WO2010151096A1

WO2010151096A1 - Therapeutic combinations of nicotinic acid and meldonium

Info

Publication number: WO2010151096A1
Application number: PCT/LV2010/000008
Authority: WO
Inventors: Ivars Kalvins; Anatolijs Birmans; Maris Veveris; Antons Lebedevs; Anatolijs Misnovs
Original assignee: Tetra, Sia
Priority date: 2009-06-25
Filing date: 2010-06-21
Publication date: 2010-12-29
Also published as: CN102802618A; EA021344B1; EA201200037A1; CN102802618B

Abstract

The present invention comprises a synergistic combination of nicotinic acid and meldonium characterized by increased efficiency in treating disorders, including platelet aggregation, dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease as chosen from the group of angina pectoris and myocardial infarction, transient and permanent ischemic attack including cerebrovascular accident and stroke and peripheral arterial occlusive disease, and by reduced flushing effect and/or increase of blood glucose levels, comprising as active ingredients nicotinic acid or pharmaceutically acceptable salt thereof and meldonium or pharmaceutically acceptable salt thereof, effective for said purpose.

Description

THERAPEUTIC COMBINATIONS OF NICOTINIC ACID AND MELDONIUM

FIELD OF INVENTION

Present invention relates to a combination medicinal product and method of prevention and/or treatment of metabolic-related disorders, including dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease as chosen from the group of angina pectoris and myocardial infarction, transient and permanent ischemic attack including cerebrovascular accident and stroke and peripheral arterial occlusive disease, prevention of platelet aggregation and thrombosis.

More precisely the invention relates to combination medicinal product containing nicotinic acid (niacin) and meldonium that synergistically enhances curing effects of nicotinic acid and ameliorates certain undesirable side effects of nicotinic acid, in particular peripheral vasodilatation (flushing) and elevations in blood glucose levels. The invention also relates to a pharmaceutical composition comprising such combination medicinal product and to the use thereof in the manufacture of a medicament for prevention and/or treatment of metabolic-related disorder.

ABBREVIATIONS USED

The following abbreviations will be used further on in the description for shortness:

ATP - adenosine triphosphate

C - cholesterol

GL - glucose

HDL-C- high density lipoprotein-cholesterol

I/R - ischemia/reperfusion

LDL-C - low density lipoprotein-cholesterol

MD - meldonium (INN)

NA - nicotinic acid

NAMg — nicotinic acid magnesium salt

PI - piracetam

RPP - rate pressure product = mean blood pressure x heart rate x IOOO ¹

SI - simvastatin

TG - triglycerides

TR - Triton WRl 339 (Tyloxapol)

VF - ventricle fibrillation

VT - ventricular tachycardia

BACKGROUND OF THE INVENTION

NA is important agent for treatment of dyslipidemias and the only agent currently available that favourably influences all components of lipid profile: lowers in blood the levels of total cholesterol, TG and LDL-C and has the most pronounced HDL-C raising activity among lipid-altering agents (Pieper JA, Am J Manag Care 2002;8(12 Suppl):S308-14).

NA in the treatment of dyslipidemias has been reported as early as 1955 (Altshul R, Hoffer A, Stephen JD, Arch Biochem Biophys 1955;54:558-559) and 1959 (Parsons Jr WB, Flinn JH, AMA Arch Intern Med 1959; 103:783-790). Since NA efficiently raises HDL-C levels (McKenney J, Arch Intern Med 2004;164(7):697-705. Carlson LA, J Intern Med 2005;258:94-114), NA is currently combined with other lipid-modifying agents that mostly act on LDL-C levels, in order to increase HDL-C levels (Rosenson RS, Am J Med 2005; 118(10): 1067-77).

NA is an effective lipid-altering agent that prevents atherosclerosis progression and reduces clinical cardiovascular events (Savel'ev AA, Shershevskii MG, Klin Med (Rus) 1996;74:48-52. Drexel H, European Heart Journal Supplements 2006;Vol 8, Suppl F: F23-F29. Brown BG, Zhao XQ, Am J Cardiol 2008;101(8A):58B-62B) by raising HDL-C levels. NA decreases morbidity and mortality rates in patients with hyperlipidemia (Canner PL et al, J Am Coll Cardiol 1986;8: 1245-55). NA is the most effective agent at increasing HDL-C in treating hyperlipidemic cases (Ellingworth DR et al, Arch Intern Med 1994; 154: 1586-95. Schectman G et al, Am J Cardiol 1993;71:758-65). NA diminishes thrombosis, reduces blood viscosity and has cardioprotective effects that may limit ischemia- reperfusion injury. (Lamping KA et al, Pharm Exp Ther 1984;231(3):532-538. Trueblood NA et al, Am J Physiol Heart Circ Physiol 2000; 279(2), H764-H771. Rosenson RS Atherosclerosis 2003;171(l):87-96).

Since low HDL-C is a risk factor in stroke (Wannamethee SG, Shaper AG, Ebrahim S, Stroke 2000;31:1882. Sacco RL, Benson RT, Kargman DE JAMA 2001;285:2729-2735. Rizos E, Mikhailidis DP, Cardiovasc Res 2001 ;52(2), 199-207. Sanossian N, Tarlov NE, Curr Treatmt Opt Cardiov Med 2008; 10(3), 195-206.), NA as agent, increasing HDL-C (Carlson LA, Curr Opin Cardiol 2006;21(4)336-344), is useful in prevention of stroke and in post-stroke condition (Koniukov SG, Liberzon SP, Klin Med (Mosk) 1975;53(9): 38-41.). It has been confirmed that HDL-C levels decrease at the time of acute ischemic stroke (Russman AN et al, J Neurol Sci 2009;279(l-2):53-56). NA had been shown to improve functional recovery after stroke (Chen J et al, Ann Neurol 2007;62(l):49-58). HDL-C itself had been proposed for treating stroke and other ischemic conditions (Kapur NK et al, Vase Health Risk Mmag 2008;4(l):39-57. EP 1 425 031).

NA is available in 3 formulations (immediate release, extended release, and long acting). Immediate-release NA is associated with adverse flushing and elevations in blood glucose levels. Long-acting NA is associated with reduced flushing, but also with risk of hepatotoxic effects. Extended-release is associated with less flushing and low hepatotoxic risk (Pieper JA, Am J Health Syst Pharm 2003;60(13 Suppl 2):S9-14. McKenney J, Arch Intern Med 2004;164(7):697-705. Knopp RH, Am J Cardiol 2008;86(Suppl);51L-56L). The use of NA sodium, potassium and magnesium salts is also described.

A major shortcoming of NA is the necessity of administering large doses to substantially alter blood lipid levels. Almost 100% of subjects treated with NA experience unpleasant side effects of flushing that in many cases prevent the therapy with NA. Cutaneous release of prostaglandin D2 was identified as the immediate cause of NA-induced flushing (Morrow JD et al, J Invest Dermatol 1992;98:812-5). Since NA-related flushing is a result of prostaglandin activity, acetylsalicylic acid as a well-established inhibitor of prostaglandin synthesis was proposed and is used to control the flushing. Besides acetylsalicylic acid other NSAIDS are also active (Oberwittler H, Baccara-Dinet M, Int J Clin Prøct 2006;60(6):707-715). However NSAIDS are not devoid of side effects themselves and can cause gastrointestinal irritation and ulceration.

