LV14963B - Corrector of endothelial dysfunction - Google Patents

Abstract

A synergistic therapeutic combination, comprising meldonium acetylsalicylic acid addition salt as corrector of endothelial dysfunction and HMG-CoA reductase inhibitor, for use in preventing and/or treating thrombosis.

Description

DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The invention relates to agents for ameliorating a morbid condition caused by endothelial dysfunction. Specifically, this relates to a second medical use of a combination of meldonium acetylsalicylic acid salt with a statin for the treatment of thrombosis.

TECHNICAL BACKGROUND One of the major problems of medicine is cardiovascular diseases such as infarction and stroke, which pose a serious threat to human health and life. Myocardial infarction and stroke are caused by disturbances in blood supply, which are usually the result of arterial thrombosis.

Thrombosis and thrombotic or thromboembolic complications are currently one of the leading causes of mortality. This highlights the urgency of the problem and the continuing lack of reliable means to address it. New antithrombotic agents with greater therapeutic width and less pronounced side effects (bleeding, etc.) are still needed.

The haemostatic system includes platelets, blood coagulation factors, and endothelial cells that lodge blood vessels. In normal condition, the integrity and proper functioning of endothelial cells prevent platelet adhesion, activation of coagulation factors and thrombus formation.

In the clinic, endothelial dysfunction causes complications in cardiovascular disease and, in addition to the risk of thrombosis and thromboembolism, increases the likelihood of sudden death (life threatening thrombus formation in vital organs of the heart and brain, and their effect on blood supply et) J Med 2010; 68 (6): 242-250); Wu KK et al., Annu Rev Med 1996; 47: 315-331; Vanhoute PM, Circ J 2009; 73 (4): 596-601; Achneck HE et al, Circulation 2010; 122: 2068-2077). The beneficial effects of aspirin on various cardiovascular diseases are usually explained by its anti-ulcer activity, but its effect on endothelial function has also been found to be an important factor (Husain S. et al., Circulation 1998; 97: 716-720. Kharbanda RR et al. al., Circulation 2002; 105: 2600-2604; Taubert D. et al., Br J Pharmacol 2004; 143 (1): 159-165; US6596708, RU 2364392). Complex salts of acetylsalicylic acid with basic amino acids and glycine have been described as parenteral agents for cardiovascular disease (WO2006128600).

There are indications that the beneficial effects of statin therapy on cardiovascular disease result from modulating endothelial function (Schaefer, K. et al., Thromb Haemost 2005; 93 (l): 145-52; Bauersachs J., Widder J.D., Pharmacol Rep. 2008; 60: 119-126; Li M. et al., Vascul Pharmacol 2007 Jan; 46 (l): 1-9, Epub 2006 Jun 21) and NO formation (Dilaveris P. et al., Curr Vasc Pharmacol 2007; 5: 227- 37; Yokoyama S. et al., J Cardiovasc Pharmacol 2005; 45: 375-381; Asahi M et al, J Cereb Blood Flow Metab, 2005; 25: 722-729; Martinez-Gonzalez J, Badimon L, Curr Pharm Des 2007 ; 13 (17): 1771-86; Laufs U, Eur J Clin Pharmacol 2003; 58 (11): 719-31, Epub 2003 Feb 18).

Stats have been reported to reduce the risk of thrombosis and alter platelet vulnerability (Libby P, Aikawa M, Clin Cardiol 2003; 26 (l Suppl 1): 111-114). The effects of statins on endothelial function and risk of thrombosis have been analyzed (Rosenson RS, Am J Cardiovasc Drugs 2001; 1 (6): 411-20) and HMG-CoA reductase inhibitors have been proposed for the normalization of vascular endothelial dysfunction (WO9513063).

There is experimental evidence that stats influence arterial and venous thrombus formation (Alfon J et al, Thromb Haemost 1999; 81: 822-827; Obi C et al, Arterioscler Thromb Vasc Biol 2009; 29 (9): 1271-1276 ) and coagulation of blood clotting (Owens AP et al, J Clin Invest 2012; 122: 558-568; Ramcharan AS et al, J Thromb Haemost 2009; 7 (4): 514-520).

