37° C. for 45 min phosphomonoglyceride, phosphatidic acid and sphin utes with ( ) free-lucensomycin and ( ) liposomal golipid. The lipids are more preferably one or more of lucensomycin. phosphatidylcholine, phosphatidylserine, phosphatidyl FIG. 6 shows the in vitro toxicity of liposomallucen glycerol, sphingomyelin or phosphatidic acid. The lip 60 somycin having varying concentrations of cholesterol ids are most preferably selected from the group consist to human RBCs. The human RBCs were incubated at ing of dimyristoyl phosphatidylcholine, dimyristoyl 37 C. for 45 minutes with ( ) cholesterol concentra phosphatidylglycerol, phosphatidylcholine and phos tion 20%, ( ) cholesterol concentration 40%, ( ) cho phatidylglycerol. lesterol concentration 60%. The liposome of the present invention also comprises 65 FIG. 7 shows the in vivo toxicity of free verses lipo a sterol, preferably cholesterol. The sterol is included in some-encapsulated large polyenes in mice. a high concentration, from about 10 to 75% by weight. FIG. 8 shows the in vivo toxicity of liposomalmepar It has been demonstrated that these high concentrations tricin with different lipid compositions. Specifically, 5,032,404 7 8 this figure represents the percent survival of mice ad preferred phosphatidylglycerol is one consisting essen ministered various doses of mepartricin encapsulated in tially of dimyristoyl phosphatidylglycerol and the most the following liposomal formulations: DEPC:PE:- preferred phosphatidylcholine is one consisting essen CHOL, PC:CHOL, DOPC:PE:CHOL, Free-mepartri tially of dimyristoyl phosphatidylcholine. When the cin, DMPC:DMPG. liposomes of the present invention comprise dimyristoyl FIG. 9 shows the survival of mice treated with multi phosphatidylglycerol and dimyristoyl phosphatidylcho ple doses of free or liposome-encapsulated large poly line they are preferably in a ratio between about 1:10 enes, including mepartricin, two days after infection and 10:1, more preferably in a ratio of about 3:7. The with Candida albicans, lipids of the present invention preferably comprise from FIG. 10 shows the in vitro toxicity of free-verses 10 about 25 to about 90 percent by weight of the liposome. liposome-encapsulated large polyenes, including mepar Most preferably, however, the lipids comprise from tricin. about 25 percent to about 45 percent by weight of the FIG. 11 shows the in vitro RBC toxicity of free liposome. verses liposome-encapsulated small polyenes (e.g., fili The liposomes of the present invention may be mul pin, lagosin and natamycin). 15 tilamellar, unilamellar or have an undefined lamellar construction. A pharmaceutical composition compris DETAILED DESCRIPTION OF THE ing the liposomes of the present invention and a phar PREFERRED EMBODIMENT maceutically acceptable carrier or diluent of the types The use of polyene macrollide compounds encapsu well known to those skilled in the art may be used for lated in liposomes including a high percentage of cho 20 the therapy of disease conditions involving local or lesterol for the treatment of disseminated fungal infec systemic fungal infections. tions is described herein as a new effective therapeutic Such liposomes may be administered parenterally, method particularly useful for treatment of systemic or topically or orally, parenterally being preferred for disseminated fungal infections. It has been demon systemic or disseminated fungal infections. Parenteral strated that hamycin, lucensomycin and mepartricin 25 dosages of polyene macrollide compounds are generally encapsulated in a liposome including a large percentage in fungicidally effective amounts between about 0.1 mg of cholesterol have lowered systemic toxicity and an /kg body weight to about 80 mg/kg body weight and enhanced therapeutic efficiency as compared to other are contemplated as adequate in most conditions. The dosage forms. particular dosages, if an infected human is being treated, Although hamycin encapsulated in liposomes having 30 will vary in each case according to the condition of the no or low cholesterol concentrations had antifungal patient, the type and extent of fungal infection, the activity in vitro, such it was toxic and noneffective in polyene macrollide compound used, and directions of an vivo (i.e., when administered intravenously.) A reduced attending physician. in vivo toxicity was observed with liposomal-hamycin Preferably, the polyene macrollide compound of the including a large percentage of cholesterol, while the 35 present invention is at least one selected from the group antifungal properties were maintained. Similarly, the consisting of hamycin, lucensomycin, nystatin, ampho toxicity of mepartricin in vivo was found to be signifi tericin B, mepartricin, candidin, filipin, lagosin, and