WO2007122636A1

WO2007122636A1 - Pharmaceutical compositions containing long chain fatty acids as excipients as well as a process for manufacturing the same

Info

Publication number: WO2007122636A1
Application number: PCT/IN2007/000161
Authority: WO
Inventors: Rajesh Jain; Kour Chand Jindal; Chandrashekhar Shankar Patil
Original assignee: Panacea Biotec Ltd
Priority date: 2006-04-25
Filing date: 2007-04-23
Publication date: 2007-11-01

Abstract

Pharmaceutical compositions comprising at least one higher primary aliphatic alcohol selected from a group of alcohols having 20 to 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor, or its pharmaceutically acceptable salts, esters, solvates or hydrates thereof, preferably clopidogrel as active agent; optionally with one or more pharmaceutically acceptable excipient(s); and process of preparation of such compositions are provided. Also described are method of treatment and use of such compositions particularly for providing antiplatelet activity optionally with lipid lowering effect and usefulness for the management of cardiovascular disease associated with hyperlipidemia. The compositions of the present invention optionally comprises at least one another organic component selected from a group comprising resins and pigments, hydrocarbons, sterols, esters, ketones, aldehydes, phenolic compounds, and mixtures thereof.

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING LONG

CHAIN FATTY ACIDS AS EXCIPIENTS AS WELL AS

A PROCESS FOR MANUFACTURING THE SAME

FIELD OF THE INVENTION The present invention relates to novel pharmaceutical compositions comprising at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor, its analogs, derivatives, or salts thereof, and process of preparation thereof. The present invention further relates to the method of treatment and use of such compositions particularly for providing antiplatelet activity optionally along with lipid lowering effect thus demonstrating usefulness for the management of cardiovascular disease optionally associated with hyperlipidemia.

BACKGROUND OF THE INVENTION

Elevated serum cholesterol levels (>200 mg/dl) have been indicated as a major risk factor for heart disease, particularly atherosclerotic vascular diseases, is the major cause of morbidity and mortality in middle age and elderly people worldwide (Pyorala et al., 1994; Sans et al., 1997). The clinical strategy for preventing CHD is based on the control of blood cholesterol levels and minimizing the thickening of the blood vessels by means of primary and secondary preventive measures.

Cholesterol is synthesized within the body for certain essential functions. But, when in excess, bad cholesterol tends to deposit on the boundaries of blood vessels (formation of plaques) leading to the increased risk of heart diseases. The liver is the site of synthesis and secretion of very low density lipoproteins (VLDL), which are subsequently metabolized to low density lipoproteins (LDL) in the circulation. LDL is the predominant cholesterol- carrying lipoproteins in the plasma and an increase in their concentration is correlated with increased atherosclerosis. Thromboxane A₂ (TXA₂) is a major product of arachidonic acid metabolism in platelets. It is a powerful inducer of platelet aggregation and for release of platelet aggregation factor (PAF). The elevated plasma levels of low-density lipoproteins (LDL) and total cholesterol (TC) alone and in tandem with platelet aggregation play a major role in atherosclerosis and CHD development.

Policosanol is the trivial name of a mixture of high molecular weight alcohols isolated particularly from sugarcane, wherein octacosanol is the main component. More particularly, policosanol is a mixture of higher primary aliphatic alcohols containing 24 to 34 carbon atoms involves in inhibiting cholesterol biosynthesis at a step located in between acetate consumption and mevalonate production, increases the number of low-density lipoprotein cholesterol (LDL-C) receptors (Menendez et al., 1994), and has a favorable effect on HDL-C (Castano et al., 1999). Exact mechanism for antiplatelet activity of policosanol is obscure, but it may be associated to significant reduction in serum Thromboxane (TXB₂) levels (Carbajal, 1998). Various studies revealed that policosanol inhibit cholesterol biosynthesis, increase the number of LDL-C receptors thereby decreases serum TC, LDL-C and increase HDL levels (Menendez et al., 1994). The effects of Policosanol treatment on rat platelet aggregation were studied, where it has showed dose dependent response. Policosanol (50-200 mg/kg, single doses) inhibited ADP-induced platelet aggregation in platelet-rich plasma, while lower doses (25 mg/kg) did not change responses to ADP significantly. However, rats treated with this dose (25 mg/kg) for 4 weeks showed a significant inhibition of platelet aggregation in PRP when a submaximal ADP concentration was administered. In one study, when administered at 5 to 20 mg/day, policosanol has been shown to decrease the risk of atheroma formation by reducing platelet aggregation, endothelial damage, and foam cell formation in animals.

Platelets are blood factors that adhere to damaged cells that line blood vessels and stick together (aggregate) to form a clot, which acts to patch the damage. "Blood-thinners" (which are also called anti-coagulants) are commonly prescribed for those at risk of cardiovascular disease. Blood thinners are intended to prevent the formation of abnormal clots, which can detach from the vessel wall and cause a stroke or heart attack. Anticoagulants such as Coumadin® (warfarin), and platelet aggregation inhibitors such as aspirin and clopidogrel (Plavix®) are often used to prevent these clots. Various animal studies have shown that policosanol decreases platelet aggregation by decreasing the synthesis of platelet-aggregating thromboxane B2 (TXB2), with no effect on prostacyclin (PGI2). Studies demonstrate policosanol reduces platelet aggregation induced by a number of experimental substances, with dose-dependent increases from 10-50 mg/day. Policosanol alone at 20 mg/day was more effective than 100 mg aspirin at reducing platelet aggregation induced by ADR and equally effective when induced by epinephrine and collagen. U.S. Patent No. 5,856,316 discloses a process for obtaining policosanol from sugarcane wax and their utilization in the treatment of hypercholesterolem ia..

