Title of Invention

A PROCESS FOR PREPARATION OF ANTI-HYPERLIPIDEMIC COMPOSITION

Abstract

This invention relates to a process for preparation of anti-hyperlipidemic composition. The invention particularly relates to the process for the preparation of composition using some pregnadienone compounds which are devoid of progestational activity as hypolipidemic drugs. The invention more particularly relates to the preparation of composition using compound 3a-hydroxypregna-5, 16-dien-20-one having the formula-8 shown in the drawing accompanying this specification

Full Text

This invention particularly relates to a process for preparation of anti-hyperlipidemic composition. The invention particularly relates to the use of some pregnadienone compounds which are devoid of progestational activity as hypolipidemic drugs for the preparation of composition. The invention more particularly relates to the use of the compound 3a-hydroxypregna-5, 16-dien-20-one having the formula-8 shown in the drawing accompanying this specification. Ever since the discovery of progesterone (1) and its proven role in the female fertility regulation, continuous efforts have been made to look for progestins (compounds with progestational activity) with maximum progestational activity (responsible for early changes in the endometrium from the proliferative to the secretory phase). One of the serious side effect of progesterone is its lipid lowering effect. Efforts have therefore been made to prepare progestins which would be totally devoid of any effect on the lipoprotein profile. The first generation progestins were Norethisterone (2) and Levonorgestrel (3), which had affinity for both progesterone and androgen receptors. Their affinity for androgen receptors may be responsible for hyperlipidemic effect (Sex hormone receptor binding, progestin selectivity and the new oral contraceptives; Am. J. Obstet. 170, 1508(1994) of D.C. Collins). The androgenic affinity has been attributed to C-17 hydroxy functionality which made these molecules to resemble androgens. In recently discovered second generation progestins such as Gestodene (4) and 3-Keto desogestrel (5), an additional olefinic bond either in or on C- and D- ring brought a dramatic change in their affinity to androgens receptors. As a result these compounds had a very high order of progestational effect with practically no affinity to androgen receptors (Table-1). Table 1. Relative binding affinity of contraceptive progestins for progesterone and androgen receptors (Formula Removed) • The higher the selectivity index, the greater the separation between the dose needed to achieve the desired progestational effect and the dose associated with the undesired androgenic effect. However, we have focussed on research work on the development of pregnane compounds which would retain hypolipidemic activity but would be devoid of progestational effect. In our endeavours we have observed that ethylacetate extract of guggul resin obtained from Commiphora mukul, a plant commonly used in Indian traditional medicine, had marked hypolipidemic activity (See the publication: G.V. Satyavati, "Effect of an indigenous drug Commiphora mukul on disorders of lipid metabolism with special reference to atherosclerosis and obesity. A Ph.D. thesis of Ayurvedic Medicine (1966), Banaras Hindu University, Varanasi, India). This activity was shown by our studies to be due to guggulsterone, a pregnadien-dione(6), and a product Guglip containing 6 is in clinical practice as a hypolipidemic agent [Guggulsterone Z- & E-, EN = 125-922, Gugulip, Drugs Fut. 13, 618(1988)]. Table 2: Cholesterol lowering activity of some pregnenolones and pregnenols as compared to clofibrate in tyloxapol treated rats. (Formula Removed) 3a-Hydroxypregna-5,16-dien-20-one (8), has earlier been reported in the literature as synthetic intermediate for making various sex hormones from diosgenin (see the publication of G. Rosenkranz; "History of steroid"; Steroid, 57, 409(1992). The compound later was isolated from Veratrum grandiflorum (K. Kancko, M. Watanabe and H. Mitsuhashi; 3a-hydroxypregna-5,16-dien-20-one from Veratrum grandiflorum; Phytochemistry, 12, 1509(1973). The other pregnane class of compounds which have low order of hypolipidemic effect are synthetic progestins: gestodene and 3-keto-desop.estrel used in ORA.' contraceptives. However, these are not used as hypolipidemic drugs. To substantiate our findings we report here the biological activity profile in detail. The present invention is based on the finding that the introduction of a double bond in A16-position of the pregnane skeleton aborts the progestational activity while retaining its