EA021344B1 - Therapeutic combinations of nicotinic acid and meldonium - Google Patents

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

The present invention relates to a combination of a drug product and a method for the prevention and / or treatment of metabolically related diseases, including dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease, selected from the group of angina pectoris and myocardial infarction, transient and permanent cerebrovascular accident, including cerebrovascular disorder and stroke, and peripheral occlusive artery disease, preventing platelet aggregation and thrombosis.

More specifically, the invention relates to a combination of a medicinal product containing nicotinic acid (niacin) and meldonium, which synergistically enhances the healing effect of nicotinic acid and to some extent improves the undesirable side effects of nicotinic acid, in particular peripheral vasodilation (rush of blood) and an increase in glucose blood. In addition, the invention relates to a pharmaceutical composition containing such a combined drug product and its use in the manufacture of a medicament for the prevention and / or treatment of metabolically related diseases.

Abbreviations Used

For brevity, the following abbreviations will be used in the description:

ATP - adenosine triphosphate

C - cholesterol

C - glucose

NEB-S - high density lipoprotein-cholesterol

Ι / K - ischemia / reperfusion

BEB-S - low density lipoprotein-cholesterol

ΜΌ - meldonium (ΙΝΝ)

ΝΑ - nicotinic acid

ΝΛΜτ - magnesium salt of nicotinic acid

ΡΙ - piracetam

ΚΡΡ - product of pressure intensity = mean blood pressure x heart rate x 1000 ^-1 8Ι - simvastatin

TS - triglycerides

TK - Triton ^ K1339 (Tu1oharo1)

V - ventricular fibrillation

UT - ventricular tachycardia

BACKGROUND OF THE INVENTION

Niacin is an important tool for the treatment of dyslipidemia and is currently the only available drug that has a beneficial effect on all components of the lipid profile: it lowers total cholesterol, TS and BEB-C in the blood and has the most pronounced activity, increases NOB-S atopd among drugs that change the content of lipids (P1eret TA., At. 1. Mapad. Sage. 2002; 8 (12 8irr1.): 8308-14).

The use of ΝΑ in the treatment of dyslipidemia was already known in 1955 (A11kb1 K., NoGGeg A., 81erbp., Arg. Wuset. Würk. 1955; 54: 558-559) and 1959 (Ratkopk 1v. \ Ν.Β .. Ρ1ίηη TH., AMA Arg. 1n1etp. Furniture. 1959; 103: 783-790).

Since NΑ effectively increases the content of NEB-C (McKeppeu 1., Argb. 1n1gp. Meb. 2004; 164 (7): 697-705. Saglkop L.A., 1. 1nlgp. Meb. 2005; 258: 94-114) , NΑ is currently combined with other lipid modifying agents, which mainly affect the content of BPBS in order to increase the level of NEB-C (Kokekkop KS, At. 1. Meb. 2005; 118 (10) : 1067-77).

NΑ is an effective agent that changes the content of lipids, which prevents the development of atherosclerosis and weakens cardiovascular events (8aeeGeu A.A., 8betkeu M.C., Klin. Honey. (Russian) 1996; 74: 48-52. Egekhe1 N ., Eigoreap Neai 1oitpa1 8irr1etep1k 2006; yo1. 8, 8irr1. Ρ: P23-P29. Vgo / up V.S., 2bao Kh.F., At. 1. Sagby1. 2008; 101 (8A): 58V-62V ) by increasing the content of NEB-S. Niacin reduces morbidity and mortality in patients with hyperlipidemia (R. Sappeg, et al., 1. At. Co11. Sagby1. 1986; 8: 1245-55). NΑ is the most effective drug for increasing the level of NOB-C in the treatment of cases of hyperlipidemic disease (Ehpdetöb, H. et al., Arg. 1nlg. Meb. 1994; 154: 1586-95. 8cb1stap S. et al., At. 1 Sagby. 1993; 71: 758-65). NΑ reduces the likelihood of thrombosis, reduces blood viscosity and has a cardioprotective effect, which can limit ischemic reperfusion injury. (Lathrshd, K.A. et al., Pag. Exp. Th. 1984; 231 (3): 532-538. Thieb. KA. Et al., At. 1. Pfu1. Neai Oge. Pfuk 2000; 279 (2) H764-H771. Kokekkop K.8. A1box1egok1K 2003; 171 (1): 87-96).

Since a low content of NOB-C is a risk factor for stroke (see appendix 8.S., 8bareg A.S., EtaStock S, 81 current 2000; 31: 1882. 8asso K.B., Vepkop K.T., Kagdtap Ό.Ε .1АМА 2001; 285: 2729-2735. Ρί / ok E., M1kLaShb1k ER., Satbyuax. Kek. 2001; 52 (2), 199-207. 8apokk1ap Ν., Tatlou Ν.Ε., Sigg. TgeaRSh.

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Θρΐ. Satyuu. May. 2008; 10 (3), 195-206.), ΝΑ as a means of increasing the content of HH-C (Sag1kop L.A., Sigg. Ορίη. Sagy1o1. 2006; 21 (4) 336-344) is useful for the prevention of stroke and conditions after a stroke (Koshikou δ.Ο., Lyb / οη 8.R., Clin. honey. (Moscow) 1975; 53 (9): 38-41). It was found that the content of HHB-C decreases during an acute ischemic stroke (Rikktap Α.Ν. et al., 1. Nito1. Δα. 2009; 279 (1-2): 53-56). It has been shown that ΝΑ improves functional recovery after a stroke (CHep 1. et al., Αηη. No. 1, 2007; 62 (1): 49-58). For the treatment of stroke and other ischemic conditions, HHB-C itself was proposed (Karig Ν.Κ. et al., Uax. Nea1kN Ryk Mapad. 2008; 4 (1): 3957. EP 1425031).

Three formulations ΝΑ are available (direct release, extended release and long-acting). Directly secreted ΝΑ is associated with an adverse rush of blood and an increase in blood glucose. ΝΑ prolonged action is associated with a low rush of blood, but also with a risk of hepatotoxic effects. Prolonged excretion is associated with a lower rush of blood and a low hepatotoxic risk (P1eret Ι.Α., Vol. 1. Nea1kN. Δνκί. RNag. 2003; 60 (13 δυρρ1.2): δ9-14. MsKeppeu 1., ΑιόΙι. 1 pkg. May 2004; 164 (7): 697-705. Kporr R.N., Rev. 1. Sagyu1. 2008; 86 (δυρρ1.); 51b-56b). The use of sodium, potassium and magnesium salts ΝΑ is also described.

The main disadvantage of ΝΑ is the need for large doses in order to significantly change the content of lipids in the blood. Almost 100% of patients treated with ΝΑ experience an unpleasant side effect of a rush of blood, which in many cases prevents treatment with ΝΑ. It was found that a sharp release of prostaglandin Ό2 is the direct cause of the ΝΑ-induced rush of blood (Mottov Ι.Ό. et al., 1. 1 unit. Nettako. 1992; 98: 812-5). Since ΝΑ-induced flushing is the result of prostaglandin activity, acetylsalicylic acid, as a well-known inhibitor of prostaglandin synthesis, has been proposed and used to control the flushing. In addition to acetylsalicylic acid, other non-steroidal anti-inflammatory drugs (ΝδΑΣΌδ) are also active (OetMk11et N., Vassata-Nshek M., Ιπί. 1. Cl. Rgask. 2006; 60 (6): 707-715). However, these ΝδΑΙΌδ have their own side effects, and they can cause gastrointestinal irritation and ulceration.

Recently, a specific antagonist for prostaglandin Ό2 has been proposed (RagNoGeg K.O., Waxi1ag Nea1kN apy Rschk Mapadedetp 2009; 5: 901-908) receptor subtype 1, laripiprant, as a means to reduce the rush of blood caused by β (E. ba1 et al. ., Syp. RNag.TNeg. 2007; 81: 849-857. Oa \ Haykop M.N., Vol. 1. Sagyu1. 2008; 101 [κυρρ1.]: 14В-19В). Although the addition of laripiprant will reduce the frequency of the rush of blood, ΕΑ cannot completely eliminate the indicated side effect. Laropiprant does not alter the effect of niacin on lipids or other side effects of nicotinic acid. Consequently, the combination of nicotinic acid with laripiprant can ensure the use of higher doses of nicotinic acid, and therefore the full potential of the drug is revealed (RagNoGeg K.O., Waxiag Nea1kN apy ΡίκΗ Mapadepetk 2009; 5: 901-908, O1kkop Α.Ο., Exrее ^ to Ορω ^ op RNagtasokNegaru 2010; 11 (10): 1715-1726).

In patients with type 2 diabetes, dyslipidemic changes are often observed, characterized by an increase in the content of TO, as well as a decrease in the content of HH-C. When considering the pharmacological effect of ΝΑ on lipid metabolism, ΝΑ should counteract dyslipidemic changes in patients with type diabetes. However, some publications indicate that ΝΑ increases insulin resistance (Oagd Α., Ogipyu δΜ., ^ ΑMΑ 1990; 264: 723-6. KaNp δ.Ε. et al., H1aekek 1989; 38: 562-8) and increases glucose content (E1at, M.V. et al., ^ ΑМΑ 2000; 284 (10): 1263-1270). Therefore, only limited doses of ΝΑ (<2 g / day) are recommended for patients with diabetes (δ ^ ρ ^ τ 1., Vekkepeide 1., Uap Oaa1 b., Sigg. May. Rec. Ορ. 2005; 21 (5): 665-682). Obviously, new drugs are needed to improve the glycemic control of diabetes patients using ΝΑ. In the clinic, the hypoglycemic ability of meldonium was registered (Kommersko M.E. et al., Klin. Med. (Rus) 2007; 85 (7): 39-42), therefore, it could be expected that this drug can be combined with ΝΑ for obtaining additional clinical benefits.

Platelets play a major role in the development of atherosclerosis and the inevitable formation of a blood clot during coronary heart disease. Antiplatelet agents become paramount for the prevention and treatment of various diseases, including cardiovascular, cerebrovascular and peripheral arterial systems (Meayo \ tk Τ.Α. et al., Sys. Rec. 2007; 100 (9): 1261-75) . ΝΑ is an effective agent that changes the content of lipids, which prevents the appearance of atherosclerosis and weakens cardiovascular events. ΝΑ has a diverse effect on lipoprotein and gives an effect against arterial thrombosis, which improves endothelial function, weakens inflammation, increases platelet stability, and reduces thrombosis (Rockepkop RS, ΑкНе ^ οκс1е ^ οκ ^ κ, 2003; 171: 87-96 )

Niacin inhibits platelet aggregation (Lact K.M., Farmakol. Toxicology, 1980; 43 (5): 581-5). Outside the body, ΝΑ affects platelet activity, moderately inhibiting aggregation, and stimulating a significant release of prostaglandin, mainly with intact expression of the main platelet receptor. The effect of ΝΑ is unique, differing from the known effect of antiplatelet agents, and suggests the possibility of therapeutic combinations (8GeGiapu, U. L. et al., Tyrnogyz 15 apy Naetoz1a51c, 2010 (in press).