cantly reduced when encapsulated in liposomes. natamycin. However, it is most preferred that the poly Liposomes have been extensively used to modify the ene macrollide compound is one or more selected from therapeutic index of polyene compounds. The present the group consisting of hamycin, lucensomycin and inventors have previously demonstrated that liposomes mepartricin. enhance the delivery of amphotericin B to infected sites A focal point of the present invention involves a (Lopez-Berestein et al., (1968) Cancer Drug Delivery, method of treating a host animal afflicted with a fungal 1:199-205), thus promoting the drug-drug carrier inter infection. This method comprises administering to the actions with systemic fungi. The observation with en 45 host an amount of a liposome of the present invention capsulated polyene drugs has been that the improve comprising a high percentage of cholesterol, a phospho ment in their therapeutic index was related to the re lipid, and an effective fungus-inhibiting amount of a duced toxicity of free-drug after encapsulation. polyene macrollide compound. The mode of administra Nevertheless, presently available liposome formula tion is preferably parenteral, i.e. by intravenous, intraar tions still do not sufficiently reduce polyene macrollide 50 terial, intramuscular, intralymphatic, intraperitoneal, toxicity. An important aspect of the present invention subcutaneous, intrapleural or intrathecal injection or involves liposomes comprising a high percentage of infusion, topical application or oral dosage. Such ad cholesterol in association with lipids and a polyene ministration is preferably repeated on a timed schedule, macrollide compound, as well as the preparation and for example twice daily for a period of two weeks. The uses of these liposomes. Liposomes of the present inven 55 treatment may be maintained until the fungus has been tion include cholesterol in a preferred range of between eliminated and may be used in conjunction with other from about 10 to about 75 percent by weight, a more forms of anti-fungal therapy or support therapy. Such preferred range being between about 30 to about 60 parenteral administration preferably involves suspen percent by weight. The polyene macrollide compound sions of polyene macrollide compounds in pharmaceuti may be part of the phospholipid lamellae, part of the cally acceptable solutions such as sterile isotonic aque encapsulated intraliposomal fluid or both. ous solutions. These suspensions may be obtained fully Preferred phospholipids of these liposomes include prepared or may be prepared from preformed compo phosphatidylglycerol, phosphatidylcholine, sphingo nents. As known to those skilled in the art, polyene myelin, phosphatidic acid or phosphatidylserine, the macrollide compounds may be prepared and mixed with more preferred phospholipids being phosphatidyl 65 pharmaceutically acceptable solutions to form suspen glycerol, phosphatidylcholine, dielaidyl phosphatidyl sions for parenteral administration. choline, phospholatidylethanolamine, dideoyl phos Topical administration of the polyene compound may phatidylcholine or a combination thereof. The most involve pharmaceutical compositions such as suspen 5,032,404 10 sions, creams or ointments which may be obtained fully vention but are not meant to limit the present invention prepared or prepared spontaneously. Such topical ad unless otherwise stated in the claims appended hereto. ministration may be near to sites of localized fungal For example, although dimyristoyl phosphatidyl infection, such as the epithelium or mucosa. glycerol and dimyristoyl phosphatidylcholine were Oral administrations of polyene macrolide com 5 used to form liposomes, these particular lipid forms are pounds preferably involve encapsulation to protect by no means the only available usable lipids known to them from gastric and intestinal digestive activities those skilled in the art. Nor do the particular formation before release from encapsulation. methods for or types of liposomes used in these exam The methods of preparation of the liposome of the ples represent the only usable methods or liposome present invention and chemotherapeutic treatment are O types. herein described in the following Examples, and are readily adapted to the production and use of analo EXAMPLE gously described liposomes by simple substitutions of Drug, Lipids and Reagents appropriate lipids or methods. Hamycin (bulk powder) was obtained from National Liposomes containing polyene macrollide compounds 15 Chemical Laboratory (India). Chromatographically described herein may be prepared from various am pure dimyristoyl phosphatidylcholine (DMPC) and phipathic substances including natural or synthetic dimyristoyl