WO2007091253A2

WO2007091253A2 - Pharmaceutical compositions comprising clopidogrel and vitamins which reduce homocysteine levels

Info

Publication number: WO2007091253A2
Application number: PCT/IL2007/000155
Authority: WO
Inventors: Shlomo Halfon; Neil Somers; Rina Yamin; Sigal First
Original assignee: Cts Ltd
Current assignee: Cts Ltd
Priority date: 2006-02-06
Filing date: 2007-02-06
Publication date: 2007-08-16
Anticipated expiration: 2008-08-06
Also published as: WO2007091253A3

Abstract

Pharmaceutical compositions comprising a combination of clopidogrel and one or more vitamins for reducing blood homocysteine levels, such as folic acid, a pyridoxine salt, preferably pyridoxine hydrochloride (vitamin B6), and cyanocobalamin (vitamin B12), are useful in the treatment or prevention of pathologies induced by platelet aggregation, preferably cardiovascular, peripheral vascular or homocysteine related disorders or diseases.

Description

PHARMACEUTICAL COMPOSITIONS COMPRISING CLOPIDOGREL AND VITAMINS WHICH REDUCE HOMOCYSTEINE LEVELS

FIELD OF THE INVENTION The present invention relates to pharmaceutical compositions comprising clopidogrel and vitamins that reduce blood homocysteine levels, particularly cyanocobalamin, pyridoxine and folic acid.

BACKGROUND OF THE INVENTION Clopidogrel, methyl (4)-(S)-α-(O-chlorophenyl)-6,7-dihydrothieno[3,2- c]pyridine-5-acetate, having the formula:

O^/^ChL

is a platelet aggregation inhibitor described for the first time in US 4,847,265. It is commonly used against thromboembolic disorders and acts by inhibiting adenosine diphosphate platelet aggregation. It is used prophilactically to prevent myocardial infarction, peripheral arterial disease and stroke. A method for prevention of secondary ischemic events with clopidogrel is described in US 5,576,328.

Clopidogrel is commonly found in the hydrogen sulfate form; it has at least five polymorphic forms labeled forms I through V. Form II is described in US

Patent Nos. 6,429,210 and 6,504,030. Forms III, IV and V and an amorphous form are described in US 6,767,913.

Various mixtures or combinations of clopidogrel with other pharmaceutically active agents are described in the art. A pharmaceutical composition comprising a combination of clopidogrel and aspirin is described in US 5,989,578. These two compounds, each with anti-platelet activity that work in a different manner, were found to have a synergistic effect together. US 6,218,403 discloses a synergistic pharmaceutical composition containing clopidogrel and a 3-hydroxy-3- methylglutaryl-CoA (HMG-CoA) reductase inhibitor such as simvastatin. Other drug combinations comprising clopidogrel and an angiotensin II antagonist such as irbesartan or an angiotensin converting enzyme inhibitor (ACE) such as omapatrilat, are described in US 6,248,729.

Although both clopidogrel and aspirin work against platelet formation, they do so utilizing different mechanisms. Clopidogrel acts as an adenosine diphosphate (ADP) antagonist, a substance released by activated platelets that amplifies platelet aggregability. This mode of action is significantly different from aspirin, which blocks thromboxane A₂ through cyclooxygenase inhibition, thereby decreasing platelet aggregability. Based on the large randomized CAPRIE study, the activity of clopidogrel (75 mg/day) was found to have a greater efficacy than aspirin (350 mg/day) in the prevention of myocardial infarction, ischemic stroke or vascular death. Additionally, the level of upper gastrointestinal discomfort and bleeding occurred more frequently during aspirin therapy than upon clopidogrel use (Schror K, Vascular Medicine 1998, 3: 247-251). Due to the different potencies, mechanisms of action and broad range of adverse effects, clopidogrel is considered to be a valuable therapeutic alternative to aspirin.

Homocysteine, an amino acid, is metabolized by the presence of the common B-vitamins which play essential roles as cofactors (Lentz S. R. "Homocysteine metabolism" J. Thromb. Haemostas. 3: 1646-1654, 2005): folic acid (otherwise known as pteroylglutamic acid; vitamin B_c; vitamin M; PGA; pteroylmonoglutamic acid; vitamin Bn; Vitamin B₉); pyridoxine hydrochloride (otherwise known as vitamin B₆ hydrochloride, adermine hydrochloride; pyridoxinium chloride; pyridoxol hydrochloride; and its related products pyridoxamine hydrochloride; pyridoxal phosphate and pyridoxine pidolate); and cyanocobalamin (otherwise known as vitamin B₁₂; cobamin; cyanocobalaminum and cycobemin as well as its related products hydroxocobalamin; hydroxocobalamin acetate; hydroxocobalamin chloride and hydroxocobalamin sulfate).