Recently a specific antagonist to prostaglandin D2 (Parhofer KG, Vascular Health and Risk Management 2009;5:901-908) receptor subtype 1, laropiprant, was proposed as agent for reducing NA-induced flushing (Lai E et al, Clin Pharm Ther 2007;81:849-857. Davidson MH, Am J Cardiol 2008;101 (suppl):14B-19B). Although the addition of laropiprant will reduce the frequence of flushing, it will not completely eliminate this side effect. Laropiprant does not change the effect of niacin on lipids or other side effects of niacin. The combination of niacin with laropiprant may therefore enable use of niacin at higher doses and therefore exploit the full potential of the drug (Parhofer KG, Vascular Health and Risk Management 2009;5:901-908, Olsson AG, Expert Opinion on Pharmacotherapy 2010;l l(10):1715- 1726).

Patients with Type 2 diabetes often have dyslipidemic changes characterized by an increase in TG levels as well as a decrease in HDL-C levels. Considering the pharmacological effects of NA on lipid metabolism, NA should counteract the dyslipidemic changes in patients with Type 2 diabetes. However, several reports indicate that NA increases insulin resistance (Garg A, Grundy SM, JAMA 1990;264: 723-6. Kahn SE et al, Diabetes 1989;38:562-8) and rises glucose levels (Elam, MB et al, JAMA 2000;284( 10): 1263- 1270). Therefore only limited doses of NA (<2 g/day) are recommended for diabetic patients. (Shepher J, Betteridge J, Van Gaal L, Curr Med Res Op 2005;21(5):665-682). It is evident that new agents are necessary to improve glycemic control for diabetic patients using NA. A hypoglycemizing ability of meldonium was noted in clinic (Statsenko ME et al, Klin Med (Rus) 2007;85(7):39- 42), therefore it was expected that this agent can be combined with NA for additional clinical benefits.

Blood platelets play a pivotal role in the development of atherosclerosis and fatal thrombus formation in the course of coronary heart disease. Antiplatelet agents have become paramount in the prevention and management of various diseases involving the cardiovascular, cerebrovascular, and peripheral arterial systems (Meadows TA et al, Circ Res 2007; 100(9): 1261-75). NA is an effective lipid-altering agent that prevents atherosclerosis and reduces cardiovascular events. NA has multifarious lipoprotein and anti-atherothrombosis effects that improve endothelial function, reduce inflammation, increase plaque stability, and diminish thrombosis (Rosenson RS, Atherosclerosis 2003; 171 :87-96)

NA inhibit platelet aggregation (Lakin KM, Farmakol Toksikol, 1980; 43(5):581-5). NA in vitro affects platelet activity by mildly inhibiting aggregation, and stimulating significant prostaglandin release, with mostly intact major platelet receptor expression. The effect of NA is unique, differs from other known antiplatelet agents, and suggests potential opportunities for therapeutic combination (Serebruany VL et al, Thrombosis and Haemostasis, 2010 (in press).

NA almost totally prevented intravascular clotting induced by thromboplastin and pituitrin, showing that it has a thrombolytic effect (Baluda VP, Kardiologija 1974; 14(11): 105-7 (Rus). Anti-thrombotic properties of NA are described by several authors (Shestakov VA, Probl Gematol Pereliv Krovi, 1977;22(8):29-35. Chekalina SI, Sov Med 1982(5): 105-8). Niacin reduces risk of blood clots (Chesney CM et al, Am Heart J, 2000;140:631-36).

MD is a medicine with certain beneficial effects on heart and vessels. A certain desirable activity of MD was discovered in animal models of atherosclerosis (Veveris M, Smilsaraja B, Baltic J Lab Anim Sci 2000; 10, 194- 199. Veveris M et al., Baltic J Lab Anim Sci 2002;12:l 16-122. Okunevich IV, Ryzhenkov VE, Patol Fiziol Eksp Ter 2002;(2):24-7), and observed in clinics (Karpov RS et al, Ter Arkh 1991;63(4):90-3), therefore it was expected that this agent can be combined with NA for additional clinical benefits. It has been also noticed that MD inhibits platelet aggregation (TsirkinVI, Ros Kardiol Zh 2002; 1 :45-52). Two weeks long therapeutic use of MD peroral administration in rabbits and dogs after experimental arterial thrombosis showed trombolytic effect (Logunova L et al, Experim Clin Pharmacoter 1991;19:91-98 (Rus). No data on prophylactic effect of MD on limitation or prevention of thrombosis are known.

PRIOR ART

The present trend to develop combination medical products to treat metabolic- related disorders may improve the efficiency of clinical care (Black DM, Curr Ther Rep 2003;5:39-32). Because fibrates, NA and statins each regulate serum lipids by different mechanisms, combination therapy may offer particularly desirable benefits in patients as compared with monotherapy. Since progress in developing new agents for lowering LDL-C levels has slowed down, the research has turned to developing better agents for raising HDL-C levels. There has been increasing use of combination therapy comprising NA, fibrates, statins and bile acid sequestrants for the treatment of metabolic-related disorders, due to the additive profiles of combined products (Miller M, Mayo Clin Proc 2003;78(6):735-42. Backes JM et al, Vase Health Risk Manag 2005;l(4):317-331. Belsey L et al, Curr Med Res Opin 2008;24(9),2703-9. Rosenson RS, Pitt B, Nat Clin Pract Cardiovasc Med 2009;6(2):98-100). There has been increasing use of combination therapy comprising NA and other agents, e.g., acetylsalicylic acid and other NSAIDs as platelet inhibitors (USP 5,981,555). These combinations however do not increase the activity of NA, i.e., no synergy has been reported. Therefore any agents that could enhance the curative effects of NA in metabolic-related diseases without increasing the undesirable side effects of NA would have beneficial effect in clinic.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a combination medicinal product for the prevention and/or treating of metabolic-related disorders and a method of prevention and/or treating metabolic-related disorders in a subject in need of the same, by the employment of NA in combination with MD, which has the further beneficial effect of alleviating the undesirable side effects of NA. The combination medicinal product of the present invention is anticipated to have a synergistic effect in a method for treatment and/or prevention of metabolic-related disorders, including dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease as chosen from the group of angina pectoris and myocardial infarction, transient and permanent ischemic attack including cerebrovascular accident and stroke and peripheral arterial occlusive disease, preventing platelet aggregation ant thrombosis.

According to the present invention, a combination medicinal product is defined as having a synergistic effect if the effect is therapeutically superior to that of NA or MD separately. It is understood that a combination pharmaceutical product as used herein means simultaneous, sequential or separate administration of the agents of the combination. An additional object of the invention is the provision of a pharmaceutical composition comprising both NA and MD for the aforesaid purpose. Further objects of the invention will become apparent hereinafter, and still others will be obvious to one skilled in the art. DESCRIPTION OF THE INVENTION

The invention comprises the combination of NA and MD with the result that an effective synergistic combination for treating metabolic-related disorders is provided, preferably in a single-dosage unit form. Alternatively, the two ingredients may be administered separately simultaneously or sequentially in any order. The exact form in which the active ingredients are administered is not important, so long as the desired effect of the invention is obtained. The active ingredients may take the form of capsules, suspensions, dispersions, elixirs, syrups, or the like, whether administered separately or in single composition.

Now we have surprisingly found that NA and MD have synergistic action on metabolic-related disorders and other beneficial effects. It was unexpectedly discovered that MD is the first agent that enhances the beneficial effects of NA, namely lowering the TG and LDL-C levels and increasing the HDL-C levels, enhances the antiaggregating effect of NA and ameliorates the undesirable side effects of NA, in particular flushing and elevations in blood glucose levels.

Therefore said combination is anticipated to be a preferable agent for treating dyslipidemia in patients with diabetes. We have also surprisingly found that the combination of present invention ameliorates the aftereffects of experimental infarction and stroke.

