Title of Invention	"PHARMACEUTICAL COMPOSITIONS COMPRISING BETULINIC ACID AND DERVATIVES AND A METHOD FOR THE PREPARATION THEREOF"
Abstract	PHARMACIUTICAL COMPOSITION COMPRISING BETULINICACHO AND DERIVATIVE AND METHOD OF PREPARATION THEREOF The present invention provides a pharmaceutical composition comprising from 0.1 to 40mg/ml of betulinic acid and its derivatives, from 20% to 50% by vol. of dimethylacetamide (DMA), from 20% to 40% by vol. of polyethylene glycol (PEG) and from 5% to 30% by wt of polyoxyethylene sorbitan fatty acid ester (tween) optionally diluted with a conventional aqueous intravenous diluting fluid. The present invention also provides a method for the preparation of said composition.

Title of Invention

"PHARMACEUTICAL COMPOSITIONS COMPRISING BETULINIC ACID AND DERVATIVES AND A METHOD FOR THE PREPARATION THEREOF"

Abstract

PHARMACIUTICAL COMPOSITION COMPRISING BETULINICACHO AND DERIVATIVE AND METHOD OF PREPARATION THEREOF The present invention provides a pharmaceutical composition comprising from 0.1 to 40mg/ml of betulinic acid and its derivatives, from 20% to 50% by vol. of dimethylacetamide (DMA), from 20% to 40% by vol. of polyethylene glycol (PEG) and from 5% to 30% by wt of polyoxyethylene sorbitan fatty acid ester (tween) optionally diluted with a conventional aqueous intravenous diluting fluid. The present invention also provides a method for the preparation of said composition.