Elevated plasma levels of homocysteine are a well-known risk factor for cardiovascular diseases, stroke, and venous thromboembolism. A single dosage form containing the three vitamins folic acid, B₆ and B₁₂ and its usage to lower homocysteine is disclosed in Israeli Patent No. 124295. The pathophysiological mechanism that associates homocysteine with cardiovascular diseases has not yet been definitively elucidated. However, based on a number of studies, there seems to be a correlation between homocysteine and the coagulation system. Specifically, in vitro studies have shown homocysteine pro-coagulant effects by the activation of factor V, inhibition of protein C activation, impairment of von Willebrand factor secretions and monocyte tissue factor expression (Khajria A. and Houston D. S. "Haemostasis", Blood, 96 (3): 966-972, 2000).

Combination products containing vitamins are well known and have been available for many years. The addition of anti-platelet agents to these combinations has also been disclosed. For example, US 6,323,188 describes the use of folic acid, pyridoxine hydrochloride, cyanocobalamin and aspirin, utilizing multiple tablets within a single administration pack for the treatment and prevention of cardiovascular diseases. IL 124295 discloses a single unit dosage containing these three vitamins and optionally further containing aspirin. US 6,121,249 describes the use of the above four active ingredients plus the addition of niacin and an antioxidant (preferably vitamin E) for the treatment and prevention of cardiovascular diseases. US 6,669,955 describes an orally administrable formulation that combines unit dosage forms of folic acid, pyridoxine hydrochloride, cyanocobalamin and aspirin as well as a cholesterol lowering agent and a renin-angiotensin inhibitor to reduce the risk of cardiovascular events. US 5,770,215 discloses a vitamin supplement for treatment of cardiac diseases, which comprises an array of vitamins including folic acid, pyridoxine hydrochloride, cyanocobalamin as well as numerous other vitamins, aspirin, and minerals such as zinc, magnesium, calcium and many others. US 6,274,170 discloses a compound dosage for the treatment of atherosclerotic cardiovascular diseases comprising ascorbic acid, folic acid, vitamins B₆ and B₁₂, vitamin E, zinc, and an anti-platelet aggregating agent, but clopidogrel is not mentioned among said agents.

It is well known to provide clopidogrel and the combination of folic acid, cyanocobalamin and pyridoxine hydrochloride separately as a therapy against cardiovascular disease, but there have been no attempts to produce a dosage form containing all these materials together in a single easy to use dosage unit.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition comprising a combination of clopidogrel or a pharmaceutically acceptable salt thereof and one or more vitamins for reducing blood homocysteine levels.

The one or more vitamins for reducing blood homocysteine levels are the vitamins folic acid, cyanocobalamin (vitamin B₁₂) and a pyridoxine salt, preferably pyridoxine hydrochloride (vitamin B₆). In one embodiment, the composition comprises clopidogrel and one vitamin, for example pyridoxine hydrochloride. In another embodiment, the composition comprises clopidogrel and two of the vitamins, for example folic acid and pyridoxine hydrochloride. In a more preferred embodiment, the composition comprises clopidogrel, folic acid, cyanocobalamin and pyridoxine hydrochloride. The pharmaceutical composition of the invention is preferably in the form of a single unit dosage for oral administration, preferably presented as tablets, capsules, pharmaceutical sachets or powdered mixture for reconstitution as syrup.

The pharmaceutical composition of the invention is useful for prevention or treatment of a cardiovascular disorder or disease, a peripheral vascular disorder or disease, a homocysteine related disorder or disease or other pathologies induced by platelet aggregation.

The present invention further provides a method for treatment of a subject susceptible to or suffering from a pathology induced by platelet aggregation, which comprises administering to said subject an effective amount of a combination of clopidogrel and one or more vitamins for reducing blood homocysteine levels. In a preferred embodiment, the combination comprises clopidogrel and the vitamins cyanocobalamin, pyridoxine hydrochloride and/or folic acid.

The present invention further provides the use of combination of clopidogrel and one or more vitamins for reducing blood homocysteine levels for the preparation of a pharmaceutical composition for the treatment of a cardiovascular disorder or disease, a peripheral vascular disorder or disease, a homocysteine related disorder or disease, or another pathology induced by platelet aggregation. In a preferred embodiment, the combination comprises clopidogrel and the vitamins cyanocobalamin, pyridoxine hydrochloride and/or folic acid.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical composition provided by the present invention comprises the antiplatelet agent clopidogrel or a pharmaceutically acceptable salt thereof, and vitamins for reducing blood homocysteine level

In one preferred embodiment, the four active agents are combined in a single dosage unit form. In another preferred embodiment, clopidogrel and a combination of the three vitamins are presented as two separate formulations. As used herein, the term "clopidogrel" refers to each and all of the five polymorphic forms I to V of clopidogrel as described in US 4,847,265 (Form I), US 6,429,210 and US 6,504,030 (Form II) and US 6,767,913 (Forms III, IV and V), as well as the amorphous form (US 6,767,913). The vitamins folic acid, cyanocobalamin and pyridoxine hydrochloride are involved in the metabolism of homocysteine and are known to be effective in reducing blood homocysteine level.

