WO1985000968A1

WO1985000968A1 - Liposome delivery method for decreasing the toxicity of an antitumor drug

Info

Publication number: WO1985000968A1
Application number: PCT/US1984/001431
Authority: WO
Inventors: Eric Mayhew; Youcef M. Rustum; Fred C. Olson; David E. Maslow; Francis C. Szoka
Original assignee: Health Research, Inc.
Priority date: 1983-09-06
Filing date: 1984-09-06
Publication date: 1985-03-14
Also published as: EP0153955A1

Abstract

A method for decreasing the toxicity of an antitumor drug. The drug is entrapped in liposomes also containing a drug-protective compound.

Description

Liposome Delivery Method for Decreasing the Toxicity of an Antitumor Drug

Background and Summary

The following references are referred to by corresponding number in this application: 1. Forssen, E. A. and Tokes, Z. A. In vitro and in vivo studies with adriamycin liposomes. Biochem Biophvs Res Comm 91:1295-1301 (1979).

2. Forssen. E. A. and Tokes, Z. A. Use of anionic liposomes for the reduction of chronic doxorubicin induced cardiotoxicity. Proc Nat Acad Sci USA 18:1873-1877 (1981).

3. Maslow, D. E., Mayhew. E., Olson, F.. and Rustum,

Y. Reduction of the inhibitory effect of Adriamycin on myocardial contraction jLn vitro by entrapment in liposomes. Proc Am Assoc Cancer Res 21:281 (1980).

4. Olson, F., Mayhew, E. , Haslow, D. , Rustum, Y. and SzoJca, F. Characterization, toxicity and therapeutic efficacy of adriamycin encaparjlated in liposomes. Eur J Cancer Clin Oncol 18(2) :167(1982) . 5. Rahman, A., essler, A., More, N. , Sikie, B.,

Rowden, G., Woolley, P. and Schein, P. S. Liposomal protection of adriamycin-induced cardiotoxicity in mice. Cancer Res 40:1532-1537 (1980). 6. Diplock, A. T., Lucy, J. A., Verrirder, M. and Zieliniewski, A. Alpha-tocopherol and the permeability to glucose and chromate of unsaturated liposomes. Febs Lett 82:341-344 (1977).

7. Fukazawa, K. , ∑keno, H. , Tokumura, A., and Tsukatani, H. Effect of alpha-tocopherol incorporation on glucose permeability and phase transition of lecithin liposomes. Chem Phys Lip 21:13-22 (1979).

8. Myers, E., McGuire, W. and Young, R. , Adriamycin: amelioration of toxicity by alpha-tocopherol. Cancer Treatment Reports 60:961-962 (1976).

9. Sonneveld, P. Effect of alpha-tocopherol on the cardiotoxicity of adriamycin in the rat. Cancer Treatment Reports 62:1033-1036 (1978).

10. Hang, Y.-M. , Madanat, F. F., Kimball, J. C. , Gleiser, C. A., Ali, M. K. , Kaufman. M. Q. and van Eyes, J. Effect of vitamin E against Adriamycin-induced toxicity in rabbits. Cancer Res 49.:1022-1027 (1980).

11. Mayhew, E. and Rustum, Y. M. Effects of liposome entrapped adriamycin (AM) against ovarian tumor

M5076 "metastatic" to the liver. Proc Am Assoc Cancer Res 23:170 (Abstract #668) (March. 1982).

12. Hunt, C. A. and Tsang, S. Alpha-tocopherol retards auto-oxidation and prolongs the shelf life of liposomes. Int J Pharmaceutics 8:101-110 (1981).

13. Konigs. A. W. T., Darren, J. and Trieling, W. B. Protection of liposomal lipids against radiation induced oxidative damage. Int J Radiat Biol

3J5:343-350 (1979). 14. Mayhew, E., Rustum, Y. M. , Szoka, F. and

Papahadjopoulos, D. Role of cholesterol in enhancing the anti-tumor activity of

OMPI fa WIPO Λ,_. 1-alpha-D-Arabinofuranosylcytosine entrapped in reverse phase evaporation vesicles. Cancer Treatment Reports 63:1923-1928 (1979). 15. Doroshaw, J. H. , et al, J Clin Invest 68:1053 (1981) 516. Scheulen, M. E., et al, Proc Am Assoc Cancer Res 23:992(1983).

17. Rogan, A. M.. et al. Science 224:994 (1984).

18. Juhl, H., et al, Biochem Biophys Res Commun 106(1):210 (1982).

1019. Weiss, B.. Annals N Y Acad Sci 356:319 (1980).

Entrapment of adriamycin (AM) in lipid bilayer vesicles, or liposomes has been reported to reduce the toxicity of this drug in animals, probably by 5 cardiotoxicity reduction (references 1-5). In addition, alpha-tocopherol (vitamin Ξ), which has been reported to stabilize phospholipid membranes (references 6,7), has also been reported to reduce the cardiotoxicity of AM in animals when both compounds are administered in the free 0 form (references 8-10). A study reported by two of the inventors herein suggests that AM entrapped in liposomes containing entrapped alpha-tocopherol is more effective against tumor growth in mice than is free AM (reference 11). Studies conducted in support of the parent 5 application and reported herein, indicate that at the AM dosage where increased effectiveness of liposome- entrapped AM is observed, the increase is about the same whether or not the entrapping liposomes contain alpha-tocopherol. None of the above-mentioned studies 0 suggest that liposomes containing both entrapped AM and alpha-tocopherol have important therapeutic properties not found in liposomes containing entrapped AM only.

An important feature of the invention, therefore, is the finding that liposomes containing both AM and alpha-tocopherol are substantially less toxic than other AM formulations whose toxicity has been studied. These findings have led to an improved method for decreasing the toxicity of drugs generally. The method comprises trapping the drug and a drug-protective compound, at a selected ratio, in the same lipid bilayer vesicles. The drug-protective compound is one which itself decreases the toxicity of the drug when both compounds are administered in free form. In a specific embodiment of the invention, the anti-tumor drug is AM, the drug-protective compound is alpha-tocopherol and the toxicity of AM when encapsulated in liposomes containing alpha-tocopherol. is decreased more than about 60% over that of AM entrapped in vesicles containing no alpha-tocopherol. It is one object of the invention, therefore, to provide a novel method for reducing the toxicity of drugs.

A more particular object of the invention is to provide a method for reducing the toxicity of an anti-tumor drug, such as AM. by including the drug in liposomes also containing a drug-protective compound, such as alpha-tocopherol.

Still another object of the invention is to provide such a method which is applicable to a wide range of drugs and drug-protective compounds, both soluble and lipophilic.

A further object of the invention is to provide a therapeutic agent comprising liposomes containing an entrapped anti-tumor drug, such as AM, and a coentrapped drug-protective compound. 5 These and other objects and features of the invention will become more fully apparent from the following detailed description of the invention.

Detailed Description of the Invention Phospholipids and purified cholesterol were

10 prepared as described previously (reference 13). Multilamellar liposomes were made by first mixing 60 μ oles of a 1:4:4 molar ratio mixture of phosphatidylglycerol; phophatidylcholine and cholesterol (in chloroform) with 1.5 mg AM (in methanol) and 0.6

15 μmoles alpha-tocopherol (in chloroform). AM

(doxorυbicin) was obtained from Adria Corp. (Columbus, OH). The AM-containing lipid mixture was evaporated by a rotary evaporation at room temperature. Phosphate buffered saline free of calcium and magnesium (PBS), pH

207.4 was added (1 ml/60 μmoles lipid) at 37°C and the suspension was shaken at 37°C overnight. The heterogeneous multilamellar liposome suspension which formed was extruded through a 0.4 micron nucleopore filter under 40-80 psi nitrogen pressure at room

25 temperature and centrifuged at 130,000 x g for 1 hr at 20°C to concentrate the liposomes and to remove much of the non-entrapped AM. The liposomes were extensively dialyzed against 100-200 volumes of PBS with stirring at 37°C. The percent of AM entrapped, determined by 0 fluorescence spectrophotometry. was between about 65% and 70%. The calculated molar ratio of alpha-tocopherol to total lipids in the liposomes is about 1:100. Substantially all of the alpha-tocopherol was trapped in the liposomes. The same procedure was used to prepare liposomes entrapping AM but containing no alpha-tocopherol (by omitting alpha-tocopherol from the preparation) as described previously (4).

The therapeutic effects of AM were tested using DBA 2J mice injected intraperitoneally with 10 L1210 leukemia cells. The animals were treated one day later by intravenous injection of AM, either in the form of free AM, liposomes entrapping AM only (AM/liposomes). or liposomes entrapping both AM and alpha-tocopherol (AM-aT/liposomes) . The dosages of AM administered, expressed in milligrams AM per kilogram animal body weight, are given at the left in Table I below. The day of death of the animals was recorded, and the mean survival time of each group was calculated. Each group contained from between 6 and 10 mice. The mean survival time and calculated standard deviations are shown at the three columns at the right in Table I.

Table I dose AM Mean Survival Time (days) 1 +. S.D.

(mg/kg) free AM AM/liposomes AM- -aT/liposomes

0 7.2 +0.4 - - —

10 18.4 +1.8 16.8 +2.4 17.6 +3.0

20 12.2 +1.6 17.6 +2.0 16.8 +1.6

50 _ 13.0 +4.3 17.0 +4.0

The data in Table I show that at a dosage of 10 mg/kg, the anti-leukemic effectiveness of free AM was maintained, but not improved, by entrapment in liposomes alone or liposomes containing alpha-tocopherol. At the

20 mg/kg dosage level, the effectiveness of liposome- entrapped AM, either in the presence or absence of alpha-tocopherol was better than that of free AM at the same concentration, similar to what was reported in reference 11. However, at no AM dosage level was the effectiveness of AM entrapped in liposomes, either in the presence of absence of alpha-tocopherol, greater than the optimal, 10 mg/kg dosage level of free AM.

Similar studies were performed to determine the effect of the various AM formulations on long-term survival in mice which had been injected intra-

5 peritoneally with 10 L1210 leukemia cells. The infected animals were treated with intraperitoneal injections of AM (10 mg/kg body weight) administered either as free AM, AM encapsulated in liposomes, AM encapsulated in liposomes also containing alpha- tocopherol, or a mixed population of liposomes containing AM only and liposomes containing alpha- tocopherol only. Significantly, the only AM formulation that gave long-term survival (2 of 6 mice survived to 172 days after initial infection) was the lipoβome formulation containing conentrapped AM and alpha-tocopherol.

At least under certain therapeutic conditions, then, supplying the drug and drug-protective compound in the same liposome population produced therapeutic results which are superior to those obtained by administering the drug and drug protective compound in separate liposome populations. The results suggest that release of a drug (in this case, an anthracycline anti-tumor drug) and a drug-related compound (in this case, a drug protective compound) in the same localized liposome-target region may have important therapeutic consequences not realized heretofore. The toxicity of the three AM preparations described with respect to Table I was tested against healthy DBA 2J mice. Groups of mice (6-10 mice/group) were injected with a single iv dose of free AM, liposomes entrapping AM only (AM/liposomes), and liposomes entrapping both AM and alpha-tocopherol

(AM-aT/liposomes) in doses of 2, 5, 10, 15, 20, 25, 30. 50, 75, 100 mg AM per kg animal body weight.

In the experiment, which is reported in Table II below, it was found that the mice died at two distinct time periods after administration: within 3-14 days, and between approximately 8-12 weeks after drug administration. The data are expressed in terms of LD_ςo, i.e., the dosage (in mg drug per kilogram of animal body weight) which produces death in half the animals receiving the drug. The upper row in Table II gives the D₅_ data for mice dying within 14 days after drug administration (acute), the lower row, for mice dying between 50 and 120 days after drug administration (chronic). The number of mice available for determination of chronic toxicity (survival of acute toxicity) varied from between about 2 and 10 mice per group.

Table II

Mean LD₅_ (mg/kg) +.S.D. free AM AM/liposomes AM-aT/liposomes acute 20 +5 45 ±5 >75 chronic 12 +5 30 +5 50

The data in Table II confirm that both acute and chronic toxicity of AM are reduced more than 2-fold by encapsulating the drug in liposomes containing no alpha-tocopherol, as has been reported previously. According to an important finding of the present invention, entrapping the drug in liposomes which also contain entrapped alpha-tocopherol further reduces acute and chronic toxicity more than about 60% with respect to liposomes entrapping AM alone. It is noted that 75 mg/kg is about the highest drug dosage which could be administered with a single intravenous injection. Therefore the D_5Q value for acute toxicity from AM-aT/liposomes may be somewhat higher than shown.

The results presented herein suggest that it may be possible to use a total cumulative dose of AM, after co-entrapment with alpha-tocopherol in liposomes, several times greater than that possible with the free drug. Alternatively, similar doses of the liposome-entrapped drug only could be used with reduced risk of cardiotoxicity.

Further reduction in drug toxicity may be achieved by increasing the relative amount of drug protective compound in the drug-containing liposomes. As noted above, drug-protective liposomes described herein had an alpha-tocopherol to total lipid ratio of about 1:100. Using an alpha-tocopherol succinate, the ratio of alpha-tocopherol to total liposome lipids can be made much greater, preferably in the molar ratio range of 1:20 to 1:5, i.e.. between about 5 and 20 mole percent alpha-tocopherol. A preferred therapeutic agent of the invention comprises liposomes containing phospholipid. cholesterol, alpha-tocopherol succinate and AM at molar percentages of between about 30% and 70%, 20% and 50%, 5% and 20% and 0.2% and 15%, respectively. The data supports the concept of using liposomes to carry more than one agent simultaneously, where one of the agents is a drug and the other agent is either a drug-protective compound, such as disclosed herein, or a drug-potentiating compound which promotes the action of the drug at the site of drug delivery. Examples of other drug-protective compounds, which have been shown to reduce anthracycline cardiac toxicity when administered in free form, include hydroxybutylated toluene, N-acetylcysteine (reference 15) and niacin and isocitrate (reference 16). In practicing the method of the present invention, these compounds would be coentrapped, for example, at encapsulated concentrations of between about 5-100 mg/ml in anthracycline-containing liposomes. to produce an enhanced reduction in drug toxicity.

Compounds that potentiate anthracycline activity include agents that block calcium uptake, such as verapamil (reference 17). compounds that interfere with calcium mobilization from an intracellular store, such as 8-(N,N-diethylamino)-octyl-3. 4,5-trimethoxy- benzoate (TMB-8) (reference 18), or compounds that interfere with calcium binding to the protein

OMPΓ '• WIPO $ calmodulin, such as trifluoroperazine. thioridozine. and other compounds noted in reference 19. These compounds would be included, at normal therapeutically effective doses, and/or maximum concentrations consistent with liposome membrane stability, in liposomes also formulated to contain encapsulated an anthracycline drug, and administered in a suitable manner, such as is detailed above.

While the present invention has been illustrated with respect to one particular embodiment, it will be appreciated by those skilled in the art that various changes and modificatios can be made without departing from the scope and spirit of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method for decreasing the toxicity of a drug substantially below that produced when the drug alone is entrapped in lipid bilayer vesicles, or that produced when the drug and a drug-protective compound are both administered in free form, the drug-protective compound being one which itself decreases the toxicity of the drug when administered with the drug in free form, said method comprising entrapping the drug and the compound, at a selected drug-to-compound ratio, in the same lipid bilayer vesicles.

2. The method of claim 1, wherein the drug-protective compound includes an anthracycline drug.

3. The method of claim 2. wherein the drug protective compound is selected from the group consisting of alpha-tocopherol, alpha-tocopherol succinate, N-acetylcysteine, niacin, isocitrate, and β-hydroxybutylated toluene.

4. The method of claim 2. wherein the drug includes adriamycin, the drug-protective compound includes alpha-tocopherol, and the toxicity of the drug entrapped in lipid vesicles also containing alpha-tocopherol, as measured in mice, is decreased at least about 50% below the toxicity of the drug entrapped in vesicles containing no alpha-tocopherol.

5. A therapeutic agent comprising liposomes containing phospholipid, cholesterol, alpha-tocopherol

^NATIO≤^ succinate, and adriamycin at molar percentages between about 30% and 70%, 20% and 50%, 5% and 20% and 0.2% and 15%, respectively.