Niacin almost completely prevents intravascular coagulation induced by thromboplastin and pituitrin, which demonstrates the thrombolytic effect of niacin (Baillia UR, Cardiology 1974; 14 (11): 105-7 (rus). Antithrombolytic properties ΝΑ are described by several authors (§6e81akou UA, Probl. Hematol. Blood transfusion, 1977; 22 (8): 29-35. Syekipe δ.Ι., δον. May. 1982 (5): 105-8). Niacin reduces the risk of blood coagulation (SNespeu S.M. et al., At. Neai 1., 2000; 140: 631-36).

Meldonium is a drug with a certain beneficial effect on the heart and blood vessels. Some desirable MO activity has been found in animal models of atherosclerosis (M. Uemeps, διηίΐ5; B. ιπφι, B. Bais 1. La. Att. 8c1. 2000; 10.194-199. M. Veemeps et al., Baangs 1. L.a. Att. . 8c1. 2002; 12: 116-122. OkeepemuN 1.U., ^ ζ ^ η ^ ν U.E., Patol. Fiziol. Exper. Ter. 2002; (2): 247), and was observed in clinics ( Κ ^ ν Κδ. Et al., Ter. Arch. 1991; 63 (4): 90-3), therefore, it was expected that this drug could be combined with ΝΑ for additional clinical benefits.

In addition, it was noted that MO inhibits platelet aggregation (Tzksh U.1., Ros. Cardiol. Zh. 2002; 1: 45-52). With a two-week therapeutic use of oral administration of MO to rabbits and dogs, after experimental arterial thrombosis, a thrombolytic effect was observed (iodipoua et al., Experimental wedge, pharmacoter. 1991; 19: 91-98 (rus). Data on the preventive effect of MO for limitations or warnings of thrombosis are not known.

State of the art

The current trend towards the development of combination drugs for the treatment of metabolically related diseases can increase the effectiveness of clinical care (Blask EM. Sigg. TN. Ver. 2003; 5: 39-32). Due to fibrates (amphipathic carboxylic acids), ΝΑ and statins each regulate serum lipids according to different mechanisms, and combination therapy can provide patients with especially desirable advantages compared with monotherapy. As progress in the development of new drugs to reduce Lb-C content has slowed, research has focused on the development of improved drugs that increase Lb-C content. For the treatment of metabolically related diseases, there is an increased use of combination therapy, including ΝΑ, fibrates, statins and substances that enhance the excretion of bile acids, thanks to the additional profiles of the combined products (M.Szeg M., Mauo Syp. Rgos. 2003; 78 (6): 735-42 Vasquez! M. et al., UAE Neayy B1zk Mapad. 2005; 1 (4): 317-331. Ve1zeu L. et al., Sigg May. Vez. Orsh. 2008; 24 (9), 2703-9 Vozepzop V.S., RSh V., No. k Syp. Rgaek Sagyuuazei May. 2009; 6 (2): 98-100). There is an increased use of combination therapy, including ΝΑ and other drugs, for example, acetylsalicylic acid and other ΝδΑΙΌδ, as platelet inhibitors (TOR 5981555). However, these combinations do not increase the activity of ΝΑ, that is, a synergistic effect is not observed. Therefore, any drugs that could enhance the therapeutic effect of ΝΑ in metabolically related diseases, without enhancing the undesirable side effects of ΝΑ, can give a positive result in clinics.

SUMMARY OF THE INVENTION

The purpose of the present invention is to develop a combination drug product for the prevention and / or treatment of metabolically related diseases and a method for the prevention and / or treatment of metabolically related diseases for a patient in need, using ΝΑ in combination with MO, which gives an additional positive result in alleviating undesirable side effects ΝΑ . It is expected that the combined drug product of the present invention will provide a synergistic effect in a method of treating and / or preventing metabolically related diseases, which include dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease selected from the group of angina pectoris and myocardial infarction, transient and permanent disturbance of cerebral circulation, including cerebrovascular disorder and stroke, and peripheral occlusive artery disease, preventing platelet aggregation and tr mboz.

According to the present invention, a combined drug product is defined as having a synergistic effect if the therapeutic effect is higher than the effect of ΝΑ or MO taken separately. It is understood that the combined pharmaceutical product used in the invention means the simultaneous, sequential or separate administration of combination drugs. In addition, the purpose of the invention is to obtain a pharmaceutical composition that contains ΝΑ, as well as MO for the above purpose. Other objectives of the invention will become apparent in the future to a person skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes a combination of ΝΑ and MO, which results in an effective synergistic combination for the treatment of metabolically related diseases, preferably in the form of a single dosage. Alternatively, these two components may be administered separately, simultaneously or sequentially in any order. The exact form in which these active ingredients are administered is not relevant, provided that the desired effect of the invention is obtained.

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The active components can be used in the form of capsules, suspensions, dispersions, elixirs, syrups, or the like, regardless of purpose: single or in a single composition.

Currently, the inventors have unexpectedly discovered that ΝΑ and ΜΌ have a synergistic effect on metabolically related diseases and other positive effects. It was unexpectedly found that ΜΌ is the first drug that enhances the positive effects of ΝΑ, namely, it reduces the content of TS and BEB-C and increases the content of IOB-C, increases the anti-aggregating effect ΝΑ and eliminates undesirable side effects ΝΑ, in particular blood flow and increase blood glucose.

Therefore, it can be expected that this combination will be the preferred drug for the treatment of dyslipidemia in patients with diabetes. In addition, the authors unexpectedly found that the combination of the present invention eliminates the aftereffect of experimental heart attack and stroke.

The inventors also found that ΝΑ and ΜΌ have a synergistic effect on platelet aggregation. It was unexpectedly found that ΜΌ is the first drug that enhances the antiplatelet effect of ΝΑ.

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

In addition, the combination product according to the present invention includes a combination of separate pharmaceutical compositions for active preparations containing the first composition ΝΑ or a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient or carrier, and a second composition containing ΜΌ or a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient . The specified combination of pharmaceutical compositions allows to obtain a combined medicinal product according to the invention for simultaneous or sequential use. The advantage of this combination is the ability for the attending physician to adjust the ratio of active drugs for individual patients. It can be expected that the combined drug product of the present invention also includes delayed release and extended release formulations of ΝΑ, as well as pharmaceutically acceptable salts of ΝΑ (sodium, potassium or magnesium) and ΜΌ, and its salts.

In addition, the combined drug product of the present invention may include other drug products having known activity in metabolically related diseases, namely statins, in particular simvastatin.

The combination drug product of the present invention may also include other drug products having a known activity in metabolically related diseases, namely platelet aggregation inhibitors, in particular clopidogrel or dipyridamole.

The pharmaceutical composition of the invention can be obtained using traditional methods, using traditional pharmaceutically acceptable excipients or carriers and methods.

The following examples are provided to illustrate, but not to limit the scope of the invention.

Examples

Tests: The pharmacological activity of the test substances was investigated by standard methods used in the prior art. Animals were kept in groups of 6 animals in appropriate cages, in artificial climate chambers, at 22 ± 1 ° С, relative humidity 60 ± 5% and 12/12 hour cycles of alternating light and dark, with free access to food and water. All experiments were carried out in accordance with the Directive of the Council of the EEC of November 24, 1986 (86/609 / EEC) relating to the maintenance of experimental animals. Every effort has been made to minimize animal suffering and reduce the number of animals used.

Substances: Cholesterol (Αοτοδ Ogdashsk company), meldonium (Ogshbeh), animal feed (K 70 Bas1ash1i), oil (commercially available), холα cholate (Lsgok Ogdashsk), ΝΑ (Lsgo5 Ogdashsk), Laropiprant (Си 0524, С 0524 ) All other chemicals were obtained from industrial sources.

Example 1. Determination of activity against atherosclerosis

Methodology We used a model of atherosclerosis in C57BB161 mice that are genetically susceptible to atherosclerosis, which is described in the literature (διηίΐΐι ΕΌ., Blek1oБ EB., 1. 1i1egi. Μеά. 1997; 242: 99109). The control group of animals received standard food for laboratory animals. Experimental atherosclerosis was induced by adding 1.15% cholesterol, 15% butter and 0.5% chol No. v to the standard diet (R. Νίκΐιίηη et al., 1. EtrHT Kek. 1993; 34: 1413-1422). The experimental groups received the test substances separately and in combination in doses that are active, as established from pilot experiments. At the 22nd week, the level of atherosclerotic changes is assessed morphologically, biochemically and histologically using standard methods and criteria (Raldep V. et al., L1yego8s1ego818. 1987; 68: 231-240). Serum cholesterol, HBb-C, HBb-C and TC in serum were determined using standard test kits. The ratio Lb-C / NBE-C was taken as a standard criterion for assessing the risk of cardiovascular disease (Teppappbe / M.L., \ B.Le B., 1. At. Co11. ΝιιΙγ. 2008; 27 (1): 1- 5).

The atherosclerosis index (reflects coronary atherosclerosis and is a detector of peripheral atherosclerosis) was calculated as follows: Index = LBE-C / NBI-C.

The atherosclerosis coefficient (reflecting coronary atherosclerosis) was calculated as follows:

Coefficient = Total cholesterol / NBI-C.

Statistics.

Data were presented as mean ± standard error of measurement (SDI) of 7-10 measurements for individual animals. The differences between the experimental groups were compared using one-way analysis of variance (ΑΝΟνΑ) with repeated comparison (Tikeu's test). A probability of P <0.05 was considered significant.

Results.

The first series of experiments was planned in order to determine the effect of various ratios and doses of drugs in combination. As shown in the table. 1, after 22 weeks, control animals of group C fed with lipid and cholesterol-rich nutrition develop atherosclerotic changes in the aorta, especially in the aortic arch. When used separately for ΝΑ and MB, there is a tendency to decrease the lesion area. Unexpectedly, the combination of ΝΑ and MB gives a significantly higher and statistically significant protective effect against atherosclerotic damage than each substance separately, that is, a synergistic effect is observed.

