Title of Invention	PACLITAXEL LIPOSOME COMPOSITION FOR TREATMENT OF CANCER AND PREPARATION THEREOF
Abstract	The present invention relates to a paclitaxel-based liposome composition for treatment of cancer, which consists substantially of the following materials by weight: paclitaxel 2-5 parts, phosphatide 20-200 parts, cholesterol 2-30 parts, amino acide 0.3-4 parts, and lyophilized excipient 10-27 parts. The products according to the present invention do not contain polyoxyethlated castor oil, and obtained adjuvant, and can be actualized e in industry. they have the advantages of low toxicity good tolerance for patient, good water solubility and better stability.

Title of Invention

PACLITAXEL LIPOSOME COMPOSITION FOR TREATMENT OF CANCER AND PREPARATION THEREOF

Abstract

The present invention relates to a paclitaxel-based liposome composition for treatment of cancer, which consists substantially of the following materials by weight: paclitaxel 2-5 parts, phosphatide 20-200 parts, cholesterol 2-30 parts, amino acide 0.3-4 parts, and lyophilized excipient 10-27 parts. The products according to the present invention do not contain polyoxyethlated castor oil, and obtained adjuvant, and can be actualized e in industry. they have the advantages of low toxicity good tolerance for patient, good water solubility and better stability.