Reduction of thrombotic complications by statins has been observed in the clinic (Yamawaki T et al, Fukuoka Igaku Zasshi 2007; 98 (6): 260-269; Lowenberg EC et al, J Thromb Haemost 2008; 72 (2): 232-237; Rodriguez AL, J Thromb Thrombolysis. 2012; 33 (4): 371-82). [0011] The stool is believed to have a beneficial effect on both arterial and venous thrombosis (Lijfering WM et al, Semin Thromb Hemost 2011; 37: 885-896). The beneficial effects of statins on venous thrombosis and thromboembolism have attracted serious attention in clinical trials (Ray JG et al, Arch Intem Med 2001; 161 (ll): 1405-10; Glynn RJ et al, N Engl J Med 2009; 360 (18): 1851 -1861; Khemasuwan D et al, Am J Med 2011; 124 (9): 852-859; Paraskevas KI et al, Current Medical Research and Opinion 2009; 25 (7): 1807-1809; Lacut K et al, Fundam Clin Phaimacol 2004; 18: 477-82; Undas A et al, Arterioscler Thromb Vasc Biol 2005; 25: 287-94), which was unfortunately not fully supported by a subsequent meta-analysis of randomized studies of 105,759 patients on statin monotherapy (Rahimi K et al. al, PLoS Med 2012; 9 (9): el001310). Subsequent experiments have shown that in vitro statins enhance the anti-adhesive action of aspirin (Luzak B et al., Eur J Clin Invest, 2012; 42: 864-72). Staging treatment can lead to a significant reduction of the components of the blood coagulation cascade (Szcezklik A et al, Med Sci Monit 2001; 7 (6): 1381-5. Undas A, Brummel-Ziedins KE, Mann KG, Arterioscler Thromb Vasc Biol 2005; 25 (2): 2878- 94. Epub 2004, Nov. 29). [0013] Combination therapy of statins and aspirin is intensively studied. Stats and aspirin have additive effects in the reduction of cardiovascular events (Athyros VG et al, Platelets 2005; 16 (2): 65-71. Musial J et al, Thromb Haemost 2001; 85 (2): 221-5). WO9811896 describes pharmaceutical combinations comprising an HMG-CoA reductase inhibitor in combination with a platelet adhesion inhibitor (aspirin, clopidogrel, dipyridamole, etc.) for the inhibition of thrombotic occlusion and for the treatment and prevention of cardiovascular and cerebrovascular events. US 6,235,311 describes the combination of aspirin and a statin to reduce the risk of myocardial infarction. WO2009022821 and WO2012081905 disclose a combined preparation for the treatment of cardiovascular diseases comprising an HMG-CoA reductase inhibitor and aspirin. US 7,598,233 describes a pharmaceutical formulation for the treatment of thrombosis which comprises an HMG-CoA reductase inhibitor and aspirin. WO2007045353 describes the combination of aspirin salts with basic amino acids and glycine with an HMG-CoA inhibitor.

Meldonium is a known cardiovascular agent with experimentation (Veveris M et al, Baltie J Lab Anim Sci 2002; 12 (l-2): l 16-122. Artyushkova EV et al, Chelovek i ego zdorovye (in Russian) 2010; No. 3: 5-10 Wilkersst et al., Pharmacological Reports 2011; 63: 752-762) and clinically proven (Geichenko VP, Kuryata AV, Muzhchil OV, Rossiyskiy Cardiologicheskiy zhumal (in Russian) 2005; No.4: 64- 67. Khlebodarov FE, Mikhin VP, Gorlova AV, Rossiyskiy Kardiologicheskiy zhumal (in Russian) 2009; No.4: 37-42 Khlebodarov FE, Turikov PYu, Mikhin VP Rossiyskiy Kardiologicheskiy Zhumal (in Russian) 2009; -40. Shabalin AV et al, Adv Gerontol (in Russian) 2006; 19: 116-19) for endothelial dysfunction. Combination of mildronate with treadmills for the treatment of endothelial dysfunction, angina and diabetes is described in WO2006099244; US20110275649 discloses it for the treatment of statin-induced diabetes (without experimental data).