phosphatidylglycerol (DMPG) were pur phospholipids. The phospholipids usable to produce chased from Avanti Polar Lipids (Birmingham, Ala.). liposomes are numerous and are not exhaustively listed Methanol for high-performance liquid chromatography herein because they are generally well known in the art. 20 (HPLC), dimethyl sulfoxide (DMSO), and N,N-dime These phospholipids include but are not limited to: thylformamide (DMFA) were purchased from Fisher lecithin, phosphatidylethanolamine, lysolecithin, lyso Scientific (Fair Lawn, N.J.). Human AB serum was phatidyl ethanolamine, phosphatidylserine, phos from MA Bioproducts (Walkersville, Md.). Human phatidylinositol, sphingomyelin, cardiolipin, phospha RBCs were obtained from normal volunteers. tidic acid and the cerebrosides. Most preferable phos 25 pholipids for the practice of aspects of the present in EXAMPLE 2 vention include dimyristoyl phosphatidylglycerol Liposome Preparation and Standardization (DMPG) and dimyristoyl phosphatidylcholine Multilamellar vesicles (MLV) were prepared as de (DMPC). The liposome may further comprise a sterol scribed previously (Lopez-Berestein et al., J. Infect. such as cholesterol. The sterol is to be included in pro 30 Dis, 278-283 (1984)). Cholesterol, phospholipids, and portions ranging from about 10% to about 75% by the phospholipids DMPC:DMPG (7:3), were mixed weight with the phospholipids and polyene compound with increasing amounts of the hamycin and the organic to produce liposomes of the present invention. A prefer solvents evaporated under vacuum using a rotary evap able but not limiting combination of DMPG and orator. The dried cholesterol-lipid-drug film was sus DMPC has been found to be a ratio of 3 to 7, although 35 pended in phosphate-buffered saline (PBS) and hand ratios between 1:10 and 10:1 are contemplated as satis shaken, allowing the film to form liposomes. The sus factory. Either unilamellar, multilamellar or other poly pensions were then recovered from the flasks and cen ene compound-containing mixed micellar preparations trifuged at 20,000 rpm for 1 hr. The pellets were resus may be used in the practice of the present invention. pended in PBS and the percentage of hamycin incorpo The liposome-encapsulated polyene compounds of 40 rated in liposomes was determined by absorbance at 380 the present invention also may prove useful in the pro nm. Similarly, hamycinliposomes composed of phos phylaxis and/or treatment of disease caused by human pholipids and without sterols were also prepared. The T lymphotropic retrovirus, designated HTLV-III/- following Table 3 shows the characteristic properties of LAV. As Gallo recently pointed out, HTLV-III/LAV these liposomes. may be carried in vivo by monocytes and macrophages 45 The stability of hamycin-liposomes was assessed by (in Scientific American, (1987) January, pp. 47-56). These incubating equal amounts of hamycin-liposomes with cell types may thus serve as potentially infectious and PBS and human AB serum at 37 C. At indicated time deadly HTLV-III/LAV reservoirs. - In a recently published study, Schaffner et al. (1986), intervals, samples were taken out, centrifuged at 10,000 Biochem. Pharmacol, 35:4110-4113) showed data indi 50 X g for 15 min and hamycin concentration in the pellet cating that the replication of HTLV-III/LAV in the was measured. Hamycin-liposomes were stable up to monocyte-related cell line H9 was inhibited by several 2-3 months and retained 60 to 80 percent of the drug antifungal polyene macrolides. These polyene macro after three months or storage at 4 C. lides included amphotericin B and amphotericin B TABLE 3 methyl ester ascorbate. 55 LIPOSOME PREPARATION The phagocytes of the blood-monocytes, macro Name of the drug Hamycin phages and polymorphonuclear leukocytes-charac Source National Chemical Laboratory teristically bind and ingest foreign substances, even Pune, INDIA Chemical structure Polyene prior to an immune response. These phagocytes also are mg/ml Maximum solubility among the first cells to take up circulating liposomes. It Solubility 1. Water/saline Nil --- appears likely that parenteral administration to an ani 2. Ethanol Soluble 40 ug/ml mal of liposomes comprising a polyene macrollide 3. Methanol Partia 100 ug/ml should be useful to inhibit intracellular HTLV-III/- 4. DMFA Yes 10 mg/ml 5. DMSO Yes 20 mg/ml LAV proliferation. The liposomeinduced increased 6. Chloroform N - bioactivity of nystatin may prove important in the con 65 Quantitation UV absorption at 380 mm trol of disease caused by HTLV-III/LAV infection. Encapsulation 1. DMPC:DMPG (7:3) 70% These following examples are presented to describe efficiency 2. DMPC:DMPG:cholesterol (6:3:1) 68% preferred embodiments and utilities of the present in Time % retention 