LV14606B - Novel factor xii inhibitor - Google Patents

Abstract

The present invention provides meldonium acetylsalicylate as novel factor XII inhibitor and use thereof as anticoagulant agent. Further the present invention provides for synergistic therapeutic combination comprising meldonium acetylsalicylate and warfarin sodium for use as anticoagulant agent.

Description

The invention described in a broader sense relates to a novel blood coagulation intracellular inhibitor and its combination with warfarin and their use as an anticoagulation agent.

TECHNICAL BACKGROUND Thromboembolic diseases are one of the leading causes of death despite the availability of anticoagulants such as warfarin and anti-aggregants such as acetylsalicylic acid and clopidogrel.

There are two convergent pathways of the coagulation cascade initiated by either external (vascular wall damage) or internal (blood-derived) components of the circulatory system. Intrinsically activated factor XII (XIIa) activates factor XI, whereas factor XIa activates factor IX. Both paths converge by activating factor X.

The external pathway is initiated by a factor VII complex with membrane protein tissue factor (AF). An internal or contact activation pathway is suggested when factor XII comes into contact with negatively charged surfaces.

Many anti-thrombotic agents act on key elements of the plasma coagulation mechanism, such as thrombin or factor Xa. This approach is based on the notion that these factors are primary from those involved in hemostasis. Warfarin inhibits the synthesis of extrinsic pathway coagulation factors VII, IX and X and is an effective oral anticoagulant. However, its use is limited by its short therapeutic width, slow onset of action and individual variability in activity, which requires dose adjustment and monitoring.

Therefore, there is a strong demand for novel, well-tolerated and effective oral anticoagulants that do not require frequent dose adjustments and monitoring of daily coagulation rates (Hylek EM. J Cardiovasc Med 2009; 10: 605-9). Accordingly, the object of the present invention is to satisfy such a request.

The biggest unmet demand for anticoagulant therapy is the replacement of warfarin with an orally active agent that could be administered at certain doses without daily coagulation monitoring. Most of the current research is devoted to the search for novel oral anticoagulants (Mackman N, Becker RC. Arterioscler Thromb Vasc Biol. 2010; 30: 369-71).

If the inventive anticoagulants have the same goal of action as warfarin, it is to be expected that they may cause a disturbance of hemostasis, i.e., by preventing thrombosis, they may also cause bleeding. The ideal drug for the prevention and treatment of thrombotic disease would be the agent that inhibits thrombosis but not hemostasis.

The logical target for the search for selective inhibitors that would not affect the higher levels of the cascade was factor X as a common element that unites the outer and inner branches of the coagulation cascade.

In rabbit experiments, the combination of apixaban, a factor Xa inhibitor, with aspirin has been shown to reduce the formation of occlusive arterial thrombi without excessive bleeding time (Wong PC, Watson CA and Crain EJ. J Thromb Haemost 2008; 6: 1736-41). Apixaban, an oral direct action inhibitor, was found to inhibit human factor Xa in vitro. (Jiang X et al. J Thromb Haemost 2009; 101 (4): 780-2). Apixaban was also effective in rat thrombosis models at doses that caused a slight increase in bleeding time in several models. (Schumacher WA et al., J Cardiovasc Pharmacol 2010; 55 (6): 609-16). In one clinical study in patients with atrial fibrillation who did not receive vitamin K antagonist therapy, apixaban reduced the risk of stroke or systemic embolism without significantly increasing the risk of major bleeding or intracranial haemorrhage (Connolly SJ et al. N Engl J Med 2011; 364: 806-17). . Another phase 3 study in patients with acute coronary syndrome (ACS) was discontinued due to a clinically significant increase in bleeding in patients randomized to apixaban: (http://www.europeanpharmaceuticalreview.com/news/industry-news/appraise-2study-with -investigational-compound-apixaban-in-acute-coronary-syndromediscontinued /).

Although several other orally active Factor Xa inhibitors are present in Phase 2 or 3 studies, these inhibitors are not currently considered to be ideal substitutes for warfarin.

[0012] A novel and promising approach for the development of new anticoagulants is the selection of agents that primarily act on coagulation factors other than warfarin prior to the total intrinsic factor X. Experiments in mice with genetic deficiency of factors XI and XII indicate that these factors do not cause hemostatic abnormalities but interfere with thrombus formation in arterial injury models. If the same mechanism works in humans, factor XI or XII could be an ideal target for drugs for the prevention or treatment of thromboembolic disease without interfering with hemostasis (Gailani D, Renne T. Arterioscler Thromb Vasc Biol 2007; 27: 2507-13. J Thromb Haemost 2007; 5 ( 6): 110612).