Meldonium acetylsalicylic acid addition salt is described in WO2010151095 as an anti-caking agent for the treatment of various pathologies induced by platelet adhesion, as an anti-inflammatory and antihyperlipidemic agent. WO2012158003 describes the addition of meldonium acetylsalicylic acid as an inhibitor of coagulation factor XII and its combination with warfarin as an anticoagulant.

Combination of Meldonium acetylsalicylic acid addition salt with a stalk for use in the prevention and / or treatment of dyslipidemia, hyperlipidemia and atherosclerosis is described in WO2010151095.

SUMMARY OF THE INVENTION

TECHNICAL PROBLEM There is still an unmet demand for new correctors of endothelial dysfunction that would be useful as antithrombotic agents.

PROBLEM SOLUTION A new combination of synergistic anti-caking agent and HMG-CoA reductase inhibitor stator can significantly reduce the risk of thrombosis.

Advantageous Effects of the Invention The present invention provides an antithrombotic agent for the treatment of thrombosis which comprises the addition of a salt of meldonium acetylsalicylic acid in combination with a spindle.

BRIEF DESCRIPTION OF THE INVENTION A synergistic therapeutic combination for the prophylaxis and treatment of thrombotic conditions comprising a therapeutically active amount of Meldonium acetylsalicylic acid addition salt as a corrector of endothelial dysfunction and a therapeutically active amount of a reductase HMG CoA is described.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and terms used below and their meanings:

Ach - Acetylcholine Chloride (Sigma)

ASA - Acetylsalicylic acid (Sigma-Aldrich)

L-NAME - N-Nitro-L-Arginine Methyl Ester Hydrochloride (Sigma-Aldrich)

MASA - Meldonium acetylsalicylic acid addition salt (obtained according to WO2010151095)

MD - Meldonium (Mildronate, Grindeks)

NO - Nitrogen monoxide

NP - Sodium nitroprusside (Sigma-Aldrich)

LLO - Time to Occlusion

FeCb (ferric trichloride) - Sigma-Aldrich

Simvastatin - (Artemis Biotech Ltd.)

HMG-CoA - 5-Hydroxy-3-methylglutaryl-Coenzyme A

THE EXPERIMENTAL PART

Effects of substances on experimental endothelial dysfunction and the associated increased risk of thrombosis

Method Experimentally generated NO deficiency caused by repeated administration of NO synthesis to blocked L-NAME was used to model cellular endothelial dysfunction (Pokrovskiy M et al., Experim Klin Pharmacol 2008; 71 (2): 29-31. Pokrovskiy M et al. Int J Hypertens 2011; 2011: 515047). Wistar male males (280-330 g) were used in the experiments. The animals were housed in groups of 7 to 8 in appropriate air-conditioned cages

At 22 ± 1 ° C with a relative humidity of 60 ± 5%, a 12/12-hour light / dark cycle and free access to food and water.

All experiments were carried out in accordance with Council Directive 86/609 / EEC of 24 November 1986 on the care of experimental animals. Every effort was made to reduce the suffering of the animals and to reduce the number of animals used.

[0025] Animals were randomized to the following groups: a) Intact animals - received water; b) Experimental Endothelial Dysfunction Group - animals were administered i.p. NO synthase inhibitor L-NAME at a dose of 25 mg / kg and p.o. water according to the protocol, c) treated endothelial dysfunction group - animals received L-NAME and test substance.

The experiment was performed according to two protocols: 1) prophylaxis and 2) treatment. 1) According to the protocol of the prevention experiment, the animals in the prevention group received, in addition to L-NAME, once daily for 7 days, a test substance in the stomach, intact and water in the L-NAME group. 2) In accordance with the treatment protocol, the test substance was administered p.o. on day 5, 6 and 7 of the endothelial dysfunction formed immediately after day 5, 6 and 7 of the experiment. the test substance or water (intact group or L-NAME group) was administered.