Full Text	Field of the Invention The invention relates to method and compositions for solubilisation of pentacyclic triterpenes More particularly, the present invention relates to betulinic acid and its derivatives that are useful for the treatment and suppression of leukemias, lymphomas, prostate and ovarian cancers. Solubilised .compositions comprising pentacyclic triterpenes and, more particularly, betulinic acid and its derivatives, and solvents and co-solvents are also described. Background of the Invention Under the auspices of a National Cooperative Natural Product Drug Discovery Group supported by the National Cancer Institute, the potential anti-tumor activity of approximately 2500 extracts derived from globally collected plants was evaluated in a panel of enzyme based assays and in a battery of cultured human tumor cell lines. One such extract, prepared from the stem bark of Ziziphus mauritiana Lam. (Rhamnaceae), displayed selective cytotoxicity against cultured human melanoma cells (Nature Medicine, Vol. 1 (10), 1995, WO 96/29068). As a result of bioactivity guided fractionation, betulinic acid, a pentacyclic triterpene, was identified as a melanoma-specific cytotoxic agent. In follow-up studies conducted with athymic mice carrying human melanomas, tumor growth was completely inhibited without toxicity. As judged by a variety of cellular responses, anti-tumor activity was mediated by the induction of apoptosis. A number of triterpenoids, including betulinic acid, have several known medical applications, including use as an anticancer drug. Anderson et al., in WO 95/04526, discuss derivatives of triterpenoids which have been used in cancer therapy, including their activity against polyamines which are required by cells to grow at an optimal rate. Some of these triterpenoids have been found to interfere with enzymatic synthesis of polyamines required for optimal cell growth, and thus inhibit the growth of cancer cells, particularly by inhibiting ornithine decarboxylase (Yasukawa, K. et al. Oncology 48 : 72-76,1991). The anti-cancer activity of betulinic acid and some derivatives has been demonstrated using mouse sarcoma 180 cells implanted subcutaneously in nude mice (JP 87,301,580). Choi et al have shown that betulinic acid 3-monoacetate, and betulinic acid methyl ester exhibit ED50 values of 10.5 and 6.8 µg/ml, respectively, against p388 lymphocytic leukemia cells (Choi, Y-H et al., Planta Medica vol XLVII, pages 511-513, 1988). The selective cytotoxicity of betulinic acid and its various derivatives, and their lack of toxicity towards normal cells, afford a favorable therapeutic index. However, the poor solubility of betulinic acid and its derivatives has been a limiting aspect on research on i other activities of betulinic acid and its derivatives. This is also reflected in the fact that jexcept for a few topical preparations no systemic preparations for the administration of the betulinic acid and/or its derivatives are reported. This is due to very poor aqueous solubility of the betulinic acid and its derivatives. Till date there is no formulation reported for the administration of the drug for human use. There is need, therefore, for solubilisation of betulinic acid and its derivatives into pharmaceutically acceptable compositions which are miscible with aqueous intravenous diluting fluids. t is therefore an important object of the present invention to overcome the poor solubility, of the betulinic acid and its derivatives by solubilising them in a pharmaceutically aceeptable non-toxic solvent system. Another very important object of the present invention is to provide a pharmaceutically acceptable composition of poorly soluble betulinic acid and its derivatives that can be utilized for the treatment of malignant diseases. Yet another object of the present invention is to solubilise new derivatives of betulinic acid having a highly selective activity against melanoma cells but have limited utility due to poor solubility. The above objects of the present invention are achieved by the present invention by providing a synergistic composition of DMA, Tween 80 and PEG Summary of the Invention The present embodiment aims to overcome the above and other objects of the present invention by providing a novel, pharmaceutically acceptable solvent system based, inter alia, upon the principles of co-solvency. In particular, the present invention relates to compositions and methods for solubilising poorly water-soluble drugs taking triterpengs such as betulinic acid and its derivatives as prototype substances. As an example of the present invention, betulinic acid or any of its derivatives is dissolved in an organic solvent as the primary vehicle i.e., dimethylacetamide (DMA), followed by the addition of co-solvent(s) such as polyethylene glycol 300 (PEG-300) and a solubiliser Polyoxyethylene 20 sorbitan monooleate (Tween 80) to retain the drug in the solution form on further dilution with an aqueous vehicle. Although the examples described herein utilize polyethylene glycol (PEG) having an average molecular weight of about 300, the molecular weight of PEG may vary from about 300 to about 10,000. In addition, other co-solvents that may be effective include such as e.g. ethanol, propylene glycol, various amides examples of which are 2-pyrolodinone, 1 methyl -2- pyrolodinone and further sulfur containing compounds such as sulfolane, dimethylasulfoxide, tetramethylene sulfoxide, although many of them have little or no previous established records as pharmaceutical solvents. Further besides tween 80, other solubiliser could be used e.g. pluronics, myrj 52, lecithin. It should be possible to extrapolate the use of these novel solvent compositions for solubilisation of other poorly soluble derivatives of Betulinic acid. The present invention provides compositions and methods for the solubilisation of Betulinic acid and derivatives in pharmaceutically acceptable liquid vehicles that avoid the use of potentially toxic solvents reserved for the solubilisation of poorly soluble drugs, such that the drugs remain physically and chemically stable and can be administered intravascularly without undue toxicity from undissolved drug and/or from the solvent vehicles at drug doses meant to be effective to exhibit clinically significant anticancer activity. The preferred composition utilise combinations of anhydrous dimethylacetamixe (DMA), and polyethylene glycol 300 (PEG) as the primary (stock) solvents and Polysorbate 80 as the solubiliser that are miscible in secondary/final solvents such as 5% dextrose solution, 10% dextrose solution or the normal saline. The latter are commonly used fluids in intravenous fusion. These solvents are examples of vehicles in which betulinic acid and its derivatives are effectively solubilised and could be administered in human, alone or in combination with other drugs. The present invention is based on the unexpected finding that when certain solvents are mixed together in a specified proportion, they interact with each other in a synergistic manner to display improved and unexpected properties. Thus, the composition of the present invention is neither a simple admixture nor a product of a chemical reaction, but a synergistic composition having improved and unexpected properties. Accordingly, the present invention provides a novel betulinic acid based pharmaceutical composition comprising 0.1 to 40 mg/ml of betulinic acid and its derivatives as herein described, from 20% to 50% by vol. of dimethylacetamide (DMA), from 20% to 40% by vol. of polyethylene