5,032,404 11 12 supernatants at 540 nm, as described previously (Mehta TABLE 3-continued et al., Biochem. Biophys, Acta., Vol. 770, pp. 230-234 LEPOSOME PREPARATION (1984)). Various doses of liposomal-lucensornycin pre Stability (a) Saline 3 months 60-80% (b) Serum pared without cholesterol were incubated with fresh Drug/Lipid ratio 1:10 washed human RBCs at 37 C. for 45 min. Free-hany cin, dissolved in dimethyl formamide (DMFA), was added to the assay at a 3% final solvent concentration. EXAMPLE 3 Release of hemoglobin by hypotonic lysis of the same Encapsulation Efficiency of Hamycin in Liposomes 10 number of human RBCs by water was taken as 100% positive control, while cells treated with PBS were The encapsulation efficiencies were calculated for taken as negative controls. different batches of liposomes prepared with a fixed As shown in FIG. 5, free-lucensomycin produced amount of liposome and increasing doses of hamycin. 70% lysis at 10 mg/ml concentration whereas liposomal The maximum incorporation was 68% obtained at a 15 drug had a substantially less toxic effect at the equiva drug/phospholipid ratio of 1:10. lent concentration. However, a 30 mg/ml concentration EXAMPLE 4 of both free-lucensomycin and liposomal-lucensomycin In vitro fungal inhibition prepared without cholesterol produced approximately The antifungal activity of free- verses liposomalha 100% lysis. mycin against Candida albicans (strain 336) was deter EXAMPLE 7 mined in vitro. All strains of yeast were grown over In vivo Toxicology of Free-Hamycin and night at 37° C. on Sabouraud dextrose agar (SDA) Liposomal-Hamycin plates. All molds were grown at 30° C. on SDA for 3 to 10 days prior to collection of spores. The inoculum was Groups of eight Hale-Stoner mice (6-8 weeks old, then processed for susceptibility testing as described 25 body wit=20-25 g; Univ. of TX Science Park, Bastrop, earlier (Hopfer et al. (1984), Antimicrob, Agents Chemo TX) each were injected with various doses of free therap, 25:387-389). A twofold serial dilutron method hamycin (in 5% DMSO diluted with saline), liposomal (Shadomy et al., In E. H. Lennette, et al., (eds.) Manual hamycin without cholesterol, and liposomal-hamycin of Clinical Microbiology, 3rd ed. American Society for 30 having a gradually increasing percentage of cholesterol. Microbiology, Washington, D.C., pp. 647-653 (1980)) (FIG. 2) The mice were observed for acute, subacute, adapted to microtiter plates was used to determine the and chronic toxicity and the survival time of each ani minimal inhibitory concentration (MIC) of the drugs. mal in different groups was noted. After 90 days, the The MIC of free-hamycin was compared with that of surviving animals were sacrificed and blood and tissue liposomal-hamycin. 35 samples were obtained. Blood bioohemistry examina The MIC of free-hamycin was 0.25 microgram/ml tion included blood urea nitrogen, alkaline phosphatase, (Table 3) and the MIC for liposomal-hamycin was 0.36 and lactic dehydrogenase (LDH). The organs (liver, microgram/ml. The antifungal activity was thus main spleen, lungs and kidneys) were obtained and preserved tained in liposomal-hamycin with or without choles in 10% formalin. Tissue slices were processed for hema terol. toxylin-eosin and Gomori methenamine silver stains. EXAMPLE 5 The maximal tolerated dose (MTD) of both liposo Toxicity of Free-Hamycin and Liposomal Hamycin mal-hamycin without cholesterol, and free-hamycin was to human RBC's in vitro 20 micrograms/mouse. Liposomal-hamycin containing Lysis of human red blood cells (RBCs) was quanti cholesterol, on the other hand, showed a maximum tated by measuring the release of hemoglobin in the 45 tolerated dose of 62 micrograms/mouse using liposomes supernatants at 540 nm, as described previously (Mehta containing 60% cholesterol. The MTD increased as the et al., (1984) Biochem. Biophys, Acta., 770;230-234). percent of cholesterol increased. No subacute or Various doses of liposomalhamycin were incubated chronic toxic reactions were observed in the surviving with iresh washed human RBCs at 37 C. for 45 min. 50 animals. Nor were there any significant changes in the Free-hamycin, dissolved in dimethyl formamide blood biochemistry pattern for the surviving animals. (DMFA), was added to the assay at a 3% final solvent concentration. Release of hemoglobin by hypotonic EXAMPLE 8 lysis of the same number of human RBCs by water was Therapy with Single Dose Free-Hamycin for taken as 100% positive control, while cells treated with 55 Disseminated Fungal Infection with Candida albicans PBS were taken as negative controls. A linear increase in lysis of human RBCs was ob Hale-Stoner mice, six to eight weeks old (body served with free-hamycin ranging from 0.4 to 4.0 weight, 20-25 g) were