On day 7, two hours after the test substances were administered, animals receiving water and / or L-NAME were drugged (etaminal sodium, 50 mg / kg ip) and a catheter was placed in the left carotid artery for blood pressure and heart rate. , a second catheter was inserted into the right leg vein for administration of the vasoactive substance. Blood flow parameters were recorded using the Polygraph RM 6100 system (Nihon Kohden, Japan).

A known method (Kochkarov V et al, Fundamental Pharmacology and Pharmacy: Material of 2 ^nd Russia-Chinese Intemational Scientific Conference) was used to evaluate the corrective effect of endothelial dysfunction and phthalate on total arterial blood pressure changes caused by endothelial-dependent and independent vasodilation. of Pharmacology Perm, 2006: 98-99. Pokrovskiy M et al, Int J Hypertens 2011; 2011: 515047). Acetylcholine (Ach) at a dose of 40 mcg / kg iv was used as a measure of endothelial-dependent vasodilatation, while endothelial-independent vasodilation was evaluated with sodium nitroprusside (NP) at a dose of 30 mcg / kg iv.

The level of endothelial dysfunction was expressed as the endothelial dysfunction coefficient (EDK), which shows the ratio of NP-induced changes to ACh-induced changes (Pokrovskiy M et al, Int J Hypertens 2011; 2011: 515047). The resulting data were mathematically processed using Microsoft Excel and the results expressed as mean ± mean standard error (Mean ± WB). The results of the different groups were compared using one-way analysis according to ANOVA and Student's t-test. P <0.05 was considered significant.

Results Administration of Ach or NP vasoactive substances to intact animals resulted in similar changes in blood pressure, i.e., equal vasodilation. The endothelial dysfunction coefficient (EDC) was close to one (Table 1).

Repeated administration of L-NAME resulted in an increase in arterial pressure and an increased response to NP (endothelial-dependent vasodilation), but the response to Ach (endothelial-dependent vasodilation) was significantly reduced, which increased the EDK by approximately five-fold. The prophylactic effect of the test substances resulted in a certain reduction in LNAME-induced endothelial dysfunction, which was

EDC reduction (comparing L-NAME + MD100, L-NAME + ASA5 and L-NAME + MASS at 5, 10 and 25 mg / kg / day; see Table 1).

Administration of MASA was significantly more effective in reducing EDC than MD or ASA (Table 1). Attention is drawn to the fact that the 5 mg / kg / day dose of MASA produced a similar effect to the MD dose of 100 mg / kg / day (EDC, 3.03 and 3.01, Table 1, respectively).

Table 1. Prophylactic effect of test substances on endothelial-independent / dependent vascular response and endothelial dysfunction coefficient (EDC) in animals with the L-NAME-induced NO deficiency model; Mean ± VSK; N 7-8

Group From the endothelium independent vascular response to NPs, units Endothelium-dependent vascular response to Ach, units EDK NP / Ach Intact 1387 ± 239.2 ** 1303 ± 154.5 * 1.07 ± 0.16 *** L-NAME 3619 ± 193.6 685 ± 95.8 5.28 ± 0.79 L-NAME + MD5 3492 ± 159.3 820 ± 132.5 4.25 ± 0.32 L-NAME + MD25 3532 ± 274.0 952 ± 114.0 3.71 ± 0.58 L-NAME + MD100 3618 ± 283.7 1204 ± 121.2 * 3.01 ± 0.50 * L-NAME + ASA5 4070 ± 350.2 1251 ± 99.4 * 3.25 ± 0.43 * L-NAME + MASA5 3632 ± 162.8 1265 ± 127.3 * 3.03 ± 0.39 ** ^a L-NAME + MASA10 3576 ± 212.3 1625 ± 153.6 ** 2.20 ± 0.44 *** ^abc L-NAME + MASA25 3471 ± 204.7 1752 ± 169.4 ** l, 98 ± 0.52 *** ^abc * P <0.05 versus L-NAM3 -t

** P <0.005 versus L-NAME *** P <0.0005 versus L-NAME ^a P <0.05 versus L-NAME + MD 5 ^b P <0.05 versus L-NAME + MD 25 ^c P <0, 05 versus L-NAME + ASA 5 The treatment regimen (treatment initiated when endothelial dysfunction developed) significantly reduced EDK only in MASA, with a significantly greater effect than MD 5, MD 25 and ASA (Table 2).