As per the various in vitro studies, policosanol inhibit cholesterol biosynthesis at a step located in between acetate consumption and mevalonate production In addition, in vitro studies also showed that policosanol increase the number of LDL-C receptors (Menendez et al., 1994). This accounts for the ability of policosanol not only to decrease total cholesterol, but also to decrease LDL serum levels and increase HDL levels. In vivo studies in correlation with in vitro studies demonstrated that policosanol inhibited TC and LDL-C induced by atherogenic diet suggesting possible inhibition of cholesterol biosynthesis (Menendez et al., 1996).

Clopidogrel (Plavix®) is a thienopyridine derivative co-developed by Sanofi-Synthelabo and Bristol-Myers Squibb. Clopidogrel hydrogen sulfate, the currently marketed form, was described for the first time in EP 281459. It is selective for ADP (adenosine diphosphate) induced platelet aggregation and is a prodrug that must undergo hepatic metabolism to become active. It acts by irreversibly modifying the platelet ADP receptors, and selectively inhibits the binding of ADP to its platelet receptors & the subsequent anteroposterior mediated activation of glycoprotein GP Ilb/IIIa complex, thereby inhibiting platelet aggregation., however it does NOT affect the cyclooxygenase pathway and thromboxane- A2 like aspirin.

It can be seen from the scientific literature that there is still an unmet need for cost-effective and easier to formulate compositions preferably as fixed dose combinations (FDCs) comprising of platelet aggregation inhibitor(s) and/or antihyperlipidemic agent(s) with possible additive or potentiating or synergistic action and a method of administration which would provide an antiplatelet activity preferably with a lipid lowering effect as well, particularly, for the management of cardiovascular disease associated with hyperlipidemia, with an acceptable safety profile.

OBJECTIVE OF THE INVENTION

It is an objective of the present invention to provide novel pharmaceutical composition comprising at least one higher primary aliphatic alcohol preferably selected from a group comprising alcohols having about 20 to about 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor; and optionally one or more pharmaceutically acceptable excipient(s).

It is an objective of the present invention to provide novel pharmaceutical compositions comprising at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof and a platelet aggregation inhibitor as active agent; optionally with one or more pharmaceutically acceptable excipient(s), wherein the platelet aggregation inhibitor is clopidogrel or its pharmaceutically acceptable salts, esters, prodrugs, solvates, hydrates, or derivatives thereof.

It is another objective of the present invention to provide a novel pharmaceutical composition comprising at least one higher primary aliphatic alcohol preferably selected from a group comprising alcohols having about 20 to about 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor; and optionally one or more pharmaceutically acceptable excipient(s), wherein the mixture of higher primary aliphatic alcohols additionally comprises of at least one another organic component selected from a group comprising resins and pigments, hydrocarbons, sterols, esters, ketones, aldehydes, phenolic compounds, and the like or mixtures thereof.

It is another objective of the present invention to provide process of preparation of novel pharmaceutical composition which comprises of the following steps: i) isolating at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof. ii) treating at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor,, iii) optionally adding one or more pharmaceutically acceptable excipient(s), and iv) formulating into a suitable dosage form.

It is another objective of the present invention to provide process of preparation of novel pharmaceutical composition wherein at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof, is isolated from natural sources or synthetic sources.

It is yet another objective of the present invention to provide a novel pharmaceutical composition comprising of a fixed dose combination of at least one higher primary aliphatic alcohol preferably selected from a group comprising alcohols having about 20 to about 39 carbon atoms or mixtures thereof; and clopidogrel or its pharmaceutically acceptable salts, esters, prodrugs, solvates, hydrates, or derivatives thereof. It is yet another objective of the present invention to provide a method of using such composition which comprises administering to a subject in need thereof an effective amount of the composition.

5 It is another objective of the present invention to provide method of treatment and use of such compositions preferably for providing an antiplatelet activity optionally along with a lipid lowering effect, which are useful particularly for the management of cardiovascular disease optionally associated with hyperlipidemia. The compositions of the present invention preferably provide a potentiating or a synergistic combination of antiplatelet agents.

10

It is also an objective of the present invention to provide a method for lowering serum cholesterol, preventing or inhibiting or treating atherosclerosis and/or reducing risk of or treating a cardiovascular event or disease including coronary artery disease and the like.

15 It is still further objective of the present invention to provide compositions comprising at least one higher primary aliphatic alcohol or mixtures thereof and at least one platelet aggregation inhibitor, its analogs, derivatives, or salts thereof preferably clopidogrel; optionally with pharmaceutically acceptable excipient(s); wherein the combination is effective at low concentrations of either or both the active agents, and provides increased patient compliance.

20

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel pharmaceutical compositions comprising at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor, or its pharmaceutically

25 acceptable salts, esters, prodrugs, solvates, hydrates, or derivatives thereof as active agent; optionally with one or more pharmaceutically acceptable excipient(s). Preferably the platelet aggregation inhibitor is clopidogrel. In an embodiment, the compositions of the present invention additionally comprises at least one another organic component selected from a group comprising resins and pigments, hydrocarbons, sterols, esters, ketones, aldehydes,

30. phenolic compounds, and the like or mixtures thereof.

