* — mi ii ii ii ii 111 iiiii iiiii ii i ii OJII Eur°Pean Patent Office <*S Office europeen des brevets (11) EP 0 719 546 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51 ) |nt. CI.6: A61 K 9/1 27 03.07.1996 Bulletin 1996/27 (21) Application number: 95117084.4 (22) Date of filing: 04.03.1988 (84) Designated Contracting States: • Ginsberg, Richard S. AT BE CH DE ES FR GB GR IT LI LU NL SE Belle Mead, NJ 08502 (US) • Mayer, Lawrence D. (30) Priority: 05.03.1987 US 22154 Vancouver, British Columbia V6N2P3 (CA) • Mitilenes, George N. (62) Application number of the earlier application in Washington, NJ 07882 (US) accordance with Art. 76 EPC: 88400521.6 (74) Representative: Warcoin, Jacques et al (71 ) Applicant: THE LIPOSOME COMPANY, INC. Cabinet Regimbeau, Princeton, NJ 08540 (US) 26, avenue Kleber F-751 16 Paris (FR) (72) Inventors: • Bally, Marcel B. Remarks: Vancouver, British Columbia V6T1 M8 (CA) This application was filed on 30 - 1 0 - 1 995 as a • Culis, Pieter R. divisional application to the application mentioned Vancouver, British Columbia V6J3Rs (CA) under INID code 62. (54) LUV comprising saturated phospholipids and method of making the same (57) A method for encapsulation of antineoplastic agents in liposomes is provided, having preferably a high drug:lipid ratio. Liposomes may be made by a process that loads the drug by an active mechanism using a transmembrane ion gradient, preferably a transmem- brane pH gradient. Using this technique, trapping effi- ciencies approach 100%, and liposomes may be loaded with drug immediately prior to use, eliminating stability problems related to drug retention in the liposomes. Drug:lipid ratios employed are about 3-80 fold higher than for traditional liposome preparations, and the release rate of the drug from the liposomes is reduced. An assay method to determine free antineoplastic agents in a liposome preparation is also disclosed. CO LO o Q_ LU Printed by Rank Xerox (UK) Business Services 2.12.4/3.4 1 EP 0 719 546 A1 2 Description Techniques for producing large unilamellar vesicles (LUVs), such as, reverse phase evaporation, infusion BACKGROUND OF INVENTION procedures, and detergent dilution, can be used to pro- duce liposomes. A review of these and other methods The present invention is directed to formulations and 5 for producing liposomes may be found in the text Lipo- methods for making antineoplastic agent-containing somes. Marc Ostro, ed., Marcel Dekker, Inc., New York, liposomes at high drug:lipid weight ratios. Such formula- 1983, Chapter 1, the pertinent portions of which are tions are generally higher than or substantially equiva- incorporated herein by reference. See also Szoka, Jr. et lent in efficacy to the same drug in their free form, yet al., (1980, Ann. Rev. Biophys. Bioeng., 9:467), the perti- generally have lower toxicity. Additionally, methods for 10 nent portions of which are also incorporated herein by the formation of such liposomes having unique release reference. A particularly preferred method for forming characteristics, are disclosed, as well as an assay to LUVs is described in Cullis et al., PCT Publication No. determine free and entrapped antineoplastic agents 86/00238, January 16, 1986, entitled "Extrusion Tech- such as doxorubicin, in a liposome preparation. More nique for Producing Unilamellar Vesicles" incorporated particularly, the invention is directed to the use of these 15 herein by reference. Vesicles made by this technique, high drug:lipid liposomes with toxic ionizable antineo- called LUVETS, are extruded under pressure through a plastic agents, such as doxorubicin, vinblastine, vincris- membrane filter. Vesicles may also be extruded through tine, 5-fluorouracil (5-FU), daunorubicin, epirubicin, a 200 nm filter; such vesicles are known as VET20oS. mitoxanthrone, and cyclophosphamide. LUVETs may be exposed to at least one freeze and thaw Doxorubicin is a widely used antineoplastic drug 20 cycle prior to the extrusion technique; this procedure is belonging to the anthracycline class of antibiotics pro- described in Mayer, et al., (Biochim. Biophys. Acta., duced by the fungi, Streptomyces peucetius. Doxoru- 1985, 817:193-196), entitled "Solute Distributions and bicin has been utilized against a variety of tumors, Trapping Efficiencies Observed in Freeze-Thawed Mul- leukemias, sarcomas, and breast cancer. Toxicities seen tilamellar Vesicles"; such vesicles are known as FATM- with commonly administered doses of doxorubicin (as 25 LVs. well as other antineoplastic agents) include myelosup- Other techniques that are used to prepare vesicles pression, alopecia, mucositis, and gastrointestinal toxic- include those that form reverse-phase evaporation ves- ities including nausea, vomiting, and anorexia. The most icles (REV), Papahadjopoulos et al., U. S. Patent No. serious doxorubicin toxicity is cumulative dose-depend- 4,235,871 . Another class of liposomes that may be used ent irreversible cardiomyopathy leading to congestive 30 are those characterized as having substantially equal heart failure in 1-10 percent of