Now we have surprisingly found that NA and MD have synergistic action on platelet aggregation. It was unexpectedly discovered that MD is the first agent that enhances the antiplatelet effects of NA.

The combination of the invention may be in a form suitable for oral use (for example as tablets, capsules, aqueous suspensions or dispersible powders or granules), for parenteral administration (for example as a sterile aqueous solution for intravenous, subcutaneous, or intramuscular dosing) or as a suppository for rectal dosing. Preferably the composition of the invention is in a form suitable for oral use, for example as tablets or capsules.

A combination product according to the present invention also includes combination of separate pharmaceutical compositions of active agents comprising a first composition of NA or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or carrier, and a second composition comprising MD or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or carrier. Such a combination of pharmaceutical compositions provides the combination medicinal product of the invention for simultaneous or sequential use. The advantage of such combination is in the possibility for the physician to adjust the proportion of active agents for the individual patient. The combination medicinal product of the present invention is anticipated to include also sustained-release and extended release formulations of NA, as well as pharmaceutically acceptable salts of NA (sodium, potassium or magnesium) and MD and salts thereof.

The combination medicinal product of the present invention can also include other medicinal products with known activity in metabolic-related disorders, namely statins, in particular, Simvastatin.

The combination medicinal product of the present invention can also include other medicinal products with known activity in metabolic-related disorders, namely inhibitors of platelet aggregation, in particular, clopidogrel or dipyridamole.

The pharmaceutical composition of the invention may be obtained by conventional procedures using conventional pharmaceutically acceptable excipients or carriers and technologies. The following examples are provided to illustrate but not limit the invention.

EXAMPLES

Tests: The pharmacological activity of tested substances was investigated by standard methods used in the art. Animals were kept in groups of 6 in adequate cages in climatized rooms at 22±1 ⁰C, relative humidity 60 ±5% and 12/12 light/darkness cycle with free access to feed and water. All experiments were carried out in accordance with the European Community Council's Directive of 24 November 1986 (86/609/EEC) relative to experimental animal care. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Substances: Cholesterol (Acros Organics), meldonium (Grindex), animal nutrition (R 70 Lactamin), butter (commercial), Na cholate (Acros Organics), NA (Acros Organics), Laropiprant (MK 0524, Cayman Chemicals). All other chemicals were from commercial sources.

Example 1. Determination of anti-atherosclerotic activity

Method. An atherosclerosis model on C57BL/6J mice genetically susceptible to atherosclerosis, proposed in literature was used (Smith J D, Breslow JL, J Intern Med 1997;242:99-109). Control group received standard laboratory animal nutrition. The experimental atherosclerosis was induced by adding to standard nutrition 1.25% cholesterol, 15% dairy butter and 0.5% Na cholate (Nishina P et al, J Lipid Res 1993;34:1413-1422). Experimental groups received the test substances separately and in combination, in doses established as active in pilot experiments. On week 22 the level of atherosclerotic changes was evaluated morphologically, biochemically and histologically by standard methods and criteria (Paigen B et al, Atherosclerosis 1987;68:231-240). Total C, HDL-C, LDL-C and TG in serum were determined by commercial test kits. LDL-C/HDL-C ratio is accepted as standard tool to evaluate cardiovascular risk (Fernandez ML, Webb D, JAm Coll Nutr, 2008;27(l):l-5). Atherosclerosis index (reflecting coronary atherosclerosis and discriminator for peripheral atherosclerosis) was calculated as follows: Index = LDL-C/HDL-C. Atherosclerosis coefficient (reflecting coronary atherosclerosis) was calculated as follows: Coefficient =Total C/HDL-C.

Statistics. Data are presented as means ± SEM of 7 to 10 separate animals/measurement. Differences between experimental groups were compared using one-way ANOVA with repeated comparisons (Tukey's test). P<0.05 was considered as significant.

Results. The first series of tests was scheduled to determine the effects of various proportions and doses of agents of combination. As shown in Table 1 after 22 weeks C control group animals receiving nutrition rich in lipids and C developed atherosclerotic changes in aorta, especially on arch of aorta. NA and MD, when used separately, displayed a tendency to decrease the area of damage. Surprisingly, the combination of NA and MD caused considerably higher and statistically significant protective effect against the atherosclerotic damage than each substance separately, i.e., it had a synergistic effect. Table 1

The influence of NA and MD on atherosclerotic dama e of aorta

**P<0 005 vs C control $P<0 05 vs NA in same dose *P<0 05 vs MD in same dose

As shown in Table 2 below, the combination of NA and MD exhibits statistically significant effect on lowering LDL-C and TG levels and increasing the HDL-C levels. At the dose combination NA50+MD150, where NA is used in a dose that itself had not a significant effect on lipid levels, the summary effect was surprisingly higher than the effect of each substance alone. It is particularly evident in atherosclerotic index and total C/HDL-C rate, where a synergy of NA and MD effects is observable.

Table 2 The influence of test substances on lipids in serum; n=7-10; Mean±SEM

NA5O+MD5O = NA 50 mg/kg + MD 50 mg/kg NA50+MD150 = NA 50 mg/kg + MD 150 mg/kg NA200+MD200 = NA 200 mg/kg + MD 200 mg/kg ♦P<005 vs C control **P<0 005 vs C control *P<00005 vs C control sP<005 vs NA in same dose *P<005 vs MD in same dose

In another series of experiments the anti-atherosclerotic activity of the combination of present invention was evaluated in more detail, and further also the known anti-lipidemic agent, SI was added to the combination. NA and MD, when used separately, displayed a tendency to decrease the area of damage (Table 3). Surprisingly, the combination of NA+MD caused considerably higher and statistically significant protective effect against the atherosclerotic damage than each substance separately. Adding of the combination of the present invention to SI further increased its protective activity against atherosclerotic damage.

Table 3

The influence of test substances on atherosclerotic damage of aorta; n=7-9; Mean±SEM I Group I Damaged area, mkm² , arch of aorta [ Damaged area, % of aorta area

NA₅₀+MDl₅0 = NA ₅0 mg/kg + MD I₅₀ mg/kg

NA₅₀+MD₅O+S110 = NA ₅0 mg/kg + MD ₅0 mg/kg + SI lO mg/kg

*P<0 O₅ vj C control

**P<0 0O₅ vs C control

*P<0 O₀₀₅ vj C control

*P<0 05 ra NA

^&P<0 O₅ vs MD

In the same experimental series total C, HDL-C, LDL-C and TG in serum were determined. As shown in Table 4 below, NA and MD, when used separately only marginaly improved the rate of cholesterol fractions and the atherosclerosis index without statisticall significance. Surprisingly the NA and MD combination considerably improved the ratio of cholesterol fractions and statistically significantly lovered the atherosclerosis index and total C/HDL-C ratio. The combination also prevented the increase of LDL-C and TG in serum. Thus the combination of the invention had significantly higher protective action against the changes in lipid metabolism than the components thereof used separately. Pronounced protective effect was retained also in triple combination with SI.

Table 4

In still another series of experiments NAMg and NA+SI combination were included in the comparative evaluation (Table 5). NAMg had been compared with NA in cockerels (Burstein J, Telkka A, Acta Pathol Microbiol Scand 1962;56:261-265). The combination of NA (50 mg/kg) and MD (150 mg/kg) had the most pronounced beneficial effect against aorta, better than NA and NAMg, as well as surpassing that of SI and NA combination, used in rate based on clinical experience (Pandian A et al, Vase Health Risk Manag 2008;4(5): 1001-1009).