glycol (PEG) and from 5% to 30% by wt. of poly oxyethylene sorbitan fatty acid ester (TWEEN) optionally diluted with a conventional intravenous diluting fluid as herein described. The aqueous diluting fluid is selected from 5% dextrose solution, 10% dextrose solution, normal saline or dextrose-normal saline. In a further preferred embodiment, said FED has a molecular weight between 300-10,000. In another embodiment, said polyoxyethylene sorbitan fatty acid ester is polyoxyethylene 20 sorbitan monooleate (Polysorbate 80, Tween 80). To obtain optimum solubility, it is preferred that said DMA, PEG and Tween are present at a ratio of about 1:4:2 (v/v) although, it will understood by a skilled artisan that the present invention will work even outside such parameters. In a more preferred embodiment, said DMA, and Tween 80 are present at a ratio of about 1:2 (v/v). In another preferred embodiment, said DMA, and PEG 300 are present at a ratio of about 1:4 (v/v) The present invention also provides a method for solubilising triterpenes which comprises the steps of: Dissolveing said triterpenes in from 20% to 50% by vol. Of dimethylacetamide (DMA), from 20% to 40% by vol. Of polyethylene glycol (PEG) and from 5% to 30% by wt of polyoxyethylene sorbitan fatty acid ester (tween) and if desired, diluting said composition with an aqueous intravenous diluting fluid. Preferably, said triterpenes are selected from betulinic acid and its derivatives, which preferably is at a concentration of upto 300 mg/ml. More preferably, PEG is added to the solution of betulinic acid or its derivatives at a ratio of DMA:PEG of about 1 : 4 (v/v), followed by the addition of Tween at a ratio of DMA:Tweem of about 1:2 (v/v) to achieve a betulinic acid or its derivatives concentration of about 0.1-40 mg/ml. The composition of the invention is a synergistic admixture evincing enhanced and unexpected properties over the aggregate properties over the individual ingredients. Given below is the list of some of the representative derivatives of Betulinic acid solubilised using the above compositions. In a further preferred embodiment, said FED has a molecular weight between 300-10,000. In another embodiment, said polyoxyethylene sorbitan fatty acid ester is polyoxyethylene 20 sorbitan monooleate (Polysorbate 80, Tween 80). To obtain optimum solubility, it is preferred that said DMA, PEG and Tween are present at a ratio of about 1:4:2 (v/v) although, it will understood by a skilled artisan that the present invention will work even outside such parameters. In a more preferred embodiment, said DMA, and Tween 80 are present at a ratio of about 1:2 (v/v). In another preferred embodiment, said DMA, and PEG 300 are present at a ratio of about 1:4 (v/v) The present invention also provides a method for solubilising triterpenes which comprises the steps of: Dissolveing said tripterpenes in from 20% to 50% by vol. Of dimethylacetamide (DMA), from 20% to 40% by vol. Of polyethylene glycol (PEG) and from 5% to 30% by wt of polyoxyethylene sorbitan fatty acid ester (tween) and if desired, diluting said composition with an aqueous intravenous diluting fluid. Preferably, said triterpenes are selected from betulinic acid and its derivatives, which preferably is at a concentration of upto 300 mg/ml. More preferably, PEG is added to the solution of betulinic acid or its derivatives at a ratio of DMA:PEG of about 1 : 4 (v/v), followed by the addition of Tween at a ratio of DMA:Tweem of about 1:2 (v/v) to achieve a betulinic acid or its derivatives concentration of about 0.1-40 mg/ml. The composition of the invention is a synergistic admixture evincing enhanced and unexpected properties over the aggregate properties over the individual ingredients. Given below is the list of some of the representative derivatives of Betulinic acid solubilised using the above compositions. REPRESENTATIVE LIST OFBETULINIC ACID DERIVATIVES SOLUBLLISED (Formula & Table Removed)The above list provides only a few representative samples of the betulinic acid derivatives obtained by derivatization of functional groups present in isolation or in combinations at the carbon sites shown in the table above (a "+" sign indicates derivatization of functional group at respective carbon site) The few examples of the functional groups used for the various substitutions in isolation or in combinations at the carbon sites shown above include : H , =O, -OH, Br, -CH(CH3)2, CH2=CCH3, -CH2COOCH3, BrCH2C(Br)CH3> -COCH=CH2, - CH(CH3)2, -CH2CH2COOCH3, -O COC6H3F2 (2,4), -N=CHC6H3F2 (3,4), =NOCH2C6H4NO2 (4), - N=CHC6H3F2(2,4), -OCO C6H4(C5H11X4) The present invention will now be described in greater deatil with refernce to the following examples, ol which are included to demonstrate preferred embodiment of the invention It should be understood that the specific forms of the invention herein illustrated and described are intended to be representative only. Changes, including but not limited to those suggested in this specification, may be made in the illustrated embodiments without departing from the concept, spirit and scope of the invention. Examplel Enhanced solubility of betulinic acid in physiologically acceptable solvents The solubility of betulinic acid and its derivatives was determined in various individual solvents. Based on the betulinic acid and its solubility in these particular vehicles, it was then attempted to enhance the solubility by mixing different solvents according to the principles of cosolvency (Spiegel and Noseworthy, 1963: Yalkowsky and Roseman, 1981). Different solvent systems were evaluated to the above estimates to arrive at a clinically Therefore in further trials attempt was made to increase the proportion of DMA to enhance the solubility of the drug on dilution. Example 4 Betulinic acid/ Derivatives 50 mg DMA 50%v/v PEG 300 50%v/v The 1:1 ratio of DMA and PEG improved the solubility of betulinic acid or its derivatives on dilution with aqueous diluting fluid like 2- 25% dextrose, normal saline. However 1:1 ratio of DMA and PEG in presence of betulinic acid or its derivatives and on dilution with aqueous diluting fluid gave a viscous solution which gels on keeping though there are no signs of any precipitation of drug. Example 5 To further improve the solubility of betulinic acid and its derivatives on dilution with aqueous system additions of surfactants was investigated. The surface-active agents most commonly used in the prior art for enhancing the solubility of poorly soluble drugs are polyoxyethylene sorbitan monooleate (Polysorbate 80) and polyoxyethylene polyoxypropylene ethers (Pluronic 68). Less commonly used surface active agents in parentral products are lecithins, Polyoxyl 40 stearate (Myrj 52). In the following examples one or more co-solvents with a surfactant or a combination of surfactants were added to keep the drug in solution after diluting with an aqueous intravenous diluting fluid. "relevant stock composition. The stock composition would then be diluted with a final solvent to yield a working formulation 0,001- 40 mg/ml. Example! Betulinic acid and its derivatives solubility Several methods and compositions were evaluated to solubilise betulinic acid and its derivatives in water miscible, physiologically acceptable vehicles. The examined solvents included DMA, DMSO, Absolute EthanoL, glycerin, polyethylene glycols (PEGs) and propylene glycol (PG), in addition to the aqueous diluting fluids as normal saline, 2-25% dextrose in solution. The solubilities these solvents are tabulated below (Table Removed) DMA and DMSO