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Title:
PROCESS FOR THE PREPARATION OF DOXORUBICIN LIPOSOMES
Document Type and Number:
WIPO Patent Application WO/2010/092590
Kind Code:
A2
Abstract:
The present invention discloses a novel process of preparation of doxorubicin liposomal suspension having entrapment efficiency greater than or equal to 95 %.

Inventors:
BHOWMICK SUBHAS BALARAM (IN)
NAMDEO ALOK B (IN)
NATARAJAN JAYAGANESH (IN)
JAIN PANKAJ (IN)
Application Number:
PCT/IN2009/000339
Publication Date:
August 19, 2010
Filing Date:
June 11, 2009
Export Citation:
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Assignee:
SUN PHARMA ADVANCED RES CO LTD (IN)
BHOWMICK SUBHAS BALARAM (IN)
NAMDEO ALOK B (IN)
NATARAJAN JAYAGANESH (IN)
JAIN PANKAJ (IN)
Foreign References:
US20050129750A12005-06-16
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Claims:
Claims:

1. A process of preparation of doxorubicin liposomal suspension having entrapment efficiency greater than or equal to 95%, said process comprising: a), heating and dissolving Hydrogenated Soy phosphatidylcholine (HSPC), cholesterol and N-(Carbonyl- methoxypolyethylene glycol 2000)-l,2-distearyl-.sn-glycero-3- phosphoethanolamine (mPEG-2000- DSPE) in alcohol at a temperature of 500C to 750C and injecting said solution through an orifice of the nozzle of an injectable system into an aqueous solution of ammonium sulphate maintained at a temperature of 500C to 750C; b). passing the suspension formed in step (a) through a polycarbonate membrane having a pore diameter ranging from about 50 nm to about 200nm, while maintaining the temperature of suspension at 500C to 750C; c) subjecting the extruded suspension obtained in step (b) to dialysis using 10%w/v sucrose solution as an exchange buffer till the external ion concentration is less than or about lOOppm d) adding doxorubicin hydrochloride and histidine to the suspension and maintaining the temperature at 600C to 750C for about half hour to two hours; e) cooling the suspension to a temperature less than 150C; f) adjusting the pH with a buffer or an alkali or an acid or a combination thereof and making up the volume with 10% sucrose solution; g) sterilizing the doxorubicin liposomal suspension by passing through a filter. 2. A process of preparation of doxorubicin liposomal suspension as claimed in claim 1, wherein the doxorubicin liposome suspension has an average mean size of about lOOnm as measured by dynamic light scattering instrument.

3. A process of preparation of doxorubicin liposomal suspension as claimed in claim 1, wherein the doxorubicin liposomes have a polydispersity index of less than 0.1 as measured by dynamic light scattering instrument.

Description:
PROCESS FOR THE PREPRATION OF LIPOSOMES

The present invention relates to liposomes and more particularly to a process for the preparation of liposomes of an antitumor active agent with high entrapment efficiency.

Liposomes are colloidal, vesicular structures composed of lipid bilayers, which enclose part of the surrounding solvent into their interior. The size of a liposome ranges from 20 nm up to several micrometers and they may be composed of one or several concentric membranes. The liposome can be of unilamellar vesicle (having a single bilayer) or multilamellar vesicles (having multiple bilayers). The bilayer is composed of two lipid monolayers having a hydrophobic "tail region" and a hydrophilic "head" region". The structure of the membrane bilayer is such that the hydrophobic "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "head" orient towards the aqueous phase.

Liposomes have been investigated over the past few decades as a system for the delivery or targeting of therapeutic agents to specific sites in the body. Drugs encapsulated in liposomes are relatively successful in reaching primary tumors and their metastases after intravenous injection to humans, and show greater therapeutic efficacy and reduced side effects than non-liposomal drugs. One of the most commonly known liposome formulations for cancer treatment is available under the brand name DOXIL ® (Doxorubicin HCl liposome injection), marketed by ALZA in United States of America.

The initial methods for producing liposomes have been described by Bangham, et al. (J. MoI. Bio., 1965, 13:238-252). The method involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipids film on the vessel. Next, an aqueous phase is added, the mixture is allowed to "swell", and the resulting liposomes, which consist of multilamellar vesicles (MLVs), are dispersed by mechanicaj means to obtain small unilamellar vesicles (SUVs).