hypolipidemic activity. Accordingly such compounds have the potential to be used as hypolipidemic agents. The hypolipidemic activity of these compounds were tested in tyloxapol induced hyperlipidemia in Charles Foster rats (Table-2). The compound 3a-hydroxypregna-5, 16-dien-20-one exhibited significant lipid lowering effect and was therefore selected for detailed evaluation. Accordingly, the present invention provides a process for the preparation of an anti-hyperlipidemic composition comprising mixing pregnane compound of general formula 14 as given in the accompanying drawing which has no hydroxyl group at C-17 position and contain a double bond in or on the D-ring optionally along with other drug molecule selected from anti platelets, antithrombotic, hypolipoproteinemic and pharmaceutically acceptable exciepient , additive or a carrier such as herein described in the ratio, of 1:4 of compared to other ingredients at temperature ranging between 20 to 50 °C to get the homogenous mixture of desired composition. In an embodiment of the present invention the pregnane compound used is selected from group consisting of 3P-Hydroxypregna-5,16-dien-20-one-acetate (7); 3ß-Hydroxypregna-5,16-dien-20-one (8) ; 3(3-Hydroxypregna-5-en-20-one-acetate (9); 3p-Hydroxypregna-5-en-20-one-(10); 5,17(20)-Pregnadien -3,16-diol-diacetate ( 11); 5,17(20)-Pregnadien-3,16-diol-diacetate (12); 16-Dehydroprgestrone (13); or Guggulsterone (6). In another embodiment of the present invention the other drug molecules is selected from group consisting of anti platelets e.g. aspirin, anti-thrombotic e.g. heparin, other hypolipoproteinemic drug such as gemfibrozil, clofibrate and guglip. In yet another embodiment of the present invention the pharmaceutically acceptable additive is selected from the known additives and carriers. In yet another embodiment of the present invention the effective dose of pregnane compound is ranging between 50mg/kg to 650 mg/kg. In yet another embodiment of the present invention the dose of 3ß-Hydroxypregna-5,16-dien-20-one (8) in human is ranging between 400 to 2000 mg single dose. In yet another embodiment of the present invention the composition reduces the cholesterol level ranging between 25 to 53%. In yet another embodiment of the present invention the composition reduces the serum trigycerides ranging between 24 to 62% In yet another embodiment of the present invention the composition showed a decrease of low density lipoprotein (LDL) ranging between 26 - 90 %. In yet another embodiment of the present invention the composition is administered orally, peritonially or any other mode. In yet another embodiment of the present invention the composition is significant hypolipidimic effect and is relatively safe and well tolerated. In yet another embodiment of the present invention the composition is used in the form of tablets, capsules and injectables. In yet another embodiment of the present invention the composition is used for the treatment of animals selected from rat, mice, cat, guinea pig and mammals such as rabbit, monkey including human being. Accordingly, the present invention provides a method for the treatment of hyperlipidemia comprising to a subject suffering therefrom, a pharmaceuticaily effective amount of a composition comprising comprising an effective amount of pregnane compound of general formula 14 which has no hydroxyl group at C-17 position and contain a double bond in or on the D-ring optionally along with other drug molecules and pharmaceuticaily acceptable additive or a carrier. in an embodiment of the present invention the pregnane compound used is selected from group consisting of 3ß~Hydroxypregna-5,16-dien-20-one-acetate (7); 3ß-Hydroxypregna-5.16-dien-20-one (8) ; 3p-Hydroxypregna-5-en-20-one-acetate (9); 3ß-Hydroxypregna-5-en-20-one-(10); 5,17(20)-Pregnadien -3,16-diol-diacetate (11); 5,17(20)-Pregnadien-3,16-dioI-diacetate (12); 16-Dehydroprgestrone (13); or Guggulsterone (6). In another embodiment of the present invention the other drug molecules is selected from group consisting of anti platelets e.g. aspirin, anti-thrombotic e.g. heparin, other hypolipoproteinemic drug such as gemfibrozil, clofibrate and guglip. In yet another embodiment of the present invention wherein the other drug molecules (at their effective doses) is selected from group consisting of anti platelets e.g. aspirin (-75-150 mg/day), anti-thrombotic e.g. heparin (15000 unit 3-4 times a day), other hypolipoproteinemic drug such as gemfibrozil (300 mg/BD), clofibrate (1.5-25 gm/day) and guglip (1500 mg/day). In yet another embodiment of the present invention the pharmaceutically acceptable additive is selected from the known