Thus, in a preferred embodiment, these three vitamins are combined with clopidogrel in the pharmaceutical composition of the invention. Pyridoxine salts other than the hydrochloride salt are also suitable according to the invention. These salts include, but ate not limited to, acid addition salts such as nitrate, citrate, sulfate and phosphate salts.

Compositions combining clopidogrel, folic acid, cyanocobalamin and pyridoxine hydrochloride are advantageous because they greatly enhance patient compliance. These compositions are distinctly advantageous over compositions comprising aspirin and said vitamins such as the compositions described in US 6,323,188, since there would be a greatly enhanced safety profile with a decreased level of upper gastrointestinal discomfort and bleeding.

While the clopidogrel and the vitamins folic acid, cyanocobalamin and pyridoxine hydrochloride have anti-thrombotic effects, they have different mechanisms of action. Clopidogrel acts as an adenosine diphosphate antagonist while folic acid, cyanocobalamin and pyridoxine hydrochloride reduce homocysteine levels and help in the upregulation of regulatory proteins utilized to form clots. The effect of combined administration of the three vitamins and clopidogrel on inhibition of platelet function as compared to the effect of clopidogrel and vitamins administered alone was studied according to the present invention, in rats. The tests conducted were an in vivo bleeding time test and an ex vivo platelet aggregation test. It was found that the effect on bleeding time of co-administration of both clopidogrel and vitamins was almost two-fold higher than the effect of clopidogrel alone, and almost 6-fold higher than the effect of administrating a cocktail comprising the three vitamins alone. In the platelet aggregation test, coadministration of vitamins and clopidogrel resulted in maximal aggregation rates much lower compared with clopidogrel or vitamins administered alone. The effect of co-administration on bleeding time was far more enhanced than a simple additive effect, which may result from adding the effects of the vitamins administered alone and clopidogrel administered alone, and indicates that coadministration has a synergetic effect on bleeding.

As demonstrated in accordance with the present invention, when clopidogrel and the three vitamins are administered to an individual, a synergistic effect may be obtained. Thus, these results indicate that the co-administration of these compounds can significantly decrease the incidence of thrombosis in individuals at risk for ischemic events.

Thus, in one embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a combination of clopidogrel or a pharmaceutically acceptable salt thereof together with folic acid, a pyridoxine salt, preferably pyridoxine hydrochloride (vitamin B₆) and cyanocobalamin (vitamin B₁₂).

Pharmaceutically acceptable salts of clopidogrel include salts with inorganic or organic acids such as the hydrochloride, hydrobromide, hydrogen sulfate, acetate, maleate, succinate, formate and ascorbate salts. The preferred inorganic salt for use according to the invention is clopidogrel hydrogen sulfate in any of its polymorphic forms I to V or in amorphous form, most preferably form II.

In one preferred embodiment, the pharmaceutical composition of the invention is in the form of a single unit dosage.

In another preferred embodiment, the pharmaceutical composition is in the form of a pack comprising two separate unit dosages, one comprising clopidogrel and a second comprising a combination of folic acid, pyridoxine hydrochloride and cyanocobalamin. Both the single unit dosage and the pack of two separate unit dosages may be presented in a pack herein referred to as "a daily administration pack".

The pharmaceutical composition of the invention, whether presented as a single unit dosage or as two separate unit dosages, one comprising clopidogrel and a second comprising a combination of the three vitamins, comprises 10 to 1000 mg clopidogrel, 0.1 to 20 mg folic acid, 1 to 100 mg pyridoxine hydrochloride and 10 to 1000 μg cyanocobalamin. More preferably, the combined composition comprises

25 to 600 mg clopidogrel, 0.4 to 5 mg folic acid, 4 to 30 mg pyridoxine hydrochloride and 100 to 600 μg cyanocobalamin. Most preferably, the amounts range from 50 to 100 mg clopidogrel, 0.7 to 2 mg folic acid, 7 to 15 mg pyridoxine hydrochloride and 300 to 500 μg cyanocobalamin.

The pharmaceutical composition of the invention is preferably administered orally on a daily basis as an immediate release or modified release dosage form.

The final dosage form of the combination of clopidogrel and three vitamins, either as a single unit dosage or as two separate unit dosages, may be any of the many variations known in the art. These include, but are not limited to, tablets, pills, hard capsules, soft capsules, pharmaceutical sachets and powders for reconstitution as syrup or for addition onto food.

The formulations of the invention may further contain water insoluble permeable polymers, herein defined as "modified release polymers", to adjust their release profile. These polymers may either be coated onto formulations such as tablets, microgranules, capsules or pills, or be mixed together with the other ingredients of any of the formulations listed above.