Table 5

NA50+MD150 = NA 50 mg/kg + MD 150 mg/kg NA50+SI2 = NA 50 mg/kg +SI 2 mg/kg *P<005 vi C control **P<0 005 vs C control ***P<0 0005 vs C control $P<0 05 VJ NA ^@P<0 05 vs NAMg 4P<005 vs MD *P<005 vs NA50+SI2

Similarly to morphometric data the biochemical tests confirmed that the combination of NA+MD is significantly better than NA or NAMg in normalizing lipid levels (Table 6).

Table 6

The influence of test substances on li ids in serum n=7-9 Mean±SEM

NA50+MD150 = NA 50 mg/kg + MD 150 mg/kg NA50+SI2 = NA 50 mg/kg +Sl 2 mg/kg *P<005 vs C control **P<0 005 vs C control ***P<0 0005 vs C Control sP<005 ra NA @P<005 vs NAMg *P<005 vs MD

The combination of NA+MD is similar to combination of NA+SI in lowering total C and LDL-C, but substantially surpass it in influence on beneficial effect on HDL-C and TG levels, as well as has more pronounced effect on atherosclerotic index and total C/HDL-C rate.

This series of tests confirmed that NA and MD combination had significantly better beneficial effect on experimental atherosclerosis than NA or NAMg and better than clinically used combination of NA and SI.

Example 2. Influence of NA and MD, separately and in combination on lipids in rat hvperlipidemia model

Method. Experimental chronical hyperlipidemia/hypercholesterolemia was induced by

TR using the method described by Levine and Saltzman (Levine S, Saltzman A,

J Pharmacol Toxicol Meth 2007;55:224-226). Animals received 250 mg/kg TR solution via the tail vein three times a week for 3 weeks. Solutions of test substances for experimental groups or water for Control group was introduced p.o. once a day one hour before injection of TR solution or taking a blood sample.

Male Wistar rats weighing 220-240 g were distributed in the following 8 groups

(group, n of animals):

1. Control 10

2. TR (TR 250 mg/kg) 14

3. NA (TR 250 mg/kg + NA 50 mg/kg/d) 14

4. MD (TR 250 mg/kg + MD 150 mg/kg/d) 14

5. NA+MD (Triton 250 mg/kg + NA 50+MD 150 mg/kg/d) 14

6. SIlO (TR 250 mg/kg + SI 10 mg/kg/d) 12

7. NA+SI2 (TR 250 mg/kg + SI 2 + NA 50 mg/kg/d) 12

8. NA+SI2+MD (TR 250 mg/kg + SI 2 + NA 50 + MD 150 mg/kg/d) 12 Blood for biochemical analyses was obtained after 1 , 2 and 3 weeks (on the next day after TR injection) by cardiac punction under ether narcosis. Serum was separated by centrifugation and analyzed for total C, HDL-C, LDL-C and TG levels by commercial kits. Statistics. The data obtained were mathematically processed using Microsoft Excel program and the results were expressed as mean ± standard error of the mean (SEM). Mean results of different groups were compared using one-way ANOVA and Student's t-test. Difference of the results were considered significant at P<0.05. Results. Repeated injections of TR developed pronounced and stable hypercholesterolemia and hyperlipidemia, characterized by significant increase of total C, LDL-C and TG levels compared to Control. NA therapy, more significantly in the first week, limited the increase of total C, LDL-C and TG, but significantly increased the HDL-C levels in 2 and 3 week only. MD was slightly less active in lowering total C, LDL-C and increasing HDL-C levels, but did not prevent the increasing of TG levels. Surprisingly the combined use of NA+MD more efficiently than the components separately lowered the levels of LDL-C and TG and further increased levels of HDL-C. Moreover, prolonged use of NA+MD (2 or 3 weeks in our experiment) demonstrated substantially more pronounced lowering of LDL-C and TG levels and increase of HDL-C than use of NA+SI (see below). Thus the combination NA+MD is expected to be useful in clinic for preventing and/or treating hypercholesterolemia and hyperlipidemia.

Use of SI in dose 10 mg/kg considerably lowered the TR-induced increase of total C and LDL-C, but only slightly influenced the levels of HDL-C and TG. Combined use of SI and NA in clinically acceptable ratio demonstrated significant protection against increase of total C, LDL-C and TG in serum and increased HDL-C levels. Surprisingly the combined use of NA, SI and MD demonstrated still better protection against TR-induced changes that was better than effects of each component separately and significantly better than NA+SI in lowering TG and LDL-C levels. Particularly important for clinical practice was the observed fact that the combination of NA, SI and MD increased the levels of HDL-C substantially better than SI or NA alone. Thus combinations of NA+MD and NA+SI+MD are expected to be useful in clinic for preventing and/or treating hypercholesterolemia and hyperlipidemia.

Table 7

Influence of NA, SI and MD, separately and in combination on lipids in rat hyperlipidemia model; n=9-14; Mean±SEM

Table 7 (cont.)

Table 7 (cont.)

*P<0.05 vs TR TR ***P<0.0005 VJ TR $P<0.05 vs NA &P<0.05 vs MD #P<0.05 vs NA+SI

Example 3. Determination of cardioprotective properties

Method. Male Wistar rats were distributed in 6 groups (12 to 16 animals each):

1) Control group received p.o. 0.9% saline;

2) NA50 group received p.o. 50 mg/kg/day of NA;

3) MD50 group received p.o. 50 mg/kg/day of MD;

4) NA50+MD50 group received p.o. 50 mg/kg/day NA plus 50 mg/kg MD;

5) MD 150 group received p.o. 150 mg/kg/day of MD;

6) NA50+MD150 group received p.o. 50 mg/kg/day of NA plus 150 mg/kg/day of MD.

Animals of test groups received the experimental substances as aqueous solutions via canula in stomach 48, 24 and 1 hour before the experiment. Animals of the control group received the same volume of saline. Animals were narcotized (pentobarbital sodium 60 mg/kg Lp.) and under mechanical ventilation prepared for blocking of left coronary artery. Experimental infarction was induced by 45 min. long occlusion of coronary artery with following 2 h long reperfusion. The following data were registered: the number of animals with VT, VF, lethality, mean arterial pressure, RPP that characterizes the functional status of myocardium, reflects the production of ATP in myocardium and is a widely used index in analysis of clinical and experimental data (Broderick TL, Drugs R D 2008; 9(2):83-91). After the experiment the ischemic and necrotic area were detected by triphenyltetrazolium-Evans blue perfusion-staining method. Left ventricle was dissected and weighed and the morphological criteria calculated: percentage of the ischemic zone of the left ventricle, percentage of the necrotic zone of the left ventricle and ratio of the necrosis zone to ischemic zone (necrosis index).

Statistics. The results are presented as means ± SEM for each group. Statistical analysis within groups was performed by Student t-Test. Data obtained from registration of blood pressure and heart rate were calculated from the survival animals in the ischemia-reperfusion experiment. Number of incidences of arrhythmia (VT and VF) and lethality were calculated in all animals. Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). P <0.05 was considered as significant. Results. Cardiac ischemia with the following reperfusion caused serious heart rhythm disturbances in the control group with 7 lethalities in the group of 16 animals. NA group was not significantly different from the control group regarding the rhythm disturbances. MD and NA+MD groups had less pronounced life-threatening rhythm disturbances (VF and VT) versus control, but the lethality was significantly lower only in the NA50+MD50 and NA50+MD150 groups (Table 8 below).