additives and carriers. In yet another embodiment of the present invention the effective dose of pregnane compound is ranging between 50mg/kg to 650 mg/kg. In yet another embodiment of the present invention the dose of 3ß-Hydroxypregna-5,16- dien-20-one (8) in human is ranging between 400 to 2000 mg single dose. In yet another embodiment of the present invention the composition reduces the cholesterol level ranging between 25 to 53%. In yet another embodiment of the present invention the composition reduces the serum trigycerides ranging between 24 to 62% In yet another embodiment of the present invention the composition showed a decrease of low density lipoprotein (LDL) ranging between 26 - 90 %. In yet another embodiment of the present invention the composition is administered orally, peritonially or any other mode. In yet another embodiment of the present invention the composition is significant hypolipidimic effect and is relatively safe and well tolerated. In yet another embodiment of the present invention the composition is used in the form of tablets, capsules and injectables. In yet another embodiment of the present invention the composition is used for the treatment of animals selected from rat, mice, cat, guinea pig and mammals such as rabbit, monkey including human being. HYPOLIPIDEMIC ACTIVITY Compound 3a-hydroxy pregna-5,16-dien-20-one (8) was found to possess marked lipid lowering activity in rats, rabbits and rhesus monkeys. Hypolipidemic activity was evaluated in normal, tyloxapol and diet induced hyperlipidemic rats, hyperlipidemic rabbits and normal rhesus monkeys. The rats were divided into control, hyperlipidemic and hyperlipidemic plus drug treated groups containing six animals in each group. Hyperlipidemia was induced by administration of tyloxapol (400 mg/kg i.p.) and high fat diet. All animals were maintained on a special diet containing cholesterol (5%), cholic acid (1.5%), casein (20%), fat (hydrogenated vegetable oil) (15%), vitamin mixture (1%), salt mixture (1%), gram (15%), wheat (30%) and wheat bran for a period of 36 days. Compound 3a-hydroxypregna-5, 16-dien-20-one and standard drugs (-clofibrate, progesterone and gugulipid) were macerated with 0.2% aqueous gum acacia suspension. The suspension was fed orally simultaneously with high fat diet at the doses and periods mentioned in the tables. The animals of control group received same amount of gum acacia or normal saline by similar route of administration. At the end of the experiments, blood was withdrawn from aorta and animals were sacrificed and livers immediately taken out for other detailed examinations like tissue lipids. Lipid Estimation Serum cholesterol Serum cholesterol was estimated by enzymatic colorimetric method described by P. Trinder, Ann. Clin. Biochem., 6,24(1969). Principle The esterified cholesterol is broken down into free cholesterol and fatty acids by enzymatic cleavage with cholesterol esterase, then free cholesterol is oxidised with cholesterol oxidase to give delta4-cholesterone. The formation of cholesterone is proportional to the quantity of cholesterol present. Procedure Following reagents supplied in the kit by Boehringer Mannheim, West Germany, were used: Solution No. 1 .Buffer potassium phosphate phenol and methanol; Solution No. 2.4-aminophenazone, methanol, hydroxypolyethoxydodecane. Solution No. 3.Cholesterol esterase, cholesterol oxidase and peroxidase enzymes were present in Solution No. 4.(Final Reagent): To make final reagent, 5 ml of solution 1 and 5 ml of solution 2 were mixed and 50 ael of solution 3 was added to this solution (10 ml of solution 4 was sufficient for 4 samples), 0.02 ml of serum added to 2 ml of solution 4, was mixed well and incubated in water bath at 30° C for 15 mts. Absorbance was then read against reagent blank (solution No. 5) at 546 nm on Eppendorf Analytical System Photometer 6210. The results of the experiments are shown in table 2 to 13. Serum triglycerides Serum triglyceride was estimated by enzymatic method described by Wahlefeld,A.W. (1974) (Wahlefeld, A.W., in : Triglycerides Determination after Enzymatic hydrolysis, Page 183Iff in H.U. Bergameyer, ed. Methods of Enzymatic analysis: 2nd English Edition, Academic Press, Inc., NY.). Principle The determination of triglyceride depends on a double enzymatic determination of glycerol. The free glycerol is subtracted from the total glycerol measured after alkaline hydrolysis of serum or plasma, the difference corresponds to the glyceride glycerol or the natural fat content (mole/mole), only traces of mono and diglycerides being present in the plasma. The decrease of extinction due to oxidation of nicotinamide adenine dinucleotide reduced form (NADH) is measured. Procedure Following reagents supplied in the kit by Boehringer Mannheim, Germany, were used. (1) Substrate/enzymes (2) Glycerol kinase. Contents of the bottle 1 were dissolved in 14 m! of redistilled water. 