were found to be the best solvents, whereas betulinic acid and its derivatives were insoluble in most of the aqueous solvents. Only with DMA and DMSO solubility in excess of 10 mg/ml was achieved. In fact both these solvents yielded 100-200 mg/ml drug in solution form. Although DMSO has been used as solvent for parentral formulations of experimental anti cancer agents; it is not a preferred vehicle for dissolving the drugs due to its toxicity and potential incompatibilities with plastic administration Devices. Therefore DMA was chosen as the solvent of choice for dissolving the drug, DMA, per se, could dissolve a concentration of betulinic acid and its derivatives upto 250 mg/ml. However, a concentrated solution of betulinic acid or its derivatives in DMA alone can't be administered as such in the body as the total volume of the solvent administered in the body would exceed the toxicity limit. Moreover the solution on dilution with aqueous fluid in the body may lead to precipitation of drug from the solution leading to increased drug toxicity. The following sets of studies were performed to arrive at physiologically acceptable formulation for intravenous administration. Example 3 Solubilising of Betulinic Acid and Derivatives using DMA & PEG A concentrated solution of betulinic acid or its derivatives was prepared by dissolving 50-200 mg of betulinic acid or its derivatives in 1 ml of DMA. The above stock solution was diluted with PEG (Polyethylene Glycol, MW 300) in a ratio of 1:4 to a concentration of 10-40 mg/ml. Betulinic acid/ Derivatives 200 mg DMA 20%v/v PEG 300 80%v/v However, on dilution with the aqueous diluting fluid, the drug immediately precipitated out. Therefore in further trial we tried to reduce the drug concentration.Betulinic acid/ Derivatives 50 mg DMA 20%v/v PEG 300 80%v/v The above solution also on dilution with the aqueous diluting fluid, led to immediate precipitation of drug even at concentrations as low as 0.001- 0.1 mg/ml. Example 6 Betulinic acid/ Derivatives 10- 20 mg DMA 50%v/v Tween 80 50% w/v The above combination could dissolve upto 20 mg/ml of the drug. But the drug immediately precipitated out on dilution with aqueous diluting fluid. Example 7 Betulinic acid/ Derivatives 10- 20 mg Absolute Ethanol 20-40 % v/v Tween 80 60-80 % w/v The above compositions also could dissolve upto 20 mg/ml of the drug. However, the drug solution remained clear on dilution with aqueous diluting fluid to a concentration of 0.3-1.4 mg/ml for up to 3 hours. Example 8 Betulinic acid/ Derivatives 60 Absolute Ethanol 65 %v/v Tween 80 5% w/v PEG 300 30 %v/v The above composition could dissolve up to 6 mg/ml of the drug. The drug solution remained clear on dilution with aqueous diluting fluid to a concentration of 0.08- 0.4 mg/ml. "Example 9 Attempt was made to reduce the content of organic solvent Betulinic acid/ Derivatives 40 mg Absolute Ethanol 35 % v/v Tween 80 15%w/v PEG 300 50 %v/v The above composition could dissolve only up to 4 mg/ml of the drug. The drug solution remained clear on dilution with aqueous diluting fluid to a concentration of 0.08- 0.4 mg/ml. However, outside this range there was immediate precipitation. Example 10 Betulinic acid/ Derivatives 30 mg DMA 10 %v/v Propylene Glycol 20 %v/v Absolute Ethanol 35 %v/v Tween 80 35 %w/v Example 11 Betulinic acid/ Derivatives 150mg DMA 20%v/v Propylene Glycol 1 0 %v/v Absolute Ethanol 30 %v/v Tween 80 40 %w/v Betulinic acid and almost all of the its derivatives were found to be soluble in the above system in a concentration range of 2- 15 mg/ml which on dilution with an aqueous diluting fluid remains clear at as lower concentration as 0.012- 2 mg/ml. However, these compositions had quite high amount of the organic solvents which is not desirable as these may prove toxic on administration in the body. Therefore, attempts were undertaken to solubilise the maximum quantity of the drug in minimum amount of the organic solvents with a less toxic co-solvent and surfactant combination. Example 12 Betulinic acid/ Derivatives 5- 20 mg/ml DMA 20 % v/v Myrj 52 30 % w/v Tween 80 50 % w/v Example 13 Betulinic acid/ Derivatives 5- 20mg/ml DMA . 20 % v/v Myrj 52 20 % w/v Tween 80 20 % w/v PEG 300 40 % v/v The above two compositions containing Myrj 52 could dissolve upto 20 mg/ml of the drug but on dilution with aqueous diluting fluid to a concentration to 0.1- 1.0 mg/ml, the drug precipitated out in a few hours only. Moreover preparations containing Myrj 52 tend to solidify at lower temperatures. Also myrj 52 is not a preferred surfactant for parentral products because of its higher toxicity. Therefore it was attempted to replace myrj 52 with Pluronic 68 and lecithin, which are comparatively safer for intravenous administration. Example 14 Betulinic acid/ Derivatives 2- 15mg/ml DMA 20 % v/v Tween 80 20 % w/v PEG 300 57%v/v Poloxamer 407 3 % w/v Example 15 Betulinic acid/ Derivatives 2- I'Smg/ml DMA 20 % v/v Tween 80 20 % w/v Lecithin 5 % w/v PEG 300 55 % v/v The above compositions could dissolve 2-15 mg/ml of betulinic acid and its derivatives. However, on dilution with the aqueous diluting fluid the drug remained soluble only at lower dilutions (0.001-1 mg/ml). Example 16 After conducting number of trials as described above to solubilise betulinic acid and its derivatives the most preferred composition was the one containing DMA, PEG 300 and Tween 80. The three components were tried in varying proportions as mentioned in the following example and arrived at the preferred composition which contains DMA, PEG 300 and tween 80 in a ratio of 1:4:2 and could dissolve betulinic acid and its derivatives at a concentration of upto 22 mg/ml which on dilution with an aqueous diluting fluid to a concentration of 0.1- 10 mg/ml remained clear without any precipitation of the drug for more than 5 days. Betulinic acid/ Derivatives 10- 20mg/ml DMA 20-50% v/v PEG 300 20- 40% v/v Tween 80 5- 30% w/v We Claim: 1. A pharmaceutical composition comprising from 0.1 to 40mg/ml of betulinic acid and its derivatives as herein described, from 20% to 50% by vol. of dimethylacetamide (DMA), from 20% to 40% by vol. of polyethylene glycol (PEG) and from 5% to 30% by wt of polyoxyethylene sorbitan fatty acid ester (tween) optionally diluted with a conventional aqueous intravenous diluting fluid as herein described. 2. The composition as claimed in claim 1 wherein the said aqueous diluting fluid is selected from 5% dextrose solution, 10% dextrose solution, normal saline and dextrose- normal saline. 3. The composition as claimed in claim 1 or 2 wherein said PEG has a molecular weight between 300-10,000. 4. The composition as claimed in any preceding claim wherein said polyoxyethylene sorbitan fatty acid ester is polyoxyethylene 20 sorbitan monooleate (Polysorbate 80, Tween 80). 5. A method for the preparation of a pharmaceutical composition as claimed in any preceding claim which comprises the steps of : dissolving 0.1 to 40 mg/ml betulinic acid or its derivatives as herein described in from 20% to 50% by vol. of dimethylacetamide (DMA), from 20% to 40% by vol. of polyethylene glycol (PEG) and from 5% to 30% by wt. of polyoxyethylene sorbitan fatty acid ester (tween) and optionally, diluting said composition with a conventional aqueous intravenous diluting fluid. 6. A pharmaceutical composition as claimed in claim 1 substantially as herein described with reference to the foregoing examples. 7. A method for the preparation of pharmaceutical composition as claimed in claim 1 susbtantially as herein described with reference to the foregoing examples.