Table 2. Therapeutic effect of test substances on endothelial-independent / vascular response and endothelial dysfunction coefficient (EDC)

animals with L-NAN E call the NO deficiency model; Mean ± VSK; N 7-8 Group From the endothelium independent vascular response to NPs, units From the endothelium dependent vascular response to Ach, units EDK NP / Ach Intact 1395 ± 193.5 ** 1294 ± 137.8 ** l, 08 ± 0.17 *** L-NAME 3667 ± 295.6 693 ± 101.3 5.29 ± 0.58 L-NAME + MD5 3628 ± 285.3 780 ± 98.3 4.65 ± 0.41 L-NAME + MD25 3678 ± 273.7 865 ± 138.5 4.18 ± 0.35 L-NAME + MD100 3651 ± 320.8 915 ± 121.7 * 3.99 ± 0.49 L-NAME + ASA5 3763 ± 374.1 833 ± 114.6 4.26 ± 0.36 L-NAME + MASA5 3650 ± 289.5 1133 ± 152.8 * ^a 3.22 ± 0.30 ** ^ac L-NAME + MASA10 3629 ± 254.9 1222 ± 158, l ** ^ac 2.97 ± 0.44 ** ^abc

L-NAME + MASA25 3535 + 270.4 1218 + 177.5 * ^a 2.90 + 0.50 ** ^abc

* P <0.05 versus L-NAME ** P <0.005 versus L-NAME *** P <0.0005 versus L-NAME ^a P <0.05 versus L-NAME + MD 5 ^b P <0.05 versus L-NAME + MD 25 ^c P <0.05 versus L-NAME + ASA 5 In the therapeutic regimen, MASA at a dose of 5 mg / kg / day induced a change in EDK greater than that at MD at 100 mg / kg / day (EDK, 3.22 and 3.99 respectively (Table 2). Comparing the efficacy of MD, ASA, and MASA in prophylaxis and therapy regimens, it is important to note that only MASA reduced the expression of endothelial dysfunction in the therapeutic regimen (see effects on EDC and endothelial-dependent vascular response to Ach, which was essentially similar to the intact group). table). Such an effect of MASA could be of benefit to the clinic for the treatment of endothelial dysfunction and for the restoration of endothelial function.

[0037] A further series of experiments focused on the testing of test substances in experimental thrombosis.

Functional status of endothelial cells is one of the key factors in maintaining normal blood rheology and in developing or preventing thrombosis. Therefore, endothelial dysfunction significantly worsens cardiovascular disease with an increased risk of thrombosis (Lowenberg EC et al., The Netherlands J of Medicine 2010; 68 (6): 242250).

An animal with experimentally (L-NAME) suppressed endothelial NO synthase has been found to be significantly less resistant to an ischemic episode (Gaballa M et al, Cardiovasc Res 1999; 42: 627-635).

In order to objectively evaluate the effect of endothelial dysfunction on thrombosis and the effects of the test substances, parallel experiments were performed with an intact group of animals and animals with endothelial dysfunction (called repeated administration of LNAME). Based on the results of orientation experiments (Tables 1 and 2) in which MASA was effective in reducing the development of endothelial dysfunction in a therapeutic regimen, the same protocol was used in the thrombosis experiment.

Method A model of experimental rat thrombosis based on FeCb-induced arterial thrombosis (Kurz K et al, Thromb Res 1990: 269-280. Wang X, Xu L, Thromb Res 2005: 115-100) was used.