Alternative methods have been developed for preparing liposomes which have shown to have improved properties such as, for example higher drug entrapping efficiency and loadability, better stability and greater ease of production and are described in U.S.Patent No.4,522,803 (the '803 patent). The '803 patent discloses new and substantially improved type of lipid vesicles .which hereinafter will be referred to as stable plurilamellar vesicles (SPLVs). Aside from being structurally different than multilamellar vesicles (MLVs), SPLVs are also prepared differently than MLVs, possess unique properties when compared to MLVs, and present a variety of different advantages when compared to such MLVs. A general review of various methods for producing liposomes is described in Deamer and Uster, "Liposome Preparation: Methods and Mechanisms", Liposomes, edited by M.Ostro, pp.27-51 (1983), incorporated herein by reference.

Free or unentrapped drug present in the liposomal suspension poses safety and toxicity issues in the liposomal formulations when administered to humans and is of great concern. The free or unentrapped drug present in the formulations will act on non-tumor cells because of differences in pharmacokinetic parameters of the free drug upon administration which leads to severe toxicity and side effects which are mostly undesired. Hence precautions have been taken and several methods have been developed to remove free or unentrapped drug from the liposomal suspension. Some of the commonly employed methods for removing free drug are described herein below:

United States Patent No. 5,192,549 (Yissum Research, 1993) discloses simple, efficient, safe, economical fast transmembrane loading of weak amphiphatic drugs into liposomes using transmembrane gradient. The amount of unentrapped drug present in the liposomal suspension after the drug loading was removed by absorption on a dowex 5OW column.

United States Patent No.6,083,530 (Liposome company, 2000) discloses a method for encapsulation of antineoplastic agents in liposomes having a high drug to lipid ratio. The amount of unentrapped drug was removed by passing the suspension over Sephadex G-50 column equilibrated in buffered saline.

United States Patent No.6,355,268 (Alza Corporation, 2002) discloses a composition for administration of a therapeutically effective dose of a topoisomerase inhibitor I or topoisomerase II inhibitor. The unencapsulated drug was removed from the liposomal suspension by using a hollow fiber tangential flow diafiltration unit.

Thus, the currently known methods for producing liposomes has several disadvantages. These methods employ an additional step of removal of free drug in the final product which is uneconomical as well as time consuming.

It would, therefore, be a significant advance in the art to provide an economically viable process for preparing doxorubicin liposomes having high entrapment efficiency without the need for an additional step of removal of free or unentrapped drug. SUMMARY OF THE INVENTION

The present invention can be summarized as given below:

A). A process of preparation of doxorubicin liposomal suspension having entrapment efficiency greater than or equal to 95%, said process comprising: a), heating and dissolving Hydrogenated Soy phosphatidylcholine (HSPC), cholesterol and N-(Carbonyl- methoxypolyethylene glycol 2000)-l,2-distearyl-5n-glycero-3- phosphoethanolamine (mPEG-2000-

DSPE) in alcohol at a temperature of 50 0 C to 75 0 C and injecting said solution through an orifice of the nozzle of an injectable system into an aqueous solution of ammonium sulphate maintained at a temperature of 50 0 C to 75 0 C ; b). passing the suspension formed in step (a) through a polycarbonate membrane having a pore diameter ranging from about 50 nm to about 200nm, while maintaining the temperature of suspension at 50 0 C to

75 0 C; c) subjecting the extruded suspension obtained in step (b) to dialysis using 10%w/v sucrose solution as an exchange buffer till the external ion concentration is less than or about lOOppm d) adding doxorubicin hydrochloride and histidine to the suspension and maintaining the temperature at

60 0 C to 75 0 C for about half hour to two hours; e) cooling the suspension to a temperature less than 15 0 C; f) adjusting the pH with a buffer or an alkali or an acid or a combination thereof and making up the volume with 10% sucrose solution; g) sterilizing the doxorubicin liposomal suspension by passing through a filter.

B. A process of preparation of doxorubicin liposomal suspension as in A above, wherein the doxorubicin liposome suspension has an average mean size of about lOOnm as measured by dynamic light scattering instrument.