Full Text	FORM-2 THE PATENTS ACT, 1970 COMPLETE SPECIFICATION [Section 10] 1. "Paclitaxel, Liposome Composition For Treatment of Cancer and Preparation. Thereof" 2. (a) NANJING ZHENZHONG BIOENGEVEERING COMPANY LTD (b) NANJING NEW@HIGH TECHNOLOGY INDUSTRY DEVELOPMENT ZONE, Jiangsu 210061, P. R CHINA. (c) P. R. CHINA The following specification particularly describes the nature of the invention and the manner in which it is to be performed. 1 The present invention relates to a pharmaceutical composition for treatment of cancer, especially relates to a paclitaxel liposome composition for treatment of cancer and method of preparation thereof. Description of the Related Art Paclitaxel is an anticarcinogen found by the National Cancer Institute, USA through screening thousands of plants in the 1960s. It is generally used for treatment of oophoroma, breast carcinoma and non-small-cell lung carcinoma. Because paclitaxel is difficult to be dissolved in water and many officinal solvents, all of the paclitaxel injections provided presently in the domestic and overseas market were prepared with the complex dissolvent of polyoxyethylated castor oil and anhydrous ethanol. The polyoxyethylated castor oil in the complex dissolvent will result in the release of histamine when degraded in body, and thus may cause serious hyperallergic reaction/pleoergy. A paclitaxel injection (trade name is Taxol) is registered in China by Bristol-Myers Squibb Company, USA, and it was pointed out in the specification that "all patients who receive the Taxol should be given in advance corticosteroid (such as dexamethasone), diphenhydramine and H2 receptor antagonist (such as cimitidine, ranitidine) in order to prevent the occurrence of serious hyperallergic reaction." And it also said that "precipitation (crystal) may appear when diluted with physiological saline or 5% glucose before injection. So it is necessary to be filtrated with 0.22 n m millipore filter to assure the safety in administration." Also paclitaxel itself has haematics toxicity, bone marrow depression, leukopenia, thrombocytopenia, anemia and other toxicities. Therefore, change of paclitaxel"s dosage form are demanded urgently to increase its solubility, improve its stability and clinical compliance, and avoid the allergic reaction caused by the above complex dissolvent and other toxic side effect. Practicable progress was achieved from the basic researches of paclitaxel liposome in the early 1990s and the recent preclinical study. The experiments indicated that the paclitaxel liposome preparation had similar 2 therapeutic effectiveness with reduced toxicity and improved tolerance compared to the paclitaxel injection preparation. SharmaA reported a paclitaxel liposome (code name TTL) made from three synthetic phosphatides (Int. J. Cancer 1997, 71, 103-107) with better stability than the paclitaxel liposome (code name ETL) made from single lecithin. But it did not say how much the difference of stability was between the two preparations, and only mentioned that the ETL preparation maintained its stability for 24 hours after dissolved in water and that the solvent used was benzene or butanol. Benzene has much toxicity when used as solvent. In US patent No. 5,415,869 (1995), it was mentioned that aggregation occurred in the paclitaxel liposome preparation made from single lecithin. Thus electronegative or electropositive ingredient such as phosphatidyl glycerol (PG) was introduced. The ratio of lecithin to PG adopted by the patent was 9:1 or 3:7 while chloroform was used as solvent, the paclitaxel liposome did not aggregation. But when the ratio was 5:5 the paclitaxel liposome crystal precipitated. PG is difficult to obtain due to its low content in lecithin. And there are only expensive products of PG of reagent grade overseas. Also chloroform has too much toxicity when used as solvent. SUMMARY OF THE PRESENT INVENTION One object of the present invention is to provide a paclitaxel liposome composition for treatment of cancer without polyoxyethylated castor oil. The second object of the present invention is to provide a paclitaxel liposome composition for treatment of cancer, which has good colliquefaction with the water soluble transfusion and can be infused directly into 5% glucose solution for intravenous drip after shaking, and has reduced toxicity and can avoid the hyperallergic reaction caused by the complex dissolvent and has improved stability and relatively lower cost. The third object of the present invention is to provide a method of preparation of paclitaxel liposome composition for treatment of cancer without polyoxyethylated castor oil. The fourth object of the present invention is to provide a method of preparation of paclitaxel liposome composition for treatment of cancer with 3 improved water solubility and stability using liposome imbedding technique. These and other objects will be further described and represented through the following detailed illustration. DETAILED DESCRIPTION OF THE INVENTION In the present invention, the paclitaxel liposome composition for treatment of cancer consists substantially of the following materials by weight: Paclitaxel 2-5 parts, Phosphatide 20-200 parts, Cholesterol 2-30 parts, Amino acids 0.3-4 parts, Lyophilized excipient 10-75 parts. Furthermore, the paclitaxel liposome composition for treatment of cancer of the present invention consists substantially of the following materials by weight: Paclitaxel 3-5 parts, Phosphatide 40-160 parts, Cholesterol 5-25 parts, Amino acids 0.8-3 parts, Lyophilized excipient 20-65 parts. The paclitaxel liposome composition for treatment of cancer of the present invention may also consist substantially of the following materials by weight: Paclitaxel 3-5 parts, Phosphatide 60-120 parts, Cholesterol 8-20 parts, Amino acids 1.0-2 parts, Lyophilized excipient 30-60 parts. The paclitaxel liposome composition for treatment of cancer of the present invention does not contain polyoxyethylated castor oil. Furthermore, in the paclitaxel liposome composition for treatment of cancer of the present invention, the said lyophilized excipient is mannitol, sucrose, glucose, or lactose which can be solid suited for injection. The said amino acid can be lysine, threonine or methionine. The said phosphatide is egg yolk lecithin or soy bean lecithin for injection. If necessary, the composition of the present invention can also be added 4 solution. Then the solution is placed in a constant temperature water bath with a temperature of 50-60 °C. After the solvent is removed with a rotatory evaporator under reduced pressure, a membrane is formed therefrom. The aqueous solution of amino acids and lyophilized excipient dissolved in the above ratios is introduced. And then hydration, sonication or homogenization is conducted to attain the liposome size 0.1-5 u m. After sterilization filtration, the gain is subpackaged into containers such as ampoules or vials etc., and lyophilized to obtain white lumpy paclitaxel liposome. Nitrogen, helium or argon gas can be aerated when sealing or capping. The paclitaxel liposome composition for treatment of cancer of the present invention can also be prepared by the following method, and includes substantially the following materials by weight: Paclitaxel 3-5 parts, Phosphatide 60-120 parts, Cholesterol 8-20 parts, Amino acids 1.0-2 parts, Lyophilized excipient 30-60 parts. Paclitaxel, phosphatide, cholesterol are agitated and dissolved successively in isopropanol or ethanol in the above ratios to obtain a clear solution. Then the solution is placed in a constant temperature water bath with a temperature of 50-60 °C. After the solvent is removed with a rotatory evaporator under reduced pressure, a membrane is formed therefrom. The aqueous solution of amino acids and lyophilized excipient dissolved in the above ratios is introduced. And then hydration, sonication or homogenization is conducted to attain the liposome size above 0.1 P m. After sterilization filtration, the gain is subpackaged into containers such as ampoules or vials etc., and lyophilized to obtain white lumpy paclitaxel liposome. Nitrogen, helium or argon gas can be aerated when sealing or capping. In the above method, the said lyophilized excipient is mannitol, sucrose, glucose, or lactose. The said amino acid can be lysine, threonine or methionine. The said phosphatide is egg yolk lecithin or soy bean lecithin for injection. 6 The products of the present invention do not contain polyoxyethylated castor oil and substitute the toxic dissolvent and expensive adjuvant with nontoxic dissolvent and easily obtained adjuvant, and can be executed in industry scale. So they have advantages of low toxicity, good water solubility and better stability, etc. They can be infused directly into 5% glucose solution for intravenous drip after shaking and avoid the hyperallergic reaction caused by the complex dissolvent, and have relatively lower cost. The paclitaxel liposome preparation prepared according to the present invention has similar anti-cancer effectiveness to the commercial paclitaxel injection, as shown in table I. Table 1. The inhibitory effect of paclitaxel liposome for injection and commercial paclitaxel injection on S-180 Group Dosag e (mg/k 8) Number of animals Tumor weight (g) Inhibition ratio (%) Before administrat ion After administrat ion Paclitaxel liposome Paclitaxel liposome Paclitaxel liposome Paclitaxel injection Paclitaxel injection Control 20.0 14.0 9.8 14.0 9.8 10 10 10 10 10 10 10 10 10 10 9 10 0.826+ 0.204 0.896+ 0.293 1.114 + 0.425 0.860+ 0.177 1.180 + 0.299 1.612 + 0.705 48.79 44.44 30.89 46.65 26.80 7 P In the present invention, the single lecithin together with cholesterol and ammo acids is used as the stabilizing agent. Amino acids are amphoteric material, which can show electrification at given pHs such as in the present technical scheme. So this will prevent aggregation and precipitations of taxol crystal. The method uses easily obtained material so that the cost is low compared to the reported methods, in which electronegative ingredient such as PG or electropositive ingredient were added. And the obtained paclitaxel liposome has better stability than that only with single lecithin. Aggregation does not occur and crystal does not precipitate in the obtained paclitaxel liposome when standing after dissolved with water (table 2). Table 2. Data of paclitaxel liposome"s stability at low temperature (2-10°C) Lot number Time (month) Appearance Microscopic examination (1X1600) No. 1 0 Loose offwhite lump No crystal after dissolved, no aggregation 1 Loose offwhite lump No crystal after dissolved, no aggregation 6 Loose offwhite lump No crystal after dissolved, no aggregation 12 Loose offwhite lump No crystal after dissolved, no aggregation No. 2 0 Loose offwhite lump No crystal after dissolved, no aggregation 1 Loose offwhite lump No crystal after dissolved, no aggregation 6 Loose offwhite lump No crystal after dissolved, no aggregation 12 Loose offwhite lump No crystal after dissolved, no aggregation 8 The pachtaxel liposome preparation according to the present invention has better colliquefaction with the glucose for injection and physiological saline. The content of paclitaxel in the obtained preparation is 4-6 mg/ml, which is a suitable concentration for clinical administration and can be used in clinical therapy with the same amount as the commercial paclitaxel injection. According to body surface area, the dosage is 175 mg/m2 intravenous drip. In the present invention, all materials and adjuvants can be purchased in the market. The present invention will be illustrated further with several examples. But these examples are used for illustration of the present invention, and do not limit the scope of the present invention. If not being pointed out specially, all parts or measurements are weight units based on the total weight. Example 1 In aseptic condition, paclitaxel for injection (2.5g), refined egg yolk lecithin for injection (30g) and cholesterol (2.7g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 300ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (50 °C) to form a membrane. 5% mannitol solution containing 2.8g lysine was added to dissolve the membrane, and a sonifier was used for ultrasonic pulverization. After being filtrated with 0.22 U m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 25mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 2 In aseptic condition, paclitaxel for injection (5.0g), refined soy bean lecithin for injection (72g) and cholesterol (6g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 800ml) was added 9 to make the mixture dissolved completely as clean solution lnen and pressure drying was conducted with a rotary evaporator in a constant temperature water bath (50°C) to form a membrane. 