Wistar male males weighing 340-400 g were used. The animals were randomized into two parallel groups: 1) without endothelial dysfunction, 2) with endothelial dysfunction. Animals were induced endothelial dysfunction by administration of L-NAME (25 mg / kg / day i.p.) for 7 days; intact animals received a similar volume of sterile 0.9% NaCl solution. According to the protocol, starting on day 5, the animals received p.o. for 3 days. either test substance or water immediately after administration of L-NAME or NaCl solution.

On day 7, two hours after administration of L-NAME, test substance, or water, rats were anesthetized with pentobarbital sodium (50 mg / kg, ip and then 10 mg / kg / hr) and placed on a heated operating table, to maintain body temperature around +37 ° C. After the incision in the neck, the left carotid artery was removed from the surrounding tissue, the adjacent vein and the vagus nerve on which a flow probe was placed (electromagnetic blood flow meter MFV 1200, Nicon Kohden,

Japan) to record blood flow. After a 15-minute stabilization period, thrombosis was induced by applying two (2x1 mm) pieces of filter paper impregnated with 15% FeCl 2 solution to the arteries.

The time of thrombosis was recorded as the time at which the blood flow was completely stopped and expressed as the time to occlusion (LLO). The results were analyzed with Microsoft Excel 2007. Data were expressed as mean ± standard error of the mean (Mean ± SD) for 7-8 animals. Differences between different experimental groups were compared using one-way ANOVA with repeated comparisons (Tukey's test). P <0.05 was considered significant.

Results In the control group, FeCb-induced thrombosis induced carotid artery occlusion with LLO for 25.3 min. Experimentally induced endothelial dysfunction significantly reduced LLO for 18.1 min; the difference from the control group was 28.5%. See above. Table 3.

3. table. Effect of experimental endothelial dysfunction on LLO and effect of test substances; Mean ± VSK; N = 7-8.

Group Baseline condition (without endothelial dysfunction) Endothelial dysfunction (with L-NAME) L-NAME effect LLO, min% LLO, min% Effect decrease% Control 25.3 ± 1.97 100 18 ^ 2.58 ¹ 100 28.5 MD 5 mg / kg x 3 27.6 ± 2.11109 20.5 ± 2.56 ¹ 113 26th MD 25 mg / kg x 3 32.3 ± 3.34 128 25.1 ± 3.13 139 22.3 ASA 5 mg / kg x 3 37.7 ± 2.63 ** 149 29, l ± 2.80 * '161 22.8 MASS 5 mg / kg x 3 39.3 ± 3.26 ** ^a 156 32.6 ± 2.78 ** ^a 180 17.3 MASS 10 mg / kg x 3 46.7 ± 3.50 ** ^ab 185 39.7 ± 2.76 *** ^abc 219 15th

* P <0.05 versus control ** P <0.005 versus control *** p <0.0005 versus control ^J P <0.05 versus baseline / in animals without endothelial dysfunction ^a P <0.05 versus MD 5 ^b P <0.05 versus MD 25 ^c P <0.05 versus ASA 5 Administration of test substances increases LLO in both groups without endothelial dysfunction and in the group with L-NAME induced endothelial dysfunction. It should be noted that MASA is relatively more effective than MD and ASA at prolonging LLO in endothelial dysfunction, cf. Table 3.

MASA at a dose of 10 mg / kg significantly reduced the effect of L-NAME on LLO, with P <0.0005, exceeding the effect of MD5, MD 25 and ASA 5 on LLO.

In this way, we showed that MASA is more effective than ASA or MD in reducing thrombosis (increasing LLO), and in endothelial dysfunction, the therapeutic effect of MASA is significantly increased, in contrast to ASA or MD (Table 3).

Conclusion MASA significantly reduces endothelial dysfunction caused by experimentally lowering NO levels when administered in both prophylactic and therapeutic regimens. When administered in the background of endothelial dysfunction, MASA was more effective than MD or ASA in reducing the risk of endothelial dysfunction and vascular thrombosis. The findings indicate a benefit to the MASA clinic for the prevention and / or treatment of thrombosis and other diseases associated with endothelial dysfunction.