Table 8

Effect of test substances on heart rhythm disturbance and lethality during coronar arter occlusion and re erfusion

*P<0 05 vs I/R control

Mean arterial pressure and heart rate was similar in all experimental groups, but the fall of RPP, observed in the control group, was statistically significantly prevented only in the NA50+MD50 and NA50+MD150 groups during reperfusion (Table 9). This indicates a considerable protection against the infarction caused functional depletion already after a short (3 days long) pre-treatment with the combination medicinal product of invention.

Table 9

Effect of test substances on rate-pressure-product (RPP) during coronar arter occlusion and re erfusion, n=9-12, Mean±SEM

*P<005 vs I/R control **P<0 005 vs I/R control *P<0 05 vs Initial *P<001 vs Initial

This was also confirmed by statistically significant reduction of the necrosis zone in percentage to left ventricle and to ischemic zone (Table 10) for combination medicinal products, containing NA+MD in both dosage combinations.

Table 10

Effect of test substances on heart morphology of rats undergoing coronary artery occlusion and

**P<0 005 vs 1/R control

"P<0 0005 vs I/R control

*P<005 vs MD at the same dose

^$P<0 O₅ vs NA

Necrotic index = Necrotic zone/Ischemic zone x 100 Thus we have surprisingly discovered a high level protection against the development of myocardial infarction by the synergic action of NA and MD combination medicinal product. NA+MD combination significantly preserved the function of myocardium, increasing the survival rate of animals (Tables 8, 9) and more significant than NA and MD alone protected myocardium from necrosis caused by ischemia and reperfusion (Table 10).

Example 4. Determination of antihypoxic and antiishemic effects on brain Further experiments were conducted to determine the effect of NA and MD combination medicinal product in experimental CNS ischemia, hypoxia and stroke models in comparison with effects of separate components.

4.1. Mice cerebral circulatory hypoxia model

Method. Experimental circulatory hypoxia was induced by MgCl₂ (Berga P et al,

Arzneimittelforschung 1986;36(9): 1314-1320) introduced during 3 s into the tail vein

(2% MgCl₂, dose 200 mg/kg) of male ICR mice. Animals received the test substance either as single dose or the same dose once daily for 7 days. The test substances were introduced via intragastric catheter. Animals were randomized in 7 groups (6 to 10 animals each):

Control group received water 0.01 mL/g

PI500 group (active control) received 500 mg/kg dose of piracetam

NA50 group received 50 mg/kg dose of NA

MD50 group received 50 mg /kg dose of MD

MDl 50 group received 150 mg/kg dose of MD

NA50+MD50 group received a dose of 50 mg/kg NA plus 50 mg/kg MD

NA50+MD150 group received a dose of 50 mg/kg NA plus 150 mg/kg MD

The last dose of test substance was given 1 hour before the test. The period between the end of the MgCl₂ injection and the cessation of the last respiratory movements was registered as survival time.

Statistics. The results are presented as means ± SEM for each group. Statistical analysis within groups was performed by Student t-Test. Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). P <0.05 was considered as significant. Results. The results are summarized in Table 11. When introduced repeatedly the protective antihypoxic effect was shown both by the clinically used agent PI and MD (Table 11). Unexpectedly the combination NA+MD showed significant protection already after a single application, particularly in the dosage NA50 + MD 150, where the effect was better than for each substance separately. Repeated application of this combination had still more pronounced effect. The results obtained in this test indicate a possible use of NA+MD combination for treating hypoxic conditions in clinic.

Table 1 1

Effect of test substances in mice cerebral circulatory h oxia model; n=6-10; Mean±SEM

*P<0.05 vi Control *P<0.005 vs Control ^@P<0.000S vs Control $P<0.05 vs MD in same dose *P<0.₀₅ VJ NA §P<0.0Q₅ vsNA

4.2. Middle cerebral artery occlusion model

Method. Male ICR mice with body mass 21 to 25 g were used. Middle cerebral artery (MCA) origin was occluded by intraluminal filament technique according to known method (Longa EZ et al, Stroke 1989;20:84-91) adapted for mice by Zhang Q et al. (Behavioural Brain Research 2006; 169:66-74). Preventive (once per day for 7 days, for permanent MCA occlusion model) and therapeutic (treatment started 1 h after transitional MCA occlusion) treatment protocols were used.

Control animals received only saline. This test is a good model of a real brain insult and stroke clinical situation, where the middle cerebral artery occlusion is often encountered. Further the experiment was continued according to 2 protocols. In the first the occlusion was permanent. In the second the occlusion by the intraluminal filament was transitional and the filament was removed after 2 hours and introduced reperfusion. Neurological state of all animals was evaluated 24 hours after the occlusion. It was rated by a point scale, where 0 points are given to animal without pathology, 4 points to animal incapable of spontaneous movement (Longa EZ et al, Stroke 1989;20:84-91). After the evaluation of neurological deficit, the animals received an overdose of sodium pentobarbital, brains were isolated and sliced in 6 layers of about 1.5 mm thickness. Slices were stained with 2% triphenyltetrazolium sodium at 37 ⁰C for 30 min. and photographed. As the most adequate for calculating the brain ischemic damage the 3rd slice from cranial side on the chiazma opticum level was selected, since it was completely supplied by blood from the middle cerebral artery.

Statistics. Data are presented as means ± SEM of 7 to 9 separate animals. Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). Differences of neurological scores between groups were analyzed by Student's t-Test. PO.05 was considered as significant. Results. 24 hours after the occlusion all 8 control animals displayed neurological disturbance with average of 2.63 points (Table 12 below).

Table 12

Influence of test substances on neurological state of permanent occlusion group animals; n=7-9; Mean±SEM

*P<0.05 vs Control •p<0.0005 vs Control *P<0.0₅ vi NA

In the sham group only 1 animal displayed slight disturbance. Repeated use of MD for 7 days gave a partial prevention from deterioration of neurological state, caused by MCA occlusion. Surprisingly, NA+MD combination displayed significant protection against the neurological disturbances, better than the effect of NA (Table 12).

Middle cerebral artery occlusion caused ischemic damage that occupied 22.2% of brain tissue at chiazma opticum level for control group animals (Table 13). NA+MD combination applied for 7 days displayed significant protection against the brain tissue damage, better also than the effect of NA alone.

Table 13

Effect of 7 day application of test substances on size of brain ischemic-infarction damage zone of permanent occlusion rou animals; n=7-9; Mean±SEM

*P<0005 vs Control 4P<0 05 v5 NA

In the next experiment middle cerebral artery transitional occlusion for 2 hours with a following reperfusion caused serious brain function disturbances for control group animals tested after 24 hours (Table 14 below). Neither NA25 (25 mg/kg x 3) group nor MD75 (75 mg/kg x 3) group with test substances introduced 1, 3 and 6 hours after the occlusion provided significant protection. Surprisingly, only the group NA25+MD75 (25 mg/kg + 75 mg/kg) with test combination introduced 3 times after the occlusion, received substantial protection of brain functions, significantly better than that from NA and MD alone (Table 14 below).

Table 14

Therapeutic effect of test substances on neurological functions after transitional occlusion; n=7-9; Mean±SEM

*P<0005 vs Control •p<00005 vs Control sP<0 05 vs MD *P<005 vs NA

Morphometry analysis revealed that NA or MD alone did not give significant protection against the brain tissue damage caused by transitional occlusion and reperfusion of MCA (Table 15 below). Surprisingly, the combination NA+MD (25 mg/kg + 75 mg/kg), with treatment starting after the occlusion, revealed substantial protection against brain tissue damage, significantly better than that of NA and MD alone (Table 15).