Full Text

Field of the Invention
The invention relates to method and compositions for solubilisation of pentacyclic triterpenes More particularly, the present invention relates to betulinic acid and its derivatives that are useful for the treatment and suppression of leukemias, lymphomas, prostate and ovarian cancers. Solubilised .compositions comprising pentacyclic triterpenes and, more particularly, betulinic acid and its derivatives, and solvents and co-solvents are also described.
Background of the Invention
Under the auspices of a National Cooperative Natural Product Drug Discovery Group supported by the National Cancer Institute, the potential anti-tumor activity of approximately 2500 extracts derived from globally collected plants was evaluated in a panel of enzyme based assays and in a battery of cultured human tumor cell lines. One such extract, prepared from the stem bark of Ziziphus mauritiana Lam. (Rhamnaceae), displayed selective cytotoxicity against cultured human melanoma cells (Nature Medicine, Vol. 1 (10), 1995, WO 96/29068). As a result of bioactivity guided fractionation, betulinic acid, a pentacyclic triterpene, was identified as a melanoma-specific cytotoxic agent. In follow-up studies conducted with athymic mice carrying human melanomas, tumor growth was completely inhibited without toxicity. As judged by a variety of cellular responses, anti-tumor activity was mediated by the induction of apoptosis.
A number of triterpenoids, including betulinic acid, have several known medical applications, including use as an anticancer drug. Anderson et al., in WO 95/04526, discuss derivatives of triterpenoids which have been used in cancer therapy, including their activity against polyamines which are required by cells to grow at an optimal rate. Some of these triterpenoids have been found to interfere with enzymatic synthesis of polyamines required for optimal cell growth, and thus inhibit the growth of cancer cells, particularly by inhibiting ornithine decarboxylase (Yasukawa, K. et al. Oncology 48 : 72-76,1991). The
anti-cancer activity of betulinic acid and some derivatives has been demonstrated using mouse sarcoma 180 cells implanted subcutaneously in nude mice (JP 87,301,580). Choi et al have shown that betulinic acid 3-monoacetate, and betulinic acid methyl ester exhibit ED50 values of 10.5 and 6.8 µg/ml, respectively, against p388 lymphocytic leukemia cells (Choi, Y-H et al., Planta Medica vol XLVII, pages 511-513, 1988).
The selective cytotoxicity of betulinic acid and its various derivatives, and their lack of
toxicity towards normal cells, afford a favorable therapeutic index. However, the poor
solubility of betulinic acid and its derivatives has been a limiting aspect on research on
i
other activities of betulinic acid and its derivatives. This is also reflected in the fact that jexcept for a few topical preparations no systemic preparations for the administration of the

betulinic acid and/or its derivatives are reported. This is due to very poor aqueous

