US006086851A United States Patent (19) 11 Patent Number: 6,086,851 Boni et al. (45) Date of Patent: *Jul. 11, 2000 54 PHARMACEUTICAL COMPOSITIONS OTHER PUBLICATIONS CONTAINING INTERDIGITATION-FUSION 66 LIPOSOMES AND GELS Ames, et al., “The Role of Polyamines in the Neutralization of Bacteriophage Deoxyribonucleic Acid”, 1960, J. Biol. 75 Inventors: Lawrence T. Boni, Monmouth Chem. 235, 3,769–775. Junction, N.J.; Andrew S. Janoff, Bally, et al., “Dopamine accumulation in large unilamellar Yardley, Pa.; Sharma R. Minchey; vesicles Systems induced by transmembrane ion gradients', Walter R. Perkins, both of Monmouth S. Chen Phys' lipids, 47 97.07. Junction, N.J.; Christine E. Swenson, Boni, et al., “Aggregation and Fusion of Unilamellar Princeton Junction, N.J.; Patrick L. Sis by Poly(Ethylene Glycol)”, BBA 775, 1984, Ahl, Princeton, N.J.; Thomas S. Davis, Chapman, et al., “Physical Studies of Phospholipids..., X. The Valhalla, N.Y. Effect of Sonication on Aqueous Dispersions of Egg Yolk Lecithin', BBA, 163, 255, 1968. 73 ASSignee: The Liposome Company, Inc., Chen, et al., “Micro-determination of Phosphorous”, 1956, Princeton, N.J. Analytical Chem., 28, 11, 1756. Chong, P., et al. “A Differential Polarized Phase Fluoromet (*) Notice: This patent is subject to a terminal dis- ric Study of the Effects of High Hydrostatic Pressure upon claimer. the Fluidity of Cellular Membranes”, Biochemistry 1983, 22, 409–415. 21 Appl. No.: 08/881,651 Eum, et al., “Temperature-Induced Fusion of Small Unila mellar Vesicles Formed from Saturated Long-Chain Leci 22 Filed: Jun. 24, 1997 thins and Diheptanolyphosphatidylcholine”, Biochem., O O 1989, 28:8206–8213. Related U.S. Application Data Liposomes, Marc J. Ostrol, es. Marcel Dekker, Inc., New York, 1983, Chapter 1, “Liposome Preparation: Methods 63 Continuation of application No. 08/315.988, Sep. 30, 1994, and Mechanisms’. cationPat. No. No. 5,820,848, 08/136,470, which Oct. is a 13,continuation-in-part 1993, abandoned, of andappli- a Lopez-Bernstein, et al., "Liposomal&&T Amphotericin B for the continuation-in-part of application No. 08/066,539, May 24, Treatment of Systemie Fungal Infections in Patients with 1993, abandoned, and a continuation-in-part of application Cancer: A Preliminary Study”, 1985, J. Infect. Dis., 151, No. 07/961.277, Oct 14, 1992, abandoned, which is a 704-71 O. continuation-in-part of application No. 07/664,576, Mar. 5, Mayer, etal., Influence of Ion Gradients on the Transbilayer cation1991, abandoned,No. 07/464,528, which Jan. is a12, continuation-in-part 1990, abandoned. of appli- Ritution- Plysis, as Unilamellar vesicles,-1, - . 