Table 15

Therapeutic effect of test substances on size of brain ischemic damage zone of transitional occlusion rou animals n=7-9; Mean±SEM

P<0005 vi Control $P<005 vs MD *P<0 05 v_- NA

Thus we have surprisingly found that the combination of NA plus MD provides a significantly better protection against functional and morphological damage of brain tissue than separate components when used therapeutically either before or after the occlusion of MCA. These results indicate that the combination medicinal product may be beneficial in treating and/or preventing CNS ischemic-hypoxic conditions including stroke, also due to its inhibiting activity in platelet aggregation and thrombosis tests, as outlined below.

Platelet antiaggregant and antithrombotic activity

Tests: Platelet aggregation in vitro; rat thrombosis model in vivo; registration of changes in skin temperature in vivo were performed.

Example 5. Influence of MD and NA on platelet aggregation

Method. Platelet aggregation was studied in whole blood obtained from healthy donor B. (37 y. old) who did not use ASA or other antiplatelet agents, using Multiplate (Multiple Platelet Function Analyzer, Dynabyte Medical, Germany) with established method (Toth O et al, Thromb Haemost, 2006;96:781-788. Velik-Salchner C et al, Anesth Analg 2008;107:1798-1806). Blood samples were collected into plastic tubes covered with hirudin (Dynabyte Medical, Germany) and used for measurement between 30 min and 4 h after collection. The measurements were performed according to modified Dynabyte Medical protocol. Isotonic sodium chloride solution (0.3 ml, or saline with test substance (in final concentration 10^"6 to 10^"4 mmol/ml)) was pre-heated to 37 ⁰C, pipetted into the test cells and 0.3 ml of whole blood sample anticoagulated with hirudin was added. After 5 min incubation and stirring at 37 C, measurements were initiated by adding of the appropriate agonist solution (sourced from Dynabyte Medical, Germany): 1) adenosine diphosphate (ADP) - ADP-Test. ADP stimulates platelet activation by the ADP receptors (P2Y12 and other). 2) ADP HS test (prostaglandin Ei in combination with ADP). The addition of the endogenous inhibitor PGEi makes ADP HS test more sensitive compared to ADP test. Aggregation curves were recorded for 6 min and analyzed using Dynabyte Medical software. We calculated the following parameters of platelet aggregation:

1) Amax, the maximal value of platelet aggregation expressed in arbitrary units (AU) of aggregation;

2) AUC, total area under the aggregation curve (AU*min). It is affected by the total height of the aggregation curve as well as by its slope and is best suited to express the overall platelet activity.

Statistics. The results were expressed as the mean and standard error of the mean (Mean ± SEM). To estimate significance of differences, one-way ANOVA was used. If null hypothesis had been rejected, the post-hoc Student-Newman-Keuls test was employed.

Results. The first series of tests was scheduled to determine the effects of various concentrations of agents. As shown in Table 16, MD in wide range of concentrations provided significant protection against the platelet aggregation caused by ADP+PGEi. Amax was reduced from 100% in control to 55-58 % in MD 10^"5 and 10 groups. NA (in 10^"4 and 10^"3 mmol/ml group) also reduced aggregation caused by ADP. The combined action of both substances provided significantly higher and pronounced reduction of platelet aggregation caused by ADP or ADP+PGEi, manifested both in AUC and Amax data. Table 16 MD, NA and combined influence on ADP and PGE +ADP induced latelet a re ation; Mean ± SEM; N= 5-8.

*P<0.05 vs Control **P<0.005 VJ Control ♦**P<0.0005 vs Control ****P<0.00005 vs Control *P<0.005 vs MD 10^"" ^0.0005 Vs MD lO^"4 $P<0.005 vs NA 10^"" 1^COOOS VS NA IO^"4

Example 6. Influence of MD and NA on thrombosis

Method. We chose an experimental model based on rat arterial thrombosis induced by FeCl₃ (Kurz K et al, Thromb Res 1990;60:269-280. Wang X, Xu L, Tromb Res 2005;115: 95-100). Tissue damage initiated by iron-mediated chemical oxidation predisposes the injured area to platelet adherence and aggregation followed by coagulation activation and fibrin deposition. Male Wistar rats with mass 350-420 g were used in experiments. Rats were randomly divided into various experimental groups, each consisting of not less than seven animals. The vehicle or test compound MD (25 mg/kg), NA (25 mg/kg) and combination MD+NA (25+25 mg/kg) were administered by oral route 2h before the initiation of thrombosis.

Rats were anaesthetized with pentobarbital sodium 50 mg/kg, Lp. and were placed on a heat controlled operating table throughout the experiment to maintain a body temperature at 37 ⁰C. One of the carotid arteries was exposed by cervical incision, separated from the adherent tissue, vagus nerve, and a flow probe (electromagnetic blood flow-meter MFV 1200 Nicon Kohden, Japan) was placed on the exposed segment of common carotid artery to record the blood flow. After a stabilization period of 15 min, thrombosis was induced by topically applying (in contact with the adventitial surface of vessel) two pieces (2x1 mm) of Whatman filter paper, soaked in 15% solution of FeCl₃. Thrombosis time of carotid artery was recorded as time taken for the complete cessation of the blood flow and has been reported as time till occlusion (TTO).

Additionally during the thrombosis experiment rat tail bleeding time was measured. The tail was transected 5 mm from the tip with scalpel and the tail was immediately immersed into 37 ⁰C warm isotonic saline until termination of bleeding was noted. Termination of bleeding was defined as the time of complete stop of bleeding with no recurrence of bleeding within the next 30 s.

Statistics. The results were analyzed by Microsoft Excel 2007 software. Data are presented as means ± SEM of 7 to 8 separate animals/measurement. Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). P<0.05 was considered as significant. Results. The average time for FeCl₃ caused vessel thrombosis and the resulting arterial flow arrest in control group was 24.4 min (Table 17).

Table 17

MD, NA and combined influence on FeCl₃ induced thrombosis Mean± SEM; N= 7-8

*P<0.05 vs Control

MD in 25 mg/kg dose did not provide significant increase in TTO. NA caused a small increase in TTO that was not significant. The influence of NA on tail bleeding time was similar. The combined use of MD and NA unexpectedly caused considerable significant increase of TTO (39%) without significant increase of tail bleeding time.

Considering the positive influence of MD and NA combination on platelet aggregation in vitro and extension of TTO in vivo this combination can find application for reducing thrombosis risk in patients with pronounced atherosclerosis, possible myocardial infarction and insult, as well as in postoperative period. The fact that MD and NA combination does not prolong bleeding time indicates the possible use of this combination for patients with increased bleeding risk in pre- and postoperative period.

Example 7. Comparative investigation of combined application of MD/NA and LA/NA for reduction of flushing

Nicotinic acid (niacin, NA) effectively lowers serum cholesterol, LDL and triglycerides, while raising HDL. However a limiting adverse effect in patients receiving immediate-or sustained-release niacin is the rapid development of significant cutaneous warmth and vasodilatation, referred to as "flush" which severely leading to discontinuation (Gupta EK, Ito MK, Heart Dis 2002;4: 124-137). Laropiprant (MK-0524) has been proposed as one of the most active and perspective agent for reducing niacin flushing (Cheng K et al, PNAS 2006; 103:6682-6687). The objective of our investigation was comparing the effect of MD and LA on flushing (changes of skin temperature and blood flow) caused by NA in experiment.