purchased from The University ug/ml, with 100% lysis produced at 4.0 ug/ml (FIG. 2). of Texas Science Park (Bastrop, TX). The mice (eight In contrast, liposomal-haxycin did not cause any lysis per group) were injected with 0.2 ml of C, albicans cell with doses up to 0.7 mg/ml. Both hamycin preparations suspension containing 7x105 colony-forming units (cfu) were equally toxic at doses higher than 5 mg/ml. via the tail vein. This concentration of cells was consis tent in producing a disseminated infection after 48 hr, EXAMPLE 6 affecting primarily the liver, spleen, lungs and kidneys. Toxicity of Free-Hamycin and Liposomal-Hamycin 65 Infected mice were treated with increasing doses of Prepared without Cholesterol to human RBC's in vitro free-hamycin from 5 microgram/mouse (FIG. 4). None Lysis of human red blood cells (RBCs) was quanti of the doses tested improved the survival of infected tated by measuring the release of hemoglobin in the mice as compared to the control untreated group. 5,032,404 13 14 EXAMPLE 9 EXAMPLE 12 Therapy with Single Dose Liposomal-Hamycin for Encapsulation Efficiency of Lucensomycin in Disseminated Fungal Infections with Candida albicans Liposones Mice were infected with C. albicans as described in The encapsulation efficiencies were calculated for Example 7. Groups of eight mice each were injected different batches of liposomes prepared with a fixed (iv) with various doses of liposomal-hamycin two days amount of liposome and increasing doses of lucensony after the injection of C. albicans. The survival of the cin. The maximum incorporation was 75% obtained at a animals in each group was recorded. (FIG. 4). 10 drug/phospholipid ratio of 1:10. Differences in survival of mice was observed with all EXAMPLE 13 doses of liposomal-hamycin as compared with free hamycin. Significant improvement in survival was ob In vitro fungal inhibition served in the liposomal-hamycin treated groups when The antifungal activity of free- versus liposomal compared with the free-hamycin group. However, all 15 lucensomycin against Candida albicans (Strain 336) was mice were sacrificed at 90 days, regardless of treatment. determined in vitro. All strains of yeast were grown Results of culture and histopathology showed approxi overnight at 37 C. on Sabouraud dextrose agar (SDA) mately 50% of animals free from infection. plates. All molds were grown at 30° C. on SDA for 3 to 10 days prior to collection of spores. The inoculum was EXAMPLE 10 20 then processed for susceptibility testing as described Drug, Lipids and Reagents earlier (Hopfer et al., (1984), Antimicrob, Agents Chemo Lucensomycin (bulk powder) was obtained from therap, 25:387-389). A twofold serial dilution method CNR (Italy). Chromatographically pure dimyristoyl (Shadomy et al., E. H. Lennette, (eds.) Manual of Clini phosphatidylcholine (DMPC) and dimyristoyl phos cal Microbiology, 3rd ed. American Society for Micro 25 biology, Washington, D.C. , pp. 647–653 (1980)) phatidylglycerol (DMPG) were purchased from Avanti adapted to microtiter plates was used to determine the Polar Lipids (Birmingham, Ala.). Methanol for highper minimal inhibitory concentration (MIC) of the drugs. formance liquid chromatography (HPLC), dimethyl The MIC of free-lucensomycin was compared with that sulfoxide (DMSO), and N,N-dimethylformamide of liposomal-lucensomycin. (DMFA) were purchased from Fisher Scientific (Fair 30 The MIC of free-lucensomycin and the MIC for Lawn, N.J.). Human AB serum was from MA Bio liposomal-hamycin demonstrated high antifungal activ products (Walkersville, Md.) Human RBCs were ob ity. The antifungal activity was maintained in liposo tained from normal volunteers. mallucensomycin with or without cholesterol. EXAMPLE 11 35 EXAMPLE 14 Liposome Preparation and Standardization Toxicity of Free-Lucensomycin and Liposomal Multilamellar vesicles (MLV) were prepared as de Lucensomycin Prepared with Cholesterol to Human scribed previously (Lopez-Berestein et al., J. Infect. RBC's in vitro Dis, 120; 278-283 (1984)). Cholesterol, and the phos Lysis of human red blood cells (RBCs) was quanti pholipids DMPC:DMPG (7:3), were mixed with in tated by measuring the release of hemoglobin in the creasing amounts of the lucensomycin and the organic supernatants at 540 nm, as described previously (Mehta solvents evaporated under vacuum using a rotary evap et al., (1984) Biochem. Biophys. Acta., 770:230-234). orator. The dried cholesterol-lipid-drug film was sus Various doses of liposomal-lucensonycin prepared pended in phosphate-buffered saline (PBS) and hand with cholesterol were incubated with various concen shaken, allowing the film to form liposomes. The sus 45 trations of fresh washed human RBCs at 37 C. for 45 pensions were then recovered from the flasks and