5% mannitol solution containing 3.4g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 30mg pachtaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 3 In aseptic condition, paclitaxel for injection (2.5g), refined soy bean lecithin for injection (30g) and cholesterol (2.7g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 400ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (50°C) to form a membrane. 7.5% sucrose solution containing 2.0g methionine was added to dissolve the membrane, and a sonifier was used for ultrasonic pulverization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 4 In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (72g) and cholesterol (6g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (50°C) to form a membrane. 7.5% mannitol solution t containing 3.5g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 10 0.22 P m filter membrane to degerm, the solution was subpackaged into ampoules (OR vials) so that 30mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 5 In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (65g) and cholesterol (6.5g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (50°C) to form a membrane. 7.5% lactose solution containing 3.8g lysine was added to dissolve the membrane, and a high"pressure refiner was used for homogenization. After beingfiltrated with 0.22 p m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 6 In aseptic condition, paclitaxel for injection (2.0g), refined egg yolk lecithin for injection (42g) and cholesterol (4g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 260ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (60 °C) to form a membrane. 5% mannitol solution (about 400ml) containing 2.0g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. 11 Example 7 In aseptic condition, paclitaxel for injection (2.8g), refined egg yolk lecithin for injection (60g) and cholesterol (4g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 350ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (55 °C) to form a membrane. 5% mannitol solution (about 300ml) containing 2.4g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 U m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 8 In aseptic condition, paclitaxel for injection (3.5g), refined soy bean lecithin for injection (lOOg) and cholesterol (4.5g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 1000ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (58 °C) to form a membrane. 5% sucrose solution (about 500ml) containing 3.1g methionine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 n m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 9 In aseptic condition, paclitaxel for injection (4.5g), refined soy bean lecithin for injection (122g) and cholesterol (8g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant 12 temperature water bath (52 °C) to form a membrane. 5% mannitol solution (about 1000ml) containing 3.1g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 10 In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (lOOg) and cholesterol (15g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (53 °C) to form a membrane. 5% mannitol solution (about 1000ml) containing 3.8g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 11 In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (120g) and cholesterol (21g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (59 °C) to form a membrane. 5% glucose solution (about 1400ml) containing 2.4g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 V- m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 30mg paclitaxel was contained in each bottle. 13 Lyophilization was conducted followed by sealing in men gases 10 obtain while lumpy paclitaxel liposome preparation. Example 12 In aseptic condition, paclitaxel for injection (4.0g), refined egg yolk lecithin for injection (120g) and cholesterol (18g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (52°C) to form a membrane. 5% lactose solution (about 1000ml) containing 3.0g methionine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 25mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 13 In aseptic condition, paclitaxel for injection (5.0g), soy bean lecithin (200g) and cholesterol (30g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 2000ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (57 °C) to form a membrane. 5% glucose solution (about 1400ml) containing 4.0g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 14 In aseptic condition, paclitaxel for injection (3.0g), refined egg yolk lecithin for injection (80g) and cholesterol (5.0g) were introduced into a 14 round-bottomed flask. And proner amount of isopropano! (about 700ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (55 °C) to form a membrane. 5% mannitol solution (about 1000ml) containing 2.4g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 15mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. 15 We Claim: 1. A paclitaxel liposome composition for treatment of cancer, comprising Paclitaxel 2-5 parts by weight, "Phosphatide 20-200 parts by weight, Cholesterol 2-30 parts by weight, Amino acids 0.3-4 parts by weight, Lyophilized excipient 10-75 parts by weight. 2. The paclitaxel liposome composition for treatment of cancer according to Claim 1, comprising Paclitaxel 3-5 parts by weight, Phosphatide 40-160 parts by weight, Cholesterol 5-25 parts by weight, Amino acids 6.8-3 parts by weight, Lyophilized excipient 20-65 parts by weight. 3. The paclitaxel liposome composition for treatment of cancer according to Claim 1, comprising Paclitaxel 3-5 parts by weight, Phosphatide 60-120 parts by weight, Cholesterol 8-20 parts by weight, Amino acids 1.0-2 parts by weight, Lyophilized excipient 30-60 parts by weight. 4. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 3, wherein the composition does not contain polyoxyethylated castor oil. 5. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said lyophilized excipient is solid mannitol, sucrose, glucose, or lactose for injection. 6. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said amino acid can be lysine, threonine or methionine. 7. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said phosphatide is egg yolk lecithin or soy bean lecithin for injection. Dated this 17* Day of April 2003 Dr. Rajeshkumar H. Acharya Advocate & Patent Agent For and on Behalf of Applicant 16 FORM-2 THE PATENTS ACT, 1970 COMPLETE SPECIFICATION [Section 10] 1. "Paclitaxel, Liposome Composition For Treatment of Cancer and Preparation. Thereof" 2. (a) NANJING ZHENZHONG BIOENGEVEERING COMPANY LTD (b) NANJING NEW@HIGH TECHNOLOGY INDUSTRY DEVELOPMENT ZONE, Jiangsu 210061, P. R CHINA. (c) P. R. CHINA The following specification particularly describes the nature of the invention and the manner in which it is to be performed. 1 The present invention relates to a pharmaceutical composition for treatment of cancer, especially relates to a paclitaxel liposome composition for treatment of cancer and method of preparation thereof. Description of the Related Art Paclitaxel is an anticarcinogen found by the National Cancer Institute, USA through screening thousands of plants in the 1960s. It is generally used for treatment of oophoroma, breast carcinoma and non-small-cell lung carcinoma. Because paclitaxel is difficult to be dissolved in water and many officinal solvents, all of the paclitaxel injections provided presently in the domestic and overseas market were prepared with the complex dissolvent of polyoxyethylated castor oil and anhydrous ethanol. The polyoxyethylated castor oil in the complex dissolvent will result in the release of histamine when degraded in body, and thus may cause serious hyperallergic reaction/pleoergy. A paclitaxel injection (trade name is Taxol) is registered in China by Bristol-Myers Squibb Company, USA, and it was pointed out in the specification that "all patients who receive the Taxol should be given in advance corticosteroid (such as dexamethasone), diphenhydramine and H2 receptor antagonist (such as cimitidine, ranitidine) in order to prevent the occurrence of serious hyperallergic reaction." And it also said that "precipitation (crystal) may appear when diluted with physiological saline or 5% glucose before injection. So it is necessary to be filtrated with 0.22 n m millipore filter to assure the safety in administration." Also paclitaxel itself has haematics toxicity, bone marrow depression, leukopenia, thrombocytopenia, anemia and other toxicities. Therefore, change of paclitaxel"s dosage form are demanded urgently to increase its solubility, improve its stability and clinical compliance, and avoid the allergic reaction caused by the above complex dissolvent and other toxic side effect. Practicable progress was achieved from the basic researches of paclitaxel liposome in the early 1990s and the recent preclinical study. The experiments indicated that the paclitaxel liposome preparation had similar 2 therapeutic effectiveness with reduced toxicity and improved tolerance compared to the paclitaxel injection preparation. SharmaA reported a paclitaxel liposome (code name TTL) made from three synthetic phosphatides (Int. J. Cancer 1997, 71, 103-107) with better stability than the paclitaxel liposome (code name ETL) made from single lecithin. But it did not say how much the difference of stability was between the two preparations, and only mentioned that the ETL preparation maintained its stability for 24 hours after dissolved in water and that the solvent used was benzene or butanol. Benzene has much toxicity when used as solvent. In US patent No. 5,415,869 (1995), it was mentioned that aggregation occurred in the paclitaxel liposome preparation made from single lecithin. Thus electronegative or electropositive ingredient such as phosphatidyl glycerol (PG) was introduced. The ratio of lecithin to PG adopted by the patent was 9:1 or 3:7 while chloroform was used as solvent, the paclitaxel liposome did not aggregation. But when the ratio was 5:5 the paclitaxel liposome crystal precipitated. PG is difficult to obtain due to its low content in lecithin. And there are only expensive products of PG of reagent grade overseas. Also chloroform has too much toxicity when used as solvent. SUMMARY OF THE PRESENT INVENTION One object of the present invention is to provide a paclitaxel liposome composition for treatment of cancer without polyoxyethylated castor oil. The second object of the present invention is to provide a paclitaxel liposome composition for treatment of cancer, which has good colliquefaction with the water soluble transfusion and can be infused directly into 5% glucose solution for intravenous drip after shaking, and has reduced toxicity and can avoid the hyperallergic reaction caused by the complex dissolvent and has improved stability and relatively lower cost. The third object of the present invention is to provide a method of preparation of paclitaxel liposome composition for treatment of cancer without polyoxyethylated castor oil. The fourth object of the present invention is to provide a method of preparation of paclitaxel liposome composition for treatment of cancer with 3 improved water solubility and stability using liposome imbedding technique. These and other objects will be further described and represented through the following detailed illustration. DETAILED DESCRIPTION OF THE INVENTION In the present invention, the paclitaxel liposome composition for treatment of cancer consists substantially of the following materials by weight: Paclitaxel 2-5 parts, Phosphatide 20-200 parts, Cholesterol 2-30 parts, Amino acids 0.3-4 parts, Lyophilized excipient 10-75 parts. Furthermore, the paclitaxel liposome composition for treatment of cancer of the present invention consists substantially of the following materials by weight: Paclitaxel 3-5 parts, Phosphatide 40-160 parts, Cholesterol 5-25 parts, Amino acids 0.8-3 parts, Lyophilized excipient 20-65 parts. The paclitaxel liposome composition for treatment of cancer of the present invention may also consist substantially of the following materials by weight: Paclitaxel 3-5 parts, Phosphatide 60-120 parts, Cholesterol 8-20 parts, Amino acids 1.0-2 parts, Lyophilized excipient 30-60 parts. The paclitaxel liposome composition for treatment of cancer of the present invention does not contain polyoxyethylated castor oil. Furthermore, in the paclitaxel liposome composition for treatment of cancer of the present invention, the said lyophilized excipient is mannitol, sucrose, glucose, or lactose which can be solid suited for injection. The said amino acid can be lysine, threonine or methionine. The said phosphatide is egg yolk lecithin or soy bean lecithin for injection. If necessary, the composition of the present invention can also be added 