solubility of the betulinic acid and its derivatives. Till date there is no formulation reported for the administration of the drug for human use.
There is need, therefore, for solubilisation of betulinic acid and its derivatives into pharmaceutically acceptable compositions which are miscible with aqueous intravenous diluting fluids.
t is therefore an important object of the present invention to overcome the poor solubility, of the betulinic acid and its derivatives by solubilising them in a pharmaceutically aceeptable non-toxic solvent system.
Another very important object of the present invention is to provide a pharmaceutically acceptable composition of poorly soluble betulinic acid and its derivatives that can be utilized for the treatment of malignant diseases.
Yet another object of the present invention is to solubilise new derivatives of betulinic acid having a highly selective activity against melanoma cells but have limited utility due to poor solubility.
The above objects of the present invention are achieved by the present invention by providing a synergistic composition of DMA, Tween 80 and PEG
Summary of the Invention
The present embodiment aims to overcome the above and other objects of the present invention by providing a novel, pharmaceutically acceptable solvent system based, inter alia, upon the principles of co-solvency.
In particular, the present invention relates to compositions and methods for solubilising poorly water-soluble drugs taking triterpengs such as betulinic acid and its derivatives as prototype substances. As an example of the present invention, betulinic acid or any of its derivatives is dissolved in an organic solvent as the primary vehicle i.e., dimethylacetamide (DMA), followed by the addition of co-solvent(s) such as polyethylene glycol 300 (PEG-300) and a solubiliser Polyoxyethylene 20 sorbitan monooleate (Tween 80) to retain the drug in the solution form on further dilution with an aqueous vehicle.
Although the examples described herein utilize polyethylene glycol (PEG) having an average molecular weight of about 300, the molecular weight of PEG may vary from about 300 to about 10,000. In addition, other co-solvents that may be effective include such as e.g. ethanol, propylene glycol, various amides examples of which are 2-pyrolodinone, 1 methyl -2- pyrolodinone and further sulfur containing compounds such as sulfolane, dimethylasulfoxide, tetramethylene sulfoxide, although many of them have little or no previous established records as pharmaceutical solvents. Further besides tween 80, other solubiliser could be used e.g. pluronics, myrj 52, lecithin.
It should be possible to extrapolate the use of these novel solvent compositions for solubilisation of other poorly soluble derivatives of Betulinic acid.
The present invention provides compositions and methods for the solubilisation of Betulinic acid and derivatives in pharmaceutically acceptable liquid vehicles that avoid the use of potentially toxic solvents reserved for the solubilisation of poorly soluble drugs, such that the drugs remain physically and chemically stable and can be administered
intravascularly without undue toxicity from undissolved drug and/or from the solvent vehicles at drug doses meant to be effective to exhibit clinically significant anticancer activity.
The preferred composition utilise combinations of anhydrous dimethylacetamixe (DMA), and polyethylene glycol 300 (PEG) as the primary (stock) solvents and Polysorbate 80 as the solubiliser that are miscible in secondary/final solvents such as 5% dextrose solution, 10% dextrose solution or the normal saline. The latter are commonly used fluids in intravenous fusion. These solvents are examples of vehicles in which betulinic acid and its derivatives are effectively solubilised and could be administered in human, alone or in combination with other drugs.
The present invention is based on the unexpected finding that when certain solvents are mixed together in a specified proportion, they interact with each other in a synergistic manner to display improved and unexpected properties. Thus, the composition of the present invention is neither a simple admixture nor a product of a chemical reaction, but a synergistic composition having improved and unexpected properties.
Accordingly, the present invention provides a novel betulinic acid based pharmaceutical composition comprising 0.1 to 40 mg/ml of betulinic acid and its derivatives as herein described, from 20% to 50% by vol. of dimethylacetamide (DMA), from 20% to 40% by vol. of polyethylene glycol (PEG) and from 5% to 30% by wt. of poly oxyethylene sorbitan fatty acid ester (TWEEN) optionally diluted with a conventional intravenous diluting fluid as herein described.
The aqueous diluting fluid is selected from 5% dextrose solution, 10% dextrose solution, normal saline or dextrose-normal saline.
In a further preferred embodiment, said FED has a molecular weight between 300-10,000.
In another embodiment, said polyoxyethylene sorbitan fatty acid ester is polyoxyethylene 20 sorbitan monooleate (Polysorbate 80, Tween 80).
To obtain optimum solubility, it is preferred that said DMA, PEG and Tween are present at a ratio of about 1:4:2 (v/v) although, it will understood by a skilled artisan that the present invention will work even outside such parameters. In a more preferred embodiment, said DMA, and Tween 80 are present at a ratio of about 1:2 (v/v). In another preferred embodiment, said DMA, and PEG 300 are present at a ratio of about 1:4 (v/v)
The present invention also provides a method for solubilising triterpenes which comprises the steps of:
Dissolveing said triterpenes in from 20% to 50% by vol. Of dimethylacetamide (DMA), from 20% to 40% by vol. Of polyethylene glycol (PEG) and from 5% to 30% by wt of polyoxyethylene sorbitan fatty acid ester (tween) and if desired, diluting said composition with an aqueous intravenous diluting fluid.
Preferably, said triterpenes are selected from betulinic acid and its derivatives, which preferably is at a concentration of upto 300 mg/ml. More preferably, PEG is added to the solution of betulinic acid or its derivatives at a ratio of DMA:PEG of about 1 : 4 (v/v), followed by the addition of Tween at a ratio of DMA:Tweem of about 1:2 (v/v) to achieve a betulinic acid or its derivatives concentration of about 0.1-40 mg/ml.
The composition of the invention is a synergistic admixture evincing enhanced and unexpected properties over the aggregate properties over the individual ingredients.
Given below is the list of some of the representative derivatives of Betulinic acid solubilised using the above compositions.
In a further preferred embodiment, said FED has a molecular weight between 300-10,000.
In another embodiment, said polyoxyethylene sorbitan fatty acid ester is polyoxyethylene 20 sorbitan monooleate (Polysorbate 80, Tween 80).
To obtain optimum solubility, it is preferred that said DMA, PEG and Tween are present at a ratio of about 1:4:2 (v/v) although, it will understood by a skilled artisan that the present invention will work even outside such parameters. In a more preferred embodiment, said DMA, and Tween 80 are present at a ratio of about 1:2 (v/v). In another preferred embodiment, said DMA, and PEG 300 are present at a ratio of about 1:4 (v/v)
The present invention also provides a method for solubilising triterpenes which comprises the steps of:
Dissolveing said tripterpenes in from 20% to 50% by vol. Of dimethylacetamide (DMA), from 20% to 40% by vol. Of polyethylene glycol (PEG) and from 5% to 30% by wt of polyoxyethylene sorbitan fatty acid ester (tween) and if desired, diluting said composition with an aqueous intravenous diluting fluid.
Preferably, said triterpenes are selected from betulinic acid and its derivatives, which preferably is at a concentration of upto 300 mg/ml. More preferably, PEG is added to the solution of betulinic acid or its derivatives at a ratio of DMA:PEG of about 1 : 4 (v/v), followed by the addition of Tween at a ratio of DMA:Tweem of about 1:2 (v/v) to achieve a betulinic acid or its derivatives concentration of about 0.1-40 mg/ml.
The composition of the invention is a synergistic admixture evincing enhanced and unexpected properties over the aggregate properties over the individual ingredients.
Given below is the list of some of the representative derivatives of Betulinic acid solubilised using the above compositions.
REPRESENTATIVE LIST OFBETULINIC ACID DERIVATIVES SOLUBLLISED
(Formula & Table Removed)The above list provides only a few representative samples of the betulinic acid derivatives obtained by derivatization of functional groups present in isolation or in combinations at the carbon sites shown in the table above (a "+" sign indicates derivatization of functional group at respective carbon site)
The few examples of the functional groups used for the various substitutions in isolation or
in combinations at the carbon sites shown above include :
H , =O, -OH, Br, -CH(CH3)2, CH2=CCH3, -CH2COOCH3, BrCH2C(Br)CH3> -COCH=CH2, -
CH(CH3)2,
-CH2CH2COOCH3, -O COC6H3F2 (2,4), -N=CHC6H3F2 (3,4), =NOCH2C6H4NO2 (4), -
N=CHC6H3F2(2,4),
-OCO C6H4(C5H11X4)
The present invention will now be described in greater deatil with refernce to the following examples, ol which are included to demonstrate preferred embodiment of the invention It
should be understood that the specific forms of the invention herein illustrated and described are intended to be representative only. Changes, including but not limited to those suggested in this specification, may be made in the illustrated embodiments without departing from the concept, spirit and scope of the invention.
Examplel
Enhanced solubility of betulinic acid in physiologically acceptable solvents
The solubility of betulinic acid and its derivatives was determined in various individual solvents. Based on the betulinic acid and its solubility in these particular vehicles, it was then attempted to enhance the solubility by mixing different solvents according to the principles of cosolvency (Spiegel and Noseworthy, 1963: Yalkowsky and Roseman, 1981). Different solvent systems were evaluated to the above estimates to arrive at a clinically
Therefore in further trials attempt was made to increase the proportion of DMA to enhance the solubility of the drug on dilution.
Example 4
Betulinic acid/ Derivatives 50 mg
DMA 50%v/v
PEG 300 50%v/v
The 1:1 ratio of DMA and PEG improved the solubility of betulinic acid or its derivatives on dilution with aqueous diluting fluid like 2- 25% dextrose, normal saline. However 1:1 ratio of DMA and PEG in presence of betulinic acid or its derivatives and on dilution with aqueous diluting fluid gave a viscous solution which gels on keeping though there are no signs of any precipitation of drug.
Example 5
To further improve the solubility of betulinic acid and its derivatives on dilution with aqueous system additions of surfactants was investigated. The surface-active agents most commonly used in the prior art for enhancing the solubility of poorly soluble drugs are polyoxyethylene sorbitan monooleate (Polysorbate 80) and polyoxyethylene polyoxypropylene ethers (Pluronic 68). Less commonly used surface active agents in parentral products are lecithins, Polyoxyl 40 stearate (Myrj 52).
In the following examples one or more co-solvents with a surfactant or a combination of surfactants were added to keep the drug in solution after diluting with an aqueous intravenous diluting fluid.
"relevant stock composition. The stock composition would then be diluted with a final solvent to yield a working formulation 0,001- 40 mg/ml.
Example!
Betulinic acid and its derivatives solubility
Several methods and compositions were evaluated to solubilise betulinic acid and its derivatives in water miscible, physiologically acceptable vehicles. The examined solvents included DMA, DMSO, Absolute EthanoL, glycerin, polyethylene glycols (PEGs) and propylene glycol (PG), in addition to the aqueous diluting fluids as normal saline, 2-25% dextrose in solution. The solubilities these solvents are tabulated below (Table Removed)
DMA and DMSO were found to be the best solvents, whereas betulinic acid and its derivatives were insoluble in most of the aqueous solvents. Only with DMA and DMSO solubility in excess of 10 mg/ml was achieved. In fact both these solvents yielded 100-200 mg/ml drug in solution form. Although DMSO has been used as solvent for parentral formulations of experimental anti cancer agents; it is not a preferred vehicle for dissolving the drugs due to its toxicity and potential incompatibilities with plastic administration
Devices. Therefore DMA was chosen as the solvent of choice for dissolving the drug, DMA, per se, could dissolve a concentration of betulinic acid and its derivatives upto 250 mg/ml. However, a concentrated solution of betulinic acid or its derivatives in DMA alone can't be administered as such in the body as the total volume of the solvent administered in the body would exceed the toxicity limit. Moreover the solution on dilution with aqueous fluid in the body may lead to precipitation of drug from the solution leading to increased drug toxicity. The following sets of studies were performed to arrive at physiologically acceptable formulation for intravenous administration.
Example 3
Solubilising of Betulinic Acid and Derivatives using DMA & PEG
A concentrated solution of betulinic acid or its derivatives was prepared by dissolving 50-200 mg of betulinic acid or its derivatives in 1 ml of DMA. The above stock solution was diluted with PEG (Polyethylene Glycol, MW 300) in a ratio of 1:4 to a concentration of 10-40 mg/ml.
Betulinic acid/ Derivatives 200 mg
DMA 20%v/v
PEG 300 80%v/v
However, on dilution with the aqueous diluting fluid, the drug immediately precipitated out. Therefore in further trial we tried to reduce the drug concentration.Betulinic acid/ Derivatives 50 mg
DMA 20%v/v
PEG 300 80%v/v
The above solution also on dilution with the aqueous diluting fluid, led to immediate precipitation of drug even at concentrations as low as 0.001- 0.1 mg/ml.
Example 6
Betulinic acid/ Derivatives 10- 20 mg
DMA 50%v/v
Tween 80 50% w/v
The above combination could dissolve upto 20 mg/ml of the drug. But the drug immediately precipitated out on dilution with aqueous diluting fluid.
Example 7
Betulinic acid/ Derivatives 10- 20 mg
Absolute Ethanol 20-40 % v/v
Tween 80 60-80 % w/v
The above compositions also could dissolve upto 20 mg/ml of the drug. However, the drug solution remained clear on dilution with aqueous diluting fluid to a concentration of 0.3-1.4 mg/ml for up to 3 hours.
Example 8
Betulinic acid/ Derivatives 60
Absolute Ethanol 65 %v/v
Tween 80 5% w/v
PEG 300 30 %v/v
The above composition could dissolve up to 6 mg/ml of the drug. The drug solution remained clear on dilution with aqueous diluting fluid to a concentration of 0.08- 0.4
mg/ml.
"Example 9
Attempt was made to reduce the content of organic solvent
Betulinic acid/ Derivatives 40 mg
Absolute Ethanol 35 % v/v
Tween 80 15%w/v
PEG 300 50 %v/v
The above composition could dissolve only up to 4 mg/ml of the drug. The drug solution remained clear on dilution with aqueous diluting fluid to a concentration of 0.08- 0.4 mg/ml. However, outside this range there was immediate precipitation.
Example 10
Betulinic acid/ Derivatives 30 mg
DMA 10 %v/v
Propylene Glycol 20 %v/v
Absolute Ethanol 35 %v/v
Tween 80 35 %w/v
Example 11
Betulinic acid/ Derivatives 150mg
DMA 20%v/v
Propylene Glycol 1 0 %v/v
Absolute Ethanol 30 %v/v
Tween 80 40 %w/v
Betulinic acid and almost all of the its derivatives were found to be soluble in the above system in a concentration range of 2- 15 mg/ml which on dilution with an aqueous diluting fluid remains clear at as lower concentration as 0.012- 2 mg/ml. However, these