In one embodiment, the pharmaceutical composition is in the form of tablets prepared by mixing the four active agents with excipients. Typical excipients include diluents, fillers, binders, lubricants, disintegrants, glidants, colorants, pigments, taste masking agents, modified release polymers, sweeteners, plasticizers, and any acceptable auxiliary substances such as absorption enhancers, penetration enhancers, surfactants, co-surfactants, and specialized oils. Examples of excipients include calcium phosphates, such as dibasic calcium phosphate, anhydrous dibasic calcium phosphate, tribasic calcium phosphate, etc.; microcrystalline cellulose, powdered cellulose; starch, pre-gelatinized starch; sodium starch glycolate; dextrates; mannitol, sorbitol; povidone; ethyl cellulose; lactose; kaolin; silicic acid; lubricants such as magnesium stearate, calcium stearate, stearic acid, mineral oil, glycerin, sodium lauryl sulfate, polyethylene glycol; and/or talc. Sodium starch glycolate, talc and the lubricant magnesium stearate are used according to the invention to aid in the tablet manufacture.

In a preferred embodiment, the tablets of the invention comprise clopidogrel, folic acid, pyridoxine hydrochloride and cyanocobalamin and as excipients lactose, microcrystalline cellulose, polyethylene glycol 6000, magnesium stearate and sodium starch glycolate.

In another preferred embodiment, the tablets of the invention are prepared as modified release tablets and comprise the above-mentioned four active agents and the excipients mannitol, microcrystalline cellulose, ethyl cellulose, povidone, sodium starch glycolate and talc. In a more preferred embodiment, the modified release tablets contain the modified release polymer ethyl cellulose mixed together with the other excipients.

In one embodiment, the tablets of the invention are prepared by mixing the active agents with microcrystalline cellulose, lactose or mannitol in the powder form followed by the addition of a solution of polyethylene glycol or a solution of ethyl cellulose, povidone and cyanocobalamin to form a granulated mixture. This granulate is then dried and mixed with sodium starch glycolate and magnesium stearate or talc and prepared as tablets using a rotary punch manufacturing process.

Materials and preparation techniques for tablet manufacture useful in the invention can be found in e.g., Remington Pharmaceutical Sciences, 21^st Edition, 2005,

Lippincott Williams and Wilkins eds.

In another preferred embodiment, the tablets of the invention are formed by mixing all the ingredients in solid state and then directly compressing them into a tablet form, i.e. a so-called direct compression technique. Tablets prepared according to the direct compression technique comprise clopidogrel hydrogen sulfate, folic acid, pyridoxine hydrochloride and triturated cyanocobalamin and as excipients starch, microcrystalline cellulose, lactose and magnesium stearate.

According to one preferred embodiment of the invention, the daily administration pack is a blister pack comprising a tablet of clopidogrel hydrogen sulfate and a tablet of vitamins packed side by side. In a more preferred embodiment, the clopidogrel tablet is prepared by the rotary punch manufacturing process via granulate formation as described above, and the vitamins tablet is formed by the direct compression technique.

The palatability of the tablets can be improved by coating the tablets with a taste-masking agent such as a methyacrylic acid copolymer (e.g. Eudragit), methylcellulose or methylhydroxypropyl cellulose. In a preferred embodiment, the tablets of the invention are coated with methylhydroxypropyl cellulose.

In another embodiment, the pharmaceutical composition is in the form of capsules, preferably prepared by mixing the four active agents with excipients such as lactose and microcrystalline cellulose, adding a solution of e.g. polyethylene glycol to form a granulated mixture, which is then dried. An excipient, preferably talc, is added to the granulate and the mixture is then filled into capsules.

In another embodiment, the pharmaceutical composition is in the form of pharmaceutical sachets, preferably prepared by mixing the four active agents with excipients such as lactose, microcrystalline cellulose, silicone dioxide, talc, sodium starch glycolate, starch, etc.

In another preferred embodiment, the present invention provides a powder mixture for reconstitution as syrup. According to this embodiment, folic acid, pyridoxine hydrochloride, cyanocobalamin and clopidogrel hydrogen sulfate are mixed with an artificial sweetener, preferably sucralose, microcrystalline cellulose and a powder-based flavoring agent, and the mixture is filled into a bottle for a later reconstitution as syrup.

Any combination of excipients known in the art is suitable for use according to the invention for the preparation of syrup for reconstitution. The excipients may be any of the excipients mentioned above suitable for the preparation of a liquid composition such as sweeteners, taste-masking agents, colorants, diluents etc.

The pharmaceutical composition or the daily administration pack provided by the invention is useful for the prevention or treatment of cardiovascular disorders or diseases, peripheral vascular disorders or diseases, homocysteine-related disorders or diseases or other pathologies induced by platelet aggregation.

Cardiovascular disorders, which can be treated and/or prevented according to the invention comprise, without being limited to, myocardial infarction (MI; heart attack), ischemic encephalic infarction (stroke), blood clots (thrombosis), and an acute coronary syndrome (ACS) such as unstable angina and evolving myocardial infarction.