4 solution. Then the solution is placed in a constant temperature water bath with a temperature of 50-60 °C. After the solvent is removed with a rotatory evaporator under reduced pressure, a membrane is formed therefrom. The aqueous solution of amino acids and lyophilized excipient dissolved in the above ratios is introduced. And then hydration, sonication or homogenization is conducted to attain the liposome size 0.1-5 u m. After sterilization filtration, the gain is subpackaged into containers such as ampoules or vials etc., and lyophilized to obtain white lumpy paclitaxel liposome. Nitrogen, helium or argon gas can be aerated when sealing or capping. The paclitaxel liposome composition for treatment of cancer of the present invention can also be prepared by the following method, and includes substantially the following materials by weight: Paclitaxel 3-5 parts, Phosphatide 60-120 parts, Cholesterol 8-20 parts, Amino acids 1.0-2 parts, Lyophilized excipient 30-60 parts. Paclitaxel, phosphatide, cholesterol are agitated and dissolved successively in isopropanol or ethanol in the above ratios to obtain a clear solution. Then the solution is placed in a constant temperature water bath with a temperature of 50-60 °C. After the solvent is removed with a rotatory evaporator under reduced pressure, a membrane is formed therefrom. The aqueous solution of amino acids and lyophilized excipient dissolved in the above ratios is introduced. And then hydration, sonication or homogenization is conducted to attain the liposome size above 0.1 P m. After sterilization filtration, the gain is subpackaged into containers such as ampoules or vials etc., and lyophilized to obtain white lumpy paclitaxel liposome. Nitrogen, helium or argon gas can be aerated when sealing or capping. In the above method, the said lyophilized excipient is mannitol, sucrose, glucose, or lactose. The said amino acid can be lysine, threonine or methionine. The said phosphatide is egg yolk lecithin or soy bean lecithin for injection. 6 The products of the present invention do not contain polyoxyethylated castor oil and substitute the toxic dissolvent and expensive adjuvant with nontoxic dissolvent and easily obtained adjuvant, and can be executed in industry scale. So they have advantages of low toxicity, good water solubility and better stability, etc. They can be infused directly into 5% glucose solution for intravenous drip after shaking and avoid the hyperallergic reaction caused by the complex dissolvent, and have relatively lower cost. The paclitaxel liposome preparation prepared according to the present invention has similar anti-cancer effectiveness to the commercial paclitaxel injection, as shown in table I. Table 1. The inhibitory effect of paclitaxel liposome for injection and commercial paclitaxel injection on S-180 Group Dosag e (mg/k 8) Number of animals Tumor weight (g) Inhibition ratio (%) Before administrat ion After administrat ion Paclitaxel liposome Paclitaxel liposome Paclitaxel liposome Paclitaxel injection Paclitaxel injection Control 20.0 14.0 9.8 14.0 9.8 10 10 10 10 10 10 10 10 10 10 9 10 0.826+ 0.204 0.896+ 0.293 1.114 + 0.425 0.860+ 0.177 1.180 + 0.299 1.612 + 0.705 48.79 44.44 30.89 46.65 26.80 7 P In the present invention, the single lecithin together with cholesterol and ammo acids is used as the stabilizing agent. Amino acids are amphoteric material, which can show electrification at given pHs such as in the present technical scheme. So this will prevent aggregation and precipitations of taxol crystal. The method uses easily obtained material so that the cost is low compared to the reported methods, in which electronegative ingredient such as PG or electropositive ingredient were added. And the obtained paclitaxel liposome has better stability than that only with single lecithin. Aggregation does not occur and crystal does not precipitate in the obtained paclitaxel liposome when standing after dissolved with water (table 2). Table 2. Data of paclitaxel liposome"s stability at low temperature (2-10°C) Lot number Time (month) Appearance Microscopic examination (1X1600) No. 1 0 Loose offwhite lump No crystal after dissolved, no aggregation 1 Loose offwhite lump No crystal after dissolved, no aggregation 6 Loose offwhite lump No crystal after dissolved, no aggregation 12 Loose offwhite lump No crystal after dissolved, no aggregation No. 2 0 Loose offwhite lump No crystal after dissolved, no aggregation 1 Loose offwhite lump No crystal after dissolved, no aggregation 6 Loose offwhite lump No crystal after dissolved, no aggregation 12 Loose offwhite lump No crystal after dissolved, no aggregation 8 The pachtaxel liposome preparation according to the present invention has better colliquefaction with the glucose for injection and physiological saline. The content of paclitaxel in the obtained preparation is 4-6 mg/ml, which is a suitable concentration for clinical administration and can be used in clinical therapy with the same amount as the commercial paclitaxel injection. According to body surface area, the dosage is 175 mg/m2 intravenous drip. In the present invention, all materials and adjuvants can be purchased in the market. The present invention will be illustrated further with several examples. But these examples are used for illustration of the present invention, and do not limit the scope of the present invention. If not being pointed out specially, all parts or measurements are weight units based on the total weight. Example 1 In aseptic condition, paclitaxel for injection (2.5g), refined egg yolk lecithin for injection (30g) and cholesterol (2.7g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 300ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (50 °C) to form a membrane. 5% mannitol solution containing 2.8g lysine was added to dissolve the membrane, and a sonifier was used for ultrasonic pulverization. After being filtrated with 0.22 U m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 25mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 2 In aseptic condition, paclitaxel for injection (5.0g), refined soy bean lecithin for injection (72g) and cholesterol (6g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 800ml) was added 9 to make the mixture dissolved completely as clean solution lnen and pressure drying was conducted with a rotary evaporator in a constant temperature water bath (50°C) to form a membrane. 5% mannitol solution containing 3.4g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 30mg pachtaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 3 In aseptic condition, paclitaxel for injection (2.5g), refined soy bean lecithin for injection (30g) and cholesterol (2.7g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 400ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (50°C) to form a membrane. 7.5% sucrose solution containing 2.0g methionine was added to dissolve the membrane, and a sonifier was used for ultrasonic pulverization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 4 In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (72g) and cholesterol (6g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (50°C) to form a membrane. 7.5% mannitol solution t containing 3.5g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 10 0.22 P m filter membrane to degerm, the solution was subpackaged into ampoules (OR vials) so that 30mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 5 In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (65g) and cholesterol (6.5g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (50°C) to form a membrane. 7.5% lactose solution containing 3.8g lysine was added to dissolve the membrane, and a high"pressure refiner was used for homogenization. After beingfiltrated with 0.22 p m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 6 In aseptic condition, paclitaxel for injection (2.0g), refined egg yolk lecithin for injection (42g) and cholesterol (4g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 260ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (60 °C) to form a membrane. 5% mannitol solution (about 400ml) containing 2.0g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. 11 Example 7 In aseptic condition, paclitaxel for injection (2.8g), refined egg yolk lecithin for injection (60g) and cholesterol (4g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 350ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (55 °C) to form a membrane. 5% mannitol solution (about 300ml) containing 2.4g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 U m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 8 In aseptic condition, paclitaxel for injection (3.5g), refined soy bean lecithin for injection (lOOg) and cholesterol (4.5g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 1000ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (58 °C) to form a membrane. 5% sucrose solution (about 500ml) containing 3.1g methionine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 n m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 9 In aseptic condition, paclitaxel for injection (4.5g), refined soy bean lecithin for injection (122g) and cholesterol (8g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant 12 temperature water bath (52 °C) to form a membrane. 5% mannitol solution (about 1000ml) containing 3.1g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 10 In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (lOOg) and cholesterol (15g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (53 °C) to form a membrane. 5% mannitol solution (about 1000ml) containing 3.8g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 11 In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (120g) and cholesterol (21g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (59 °C) to form a membrane. 5% glucose solution (about 1400ml) containing 2.4g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 V- m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 30mg paclitaxel was contained in each bottle. 13 Lyophilization was conducted followed by sealing in men gases 10 obtain while lumpy paclitaxel liposome preparation. Example 12 In aseptic condition, paclitaxel for injection (4.0g), refined egg yolk lecithin for injection (120g) and cholesterol (18g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (52°C) to form a membrane. 5% lactose solution (about 1000ml) containing 3.0g methionine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 25mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 13 In aseptic condition, paclitaxel for injection (5.0g), soy bean lecithin (200g) and cholesterol (30g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 2000ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (57 °C) to form a membrane. 5% glucose solution (about 1400ml) containing 4.0g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. Example 14 In aseptic condition, paclitaxel for injection (3.0g), refined egg yolk lecithin for injection (80g) and cholesterol (5.0g) were introduced into a 14 round-bottomed flask. And proner amount of isopropano! (about 700ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water bath (55 °C) to form a membrane. 5% mannitol solution (about 1000ml) containing 2.4g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 15mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation. 15 We Claim: 1. A paclitaxel liposome composition for treatment of cancer, comprising Paclitaxel 2-5 parts by weight, "Phosphatide 20-200 parts by weight, Cholesterol 2-30 parts by weight, Amino acids 0.3-4 parts by weight, Lyophilized excipient 10-75 parts by weight. 2. The paclitaxel liposome composition for treatment of cancer according to Claim 1, comprising Paclitaxel 3-5 parts by weight, Phosphatide 40-160 parts by weight, Cholesterol 5-25 parts by weight, Amino acids 6.8-3 parts by weight, Lyophilized excipient 20-65 parts by weight. 3. The paclitaxel liposome composition for treatment of cancer according to Claim 1, comprising Paclitaxel 3-5 parts by weight, Phosphatide 60-120 parts by weight, Cholesterol 8-20 parts by weight, Amino acids 1.0-2 parts by weight, Lyophilized excipient 30-60 parts by weight. 4. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 3, wherein the composition does not contain polyoxyethylated castor oil. 5. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said lyophilized excipient is solid mannitol, sucrose, glucose, or lactose for injection. 6. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said amino acid can be lysine, threonine or methionine. 7. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said phosphatide is egg yolk lecithin or soy bean lecithin for injection. Dated this 17* Day of April 2003 Dr. Rajeshkumar H. Acharya Advocate & Patent Agent For and on Behalf of Applicant 16