7.1.1. Determination of cutaneous vasodilatation

Model. Male Wistar rats were narcotized by sodium pentobarbital (50 mg/kg i.p.) and kept under narcosis by additional doses (10 mg/kg) each hour. Blood pressure was measured in left carotid artery, ECG recorded by standard II lead. Blood flow in the right ear artery was measured by laser Doppler flow meter (OXYFLOW 2000, USA). Blood flow, ECG and arterial pressure were registered byADInstruments PowerLab systems and stored in computer for further processing. After 10 min. long registration of baseline test substances were injected s.c. into withers area and registration continued for 30 min. The average blood flow data for each animal were calculated taking into account the average blood pressure and compared with initial and control. Results were calculated from 5 to 8 separate experiments and expressed in % as maximal change in blood flow to baseline [Carballo-Jane E et al, J Pharmacol Toxicol Methods 2007;56(3): 308-316]. Statistics. The results are presented as means ± SEM for each group. Statistical analysis within groups was performed by Student t-Test for unpaired data and Chi- square Test. Differences between each experimental group were compared using oneway ANOVA with repeated comparisons (Tukey's test). P <0.05 was considered as significant.

Results. As can be observed from Table 18 below, NA in dose of 15 mg/kg caused significant increase in blood flow in ear artery in this animal model. MD similarly to control, caused non-significant variation in blood flow. NA together with MD caused delayed (slowly increasing) and statistically significant less pronounced absolute increase in blood flow as compared to NA alone (Table 18). The potential of MD to antagonize the peripheral vasodilatation, caused by NA may have beneficial effect in clinic for diminishing the cutaneous effects (flushing) of niacin, therefore it was investigated in more detail (see below).

Table 18

Effect of experimental substances on cutaneous vasodilatation, n=5-8, Mean ±SEM

P<0 01 vs Control sP<0 0₅ rø NA

7.1.2. Determination of cutaneous vasodilatation

Materials and methods. See Section 7.1.

Experimental schedule

GROUP TREATMENT Number of animals

Solvent for NA and MD 5

NA NA 15 mg/kg 7

Solvent for LA 5

LA LA 0.3 mg/kg 6

LA+NA [0] LA 0.3 mg/kg+NA 15 mg/kg 6

LA+NA [30] LA 0.3 mg/kg+NA 15 mg/kg 7

MD MD 45 mg/kg 6

NA+MD [0] NA 15 mg/kg+MD 45 mg/kg 6

NA+MD [30] NA 15 mg/kg+MD 45 mg/kg 6

Statistics. The results are presented as means ± SEM for each group. StJ analysis within groups was performed by Student t-Test. Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). P<0.05 was considered as significant.

Results. MD when administered alone, similarly to LA caused only insignificant changes in blood flow. When NA was given simultaneously with MD (advance time = 0), the rise in blood flow was slower and less pronounced than in case of simultaneous administration of NA and LA (LA+NA [0], see Table 19). There was no significant difference on ear blood flow between the MD+NA [0] (when MD was added together with NA) and MD+NA [30] (pretreatment 30 min in advance of NA with MD 45 mg/kg). In our experiment only pretreatment with LA 30 min before NA (NA+LA [30]) significantly reduced the NA induced rise of blood flow in rat ear vessels (Table 19). Table 19

Effect of MD and LA on NA induced cutaneous vasodilatation;

The potential of MD to antagonize the peripheral vasodilatation, caused by NA may have beneficial effect in clinic for diminishing the cutaneous effects (flushing) of NA. In our experiments we could not establish any advantage of simultaneous application of NA and LA combination in comparison with simultaneous application of NA and MD.

7.2. Assessment of niacin-induced skin temperature changes

Materials and methods. For registration of changes in skin temperature of intact rats contactless temperature recording method was used (Papaliodis D et al, Br J Pharmacol 2008;153:1382-1387). Temperature measurements were performed with a hand-held infrared thermometer (Model ProScan 510, TFA-Dostman). Animals were habituated to handling and to the infrared probe for 3 days before use. Three temperature readings from the dorsal side of each ear were recorded without anaesthesia immediately before animals were injected s.c. with either NA (withers area) or solvent/test compound (tail area). The ear temperature was then measured every 5 min. for a period of up to 60 min. The animals were returned to their cages between measurements. Six ear temperature measurements (three from each ear) were averaged for each time point. Laropiprant [MK 0524, Cayman Chemicals] (LA) was first dissolved in DMSO and then freshly diluted with 0.9% NaCl, on each day of the experiment. The rate of NA and LA combination was based on Summary of Product Characteristics for Tredaptive™ (nicotinic acid/laropiprant) 1000 mg/20 mg modifϊed-release tablets.

Experimental schedule

I Testing of time and solvent influence on skin temperature

GROUP TREATMENT Number of animals

SoIvLA Solvent for LA 6

SoIvNA Solvent for NA 6

NA NA 15 mg/kg sc 6 II Investigation of the effect of NA/LA and NA/MD combination on skin temperature

LA was introduced simultaneously with NA as NA+LA [0] or 30 min in advance of NA as NA+LA [30], MD was introduced simultaneously with NA as NA+MD [0] or 30 min in advance of NA as NA+MD [30]; also the effects of LA and MD alone on skin temperature were checked.

GROUP TREATMENT Number of animals

Control/solvent 6

NA NA 15 mg/kg 6

LA LA 0.3 mg/kg 6

NA+LA NA 15 mg/kg+LA 0.3 mg/kg 6*

MD MD 45 mg/kg 6

NA+MD NA 15 mg/kg+MD 45 mg/kg 6*

*in each time group

Statistics. Data were analyzed by Microsoft Excel software and results expre mean +/- mean standard error of mean. Mean results of different groups were compared using single-factor analysis according to ANOVA and t-Student's test. PO.05 was considered as significant.

Results. The baseline mean ear temperature was 28.1-30.2 ⁰C recorded at 10 AM to 2 PM. A time response study for NA (15 mg/kg s.c.) showed a maximal temperature increase of 2.32±0.37 ⁰C from baseline and 2.57±0.43 in comparison with Solvent group (PO.005) at 10 min (below). It was established that the effect of the LA solvent on ear temperature was substantially different from that of NA and MD solvent only in the first 5 min after injection, therefore only one control group was used.

SoIvLA

Subcutaneous injection of MD or LA did not cause significant changes in rat ear skin temperature (Table 21). Simultaneous administration of NA and MD (NA+MD [0] group; advance time = 0) caused reduction of NA flushing that was similar to that caused by simultaneous administration of NA and LA. The increase of temperature, caused by NA was reduced, correspondingly, to 69% and 67 % (Table 21). There was no significant difference on temperature between the MD+NA [0] (when MD was added together with NA) and MD+NA [30] (pretreatment 30 min in advance with MD 45 mg/kg). In our experiment only pre-treatment with LA, when given s. c. at dose 0.3 mg/kg 30 min before the injection of NA, caused significant protection against the increase of skin temperature, induced by NA (Table 21).

Table 21

*P<0.05 vs Control **P<0.005 vs Control ♦**P<0.0005 vs Control sP<0.05 vs NA M^$P<0.0005 vs NA

Thus we have established that the combination of NA with MD has not only the unexpected synergistic antiplatelet activity, but also has a reduced side effect - flushing.

Example 8. Determination of influence on blood sugar levels

It is well-known that even single large oral dose of NA increases the GL level in blood of animals (Thornton JH, Schultz LH, J Dairy Sci 1980;63 ,262-268) and using NA in treating diabetes patients requires monitoring of sugar levels (Goldberg RB, Jacobson TA, Mayo Clin Proc 2008;83(4):470-8).

Method. Male adult Wistar rats were used in experiments. Animals were fasted from the night before experiment to stabilize the sugar level in blood. GL levels in venous blood were determined before the experiment and 45 min. after oral administration of blank or test substance. GL levels were determined by a commercial kit (Optium, Abbott Diabetes Care Ltd, USA). NA was used in a high dose (300 mg/kg p.o.), that is known to cause a stable and long-lasting rise of GL levels in blood. The same dose of MD (300 mg/kg) was used.