Cardiovascular diseases, which can be treated according to the invention comprise, without being limited to, stable angina pectoris, atherosclerotic vascular diseases such as coronary artery disease (CAD), silent ischemia, heart failure and arrhythmias.

Other pathologies induced by platelet aggregation can also be treated or prevented using the composition of the invention. These pathologies include, but are not limited to, a secondary ischemic condition or disorder, hypertension, presenile dementia of ischemic origin, claudication and thromboembolic disorders associated with diabetes or haemodialysis. The secondary ischemic condition or disorder as referred to herein is a condition selected from the group consisting of a subsequent acute ischemic event in patients with established atherosclerotic vascular disease involving the coronary, cerebral or peripheral circulation such as a secondary stroke, acute myocardial infarction (AMI) and vascular death; rethrombosis following thrombolysis; restenosis; events following percutaneous coronary intervention in acute coronary syndromes such as elective stent or drug- eluting stent insertion and aortocoronary bypasses; vascular prostheses insertion; angioplasty such as endarectomy; and ischemic events in survivors of MI and stroke. In preferred embodiments, the pharmaceutical compositions of the invention are useful for treating or preventing ischemic events. Ischemia of the heart and brain rank among the most prevalent diseases in the Western world. Major ischemic events include myocardial infarction (heart attack) and ischemic encephalic infarction (stroke).

In another aspect, the present invention provides a method for treatment of a subject susceptible to or suffering from a cardiovascular disorder or disease, a peripheral vascular disorder or disease, a homocysteine-related disorder or disease or other pathologies induced by platelet aggregation, comprising administering to the subject an effective amount of a combination of clopidogrel, cyanocobalamin, a pyridoxine salt, preferably pyridoxine hydrochloride and folic acid.

In a further aspect, the invention provides the use of a combination of clopidogrel, cyanocobalamin, pyridoxine hydrochloride and folic acid for the preparation of a pharmaceutical composition for the treatment of a cardiovascular disorder or disease, a peripheral vascular disorder or disease, a homocysteine- related disorder or disease or other pathologies induced by platelet aggregation.

The invention will now be exemplified by the following non-limiting examples.

EXAMPLES

Example 1. Preparation of tablets containing a combination of folic acid, cyanocobalamin, pyridoxine hydrochloride and clopidogrel

Tablets (1000 units) were prepared by mixing 75 g clopidogrel hydrogen sulfate with 1O g pyridoxine hydrochloride, 200 g lactose, 1 g folic acid, 40 g 1% triturated cyanocobalamin and 100 g microcrystalline cellulose. A 2% solution of polyethylene glycol 6000 was added slowly to the above mixture of powders until a granulation mixture resulted. The resulting granulate was dried using a fluid bed process. Finally, 3 g magnesium stearate and 8 g sodium starch glycolate were mixed into the granulate. The resulting combination was then prepared as tablets using a rotary punch manufacturing process.

Example 2. Preparation of coated tablets containing a combination of folic acid, cyanocobalamin, pyridoxine hydrochloride and clopidogrel

To improve the palatability of the tablets obtained in Example 1 above, the tablets were placed inside a rotary coating pan and heated to 40⁰C, and a suspension of 1% polyethylene glycol 8000 and 7% methylhydroxypropyl cellulose in water was prepared and coated onto the tablets.

Example 3. Preparation of modified release tablets containing a combination of folic acid, cyanocobalamin, pyridoxine hydrochloride and clopidogrel

Tablets (10,000 units) were prepared by mixing 750 g clopidogrel hydrogen sulfate with 100 g pyridoxine hydrochloride, 2.5 kg mannitol, 1O g folic acid and 1 kg microcrystalline cellulose. A solution of 3% povidone, 3% ethyl cellulose was added slowly to the above mixture of powders until a granulation mixture resulted. The resulting granulate was dried in an oven, and a mixture of 400 g 1% triturated cyanocobalamin, 400 g sodium starch glycolate and 30O g talc was mixed into the dried granulate. The resulting combination was then prepared as modified release tablets using a rotary punch manufacturing process.

Example 4. Preparation of tablets containing a combination of folic acid, pyridoxine hydrochloride and clopidogrel

Tablets (10,000 units) were prepared by mixing 750 g clopidogrel hydrogen sulfate with 100 g pyridoxine hydrochloride, 5 kg microcrystalline cellulose, 1O g folic acid, 0.5 kg starch, 1.5 kg lactose and 50 g magnesium stearate in a direct compression format. The resulting combination was then prepared as tablets using a rotary punch manufacturing process. Example 5. Preparation of pharmaceutical packs comprising tablets of clopidogrel and tablets of vitamins

Tablets comprising clopidogrel were prepared by mixing clopidogrel hydrogen sulfate with mannitol and microcrystalline cellulose. A solution of povidone was added slowly to the above mixture of powders until a granulation mixture resulted. The resulting granulate was dried using a rotary bed process, and a mixture of sodium starch glycolate and magnesium stearate was mixed into the dried granulate. The resulting combination was then prepared as tablets using a rotary punch manufacturing process. The vitamins tablets were prepared by mixing pyridoxine hydrochloride, microcrystalline cellulose, folic acid, triturated cyanocobalamin, starch and magnesium sterate in a direct compression format. The resulting combination was then prepared as tablets using a rotary punch manufacturing process.