compositions had quite high amount of the organic solvents which is not desirable as these may prove toxic on administration in the body. Therefore, attempts were undertaken to solubilise the maximum quantity of the drug in minimum amount of the organic solvents with a less toxic co-solvent and surfactant combination.
Example 12
Betulinic acid/ Derivatives 5- 20 mg/ml
DMA 20 % v/v
Myrj 52 30 % w/v
Tween 80 50 % w/v
Example 13
Betulinic acid/ Derivatives 5- 20mg/ml
DMA . 20 % v/v
Myrj 52 20 % w/v
Tween 80 20 % w/v
PEG 300 40 % v/v
The above two compositions containing Myrj 52 could dissolve upto 20 mg/ml of the drug but on dilution with aqueous diluting fluid to a concentration to 0.1- 1.0 mg/ml, the drug precipitated out in a few hours only. Moreover preparations containing Myrj 52 tend to solidify at lower temperatures. Also myrj 52 is not a preferred surfactant for parentral products because of its higher toxicity. Therefore it was attempted to replace myrj 52 with Pluronic 68 and lecithin, which are comparatively safer for intravenous administration.
Example 14
Betulinic acid/ Derivatives 2- 15mg/ml
DMA 20 % v/v
Tween 80 20 % w/v
PEG 300 57%v/v
Poloxamer 407 3 % w/v
Example 15
Betulinic acid/ Derivatives 2- I'Smg/ml
DMA 20 % v/v
Tween 80 20 % w/v
Lecithin 5 % w/v
PEG 300 55 % v/v
The above compositions could dissolve 2-15 mg/ml of betulinic acid and its derivatives. However, on dilution with the aqueous diluting fluid the drug remained soluble only at lower dilutions (0.001-1 mg/ml).
Example 16
After conducting number of trials as described above to solubilise betulinic acid and its derivatives the most preferred composition was the one containing DMA, PEG 300 and Tween 80. The three components were tried in varying proportions as mentioned in the following example and arrived at the preferred composition which contains DMA, PEG 300 and tween 80 in a ratio of 1:4:2 and could dissolve betulinic acid and its derivatives at a concentration of upto 22 mg/ml which on dilution with an aqueous diluting fluid to a concentration of 0.1- 10 mg/ml remained clear without any precipitation of the drug for more than 5 days.
Betulinic acid/ Derivatives 10- 20mg/ml
DMA 20-50% v/v
PEG 300 20- 40% v/v
Tween 80 5- 30% w/v