One of the first factors that is activated in the blood thrombus formation process is factor XII. Activated factor XIIa then activates factor XI and then activates factor IX. Factor IXa then combines with Villa factor to activate Factor X. It has now been established that intrinsic pathways XII and XI play a functional role in thrombus formation and stabilization in stroke, as their deficiency prevents cerebral ischemia without a significant effect on hemostasis. Based on the accumulated evidence that clot formation and hemostasis are inseparably linked, a new approach to treating ischemic stroke has emerged (Stoll G, Nieswandt B. Blood 2008; 112 (9): 3555-62).

[0014] The risk of venous thrombosis increases significantly if the level of factor XI is elevated. Increased Factor XII activity may also be a new risk factor for venous thrombosis, whereas reduced Factor XII activity appears to play no important role in the development of venous thrombosis. (Schmitt J et al. J Thromb Haemost 2005; 3 (S 1) abstract number: P1849) Since mice lacking factor XI are resistant to experimental thrombosis but have normal haemostasis, targeted inhibition of factor XIa may be a useful strategy for combating thrombosis without a significant increase in the risk of bleeding (Cheng Q, Tucker EI, Pine MS et al Blood 2010; 116 (19): 3981-89).

Inhibition of the intrinsic coagulation pathway by selective inhibition of factor XI does not interfere with the extrinsic and total pathway, making factor XI a promising therapeutic target (Schumacher WA et al., Arterioscler Thromb Vasc Biol. 2010; 30: 388-92).

Therapeutic inhibition of factor XI has been proposed as a novel strategy for treating or preventing thrombus formation (Wang X, Cheng Q, Xu L et al J Thromb Hemost 2005; 3: 695-702). Recent studies have confirmed this approach, showing that thrombus formation can be suppressed by maintaining hemostasis (Schumacher WA et al. Arterioscler Thromb Vasc Biol 2010; 30: 388-92).

Several different factor XI inhibitors have been patented (WO / 2007/070826; WO / 2008/076805; ΨΟ / 2006/076575; WO / 2007/070816; WO / 2009/114677).

In vivo studies of thrombus formation, platelet accumulation, and thrombin generation have provided evidence for the involvement of new proteins and have shown new pathways and mechanisms (Furie B, Furie BC. J Thromb Haemost 2007; 5 (Suppl. 1): 12- 7). Consequently, activated factor XII may be a target for the development of novel inhibitors of factor Xa or thrombin formation (Furie B, Furie BC. N Engl J Med 2008; 259: 938-49).

[0019] There are two approaches to regulating factor XII activity, either inhibiting its activation to factor XIIa or inhibiting the function of the activated form XIIa (Schousboe I. Biochem Pharmacol 2008; 75 (5): 1007-13).

Experimental data support the hypothesis that factor XIIa activation by factor XIIa is involved in the formation of thrombi in primates. Since factor XII deficiency does not affect human hemostasis, targeted inhibition of factor XIIa might be a useful strategy to reduce the risk of bleeding complications (Cheng Q et al. Blood 2010; 116 (19): 3981-9. Stavrou E, Schmaier AH. Thromb Res 2010; 125 (3): 2105).

Recently, Factor XII has been identified as a novel (ideal) target for anti-thrombotic therapy, particularly in arterial thrombosis, with minimal risk of bleeding (RenneT et al., J Exp Med 2005; 202 (2) 271-81. Blood Celis Mol Dis 2006; 36 (2): 148- 51. Expert Rev Cardiovasc Ther 2007; 5 (4): 733-41).

These facts raised the question of whether factor XII could be a unique target for a drug that blocked thrombosis but without increased bleeding as a side effect (Colman RW. J Exp Med 2006; 203 (3): 493-5). .

The first selective non-peptide factor XII inhibitor has recently been described (Robert S et al. J Med Chem 2008; 51: 3077-80).

It follows from the above that it is desirable to find novel inhibitors of the intrinsic coagulation pathway Factors XII and XI for safer anticoagulants.

Although efforts are currently focused on the development of new oral anticoagulants, it may be found that various combinations of known anticoagulants and anti-caking agents can also be used as beneficial remedy drugs.

More than 20 years ago, a low-dose combination of warfarin and aspirin was described in USP 5,041,430.