Table 1. The effect of ΝΑ and MB on atherosclerotic damage to the aorta; n = 7-10;

Mean ± SDI

Group Damage area in the aortic arch, μm ² Area of damage to the aorta,% Control 44 ± 25.8 " 0.02 ± 0.01 " C control 6076 ± 1282 5.44 ± 1.12 ΝΑ 50 mg / kg 5867 ± 975 5.21 ± 1.07 ΝΑ200 mg / kg 4153 ± 742 3.39 ± 0.69 MO 50 mg / kg 4741 ± 786 3.87 ± 0.64 MO 150 mg / kg 3801 ± 573 3, ZOE, 49 MO 200 mg / kg 3098 ± 547 2.74 ± 0.47 *

ΝΑ50 + Μ050 3106 ± 368 2.58 ± 0.31 ** ΝΑ50 + Μ0150 223I390 ¹⁴ 1.9310.40 ^ 4A200 + M0200 1602 ± 404 " 1.1910.38 ^-14

NΑ50 + M ^ 50 = ΝΑ 50 mg / kg + MB 50 mg / kg NΑ50 + M ^ 150 = ΝΑ 50 mg / kg + MB 150 mg / kg NΑ200 + M ^ 200 = ΝΑ 200 mg / kg + MB 200 mg / kg * P <0.05 relative to C control ** P <0.005 relative to C control $ P <0.05 relative to ΝΑ in the same dose & P <0.05 relative to MB in the same dose

As shown below in the table. 2, the combination of ΝΑ and MB gives a statistically significant effect of reducing the content of EB-C and TC and increasing the content of NB-C. For a combined dose of NΑ50 + M ^ 150, where ΝΑ was administered in a dose that does not have a significant effect on the lipid content, the total effect unexpectedly turned out to be higher than the effect of each substance separately. This is especially noticeable in the atherosclerotic indicator and the ratio of total cholesterol / NBI-C, where synergism of the action of ΝΑ and MB was observed.

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Table 2. The effect of the test substances on lipids in serum n = 7-10, the average value ± SOI

Group Total C, mg / dl Noah mg / dl sir, mg / dl Index, IX-C / NOC General C / Hbb-C ratio those, mg / dl Control 66,514,5 5713.4 9,712.1 0.17 ± 0.04 1,1710,04 22.1 12.19 C control 161110,4 5015.0 11019.9 2.12J, 25 3.1310.23 48,814,93 ΝΑ5Ο mg / kg 15619.4 5615.2 10016.9 1.7810.19 2.7710.18 37,814.56 ΝΑ200 153,111.1 5914.7 93 ± 8.3 1.6310.15 * 2,6010,2 * 31,714,21 1UY50 mg / kg 15719.5 5415.0 10217.5 1.9710.20 2.9410.2 44,314,84 Μϋ 150 mg / kg 153111.8 59 ± 5.0 9419.1 1.6510.18 * 2.6310.18 * 42,914.62 Μϋ200 mg / kg 151111.1 5613.9 95110.0 1.7210.20 2.70J.2 42,113,35 ΝΑ50 + ΜΡ50 156,110.2 58 ± 5.5 9717.9 1.7110.23 2.72J.23 40,113,75 ΝΑ50 + Μ0150 14818.9 6515.7 8314,4 ^ 1.3110.09 ** 2, ZOYA, 08 * ” 36,112,23 ΝΑ200 + Μϋ200 15217.1 6715.2 * 8515.1 * 1.2910.11 ** 2.31J, 12 * 33,612,45 ⁴

ΝΑ50 + ΜΌ50 = ΝΑ 50 mg / kg + ΜΌ 50 mg / kg ΝΑ50 + ΜΌ150 = ΝΑ 50 mg / kg + ΜΌ 150 mg / kg ΝΑ200 + ΜΌ200 = ΝΑ 200 mg / kg + ΜΌ 200 mg / kg * P <0, 05 relative to C control ** P <0.005 relative to C control P <0.0005 relative to C control $ P <0.05 relative to ΝΑ at the same dose & P <0.05 relative to ΜΌ at the same dose

In another series of experiments, the anti-atherosclerosis activity of the combination of the present invention was evaluated in more detail, and in addition, the well-known antilipidemic drug, §1, was also added to the combination. With the separate use of ΝΑ and ΜΌ, a tendency was shown to decrease the area of damage (Table 3). Unexpectedly, the combination of ΝΑ + ΜΌ gives a significantly higher and statistically significant protective effect against atherosclerotic damage than each substance separately. Adding a combination of the present invention to §1 further increases the activity of the protective action against atherosclerotic damage.

Table 3. The effect of the tested substances on atherosclerotic damage to the aorta n = 7-9; mean ± SDI

Group Damage area in the aortic arch, μm ² Area of damage to the aorta,% Control 29.4114.8 ** 0,0110,01 ’ C control 64161860 5.97J, 89 ΝΑ 50 mg / kg 50011726 4.5210.64 Μϋ 150 mg / kg 41891628 3.5810.67 * 3110 mg / kg 28191447 ** 2.67 J, 36 ** ΝΑ50 + Μϋ150 24131442 ** ® * 2.2810.40 *** ΝΑ50 + Μϋ50 + 5Ι10 22631314 ** ” 2.1910.30 ** ”

ΝΑ50 + ΜΌ150 = ΝΑ 50 mg / kg + ΜΌ 150 mg / kg.

ΝΑ50 + ΜΌ50 + §Ι10 = ΝΑ 50 mg / kg + ΜΌ 50 mg / kg + §1 10 mg / kg * P <0.05 relative to the C control.

** P <0.005 relative to the C control.

# P <0,0005 relative to C control.

$ P <0.05 with respect to ΝΑ.

& P <0.05 with respect to ΜΌ

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In the same experimental series, total cholesterol, NEB-C, LEB-C and TO in serum were determined. As shown below in the table. 4, when used separately, ΝΑ and ΜΌ only slightly improve the ratio of cholesterol fractions and the rate of atherosclerosis without statistical significance. Unexpectedly, the combination of ΝΑ and ΜΌ significantly improves the ratio of cholesterol fractions and statistically significantly reduces the rate of atherosclerosis and the ratio of total C / HH-C. In addition, this combination prevents the increase of Lb-C and TO in serum. Thus, the combination according to the invention has a significantly higher protective effect against changes in lipid metabolism than the components of the combination taken separately. The pronounced protective effect is also preserved in a triple combination with 81.

Table 4. The effect of the test substances on lipids in serum n = 7 - 9, average value ± SOI

Group Total C, mg / dl NIGHT-C mg / dl IE-s mg / dl Index, them-with / noah General C / NOA ratio tf mg / dl Control 66.8 * 3.7 ’ 58 ± 3.4 8.7 * 1.94 ' 0.15 * 0.03 ’ 1.15 * 0.03 * '22, 8 * 1.82 * C control 167 * 8.64 54 ± 4.7 113 ± 8.4 2.24 * 0.25 3.23 * 0.24 52.2 * 3.98 ΝΑ 50 mg / kg 158 * 9.9 57 ± 5.2 100 * 7.6 1.76 * 0.21 2.75 * 0.19 41.0 * 4.62 150 mg / kg 159 * 7.8 62 ± 4.1 95 ± 8.2 1.58 * 0.21 2.60 * 0.21 44.2 * 4.05 5Ι 10 mg / kg 132 ± 9.9 ‘ 55 ± 5.0 77 * 6.7 * 1.4 * 0.08 * 2.4 * 0.08 * 47.7 * 3.73 ΝΑ50 + Μ0150 139 * 7.5 * 60 ± 6.1 79 ± 6,2 ‘ 1.32 * 0.20 ** 2.30 * 0.20 * 39.5 * 3.96 * ΝΑ50 + ΜΟ50 + 3Π0 141 * 7.3 * 61 ± 5.5 80 ± 5.7 ‘ 1.31 * 0.16 ** 2.30 * 0.16 ** 42.2 * 3.65

ΝΑ50 + ΜΌ150 = ΝΑ 50 mg / kg + ΜΌ 150 mg / kg

ΝΑ50 + ΜΌ50 + 8Ι10 = ΝΑ 50 mg / kg + ΜΌ 50 mg / kg + 81 10 mg / kg) * P <0.05 relative to the C control.

# P <0,0005 relative to C control.

$ P <0.05 relative to ΝΑ

In another series of experiments, the combinations ΝΑΜτ and ΝΑ + 8Ι were included in the comparative evaluation (Table 5). The combination ΝΑΜ was compared with ΝΑ on the cockerels (VshGesh 1., Te1kka Α., Lba Ra1o1. ΜίοΓοόίαΙ 8 Sep 1962; 56: 261-265). The combination of ΝΑ (50 mg / kg) and ΜΌ (150 mg / kg) showed the most pronounced positive effect on the aorta, better than ΝΑ and ΝΑΜτ ^- , and also superior to the effect of the combination of 8Ι and ΝΑ used in a dose based on clinical experience (RapShap A. et al., Uax. Nea Kgk Uapad. 2008; 4 (5): 1001-1009).

Table 5. The effect of the tested substances on atherosclerotic damage to the aorta n = 7-9; mean ± SDI

Group Damage area in the aortic arch, μm ² Area of damage to the aorta,% Control 35 * 19.8 * “ 0.02 * 0.01 "* C control 6216 * 839 5.75 * 0.82 ΝΑ 50 mg / kg 4978 * 688 4.23 * 0.67 ΝΑΜγ 60 mg / kg 4053 * 518 * 3.39 * 0.44 * ME 150 mg / kg 3904 * 489 * 3.47 * 0.45 * ΝΑ50 + Μ0150 2105 * 215 “’ ® " 1.95 * 0.30 “’ ® " ΝΑ50 + 3Ι2 3068 * 297 * 2.67 * 0.48 * ΝΑ50 + ΜΌ150 = ΝΑ 50 mg / kg + ΜΌ 150 mg / kg

ΝΑ50 + 8Ι2 = ΝΑ 50 mg / kg + 8Ι 2 mg / kg * P <0.05 relative to the C control.

** P <0.005 relative to the C control. *** P <0.0005 relative to C control. $ P <0.05 with respect to ΝΑ.

@ P <0.05 with respect to ΝΑΜκ & P <0.05 with respect to ΜΌ.

# P <0.05 relative to ΝΑ50 + 8Ι2

- 7 021344

Like morphometric data, biochemical tests confirmed that the combination of ΝΑ + ΜΌ is much better than ΝΑ or ΝΑΜ§ normalizes the lipid content (Table 6).