51) Int. Cl." .......................... A61K 49/04; A61K 9/127; Mayer et al., “Techniques for Ecapsulating Bioactive Agents B01J 13/02; B32B5/16 into Liposomes”, 1986, Chem. Phys. Lipids, 40, 333-345. 52 U.S. Cl. ............................ 424/9.4; 424/450; 264/41; Mayer, et al. "Uptake of Adriamycin into Large Unilamellar 264/4.3; 264/4.32; 428,4022. 438,402.24 Vesicles in Response to a pH Gradient.”, BBA, 857 (1986), 58 Field of Search ..................................... 424/94, 9.45, McConnell,20. et al., “Phospholipid vesicles fusion and drug 424/450, 812; 428/402.2, 402.24; 264/4, loading: tempature, Solute and cholestorol effects, and, a 4.1, 4.3, 4.6, 4.32; .i. i. is: SE pigration for Solute-loaded vesicles', BBA, 1985, Ostro, et al., Amer. J. Hesp. Pharm., “Use of liposomes as 56) References Cited injectable-drug delivery systems”, Aug. 1989. Perkins, et al., “The captured volume of multimlamellar U.S. PATENT DOCUMENTS Vesicles”, BBA, 1988,943, 103-107. 3,993,754 11/1976 Rahman et al. ........................ 424/177 Rahman, et al., "Liposomal Protection of Adriamycin-in 4,125,410 11/1978 Sears et al. .................. - - - - - 424/19 duced Cardiotoxity in Mice”, 1980, Cancer Res., 40, 4,235,871 11/1980 Papahadjopoulos et al. - - - - - 424/19 1532-1537. 4,515,736 5/1985 Deamer ........................ - - - - - 264/4.3 Slater, et al., “Interdigitated Bilayer Membranes', Progress 4,522,803 6/1985 Lenk et al. ... 424/1.1 Lipid Res., 27, 325-359, 1988. 4,544.545 10/1985 Ryan et al. ... ... 424/1.1 4,560,665 12/1985 Nakae et al... ... 436/829 Primary Examiner Jose' G. Dees 4,588,578 5/1986 Fourntain ................................. 424/1.1 Assistant Examiner Michael G. Hartley 4,789,633 12/1988 Huang et al. ... 424/450 Attorney, Agent, or Firm-Rosanne Goodman 4,877.561 10/1989 Iga et al. ....... ... 264/4.3 4,921,706 5/1990 Roberts et al. ... 424/450 57 ABSTRACT 4,944,948 7/1990 Uster et al. ....... ... 424/450 4,962,022 10/1990 Fleming et al. .. ... 435/7 This invention provides a composition containing a sized 5,078.986 1/1992 Bosworth et al. ... 424/9 liposome comprising a lipid and an inducer; the sized 5,312,615 5/1994 Schneider et al. ....................... 424/9.4 liposome has a diameter of at most about 1 micron and the inducer is present in an amount effective to induce FOREIGN PATENT DOCUMENTS interdigitation-fusion of the sized liposome. Also provided O 190 050 8/1986 European Pat. Off herein are interdigitation-fusion liposomes and gels, and 86/OO238 1/1986 WTE) Y . methods of making the Same. 87/OOO43 1/1987 WIPO. 91/10422 7/1991 WIPO. 14 Claims, 33 Drawing Sheets U.S. Patent Jul. 11, 2000 Sheet 1 of 33 6,086,851 FIG.1A U.S. Patent Jul. 11, 2000 Sheet 2 of 33 6,086,851 iii. TEMPERATURE ill. TEMPERATURE