Animals were randomized in 4 groups (n=8):

1) Control group (received 1 % NaCl solution, dose 2 ml/kg)

2) NA group (received NA, dose 300 mg/kg)

3) MD group (received MD, dose 300 mg/kg)

4) NA+MD group (received 300 mg/kg NA and 300 mg/kg MD).

Statistics. The results are presented as means ± SEM for each group. Statistical analysis within groups was performed by Student t-Test. Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). P <0.05 was considered as significant. Results. NA caused statistically significant rise of GL levels vs baseline and control (Table 21 below). MD caused an insignificant decrease in GL levels that did not differ from control. The combination of NA and MD caused a noticeably less pronounced rise in GL levels (change to baseline 27.5 %, compared with 61.1 % caused by NA). This positive effect indicates that combination medicinal product NA+MD may cause less pronounced side effects in patients with unstable or disturbed glycemic control.

Table 21

Effect of test substances on GL level in rat blood n=8 Mean±SEM

*P<005 vs Control 4PO Ol vs Baseline

Summary conclusions

It was unexpectedly discovered that a combination of NA and MD enhanced the curative effect of NA on disorders, including dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease as chosen from the group of angina pectoris and myocardial infarction, transient and permanent ischemic attack including cerebrovascular accident and stroke and peripheral arterial occlusive disease, improved the condition of heart and brain under ischemic-hypoxic conditions. The combination also ameliorated the peripheral vasodilatation caused by NA. Therefore the novel combination medicinal product is expected to exhibit improved activity compared to NA in treating metabolic-related disturbances, allowing to reduce daily doses of NA, and have less pronounced undesirable side effects.

Modes of carrying out the invention

The term "a combination medicinal product" used herein provides for the simultaneous, sequential or separate administration of the components of the combination. Thus the present invention provides a combination medicinal product comprising NA and MD or a pharmaceutically acceptable salt thereof for use simultaneously, sequentially or separately for the prevention of platelet aggregration. A combination medicinal product of the invention may be administered in the form of a pharmaceutical composition. According to this aspect of the invention there is provided a pharmaceutical composition which comprises NA or a pharmaceutically acceptable salt thereof and MD or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.

A pharmaceutical composition according to the present invention also includes separate compositions comprising a first composition of NA or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier, and a second composition comprising MD or pharmaceutically acceptable salt thereof and a pharmaceutically-acceptable diluent or carrier. Such a composition provides for sequential or separate use. Since the treatment or prevention of metabolic-related disorders presumes prolonged use of medicinal product, the most preferable mode of carrying out the invention is provided by a form suitable for oral use, for example as tablets or capsules. The amount of each active ingredient of the present combination medicinal product in a pharmaceutical composition will vary depending upon the condition treated. Those skilled in the art of treating metabolic-related disorders patients can readily select the appropriate amounts of each active ingredient and a suitable dosing schedule. The preferable ratio of the active ingredients NA and MD or salt thereof is from 3:1 to 1:3.

According to a further aspect of the invention there is provided the use of the combination medicinal product as defined herein before or of the pharmaceutical composition as defined hereinbefore, for the manufacture of a medicament for administration simultaneously, sequentially or separately for the prevention of platelet aggregation/thrombosis.

Claims

1. A combination medicinal product having increased activity and reduced side effects in preventing and/or treating metabolic-related disorders or a pathology induced by platelet aggregation, comprising an effective amount of nicotinic acid or pharmaceutically acceptable salt thereof and an effective amount of meldonium or pharmaceutically acceptable salt thereof.

2. The product of Claim 1, wherein the nicotinic acid or pharmaceutically acceptable salt thereof is in the form of immediate-release, sustained-release or extended-release formulation.

3. The product of Claim 1, wherein the meldonium or pharmaceutically acceptable salt thereof is in the form of immediate-release, sustained-release or extended-release formulation.

4. The product of Claim 1 , wherein the side effects are peripheral vasodilatation (flushing) and/or increase of blood glucose levels.

5. Use of a combination medicinal product of Claim 1, wherein the metabolic disorder is selected from the group, consisting of dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease selected from the group of angina pectoris and myocardial infarction, transient or permanent ischemic attack including cerebrovascular accident and stroke, and peripheral arterial occlusive disease.

6. Use of a combination medicinal product of Claim 1 for preventing and/or treating a pathology induced by platelet aggregation.

7. A pharmaceutical composition suitable for use in treatment of metabolic- related disorder or a pathology induced by platelet aggregation which comprises an effective amount of nicotinic acid or pharmaceutically acceptable salt thereof and an effective amount of meldonium or pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable excipient or carrier.

8. The composition of Claim 7, wherein the nicotinic acid or a pharmaceutically acceptable salt thereof is in the form of immediate-release, sustained-release or extended-release formulation.

9. The composition of Claim 7, wherein the meldonium or a pharmaceutically acceptable salt thereof is in the form of immediate-release, sustained-release or extended-release formulation.

10. The composition of Claim 7, comprising about 50-500 mg of nicotinic acid or pharmaceutically acceptable salt thereof and about 50-500 mg of meldonium or pharmaceutically acceptable salt thereof.

11. Use of a combination medicinal product of Claim 1 or a pharmaceutical composition of Claim 7, for the treatment and/or prophylaxis of metabolic- related disorder or a pathology induced by platelet aggregation.

12. Use of a combination medicinal product of Claim 1 or a pharmaceutical composition of Claim 7, for the manufacture of a medicament for administration simultaneously, sequentially or separately to a patient for treating or preventing of metabolic-related disorders or a pathology induced by platelet aggregation.

13. The use of a combination medicinal product of Claim 12, wherein the metabolic disorder is selected from a group, consisting of dyslipidemia, hyperlipidemia and atherosclerosis, coronary heart disease selected from the group of angina pectoris and myocardial infarction, transient or permanent ischemic attack including cerebrovascular accident and stroke, and peripheral arterial occlusive disease.

14. The use of a combination medicinal product of Claim 12, wherein a pathology induced by platelet aggregation includes ischemic events such as myocardial infarction or stroke, thrombosis and thromboemolism.

15. A method for the preventing or treating of metabolic-related disorder comprising simultaneously, sequentially or separately administering an effective amount of a combination therapeutic product of Claim 1 or pharmaceutical composition of Claim 7 to a patient in need of such treatment.

16. The method of Claim 15, wherein the disorder is selected from the group, consisting of dyslipidemia, hyperlipidemia and atherosclerosis, coronary heart disease selected from the group of angina pectoris and myocardial infarction, transient or permanent ischemic attack including cerebrovascular accident and stroke, and peripheral arterial occlusive disease.

17. A method for the preventing and/or treating a pathology induced by platelet aggregation comprising simultaneously, sequentially or separately administering an effective amount of a combination therapeutic product of Claim 1 or pharmaceutical composition of Claim 7 to a patient in need of such treatment.

18. The method of Claim 17, wherein the pathology includes ischemic events such as myocardial infarction or stroke, or thrombosis and thromboemolism.

19. The method of Claim 15 or Claim 17, wherein the combination is administered as a single pharmaceutical composition, containing both nicotinic acid or pharmaceutically acceptable salt thereof and meldonium or a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable excipient or carrier.

20. The composition of Claim 7, further comprising a statin, selected from the group of atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin.

21. Use of a combination medicinal product according Claim 20 for preventing and/or treating of disorder, selected from the group of dyslipidemia, hyperlipidemia and atherosclerosis.