cen min. Free-lucensomycin, dissolved in dimethyl form trifuged at 20,000 rpm for 1 hr. The pellets were resus amide (DMFA), was added to the assay at a 3% final pended in PBS and lucensoxycin incorporated in lipo solvent concentration. Release of hemoglobin by hypo somes was determined by absorbance at 303 nm. Simi tonic lysis of the same number of human RBCs by water larly, liposomes composed of phospholipids and with 50 was taken as 100% positive control, while cells treated out cholesterol were also prepared. The following with PBS were taken as negative controls. Table 4 shows the characteristic properties of these Lucensomycin-liposomes containing 3 mg choles liposomes. terol per 10 mg phospholipids (60% by weight) were TABLE 4 found to be less toxic than those with no cholesterol or 55 LIPOSOME PREPARATION those containing 1 or 2 mg cholesterol. (FIG. 5). Name of the drug Lucensonycin EXAMPLE 1.5 Batch No. P.H. 59659 Source CNR, Milano, Italy In vivo Toxicity of Liposomal-Mepartricin with Chemical structure Polyene Different Lipid Compositions Solvent 1 mg/ml Maximum 60 Solubility 1. water/saline ni The in vivo toxicity of liposomal-mepartricin was 2. Methanol Partial 500 ug/ml evaluated in groups of 8 Hale-Stone mice (6-8 weeks 3. DMSO Yes 20 mg/ml old, body weight = 20-25g; University of Texas Sci 5. DMFA Yes 10 mg/ml ence Park, Bastrop, Tex.). Liposomal-mepartricin was Quantitation UV absorption at 303 nm Encapsulation 1. DMPC:DMPG (7:3) 75% 65 prepared following the protocol set forth in Example 2. efficiency However, the composition of the liposome was varied Drug/lipid ratio 1:10 in order to determine the optimal formulation to buffer the toxicity of mepartricin. The composition of the 5,032,404 - 15 - 16 liposomes formulated and evaluated in this study are C. albicans according to the protocol set forth in Exam listed in Table 5. Each liposomal-mepartricin prepara ple 6. tion was administered to a group of normal and a group The data on MIC of various liposomal-mepartricin of infected mice. These infected mice were injected preparations in normal and infected mice is presented with 0.2 ml of C. albicans according to the protocol set for each of the small polyenes (filipin, lagasin natamysin forth in Example 6. Percent survival was also evaluated and nystatin) and large polyenes in Table 6. in days after treatment with liposomal mepartricin. As shown in FIG. 8, it was surprising to note that liposome TABLE 6 ANTIFUNGALACTIVITY OF FREE VERSUS mepartricin (DMPC:DMPG, 7:3) was more toxic than LIPOSOMAL POLYENES the free drug. This observation prompted manipulation O of the liposome composition to be used. The data on MIC (ug/ml) MTD of various liposomal-mepartricin preparations in Polyene Free Liposomal normal and infected mice is presented in Table 5. Of Small these, three lipid preparations were found to buffer the Filipin 8.0-16.0 6.0 toxicity of mepartricin significantly; PC:chol(9:1), 15 Lagosin 4.0 3.0 Natamycin 4.0 8.0 DOPC:PE:chol(6:3:1) and DEPC:PE:chol(6:3:1). Nystatin 1.0 1.0 The respective MTD's (mg/kg) obtained for the vari Large ous liposomal-mepartricin indicate the inclusion of cho Amphotericin B 0.4 0.4 lesterol precludes an immediate toxicity, with the ex Candidin S.0 5.5 ception of the DEPC:chol (9:1) mepartricin liposome. Mepartricin 2.0-4.0 2.0 Of these, particular lipid compositions were found to buffer the toxicity significantly. These were PC:chol Of these, it appears the MIC of liposomal-natamycin is (9:1), DOPC:PE:chol (7:3:1) and DEPC:PE:chol significantly increased from that of its free-form (i.e., (7:3:1). FIGS. 9 and 10 show the survival rate. and in free=4.0 MIC vs. liposomal=8.0 MIC). The MIC of vitro toxicity of free versus liposome-encapsulated large 25 lagosin was reduced 25% (4.0 MIC-3.0 MIC) by en polyenes, including mepartricin. capsulation. TABLE 5 IN VIVO TOXICITY OF LIPOSOMAL MEPARTRICN EXAMPLE 6 WTHDIFFERENT LIPED COMPOSITIONS Toxicity of Free-Small Polyenes and Liposomal-small Immediate MTD (ng/kg) polVenes to Human RBC's in vitro reaction Normal infected Lipid composition at 8 mg/kg dose nice See Lysis of human red blood cells (RBCs) was quanti DMPC:DMPG (7:3) Yes <8.0 o tated by measuring the release of hemoglobin in the DMPC:DMPG:cho No 8.0 - supernatants at 540 nm, as described previously (Mehta, (6:3:1) : ... - et al. (1984) Biochem. Biophys. Acta, 770:230-234). Vari DMPC alone Yes C8.0 35 DMPC:chol (9:1) No 12.0 .. ous doses of the liposomal-small polyenes described in EGG PC alone Yes <8.0 O Example-16 were prepared and incubated with various Egg PC:chol (9:1) No 20.0 12.0 concentrations of fresh washed human RBC's at 37 C. DPPC alone No <8.0 for 45 min. Release of hemoglobin by hypotonic lysis of DPPC:chol (9:1) No 8.0 V DPPC:PE:chol (6.5:2.5:1) No 6.0 40 the same number of human RBCs by water was taken as DSPC alone No <89. . . . 