In an embodiment, the pharmaceutical compositions of the present invention comprises a low

. dose of platelet aggregation inhibitor, its analogs, derivatives, or salts thereof, preferably clopidogrel and is intended to show a synergistic, additive or a potentiated antiplatelet activity

35 with the higher primary aliphatic alcohol(s) or mixtures thereof. The ability of higher primary aliphatic alcohol(s) to reduce induced platelet aggregation and the ability of clopidogrel to inhibit platelet aggregation; when combined in the present invention preferably results in an enhanced antiplatelet activity.

In an embodiment, the compositions for inhibiting platelet aggregation optionally with lowering cholesterol in blood of a mammal, particularly humans or animals, comprise higher primary aliphatic alcohol(s) or esters thereof along with clopidogrel, its analogs, derivatives or salts thereof. In another embodiment, a method for inhibiting platelet aggregation and optionally lowering cholesterol level in blood of a mammal comprises orally administering to the said mammal, a composition comprising a therapeutically effective amount of higher primary aliphatic alcohol(s) in combination with at least one platelet aggregation inhibitor such as clopidogrel, its analogs, derivatives, or salts thereof. Other platelet aggregation inhibitors (PAIs) useful in the present invention include aspirin, statin such as pravastatin, or any other PAIs known to the art.

In an embodiment, the present invention relates to novel pharmaceutical composition comprising a mixture of higher primary aliphatic alcohols selected from high molecular weight alcohols having about 20 to about 39 carbon atoms in an amount of about 0.1 % to about 99.9 % by weight of the composition and at least one platelet aggregation inhibitor, its salts, analogs or derivatives thereof, preferably clopidogrel in an amount of about 0.1 % to about 99.9 % by weight of the composition; additionally comprising at least one another organic component selected from a group comprising resins and pigments, hydrocarbons, esters, ketones and aldehydes, and phenolic compounds in an amount of about 0.1 % to about 70 % by weight of the composition; optionally with pharmaceutically acceptable excipient(s) in an amount of about 0 % to about 99.9% by weight of the composition.

In an embodiment, the higher primary aliphatic alcohol(s) in the present invention is selected from but not limited to a group comprising 1-tetracosanol, 1-hexacosanol, 1-heptacosanol, 1- octacosanol, 1-nonacosanol, 1-tetratriacontanol, 1-triacontanol, 1-hexacontanol, eicosanol, 1- hexacosanol, 1-tetracosanol, 1-dotriacontanol, 1-tetracontanol, and the like or mixtures thereof. Preferably the mixture of higher primary aliphatic alcohols comprises 1-tetracosanol, 1-hexacosanol, 1-heptacosanol, 1-octacosanol, and 1-triacontanol, and the like. In a further embodiment, the present invention provides a composition, wherein the mixture of higher primary aliphatic alcohol(s) is selected from a group comprising such alcohols having about 20 to about 39 carbon atoms preferably comprising 1-tetracosanol, 1-hexacosanol, 1- heptacosanol, 1-octacosanol, and 1-triacontanol, are present in an amount of at least 10% by weight of the composition. In another embodiment of the present invention, the higher primary aliphatic alcohol or mixture of higher primary aliphatic alcohols is isolated from a number of different sources including plant sources such as sugar cane wax and rice bran wax, or animal sources or insects or honeycomb wax. The higher primary aliphatic alcohol(s) used in the preferred embodiment of the invention is obtained from sugar cane wax. It should be understood, however, that the invention is not limited in this regard and that higher primary aliphatic alcohol(s) commonly available from other naturally occurring and/or synthetic sources may be utilized.

In a still further embodiment of the present invention, the composition comprises a mixture of higher primary aliphatic alcohols selected from a group comprising such alcohols having from about 20 to about 39 carbon atoms and other organic component(s) selected from resins and pigments, hydrocarbons, sterols, esters, ketones, aldehydes, and phenolic compounds in the following amounts:

1 -tetracosanol 0.0-2.0 %

1-hexacosanol 0.2-2.0 %

1 -heptacosanol 0.0-1.0 %

1 -octacosanol 30.0-40.0 % 1-triacontanol 6.0-9.5 %

Phytosterols 0.1-1.0 %

Resins and pigments 5.0-10.0 %

Hydrocarbons 1.0-10.0 %

Esters 1.0-10.0 % Ketones and Aldehydes 1.0-10.0 %

Phenolic compounds 0.0-5.0 %

The higher primary aliphatic alcohol or mixtures thereof useful in the present invention are obtained from a natural source such as a naturally occurring wax, a synthetic source or semi- synthetic source or a combination of such sources. The mixture of high-molecular weight aliphatic alcohols of the present invention occur naturally in wax form and are characterized by fatty alcohol chains ranging preferably from 20 to 39 carbon atoms in length. The other organic components such as resins, pigments, hydrocarbons, esters, ketones, aldehydes, phytosterols, phenolic compounds, and the like or mixtures thereof may also be extracted 0161 alongwith the higher primary aliphatic alcohols from the same natural source or extracted from a different natural source and added to the composition or separately synthesized and then added to the composition. It should be understood, however, that the invention is not limited in regard to the source and/or the number or amount of different higher primary aliphatic alcohol(s) used and/or other additional components used in the composition. In another embodiment of the present invention, additional pharmaceutically active agents or other natural components can be used preferably selected from those known to the art as useful agents for providing lipid lowering effect as well as an antiplatelet activity and useful for the management of cardiovascular disease associated with hyperlipidemia.