C. A process of preparation of doxorubicin liposomal suspension as in A above, wherein the doxorubicin liposomes have a polydispersity index of less than 0.1 as measured by dynamic light scattering instrument.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process of preparation of doxorubicin liposomal suspension having an entrapment of efficiency greater than or equal to 95%, said process comprising: a). Heating and dissolving Hydrogenated Soy phosphatidylcholine (HSPC), cholesterol and N-(Carbonyl- methoxypolyethylene glycol 2000)-l,2-distearyl-.?«-glycero-3- phosphoethanolamine (mPEG-2000- DSPE) in alcohol at a temperature of 50 0 C to 75 0 C and injecting said solution through an orifice of the nozzle of an injectable system into an aqueous solution of ammonium sulphate maintained at a temperature of 50 0 C to 75 0 C ; b). Passing the suspension formed in step (a) through a polycarbonate membranes having a pore diameter ranging from about 50 nm to about 200nm, while maintaining the temperature of the suspension at 50 0 C to 75 0 C; c) Subjecting the extruded suspension obtained in step (b) to dialysis using 10%w/v sucrose solution as an exchange buffer till the external ion concentration is less than or about 100 ppm d) Adding doxorubicin hydrochloride and histidine to the suspension and maintaining the temperature at 60 0 C to 75 0 C for about half hour to two hours; e) Cooling the suspension to a temperature less than 15 0 C; f) Adjusting the pH with a buffer or an alkali or an acid or a combination thereof and making up the volume with 10% sucrose solution; g) Sterilizing the doxorubicin liposomal suspension by passing through a filter.

The term "polydispersity" or "polydispersity index (PDI)" as used herein, is a dimension less size- distribution measure relates to an estimate of the width of the distribution. It is a measure of dispersion homogeneity, which ranges from 0 (homogeneous dispersion) to 1 (high heterogenicity). Generally, the polydispersity calculation is performed automatically by a microprocessor associated with a particle-size analyzer, for example, a Zeta sizer (Malvern Instruments, Herrenberg, Germany).

The term "entrapment efficiency" refers to the percentage of drug encapsulated into the interior cavity of liposome or associated with lipid bilayer membrane of the liposome. The percentage (%) drug entrapped into the liposome is calculated by using the formula:

%Drug encapsulated = (% Assay of the drug content) - (% Free drug content)

The alcohol solvent used in the step (a) in the process of the present invention is selected from the group consisting of fatty alcohol, glycol, methanol, ethanol, isopropanol, ethylene glycol, propylene glycol and mixtures thereof. In a preferred embodiment the alcohol solvent is ethanol and is used in a concentration so as to obtain a lipid mixture concentration of about 150 mg/ml to about 300mg/ml.

In an embodiment, the hydrogenated soy phsophatidylcholine (HSPC) is present in a concentration ranging from about 8 mg/ml to about 11 mg/ml, N-(Carbonyl-methoxypolyethylene glycol 200O)-1, 2- distearyl-.sH-glycero-3-phosphoethanolamine (mPEG-2000-DSPE) is present in a concentration ranging from about 2 mg/ml to 5 mg/ml and Cholesterol is present in a concentration ranging from about 2mg/ml to about 5 mg/ml, in the liposome suspension composition.

In the process of the present invention, the lipid mixture comprising HSPC, cholesterol and mPEG-2000- DSPE are dissolved in ethanol to obtain a lipid mixture. In a preferred embodiment, the process is carried out at a temperature of about 5O 0 C to about 75 0 C, more preferably, carried out at a temperature of 55 0 C to about 70 0 C.

In the process of the present invention, the lipid mixture is added to the aqueous ammonium sulfate buffer system using solvent injection method. The design of the injection system can be of various types including single nozzle multiple holes, multiple nozzles or multiple needle assembly. The diameter of orifice of the nozzle may be selected and the spray rate of the lipid mixture is controlled so as to produce liposomes of a desired size. Preferably, the process of injecting lipid mixture into the aqueous ammonium sulfate solution is carried out at a temperature of about 5O 0 C to about 75 0 C, more preferably, at a temperature of 55 0 C to about 70 0 C.