The mixture of two tablets is packaged for ease of use by the patient in a blister pack, whereby in each pack one tablet of clopidogrel and one tablet of vitamins are presented side by side.

Example 6. Preparation of capsules containing a combination of folic acid, cyanocobalamin, pyridoxine hydrochloride and clopidogrel Capsules (1000 units) were prepared by mixing 5O g clopidogrel hydrogen sulfate with 15 g pyridoxine hydrochloride, 200 g lactose, 1.5 g folic acid, 5O g 1% triturated cyanocobalamin and 100 g microcrystalline cellulose and 3O g talc. The resulting mixture was filled into hard gelatin capsules.

Example 7. Preparation of syrup for reconstitution containing a combination of pyridoxine hydrochloride and clopidogrel

For the preparation of the titled formulation, 100 mg pyridoxine hydrochloride and 75 mg clopidogrel hydrogen sulfate were mixed together along with 2 mg sucralose (a low-calorie sweetener made from sugar), 1O g mannitol and a 2 mg flavoring agent. The resulting mixture was filled into a bottle for later use. Immediately before use the bottle is filled with water up to a pre-marked level and the syrup is shaken and ingested.

Example 8. Preparation of packages containing a powdered combination of folic acid, cyanocobalamin, pyridoxine hydrochloride and clopidogrel for inclusion in food

Powdered mixture (1000 packages) was prepared by mixing 75 g clopidogrel hydrogen sulfate with 1O g pyridoxine hydrochloride, 15O g lactose, 1 gram folic acid, 0.4 g cyanocobalamin and 100 g microcrystalline cellulose. The ensuing mixture was filled into packages (bags). Individual packages are employed by pouring onto food, for example applesauce, and ingested along with the food.

Example 9. Preparation of pharmaceutical sachets containing a combination of folic acid, cyanocobalamin, pyridoxine hydrochloride and clopidogrel Pharmaceutical sachets were prepared by mixing 100 mg clopidogrel hydrogen sulfate with 15 mg pyridoxine hydrochloride, 100 mg lactose, 4 mg folic acid, 200 mg 0.1% triturated cyanocobalamin and 50 mg microcrystalline cellulose. Finally, 1 mg silicone dioxide and 3 mg sodium starch glycolate were added. The ensuing mixture was filled into pharmaceutical sachet for oral administration.

Example 10. Dose-response effect of combined treatment of clopidogrel hydrogen sulfate and vitamins on bleeding time

The effect of combined administration of an aqueous solution of the vitamins folic acid, pyridoxine hydrochloride and cyanocobalamin at constant concentrations and varying doses of clopidogrel on inhibition of platelet function as compared to the effect of clopidogrel administered alone, was studied. The rat-tail transaction bleeding time test was performed to study effects of this combination on platelet function in vivo. Bleeding time test measures the overall manifestation of effects on platelet function in vivo. The vitamin cocktail was prepared by dissolving 0.04 mg folic acid, 0.1 mg vitamin B₆ and 6 mg vitamin B₁₂ in 1 ml of water. An aqueous solution of clopidogrel at a concentration of 1 mg/ml was also prepared. The solutions were kept frozen until usage. Water alone was used as a control.

Sprague Dawley female rats weighting 180-220 g (Harlan Jerusalem) were weighted each day and fed by gavage with either vitamins (in volume of 1 ml per 200 g weight) or water as a control.

On day 28, vitamins or water were given to 16 h fasted animals and one hour later clopidogrel (in various quantities between 1.2 to 5 mg/kg weight) or water were administered. Two hours after administration of clopidogrel, bleeding time was tested. Treatment groups included 8 groups as indicated herein:

Tail transaction bleeding time was performed according to Dejana et al.

(Dejana E., Villa S. and de Gaetano G. "Bleeding time in Rats: a comparison of different experimental conditions".!. Thromb. Homeostas., 48(1): 108-111, 1982.), by section of the extremity of the tail, 3 mm from the tip, with scissors. The tails were maintained in contact with air and gently blotted with filter paper each minute during a 10-min period, and then every 2 min. The time in minutes to cessation (no rebleeding for 1 min) was noted. The observation time was limited to 90 min. Care was taken that no pressure was exerted on the tail tip that could affect hemostasis.

Statistical analysis: the results are expressed as mean ± standard error (SE). The Mann-Whitney-U test was used to analyze the difference between vehicle- and drug-treated groups. The corresponding p values were calculated, taking p<0.05 to indicate a significant difference. ANOVA was used to test for overall differences between the effects of different concentrations of clopidogrel, vitamins and controls. P value below 0.05 was considered to indicate a significant difference. The experimental results and statistical analysis are summarized in Tables 1 to 3.