Table 6. The effect of the tested substances on lipids in serum n = 7-9, the average value ± SOI

Group Total C, mg / dp NBB-S mg / dl BBB-S mg / dl Index, BBB-S / NBB-S General C / NbE-S ratio tf mg / dl Control 67 ± 3.9 ' 56 ± 3.0 7,811.83 ' 0.1410.03 ” 1.19 ± 0.04 26,512,13 ” C control 164 ± 9.2 50 ± 3.8 10717,4 2.2110.18 3.3310.19 54,815,56 ΝΑ 50 mg / kg 158 ± 8.7 57 ± 4.7 96 ± 6,2 1.7410.15 2.82 ± 0.16 40,813,46 ' IAMd 60 mg / kg 151 ± 9.6 55 ± 4.0 9117.7 1.6710.12 2.7310.14 41,411,06 ' MB 150 mg / kg 15418.1 55 ± 3.3 9515.5 1.7510.14 2.74 ± 0.11 43,211,25 ΝΑ50 + Μ6150 148 ± 7.4 63 ± 3.9 81 ± 4.1 ' 1.31 ± 0.1 T * ® ⁴ 2,3510,1ο ”* ® ⁴ 37.5 ± 0.97'® ⁴ ΝΑ50 + 3Ι2 14414.7 5814.4 82 ± 4.8 ' 1.4910.12 2.5210.12 ” 42,411,74

ΝΆ50 + ΜΌ150 = ΝΑ 50 mg / kg + ΜΌ 150 mg / kg.

ΝΑ50 + 8Ι2 = ΝΑ 50 mg / kg +81 2 mg / kg * P <0.05 relative to the C control.

** P <0.005 relative to the C control.

*** P <0.0005 relative to C control.

$ P <0.05 with respect to ΝΑ.

@ P <0.05 with respect to ΝΑΜτ.

& P <0.05 with respect to ΜΌ

The combination of ΝΑ + ΜΌ is similar to the combination of ΝΑ + 8Ι while lowering total cholesterol and BEB-C. but it significantly exceeds it in a positive effect on the content of NEB-S and TS, and also has a more pronounced effect on the atherosclerotic index and the ratio of total cholesterol / NEB-S.

This series of tests confirms that the combination of ΝΑ and ΜΌ gives a significantly improved positive result for experimental atherosclerosis than оза or NΑΜg and better than the clinically used combination of комбина and 8Ι.

Example 2. The effect of ΝΑ and ΜΌ, taken separately and in combination, on lipids in a rat model of hyperlipidemia

Methodology

Experimental chronic hyperlipidemia / hypercholesterolemia was induced using TK using the method described by Leuche and 8aH / tap (Leuche 8., 8aI / tap,., 1. Rägtaeo 1. Τοχίοοί. Mar. 2007; 55: 224-226). Animals received a solution of 250 mg / kg TK through the tail vein three times a week for 3 weeks. The solutions of the test substances for the experimental groups or water for the Control group was administered l / o, once a day, one hour before the injection of the TC solution or taking a blood sample. Male HuyGag rats weighing 220-240 g were distributed in the following 8 groups:

Group The number of animals, η 1. Control 10 2. TV (TV 250 mg / kg) 14 3. ΝΑ (TB 250 mg / kg + ΝΑ 50 mg / kg / d) 14 4. Mb (TB 250 mg / kg + Mb 150 mg / kg / d) 14 5. ΝΑ + Mb (ΤπΙοη 250 mg / kg + ΝΑ 50 + Mb 150 mg / kg / d) 14 6.8110 (TW 250 mg / kg + 3110 mg / kg / d) 12 7. ΝΑ + 3Ι2 (TB 250 mg / kg + 5Ι 2 + ΝΑ 50 mg / kg / d) 12 8. ΝΑ + 3Ι2 + Μ6 (TV 250 mg / kg + 8Ι 2 + ΝΑ 50 + Mb 150 mg / kg / d) 12

A biochemical blood test was performed after 1, 2, and 3 weeks (the day after the injection of TC) by heart puncture under ether anesthesia. Serum was separated by centrifugation and analyzed for total cholesterol, NEB-C. BEB-S and TS using kits commercially available.

- 8 021344

Statistics.

The obtained data were mathematically processed using the program Exce1 and the results were expressed as the average value ± SDI. The average results for different groups were compared using a one-way analysis of ΑΝΟν С and Student's criterion.

The difference in results was considered significant at P <0.05.

Results. Repeated injections of TC led to the development of severe and stable hypercholesterolemia and hyperlipidemia, characterized by a significant increase in the total content of cholesterol, BOB and TS compared with the control group. The therapeutic treatment of ΝΑ, more significantly in the first week, limited the increase in total cholesterol, BOB-C and TS, but significantly increased the content of HO-C only after 2 and 3 weeks. Meldonium (ΜΌ) was slightly less active in lowering total cholesterol, ΕΌΕ-C, and increasing HOB-C, but it did not prevent an increase in TS. Unexpectedly, the combined use of ΝΑ + ΜΌ is more effective than the individual components, reduced the content of BOB-C and TS and, in addition, increased the content of HOB-C. Moreover, with prolonged use of ΝΑ + ΜΌ (2 or 3 weeks in the indicated experiment), a much more pronounced decrease in the content of BOB-C and TS and an increase in the level of HOB-C were demonstrated than when using ΝΑ +81 (see below). Thus, it can be expected that the ΝΑ + ΜΟ combination will be used in the clinic to prevent and / or treat hypercholesterolemia and hyperlipidemia.

The use of 81 at a dose of 10 mg / kg significantly reduces the increase in total cholesterol and BOB-C induced by TK, but only slightly affects the content of HOB-C and TS. The combined use of 81 and ΝΑ in a clinically acceptable ratio showed significant protection against an increase in total cholesterol, BOB-C and TS in serum and an increase in the content of HOB-C. Unexpectedly, the combined use of ΝΑ, 81 and ΜΌ showed even better protection against changes induced by TC, that is, better than the effect of each individual component, and significantly better than ΝΑ + 8Ι to reduce the content of TS and BOB-S. A particularly important observation for clinical practice is that the combination of ΝΑ, 8Ι and ΜΌ increases the content of HOB-C significantly better than 8Ι or ΝΑ separately. Thus, it can be expected that the combinations ΝΑ + ΜΌ and ΝΑ + 8Ι + ΜΌ will be used in the clinic to prevent and / or treat hypercholesterolemia and hyperlipidemia.

Table 7. The effect of ΝΑ, 8Ι and ΜΌ, taken separately and in combination, on lipids in the rat model of hyperlipidemia n = 9-14: average value ± SDI

Group Total cholesterol after 1, 2 and 3 weeks, mg / dl C1 C2 Sz Control 77,614.9 ” 75.1 ± 5.1 72,712.5 ” TC 487.6125.4 501116.7 513,141.1 ΝΑ 345 115.7 *** 401.1125.1 ** 405.5125.9 * Μϋ 412124.7 414.7123.2 * 401 ± 22.8 * ΝΑ + Μϋ 340.5119.5 “* ® 360121.7 *** 346.4132.8 ** 81 314,129.6 *** 329.5136.2 ** 335.7131.6 ** ΝΑ + 31 345.9127.5 ** 388.7132.3 * 384.5133.9 * ΝΑ + 31 + Μϋ 333.5126.2 *** ® 376,4123,1 *** ZZZDIE.Z **** ®

Table 7 (continued)

Group HOC-C after 1, 2 and 3 weeks, mg / dl H2O-C1 NOOS2 NOB-SZ Control 54,611.9 * 54,111.3 53,711.0 * TC 76,316,9 76.2 ± 11.4 77110,2 ΝΑ 111,319.1 * 144.7113.5 ” 127.3110.9 " MO 107,318.4 * 118.6110.3 * 115.9 ± 9.6 * ΝΑ + Μϋ 126,2111,1 " 150,4111,2 * “®’ 136.9118.1 * 31 84.5112.5 98.5120.5 94.4110.2 ΝΑ + 5Ι 99 ± 12.9 116.2 ± 11.7 * 114.9111.9 * ΝΑ + 51 + Μϋ 124.8118.2 * Ϊ5731Ϊ5 ⁷ * ® * 143,219.7 * “

- 9 021344

Group Yu after 1, 2 and 3 weeks, mg / dl 1.0001 YuOS2 ίΟί-sz Control 18,713.8 ” 19,714,4 16,312.0 “ TR 388.7126.7 402.1119.2 405.1141.7 ΝΑ 216.3114.0 * “ 250.3120.6 ' 265.1118.4 * Μϋ 290.8121 * 275122.3 270116.9 * ΝΑ + Μϋ 209.6116.7 **** 197.6115.4 ****** 203.3120.3 “*” 51 212,144.1 *** 228.9120.6 *** 232.7 ± 22.8 “ ΝΑ + 51 227.2126.1 "* 272.5121.3 " 268 123,2 * ΝΑ + 51 + Μϋ 193.2116.5 **** 216.6117 “**” 197.1116.6 “’ ** Group TS after 1, 2 and 3 weeks, mg / dl TO1 ΤΘ2 thesis Control 3812.9 ' 3713,2 ' 3814.4 ” TR 1240180,1 1297178.3 12341114.1 ΝΑ 734181.6 ' 860173.8 " 828,144.7 * MO 1040191 1081163,2 1010172.5 ΝΑ + Μϋ 785162.9 * “* 792173.8 * “* 774139.2 “** 31 849,196.8 * 879,172.1 “ 8911129.1 ΝΑ + 51 769 169.2 * “ 868136.1 "* 879,131 * ΝΑ + 51 + Μϋ 734.4 ± 95.7 * "* 763136 *** ' ⁴ * 692145 *** ”

* P <0.05 relative to TP.

** P <0.005 relative to TP.

*** P <0.0005 relative to TP $ P <0.05 relative to ΝΑ. & P <0.05 with respect to ΜΌ.

# P <0.05 with respect to ΝΑ + 8Ι

Example 3. Determination of cardioprotective characteristics

Methodology

HUMag male rats were divided into 6 groups (from 12 to 16 animals in the group):

1) The control group received n / a 0.9% saline;

2) group ΝΑ50 received n / a 50 mg / kg / day ΝΑ;

3) group ΜΌ50 received n / a 50 mg / kg / d ΜΌ;

4) the ΝΑ50 + ΜΌ50 group received n / a 50 mg / kg / d ΝΑ plus 50 mg / kg ΜΌ;

5) the ΜΌ150 group received p / o 150 mg / kg / d ΜΌ;

6) the ΝΑ50 + ΜΌ150 group received p / o 50 mg / kg / d ΝΑ plus 150 mg / kg / d ΜΌ.