Combined warfarin-aspirin or warfarin-clopidogrel therapy is widely used for the long-term prevention of primary and secondary atherothrombotic and thromboembolic diseases. These combinations have known drawbacks and cannot be used in all cases (Donadini MP, Douketis JD. J Thromb Thrombolysis 20I0; 29 (2): 208-13. Http://www.drugs.com/drug-interactions/plavix-with -warfarin-705-360-2311 -0.html). The efficacy of Aspirin plus warfarin combination therapy in high risk patients with atherosclerotic heart disease has been confirmed in extensive clinical trials (Arjomand H, Cohen M, Ezekowitz MD. J Invasive Cardiol. 2004; 16 (5): 271-8). Addition of warfarin to standard aspirin therapy after acute coronary syndrome reduces the incidence of myocardial infarction and stroke (Rothberg MB et al. J Exp Med 2005; 143 (4): 241-50). However, in other cases, clinical results were not as conclusive (Flaker GC et al., Am Heart J. 2006; 152 (5): 967-73. Anand S et al., N Engl J Med. 2007; 357 (3): 217-27. Marcusson LL et al., Rev Med Suisse 2010; 6 (267): 194250).

A new anti-caking agent, Meldonium acetylsalicylate (WO / 2010/151095), which could be used in combination therapy has recently been described.

SUMMARY OF THE INVENTION TECHNICAL PROBLEM The demand for novel anticoagulants acting by a mechanism other than warfarin is unmet.

PROBLEM SOLUTION The present invention provides meldonium acetylsalicylate as a novel factor XII inhibitor and for its use as an anticoagulant.

The present invention also describes a synergistic therapeutic composition comprising meldonium acetylsalicylate and warfarin for use as an anticoagulant agent. The present invention also provides a method of treating and / or preventing a blood coagulation disorder, comprising administering to a subject in need thereof a therapeutically effective amount of meldonium acetylsalicylate with or without a pharmaceutically acceptable carrier, excipient or diluent.

The present invention also provides a method of treating and / or preventing a blood coagulation disorder, comprising administering to a subject in need thereof a therapeutically effective amount of meldonium acetylsalicylate, with or without a pharmaceutically acceptable carrier, excipient or diluent, and a therapeutically effective amount of warfar. , with or without a pharmaceutically acceptable carrier, excipient or diluent, as a single dosage form or as separate dosage forms of these active substances.

POSITIVE EFFECTS OF THE INVENTION The present invention provides meldonium acetylsalicylate as a novel oral anticoagulant with reduced risk of bleeding and a synergistic effect in combination with warfarin.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions The following abbreviations and their meanings are used:

WF - warfarin sodium (as clathrate)

ASA - acetylsalicylic acid

MASA - Meldonium acetylsalicylate (Meldonium acetylsalicylic acid addition salt) aPTT - Activated partial thromboplastin time PT - Prothrombin time

TBT - duration of tail bleeding

TTO - Time to Occlusion

Sources of Substances The substances used in the studies originated from:

ASA - Sigma-Aldrich

FeCL (ferric trichloride) - Sigma-Aldrich

MASS - Obtained by W0 / 2010/151095

Thromboplastin - Sigma-Aldrich

Triton Χ-100 - Sigma-Aldrich

Warfarin Sodium (as Clathrate) - Taro Pharmaceutical Industries

EXPERIMENTAL PART

I. Effects of Test Substances on Bleeding and Coagulation Factors The effects of repeated doses of MASA and WF on bleeding duration, total blood loss, and coagulation factors VII, X, XI, and XII were evaluated based on the above.

Wistar male males (280-360 g) were used in the experiments. The animals were kept 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. All experiments were carried out in accordance with Council Directive 86/609 / EEC of 24 November 1986 on the protection of experimental animals.

The rats were randomized into two experimental groups of 10 animals each. Solvent or test compounds MASA (5 or 10 mg / kg), warfarin (0.5 mg / kg) were administered p.o. once a day for 7 days. On day 7, the rats were anesthetized with pentobarbital sodium (50 mg / kg, i.p.) for two hours after administration of the test substance and placed on a controlled operating table to maintain a body temperature of 37 ° C.

Rat tail bleeding time (TBT) and bleeding volume - lost blood volume - were determined in all animals by a known method (Sato K et al. Jap J Pharmacol. 1998; 78: 191-97). in brief: The tail was cut 5 mm from the tip with a scalpel and immediately placed in 10 ml of isotonic saline solution maintained at 37 ° C until bleeding ceased. Bleeding time was calculated as the time until the bleeding stopped and did not recur within the next 30 s.

The collected blood was hemolyzed by addition of Triton Χ-100 and photometrically determined at 546 nm. The standard curve was constructed from the results of a pooled blood sample of three intact rats. Blood loss was calculated using a standard curve.