Effect of MASA .. ASA and MD alone and in combination with simvastatin on experimental thrombosis The prophylactic effect of simvastatin, ASA, MD or MASA alone and in combination with simvastatin was tested in experimental arterial thrombosis in rats.

Methods A modified model of iron-trichlondrhythmia-induced thrombosis in rats was used (Kurz K et al, Thromb Res 1990,60: 269-280; Wang X, Xu L, Thromb Res 2005; 115: 95-100. Optimization of ferric chloride induced thrombosis in rats: J Pharm Toxicol Methods 2010; 61: 287-291).

Wistar male males weighing 310-360 g were used. The animals were housed in an air-conditioned room at 22 ± 1 ° C with a relative humidity of 60 ± 5% and a 12/12-hour light / dark cycle with free access to food and water.

[0053] All experiments were performed in accordance with Council Directive 86/609 / EEC of 24 November 1986 on the care of experimental animals. Every effort was made to reduce the suffering of the animals and to reduce the number of animals used.

The animals were randomized in groups of 10 and 3 consecutive days once daily with water (control group) or po: MASA 15 (15 mg / kg x 3), MASA 30 (30 mg / kg x 3), MD (25 mg / kg x 3), ASA (5 mg / kg x 3), MD + ASA (25 + 5 mg / kg x 3), or simvastatin (5 mg / kg x 3). Combinations of test substances with simvastatin were also administered p.o. as follows: MASA 15 + simvastatin (15 + 5 mg / kg x 3), MASA 30 + simvastatin (30 + 5 mg / kg x 3), MD + simvastatin (25 + 5 mg / kg x 3) or ASA + simvastatin (5 + 5 mg / kg x 3). Simvastatin was used at a dose of 5 mg / kg / day as this dose was shown to be sufficient to influence a number of regulatory factors, including the NO system, ie causing a so-called pleiotropic effect (Tuuminen R et al, Circulation 2011; 124: 1138- 1150. Arora R, Hare DL, Zulli A, J Atheroscler Thromb 2012; 19: 705-711).

Rats were anesthetized with pentobarbital sodium (50 mg / kg i.p. and then 10 mg / kg / hr) and placed on a controlled operating table to maintain a body temperature of 37 ° C throughout the experiment. One of the carotid arteries was exposed by a notch in the neck, separated from the surrounding tissue and vagus nerve. A flow probe (electromagnetic blood flow meter MFV 1200, Nicon Kohden, Japan) was placed on the exposed segment of the total carotid artery to record blood flow.

After 15 min. after a long period of stabilization, thrombosis was induced by applying two (2x1 mm) Whatman paper strips impregnated with 15% FeCb solution onto the vessel surface. The time of carotid thrombosis was recorded as the time required for complete blood flow to stop and is shown as the time to occlusion (LLO). If the blood flow in the groups receiving the test substances did not stop for 90 min. time, LLO was reported as> 90 min.

Data were shown as Mean ± VSK. Differences between different experimental groups were compared using one-way ANOVA with repeated comparisons (Tukey's test) and a value of P <0.05 was considered reliable.

Results Following contact with animals of the iron trichloride control group, vascular thrombosis developed and resulted in complete arrest of the blood flow to the carotid artery on average after 22.5 minutes (Table 4).

Table 4. Effects of test substances and their combinations on carotid thrombosis in rats - time to occlusion (LLO). Average + VSK; N = 8-10.