Table 1. Summary of bleeding test results: means and SE

Table 2. Mann-Whitney-U analysis of the effect of different concentrations of clopidogrel on bleeding time in the presence and absence of vitamins

* Dose of clopidogrel in mg/kg

Table 3. Two-way ANOVA analysis of the effect of different concentrations of clopidogrel on bleeding time in the presence and absence of vitamins

Treatment Sig.

Clopidogrel (1.20,2.50,3.25 mg/kg) vs. Clopidogrel (1.20,2.50,3.25 mg/kg) + vitamins 0.017 The combined effect of clopidogrel and vitamins as compared to clopidogrel alone was statistically significant at a concentration of 3.25 mg/kg (see Table 2). In addition, as shown in Table 3, ANOVA test for the overall effect on bleeding time of the three concentrations of clopidogrel (1.20, 2.50, 3.25 mg/kg,) in the presence versus absence of vitamins, showed a significant difference: Sig=0.017.

The results suggest that combined treatment of clopidogrel and the three vitamins is more beneficial compared to treatment with clopidogrel alone in prolongation of bleeding time.

Example 11. In vivo effect of clopidogrel hydrogen sulfate, folic acid, pyridoxine hydrochloride and cyanocobalamin combined together on bleeding time.

The effect of oral administration of a cocktail consisting of a combination of the title vitamins and clopidogrel at a concentration of 4 mg/kg on the tail bleeding time in rats was tested in an additional study. The study protocol was similar to that described in Example 10.

The Mann-Whitney-U test was used to determine the difference between vehicle and drug-treated groups. The corresponding p values were calculated, taking p<0.05 to indicate a significant difference. The results and statistical analysis are summarized in Tables 4 -6.

Table 4. Summary of bleeding test results: means and SE

SE=Std. error N= rats number Table 5. Mann-Whitney-U analysis of 4 mg/kg clopidogrel on bleeding time in the presence and absence of vitamins

Table 6. Two-way ANOVA analysis of the effect of 4 mg/kg clopidogrel on bleeding time in the presence and absence of vitamins

The combined effect of clopidogrel and vitamins as compared to clopidogrel alone was statistically significant at concentration of 4 mg/kg (see Table 5). ANOVA test for the overall effect of clopidogrel (4 mg/kg), in the presence or absence of vitamins, showed a significant difference: Sig=0.021(see Table 6).

The results suggest that combined treatment of clopidogrel and the three vitamins is more beneficial compared to treatment with clopidogrel alone in prolongation of bleeding time. The results clearly show that co-administration of clopidogrel and the three vitamins results in a synergistic effect on the bleeding time compared to the effect of administering either the vitamins or clopidogrel alone.

Example 12. Ex vivo effects of clopidogrel hydrogen sulfate, folic acid, pyridoxine hydrochloride and cyanocobalamin combined together on platelet aggregation.

The ex vivo effect of clopidogrel hydrogen sulfate, folic acid, pyridoxine hydrochloride and cyanocobalamin on ADP-induced platelet aggregation of platelet-rich plasma (PRP) in rats was tested in order to study the effect of the compounds on platelet function ex vivo.

Sprague Dawley female rats were orally treated with the reagents as described in Example 11 above. Thus, rats were fed with either vitamins, clopidogrel, a combination of vitamins and clopidogrel or water (as control). The study included 4 groups as described in Example 11. On day 28, 1 hour after administration, 3-5 ml of blood were withdrawn from the abdominal aorta for platelet aggregation studies. The blood was collected into a 3.2% trisodium citrate solution (9:1 v/v). Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were obtained by blood centrifugation (50Og x 6 min for PRP and 3000g x 10 min for PPP). PRP was diluted with PPP to a concentration of 3x10⁵ platelets/μl. PRP samples were pre-incubated at 37⁰C for three minutes. Platelet aggregation of the samples was measured at 37⁰C according to turbidometric method in an aggregometer (Chrono-Log Corporation, PA, USA). Aggregation was induced by 5 μM ADP and in some samples by 2.5 μM ADP (if enough PRP was obtained). The maximal rate of aggregation was determined. The results are summarized in Table 7

Table 7. Summary of maximal aggregation rate results: means and SE

SE=Std. error N= rats number

As seen from Table 7, co-administration of both vitamins and clopidogrel resulted in a significant lowering of maximal aggregation rates.

Claims

1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a combination of clopidogrel or a pharmaceutically acceptable salt thereof and one or more vitamins for reducing blood homocysteine levels.

2. A pharmaceutical composition according to claim 1, wherein said one or more vitamins for reducing blood homocysteine levels are folic acid, a pyridoxine salt and cyanocobalamin (vitamin B₁₂).

3. The pharmaceutical composition according to claim 2, wherein said pyridoxine salt is pyridoxine hydrochloride (vitamin B₆)

4. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable salt of clopidogrel is clopidogrel hydrogen sulfate.