We Claim:
1. A pharmaceutical composition comprising from 0.1 to 40mg/ml of betulinic acid and its
derivatives as herein described, from 20% to 50% by vol. of dimethylacetamide (DMA), from 20% to 40% by vol. of polyethylene glycol (PEG) and from 5% to 30% by wt of polyoxyethylene sorbitan fatty acid ester (tween) optionally diluted with a conventional aqueous intravenous diluting fluid as herein described.
2. The composition as claimed in claim 1 wherein the said aqueous diluting fluid is
selected from 5% dextrose solution, 10% dextrose solution, normal saline and dextrose-
normal saline.
3. The composition as claimed in claim 1 or 2 wherein said PEG has a molecular weight
between 300-10,000.
4. The composition as claimed in any preceding claim wherein said polyoxyethylene
sorbitan fatty acid ester is polyoxyethylene 20 sorbitan monooleate (Polysorbate 80,
Tween 80).
5. A method for the preparation of a pharmaceutical composition as claimed in any
preceding claim which comprises the steps of : dissolving 0.1 to 40 mg/ml betulinic
acid or its derivatives as herein described in from 20% to 50% by vol. of
dimethylacetamide (DMA), from 20% to 40% by vol. of polyethylene glycol (PEG) and
from 5% to 30% by wt. of polyoxyethylene sorbitan fatty acid ester (tween) and
optionally, diluting said composition with a conventional aqueous intravenous diluting
fluid.
6. A pharmaceutical composition as claimed in claim 1 substantially as herein described
with reference to the foregoing examples.
7. A method for the preparation of pharmaceutical composition as claimed in claim 1
susbtantially as herein described with reference to the foregoing examples.

Documents:

505-del-2000-abstract.pdf

505-del-2000-claims.pdf

505-DEL-2000-Correspondence-Others-(19-08-2010).pdf

505-del-2000-correspondence-others.pdf

505-del-2000-correspondence-po.pdf

505-del-2000-description (complete).pdf

505-del-2000-form-1.pdf

505-del-2000-form-19.pdf

505-del-2000-form-2.pdf

505-del-2000-form-26.pdf

505-del-2000-form-3.pdf

505-DEL-2000-GPA-(19-08-2010).pdf

505-del-2000-petition-138.pdf

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Patent Number

216518

Indian Patent Application Number

505/DEL/2000

PG Journal Number

13/2008

Publication Date

28-Mar-2008

Grant Date

14-Mar-2008

Date of Filing

11-May-2000

Name of Patentee

DABUR RESEARCH FOUNDATION,

Applicant Address

22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	BURMAN ANAND C.	DABUR RESEARCH FOUNDATION, 22 SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PARDESH, INDIA.
2	MUKHERJEE RAMA	DABUR RESEARCH FOUNDATION, 22 SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PARDESH, INDIA.
3	BALA HONEY	DABUR RESEARCH FOUNDATION, 22 SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PARDESH, INDIA.
4	KHATTAR DHIRAJ	DABUR RESEARCH FOUNDATION, 22 SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PARDESH, INDIA.

PCT International Classification Number

A61K 31/19

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA