IL136343A - Combined chemo - immunotherapy with liposomal drugs and cytokines - Google Patents

Description

136343/3 Combined chemo-immunotherapy with liposomal drugs and cytokines Yissum Research Development Company of the Hebrew University of Jerusalem Hadasit Medical Research Services 0")>ii nwinrt J OI iprttt mi¾> iwm & Development Limited C. 125306 Field of the Invention The present invention relates to the use of liposomes technology in the preparation of pharmaceutical products for anti-tumor treatment and to such pharmaceutical products.

References Adler, A. et al., Cancer Biotherapy 10:293-306 (1995).

Curran, D.P. et al., Angew. Chem. Intl. Ed. Eng. 34(23/24):26834 (1996).

Gabizon, A. et al., Adv. Drug Delivery Reviews 24(2-3):337-344 (1997).

Kedar, E. et al., J. Immunotherapy 16:47-59 (1994).

Lasic, D. ands Martin, F., Eds., STEALTH LIPosoMEs CRC Press, Boca Raton, FL (1995).

Papahadjopoulos, D. et al., Proc. Natl. Acad. Sci. USA 88:11460-11464 (1991).

Sears, B.D., U.S. Pat. No. 4,426,330 (1984).

Sears, B.D., U.S. Pat. No. 4,534,899 (1985a).

Szoka, F., Jr. petal., U.S. Pat. No. 4,235,871 (1980b).

Szoka, F., Jr. petal., Ann. Re. Biophys. Bioeng. 9:467 (1980).

Tirosh, O. et al., J. Chem. Soc. Perk. Trans. II 2:383-389 (1997).

Woodle, M.C. petal., U.S. Pat. No. 5,013,556 (1991).

Background of the Invention Despite prolific research in the area of cancer chemotherapy, such treatment remains far from satisfactory. The inability of chemotherapeutic drugs to reach the tumor site, intrinsic and acquired cross-resistance to multiple chemotherapeutic agents, and, especially, the high toxicity of many of these agents all contribute to treatment failures.

The use of immunostimulating cytokines, such as IL-2 and interferon-a, has proven to be effective in treatment of a proportion of patients with malignancies such as melanoma and renal cell carcinoma, both alone and in combination with other therapeutic agents. However, major problems limit their wide clinical use, including rapid plasma clearance, biodistribution to nonrelevant tissues, and high toxicity. Furthermore, their efficacy has been low in treatment of the most common tumOrs, e.g. colorectal, mammary, prostate, and lung carcinomas. 01253061\33-01 Summary of Invention The present invention provides the use of an immunostimulating cytokine for the preparation of a pharmaceutical composition for the treatment of a subject in need of antitumor therapy at least three days after treatment with a chemotherapeutic drug, wherein said preparation comprises encapsulating the cytokine in multi-lamellar liposomes (MLV).

The present invention also provides the use of an immunostimulating cytokine encapsulated in multi-lamellar liposomes (MLV) for the preparation of a pharmaceutical composition for treatment of a subject in need of antitumor therapy at least three days after administration of a chemotherapeutic drug to said patient.

The present invention yet further provides the use of an immunostimulating cytokine encapsulated in multi-lamellar liposomes (MLV) for the preparation of a pharmaceutical composition for treatment of a cancer patient in combination with a chemotherapeutic drug from the group consisting of anthraquinones, platin complex and topoisomerase I inhibitors excluding camptothecin.

In accordance with one embodiment of the invention, the immunostimulating cytokine is selected from interleukin-2 (IL-2). IL-12, IL-15, IL-18, IFN-γ, IFN-o , IFN-β, TNF-cc, G-CSF, and GM-CSF. Preferably, the cytokine is IL-2.

In accordance with one embodiment of the invention, the cytokine is encapsulated in liposomes comprising at least one lipid selected from the group consisting of dimynstoyl phosphatidyl choline (DMPC), dimyristoyl phosphatidyl glycerol (DMPG), l,2distearoyl trimethylammonium propane (DSTAP), phosphatidyl choline, phosphatidyl ethanolamine, and cholesterol.