In a further embodiment, the present invention provides a process for preparing the novel composition. In another embodiment is provided a process of preparation of compositions of the present invention which comprises of the following steps: i) isolating at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof; ii) treating at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor; iii) optionally adding one or more pharmaceutically acceptable excipient(s), and iv) formulating into a suitable dosage form.

In an embodiment, the process of preparation comprises of the following steps: i) isolating the wax described hereinabove, ii) subjecting the wax to extraction with a liquid organic extractant in which primary aliphatic alcohols and other organic components are soluble, iii) recovering said soluble mixture from said extractant, iv) purifying the extract by repeated washing and crystallization, v) drying the extract at temperature preferably below 70⁰C and making it into powder, vi) adding the platelet aggregation inhibitor, vii) optionally adding pharmaceutically acceptable excipients and making it into a suitable dosage form.

The wax is preferably isolated from a number of different sources, including sugar cane wax, beeswax, and rice bran wax, more preferably sugar cane wax. The liquid organic extractant of the present invention are selected from but not limited to a group comprising hexane, heptane, petroleum ether, chlorinated hydrocarbons, methanol, ethanol, isopropyl alcohol, ethyl acetate, acetone, ethyl methyl ketone, and the like, or mixtures thereof. In the said process, the soluble mixture from the said extractant is recovered by distillation, with or without the application of vacuum. The extract is purified preferably by repeated washing and crystallization methods. The solvents used for washing are selected from but not limited to hexane, heptane, petroleum ether, methanol, ethanol, isopropyl alcohol, ethyl acetate, acetone, ethyl methyl ketone, and the like, or mixtures thereof and the solvents for crystallization are selected from but not limited to hexane, heptane, petroleum ether, chlorinated hydrocarbons, methanol, ethanol, isopropyl alcohol, ethyl acetate, acetone, ethyl methyl ketone, toluene, and like, or mixtures thereof. The extract is dried by subjecting it to hot air oven, or by a Fluid bed drier, preferably at temperature below 70⁰C.

In an embodiment, the present invention employs clopidogrel or a compound other than clopidogrel itself that the body metabolizes into clopidogrel, thus producing the same effect as described herein as the preferred platelet aggregation inhibitor: Additionally other PAIs may be employed to obtain the compositions of the present invention.

In the 'Pharmacological study' described hereinafter, the mixture of higher primary aliphatic alcohols selected from a group comprising such alcohols having about 20 to 39 carbon atoms; and other organic components such as resins, pigments, hydrocarbons, sterols, esters, ketones, aldehydes, and phenolic compounds or mixtures; is denoted as Εxtract-A'.

Pharmacological study

Platelet aggregation assay was conducted using human blood. 10 ml of blood was collected from each individual separately into tubes containing ACD (Adenosine Citrate Dextrose) buffer. The blood was pooled and PRP (Platelet Rich Plasma) was isolated by centrifugation. The platelet number was kept constant at 3x10⁵ for all the experiments. 10 μM ADP (Adenosine 5'-diphosphate) and 2 μg/ml of collagen were used to induce the platelet aggregation. 2 mg of drug (clopidogrel denoted as 'CD' or Extract-A denoted as 'PP' or their combination) was dissolved in a mixture of cyclohexane and methanol (150 μl: 45 μl) to obtain a final solution having a concentration of 3.5 mg/ml. No precipitation was observed in the solution at a temperature of 37°C or above. The inhibition of Platelet aggregation were studied using Clopidogrel and Extract-A individually and also in combination at different concentrations. Platelet aggregation experiments were performed using 96 Well ELISA plates.100 μl of PRP was taken into Well and appropriate drug was added. The plate was shaken to mix and read at 405 nm thrice for 5 minutes on a Spectrophotometer. The study results is presented in Table- 1 to Table-4 and shown graphically in Figure- 1 to Figure-4.

Table-1: Effect of Clopidogrel on ADP or Collagen induced platelet aggregation Treatment CD CD CD CD CD

(80 μg) (160 μg) (200 μg) (300 μg) (400 μg) ADPlO(μM) 100% 79.7% 69% 38.5% 3.5%

Collagen (2 μg /ml) 89% 81.8% 58% 40% 0%

Table-2: Effect of Extract-A on ADP or Collagen induced platelet aggregation

Treatment PP PP PP PP

(7μg) (14 μg) (21 μg) (35 μg)

ADP(lOμM) 95% 68% 30% 8.6% Collagen (2 μg /ml) 87.27% 87.27% 0% 0%

Table-3: Effect of combination of Clopidogrel & Extract-A on Collagen induced platelet aggregation

Treatment Percent aggregation Collagen (2 μg /ml) 100.00

PP 7 μg 86.80

CD200μg 55.90

PP 7 μg + CD 200 μg 29.40

PP 7 μg + CD 220 μg 22.03 PP 7 μg + CD 240 μg 22.05

PP 7 μg + CD 260 μg 11.86

PP 7 μg + CD 300 μg 2.90

CD 300 μg 44.89

Table-4: Effect of combination of Clopidogrel & Extract-A on ADP induced platelet aggregation Treatment Percent aggregation