2.5 ml of the solution was mixed with 0.05 ml of serum and incubated at 20-25° C for 10 minutes. Absorbance (Al) was read at 365 nm against the reagent blank (solution 1). Then 0.01 ml of glycerol kinase was added to above samples and kept for 10 minutes in water bath at 20-21° C. Absorbance (A2) was read at 365 nm on Eppendorf Analytical system photometer 6210. The results are indicated in table 3,5,7,8,9,10,11 and 13. HDL-cholesterol Principle Chylomicrons, VLDL (very low density lipoproteins) and LDL-C (low density lipoproteins cholesterol) are precipitated by adding phosphotungustic acid and magnesium chloride to the sample. Centrifugation leaves only the HDL-C (high density lipoproteins cholesterol) in the supernatant. Their cholesterol content is determined enzymatically as cholesterol (Mimation. The results are indicated in table 6,7,12 and 13. LDL-Cholesterol LDL-C is calculated after estimating serum total cholesterol (TC), HDL-C and triglycerides (TG) by the Friedewald formula (W.T. Friedewald et. al., Clin. Chem., 18, 499(1972) which is as follows: The results are indicated in table 6,7,12 and 13. LDL-C = TC - TG/5 - HDL-C mg/dl The values obtained are reliable provided that • no chylomicrons are present in the sample • the TG concentration does not exceed 400 mg/dl. Statistical analysis: Data were analysed using student's 't' test. Hyperlipidemic group was compared with control group, and drug treated group in hyperlipidemic rats. The p Hypolipidemic activity in normal rats: In normal rats, 3a-hydroxypregna-5,16-dien-20-one of the formula 8, at 50 mg/kg produced a significant lowering of serum cholesterol and triglycerides by 39.5 and 35% respectively. Clofibrate at the same dose caused lowering in serum cholesterol and triglycerides by 35 and 28% respectively. Table 3. Hypolipidemic effect of 3a-hydroxypregna-5,16-dien-20-one (8) in normal rats (Formula Removed) @ 3a-hydroxypregna-5,16-dien-20-one Values * Mean ft SD. Figure in parenthesis represent number of animals. It can be inferred from this table that the lipid lowering activity was comparable in both groups and the animals did not develop any tolerance to the compound even after administering for 30 days (Table-3). Hypolipidemic activity in tyloxapol treated rats In 20 male rats (average wt. 300 g) hypercholesterolemia was produced by tyloxapol as described above. 3a-hydroxypregna-5,16-dien-20-one of the formula 8 was administered by intraperitoneal route in the dose of 50 mg/kg, 15 minutes after subcutaneous injection of tyloxapol. Twenty hours after the treatment it was observed that animals treated with the same compound had a fall of 37.5% as compared to untreated animals (Table-4). Table 4. Hypolipidemic effect of 3a-hydroxypregna-5,16-dien-20-one in tyloxapol treated rats (Formula Removed) a) 3a-hydroxypregna-5,16-dien-20-one * Values are mean n SD. Figures in parenthesis represent number of animals. it can be inferred from the table that the compound 3a-hydroxy pregna-5,16-dien-20-one produces a significant hypolipidemic effect to the tune of 37.57 as compared to standard drug clofibrate which produces a 14.6% lipid lowering in tyloxapol induced hyperlipidemic rats. Hypolipidemic activity in diet induced hyperlipidemic rats Twenty three normal male rats average weight 110-120 g were taken for study and were divided into four groups. Group I: animals received special diet and 3a-hydroxypregna-5,16-dien-20-one 50 mg/kg p.o. in 1% gum acacia. Group II: animals received 3a-hydroxypregna-5,16-dien-20-one, 100 mg/kg p.o. in 1% gum acacia and special diet. Group III: animals received special fat diet and 1% gum acacia and served as control. Group IV: animals were fed with CDRI stock diet and served as normal control. All animals were sacrificed at the end of 36 days. Blood was drawn from the tail at 10 days and from the aorta at the time of sacrifice for estimations of serum cholesterol triglycerides and HDL-cholesterol. LDL-cholesterol was calculated as described. Table 5. Effect of 3a-hydroxypregna-5,16-dien-20-one (8) on serum cholesterol and triglycerides in hyperlipidemic rats. (Formula Removed) @ 3a-hydroxypregna-5,16-dien-20-one * HFD = High fat diet Values are ** Mean n SD. Figures in parenthesis are number of animals. Table 6. Effect of 3a-hydroxypregna-5,16-dien-20-one (8) on HDL-cholesterol and LDL-cholesterol in hyperlipidemic rats. (Formula Removed) @ 3a-hydroxypregna-5,16-dien-20-one * HFD = High fat diet, !! (Decrease compared with Group I Data) Values are ** Mean fi SD. Figures in parenthesis are number of animals. Results It can be inferred from the tables 5&6 that the animals treated with 3a-hydroxypregna-5,16-dien-20-one, at 50 and 100 mg/kg showed a significant lowering in serum cholesterol by 30.9 and 58.5%, triglycerides by 55 and 62%, LDL-cholesterol by 26.8 and 74.2% respectively whereas the HDL-C showed an increase by 18.0 and 15.7% respectively. Table 7. Effect of compound 3a-hydroxypregna-5,16-dien-20-one (8) on lipid profile in rats. (Formula Removed) @ 3 a-hydroxypregna-5,16-dien-20-one * HFD = High fat diet Values as statistical significance; NS = Not significant The mean of 36 days of diet alone and along with 3a-hydroxypregna-5,16-dien-20-one was analysed, by one way analyses of Variance for each variable separately. F ratio of cholesterol for 10 days and 36 days was found to be significant at 1%. The mean of cholesterol after 10 days and 36 days and TG at 36 days for the two dosages with and without the drug have been compared by the student's 't' test and it is evident from this table that there is a statistically significant decrease of cholesterol, TG and LDL-C with drug treatment. Effect of 3a-hydroxypregna-5,16-dien-20-one and progesterone was studied in hyperlipidemic Charls Foster male rats (ave. wt. 250-300g). Animals were fed with high fat diet for 10 days as described above. Table 8. Effect of 3a-hydroxypregna-5,16-dien-20-one (8) in hypercholesterolemic rats. (Formula Removed) @ 3a-hydroxypregna-5,16-dien-20-one * = p HFD = High fat diet Values are ** Mean fl SD. Figures in parenthesis are number of animals. It can be inferred from the table that 3a-hydroxypregna-5,16-dien-20-one at 50 mg/kg produced a significant fall in serum cholesterol and triglycerides but to achieve a comparable fall very high doses of progesterone(50 mg/kg) was needed which is an impractical dose and is never used in the clinical setting. Hypolipidemic activity in hyperlipidemic rabbits Effect of 3a-hydroxypregna-5,16-dien-20-one was studied on hypercholesterolemic albino rabbits. Serum cholesterol and triglycerides were estimated by the methods mentioned earlier. Twelve male albino rabbits (approx. 1.5-2 kg) on a CDRI stock diet were made hyperlipidemic by feeding daily cholesterol 0.5 g/kg in 2ml of groundnut oil for 45 days and then blood was drawn from the marginal vein in the ear of rabbits for serum cholesterol and triglycerides estimations. Table 9. Effect of compound 3a-hydroxypregna-5,16-dien-20-one (8) in hyperlipidemic male albino rabbits. (Formula Removed) @ 3a-hydroxypregna-5,16-dien-20-one Mean absolute values mg/dl. Two controlled experiments were carriedout for a period of three months. In one set of experiments, the control group received 0.5 g/kg of cholesterol for 90 days and 3a-hydroxypregna 5,16-dien-20-one in dose of 100 mg/kg and 50 mg/kg while in the control group (given only cholesterol) a massive rise of serum cholesterol and triglycerides were seen after 90 days, the addition of 3a-hydroxypregna-5,16-dien-20-one at 50 and 100 mg/kg doses kept the rise well under control. The decrease percentage at 100 mg dose was 28% at 30 days to 52% at 90 days for cholesterol and 45 to 81% for triglycerides. In the 50 mg/kg dose group the decrease percentage ranged from 40% at 30 days to 50% at 90 days for cholesterol and 45% at 30 days to 75% at 90 days for triglyceride (Table-9). Fable 10. Hypolipidemic activity in hyperlipidemic rabbits (120 days experiment). (Formula Removed) @ 3a-hydroxypregna-5, l6-dien-20-one In another set of experiments with hyperlipidemic rabbits after 120 days of administration of 3a-hydroxypregna-5,16-dien-20-one at dose of 100 mg/kg compound 3a-hydroxypregna-5,16-dien-20-one, serum Cholesterol showed a reduction to the tune of 74% and triglycerides to 60% while gugulipid at a comparable dose of 100 mg/kg exhibited reduction of serum cholesterol by 81% and triglycerides by 65% (Table-10). This decrease in level of serum cholesterol and triglycerides confirms the dose and duration dependent hypolipidemic efficacy of compound 3a-hydroxypregna-5,16-dien-20-one. Hypolipidemic effect in Rhesus monkeys In Rhesus monkeys: 3a-hydroxypregna-5,16-dien-20-one was administered orally daily for 90 days in doses of 62.5, 125 or 650 mg/kg to different groups of animals. Table 11. Hypolipidemic effect of compound 3a-hydroxypregna-5,16-dien-20-one (8) in rhesus monkeys. (Formula Removed) @ 3a-hydroxypregna-5,16-dien-20-one Mean + SD values mg/dl. Significant decrease in serum cholesterol was observed (47-52%). At 90 days, percentage decrease in triglycerides varied from 