Full Text

FORM-2
THE PATENTS ACT, 1970
COMPLETE SPECIFICATION
[Section 10]
1. "Paclitaxel, Liposome Composition For Treatment of Cancer
and Preparation. Thereof"
2. (a) NANJING ZHENZHONG BIOENGEVEERING COMPANY LTD
(b) NANJING NEW@HIGH TECHNOLOGY INDUSTRY DEVELOPMENT ZONE,
Jiangsu 210061, P. R CHINA.
(c) P. R. CHINA
The following specification particularly describes the nature of the
invention and the manner in which it is to be performed.
1

The present invention relates to a pharmaceutical composition for treatment of cancer, especially relates to a paclitaxel liposome composition for treatment of cancer and method of preparation thereof.
Description of the Related Art
Paclitaxel is an anticarcinogen found by the National Cancer Institute, USA through screening thousands of plants in the 1960s. It is generally used for treatment of oophoroma, breast carcinoma and non-small-cell lung carcinoma. Because paclitaxel is difficult to be dissolved in water and many officinal solvents, all of the paclitaxel injections provided presently in the domestic and overseas market were prepared with the complex dissolvent of polyoxyethylated castor oil and anhydrous ethanol. The polyoxyethylated castor oil in the complex dissolvent will result in the release of histamine when degraded in body, and thus may cause serious hyperallergic reaction/pleoergy.
A paclitaxel injection (trade name is Taxol) is registered in China by Bristol-Myers Squibb Company, USA, and it was pointed out in the specification that "all patients who receive the Taxol should be given in advance corticosteroid (such as dexamethasone), diphenhydramine and H2 receptor antagonist (such as cimitidine, ranitidine) in order to prevent the occurrence of serious hyperallergic reaction." And it also said that "precipitation (crystal) may appear when diluted with physiological saline or
5% glucose before injection. So it is necessary to be filtrated with 0.22 n m
millipore filter to assure the safety in administration." Also paclitaxel itself has haematics toxicity, bone marrow depression, leukopenia, thrombocytopenia, anemia and other toxicities. Therefore, change of paclitaxel"s dosage form are demanded urgently to increase its solubility, improve its stability and clinical compliance, and avoid the allergic reaction caused by the above complex dissolvent and other toxic side effect.
Practicable progress was achieved from the basic researches of paclitaxel liposome in the early 1990s and the recent preclinical study. The experiments indicated that the paclitaxel liposome preparation had similar
2

therapeutic effectiveness with reduced toxicity and improved tolerance compared to the paclitaxel injection preparation. SharmaA reported a paclitaxel liposome (code name TTL) made from three synthetic phosphatides (Int. J. Cancer 1997, 71, 103-107) with better stability than the paclitaxel liposome (code name ETL) made from single lecithin. But it did not say how much the difference of stability was between the two preparations, and only mentioned that the ETL preparation maintained its stability for 24 hours after dissolved in water and that the solvent used was benzene or butanol. Benzene has much toxicity when used as solvent.
In US patent No. 5,415,869 (1995), it was mentioned that aggregation occurred in the paclitaxel liposome preparation made from single lecithin. Thus electronegative or electropositive ingredient such as phosphatidyl glycerol (PG) was introduced. The ratio of lecithin to PG adopted by the patent was 9:1 or 3:7 while chloroform was used as solvent, the paclitaxel liposome did not aggregation. But when the ratio was 5:5 the paclitaxel liposome crystal precipitated. PG is difficult to obtain due to its low content in lecithin. And there are only expensive products of PG of reagent grade overseas. Also chloroform has too much toxicity when used as solvent.
SUMMARY OF THE PRESENT INVENTION
One object of the present invention is to provide a paclitaxel liposome composition for treatment of cancer without polyoxyethylated castor oil.
The second object of the present invention is to provide a paclitaxel liposome composition for treatment of cancer, which has good colliquefaction with the water soluble transfusion and can be infused directly into 5% glucose solution for intravenous drip after shaking, and has reduced toxicity and can avoid the hyperallergic reaction caused by the complex dissolvent and has improved stability and relatively lower cost.
The third object of the present invention is to provide a method of preparation of paclitaxel liposome composition for treatment of cancer without polyoxyethylated castor oil.
The fourth object of the present invention is to provide a method of preparation of paclitaxel liposome composition for treatment of cancer with
3

improved water solubility and stability using liposome imbedding technique.
These and other objects will be further described and represented through the following detailed illustration.
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the paclitaxel liposome composition for treatment of cancer consists substantially of the following materials by weight:
Paclitaxel 2-5 parts, Phosphatide 20-200 parts, Cholesterol 2-30 parts,
Amino acids 0.3-4 parts, Lyophilized excipient 10-75 parts.
Furthermore, the paclitaxel liposome composition for treatment of cancer of the present invention consists substantially of the following materials by weight:
Paclitaxel 3-5 parts, Phosphatide 40-160 parts, Cholesterol 5-25 parts,
Amino acids 0.8-3 parts, Lyophilized excipient 20-65 parts.
The paclitaxel liposome composition for treatment of cancer of the present invention may also consist substantially of the following materials by weight:
Paclitaxel 3-5 parts, Phosphatide 60-120 parts, Cholesterol 8-20 parts,
Amino acids 1.0-2 parts, Lyophilized excipient 30-60 parts.
The paclitaxel liposome composition for treatment of cancer of the present invention does not contain polyoxyethylated castor oil.
Furthermore, in the paclitaxel liposome composition for treatment of cancer of the present invention, the said lyophilized excipient is mannitol, sucrose, glucose, or lactose which can be solid suited for injection. The said amino acid can be lysine, threonine or methionine. The said phosphatide is egg yolk lecithin or soy bean lecithin for injection.
If necessary, the composition of the present invention can also be added
4

solution. Then the solution is placed in a constant temperature water bath with a temperature of 50-60 °C. After the solvent is removed with a rotatory
evaporator under reduced pressure, a membrane is formed therefrom. The aqueous solution of amino acids and lyophilized excipient dissolved in the above ratios is introduced. And then hydration, sonication or homogenization is
conducted to attain the liposome size 0.1-5 u m. After sterilization filtration, the
gain is subpackaged into containers such as ampoules or vials etc., and lyophilized to obtain white lumpy paclitaxel liposome. Nitrogen, helium or argon gas can be aerated when sealing or capping.
The paclitaxel liposome composition for treatment of cancer of the present invention can also be prepared by the following method, and includes substantially the following materials by weight:
Paclitaxel 3-5 parts, Phosphatide 60-120 parts, Cholesterol 8-20 parts,
Amino acids 1.0-2 parts, Lyophilized excipient 30-60 parts.
Paclitaxel, phosphatide, cholesterol are agitated and dissolved successively in isopropanol or ethanol in the above ratios to obtain a clear solution. Then the solution is placed in a constant temperature water bath with
a temperature of 50-60 °C. After the solvent is removed with a rotatory
evaporator under reduced pressure, a membrane is formed therefrom. The aqueous solution of amino acids and lyophilized excipient dissolved in the above ratios is introduced. And then hydration, sonication or homogenization is
conducted to attain the liposome size above 0.1 P m. After sterilization
filtration, the gain is subpackaged into containers such as ampoules or vials etc., and lyophilized to obtain white lumpy paclitaxel liposome. Nitrogen, helium or argon gas can be aerated when sealing or capping.
In the above method, the said lyophilized excipient is mannitol, sucrose, glucose, or lactose. The said amino acid can be lysine, threonine or methionine. The said phosphatide is egg yolk lecithin or soy bean lecithin for injection.
6

The products of the present invention do not contain polyoxyethylated castor oil and substitute the toxic dissolvent and expensive adjuvant with nontoxic dissolvent and easily obtained adjuvant, and can be executed in industry scale. So they have advantages of low toxicity, good water solubility and better stability, etc. They can be infused directly into 5% glucose solution for intravenous drip after shaking and avoid the hyperallergic reaction caused by the complex dissolvent, and have relatively lower cost.
The paclitaxel liposome preparation prepared according to the present invention has similar anti-cancer effectiveness to the commercial paclitaxel injection, as shown in table I.
Table 1. The inhibitory effect of paclitaxel liposome for injection
and commercial paclitaxel injection on S-180
Group Dosag
e (mg/k
8) Number of animals Tumor
weight
(g) Inhibition ratio (%)
Before
administrat
ion After
administrat
ion
Paclitaxel
liposome
Paclitaxel
liposome
Paclitaxel
liposome
Paclitaxel
injection
Paclitaxel
injection
Control 20.0
14.0
9.8
14.0
9.8 10 10 10 10 10 10 10
10
10
10
9
10 0.826+
0.204** 0.896+
0.293** 1.114 +
0.425 0.860+
0.177** 1.180 +
0.299 1.612 +
0.705 48.79 44.44 30.89 46.65 26.80
7

** P In the present invention, the single lecithin together with cholesterol and ammo acids is used as the stabilizing agent. Amino acids are amphoteric material, which can show electrification at given pHs such as in the present technical scheme. So this will prevent aggregation and precipitations of taxol crystal. The method uses easily obtained material so that the cost is low compared to the reported methods, in which electronegative ingredient such as PG or electropositive ingredient were added. And the obtained paclitaxel liposome has better stability than that only with single lecithin. Aggregation does not occur and crystal does not precipitate in the obtained paclitaxel liposome when standing after dissolved with water (table 2).
Table 2. Data of paclitaxel liposome"s stability at low temperature (2-10°C)

Lot number Time (month) Appearance Microscopic examination (1X1600)
No. 1 0 Loose offwhite lump No crystal after dissolved, no aggregation
1 Loose offwhite lump No crystal after dissolved, no aggregation
6 Loose offwhite lump No crystal after dissolved, no aggregation
12 Loose offwhite lump No crystal after dissolved, no aggregation
No. 2 0 Loose offwhite lump No crystal after dissolved, no aggregation
1 Loose offwhite lump No crystal after dissolved, no aggregation
6 Loose offwhite lump No crystal after dissolved, no aggregation
12 Loose offwhite lump No crystal after dissolved, no aggregation
8

The pachtaxel liposome preparation according to the present invention has better colliquefaction with the glucose for injection and physiological saline. The content of paclitaxel in the obtained preparation is 4-6 mg/ml, which is a suitable concentration for clinical administration and can be used in clinical therapy with the same amount as the commercial paclitaxel injection. According to body surface area, the dosage is 175 mg/m2 intravenous drip.
In the present invention, all materials and adjuvants can be purchased in the market.
The present invention will be illustrated further with several examples. But these examples are used for illustration of the present invention, and do not limit the scope of the present invention.
If not being pointed out specially, all parts or measurements are weight units based on the total weight.
Example 1
In aseptic condition, paclitaxel for injection (2.5g), refined egg yolk lecithin for injection (30g) and cholesterol (2.7g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 300ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (50 °C) to form a membrane. 5% mannitol solution
containing 2.8g lysine was added to dissolve the membrane, and a sonifier was used for ultrasonic pulverization. After being filtrated with 0.22 U m filter
membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 25mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 2
In aseptic condition, paclitaxel for injection (5.0g), refined soy bean lecithin for injection (72g) and cholesterol (6g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 800ml) was added
9

to make the mixture dissolved completely as clean solution lnen and pressure drying was conducted with a rotary evaporator in a constant temperature water
bath (50°C) to form a membrane. 5% mannitol solution containing 3.4g lysine
was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm,
the solution was subpackaged into ampoules (or vials) so that 30mg pachtaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 3
In aseptic condition, paclitaxel for injection (2.5g), refined soy bean lecithin for injection (30g) and cholesterol (2.7g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 400ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (50°C) to form a membrane. 7.5% sucrose solution
containing 2.0g methionine was added to dissolve the membrane, and a sonifier was used for ultrasonic pulverization. After being filtrated with 0.22 u m filter
membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 4
In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (72g) and cholesterol (6g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (50°C) to form a membrane. 7.5% mannitol solution
t
containing 3.5g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
10

0.22 P m filter membrane to degerm, the solution was subpackaged into
ampoules (OR vials) so that 30mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 5
In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (65g) and cholesterol (6.5g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (50°C) to form a membrane. 7.5% lactose solution
containing 3.8g lysine was added to dissolve the membrane, and a high"pressure refiner was used for homogenization. After beingfiltrated with
0.22 p m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 6
In aseptic condition, paclitaxel for injection (2.0g), refined egg yolk lecithin for injection (42g) and cholesterol (4g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 260ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water
bath (60 °C) to form a membrane. 5% mannitol solution (about 400ml)
containing 2.0g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
11

Example 7
In aseptic condition, paclitaxel for injection (2.8g), refined egg yolk lecithin for injection (60g) and cholesterol (4g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 350ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water
bath (55 °C) to form a membrane. 5% mannitol solution (about 300ml)
containing 2.4g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 U m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 8
In aseptic condition, paclitaxel for injection (3.5g), refined soy bean lecithin for injection (lOOg) and cholesterol (4.5g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 1000ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (58 °C) to form a membrane. 5% sucrose solution (about
500ml) containing 3.1g methionine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 n m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 9
In aseptic condition, paclitaxel for injection (4.5g), refined soy bean lecithin for injection (122g) and cholesterol (8g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
12

temperature water bath (52 °C) to form a membrane. 5% mannitol solution
(about 1000ml) containing 3.1g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated
with 0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 10
In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (lOOg) and cholesterol (15g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (53 °C) to form a membrane. 5% mannitol solution
(about 1000ml) containing 3.8g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated
with 0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 11
In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (120g) and cholesterol (21g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (59 °C) to form a membrane. 5% glucose solution (about
1400ml) containing 2.4g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 V- m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 30mg paclitaxel was contained in each bottle.
13

Lyophilization was conducted followed by sealing in men gases 10 obtain while lumpy paclitaxel liposome preparation.
Example 12
In aseptic condition, paclitaxel for injection (4.0g), refined egg yolk lecithin for injection (120g) and cholesterol (18g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (52°C) to form a membrane. 5% lactose solution (about
1000ml) containing 3.0g methionine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials) so that 25mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 13
In aseptic condition, paclitaxel for injection (5.0g), soy bean lecithin (200g) and cholesterol (30g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 2000ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was
conducted with a rotary evaporator in a constant temperature water bath (57 °C)
to form a membrane. 5% glucose solution (about 1400ml) containing 4.0g lysine was added to dissolve the membrane, and a high-pressure refiner was
used for homogenization. After being filtrated with 0.22 u m filter membrane to
degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 14
In aseptic condition, paclitaxel for injection (3.0g), refined egg yolk lecithin for injection (80g) and cholesterol (5.0g) were introduced into a
14

round-bottomed flask. And proner amount of isopropano! (about 700ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (55 °C) to form a membrane. 5% mannitol solution
(about 1000ml) containing 2.4g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated
with 0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials) so that 15mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
15

We Claim:
1. A paclitaxel liposome composition for treatment of cancer, comprising Paclitaxel 2-5 parts by weight, "Phosphatide 20-200 parts by weight, Cholesterol 2-30 parts by weight, Amino acids 0.3-4 parts by weight, Lyophilized excipient 10-75 parts by weight.
2. The paclitaxel liposome composition for treatment of cancer according to Claim 1, comprising Paclitaxel 3-5 parts by weight, Phosphatide 40-160 parts by weight, Cholesterol 5-25 parts by weight, Amino acids 6.8-3 parts by weight, Lyophilized excipient 20-65 parts by weight.
3. The paclitaxel liposome composition for treatment of cancer according to Claim 1, comprising Paclitaxel 3-5 parts by weight, Phosphatide 60-120 parts by weight, Cholesterol 8-20 parts by weight, Amino acids 1.0-2 parts by weight, Lyophilized excipient 30-60 parts by weight.
4. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 3, wherein the composition does not contain polyoxyethylated castor oil.
5. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said lyophilized excipient is solid mannitol, sucrose, glucose, or lactose for injection.
6. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said amino acid can be lysine, threonine or methionine.
7. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said phosphatide is egg yolk lecithin or soy bean lecithin for injection.
Dated this 17* Day of April 2003
Dr. Rajeshkumar H. Acharya Advocate & Patent Agent For and on Behalf of Applicant
16

FORM-2
THE PATENTS ACT, 1970
COMPLETE SPECIFICATION
[Section 10]
1. "Paclitaxel, Liposome Composition For Treatment of Cancer
and Preparation. Thereof"
2. (a) NANJING ZHENZHONG BIOENGEVEERING COMPANY LTD
(b) NANJING NEW@HIGH TECHNOLOGY INDUSTRY DEVELOPMENT ZONE,
Jiangsu 210061, P. R CHINA.
(c) P. R. CHINA
The following specification particularly describes the nature of the
invention and the manner in which it is to be performed.
1

The present invention relates to a pharmaceutical composition for treatment of cancer, especially relates to a paclitaxel liposome composition for treatment of cancer and method of preparation thereof.
Description of the Related Art
Paclitaxel is an anticarcinogen found by the National Cancer Institute, USA through screening thousands of plants in the 1960s. It is generally used for treatment of oophoroma, breast carcinoma and non-small-cell lung carcinoma. Because paclitaxel is difficult to be dissolved in water and many officinal solvents, all of the paclitaxel injections provided presently in the domestic and overseas market were prepared with the complex dissolvent of polyoxyethylated castor oil and anhydrous ethanol. The polyoxyethylated castor oil in the complex dissolvent will result in the release of histamine when degraded in body, and thus may cause serious hyperallergic reaction/pleoergy.
A paclitaxel injection (trade name is Taxol) is registered in China by Bristol-Myers Squibb Company, USA, and it was pointed out in the specification that "all patients who receive the Taxol should be given in advance corticosteroid (such as dexamethasone), diphenhydramine and H2 receptor antagonist (such as cimitidine, ranitidine) in order to prevent the occurrence of serious hyperallergic reaction." And it also said that "precipitation (crystal) may appear when diluted with physiological saline or
5% glucose before injection. So it is necessary to be filtrated with 0.22 n m
millipore filter to assure the safety in administration." Also paclitaxel itself has haematics toxicity, bone marrow depression, leukopenia, thrombocytopenia, anemia and other toxicities. Therefore, change of paclitaxel"s dosage form are demanded urgently to increase its solubility, improve its stability and clinical compliance, and avoid the allergic reaction caused by the above complex dissolvent and other toxic side effect.
Practicable progress was achieved from the basic researches of paclitaxel liposome in the early 1990s and the recent preclinical study. The experiments indicated that the paclitaxel liposome preparation had similar
2