Animals of the test groups received experimental substances in the form of aqueous solutions through a cannula into the stomach 48, 24 and 1 hour before the experiment. Animals of the control group received the same volume of saline. Animals were anesthetized (sodium pentobarbital 60 mg / kg, ip) and, with mechanical gas exchange in the lungs, they were prepared to block the left coronary artery. Experimental heart attack was caused by blockage of the coronary artery for 45 minutes, followed by reperfusion for 2 hours. The following data were recorded: the number of animals with ventricular tachycardia (UT), ventricular fibrillation (UR), mortality, mean blood pressure, the product of pressure intensity (PPP), which characterizes the functional state of the heart muscle, reflects the production of adenosine triphosphate (ATP) in the heart muscle and is a widely used indicator in the analysis of clinical and experimental data (Vgobspsk T., Egidh R 2008; 9 (2): 83-91). After the experiment, ischemic and necrotic regions were determined using perfusion staining with Evans blue dye (triphenyldiazolium dye). The left ventricle was cut, weighed, and morphological criteria were calculated: the percentage of the ischemic zone of the left ventricle, the percentage of the necrotic zone of the left ventricle, and the ratio of the necrotic zone to the ischemic zone (an indicator of necrosis).

Statistics.

Results were presented as mean ± SDI for each group. Statistical analysis within groups was performed using Student's criterion. The data obtained during registration of blood pressure and heart rate were calculated for animals surviving in the ischemic reperfusion experiment. The number of cases of arrhythmia (UT and UR) and mortality were calculated for all animals. The differences between the experimental groups were compared using one-way analysis of variance (ΑΝΟνΑ) with repeated comparison (Tikeu'8 test). A probability of P <0.05 was considered significant.

Results. Cardiac ischemia followed by reperfusion causes serious heart rhythm disturbances in the control group with 7 deaths in a group of 16 animals. Group ΝΑ was slightly different from the control group regarding rhythm disturbances. In groups ΜΌ and ΝΑ + ΜΌ, less pronounced life-threatening rhythm disturbances (SD and UT) were observed relative to control, however, mortality was significantly lower only in groups ΝΑ50 + ΜΌ50 and ΝΑ50 + ΜΌ150 (Table 8).

Table 8. Effect of test substances on cardiac arrhythmias and mortality during occlusion and reperfusion of the coronary artery

Group η Ventricular tachycardia Ventricular fibrillation Mortality η % η % η % 1 / K control sixteen sixteen one hundred thirteen 81.3 7 43.8 ΝΑ50 12 12 one hundred 8 66.7 3 25 Μϋ50 12 eleven 91.7 7 58.3 3 25 Μϋ150 12 10 83 7 58.3 2 17 ΝΑ50 + Μϋ50 12 10 83 6 fifty one* 8.3 ΝΑ50 + Μ0150 12 nine 75 5 42 0 * 0

* P <0.05 relative to 1 / K control

Mean arterial pressure and heart rate were similar in all experimental groups, however, the drop in CRC noted in the control group was statistically significantly prevented only in the ΝΑ50 + ΜΌ50 and ΝΑ50 + ΜΌ150 groups during reperfusion (Table 9). This indicates significant protection against heart attack caused by functional bloodletting, after a brief (within 3 days) pre-treatment with the combined drug of the invention.

Table 9. The effect of the test substances on the product of pressure intensity (CRC) during blockage of the coronary artery and reperfusion n = 9-12; mean ± SDI

Group Initial value Cork 45 min. Reperfusion 120 min. Ι / P control 44.2 ± 3.0 39.7 ± 2.3 * 34.2 ± 2.1 ” ΝΑ 43.1 ± 2.8 40.4 ± 2.7 36.0 ± 1.3 ⁵ Μϋ50 44.8 ± 1.7 41.5 ± 2.9 37,611.9 * M6150 44.3 ± 1.8 40.3 ± 2.5 37.9 ± 1.8 ΝΑ50 + Μϋ50 42.7 ± 3.1 40.5 ± 1.9 38.6 ± 1.8 * ΝΑ50 + ΜΡ150 43.9 ± 2.7 41.2 ± 1.8 40.1 ± 1.6 ‘*

* P <0.05 relative to 1 / K control.

** P <0.005 relative to 1 / K control & P <0.05 relative to the initial value.

# P <0.01 relative to the initial value

The above is also confirmed by the statistical significance of reducing the percentage of the necrotic zone in the left ventricle and in the ischemic zone (Table 10) for combined drugs containing ΝΑ + ΜΌ in both dosage combinations.

- 11 021344

Table 10. The effect of the test substances on the heart morphology of rats that underwent occlusion of the coronary artery and reperfusion n = 9-12; mean ± SDI

Group Left ventricle mg Ischemic zone / left ventricle,% Necrotic zone / left ventricle,% Necrosis rate,% 1 / (7 controls 872.9125.1 48,412,4 34,412.8 70,913,7

ΝΑ 880,4125,2 47,011,4 29,812,4 63,013.9 Μϋ50 878.9148.1 48,411,5 28,413,1 58,414,3 Μϋ150 873,2141,8 48.111.9 28,111.8 * 58,212.9 * ΝΑ50 + Μϋ50 887,6122,3 46,811.6 22.8 ± 1.9 *** 49,112.1 ** " ΝΑ50 + Μϋ150 867.0 ± 31.5 47,712,1 21,511.3 ** " 45.612.6 * "

* P <0.05 relative to the Ι / P control ** P <0.005 relative to the Ι / P control P <0.0005 relative to the Ι / P control & P <0.05 relative to ΜΌ with the same dose of $ P <0.05 relative to ΝΑ .

Necrosis rate = (necrotic zone / ischemic zone) x 100

Thus, a high level of protection against the development of myocardial infarction through the synergistic action of the combined drug ΝΑ and ΜΌ was unexpectedly established. The combination (ΝΑ + ΜΌ) largely preserves the functions of the heart muscle, increasing the survival rate of animals (Tables 8, 9), and more significantly than, and ΜΌ separately, it protects the heart muscle from necrosis caused by ischemia and reperfusion (table. 10).

Example 4. Determination of antihypoxic and anti-ischemic effects in the brain

Additional experiments were conducted to determine the effect of the combined drug ΝΑ and ΜΌ in experimental models of central nervous system (CNS) ischemia, hypoxia, and stroke compared with the effects of the individual components.

4.1. Mouse model of hypoxic cerebrovascular insufficiency

Methodology

Experimental hypoxic circulatory failure was induced by the introduction of Μβί ', Τ (2% Μ§01 ₂ , a dose of 200 mg / kg) for 3 s, into the tail vein (Always R. et al., Lg / psiphcIogesNipd 1986; 36 (9) : 1314-1320) 1CP male mice. Animals received the test substance either as a single dose, or the same dose once a day for 7 days. Test substances were administered via an intragastric catheter. The animals were randomly distributed in 7 groups (6-10 animals each): The control group received water 0.01 ml / g

The ΡΙ500 group (active control) received a dose of 500 mg / kg of piracetam

Group ΝΑ50 received a dose of 50 mg / kg ΝΑ Group ΜΌ50 received a dose of 50 mg / kg ΜΌ

The ΜΌ150 group received a dose of 150 mg / kg ΜΌ

The ΝΑ50 + ΜΌ50 group received a dose of 50 mg / kg ΝΑ plus 50 mg / kg ΜΌ

The ΝΑ50 + ΜΌ 150 group received a dose of 50 mg / kg ΝΑ plus 150 mg / kg ΜΌ

The last dose of the test substance was given 1 hour before the test. The period between the end of the injection of Μ§Ο ₂ and the termination of the last respiratory movement is recorded as the survival time.

Statistics.

Results were presented as mean ± SDI for each group. Statistical analysis within groups was performed using Student's criterion. Differences between different experimental groups were compared using one-way analysis of variance (ΑΝΟνΑ) with repeated comparison (Tikeu'8 test). A probability of P <0.05 was considered significant.

Results.

The results are summarized in table. 11. With repeated administration, a protective antihypoxic due to the clinical use of the drug ΡΙ, as well as ΜΌ was demonstrated (Table 11). Unexpectedly, the ΝΑ + ΜΌ combination showed significant protection after a single use, especially at a dosage of ΝΑ50 + ΜΌ150, where the effect was better than that of each substance separately. Repeated use of this combination gives an even more pronounced effect. The results obtained in this test indicate the possible use of the combination ΝΑ + ΜΌ for treatment

- 12 021344 hypoxic conditions in the clinic.

Table 11. The effect of the tested substances on hypoxic insufficiency of cerebral circulation in a mouse model n = 6-10; mean ± SDI

Test group Survival time, s Single dose 7 day treatment The control 25.75 ± 1.01 25.4 ± 1.07 ΡΙ500 28,541.04 29,841.32 * ΝΑ50 27,340,68 27,840,86 ME50 26,541.07 28,941.20 * Μϋ150 27,040.93 29,841.19 * ΝΑ50 + Μ050 30.8 ± 1.44 * ’ 32,641.18 '** ΝΑ50 + Μϋ150 31,141,33 *** 34,441,50® * "

* P <0.05 relative to control.

# P <0.005 relative to control.

@ P <0,0005 relative to control.

$ P <0.05 with respect to ΜΌ at the same dose.

& P <0.05 with respect to ΝΑ.

§P <0.005 relative to ΝΑ

4.2. Blockage of the middle cerebral artery

Methodology

Male 1SK mice were used with a body weight of 21 to 25 g. The beginning of the middle cerebral artery (MCA) was plugged using the technique of a filament located in the lumen according to the well-known method (Bond Ε.Ζ. et al., §1 current 1989; 20 : 84-91), adapted for mice according to the publication of Ζΐκπίβ O. et al. (Veyauyuita1 Vtash RezhegsN 2006; 169: 66-74). The treatment protocols were used in the prophylactic (once a day for 7 days, for the model of permanent blockage of the MCA) and therapeutic (start of treatment 1 hour after the temporary blockage of the MCA) mode.

Control animals received only saline. This test is a good model of real cerebral hemorrhage and the clinical situation of a stroke, when blockage of the middle artery of the large brain is common. Then the experiment was continued in accordance with two protocols. In the first, the blockage was constant. In the second, clogging with a thread located in the lumen was temporary, and the thread was removed after 2 hours and reperfusion was performed. The neurological status of all animals was evaluated 24 hours after blockage. The assessment was carried out using a point estimate, in which 0 points were obtained for animals without pathology, 4 points for animals incapable of spontaneous movement (Bond Ε.Ζ. et al., §1 current 1989; 20: 84-91). After evaluating the neurological deficit, the animals received too large a dose of sodium pentobarbital, the brain was isolated and 6 layers were cut with a thickness of about 1.5 mm. These layers were stained with 2% sodium triphenyltetrazolium at 37 ° C for 30 min and photographed. The third layer from the cranial side, at the intersection of the optic nerves, was chosen as the most representative site for calculating ischemic brain damage, since it is completely supplied with blood from the middle artery of the large brain.