ADP(lOμM) 100.00

PP 7 μg 93.70 CD 200 μg 85.90

PP 7 μg + CD lOO μg 85.90

PP 7 μg + CD 150ug 53.10

PP 7 μg + CD 200 μg 29.60 PP 7 μg + CD 220 μg 27.1 1

PP 7 μg + CD 250 μg 25.42

PP 7 μg + CD 275 μg 15.25

CD 250 μg 64.40

It was concluded that Clopidogrel and Extract-A showed concentration dependent inhibition of platelet aggregation induced by collagen or ADP. Further, the combination of Clopidogrel and Extract-A showed a synergistic inhibition of platelet aggregation.

Another study was performed to monitor the platelet aggregation using 96 well plate by using human platelets in microplate reader. ADP was used as an ideal agonist to stimulate platelet aggregation. The. aggregation pattern was found to vary between the individuals though platelet count was maintained at same concentration. The readings of samples without drug were taken as controls. Similarly samples were prepared without drugs but only with vehicle which served as vehicle control. Clopidogrel (CD) was used as positive control against the test drug (plant product or PP). The readings were taken for individual drugs as well as in combination for each concentration. The procedure to carry out the experiment is provided in the following steps:

1. Blood from volunteers free from medication was collected in 15% v/v acid-citrate- dextrose (ACD) anticoagulant.

2. Blood was centrifuged at 180 g for 10 minutes at room temperature.

3. Washed platelets were prepared and resuspended in buffer A at concentrations of 5xlO⁵/μl and supplemented with 1.8mM CaCl₂.

4. Aliquots (90μl) of platelets were dispensed into 96-well, flat-bottomed, microtitre plates and buffer A was added to bring the final volume to 98μl.

5. Plate was incubated at 37°C for 3 minutes during the run time and allows it to shake vigorously in an orbital shaking equivalent to 1000 rpm, and then the drug was added and again incubated for 3 minutes.

6. Measurements of the optical density were performed using micro plate reader at 420 nm. 7. Readings were taken at the indicated times upto 12 min.

8. The agonist ADP at a concentration of 10 mM was added and after 4 minutes reading was taken.

9. Platelet aggregation was calculated by subtracting the final reading from the initial reading of the same well.

10. Data were expressed as percentages maximal response normalized to 100%.

The following results were obtained from the study:

1. The concentration of CD was kept constant with different concentrations of PP. Lowest concentration of CD at 35μg/ml, produced minimum effect on aggregation. The various concentration of plant products were selected after performing several trials. The concentrations selected were minimum concentration of 3.5μg producing minimum effect to maximum 28μg which produced approximately 50% of activity. When CD 35μg used in combination with increasing concentrations of PP, it was observed that in lower concentrations range there was additive effect which faded as PP concentration increased. The result is shown in Figure-5.

2. The concentration of CD was kept at 70μg against different PP concentration. When compared with individual effect of CD and PP in combination at minimum concentration combination showed synergism, thereafter not showing any relative inhibition in other dose combinations. The result is shown in Figure-6.

3. When the concentration of CD was kept at 105μg and was used against various concentration of PP, the combined effect was seen to be sum of their individual potency. The result is shown in Figure-7.

4. When the concentration of CD was further increased to 140μg with 5 selected concentrations of PP, the combined effect was additive. Concentration dependent inhibition of aggregation is evident for PP. The result is shown in Figure-8.

5. At concentration 175μg of CD the combination showed variations in the additive effect. The result is shown in Figure-9.

6. Further, the concentration of PP was kept constant against different concentrations of CD. Lower concentration group shows minimum inhibition individually but same dose in combination produces synergistic effect on inhibition of platelet aggregation. The result is shown in Figure- 10.

7. When PP was used at the concentration of 7μg and CD was used at the concentration of 70μg, it was found that this specific combination had inhibit more than two times to that of individual drug effects. The result is shown in Figure-11.

8. Further, increasing the concentration of PP to 14 μg individually produced 35% inhibition of aggregation, which when combined with different concentration of CD gives rise to additive effect in most combination. The result is shown in Figure-12. 9. The platelet aggregation was inhibited upto 35% at 21 μg concentration of PP. The result is shown in Figure- 13.

10. The highest concentration selected for plant product was 28μg. The result is shown in Figure-14.

11. Drugs with two different units were analyzed directly in fixed-ratio combinations (Table 5). The serial dilutions were used at the highest concentration combination to generate the dose-response curve.

Table 5:

CD μg/ml % inhibition PP μg/ml % inhibition CD+PP (% inhibition) 35 1 1 3.5 9 27

70 20 7 12 36

105 31 14 18 45

140 46 21 42 58

175 69 28 45 73 The Median effect plot was plotted by performing the experiment at the fixed ratio of drug combination. Based on the potency of the drugs, PP to CD ratio was taken asl:10. The plot is shown in Figure-11.

It was concluded from the study that the combined action of Clopidogrel (CD) and plant product (PP) revealed thai some of the combinations preferably at low concentrations showed synergism in inhibiting platelet aggregation.

BREIF DESCRIPTION OF THE FIGURES

Figure 1: Effect of Clopidogrel on ADP or Collagen induced platelet aggregation Figure 2: Effect of Extract-A on ADP or Collagen induced platelet aggregation

Figure 3: Effect of combination of Clopidogrel & Extract-A on Collagen induced platelet aggregation

Figure 4: Effect of combination of Clopidogrel & Extract-A on ADP induced platelet aggregation Figure 5: Inhibition of platelet aggregation using drug combination with CD 35μg Figure 6: Inhibition of platelet aggregation using drug combination with CD 70 μg Figure 7: Inhibition of platelet aggregation using drug combination with CD 105 μg Figure 8: Inhibition of platelet aggregation using drug combination with CD 140 μg Figure 9: Inhibition of platelet aggregation using drug combination with CD 175 μg Figure 10: Inhibition of platelet aggregation using drug combination with PP 3.5 μg Figure 11 : Inhibition of platelet aggregation using drug combination with PP 7 μg Figure 12: Inhibition of platelet aggregation using drug combination with PP 14 μg Figure 13: Inhibition of platelet aggregation using drug combination with PP 21 μg Figure 14: Inhibition of platelet aggregation using drug combination with PP 28 μg Figure 15: Median effect plot showing inhibition of platelet aggregation

In an embodiment, the present invention provides a fixed dose combination, which can be administered by oral route in the form of tablets, pills, capsules, suspensions, solutions, emulsion, finely divided powders, sprays, aerosols, or the like. In an embodiment, the present invention provides a composition and a method for providing an antiplatelet effect and also reducing serum cholesterol levels in humans and animals by administering the composition.

The present invention provides process for preparation of a fixed dose combination as oral dosage form such as tablets, pills, capsules, gels, finely divided powders, dispersions, suspensions, solutions, emulsions, etc; pulmonary and nasal dosage form such as sprays, aerosols, etc.; topical dosage forms such as gels, ointments, creams, etc; parenteral dosage forms; controlled release formulations; fast melt formulations, lyophilized formulations, delayed release formulations, sustained release, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations. The compositions of the present invention can be formulated for administration by the route selected from the group consisting of oral, pulmonary, rectal, colonic, parenteral, local, buccal, nasal, and topical.

In a further embodiment, the pharmaceutically acceptable excipients are selected from but not limited to a group comprising diluents, disintegrants, fillers, bulking agents, vehicles, pH adjusting agents, stabilizers, anti-oxidants, binders, buffers, lubricants, antiadherants, coating agents, preservatives, emulsifiers, suspending agents, release controlling agents, polymers, colorants, flavoring agents, plasticizers, solvents, preservatives, glidants, chelating agents and the like; used either alone or in combination thereof. In an embodiment, the compositions of the present invention comprise a combination of at least one higher primary aliphatic alcohol or mixtures thereof and clopidogrel, its analogs, derivatives, or salts thereof optionally with pharmaceutically acceptable excipient(s) and formulated into a tablet. The pharmaceutically acceptable excipient(s) include, but not limited to, diluent, stabilizer, binders, lubricant, coating agent, coloring agent, disintegrant, antioxidant, plasticizer, polymer, and the like. In another embodiment of the present invention, the combination of at least one higher primary aliphatic alcohol or mixtures thereof with clopidogrel, its analogs, derivatives, or salts thereof can be incorporated into pharmaceutical compositions in the form of capsules. These capsules may also comprise pharmaceutically acceptable excipient(s) such as diluent, antioxidant, coloring agent, stabilizer, disintegrant, lubricant, and the like.

In an embodiment of the present invention, the diluent is selected from but not limited to a group comprising lactose, cellulose, microcrystalline cellulose, mannitol, diclacium phosphate, pregelatinized starch, and the like, used either alone or in combination thereof. The binder is selected from but not limited to a group comprising polyvinylpyrrolidone, cellulose derivatives such as hydroxypropyl methylcellulose, methacrylic acid polymers, acrylic acid polymers, and the like. The polymers useful in the present invention is selected from but not limited to a group comprising polyvinylpyrrolidone/polyvinylacetate copolymer (Kollidon® SR), methacrylic acid polymers, acrylic acid polymers, cellulose derivatives, gums, resins, alginates, lipophilic or hydrophilic polymers, and the like or mixtures thereof. In an embodiment, the lubricant(s) used in the present invention are selected from, but not limited to a group comprising of stearic acid, magnesium stearate, zinc stearate, glyceryl behenale, cetostearyl alcohol, hydrogenated vegetable oil, and the like used either alone or in combination thereof.

The examples given below serve to illustrate embodiments of the present invention. However they do not intend to limit the scope of present invention.

EXAMPLES

Preparation of extract Example-1

4 kg of air-dried Sugar mill Filter cake (or Press Mud) obtained as a byproduct during sugar manufacture from sugarcane was pulverized and extracted four times by boiling with 20 L of dichloroethane each time. The dichloroethane extract was filtered and the solvent was distilled off to get a dark green residue (400 g). The residue was extracted with 4 L of boiling methanol 3 times and the extract was filtered to remove the pitch while still hot (temperature above 50⁰C). The filtered extract was distilled to remove methanol till a green residue (200 g) is obtained. The residue was dissolved in 2 L of boiling ethyl methyl ketone and set aside for crystallization. After complete crystallization the solvent is filtered, concentrated to half its volume by distillation and set aside for crystallization of the second crop. Both the crops were pooled and washed with cold hexane. The crystallization and washing procedures were repeated once more. The final washed crystals were dried under a current of air at a temperature not exceeding 70⁰C. The resultant creamish yellow lumps were pulverized to a fine powder (50 g).

Exaraple-2

Beeswax obtained after extraction of honey from honeycomb was dried and pulverized and extracted four times by boiling with of ethyl alcohol each time. The alcoholic extract was filtered and the solvent was distilled off to get a residue. The residue was extracted with boiling methanol 3 times and the extract was filtered to remove the pitch while still hot (temperature above 50⁰C). The filtered extract was distilled to remove methanol till a green residue is obtained. The residue was dissolved in boiling ethyl acetate and set aside for crystallization. After complete crystallization the solvent is filtered, concentrated to half its volume by distillation and set aside for crystallization of the second crop. Both the crops were pooled and washed with cold hexane. The crystallization and washing procedures were repeated once more. The final washed crystals were dried under a current of air at a temperature not exceeding 70⁰C. The resultant lumps were pulverized to a fine powder.

ExampIe-3

4 kg of air-dried Sugar mill Filter cake (or Press Mud) was pulverized and extracted four times by boiling with 20 L of hexane each time. The hexane extract was filtered and the solvent was distilled off to get a dark green residue (350 g). The residue was extracted with 3.5 L of boiling methanol 3 times and the extract was filtered to remove the pitch while still hot (temperature above 50⁰C). The filtered extract was distilled to remove methanol till a green residue (200 g) is obtained. The residue was dissolved in 2 L of boiling acetone and set aside for crystallization. After complete crystallization the solvent is filtered, concentrated to half its volume by distillation and set aside for crystallization of the second crop. Both the crops were pooled and washed with cold hexane. The crystallization and washing procedures

- 16 - were repeated once more. The final washed crystals were dried under a current of air at a temperature not exceeding 70⁰C. The resultant creamish yellow lumps were pulverized to a Fine powder (45 g).

ExampIe-4

10 kg of air-dried Sugar mill Filter cake (or Press Mud) was pulverized and extracted four times by boiling with 50 L of methanol each time. The methanol extract was filtered and the solvent was distilled off to get a dark green residue (650 g). The residue was extracted with 6.5 L of boiling methanol 3 times and the extract was filtered to remove the pitch while still hot (temperature above 5O⁰C). The filtered extract was distilled to remove methanol till a green residue (500 g) is obtained. The residue was dissolved in 2 L of boiling ethyl acetate and set aside for crystallization. After complete crystallization the solvent is filtered, concentrated to half its volume by distillation and set aside for crystallization of the second crop. Both the crops were pooled and washed with cold hexane. The crystallization and washing procedures were repeated once more. The final washed crystals were dried under a current of air at a temperature not exceeding 70⁰C. The resultant creamish yellow lumps were pulverized to a fine powder (102 g).

Preparation of compositions Example-5 (capsule)

S. No. Ingredient tng/capsule

1. Extract-A 80.0

2. Clopidogrel 75.0

3. Microcrystalline cellulose 200.8 4 4.. M Maannnniittooll 72.0

5. Talc 3.2

6. Sodium starch glycollate 12.0

7. Colloidal silicon dioxide 12.0 Procedure: i) Extract-A, Clopidogrcl, Microcrystalline cellulose and Mannitol were sifted and mixed together, ii) Talc, Sodium starch glycollate and Colloidal silicon dioxide were passed through fine sieves individually and then mixed together, iii) The materials of step (i) and (ii) were mixed and filled into empty hard gelatin capsules.

Example-6 (Uncoated tablet)

S. No. Ingredient mg/capsule

1. Extract-A 40.0

2. Clopidogrel 75.0

3. Aspirin 10.0

4. Lactose 120.0

5. Croscarmellose sodium 10.0

66.. TTaallcc 4.0

7. Colloidal silicon dioxide 10.0 Procedure: i) Extract-A, Clopidogrel, Aspirin, Lactose and Croscarmellose sodium were sifted and mixed together. ii) The material of step (i) was compacted. iii) The compacts of step (ii) were passed through sieve and mixed. iv) Talc and Colloidal silicon dioxide were passed through fine sieve and mixed together. v) The material of step (iii) was mixed with material of step (iv). vi) The material of step (v) was compressed into tablets.

ExampIe-7 (Film-coated tablet)

S. No. Ingredient mg/tablet

Core tablet composition

1. Extract-A 100.0 2. Clopidogrel 37.5

3. Microcrystalline cellulose 120.0

4. Mannitol 80.0

5. Croscarmellose sodium 10.0

6. Lactose 66.0 7. Talc 4.0

8. Colloidal silicon dioxide 10.0

9. Croscarmellose sodium 10.0 Film coating composition

10. Hydroxypropyl methylcellulose (HPMC E-15) 12.0 11. Polyethylene glycol 400 (PEG 400) 2.4

12. Iron oxide red 0.75

13. Iron oxide yellow 0.50

14. Titanium dioxide 0.25 15. Isopropyl alcohol q.s. (lost in processing)

16. Dichloromethane q.s. (lost in processing)

Procedure: i) Extract-A, Clopidogrel, Microcrystalline cellulose, Mannitol, Croscarmellose sodium and Lactose were sifted and mixed together. ϋ) The material of step (i) was compacted. iii) The compacts of step (ii) were passed through sieve and mixed. iv) Talc, Colloidal silicon dioxide and Croscarmellose sodium were passed through fine sieve and mixed together. v) The material of step (iii) was mixed with material of step (iv). vi) The material of step (v) was compressed into tablets. vii) Hydroxypropyl methylcellulose was dispersed in a mixture of Isopropyl alcohol and

Dichloromethane with continuous mixing in a homogenizer. viii) PEG 400 was added to the above solution of step (vii) and mixed. ix) Iron oxide red, iron oxide yellow and titanium dioxide were passed through fine sieve and mixed. x) The material of step (ix) was added to material of step (viii) and mixed for 30 minutes.