24.30 to 35.5% Table 12. Effect of compound 3a-hydroxypregna-5,16-dien-20-one (8) on HDL-cholesterol and LDL-cholesterol in rhesus monkeys (Formula Removed) @ 3a-hydroxypregna-5,16-dien-20-one Mean absolute values mg/dl; * = + denotes increase; - denotes decrease The compound also caused a marked decrease in low density lipoprotein (74.8-90%) whereas changes in HDL-Cholesterol were not significant. TOXICITY DATA Acute toxicity a. Rats: The compound 3a-hydroxypregna-5,16-dien-20-one was administered in 1% gum acacia suspension by intubation to male and female Charles Foster rats in doses of 125, 250 and 500 mg/kg/day respectively for 90 days. All the rats tolerated the compound well throughout the period of the experiment. There was a steady gain in the body weight in all animals, males gained more weight than the females. No significant abnormality was detected in any of the haematological and biochemical parameters. b. Rhesus monkeys: The compound 3a-hydroxypregna-5,16-dien-20-one was administered as a suspension in 1% gum acacia orally by intubation for 90 days. Initially, all the animals received a daily dose of 62.5 mg/kg b.w. and after 7 days the dose was escalated, 125 mg/kg or 250 mg/kg in 2 different groups of animals. Control animals received 1% gum acacia only. The monkeys tolerated the compound well and no behavioral or physical abnormalities could be observed. The weight change observed in control and treated animals was similar. No significant change was detected in any of the haematological and biochemical parameters. Mutagenicity studies At the dose levels of 100, 200 and 400 mg/kg orally the compound 3a-hydroxypregna-5,16-dien-20-one is not found to be mutagenic in mice and rats as indicated by a negative micronucleus test. The dominant lethal test was done in male rats with the doses of 50, 100, 200 mg/kg/day for 70 consecutive days and after treatment. The incidence of dominant lethality in the female rats impregnated by treated animals was not significantly higher than that of controls. Teratology studies a. Rats: Compound 3a-hydroxypregna-5,16-dien-20-one was administered orally at the daily doses of 125 mg and 250 mg/kg body weight in Charles Foster rats during the period of organogenesis. In highest dose group, comparatively high resorptions were recorded. Gross, visceral and skeletal anomalies were recorded in control and treated groups. These were all incidental and insignificant findings. Hence, the compound 3a- hydroxypregna-5,16-dien-20-one is not teratogenic or foetotoxic at the dose of 125 mg/kg which is 2.5 times of ED50. b. Rabbits: The compound 3a-hydroxypregna-5,16-dien-20-one was administered orally at the daily doses of 62.5 mg/kg and 125 mg/kg body weight in rabbits during the period of organogenesis. Maternal body weight gain was low in treated rabbits as compared to controls. Initial, gross, visceral and skeletal anomalies were seen in both control and treated groups and these were insignificant in number. Hence, the compound 3a- hydroxypregnane-5,16-dien-20-one is not fetotoxic or teratogenic at the doses used in the experiment. PHARMACOLOGICAL DATA Effects on the Central Nervous System (CNS) a.Gross Effects: The compound 3a-hydroxypregnane-5,16-dien-20-one was tested on albino mice of either sex weighing between 15-20 g. At least 5 animals were used at each dose level. No CNS effects were observed upto a dose of 216.0 mg/kg i.p. b.Spontaneous motor activity: This was measured with a Bijou Actophotometer and was unaffected by 400.0 mg/kg i.p. c.Analgesic activity: This was tested by the Eddy's Hot Plate method and the phenylquinone (2 mg/kg) writhing test. The compound was devoid of analgesic activity upto a dose of 400 mg/kg i.p. d.Effect on Rectal Temperature: The rectal temperature was measured with YSI electronic thermometer and was not affected by the compound over a 5 hour observation period at a dose of 400.0 mg/kg i.p. (1/5 LD50). e.Anticonvulsant activity: The supramaximal electro-shock seizure pattern was not modified by 400.0 mg/kg i.p. of the compound 3a-hydroxypregna-5,16-dien-20-one Effect on Blood Pressure and Respiration in Anaesthetised Cats Cats (2.4 kg) anaesthetised with pentobarbitone sodium (30 mg/kg i.v.) were used in these studies. The compound 3a-hydroxypregna-5,16-dien-20-one produced hypotension which lasted for about 35 min at a dose of 10 mg/kg i.v. with practically no effect on pressor and depressor responses to adrenaline, acetylcholine, histamine and isoprenaline respectively. There was no change in the heart rate or respiration. Effect on isolated guinea pig ileum preparation The results show that the compound 3a-hydroxypregna-5,16-dien-20-one (5-20 mg/ml) causes a weak nonspecific anti-spasmodic effect on the isolated guinea pig ileum preparation. Platelet aggregation inhibition activity: Platelet aggregation inhibiting activity of 3a-hydroxypregna-5,16-dien-20-one and clofibrate was tested on a platelet aggregometer at 2x10 AM concentration which caused 30% inhibition and at 5x10AM concentration of the compound 3a-hydroxypregna-5,16-dien-20-one there was complete inhibition when adenosine diphosphate (ADP) and adrenaline were based as platelet aggregation inducing agents. PHASE-I CLINICAL TRIALS The compound 3a-hydroxypregna-5,16-dien-20-one was taken up for phase 1 clinical trials after obtaining permission from the Drugs Controller of India. The study was performed in two stages (a) single dose study and (b) multiple dose study. Initially, the single oral dose tolerance study was undertaken. The design of this study was double blind, placebo controlled, noncrossover. 30 healthy male volunteers were included in the study between the age group of 16-65 years. Placebo was given to 6 volunteers in the form of identical capsules. The drug 3a-hydroxypregna-5,16-dien-20-one was given in escalating doses of 200, 400, 800, 1200 and 2000 mg single oral dose to 4 volunteers in each dose group. None of the 30 volunteers had any significant side effect requiring specific management. Only minor nonspecific side effect was found in a single volunteer at 400 mg dose who developed constipation and mild gaseous distention which disappeared in the evening of the same day without any treatment. Thus in all the 30 volunteers the compound 3a-hydroxypregna-5,16-dien-20-one was well tolerated. The multiple dose studies were planned in two doses (a) 10 volunteers were given 400 mg daily for 4 weeks: (b) 10 volunteers were given 800 mg daily for 4 weeks. In view of liver function changes in one volunteer, the high dose studies were extended in additional 6 volunteers to confirm its safety. During the period of drug administration the volunteers were examined once a day. The investigations were done on the day zero and again on the 28th day of continuous use. All the 10 volunteers of low dose group completed the trial and the compound 3a-hydroxypregna-5,J,6-dien-20-one was well tolerated by all of them. No derangement in hematological, biochemical or electrocardiogram (EKG) parameters was observed after 4 weeks of treatment. None of these 10 volunteers had any side effects. 10 healthy or mildly hyperlipidemic volunteers were included in the high dose (800 mg/day) study for 28 days in 2 divided doses. Only one volunteer had a minor side effect in form of mild abdominal discomfort and 2-3 loose motions per day and nausea in the first 3-4 days and then was totally relieved. None of the other 9 cases showed any derangement of hematological, biochemical or EKG parameters. Six additional volunteers were given compound 3a-hydroxypregna-5,16-dien-20-one 800 mg daily in divided doses. All the six volunteers tolerated the drug very well and no side effects were observed. The hematological, biochemical and EKG parameters remained well within the normal limits. In all the twenty six cases in the multiple dose study, there was no effect on platelet aggregation, serum electrolytes. There was no significant change in the post-treatment lipid levels as compared to pre-treatment values. This may be because the effect is always less marked in normal individuals. EXPLORATORY EFFICACY CLINICAL TRIALS IN HYPERLIPIDEMIC PATIENTS Before conducting the phase II clinical efficacy studies five cases of primary hyperlipidemia were taken up for exploratory efficacy trials for a continuous period of 12 weeks to have a feel of the drug. All the 5 cases of primary hyperlipidemia and 4 of these had a combined hypercholesterolemia and hypertriglyceridemia and 1 case had isolated hypertriglyceridemia. 3a-hydroxypregna-5,16-dien-20-one was given in a dose of 400 mg/kg i.e. 200 mg capsules, one capsule in morning and one in evening for 12 weeks. The pattern of effects seen in Table-13 were as follows: all the 5 cases tolerated the drug 3a-hydroxypregna-5,16-dien-20-one well and there was no incidence of side effects or any derangement of the haematological and biochemical parameters. There was little effect in the first 8 weeks but at 12 weeks in 4 out of 5 cases, there was reduction in cholesterol by (Mean n SD, 25.54 ft 13.61%). There was no response in 1 case. In 4 out of 5 cases, there was also a reduction in triglycerides by 24.59 n 13.16% (Mean n SD). The HDL-cholesterol showed a definite increase after 12 weeks in all the 5 cases (15.66 n 9.35%). The atherosclerosis risk ratio i.e. total cholesterol/HDL-cholesterol