The ammonium sulfate solution is present in a concentration ranging from about 25mg/ml to about 35mg/ml. The ammonium sulfate solution is used to create a proton gradient across the liposome membrane for remote loading of the amphiphatic drug into the liposomes as described, for example, in U.S.Pat.No.5, 192,549 and U.S.Pat. No.5,316,771 both assigned to Yissum Research Development Company, Israel. Here the liposomes are prepared in an aqueous buffer containing an ammonium salt, typically 0.1 to 0.3M ammonium salt, such as ammonium salt, at a suitable pH, e.g., 5.5 to 7.5. The gradient can also be produced by using sulfated polymers, such as dextran ammonium sulfate or heparin sulfate. After liposome formation and sizing, the external medium is exchanged for one lacking ammonium ions, e.g., the same buffer but one in which ammonium sulfate is replaced by NaCl or sucrose that gives the same osmolarity inside and outside of the liposome.

In the process of the present invention, the liposome suspension obtained from step (b) is subjected to an extrusion process to reduce the size and to obtain a liposome having a mean average particle size of less than 400nm, preferably, having a liposome particle size distribution in the range from about 50 nm to about 300nm and more preferably, having a mean average particle size in the range 80nm to about 120 nm.

The extruder system for the extrusion process of the present invention preferably uses a polycarbonate filtration membrane. The extruder system contains a polycarbonate filtration membrane having a filter of pore diameter in the range about 0.05μm to about 0.2 μm.

In an embodiment of the invention, the polycarbonate filters are stacked serially. The number of filters that can be present may vary from two to six. Most preferably, four polycarbonate filters, each having a pore diameter of 0.080 μm are stacked one over the other and the liposomal suspension is passed through the extruder.

In another embodiment, the liposomal suspension is extruded through a series of filters having a decreasing average pore diameter. The liposomal suspension is sequentially passed through 0.2μm, O.lμm and 0.08μm filters or O.lμm and 0.08μm filters, passing through each pore size filter for a specific number of times.

In an embodiment, the extrusion process according to the present invention comprises, passing the mixture obtained in step (b) through the filtration membranes having a polycarbonate filters, each filter having a pore diameter of 0.2μm for one time, O.lμm for one tome and 0.08μm for three times, operated at a pressure of about 100 psi to about 1000 psi, more preferably, at a pressure of about 200 psi to about

800psi. The process of extrusion is repeated till the mean average particle diameter of liposomes is in the range from about 50 nm to about 300nm. The extrusion process of step (c) is carried out at a temperature of about 5O 0 C to about 75 0 C, preferably carried out at a temperature of 55 0 C to about 70 0 C.

In another embodiment, the extrusion process according to the present invention comprises, passing the mixture obtained in step (b) through the filtration membranes having a polycarbonate filters, each filter having a pore diameter of O.lμm for one time and 0.08μm for six times, operated at a pressure of about 100 psi to about 1000 psi, more preferably, at a pressure of about 200 psi to about 800psi. The process of extrusion is repeated till the mean average particle diameter of liposomes is in the range from about 50 nm to about 300nm. The extrusion process of step (c) is carried out at a temperature of about 5O 0 C to about 75 0 C, preferably carried out at a temperature of 55 0 C to about 70 0 C. After the size reduction using extrusion process, the external medium of the liposomes is treated to produce an ion gradient across the liposome membrane, which is typically lower inside and higher outside concentration gradient. In the process of the present invention, the liposomal suspension obtained in the step (c) was subjected the process of dialysis. The dialysis is carried out to remove ammonium sulfate ions present in the liposomal suspension using a nonionic sucrose solution as the exchange buffer and to create an ion gradient across the liposome membrane.

The term "exchange buffer" as defined herein, means a buffer which exchanges for ammonium ions present in the external medium of liposome suspension and that which provides osmolarity inside and outside of the liposome. Preferably, sodium chloride or sucrose solution can be used as the exchange buffer; more preferably, sucrose solution is used as the exchange buffer and may be present in a concentration ranging from about 7%w/v to about 12%w/v. More preferably, the sucrose solution is present in a concentration about 10% w/v.

The concentration of external ions present in the liposomal suspension after the step of dialysis was found be an important parameter for the entrapment efficiency of doxorubicin in the liposomes. The concentration of the external ion concentration was varied and the effect of the external ion was studied with respect to the amount of drug encapsulated into the liposomes. It was surprisingly found that as the amount of external ion concentration had a significant effect on the entrapment efficiency of the doxorubicin in to the liposome.