100% positive control, while cells with PBS were taken DSPC:chol (9:1) No 100 *-* is r as negative controls. DSPC:PE:chol (6.5:2.5:1) No 20.0 0.0 DOPC alone No 8.0 4.0 As shown in FIG. 11, the incorporation of small DOPC:chol (9:1) No 4.0 m polyenes in liposomes did not significantly reduce RBC DOPC:PE:chol (6:3:1) No 200 2.0 45 toxicity. The toxicity pattern of filipin and lagosin did DEPC alone Yes (8.0 not change with liposome encapsulation, whereas lipo DEPC:chol (9:1) Yes <8.0 DEPC:PE:chol (6.5:2.5:1) No 40 a some encapsulation of natamycin provided some pro The animals that had immediate reactions died instantly after the intravenous tection of lower doses (5-40 ug/ml dose) injection. Changes may be made in the elements and methods DMPC, dimyristoyl phosphatidylcholine; DMPG, dinnyristoyl phosphatidyl N glycerol: EGG PC, egg phosphatidylcholine; DPPC, dipalmitoyl phosphatidylcho 50 described herein or in the steps or the sequence of steps line; PE, phospholatidyl ethanolamine; DSPC, distearoyl phosphatidylcholine; of the method described herein without departing from DOPC, dideoyl phosphatidylcholine; DEPC, dielaidy phosphatidylcholine; chol, the concept and scope of the invention as defined in the cholesterol. Mice died after 24 hours. following claims. What is claimed is: 55 1. A liposomal agent for treating disseminated fungal EXAMPLE 6 infection in an animal, said agent comprising; The antifungal activity of free-verses liposomal-small (a) hamycin, - polyenes was determined in vivo. These small polyenes (b) at least 10% of a sterol by weight, and included filipin, lagosin and natamycin. m (c) at least 25% lipids by weight. The in vivo toxicity of liposomal small polyen aS 60 2. The liposomal agent of claim 1 wherein the poly evaluated in groups of 8 Hale-Stone mice (6-8 weeks ene macrolide antifungal compound comprises from old, body weight = 20-25g; University of Texas Sci about 0.1 to about 10% by weight of the liposomal ence Park, Bastrop, Tex.). Liposomal polyenes were agent. - prepared following the protocol set forth in Example 2. 3. The liposomal agent of claim 1 wherein the lipids Each liposomal-small polyene formulated and evalu are one or more of phosphomonoglyceride, phospha ated in this study, listed in table 6, was administered to tidic acid and sphingolipid. a group of normal and a group of infected mice. These 4. The liposomal agent of claim 1 wherein the lipids infected mice were prepared by injection with 0.2 m. of are one or more of phosphatidylcholine, phosphatidyl 5,032,404 17 18 serine, phosphatidylglycerol, sphingomyelin and phos 12. The liposomal agent of claim 10 wherein the ste phatidic acid. rol is cholesterol and comprises from about 10 to about 5. The liposomal agent of claim 1 wherein the lipids 75% by weight of the liposomal agent. are one or more of the group consisting of dimyristoyl 13. The liposomal agent of claim 10 wherein the ste phosphatidylcholine, dimyristoyl phosphatidylglycerol, rol is cholesteroland comprises about 60% by weight of phosphatidylcholine, phosphatidylglycerol, dideoyl the liposomal agent. phosphatidylcholine, dielaidyl phosphatidylcholine, 14. The method for treatign disseminated fungal in and phosphatidylethanolamine. fection in an animal comprising administering to an 6. The liposomal agent of claim 1 wherein the lipids animal a fungicidally effective amount of the liposomal include dimyristoyl phosphatidylcholine and dimyrist 10 agent of claim 1. oyl phosphatidylglycerol. 15. The method of claim 14 wherein the mode of 7. The liposomal agent of claim 11 wherein the administering the liposomal agent is oral, topical or weight ratio of dimyristoyl phosphatidlycholine: dimy parenteral. ristoyl phosphatidylglycerol is between about 1:10 and 16. The method of claim 14 wherein the mode of about 10:1. 15 administering the liposomal agent is parenteral. 8. The liposomal agent of claim 1 wherein the lipids 17. The method of claim 14 wherein the animal is a are comprised of dimyristoyl phosphatidylcholine and human. dimyristoyl phosphatidylglycerol in a weight ratio of 18. The method of claim 14 wherein the fungicidally about 7:3. effective amount is between about 0.1 mg /kg body 9. The liposomal agent of claim 1 defined further as 20 weight and about 80 mg/kg body weight. being a stable multilamellar vesicle. 19. The method of claim 14 wherein the mode of 10. The liposomal agent of claim 1 wherein the sterol administering the liposomal agent is intravenous, intra is cholesterol. arterial, subcutaneous, intramuscular, intralymphatic, 11. The liposomal agent of claim 1 wherein the sterol intraperitoneal or intrapleural. is ergosterol. 25 sk k sk k k