therapeutic effectiveness with reduced toxicity and improved tolerance compared to the paclitaxel injection preparation. SharmaA reported a paclitaxel liposome (code name TTL) made from three synthetic phosphatides (Int. J. Cancer 1997, 71, 103-107) with better stability than the paclitaxel liposome (code name ETL) made from single lecithin. But it did not say how much the difference of stability was between the two preparations, and only mentioned that the ETL preparation maintained its stability for 24 hours after dissolved in water and that the solvent used was benzene or butanol. Benzene has much toxicity when used as solvent.
In US patent No. 5,415,869 (1995), it was mentioned that aggregation occurred in the paclitaxel liposome preparation made from single lecithin. Thus electronegative or electropositive ingredient such as phosphatidyl glycerol (PG) was introduced. The ratio of lecithin to PG adopted by the patent was 9:1 or 3:7 while chloroform was used as solvent, the paclitaxel liposome did not aggregation. But when the ratio was 5:5 the paclitaxel liposome crystal precipitated. PG is difficult to obtain due to its low content in lecithin. And there are only expensive products of PG of reagent grade overseas. Also chloroform has too much toxicity when used as solvent.
SUMMARY OF THE PRESENT INVENTION
One object of the present invention is to provide a paclitaxel liposome composition for treatment of cancer without polyoxyethylated castor oil.
The second object of the present invention is to provide a paclitaxel liposome composition for treatment of cancer, which has good colliquefaction with the water soluble transfusion and can be infused directly into 5% glucose solution for intravenous drip after shaking, and has reduced toxicity and can avoid the hyperallergic reaction caused by the complex dissolvent and has improved stability and relatively lower cost.
The third object of the present invention is to provide a method of preparation of paclitaxel liposome composition for treatment of cancer without polyoxyethylated castor oil.
The fourth object of the present invention is to provide a method of preparation of paclitaxel liposome composition for treatment of cancer with
3

improved water solubility and stability using liposome imbedding technique.
These and other objects will be further described and represented through the following detailed illustration.
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the paclitaxel liposome composition for treatment of cancer consists substantially of the following materials by weight:
Paclitaxel 2-5 parts, Phosphatide 20-200 parts, Cholesterol 2-30 parts,
Amino acids 0.3-4 parts, Lyophilized excipient 10-75 parts.
Furthermore, the paclitaxel liposome composition for treatment of cancer of the present invention consists substantially of the following materials by weight:
Paclitaxel 3-5 parts, Phosphatide 40-160 parts, Cholesterol 5-25 parts,
Amino acids 0.8-3 parts, Lyophilized excipient 20-65 parts.
The paclitaxel liposome composition for treatment of cancer of the present invention may also consist substantially of the following materials by weight:
Paclitaxel 3-5 parts, Phosphatide 60-120 parts, Cholesterol 8-20 parts,
Amino acids 1.0-2 parts, Lyophilized excipient 30-60 parts.
The paclitaxel liposome composition for treatment of cancer of the present invention does not contain polyoxyethylated castor oil.
Furthermore, in the paclitaxel liposome composition for treatment of cancer of the present invention, the said lyophilized excipient is mannitol, sucrose, glucose, or lactose which can be solid suited for injection. The said amino acid can be lysine, threonine or methionine. The said phosphatide is egg yolk lecithin or soy bean lecithin for injection.
If necessary, the composition of the present invention can also be added
4

solution. Then the solution is placed in a constant temperature water bath with a temperature of 50-60 °C. After the solvent is removed with a rotatory
evaporator under reduced pressure, a membrane is formed therefrom. The aqueous solution of amino acids and lyophilized excipient dissolved in the above ratios is introduced. And then hydration, sonication or homogenization is
conducted to attain the liposome size 0.1-5 u m. After sterilization filtration, the
gain is subpackaged into containers such as ampoules or vials etc., and lyophilized to obtain white lumpy paclitaxel liposome. Nitrogen, helium or argon gas can be aerated when sealing or capping.
The paclitaxel liposome composition for treatment of cancer of the present invention can also be prepared by the following method, and includes substantially the following materials by weight:
Paclitaxel 3-5 parts, Phosphatide 60-120 parts, Cholesterol 8-20 parts,
Amino acids 1.0-2 parts, Lyophilized excipient 30-60 parts.
Paclitaxel, phosphatide, cholesterol are agitated and dissolved successively in isopropanol or ethanol in the above ratios to obtain a clear solution. Then the solution is placed in a constant temperature water bath with
a temperature of 50-60 °C. After the solvent is removed with a rotatory
evaporator under reduced pressure, a membrane is formed therefrom. The aqueous solution of amino acids and lyophilized excipient dissolved in the above ratios is introduced. And then hydration, sonication or homogenization is
conducted to attain the liposome size above 0.1 P m. After sterilization
filtration, the gain is subpackaged into containers such as ampoules or vials etc., and lyophilized to obtain white lumpy paclitaxel liposome. Nitrogen, helium or argon gas can be aerated when sealing or capping.
In the above method, the said lyophilized excipient is mannitol, sucrose, glucose, or lactose. The said amino acid can be lysine, threonine or methionine. The said phosphatide is egg yolk lecithin or soy bean lecithin for injection.
6

The products of the present invention do not contain polyoxyethylated castor oil and substitute the toxic dissolvent and expensive adjuvant with nontoxic dissolvent and easily obtained adjuvant, and can be executed in industry scale. So they have advantages of low toxicity, good water solubility and better stability, etc. They can be infused directly into 5% glucose solution for intravenous drip after shaking and avoid the hyperallergic reaction caused by the complex dissolvent, and have relatively lower cost.
The paclitaxel liposome preparation prepared according to the present invention has similar anti-cancer effectiveness to the commercial paclitaxel injection, as shown in table I.
Table 1. The inhibitory effect of paclitaxel liposome for injection
and commercial paclitaxel injection on S-180
Group Dosag
e (mg/k
8) Number of animals Tumor
weight
(g) Inhibition ratio (%)
Before
administrat
ion After
administrat
ion
Paclitaxel
liposome
Paclitaxel
liposome
Paclitaxel
liposome
Paclitaxel
injection
Paclitaxel
injection
Control 20.0
14.0
9.8
14.0
9.8 10 10 10 10 10 10 10
10
10
10
9
10 0.826+
0.204** 0.896+
0.293** 1.114 +
0.425 0.860+
0.177** 1.180 +
0.299 1.612 +
0.705 48.79 44.44 30.89 46.65 26.80
7

** P In the present invention, the single lecithin together with cholesterol and ammo acids is used as the stabilizing agent. Amino acids are amphoteric material, which can show electrification at given pHs such as in the present technical scheme. So this will prevent aggregation and precipitations of taxol crystal. The method uses easily obtained material so that the cost is low compared to the reported methods, in which electronegative ingredient such as PG or electropositive ingredient were added. And the obtained paclitaxel liposome has better stability than that only with single lecithin. Aggregation does not occur and crystal does not precipitate in the obtained paclitaxel liposome when standing after dissolved with water (table 2).
Table 2. Data of paclitaxel liposome"s stability at low temperature (2-10°C)

Lot number Time (month) Appearance Microscopic examination (1X1600)
No. 1 0 Loose offwhite lump No crystal after dissolved, no aggregation
1 Loose offwhite lump No crystal after dissolved, no aggregation
6 Loose offwhite lump No crystal after dissolved, no aggregation
12 Loose offwhite lump No crystal after dissolved, no aggregation
No. 2 0 Loose offwhite lump No crystal after dissolved, no aggregation
1 Loose offwhite lump No crystal after dissolved, no aggregation
6 Loose offwhite lump No crystal after dissolved, no aggregation
12 Loose offwhite lump No crystal after dissolved, no aggregation
8

The pachtaxel liposome preparation according to the present invention has better colliquefaction with the glucose for injection and physiological saline. The content of paclitaxel in the obtained preparation is 4-6 mg/ml, which is a suitable concentration for clinical administration and can be used in clinical therapy with the same amount as the commercial paclitaxel injection. According to body surface area, the dosage is 175 mg/m2 intravenous drip.
In the present invention, all materials and adjuvants can be purchased in the market.
The present invention will be illustrated further with several examples. But these examples are used for illustration of the present invention, and do not limit the scope of the present invention.
If not being pointed out specially, all parts or measurements are weight units based on the total weight.
Example 1
In aseptic condition, paclitaxel for injection (2.5g), refined egg yolk lecithin for injection (30g) and cholesterol (2.7g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 300ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (50 °C) to form a membrane. 5% mannitol solution
containing 2.8g lysine was added to dissolve the membrane, and a sonifier was used for ultrasonic pulverization. After being filtrated with 0.22 U m filter
membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 25mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 2
In aseptic condition, paclitaxel for injection (5.0g), refined soy bean lecithin for injection (72g) and cholesterol (6g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 800ml) was added
9

to make the mixture dissolved completely as clean solution lnen and pressure drying was conducted with a rotary evaporator in a constant temperature water
bath (50°C) to form a membrane. 5% mannitol solution containing 3.4g lysine
was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with 0.22 u m filter membrane to degerm,
the solution was subpackaged into ampoules (or vials) so that 30mg pachtaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 3
In aseptic condition, paclitaxel for injection (2.5g), refined soy bean lecithin for injection (30g) and cholesterol (2.7g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 400ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (50°C) to form a membrane. 7.5% sucrose solution
containing 2.0g methionine was added to dissolve the membrane, and a sonifier was used for ultrasonic pulverization. After being filtrated with 0.22 u m filter
membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 4
In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (72g) and cholesterol (6g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (50°C) to form a membrane. 7.5% mannitol solution
t
containing 3.5g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
10