In a preferred embodiment, the dialysis was carried out till an external ion concentration was less than or about 100 parts per million (ppm). More preferably, the external ammonium ion concentration was kept less than 50ppm.

In a preferred embodiment, the dialysis is carried out a lower shear rate and lower transmembrane pressure so as to prevent the increase in particle size. The mean particle size of the liposome obtained was less than lOOnm, preferably in the range between 80 nm to lOOnm.

The liposomes obtained after the process of dialysis were subjected to loading of doxorubicin hydrochloride into the liposomes. The doxorubicin used in the compositions of the present invention may be used as a base or its pharmaceutically acceptable salt. The pharmaceutically acceptable salt of doxorubicin may be selected from the group comprising hydrochloride, hydrobromide, sulfate, phosphate, nitrate, acetate, succinate, tartarate, ascorbate, citrate, glutamate, benzoate, methanesulfonate, ethanesulfonate and the like. More preferably, the doxorubicin hydrochloride is used in the present invention. The doxorubicin hydrochloride is present in a concentration ranging from about 0.1 mg/ml to about 50mg/ml, preferably about 1 mg/ml to about 40mg/ml.

In an embodiment, the doxorubicin hydrochloride solution and the liposome suspension are mixed at a temperature of about 45 0 C to about 75 0 C, more preferably, at a temperature of about 50 0 C to about 7O 0 C and most preferably at a temperature of about 60 0 C to about 65 0 C. The doxorubicin hydrochloride solution and the liposome suspension are mixed under a constant stirring speed for about half hour to about two hours to facilitate the drug loading. At this temperature, the lipid bilayer of the liposome is loose and allows the doxorubicin hydrochloride to enter the liposome particles. The temperature of the drug encapsulated liposome suspension is then decreased to a temperature below 15 0 C, preferably to a temperature of about 2 0 C to 8 0 C. At these temperature conditions, the lipid bilayer of the liposome becomes dense such that the drug is stably encapsulated in the liposome particles in the liposome.

In a preferred embodiment, the process of preparation of doxorubicin liposomal suspension of the present invention produces doxorubicin liposomes with entrapment efficiency greater than 95%, more preferably greater than 98% and thus avoids the need for removing the unencapsulated drug from the external medium.

In a preferred embodiment, the doxorubicin liposomes do not use any additional steps or methods to remove the unentrapped doxorubicin from the liposomal suspension.

The doxorubicin encapsulated liposomes of the present invention may be suspended in a vehicle containing pharmaceutically acceptable additives suitable for keeping the product stable on storage for prolonged periods of time and suitable for administration to humans. For example, the pharmaceutically acceptable additives are a cryoprotectant, stabilizer, buffers, antioxidant, a pH regulator, a dispersing agent or mixtures thereof and are well known in the art.

In an embodiment of the invention, the pH of the doxorubicin liposomal suspension is adjusted with a buffer or an alkali or an acid or a combination thereof. Preferably, histidine is used as a buffering agent, sodium hydroxide as an alkali and hydrochloric acid as the acid in the process of the present invention. The doxorubicin liposome pH was adjusted to about 6.3 to about 7.4; more preferably adjusted to a pH about 6.5 to about 7.0. The liposome suspension was subjected to the process of sterile filtration after the step of drug loading and pH adjustment before filling to containers for storage. The sterile filtration can be carried out using filters known in the art of size 0.2μm.

The liposome suspension obtained after sterile filtration are subjected to particle size analysis for measuring mean particle size and polydispersity of the suspension using Zeta sizer. The liposome are then stored in a glass vials and aseptically sealed with nitrogen purging.

In an embodiment of the invention, liposomes produced by the process of the present invention, after the step of sterile filtration, have a particle size less than lOOnm and a polydispersity index of less than 0.1 when measured by dynamic light scattering using a Zeta sizer (Malvern Instruments, Herrenberg, Germany).

It will be understood by those skilled in the art that numerous and various modifications can be made without departing from the spirit of the present invention. Therefore, it should be clearly understood that the following examples are illustrative only and are not to be construed to limitthe scope of the present invention.