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,032,404 DATED July 16, 1991 Page 1 of 6 INVENTOR(S) : Gabriel Lopez- Berestein, et all It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

In the title of the patent, "LIPSOME" should read -- LIPOSOME--. See the title page of the application. At Column 2, line 31, "bloo should read --blood, causing

At column 6, line 42, " ( ) PL" should read -- (O) PL--.

At column 6, line 43, " ( ) PL: CHOL (cholesterol) (53) it

should read -- (A) PL: CHOL (cholesterol) (53)--.

At column 6, lines 43-44, " ( ) PL: CHOL (10) " should read

-- ( ) PL: CHOL (103)--.

At column 6, line 44, " ( ) PL:CHOL (20) should read

-- (O) PL:CHOL (203)--.

At Column 6, line 44, " (A) PL: CHOL (30) " should read

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 5,032,404 OATED July 16, 1991 Page 2 of 6 NVENTOR(S) : Gabriel Lopez-Berestein, et al. it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: --(()) PL:CHOL (303)--.

At column 6, line 45, " () PL: CHOL (40%)" should read -- (Cy) PL: CHOL (403)--.

At column 6, line 45, "" ( ) PL: CHOL (603)" should read -- (O) PL:CHOL (603)--. application. At column 6, line 50, " ( ) controls" should read -- (O) Controls--.

At column 6, line 50, " ( ) free-hamycin 5 mg/mouse" should read -- (A) free-hamycin 5 mg/mouse--.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. 5,032,404 DATED July 16, 1991 Page 3 of 6 INVENTOR(S) : Gabriel Lopez-Berestein, et all It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: At column 6, lines 50-51, " ( ) free-hamycin lo mg/mouse" should read -- ( ) free-hamycin 10 mg/mouse--.

At column 6, lines 51-52, " (O) free-hamycin 15 mg/mousen should read -- (O) free-hamycin 15 mg/mouse--.

At column 6, line 52, it (A) liposomal-hamycin 15 mg/mousen should read -- ( () ) liposomal-hamycin 15 mg/mouse--.

At column 6, lines 52-53, t ( ) liposomal-hamycin 30 mg/mouse" should read -- ( () ) liposomal-hamycin 30 mg/mouse--.

At column 6, line 53, " ( ) 45 mg/mouse II should read (C ) liposomal-hamycin 45 mg/mouse--.

At column 6, line 57, it ( ) free lucensomycin" should read

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. 5,032, 404 OATE) July 16, 1991 Page 4 of 6 INVENTOR(S) : Gabriel Lopez-Berestein, et all It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: W -- () free-lucensomycin--.

At column 6, lines 57-58, " ( ) liposomal-lucensomycin." should read -- (O) liposomal-lucensomycin--. page l4, line 33 of the application. At column 6, lines 62-63, " ( ) cholesterol concentration 20" should read -- (O) cholesterol concentration 20 k--.

At column 6, line 63, " ( ) cholesterol concentration (403)" should read -- (O) cholesterol concentration (40) --.

At column 6 lines 63-64, " ( ) cholesterol concentration 60," should read -- (A) cholesterol concentration 60--.

At column 7 line 43, 1968" should read --1986--.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 5,032,404 DATED July 16, 1991 Page 5 of 6 INVENTOR(S) : Gabriel Lopez-Berestein, et all it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: At column ill, line 27, "dilutron" should read --dilution--.

At Column 11, line 43, "Liposomal Hamycin" should read -- Liposomal Hamycin--. At column 11, line 50, "iresh" should read --fresh--.

At column ll, line 59, "haxycin" should read --hamycin--.

At page 13, line 39, " (1984})" should read -- (1984))--.

At column 16, line 30, "polVenes" should read --polyenes--.

At column 18, line 7 (the first line of claim 14), "treatign" should read --treating--.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 5,032,404 DATED July 16, 1991 Page 6 of 6 NVENTOR(S) : Gabriel Lopez-Berestein, et all it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

At column l6, line 30, "poly Wenes" should read -polyenes--. At column 18, line 7 (the first line of claim 14), "treatign" should read --treating--.

Signed and Sealed this Ninth Day of March, 1993

Attest:

STEPHEN G. KUNIN

Attesting Officer Acting Commissioner of Patents and Trademarks