Statistics.

Data were presented as mean ± SDI for 7–9 individual animals. The differences between the experimental groups were compared using one-way analysis of variance (ΑΝΟνΑ) with repeated comparison (Tikeu's test). Differences in neurological scores between groups were analyzed using Student's test. A probability of P <0.05 was considered significant.

Results.

Twenty-four hours after blockage, all 8 control animals showed neurological abnormalities at an average of 2.63 points (Table 12).

- 13 021344

Table 12. The effect of the tested substances on the neurological state in the group of animals with constant blockage n = 7-9; mean ± SDI

Test group Neurological condition, points Control 2.63 ± 0.38 Simulation ~ 0.14 ± 0.14 ' ΝΑ50 x 7 2.25 ± 0.25 Μϋ150 x 7 1.63 ± 0.29 ΝΑ50 + Μ0150 x 7 1.38 ± 0.26 * ⁸

* P <0.05 relative to control.

# P <0,0005 relative to control.

& P <0.05 with respect to ΝΑ

In the simulation group, only one animal showed a slight disturbance. Repeated use of ΜΌ for 7 days partially prevented the deterioration of the neurological condition caused by the action of ISA blockage. Unexpectedly, the combination of ΝΑ + ΜΌ provided significant protection against neurological disorders, which exceeded the effect of ΝΑ (Table 12).

Blockage of the middle artery of the large brain caused ischemic damage in the control group of animals, covering 22.1% of the brain tissue, at the intersection of the optic nerves (Table 13). The use of the combination ΝΑ + ΜΌ for 7 days provided significant protection against damage to the brain tissue, which exceeded the effect of individual ΝΑ.

Table 13. The effect of the use of the test substances for 7 days on the size of the area of the brain damage with ischemic infarction with constant blockage in the group of animals n = 7-9; mean ± SDI

Test group Area of ischemic lesion,% The control 22.2 ± 2.21 ΝΑ 19.0 ± 1.75 Μϋ 16.9 ± 1.82 ΝΑ50 + Μϋ150 12.9 ± 1.54 ' ^c

* P <0.005 relative to control.

& P <0.05 with respect to ΝΑ

In the next experiment, temporary blockage of the middle artery of the large brain for 2 hours followed by reperfusion led to serious impairment of brain functions in the control group of animals tested after 24 hours (Table 14). Administration of the test substances 1, 3, and 6 hours after blockage does not provide significant protection in either the группе25 group (25 mg / kgx3) or the ΜΌ75 group (75 mg / kgx3). Unexpectedly, only in the ΝΑ25 + ΜΌ75 group (25 mg / kg + 75 mg / kg) with the test combination, administered 3 times after blockage, significant protection of brain functions was obtained, significantly exceeding the effect of individual ΝΑ and ΜΌ (Table 14).

Table 14. The therapeutic effect of the tested substances on neurological functions after temporary blockage n = 7-9; mean ± SDI

Test group Neurological condition, points The control 2.75 ± 0.31 Simulation group 0.29 ± 0.18 ’ ΝΑ25 2.25 ± 0.16 Μϋ75 2.13 ± 0.23 ΝΑ25 + ΜΟ75 1.44 ± 0.18 ‘“ *

* P <0.005 relative to control. # P <0.0005 relative to the control $ P <0.05 relative to ΜΌ. & P <0.05 with respect to ΝΑ

- 14 021344

Morphometric analysis showed that individual ΝΑ or MB do not provide significant protection against brain tissue damage caused by temporary blockage and reperfusion of the MCA (Table 15). Unexpectedly, the combination of ΝΑ + MB (25 mg / kg + 75 mg / kg), introduced immediately after blockage, provides significant protection against damage to the brain tissue, significantly exceeding the effect of individual ΝΑ and MB (Table 15).

Table 15. The therapeutic effect of the test substances on the size of the area of ischemic lesion from temporary blockage in the group of animals n = 7-9; mean ± SDI

Test group Area of ischemic lesion,% The control 22.3 ± 1.3 ΝΑ25 18.9 ± 1.8

1UY75 18,211,3 ΝΑ25 + ΜΟ75 13,311,7 ** ¹

# P <0.005 relative to control.

$ P <0.05 relative to MB.

& P <0.05 with respect to ΝΑ

Thus, the inventors unexpectedly found that the combination of ΝΑ plus MB provides significantly better protection of functional and morphological damage to brain tissue than the individual components during therapeutic use either before or after blockage of the MCA. These results indicate that the combined drug product may be useful in the treatment and / or prevention of ischemic-hypoxic conditions of the central nervous system, including stroke, also due to its own inhibitory activity in the platelet aggregation and thrombosis tests, which are described below.

Platelet Aggregation Activity and Antithrombotic Activity

Tests: Platelet Aggregation ίη νίΐτο; rat thrombosis model ίη νίνο; registration of changes in skin temperature ίη νίνο.

Example 5. The effect of MB and ΝΑ on platelet aggregation

Methodology Platelet aggregation was studied in whole blood obtained from a healthy donor B. (age 37), who did not take Α8Α or any other antiplatelet agents, using the Mi1yp1a1e device (Multifunctional platelet analyzer, Bübäüe Mebüa1, Germany) according to the method developed S. O. et al. (TOT. Nanto51. 2006; 96: 781-788) and Vе1 ^ к8а1сЬпег S. et al. (ΑηοδΐΕ Αη; · ι1§. 2008; 107: 1798-1806). Blood samples were collected in plastic tubes coated with an anticoagulant - hirudin (Bupalis mebyla, Segtapu), and used for measurements between 30 min and 4 h after blood sampling. The measurements were carried out according to a modified protocol of the company Bubalu Mebu1. An isotonic sodium chloride solution (0.3 ml, or physiological solution of the test substances (in a final concentration of 10 ^-6 to 10 ^-4 mmol / ml) was heated to 37 ° C), and a sample of 0.3 was added from the pipette to the test cells ml of whole blood containing an anticoagulant - hirudin.Measurements begin 5 minutes after incubation and stirring at 37 ° C by adding a solution of the corresponding agonist (obtained from Bupalébée Méboulet, Segtapu):

1) adenosine diphosphate (ΑΌΡ) - ΑΌΡ-test. ΑΌΡ stimulates platelet activation by ΑΌΡ receptors (Ρ2Υ12 and others).

2) test ΑΌΡ H8 (prostaglandin E ₁ in combination with ΑΌΡ). The addition of an endogenous ΡCΕ1 inhibitor makes the ΑΌΡ H8 test more sensitive than the ΑΌΡ test.

Aggregation curves were recorded for 6 min and analyzed using the software Bump-Feb-Meb-1. The following platelet aggregation parameters were calculated:

1) Αtax, the maximum value of platelet aggregation, expressed in arbitrary units (Αυ) of aggregation;

2) Αυϋ, the total area under the aggregation curve ^^ min). The total height of the aggregation curve, as well as the slope of the curve, affects the size of the area, and ΑυС best expresses the total platelet activity.

Statistics. Results were expressed as mean ± SDI. One-way analysis of ΑΝΟν одно was used to assess the significance of deviations. If the invalid hypothesis is rejected, then after that test 81ibep1-№ \ utap-1 <ei15 is used.

Results.

The first series of trials was planned in order to determine the effect of various concentrations of drugs. As shown in the table. 16, ΜΌ in a wide range of concentrations provides significant protection against platelet aggregation caused by the action of L ^ Ρ + ΡCE1. The value of xtax decreases from 100% in the control group to 55-58% in the groups ΜΌ 10 ^-5 and 10 ^-4 . Niacin (in groups 10 ^-4 and 10 ^-3 mmol / ml) also reduces aggregation caused by the action of ΑΌΡ. The combined effect of both substances provides a higher and more pronounced decrease in platelet aggregation caused by the action of ΑΌΡ or ΑΌΡ + PCE !, which is manifested in the values of ΑΌΟ, as well as Αtax.

Table 16. Individual and combined action of ΜΌ and ΝΑ on induced ΑΌΡ and ΡCE ₁ + L ^ Ρ platelet aggregation; mean ± SDI; Ν = 5-8

** P <0.005 relative to control.

*** P <0.0005 relative to control.

*** P <0.00005 relative to the control.

# P <0.005 relative to ΜΌ 10 ^-4 .

^## P <0,0005 relative to ΜΌ 10 ^-4 .

$ P <0.005 relative to ΝΑ 10 ^-4 .

$$ P <0,0005 relative to ΝΑ 10 ^-4

Example 6. The effect of ΜΌ and ΝΑ on thrombosis

Methodology The inventors chose the experimental thrombosis model based on arterial thrombosis in rats induced by the action ReS1 ₃ (Kig / Κ et al Tgot Keck 1990, 60:.... 269-280, Uaid X., Xu L, Tgot Keck 2005.. 115: 95-100). Tissue damage, initiated by chemical oxidation under the indirect action of iron, causes adhesion of the damaged area and aggregation of platelets with subsequent activation of coagulation and precipitation of fibrin. HUMag male rats weighing 350-420 g were used in the experiments. Rats were statistically divided into different experimental groups, each of which contained at least seven animals. The vehicle or test compound ΜΌ (25 mg / kg), ΝΑ (25 mg / kg) and the combination ΜΌ + ΝΑ (25 + 25 mg / kg) were administered by mouth, 2 hours before the initiation of thrombosis.

Rats were anesthetized with sodium pentobarbital (50 mg / kg, ip) and were kept on a temperature-controlled table during the experiment to maintain a body temperature of 37 ° C. One of the carotid arteries was cut on the neck, separated from the adjacent tissue, the vagus nerve, and a flow sensor (electromagnetic blood flow meter 1200ν 1200, KoMei soybean, Garai) was placed on the exposed segment of a conventional carotid artery to record blood flow. After 15 min stabilization period thrombosis was induced by topical application (in contact with the adventitial surface of the vessel) two pieces (2x1 mm) UaGtai filter paper impregnated with 15% solution of ₃ ReS1. Carotid artery thrombosis time was recorded as the time required for complete cessation of blood flow, and recorded as time to blockage (TTO).

In addition, during experimental thrombosis, the time of bleeding from the tail of rats was measured. A transverse incision was made on the tail with a scalpel (at a distance of 5 mm from the end), and the tail was immersed directly in a warm (37 ° C) isotonic saline solution until the bleeding stopped. The cessation of bleeding was defined as the time to stop bleeding completely, without resuming bleeding for the next 30 s.

Statistics.

The results were processed using the program Μ ^ s ^ ioi Exse1 2007. The data were expressed as the average value ± SDI from measurements on 7-8 individual animals. Differences between experimental groups were compared using one-way analysis of variance (ΑΝΟνΑ) with repeated comparison (Tikeu's test). A probability of P <0.05 was considered significant.

Results. The average time for induced ReCl ₃ arterial thrombosis, with the resulting stoppage of arterial blood flow, in the control group is 24.4 min (Table 17).

Table 17. The effect of ΜΌ, ΝΑ and their combination on the induced FeCl ₃ thrombosis average value ± SDI; Ν = 7-8

Group Time to blockage Tail bleeding time MIN % MICK. % Control 2L, 4 ± 1.45 one hundred 8.9 ± 1.28 one hundred Μϋ 25 mg / kg 29.8 ± 2.29 122 10.5 ± 1.01 118 ΝΑ 25 mg / kg 30.3 ± 3.12 124 11.5 * 1.39 129 Μϋ + ΝΑ (25 + 25 mg / kg) 34.0 ± 2.78 * 139 11.4 * 1.42 128

* P <0.05 relative to control

Meldonium at a dose of 25 mg / kg does not provide a significant increase in TTO time. Niacin causes a slight increase in TTO, which is not significant. The effect of ΝΑ on tail bleeding time is similar. The combined use of ΜΌ and ΝΑ unexpectedly causes a very significant increase in TTO (39%), without a significant increase in tail bleeding time.

Considering the positive effect for the combination of ΜΌ and ΝΑ against platelet aggregation ίη νίίΓΟ and prolongation of the TTO ίη νίνο, this combination can be used to reduce the risk of thrombosis in patients with severe atherosclerosis, potential myocardial infarction and stroke, as well as in the postoperative period. The fact that the combination of ΜΌ and ΝΑ does not prolong the time of tail bleeding indicates the possible use of this combination for patients with an increased risk of bleeding in the period before and after surgery.

Example 7. A comparative study of the combined use of ΜΌ / ΝΑ and ΡΑ / ΝΑ to reduce the flow of blood

Nicotinic acid (niacin, ΝΑ) effectively reduces the level of serum cholesterol, bb and triglycerides with an increase in ΗΩΡ However, the limiting adverse effect in patients receiving nicotinic acid directly or in the form of prolonged excretion is the rapid development of significant skin heat and vasodilation, called the tide, which leads to a painful termination (Sir1a E.K., Ιΐο Μ.Κ., NeaK Όίδ. 2002.4: 124-137). Laropiprant (MK-0524) was suggested as one of the most active and promising drugs for reducing blood flow under the action of niacin (K. Skepd et al., § 2006; 103: 6682-6687). The aim of this study was to compare the effect of ΜΌ on blood flow (changes in skin temperature and blood flow) caused by the action of ΝΑ in the experiment.

7.1.1. Determination of the expansion of skin vessels

Model.

Male rats \ νίδΙ; · ΐΓ were anesthetized with sodium pentobarbital (50 mg / kg, ip) and kept under general anesthesia by introducing additional doses (10 mg / kg) every hour. Blood pressure was measured in the left carotid artery, an electrocardiogram (ECC) was recorded using standard conductor II. Blood flow in the right ear artery was determined using a laser Doppler flow meter (OHUROHOU 2000, υ§Α). Blood flow, ECC, and arterial pressure were recorded using a п 1n51GyteP system (5 RotebLaB, and the data were stored in a computer for further processing. After registering the baseline for 10 minutes, the test substances were injected subcutaneously (s / c) into the scruff, and registration continued for 30 minutes, the Average blood flow for each animal was calculated taking into account the average blood pressure, and compared with the initial and control values.The results were calculated for 5-8 separate experiments and expressed in%, as the maximum e change from baseline blood flow (Saga11o-1ape E. et al, ί RKagtaso1 Tohyuo1 Μeΐοάδ 2007; 56 (3):.... 308-316).

Statistics.

The results for each group were expressed as mean ± SDI. Statistical analysis within the groups was performed using the Student criterion for unpaired data and the hiquadrit criterion. Differences between each experimental group were compared using one-way analysis of variance with repeated comparisons (Tikeu's test). A probability of P <0.05 was considered significant.

- 17 021344

Results.

As can be seen below from the table. 18, nicotinic acid at a dose of 15 mg / kg causes a significant increase in blood flow in the ear artery for this animal model. MO, like control, causes a slight variation in blood flow. NΑ together with MO causes a retarded (slowly increasing) and statistically significantly less pronounced absolute increase in blood flow compared to N одним alone (Table 18). The potential of MOs to counteract peripheral vasodilation caused by the action of NΑ can have a positive result in the clinic to reduce skin effects (rush of blood) from nicotinic acid, therefore, this will be further investigated in detail, as described below.

Table 18. The effect of experimental substances on the expansion of skin vessels; mean ± SDI, Ν = 5-8

Group Blood flow changes,% Control / solvent 2.15 ± 4.29 ΝΑ (15 mg / kg) 46.117.52 ’ Μϋ (45 mg / kg) 7,1513,72 ΝΑ + Μϋ (15 + 45 mg / kg) 19.3316.44 *

* P <0.01 relative to the control.

$ P <0.05 relative to NΑ

7.1.2. Determination of the expansion of skin vessels. Materials and methods. See section 7.1.

Experiment program

GROUP PROCESSING

Number of animals

Thinner for ΝΑ and Μϋ 5 ΝΑ ΝΑ 15 mg / kg 7 Solvent for YES 5 Ba LA 0.3 mg / kg 6 BA + ΝΑ [0] LA 0.3 μγ / κγ + ΝΑ 15 mg / kg 6 BA + ΝΑ [30] LA 0.3 μγ / κγ + ΝΑ 15 mg / kg 7 MO Μϋ 45 mg / kg 6 ΝΑ + Μϋ [0] ΝΑ 15 mg / kg + Μϋ 45 mg / kg 6 ΝΑ + Μϋ [30] ΝΑ 15 mg / kg + Μϋ 45 mg / kg 6

Statistics.

The results for each group were expressed as mean ± SDI. Statistical analysis within groups was performed using Student's criterion. Differences between each experimental group were compared using one-way analysis of variance with repeated comparisons (Tikeu'k test). A probability of P <0.05 was considered significant.

Results.

When administered individually, MO, like LA, caused only slight changes in blood flow. When NΑ was given simultaneously with MO (lead time = 0), the increase in blood flow was slower and less pronounced than in the case of simultaneous administration of NΑ and LА (L + NL [0], see Table 19). There were no significant differences in ear flow between ME + KA [0] (when MO was added with NΑ) and ME + KA [30] (pretreatment with meldonium 45 mg / kg 30 min before NΑ). In the experiment of the invention, only upon preliminary treatment with LA, 30 minutes before NL (NL + ^ L [30]), the NL-induced increase in blood flow in the ear vessels of the rat was significantly reduced (Table 19).

Table 19. The effect of MO and LA on NΑ-induced expansion of the skin vessels;

Ν = 5-7, average value ± SOI

Group Blood flow changes,% Thinner for ΝΑ and Μϋ 2.1211.66 ” ΝΑ 52.2718.5 "

- 18 021344

** P <0.005 relative to the solvent.

$ P <0.05 with respect to ΝΑ.

^$$ P <0.005 relative to ΝΑ

The potential of MOs to counteract peripheral vasodilation caused by the action of ΝΑ can have a positive result in the clinic to reduce the skin effects of ΝΑ (rush of blood). In the experiments of the invention, it is impossible to evaluate all the advantages of the simultaneous administration of the combination of ΝΑ and ΕΑ compared with the simultaneous administration of ΝΑ and MO.

7.2. Assessment of changes in skin temperature induced by nicotinic acid

Materials and methods.

To record changes in the skin temperature of intact rats, a non-contact temperature recording method was used (RaraIoFz и. Et al., Vg. 1. RNagtaso. 2008; 153: 1382-1387). Temperature measurements were carried out using a portable infrared thermometer (Moye1 Rgozsap 510, озEoz1tap). Animals were taught to manipulate and to an infrared sensor for 3 days before the study. Temperature readings on the back of each ear were recorded three times without analgesia immediately prior to sc injection or ΝΑ (in the scruff area) or solvent / test compound (in the tail area). Ear temperature was then measured every 5 minutes for a period of 60 minutes. Between measurements, animals were returned to the cells. For each point in time, the data of six ear temperature measurements were averaged (three for each ear). Laropiprant [MK 0524, Southap SNet1sa1z firm | (ΕΑ) was first dissolved in dimethyl sulfoxide, and then the fresh solution was diluted with 0.9% solution No. C1, on each day of the experiment. The proportion in the combination of ΝΑ and ΕΑ is based on the Product Characteristics Summary for Tgeyaryue ™ Modified Release Tablets 1000 mg / 20 mg (nicotinic acid / laripiprant).

Experiment program

I. Testing the effects of time and solvent on skin temperature

GROUP TREATMENT Number of animals Sol. Solvent for ίΑ 6 Solution Thinner for ΝΑ, MAZA and Μϋ 6 ΝΑ ΝΑ 15 mg / kg subcutaneously 6

II. Study of the effect of ΝΑ / ΕΑ and ΝΑ / MO combinations on skin temperature

Laropiprant was administered simultaneously with ΝΑ in the form of ΝΑ + ΕΑ [0] or 30 minutes before ΝΑ in the form of ΝΑ + ΕΑ [30], MO was administered simultaneously with ΝΑ in the form of ΝΑ + MO [0] or 30 minutes before ΝΑ in the form of ΝΑ + MO [30]; in addition, the effect of individual ΕΑ and MO on skin temperature was recorded.

GROUP PROCESSING Number of animals

Control / solvent 6

ΝΑ ίΑ

ΝΑ + ba me

ΝΑ + Μϋ

ΝΑ 15 mg / kg

LA 0.3 mg / kg

ΝΑ 15 mg / kg + LA 0.3 mg / kg Μϋ 45 mg / kg

ΝΑ 15 mg / kg + Μϋ 45 mg / kg e

6 *

6 * in each time group

Statistics.

The data were processed using the Myugoza Exce1 program and the results were expressed as mean ± standard error of the mean. The average results for different groups were compared using a one-way analysis of ОNOU С and Student's criterion. A probability of P <0.05 was considered significant.

Results.

At the basic level, the average ear temperature recorded from 10 to 14 hours was 28.1-30.2 ° C. The study of the response time to the introduction of ΝΑ (15 mg / kg, s / c) showed a maximum temperature increase for 10 minutes equal to 2.32 ± 0.37 ° C from the baseline and 2.57 ± 0.43 ° C in compared with the Solvent group (P <0.005) at the 10th minute (see below). It was found that the effect of ΕΑ / solvent on the temperature of the ear differs significantly from the effect of ΝΑ and ΜΌ / solvent only in the first 5 minutes after injection, therefore, only one control group was used.

Effect of ΝΑ, solvent / ΝΑ and solvent / ΌΑ on rat ear temperature

Subcutaneous injection of ΜΌ or ΌΑ does not cause significant changes in the temperature of the ear of rats (table. 21). Simultaneous administration of ΝΑ and ΜΌ (group ΝΑ + ΜΌ [0]; lead time = 0) causes a decrease in blood flow from ΝΑ, which is similar to the effect that is caused by the simultaneous administration of ΝΑ and ΌΑ. The temperature increase caused by the action of ΝΑ decreases by 69% and 67%, respectively (Table 21). There is no significant difference in temperature between the groups ΜΌ + ΝΑ [0] (when ΜΌ is added together with ΝΑ) and ΜΌ + ΝΑ [30] (preliminary treatment with meldonium 45 mg / kg 30 minutes before ΝΑ). In the experiment of the invention, only pretreatment with laripiprant, which is administered s.c. at a dose of 0.3 mg / kg 30 minutes before injection ΝΑ, provides significant protection against an increase in skin temperature induced by табл (Table 21).

Table 21. The effect of ΌΑ and ΜΌ on the increase in skin temperature caused by the action of ΝΑ;

Ν = 6, average value ± SOI

Group Initial temperature ° C Maximum temperature, ° C Increase % Control / Solvent 29.5 ± 0.29 29.62 ± 0.25 *** - ΝΑ 29.61 ± 0.4 32,210,42 ’ one hundred Ba 29.4310.27 29,510.35 ’** -

ΝΑ + ΙΑ [0] 29.7210.31 31,4510,4 67 ΝΑ + ΙΑ [30] 29.5110.32 30.7310.34 '* ^{** ***} 47 Μϋ 29.4210.38 29,710.31 *** - ΝΑ + Μϋ [0] 29.5310.29 31.3310.48 ' 69 ΝΑ + Μϋ [30] 29.6810.26 31,4010,39 ' 65

* P <0.05 relative to control.

** P <0.005 relative to control.

*** P <0.0005 relative to control.

$ P <0.05 with respect to ΝΑ.

$$$ P <0,0005 relative to ΝΑ

Thus, the invention found that the combination of ΝΑ with ΜΌ not only has a non-obvious synergistic antiplatelet activity, but also suppresses a side effect - a rush of blood.

Example 8. Determination of the effect on blood sugar

It is well known that even a single large oral dose ΝΑ increases the blood glucose content of animals (TDogShop TN., 8c1sh11 / b.N. I. Ό; ηγυ 8c1. 1980; 63, 262-268), and for the use of ΝΑ in the treatment of patients with diabetes, monitoring of sugar content is required (CoIBeGd K.V., Acacia T.A., Causto Cl., Proc. 2008; 83 (4): 470-8).

Methodology

Adult male rats \ νίδΙ; · ΐΓ were used in the experiments. The animals were fasted overnight before the experiment in order to stabilize their blood sugar. Venous blood glucose was determined before the experiment and 45 minutes after oral administration of the control

- 20 021344 samples or test substance. The glucose content was determined using a standard set (Orbit company, Lboy E | aLe1e5 Sage Yb., ΥδΑ /. We used a large dose of ΝΑ (300 mg / kg, p / o), which, as you know, causes a stable and long-term increase in the content of L in the blood The same dose of ΜΌ (300 mg / kg) was used.

Animals were divided into 4 groups (n = 8):

1) The control group received a 1% solution No. C1, dose 2 ml / kg)

2) group ΝΑ received ΝΑ, dose 300 mg / kg

3) group ΜΌ received ΜΌ, dose 300 mg / kg

4) the ΝΑ + группа group received 300 mg / kg ΝΑ and 300 mg / kg ΜΌ.

Statistics.

Results were presented as mean ± SDI for each group. Statistical analysis within groups was performed using Student's criterion. Differences between any experimental groups were compared using one-way analysis of variance (ΑΝΟνΑ) with repeated comparison (Tikeu's test). A probability of P <0.05 was considered significant.

Results.

Nicotinic acid causes a statistically significant increase in the content of OB relative to the base and control levels (Table 22). ΜΌ causes a slight decrease in the content of OB, which does not differ from the control. The combination of ΝΑ and ΜΌ causes a significantly less pronounced increase in the content of OI (a change from the baseline level of 27.5%, compared with 61.1%, caused by the action of ΝΑ). This positive effect indicates that the combined drug product ΝΑ + ΜΌ may cause less pronounced side effects in patients with unstable or impaired glycemic control.

Table 22. The effect of test substances on glucose in rat blood; n = 8, average value ± SOI

Group A basic level of Experiment Change from base level,% % of control Control (saline solution) 3.8440.51 3,940,53 +1.6 0 ΝΑ 3,840,344 6.1240.42 '' +61.1 +56.9 Μϋ 3.9240.34 3.7240.31 -5.1 -4.6 ΝΑ + ME 4.03 ± 0.349 5.1440.56 +27.5 +31.8

* P <0.05 relative to control.

P <0.01 relative to the baseline

Brief conclusions

It was unexpectedly found that the combination of ΝΑ and ΜΌ enhances the therapeutic effect of ΝΑ in relation to disorders including dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease, selected from the group: angina pectoris and myocardial infarction, transient and permanent cerebrovascular accident, including cerebrovascular disorder and stroke , and peripheral occlusive artery disease, improving the condition of the heart and brain in ischemic-hypoxic conditions. In addition, this combination improves the expansion of peripheral vessels caused by the action of ΝΑ. Therefore, it can be expected that the new combined drug product exhibits increased activity compared to лечении in the treatment of metabolically related disorders, which allows to reduce daily doses of ΝΑ, and has less pronounced unwanted side effects.

Embodiments of the Invention

The term combined drug product used in the invention involves the simultaneous, sequential or separate administration of the components of the combination. Thus, the present invention is a combined drug product that contains ΝΑ and ΜΌ or their pharmaceutically acceptable salts for simultaneous, sequential or separate use to prevent platelet aggregation. The combined drug product according to the invention may be administered in the form of a pharmaceutical composition. In accordance with this aspect of the invention, there is provided a pharmaceutical composition which comprises ΝΑ or a pharmaceutically acceptable salt thereof and ΜΌ or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.

The pharmaceutical composition according to the present invention also includes separate compositions containing the first composition ΝΑ or its pharmaceutically acceptable salt and a pharmaceutically acceptable diluent or carrier, and a second composition containing ΜΌ or its pharmaceutically acceptable salt and a pharmaceutically acceptable diluent or carrier. Such a composition is intended for sequential or separate use. Since the treatment or prevention of metabolically related diseases involves the continued use of a medicinal product, in a most preferred embodiment of the invention, a form suitable for oral administration is provided, for example, in the form of tablets or capsules. The amount of each active component of the present combined drug product in the pharmaceutical composition may vary depending on the condition of the patient. Specialists in the treatment of metabolically related diseases of patients will be able to easily select the appropriate amount of each active component and the appropriate dosage regimen. The preferred ratio of the active components ΝΑ and ΜΌ or their salts is in the range from 3: 1 to 1: 3.

Another object of this invention is the use of a combination drug product as described above or a pharmaceutical composition thereof as described above for the manufacture of a medicament for simultaneous, sequential or separate administration to prevent platelet aggregation / thrombosis.

Claims (11)

CLAIM 1. Therapeutic combination for the prevention and / or treatment of diseases induced by dyslipidemia and / or platelet aggregation, containing an effective amount of nicotinic acid or its pharmaceutically acceptable salt and an effective amount of meldonium or its pharmaceutically acceptable salt. 2. A combination according to claim 1 for the prevention and / or treatment of diseases induced dyslipidaemia which is selected from the group consisting of hyperlipidemia, atherosclerosis, peripheral arterial occlusive disease, angina pectoris, myocardial infarction, cerebrovascular disorders, and stroke induced by transient or permanent ischemic violation . 3. The combination according to claim 1 for the prevention and / or treatment of diseases induced by platelet aggregation and including thrombosis and thromboembolism. 4. The use of a combination according to claim 1 for the prevention and / or treatment of diseases induced by dyslipidemia and / or platelet aggregation, containing an effective amount of nicotinic acid or its pharmaceutically acceptable salt and an effective amount of meldonium or its pharmaceutically acceptable salt. 5. The use of a combination according to claim 4 for the prevention and / or treatment of diseases caused by dyslipidemia, which are selected from the group consisting of hyperlipidemia, atherosclerosis, occlusive peripheral arterial lesions, angina pectoris, myocardial infarction, cerebrovascular disorder and stroke induced by transient or permanent or induced by transient or permanent, induced cerebrovascular disorder and stroke induced by transient or persistent or occipital, permanent myocardial, cerebrovascular disorder and stroke induced by transient arteries or myocardial a violation. 6. The use of a combination according to claim 4 for the prevention and / or treatment of diseases induced by platelet aggregation, including thrombosis and thromboembolism. 7. Pharmaceutical composition for the prevention and / or treatment of diseases caused by dyslipidemia, which are selected from the group consisting of hyperlipidemia, atherosclerosis, occlusive lesion of peripheral arteries, angina pectoris, myocardial infarction, cerebrovascular disorder and stroke, induced by transient or persistent, induced cerebrovascular disorder, and stroke induced by transient or persistent orchestrated or persistent, or by post-mortem. an effective amount of nicotinic acid or its pharmaceutically acceptable salt and an effective amount of meldonium or its pharmaceutically acceptable my salts are combined with a pharmaceutically acceptable excipient or carrier. 8. The composition according to claim 7, which further comprises a statin selected from the group of atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin. 9. The composition according to claim 7, which contains from about 50 to about 500 mg of nicotinic acid or its pharmaceutically acceptable salt and from about 50 to about 500 mg of meldonium or its pharmaceutically acceptable salt. 10. The use of a composition according to claim 8 for the prevention and / or treatment of diseases induced by dyslipidemia, which are selected from the group consisting of hyperlipidemia and atherosclerosis. 11. The use of a composition according to claim 7 for the prevention and / or treatment of diseases induced by dyslipidemia, which are selected from the group consisting of hyperlipidemia, atherosclerosis, occlusive damage of peripheral arteries, angina pectoris, myocardial infarction, cerebrovascular disorder and stroke induced by transient or permanent or induced by transient or permanent, or by cerebrovascular disorder and stroke induced by transient or permanent or incendiary or myocardial infarction, cerebrovascular disorder and stroke induced by transient or permanent or incendiary myocardial infarction a violation.