EXAMPLE 1

Preparation of doxorubicin liposomal suspension TABLE 1

Process of Manufacture:

HSPC, Cholesterol and mPEG-2000-DSPE were weighed and taken in a schott-duran glass bottle. Ethanol was slowly added to the mixture of lipids in the glass bottle with constant stirring while maintaining the temperature of mixture at 65 0 C. Aqueous ammonium sulfate solution was prepared in a glass container with water for Injection. The lipid mixture was slowly injected into the ammonium sulfate solution with stirring. The liposome suspension formed was stirred for 30 minutes while maintaining the temperature of the suspension at 65 0 C.

(a) Extrusion: The liposomal suspension was then subjected to extrusion process through a thermobarrel extruder. The extrusion process comprised of passing the suspension through a 0.2μm (1 time), 0.1 μm (1 time) and 0.08μm (3 times) pore size polycarbonate membrane filter

(Whatman) sequentially, at a pressure range of 350 psi to 850 psi. After each pass, the liposomal size was measured using dynamic light scattering technique using Zeta sizer ( Malvern Instruments, Herrenberg, Germany) and the process was repeated till a particle size in the range 80 nm to lOOnm was obtained.

(b) Dialysis: The liposomal suspension was subjected to dialysis with 10%w/v sucrose solution as the exchange buffer and to remove' external phase ammonium sulfate ions. The process of dialysis was continued till the external ion concentration was less than or about 100 ppm.

(c) Drug loading: Doxorubicin hydrochloride was dissolved in a glass container with 10%sucrose solution so as to maintain a doxorubicin HCl concentration of 30mg/ml in the solution. The solution was maintained at a temperature of 60 0 C. A sufficient quantity of histidine was added to the drug solution and then transferred to the vessel containing liposomal suspension obtained after dialysis process. The suspension was then maintained at a temperature of 60 0 C for about an hour to facilitate drug loading into the liposomes. The liposomes were then cooled to a temperature of 2 0 C to 8 0 C and incubated for an hour. The volume was made up with 10% sucrose solution and histidine solution was added to and the pH was adjusted to 6.65 using NaOH/HCl.

(d) Sterile Filtration: The doxorubicin loaded liposomes were sterile filtered using a 0.2μm syringe filter. The liposome suspension is then transferred to glass vials under nitrogen purging for storage. " "

EXAMPLE 2

Doxorubicin liposomes prepared as per the process of Example 1 were measured for liposome size at different stages of manufacturing using Zeta sizer (Malvern Instruments, Herrenberg, Germany). The results of the study are given in Table 2.

TABLE 2

EXAMPLE 3

Effect of external ion concentration on the free drug content

During the process of the dialysis in the process of Example 1, a sample of 50 ml was withdrawn at different external ion concentration level of permeate i.e., at 25, 50, 100, 500 and 5000ppm. The samples having different external ion concentrations were then subjected to doxorubicin loading and the final samples were subjected to % assay, % entrapment and %free drug content by using High Performance Liquid Chromatography (HPLC). The results obtained are shown in Table 3.

EXAMPLE 4

Pharmacokinetic study of Doxorubicin liposomes in tumor bearing Balb/c mice

The pharmacokinetic study of doxorubicin liposomes prepared as per the process of example 1 was carried out on Balb/c mice. Ten mice were taken for the pharmacokinetic evaluation study in each test. A single dose of 5.0mg/kg was administered intravenously (i.v.) to each mice in the test and blood samples were collected at an interval of 0.083, 0.50, 1.0, 4.0, 8.0, 24.0, 48.0, 96.0, 168.0 and 240.0 hr following i.v administration. The doxorubicin concentration in plasma was analyzed using HPLC-Mass Spectroscopy (MS)/MS. The results obtained are shown in Table 4.

Table 4

EXAMPLE 5 In-vivo efficacy study of Doxorubicin Liposomes in syngenic fibrosarcoma bearing

Balb/c mice

BALB/c mice (13 mice per treatment group, 15 placebo-treated mice) received a four weekly intravenous administrations of 4.5 mL/kg , for a total of 9 mg/kg of doxorubicin liposome per treatment on Days 0, 7, 14, and 21. Animals were observed daily for mortality. On Days 7, 14, 21, and 28, tumor volume and body weight of each animal were measured. The results obtained are shown in Table 5

Table 5

Negative value indicates tumor regression and change in body weight from day 0.

These results clearly demonstrate highly significant anti tumor activity for doxorubicin liposomes manufactured as per process of the present invention.