The core tablets of step (vi) were charged into the coating pan and coated with the coating solution of step (x) till an average tablet weight gain of -2-3% is achieved.

Claims

We Claim:

1. A novel pharmaceutical composition comprising at least one higher primary aliphatic alcohol preferably selected from a group comprising alcohols having about 20 to about 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor; and optionally one or more pharmaceutically acceptable excipient(s).

2. The novel pharmaceutical composition according to claim 1, wherein the mixture of higher primary aliphatic alcohols comprises 1-tetracosanol, 1-hexacosanol, 1- heptacosanol, 1-octacosanol, 1 -nonacosanol, 1-tetratriacontanol, 1-triacontanol, 1- hexacontanol, eicosanol, 1-hexacosanol, 1-tetracosanol, 1-dotriacontanol, 1-tetracontanol, and the like or mixtures thereof.

3. The novel pharmaceutical composition according to claim 2, wherein the mixture of higher primary aliphatic alcohols additionally comprises of at least one another organic component selected from a group comprising resins and pigments, hydrocarbons, sterols, esters, ketones, aldehydes, phenolic compounds, and the like.

4. The novel pharmaceutical composition according to claim 1, wherein the platelet aggregation inhibitor is clopidogrel, or its pharmaceutically acceptable salts, esters, prodrugs, solvates, hydrates, or derivatives thereof.

5. The novel pharmaceutical composition according to claims 1-4, wherein the composition comprises a mixture of higher primary aliphatic alcohols selected from high molecular weight alcohols having about 20 to about 39 carbon atoms in an amount of about 0.1 % to about 99.9 % by weight of the composition and at least one platelet aggregation inhibitor, in an amount of about 0.1 % to about 99.9 % by weight of the composition, and optionally pharmaceutically acceptable excipient(s) in an amount of about 0 % to about 99.9% by weight of the composition.

6. The novel pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable excipient(s) are selected from a group comprising diluents, disintegrants, fillers, bulking agents, vehicles, pH adjusting agents, stabilizers, anti-oxidants, binders, buffers, lubricants, antiadherants, coating agents, preservatives, emulsifiers, suspending agents, release controlling agents, polymers, colorants, flavoring agents, plasticizers, solvents, preservatives, glidants, chelating agents; used either alone or in combination thereof.

7. A novel pharmaceutical composition according to claims 1-6, which is formulated as oral dosage form such as tablets, pills, capsules, gels, finely divided powders, dispersions, suspensions, solutions, emulsions, etc; pulmonary and nasal dosage form such as sprays, aerosols, etc.; topical dosage forms such as gels, ointments, creams, etc; parenteral dosage forms; controlled release formulations; fast melt formulations, lyophilized formulations, delayed release formulations, sustained release, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations.

8. The novel pharmaceutical composition according to claims 1, wherein the composition is prepared by the process comprising of the following steps: i) isolating at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof. ii) treating at least one higher primary aliphatic alcohol preferably selected from those having 20 to 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor, iii) optionally adding one or more pharmaceutically acceptable excipient(s), and iv) formulating into a suitable dosage form.

9. The novel pharmaceutical composition according to claim 8, wherein the higher primary aliphatic alcohol preferably selected from a group comprising alcohols having about 20 to about 39 carbon atoms or mixtures is isolated from natural sources or synthetic sources.

10. The novel pharmaceutical composition according to claims 1, wherein the composition comprises of a fixed dose combination of at least one higher primary aliphatic alcohol preferably selected from a group comprising alcohols having about 20 to about 39 carbon atoms or mixtures thereof; and clopidogrel or its pharmaceutically acceptable salts, esters, prodrugs, solvates, hydrates, or derivatives thereof.

11. Use of the novel pharmaceutical composition according to claim 1, comprising at least one higher primary aliphatic alcohol preferably selected from a group comprising alcohols having about 20 to about 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor; optionally with one or more pharmaceutically acceptable excipient(s) for preparing a composition for antiplatelet activity, lowering serum cholesterol, preventing or inhibiting or treating atherosclerosis and/or reducing risk of or treating a cardiovascular event or disease including coronary artery disease and the like.

12. The method for antiplatelet activity, lowering serum cholesterol, preventing or inhibiting or treating atherosclerosis and/or reducing risk of or treating a cardiovascular event or disease including coronary artery disease and the like, which comprises administering a novel pharmaceutical composition according to claim 1 comprising at least one higher primary aliphatic alcohol preferably selected from a group comprising alcohols having about 20 to about 39 carbon atoms or mixtures thereof and at least one platelet aggregation inhibitor; optionally with one or more pharmaceutically acceptable excipient(s) to a subject in need thereof.