Ii IIIi N.5. i 33 I I ls ETHANOL I TEMPERATURE LB FIG. 1B U.S. Patent Jul. 11, 2000 Sheet 3 of 33 6,086,851 A .41 LUVET A- A --9 2u LUVET A V-V V O-- - 0.05u LUVET 5 ?',/O-O- 10 Oiul LUVET 40 O 0.5 1.0 15 2.0 2.5 3.0 3.5 4.0 ETHANOL(M) FIG 2 U.S. Patent Jul. 11, 2000 Sheet 4 of 33 6,086,851 v0 0--0.05u LUVET O 1 uLUVET - Civ-v 2. LUVET w AA 4). LUVET O 0.5 10 15 2.0 2.5 3.0 3.5 4.0 ETHANOL(M) FIG. 3 U.S. Patent Jul. 11, 2000 Sheet 5 of 33 6,086,851 50 40 30 20 10 O 1 2 3 4. ETHANOL CONCENTRATION, M FIG. 4a 4 O 3O O20 O 2 3 4 ETHANOL CONCENTRATION, M FG. 4b) U.S. Patent Jul. 11, 2000 Sheet 6 of 33 6,086,851 100 80 60 40 20 O 1 2 3 4. ETHANOL CONCENTRATION, M FIG. 4C U.S. Patent Jul. 11, 2000 Sheet 7 of 33 6,086,851 30 TVNHELNI‘EWITTOAWml/?ri|d };N] ZZZZZZZZZZZZZZZZ º– ºZZZZZZZZZZZZZ) o– ZZZZZZZZZZZZZZZZZZZDHPC — DOPC (]|$ PHOSPHATIDYLCHOLINES FIG. 5 U.S. Patent Jul. 11, 2000 Sheet 8 of 33 6,086,851 CNI TWNHELNI‘EWITTONWri?ni|d ZZZZZZZZZZZZZZZZ5( — ¡?`?— LUVET DIAMETER (MICRONS ZZZZZZZZZZZZZZZZZZZZZ C)CDO ) F.G. 6 U.S. Patent Jul. 11, 2000 Sheet 9 of 33 6,086,851 50 40 30 20 10 0.0 0.2 0.4 0.6 0.8 1.0 MOLE FRACTION DPPG FIG. 7a 100 80 60 40 20 O 0.0 0.2 0.4 0.6 0.8 1.0 MOLE FRACTION DPPG FG.7b) U.S. Patent Jul. 11, 2000 Sheet 10 of 33 6,086,851 100 80 60 40 20 O 20 40 60 80 DPPC, mg/ml FIG. 8a 4O 23OO 10 O 20 40 60 80 DPPC, mg/ml FIG. 8b U.S. Patent Jul. 11, 2000 Sheet 11 of 33 6,086,851 100 X-7.58 % 50 10mg/ml DPPC 10 100 1000 PARTICLE SIZE (um) FIG. 9a 100 XE7.22 % 50 20mg/ml DPPC 1 10 100 1000 PARTICLESIZE (um) FIG.9b) U.S. Patent Jul. 11, 2000 Sheet 12 of 33 6,086,851 5O 4O 3 O 2O O O aO O 10 20 30 INITIAL CHOLESTEROL 96. FIG 10a O 10 20 30 INTIAL CHOLESTEROL% FIG. Ob U.S. Patent Jul. 11, 2000 Sheet 13 of 33 6,086,851 20 C S. S. Lif d B 10 O D - CC 2 H 2 O 0.0 0.2 0.4 0.6 0.8 1.0 MOLE FRACTION DOPC 100 80 SS a. 60 D O 40 Q Ol - 20 O Hv 0.0 0.2 0.4 0.6 0.8 1.0 MOLE FRACTION DOPC FIG 11b) U.S. Patent Jul. 11, 2000 Sheet 14 of 33 6,086,851 cro CD TWNHELNI'EWITTOAWri||mdla (— ZZZZZZZZZZZZZZ ZZZZZZZZZZZZZZZ```` INCUBATION TIMES, MINUTES F.G. 12 U.S. Patent Jul. 11, 2000 Sheet 15 of 33 6,086,851 1 8O 6 O 20 40 60 80 100 120 U.S. Patent Jul. 11, 2000 Sheet 16 of 33 6,086,851 F.G. 14 O 8 6 O 2O 40 60 80 100 Pressure (1000's psi) 64C 23C S4C dum us us ad up a o e s a Oooo a U.S. Patent Jul. 11, 2000 Sheet 20 of 33 6,086,851 l C/D O C. L. Lu Cld 2 C) C CVO - r a A U C.) He o r co O CO ChCh- OO co o r O 2 5 an cC L C L N. C - O S CN O C Cd O O O O CY S CN re v- O ld Wr/ WTON TWN IN U.S. Patent Jul. 11, 2000 Sheet 21 of 33 6,086,851 ----TOHBISHTOH0/0dd0HI —GHH|0d00/0ddC] |008'0009'0007000Z0000" 310W'N0||0WHHTOHBISHTOHOHO0&00 00'02 ld Wrl/rl WTONIWN-3LN U.S.