5. The pharmaceutical composition according to claim 4, wherein said pharmaceutically acceptable salt is any one of the polymorphic Forms I to V or the amorphous form of clopidogrel hydrogen sulfate.

6. The pharmaceutical composition according to claim 5, wherein said polymorphic form is Form II.

7. The pharmaceutical composition according to claim 1, comprising a combination of clopidogrel and pyridoxine hydrochloride.

8. The pharmaceutical composition according to claim 1, comprising a combination of clopidogrel, folic acid and pyridoxine hydrochloride. '

9. The pharmaceutical composition according to claim 1, comprising a combination of clopidogrel, folic acid, cyanocobalamin and pyridoxine hydrochloride.

10. The pharmaceutical composition according to claim 1, in the form of a single unit dosage.

11. The pharmaceutical composition according to claim 1, presented in a pack comprising two separate unit dosages, one comprising clopidogrel and a second comprising a combination of folic acid, pyridoxine hydrochloride and cyanocobalamin.

12. The pharmaceutical composition according to claim 10 or 11, comprising 10 to 1000 mg clopidogrel, 0.1 to 20 mg folic acid, 1 to 100 mg pyridoxine hydrochloride and 10 to 1000 μg cyanocobalamin.

13. The pharmaceutical composition according to claim 12, comprising 25 to 600 mg clopidogrel, 0.4 to 5 mg folic acid, 4 to 30 mg pyridoxine hydrochloride and 100 to 600 μg cyanocobalamin.

14. The pharmaceutical composition according to claim 13, comprising 50 to 100 mg clopidogrel, 0.7 to 2 mg folic acid, 7 to 15 mg pyridoxine hydrochloride and 300 to 500 μg cyanocobalamin.

15. The pharmaceutical composition according to claim 1, for oral administration.

16. The pharmaceutical composition according to claim 15, as an immediate release dosage form.

17. The pharmaceutical composition according to claim 15, as a modified release dosage form.

18. The pharmaceutical composition according to claim 15, in the form of tablets.

19. The pharmaceutical composition according to claim 18, wherein said tablets are coated with a taste-masking agent, preferably methylhydroxypropyl cellulose.

20. The pharmaceutical composition according to claim 15, in the form of capsules.

21. The pharmaceutical composition according to claim 15, in the form of pharmaceutical sachets.

22. The pharmaceutical composition according to claim 15, in the form of a powder for syrup reconstitution.

23. The pharmaceutical composition according to claim 1 for prevention or treatment of a cardiovascular disorder or disease, a peripheral vascular disorder or disease, a homocysteine related disorder or disease or other pathologies induced by platelet aggregation.

24. The pharmaceutical composition according to claim 23, wherein said cardiovascular disorder is selected from the group consisting of myocardial infarction, ischemic encephalic infarction, blood clots and an acute coronary syndrome such as unstable angina and evolving myocardial infarction.

25. The pharmaceutical composition according to claim 23, wherein said cardiovascular disease is selected from the group consisting of stable angina pectoris, an atherosclerotic vascular disease, silent ischemia, heart failure and arrhythmias.

26. The pharmaceutical composition according to claim 23, wherein said other pathologies induced by platelet aggregation are selected from the group consisting of a secondary ischemic condition or disorder, hypertension, presenile dementia of ischemic origin, claudication, and thromboembolic disorders associated with diabetes or haemodialysis.

27. The pharmaceutical composition according to claim 26, wherein said secondary ischemic condition or disorder is selected from the group consisting of a subsequent acute ischemic event in patients with established atherosclerotic vascular disease involving the coronary, cerebral or peripheral circulation such as a secondary stroke, acute myocardial infarction (AMI) and vascular death; rethrombosis following thrombolysis; restenosis; events following percutaneous coronary intervention in acute coronary syndromes such as elective stent or drug- eluting stent insertion and aortocoronary bypasses; vascular prostheses insertion; angioplasty such as endarectomy; ischemic events in survivors of MI and stroke.

28. A method for treatment of a subject susceptible to or suffering from a cardiovascular disorder or disease, a peripheral vascular disorder or disease, a homocysteine related disorder or disease, or another pathology induced by platelet aggregation, comprising administering to the subject an effective amount of a combination of clopidogrel and one or more vitamins for reducing blood homocysteine levels.

29. The method according to claim 28, wherein said one or more vitamins is selected from cyanocobalamin, pyridoxine hydrochloride and/or folic acid.

30. Use of combination of clopidogrel and one or more vitamins for reducing blood homocysteine levels for the preparation of a pharmaceutical composition for the treatment of a cardiovascular disorder or disease, a peripheral vascular disorder or disease, a homocysteine related disorder or disease, or another pathology induced by platelet aggregation.

31. The use according to claim 30, wherein said one or more vitamins is selected from cyanocobalamin, pyridoxine hydrochloride and/or folic acid.