A preferred embodiment provides the encapsulation of the cytokine in liposomes comprising dimyristoyl phosphatidyl choline (DMPC) in combination with 0-50 mole percent of at least one lipid selected from dimyristoyl phosphatidyl glycerol (DMPG) and 1,2-distearoyl triinethylammonium propane (DSTAP).

Preferably, the liposome is composed of DMPC and DMPG, more preferably, the liposome is composed of DMPC and DMPG in a molar ratio of about 9: 1.

The anthraquinone is preferably selected from doxorubicin, epirubicin, daunorubicin and mitoxanthrone; the platin complex is preferably cis-platin; and the topoisomerase I inhibitor is preferably selected from topotecan and irinotecan.

In accordance with one embodiment, the chemotherapeutic drug is encapsulated in a liposome. For example, the chemotherapeutic drug is a polyethylene glycol coated 01253061\33-01 liposomal doxorubicin.

The invention also provides a pharmaceutical product comprising an immuno stimulating cytokine encapsulated in multi-lamellar liposomes (MLV) and a chemotherapeutic drug, as a combined preparation for separate or sequential administration in antitumor therapy.

The immunostimulating cytokine, the liposomal composition comprising the cytokine, and the chemotherapeutic drug (either encapsulated or non-encapsulated) are as defined above.

Brief Description of the Drawings Figure shows the survival rate of BALB/c mice injected intraperitoneally with 5xl05 Ml 09 tumor cells (lung adenocarcinoma) and subsequently treated with free adriamycin or DOXIL®, respectively, alone or in combination with intraperitoneal IL-2 in DMPC/DMPG MLV liposomes, or with liposomal IL-2 alone; and Figure 2 shows the survival rate of BALB/c mice injected intravenously with 5x105 Ml 09 tumor cells and subsequently treated with DOXIL® (at day 7), alone or in combination with intravenous IL-2 in Stealthy PEGylated SUV liposomes (at days 11 , 14, and 17), or with liposomal IL-2 alone (at days 11, 14, and 17).

Detailed Description of the Invention I. Definitions The terms below have the following meanings unless indicated otherwise.

"Vesicle-forming lipids" refers to amphipathic lipids which have hydrophobic and polar head group moieties, and which (a) can form bilayer vesicles in water, as exemplified by phospholipids, or (b) can be stably incorporated into lipid bilayers, with the hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and the polar head group mciety oriented toward the exterior, polar surface of the membrane.

The vesicle-forming lipids of this type typically include one or two hydrophobic acyl hydrocarbon chains or a steroid group, and may contain a chemically reactive group, such as an amine, acid, ester, aldehyde or alcohol, at the polar head group. Included in this class are the phospholipids, where the two hydrocarbon chains are typically between about 14-22 carbon atoms 01253061X33-01 in length, and have varying degrees of unsaturation. Representative examples are phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatide acid (PA), phosphatidyl inositol (PI), sphingomyelin (SM), negatively charged lipids such as dimyristoyl phosphatidyl glycerol (DMPG), and positively charged lipids such as l,2^tearoyl-3-trmiethylarnmonium propane (DSTAP). The liposomes may also contain sterols, such as cholesterol, which do not form liposomes themselves but can be incorporated into, and may stabilize, liposomes containing lipids such as those described above.

A "Cetus unit" (CU) is equal to six International Units (IU) of Immunological Activity, the international reference standard of a biological preparation of interleukin-2 (Π--2). The term "unit" used herein in reference to cytokine levels refers to Cetus units. Π. Liposomal Compositions A. Lipid Components Various vesicle-fonning lipids, as defined above, may be used in the present liposomal compositions, according to methods well known in the art. Preferred lipids for the current invention allow long-term storage of the hposome-entrapped agents and effective release of these components upon adrninistration. Representative lipids include, but are not limited to, dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol, egg phosphatidylcholine (egg PC), phosphatidyl erhanolarnine (PE), distearoyl phosphatidyl ethanol-amine (DSPE), phosphatidyl inositol (PI), l,2-distearoyl-3-trimemylammoriium propane (DSTAP), l^-dimyristoyl-3-trrmemylammonium propane (DMTAP), and combinations thereof.