0.22 P m filter membrane to degerm, the solution was subpackaged into
ampoules (OR vials) so that 30mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 5
In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (65g) and cholesterol (6.5g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (50°C) to form a membrane. 7.5% lactose solution
containing 3.8g lysine was added to dissolve the membrane, and a high"pressure refiner was used for homogenization. After beingfiltrated with
0.22 p m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 6
In aseptic condition, paclitaxel for injection (2.0g), refined egg yolk lecithin for injection (42g) and cholesterol (4g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 260ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water
bath (60 °C) to form a membrane. 5% mannitol solution (about 400ml)
containing 2.0g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
11

Example 7
In aseptic condition, paclitaxel for injection (2.8g), refined egg yolk lecithin for injection (60g) and cholesterol (4g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 350ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant temperature water
bath (55 °C) to form a membrane. 5% mannitol solution (about 300ml)
containing 2.4g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 U m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 8
In aseptic condition, paclitaxel for injection (3.5g), refined soy bean lecithin for injection (lOOg) and cholesterol (4.5g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 1000ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (58 °C) to form a membrane. 5% sucrose solution (about
500ml) containing 3.1g methionine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 n m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 9
In aseptic condition, paclitaxel for injection (4.5g), refined soy bean lecithin for injection (122g) and cholesterol (8g) were introduced into a round-bottomed flask. And proper amount of isopropanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
12

temperature water bath (52 °C) to form a membrane. 5% mannitol solution
(about 1000ml) containing 3.1g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated
with 0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 10
In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (lOOg) and cholesterol (15g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (53 °C) to form a membrane. 5% mannitol solution
(about 1000ml) containing 3.8g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated
with 0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials). Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 11
In aseptic condition, paclitaxel for injection (5.0g), refined egg yolk lecithin for injection (120g) and cholesterol (21g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1500ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (59 °C) to form a membrane. 5% glucose solution (about
1400ml) containing 2.4g threonine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 V- m filter membrane to degerm, the solution was subpackaged into ampoules (or vials) so that 30mg paclitaxel was contained in each bottle.
13

Lyophilization was conducted followed by sealing in men gases 10 obtain while lumpy paclitaxel liposome preparation.
Example 12
In aseptic condition, paclitaxel for injection (4.0g), refined egg yolk lecithin for injection (120g) and cholesterol (18g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 1200ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (52°C) to form a membrane. 5% lactose solution (about
1000ml) containing 3.0g methionine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated with
0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials) so that 25mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 13
In aseptic condition, paclitaxel for injection (5.0g), soy bean lecithin (200g) and cholesterol (30g) were introduced into a round-bottomed flask. And proper amount of ethanol (about 2000ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was
conducted with a rotary evaporator in a constant temperature water bath (57 °C)
to form a membrane. 5% glucose solution (about 1400ml) containing 4.0g lysine was added to dissolve the membrane, and a high-pressure refiner was
used for homogenization. After being filtrated with 0.22 u m filter membrane to
degerm, the solution was subpackaged into ampoules (or vials) so that 20mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
Example 14
In aseptic condition, paclitaxel for injection (3.0g), refined egg yolk lecithin for injection (80g) and cholesterol (5.0g) were introduced into a
14

round-bottomed flask. And proner amount of isopropano! (about 700ml) was added to make the mixture dissolved completely as clear solution. Then underpressure drying was conducted with a rotary evaporator in a constant
temperature water bath (55 °C) to form a membrane. 5% mannitol solution
(about 1000ml) containing 2.4g lysine was added to dissolve the membrane, and a high-pressure refiner was used for homogenization. After being filtrated
with 0.22 u m filter membrane to degerm, the solution was subpackaged into
ampoules (or vials) so that 15mg paclitaxel was contained in each bottle. Lyophilization was conducted followed by sealing in inert gases to obtain white lumpy paclitaxel liposome preparation.
15

We Claim:
1. A paclitaxel liposome composition for treatment of cancer, comprising Paclitaxel 2-5 parts by weight, "Phosphatide 20-200 parts by weight, Cholesterol 2-30 parts by weight, Amino acids 0.3-4 parts by weight, Lyophilized excipient 10-75 parts by weight.
2. The paclitaxel liposome composition for treatment of cancer according to Claim 1, comprising Paclitaxel 3-5 parts by weight, Phosphatide 40-160 parts by weight, Cholesterol 5-25 parts by weight, Amino acids 6.8-3 parts by weight, Lyophilized excipient 20-65 parts by weight.
3. The paclitaxel liposome composition for treatment of cancer according to Claim 1, comprising Paclitaxel 3-5 parts by weight, Phosphatide 60-120 parts by weight, Cholesterol 8-20 parts by weight, Amino acids 1.0-2 parts by weight, Lyophilized excipient 30-60 parts by weight.
4. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 3, wherein the composition does not contain polyoxyethylated castor oil.
5. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said lyophilized excipient is solid mannitol, sucrose, glucose, or lactose for injection.
6. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said amino acid can be lysine, threonine or methionine.
7. The paclitaxel liposome composition for treatment of cancer according to Claims 1 to 4, wherein the said phosphatide is egg yolk lecithin or soy bean lecithin for injection.
Dated this 17* Day of April 2003
Dr. Rajeshkumar H. Acharya Advocate & Patent Agent For and on Behalf of Applicant
16

Documents:

422-mumnp-2003-abstract(22-06-2007).doc

422-mumnp-2003-abstract(22-06-2007).pdf

422-mumnp-2003-cancelled pages(15-04-2003).pdf

422-mumnp-2003-claims(granted)-(22-06-2007).doc

422-mumnp-2003-claims(granted)-(22-06-2007).pdf

422-mumnp-2003-corrspondence(22-06-2007).pdf

422-mumnp-2003-corrspondence(ipo)-(20-07-2007).pdf

422-mumnp-2003-form 1(21-04-2003).pdf

422-mumnp-2003-form 18(24-01-2005).pdf

422-mumnp-2003-form 2(granted)-(22-06-2007).doc

422-mumnp-2003-form 2(granted)-(22-06-2007).pdf

422-mumnp-2003-form 26(17-04-2003).pdf

422-mumnp-2003-form 3(17-04-2003).pdf

422-mumnp-2003-form 3(22-06-2007).pdf

422-mumnp-2003-form 5(17-04-2003).pdf

422-mumnp-2003-form 8(03-01-2007).pdf

422-mumnp-2003-form-pct-isa-210(02-08-2001).pdf

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Patent Number

208865

Indian Patent Application Number

422/MUMNP/2003

PG Journal Number

35/2007

Publication Date

31-Aug-2007

Grant Date

14-Aug-2007

Date of Filing

21-Apr-2003

Name of Patentee

NANJING ZHENZHONG BIOENGINEERING COMPANY LTD

Applicant Address

NANJING NEW @ HIGH TECHNOLOGY INDUSTRY DEVELOPMENT ZONE JIANGSU 210061 P.R.

Inventors:

#	Inventor's Name	Inventor's Address
1	WENG GUOYING & ZHOU, WEI & CHENG, GUANG & CHENG, PEIYUAN	NANJING NEW @ HIGH TECHNOLOGY INDUSTRY DEVELOPMENT ZONE JIANGSU 210061 P.R.
2	ZHOU WEI	NANJING NEW @ HIGH TECHNOLOGY INDUSTRY DEVELOPMENT ZONE JIANGSU 210061 P.R.
3	CHENG GUANG	NANJING NEW @ HIGH TECHNOLOGY INDUSTRY DEVELOPMENT ZONE JIANGSU 210061 P.R.
4	CHENG PEIYUAN	NANJING NEW @ HIGH TECHNOLOGY INDUSTRY DEVELOPMENT ZONE JIANGSU 210061 P.R.

PCT International Classification Number

A61K127

PCT International Application Number

PCT/CN01/00309

PCT International Filing date

2001-02-28

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	001 19039.3	2000-10-19	China