showed a definite reduction in all the 5 cases after 12 weeks of therapy. Table 13. Lipid profile of hyperlipidemic patients completing trial of 3a-hydroxypregna-5,16-dien-20-one (8) at 400 mg/day dose x 12 weeks (Formula Removed) Thus this exploratory study (Table-13) shows that 3a-hydroxypregna-5,16-dien-20-one (8) has a significant hypolipidemic effect and is relatively safe and well tolerated compound. CONCLUSIONS In order to have a feel of universal hypolipidemic activity of 3a-hydroxypregna-5,16-dien-20-one, its activity is summerised below in different animal models: Table 14: Comparative hypolipidemic effect of (8) in various animal models and man. (Formula Removed) The inference drawn from the data presented in table-14 are that: a.There is a significant fall of serum cholesterol, triglycerides and LDL in various animal models and man. b.There is a mild increase in HDL-C in the animal models as well as in man c.Compound 8 seems to possess a potent and universal hypolipidemic activity. The above findings show that 3a-hydroxypregna-5,16-dien-20-one is a potent hypolipidemic/hypolipoproteinemic agent devoid of hormonal actions unlike the pregnenolones used in oral contraceptives such as progesterone, gestodene and 3-ketodesogestrel. Some progestogens such as norethisterone and levonorgestrel in contrast show rise in lipid levels while maintaining high order of progestogenic activity. The effect on lipid and progestogenic activity in pregnenolones, thus is not interrelated and in fact it has been proposed that the effect on lipids is a manifestation of the down-regulatory action on androgen receptors. This finding shows that it is possible to get hypolipidemic agents which have no progestational activity which adds a new dimension to the dissociation of hormonal action as well as lipid levels modulating action and adds to the understanding of structural correlates of these various biological activities of pregnenolones. A process for the preparation of an anti-hyperlipidemic composition comprising mixing pregnane compound of general formula 14 as given in the accompanying drawing which has no hydroxyl group at C-17 position and contain a double bond in or on the D-ring optionally along with other drug molecule selected from anti platelets, anti-thrombotic, hypolipoproteinemic and pharmaceutically acceptable exciepient , additive or a carrier such as herein described in the ratio, of 1:4 of compared to other ingredients at temperature ranging between 20 to 50 °C to get the homogenous mixture of desired composition. A process as claimed in claim 1 wherein the pregnane compound used is selected from group consisting of 3p-hydroxypregna-5, 16-dien-20-one-acetate (7); 3ß-hydroxypregna-5,16-dien-20-one (8) ; 3ß-hydroxypregna-5~en-20-one-acetate (9); 3p-hydroxypregna-5-en-20-one-(10); 5,17(20)-pregnadien -3,16-diol-diacetate ( 11); 5,17(20)-pregnadien-3,16-diol-diacetate (12); 16-dehydroprgestrone (13); or guggulsterone (6). A process as claimed in claims 1 to 2 wherein the other drug molecules is selected from group consisting of anti platelets such as aspirin, anti-thrombotic e.g. heparin, other hypolipoproteinemic drug such as gemfibrozil, clofibrate and guglip. A process as claimed in claims 1 to 3 wherein the pharmaceutically acceptable additive is selected from the known additives and carriers such as lactose, microcrystalling cellulose, ethyl cellulose, hydroprophlmethyl cellulose, starch soluble, sodium carboxy methyl cellulose, disodium calcium phosphatae, talc and magnesium ster\arate as lubricant not more than 2%. A process as claimed in claims 1 to 4 wherein the mixing of the compound with pharmaceutically acceptable additive is carried out and compressed in tablets and thereafter crushed , sieved and then filled in the capsules for better absorption. A process as claimed in claims 1 to 5 wherein the mixing of the compound with pharmaceutically acceptable additive is carried out by dissolving the compound in organic solvent selected from ethanol, propanol and preparing the paste with additive in a ratio of 1:4 of compound to other ingredients. 7. A process for the preparation of an anti-hyperlipidemic composition as herein described with reference to examples and figures accompanying the specification.

Documents:

2302-del-1995-abstract.pdf

2302-del-1995-claims.pdf

2302-del-1995-correspondence-others.pdf

2302-del-1995-correspondence-po.pdf

2302-del-1995-description (complete).pdf

2302-del-1995-drawings.pdf

2302-del-1995-form-1.pdf

2302-del-1995-form-13.pdf

2302-del-1995-form-2.pdf

2302-DEL-1995-Form-3.pdf

2302-DEL-1995-Form-4.pdf

2302-del-1995-form-9.pdf

2302-del-1995-petition-138.pdf