The vesicle-forming lipids, preferably those making up SUV's, may contain about 1-10 mole percent of a lipid having a polar head group, typically a phosphate containing head group, derivati-zed with a polyethylene glycol (PEG) chain which has a molecular weight of between 750 and 10,000 daltons. The rate of clearance of liposomes from circulation is typically reduced by employing such PEG-derivatized, or "PEGylated", lipids. PEG coating is believed to inhibit nonspecific adsorption of serum proteins, thereby preventing nonspecific recognition of liposomes by macrophages (Papahadjopoulos, et al., 1991). Another advantage of these long-circulating liposomes is their good extravasation capacity and high accumulation in tumors (Lasic and Martin, 1995; Gabizon, et al., 1997). They are also referred to as sterically stabilized liposomes, SSL, or Stealth® liposomes.

The preparation of such lipids is described in, for example, Woodle, etal., 1991; Sears (1984, 1985); Tirosh et al. (1997) or copending and co-owned application having U.S. serial number 08/570,440. The PEG chain may be linked directly to the phosphatide acid head group of a phospholipid. Various other linkages are possible; for example, lipics connirirg a ~.z2. ~j. ethanolamine (PE) or other amino head group may be conveniently coupled to activated PEG chains via reaction with brominated PEG. PEG-modified lipids are also commercially available, e.g. from Sequus Corporation, Menlo Park, CA.

B. Preparation of Liposomes and Liposomal Compositions Liposomes may be prepared by a variety of techniques, such as those detailed in Szoka et al. (1980b). To form multilamellar vesicles (MLV's), a mixture of vesicle- forming lipids dissolved in a suitable solvent is evaporated in a vessel to form a thin film, which is then hydrated by an aqueous medium to form MLV's, typically with sizes between about 0.1 to 10 microns. Tert-butanol is a preferred solvent for the process. The MLV's may then be downsized to a desired size range by extruding the aqueous suspension through a polycarbonate membrane having a selected uniform pore size, typically 0.05 to 1.0 microns.

Preparations of MLV's or KEV's (described below) may be treated, e.g. by extrusion, sonication or high pressure homogenization, to produce unilamellar vesicles. Small unilamellar vesicles (SUV's) are characterized by sizes in the 30-100 nm range, while large unilamellar vesicles (LUV's) are defined as those having mean diameters of about 100-200 nm. SUV's may also be formed directly by high pressure homogenization of an aqueous dispersion of lipids.

Various methods are available for encapsulating other agents in liposomes. Preparation of SSL-encapsulated IL-2 is described in Kedar et al. (1994). In this procedure, generally, the lipid components, including a PEG-substituted lipid, are dissolved in t-butanol. The solution is sonicated, and IL-2 is added with further sonication. The mixture is lyophilized and rehydrated, fonning MLV's, which can then be downsized by high pressure homogenization or by successive extrusion through polycarbonate filters. These downsizing methods gave vesicles having diameters of 50-80nm and about 200nm, respectively. The procedure achieved approximately 80-90% encapsulation of the IL-2.

In the reverse phase evaporation method (Szoka, et al. , 1980a) a nonaqueous solution of vesicle-fonning lipids is dispersed with a smaller volume of an aqueous medium to form a water— in-oil emulsion. The agent to be incorporated is included either in the lipid solution, in the case of a lipophilic agent, or in the aqueous medium, in the case of a water-soluble agent. After removal of the lipid solvent, the resulting gel is converted to liposomes. These reverse phase evaporation vesicles (REVs) have typical average sizes between about 0.2-4 microns and are predominantly oligolamellar, that is, containing one or a few lipid bilayer shells. The REVs may be sized by extrusion, if desired, to give oligolamellar vesicles having a maximum selected size between about 0.05 to 1.5 microns.

Other methods for adding additional components to liposomal compcsiiici-s include z r z- phiiization with other components and redispersion of the resu!tkg solid to ¾~ .MlY's. In a method described by Adler, ex al. (1995), an aqueous solution of the agent to be encapsulated is added to a t-butanol solution of lipids. The mixture is sonicated and lyophilized, and the resulting powder is rehydrated.

Liposome compositions confining an entrapped agent may be treated after final sizing, if necessary, to remove free (non-entrapped) agent. Conventional separation techniques, such as centrifugation, diafiltration, and molecular-sieve chromatography are suitable for this purpose. The composition may also be sterilized by filtration through a conventional 0.22 or 0.45 micron depth filter.

To form the compositions of the current invention, the concentration of drug and/or cytokine in the liposomes is preferably effective to give a protein/lipid weight ratio between about 1: 100 and 1:1000.

Stabilizers may also be added to the liposomal compositions. For example, addition of a metal chelator such as Desferal™ or ciiethylenetriamine pentaacetic acid (DTPA) to the lyophilization medium, at a concentration of 100 μΜ, has been shown to reduce activity loss of entrapped EL-2 during liposome preparation and storage at 4°C. Antioxidants such as BHT or Vitamin E may also be included.

For long term storage, the compositions may be stored as the dry lyophilized powder, which is stable for at least a year at 4°C, and hydrated to form an aqueous suspension before use. ΙΠ. Combined Chemotherapy/Cvtokine Therapy A. Formulations Cytokines useful for enhancing antitumor activity of chemotherapeutic drugs include EL-2, IL- 12, IL-15, IL-18, IF -γ, IFN-a, IFN-β, TNF-a, G-CSF, and GM-CSF. A preferred cytokine for the present invention is IL-2 (interleukin 2), which acts as a growth and maturation factor for T- lymphocytes.

A variety of liposomal formulations may be used for encapsulation of the cytokine. These include MLV, LUV or SUV, as defined above, as well as OLV (oligolamellar vesicles) and MW (multivesicular vesicles), composed of vesicle-fonning lipids such as those described above.

Combinations of lipids are generally most effective (see, for example, Kedar ex al., 1994). One preferred type of formulation employs SUV or LUV, having a mean diameter of approximately 50 to 120 nm, containing about 1-10 mole percent of a lipid having a polar head group derivatized with a polyethylene glycol (PEG) chain (also referred to as a PEGylated lipid). Formulation A below is one example. Other preferred formulations employ dimyristoyl phosphatidyl choline (DM?C) and, optionally, up to 50 mole percent of at least one lipid selected from dimyr-S-oyl phosphatidyl glycerol (DMPG) and l,2niistearoyl-3-trimefeyiarj-^ rcpsrs C 5TA?}. In these formulations, the proportion of DMPG and/or DSTAP is more preferably 5 - 25 mole percent. Formulation B below is one example. In all cases, small quantities (up to about one mole percent) of stabilizers such as tocopherol or Desferal™ may be included.

For the experiments described below, liposomal IL-2 was prepared in two formulations, using IL-2 obtained from Chiron Corporation (Emeryville, CA), according to known methods such as those described above. Formulation A employed sterically stabilized (SSL) small unilamellar vesicles (SUV) composed of ^EG-DSPE (N-carbamyl-(polyethylene glycol methyl ether)-l,2-distearoyI-sn-glycen 3-phosphoethanolamine triethylammonium salt, provided by Sequus Corporation), egg phosphatidyl choline, and cholesterol in a molar ratio of about 5:55:40. The vesicles were about 50-70 nm in diameter. Encapsulation efficiency of IL-2 was greater than 80% , based on an in vitro IL-2 bioassay (z.e., > 80% of the initial amount of added IL-2 became encapsulated in liposomes).

Formulation B employed multilamellar vesicles (MLV) composed of DMPC-DMPG (dimyristoyl phosphatidyl choline - dimyristoyl phosphatidyl glycerol) in a 9: 1 molar ratio. The vesicles were approximately 500-1500 nm in size, and the encapsulation efficiency was approximately > 90%. This high efficiency of encapsulation was achieved at a lipid:IL-2 ratio (wr.wt) of 1000:1 for DMPC alone, and 100:1 for DMPC containing DMPG or DSTAP.

The chemotherapeutic drug is preferably encapsulated in liposomes having about 1-10 mole percent of a PEGylated lipid, as described above. For example, DOXIL®, a stable formulation of adriamycin in Stealth9 liposomes, is available from SEQUUS Pharmaceuticals, Inc. (Menlo Park, CA). Free adriamycin is available, e.g., from Cetus Oncology Corp. (Emeryville, CA) as a formulation of doxorubicin hydrochloride and lactose.

Other chemotherapeutic drugs which are also preferred for the present method include other aiuhraquinones, such as epirubicin, daunorubicin, and mitoxanthrone, and cis-platm. Also contemplated are topoisomerase I inhibitors such as camptothecin and its analogs, e.g. topotecan and irinotecan, also designated CPT-11. Camptothecin is isolated from the stem wood of the Chinese tree Camptotheca aciminata; preparation of me above noted analogs has been described by, e.g., Curran et al. (1996).

B. Liposomal Adriamycin - Liposomal IL-2 The effect of adriamycin, used alone or in combination with interleukin-2 (IL-2), where each component was in free or liposome-encapsulated form, on the survival rate of BALB/c mice infected with tumor cells, was tested as described below.

Bl. Lung Adenocarcinoma Model: IL-2 in MLV. Six groups of BALB/c mice were injected traperitonealry with 5 χ 10s M109 tumor cells (day 0). Free adriamycin or DOXH¾, respectively, were administered intravenously on day 7 at a dose of S iss/kg, ar_d hrapsri --^ cytokine treatment was initiated 3 days later. Liposomal IL-2 (formulation B; MLV DMPC/DMPG (9:1 mole ratio) liposomes containing IL-2) was given once daily (50,000 CU/mouse) on days 10, 13 and 16. Control groups received no treatment or received the IL-2 treatment alone.

Each group, consisting of 8-9 mice, was inspected for survival up to 100 days after tumor inoculation. Table I shows the number of survivors at the end of the experiment and the median survival time obtained; Figure 1 shows the survival curves for all groups.

TABLE I As Table I shows, adriamycin (ADR) in combination with MLV-IL-2 (liposomal IL-2, formulation B) was much more effective man either adriamycin alone or liposomal IL-2 alone, both of which showed lower survival rates than the control. When liposomal adriamycin (DOXIL®) was administered alone, or when non-Iiposomal adriamycin was combined with liposomal IL-2, five of eight mice survived for the duration of the test.

The best result, i.e. survival of all subjects for 100 days or more, was observed for the combination of liposomal ADR (DOXIL®) with liposomal IL-2. In terms of number of surviving subjects, the effect of the combination treatment was greater than a combination of the effects of the individual treatments.

B2. Metastatic Lung Adenocarcinoma Model: IL-2 in MLV (Formulation B) and PEG- Derivatized SUV (SSLV In mis were injected intravenously with 5 x 10s M109 tumor cells (day 0). Free adriamycin or DOXIL®, respectively, were administered intravenously on day 7 (8 mg/kg), followed 3 days later by intravenous cytokine treatment.

Liposomal IL-2 (Formulation A; PEGylated SUV containing IL-2) was given once daily (50,000 CU/mouse) on days 11, 14 and 17. Control groups received no treatment or received the IL-2 treatment alone.

Each group, consisting of 8-9 mice, was inspected for survival to !CO days r.z tunc? inoculation. Results are shown in Table Π and Figure 2.

TABLE Π As a comparison of groups 3-5 shows, the combined treatment with DOXDL® and liposomal IL-2 was significantly more effective than treatment with either liposomal component alone, particularly in terms of the number of subjects surviving for the duration of the test, i.e. 100 days or more (7 out of 9 compared to 0-1 out of 8). In this aspect, the combined treatment was significantly more effective than a combination of the effects derived from the individual therapies.

In a second, more extensive study, nine groups of BALB/c mice were injected intraperitoneally with 5 x 10s M109 tumor cells. Free adriamycin or DOXJL® (8 mg kg) were administered intraperitoneally 7 days later, followed 3 days later by intravenous cytokine treatment. The cytokine, given once daily (50,000 CU/mouse) on days 10, 13 and 16, consisted of free IL-2, IL-2 in Formulation A (Stealth® PEGylated SUV), or EL-2 in Formulation B (9:1 molar DMPC DMPG MLV).

Each group, consisting of 8 mice, and an untreated control group of 11 mice, were inspected for survival up to 120 days after tumor inoculation. Results are shown in Table ΙΠ.

TABLE EI In this study, adnunistrarion of free ADR and IL-2 showed little or no benefit over free ADR alone (groups 2-5). However, combinations of either free or liposomal IL-2 with the chemomerapeutic drug in liposomes (DOXIL®) showed clear benefits over administration of the drug alone (groups 6-9). Overall, the groups (8 and 9) treated with a combination of both components in liposomes showed superior results. Group 8, in particular, showed a high survival rate and almost a complete absence of tumors.

B3. Subcutaneous colon carcinoma model: IL-2 in MLV. In this test, 7 groups of BALB/c mice were injected in the footpad with 10s C26 colon carcinoma cells. Seven days later, 8 mg/kg free or liposomal adriamycin was administered i.v. Free or liposomal TL-2, as shown in Table IV, was administered i.p. according to the schedule described above. Results are shown in Table IV.

TABLE IV As the data shows, administration of liposomal drug alone was somewhat beneficial, but only the group receiving the combined liposomal treatment showed significant recovery from tumors. In this group (group 7), it was also observed that the tumors were significantly smaller than in the other groups.

IV. Adimnistration For use in humans, a therapeutically effective dose of the composition typically corresponds to 20-100 mg adriamycin m2 of body surface. For IL-2, a preferred dose corresponds to 50,000 -500,000 CU per square meter of body surface. Administration may be by intraperitoneal (ip), subcutaneous (sc), intravenous (iv), intraarterial (ia), or intramuscular (im) injection. Liposomes in the form of large multilamellar vesicles (MLV's) are preferred for intraperitoneal, subcutaneous or intramuscular administration, while SUV's are preferred for intravenous as well as intramuscular administration.

As shown above, administration of liposome-encapsulated chemotherapeutic drug is followed by administration of the liposome-encapsulated cytokine. While specific time intervals and courses of treatment have been shown in the examples above, it is understood that dosages, time intervals between courses, and the number of courses of treatment, for both drug and cytokine, may be varied depending on the extent of symptoms and the condition of the patient.

While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications ma be made without departing from the invention. 12 136343/2

Claims (26)

1. Use of an immunostimulating cytokine for the preparation of a pharmaceutical composition for the treatment of a subject in need of antitumor therapy at least three days after treatment with a chemotherapeutic drug, wherein said preparation comprises encapsulating the cytokine in multi-lamellar liposomes (MLV).

2. Use of an immunostimulating cytokine encapsulated in multilamellar liposomes (MLV) for the preparation of a pharmaceutical composition for treatment of a subject in need of antitumor therapy at least three days after administration of a chemotherapeutic drug to said patient.

3. Use of an irnmi ostimulating cytokine encapsulated in multilamellar liposomes (MLV) for the preparation of a pharmaceutical composition for treatment of a cancer patient in combination with a chemotherapeutic drug from the group consisting of anthraquinones, platin complex and topoisomerase I inhibitors excluding camptothecin.

4. Use according to any one of claims 1 to 3, wherein said immunostimulating cytokine is selected from interleukin-2 (IL-2). IL-12, IL-15, IL-18, EFN-γ, IFN-a, IFN-β, TNF-a, G-CSF, and GM-CSF.

5. Use according to Claim 4, wherein the cytokine is IL-2.

6. Use according to any one of Claims 1 to 5, wherein the cytokine is encapsulated in liposomes comprising at least one lipid selected from the group consisting of dimyristoyl phosphatidyl choline (DMPC), dimyristoyl phosphatidyl glycerol (DMPG), l,2distearoyl trimethylammonium propane (DSTAP), phosphatidyl choline, phosphatidyl ethanolamine, and cholesterol.

7. Use according to Claim 6, wherein the cytokine is encapsulated in liposomes comprising dimyristoyl phosphatidyl choline (DMPC) in combination with 0-50 mole percent of at least one lipid selected from dimyristoyl phosphatidyl glycerol (DMPG) and 1,2-distearoyl triinethylammonium propane (DSTAP). 01253061\18-02 13 136343/2

8. Use according to Claim 7, wherein the liposome is composed of DMPC and DMPG.

9. Use according to Claim 8, wherein said liposome is composed of DMPC and DMPG in a molar ratio of about 9:1.

10. Use according to any one of Claims 3 to 9, wherein said anthraquinone is selected from doxorubicin, epirubicin, daunorubicin and mitoxanthrone.

11. Use according to any one of Claims 3 to 9, wherein said platin complex is cis-platin.

12. Use according to any one of Claims 3 to 9, wherein said topoisomerase I inhibitor is selected from topotecan and irinotecan.

13. Use according to any one of the preceding claims wherein said chemotherapeutic drug is encapsulated in a liposome.

14. Use according to claim 13, wherein said chemotherapeutic drug is a polyethylene glycol coated liposomal doxorubicin.

15. A pharmaceutical product comprising an immunostimulating cytokine encapsulated in multi-lamellar liposomes (MLV) and a chemotherapeutic drug, as a combined preparation for separate or sequential administration in antitumor therapy.

16. The pharmaceutical product of Claim 15, wherein said immunostimulating cytokine is selected from interleukin-2 (IL-2). IL-12, IL-15, IL-18, IFN-γ, IFN-a, IFN-β, TNF-a, G-CSF, and GM-CSF.

17. The pharmaceutical product of Claim 16, wherein the cytokine is IL-2.

18. The pharmaceutical product of any one of Claims 15 to 17, wherein the cytokine is encapsulated in liposomes comprising at least one lipid selected from the group consisting of dimyristoyl phosphatidyl choline (DMPC), dimyristoyl phosphatidyl glycerol (DMPG), l,2distearoyl trimethylammonium propane (DSTAP), phosphatidyl choline, phosphatidyl ethanolamine, and cholesterol. 01253061U 8-02 14 136343/2

19. The pharmaceutical product of Claim 18, wherein the cytokine is encapsulated in liposomes comprising dimyristoyl phosphatidyl choline (DMPC) in combination with 0-50 mole percent of at least one lipid selected from dimyristoyl phosphatidyl glycerol (DMPG) and 1 ,2-distearoyl triinethylammonium propane (DSTAP).

20. The pharmaceutical product of Claim 19, wherein the liposome is composed of DMPC and DMPG.

21. The pharmaceutical product of Claim 20, wherein said liposome is composed of DMPC and DMPG in a molar ratio of about 9: 1.

22. The pharmaceutical product of any one of Claims 15 to 20, wherein said chemotherapeutic drug is encapsulated in liposomes.

23. The pharmaceutical product of any one of Claims 15 to 22, wherein said chemotherapeutic drug is selected from the group consisting of anthraquinones, platin complex and topoisomerase I inhibitors excluding camptothecin.

24. The pharmaceutical product of Claim 23, wherein said anthraquinone is selected from doxorubicin, epirubicin, daunorubicin and mitoxanthrone.

25. The pharmaceutical product of Claim 23, wherein said platin complex is cis-platin.

26. The pharmaceutical product of Claim 23, wherein said topoisomerase I inhibitor is selected from topotecan and irinotecan. For the Applicants REI RS