Group Time to Occlusion (LLO) min % Control 22.5 + 2.18 100 ASA (5 mg / kg x 3) 32.0 + 2.73 ¹ 142 MD (25 mg / kg x 3) 25.1 + 2.99 112 ASA + MD (5 + 25 mg / kg x 3) 40.0 + ^$ 3.97 ^{$ 2} * 178 Simvastatin (5 mg / kg x 3) 29.3 + 2.24 ¹ 130 MASS 15 (15 mg / kg x 3) 46.7 + 3.92 ^{3 $$ #} ** 207 MASS 30 (30 mg / kg x 3) 52.2 + 3.94 ^{4 $$ ##} *** ^c 232 ASA + Simvastatin (5 + 5 mg / kg x 3) 39.1 ± 3.69 ² * 174 MD + simvastatin (25 + 5 mg / kg x 3) 31.0 + 2.88 ¹ 138 MASS 15 + simvastatin (15 + 5 mg / kg x 3) 58.5 ± 5.67 ³ ** ^& @@ 260 MASS 30 + simvastatin (30 + 5 mg / kg x 3) 68.6 ± 6.37 ^4ab *** ^&& @@@ 305

^ <0.05 versus control ² P <0.005 versus control ³ P <0.0005 versus control ⁴ P <0.00005 versus control ^# P <0.05 versus ASA ^## P <0.005 versus ASA ^$ P <0.05 versus MD ^$$ P <0.005 versus MD ^a P <0.05 versus MASAI 5 ^b P <0.05 versus MASA30 * P <0.05 versus simvastatin ** P <0.005 versus simvastatin *** P <0.0005 versus simvastatin ^c P <0.05 versus ASA + MD ^& P <0.05 versus AS A + simvastatin ^&& P <0.005 versus ASA + simvastatin @P <0.05 versus MD + simvastatin @@ p <0.005 versus MD + simvastatin @ @@ p <0.0005 versus MD + simvastatin Prophylactic administration of MASA 15 (15 mg / kg x 3) resulted in a significant increase in LLO, which was more pronounced than the effects of ASA, MD or simvastatin. Increasing the MASA dose (30 mg / kg x 3) resulted in a further increase in antithrombotic effect and was significantly better than the ASA + MD effect (Table 1). In our model, MASA in combination with simvastatin caused an unexpected and surprising increase in antithrombotic effect: MASA 15 mg / kg + simvastatin 5 mg / kg prolonged the LLO by 25% compared with MASA 15 mg / kg + simvastatin 5mg / kg prolonged LLO by 31% compared to MASA 30 mg / kg. The combination of MASA 30 mg / kg + simvastatin 5 mg / kg significantly better than MASA 15 mg / kg or MASA 30 mg / kg prevented thrombotic occlusion of the carotid artery.

In the fly model, MASA + simvastatin (15 + 5 or 30 + 5 mg / kg) caused a very significant inhibition of thrombosis (260 and 305%, respectively), which is significantly higher than that induced by ASA + simvastatin or MD + simvastatin ( Table 4).

Conclusion The combined use of MASA and simvastatin results in a synergistic effect on LLO inhibition in an experimental thrombosis model. The results of the study suggest that in combination with simvastatin, MASA can provide improvements in the treatment and / or prevention of cardiovascular disease.

Claims (5)

A therapeutic combination comprising a therapeutically effective amount of a salt of Meldonium acetylsalicylic acid as a corrector of endothelial dysfunction and a therapeutically effective amount of a statin for inhibiting HMG-CoA reductase for use in the prevention and / or treatment of thrombosis. The combination of claim 1, wherein the statin is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, mvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and pharmaceutically acceptable salts thereof. The combination according to claim 1, wherein the thrombosis is associated with acute coronary syndrome, myocardial infarction, unstable angina, hypertension, vascular spasm, transient ischemic attack, atrial fibrillation, stroke, arterial sclerosis, peripheral arterial occlusion, thrombosis, thrombophlebitis, coronary artery thrombosis, arterial embolism, cerebral artery thrombosis, cerebral embolism, renal embolism, pulmonary embolism, diabetes, surgery and thickening of the blood. The combination of claim 1, wherein the Meldonium acetylsalicylic acid addition salt and the HMG-CoA reductase inhibitor are used as separate dosage forms. The combination of claim 1, wherein the Meldonium acetylsalicylic acid addition salt and the HMG-CoA reductase inhibitor are used as a single dosage form.