patients receiving doses lamellar solute distribution. This class of liposomes is greater than 550 mg per square meter of body area. denominated as stable plurilamellar vesicles (SPLV) as These toxicities severely limit the clinical utility of antin- defined in U. S. Patent No. 4,522,803 to Lenk, et al. and eoplastic agents such as doxorubicin. includes monophasic vesicles as described in U. S. Pat- Liposomes are completely closed lipid bilayer mem- 35 ent No. 4,588,578 to Fountain, et al. and frozen and branes containing an entrapped aqueous volume. Lipo- thawed multilamellar vesicles (FATMLV) as described somes may be unilamellar vesicles (possessing a single above. membrane bilayer) or multilameller vesicles (onion-like A variety of sterols and their water soluble deriva- structures characterized by multiple membrane bilayers, tives such as cholesterol hemisuccinate have been used each separated from the next by an aqueous layer). The 40 to form liposomes; see specifically Janoff etal., U.S. Pat- bilayer is composed of two lipid monolayers having a ent No. 4,721,612, issued January 26, 1988, entitled hydrophobic "tail" region and a hydrophilic "head" region. "Steroidal Liposomes." Mayhew et al., PCT Publication The structure of the membrane bilayer is such that the No. WO 85/00968, published March 14, 1985, described hydrophobic (nonpolar) "tails" of the lipid monolayers ori- a method for reducing the toxicity of drugs by encapsu- ent toward the center of the bilayer while the hydrophilic 45 lating them in liposomes comprising alpha-tocopherol "heads" orient towards the aqueous phase. and certain derivatives thereof. Also, a variety of toco- The original liposome preparation of Bangham et al. pherols and their water soluble derivatives have been (J. Mol. Biol., 1965, 13:238-252) involves suspending used to form liposomes, see Janoff et al., PCT Publica- phospholipids in an organic solvent which is then evap- tion No. 87/02219, published April 23, 1987, entitled orated to dryness leaving a phospholipid film on the reac- so "Alpha Tocopherol-Based Vesicles". tion vessel. Next, an appropriate amount of aqueous In a liposome-drug delivery system, a bioactive phase is added, the mixture is allowed to "swell", and the agent such as a drug is entrapped in the liposome and resulting liposomes which consist of multilammellar ves- then administered to the patient to be treated. For exam- icles (MLVs) are dispersed by mechanical means. This ple, see Rahman et al., U. S. Patent No. 3,993,754; preparation provides the basis for the development of the 55 Sears, U. S. Patent No. 4,145,410; Paphadjopoulos et small sonicated unilammellar vesicles described by al., U. S. Patent No. 4,235,871 ; Schneider, U. S. Patent Papahadjopoulos etal. (Biochim. Biophys, Acta., 1967, No. 4,224,179; Lenk et al., U. S. Patent No. 4,522,803; 135:624-638), and large unilamellar vesicles. and Fountain et al., U. S. Patent No. 4,588,578. Alterna- tively, if the bioactive agent is lipophilic, it may associate 2 3 EP 0 719 546 A1 4 with the lipid 5ilayer. In the present invention, the term sitions incorporating variable amounts of positively "entrapment" shall be taken to include both the drug in charged and negatively charged lipids in addition to the aqueous volume of the liposome as well as drug phosphatidylcholine (PC) and cholesterol. The varia- associated with the lipid bilayer. tions in lipid composition largely stem from the require- As has been established by various investigators, s ments for trapping doxorubicin, as systems containing cancer therapy employing antineoplastic agents can in only positive or neutral lipids exhibit low trapping efficien- many cases be significantly improved by encapsulating cies and drug to lipid ratios (Gabizon et al., 1983, supra. : the antineoplastic agent in liposomes using traditional and Shinozawa et al., supra.) In liposomes containing methods, rather than administering the free agent negatively charged lipids such as cardiolipin, higher drug directly into the body. See, for example, Forssen, et al., 10 to lipid ratios are achievable due to the association of the (1983), Cancer Res., 43:546; and Gabizon etal., (1982), positively charged doxorubicin with the negatively Cancer Res., 42:4734. Passive incorporation of such charged lipid. However, the resulting preparations are agents in liposomes can change their antitumor activi- inconsistent, exhibiting variability in vesicle size and sur- ties, clearance rates, tissue distributions, and toxicities face charge. Also, the type and amount of lipid required compared to direct administration. See, for example, is is prohibitive due to cost considerations. Rahman et. al., (1982), Cancer Res., 42:1817; Rosa, et Yet another problem with prior antineoplastic agent- al., (1982) in Transport in Biomembranes: Model Sys- containing liposomes is that none of the previous lipo- tems and Reconstitution. R. Antoline et al., ed. Raven somal formulations of doxorubicin fully satisfy funda- Press, New York. 243-256; Rosa, etal., (1983), Pharma- mental stability demands.