The abdominal cavity was opened to draw blood from the cava inferior vein with Vacutest tubes (2 ml; with 3.8% sodium citrate) for further determination of coagulation factors VII, X, XI and XII. Platelet-free plasma was obtained by centrifugation of the blood sample for 15 minutes at 2000 g. Measurements were made using Sysmex CA-1500 (Sysmex Corporation, Japan), in vitro diagnostic reagents, and appropriate control, standard and coagulation factor-free plasma (Siemens Healthcare Diagnostics Products GmbH, Germany) according to the Siemens Healthcare Diagnostics Protocol. Results were interpreted using a reference curve obtained by dilution of standard plasma with non-coagulant plasma and expressed as a percentage (100% standard).

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

Repeated administration of 0.5 mg / kg / day of WF resulted in a significant increase in TBT and lost blood volume (Table 1). Compared to the solvent-only control animals, 5 mg / kg / day and twice the 10 mg / kg / day MASA animals did not produce statistically significant changes in TBT and blood loss; it should be noted that this difference is highly reliable compared to the WF group (P <0.005 and P <0.0005), see Table 1.

Table 1. Effect of repeated (7 days) administration of MASA or WF on duration and volume of blood lost. Mean ± SEM; N = 8-10.

Group Tail bleeding duration (TBT) Blood volume min % ml Control 7.17 ± 1.05 100 0.056 ± 0.05 WF (0.5 mg / kg / d) 23.5 ± 2.40 ³ 328 1.23 ± 0.18 ³ MASS (5 mg / kg / d) 8.8 ± ^{1.54 aaa} 123 0.11 ± 0.05 ^ MASS (10 mg / kg / d) 10.7 ± 1.51 ^Μ 149 0.20 ± 0.08 ^aaa

^J P <0.0005 versus control ^ω Ρ <0.005 versus WF ^ <0.0005 versus WF Table 2 shows the effect of WF or MASA on coagulation factors VII, X, XI and XII after repeated (7 days) administration.

Table 2. Effects of test substances on coagulation factors VII, X, XI and XII

repeated (7 days) administration. Mean ± SEM; ' N = 8-10. Group F VII,% F X,% F XI% FXII% Control 107.7 ± 7.23 99.3 ± 3.12 95.0 ± 4.72 103.9 ± 8.88 WF (0.5 mg / kg / d) 1.9 ± 0.57 ⁴ 2.8 ± 0.86 ⁴ 77.9 ± 6.98 88.4 ± 7.06 MASS (5 mg / kg / d) 106, (^ 7.38 ²²² 69.4 ± 10.27 ^laaa 61.1 ± 6.54 ² 71.7 ± 6.22 ^L MASS (10 mg / kg / d) 118.84 = 11.42 ³ ^ 54.5 ± 9.43 ^Zaa 63.3 ± 7.70 ² 66.9 ± 6.75 ^za

* P <0.05 versus control ² P <0.005 versus control ⁴ P <0.00005 versus control ^a P <0.05 versus WF ^ω Ρ <0.005 versus WF ^ <0.0005 versus WF (0, 5 mg / kg / d) completely inhibited Factor VII and Factor X, but did not reliably affect Factor XI and Factor XII levels. MASA, in contrast to WF, had no effect on factor VII but reliably lowered factor XI and XII levels (Table 2). MASA (more reliably at 10 mg / kg) also lowered Factor X levels, but less reliably than WF. The effect of MASA on factors XI and X may be direct and / or indirect via factor XII.

These results suggest that the MASA clinic may be an effective means of preventing thrombosis and thromboembolism, with the advantage of reducing the risk of bleeding.

II. Effect of Compounds under Investigation on Arterial Thrombosis We chose a rat model of arterial thrombosis in which thrombosis is induced by ferric trichloride, which is widely used to evaluate one or more factor coagulation inhibitors (Elg M et al. Thromb Res 1999; 94: 187- 197. Surin WR et al., J Pharm Toxicol Methods 2010; 61: 287-291).

Wistar male males (330-410 g) were used in the experiments and the study was performed according to the method described (Kurz K et al. Thromb Res 1990; 60: 269-80; Wang X, Xu L. Thromb Res 2005; 115: 95-100). Rats were randomized to the appropriate experimental groups, each with at least 7 animals, or the test compound MASA (10 mg / kg), ASA (5 mg / kg), WF (0.25 mg / kg), MASA + WF (10). + 0.25 mg / kg) or ASA + WF (5 + 0.25 mg / kg) was administered orally (via a catheter in the stomach).

The choice of WF dose (0.25 mg / kg) was based on our data indicating that a 0.5 mg / kg dose of WF causes a significant increase in TBT and blood loss. Also, published data on WF toxicity indicate that administration of WF to rats at a dose of 0.5 mg / kg (Smith G et al. Thromb Res 1988; 50: 163-74) after 9-11 days not only shows high efficacy (protection against experimental thrombosis). and plausible changes in PT and aPTT) but also noticeable toxicity; at a dose of 0.25 mg / kg / d, it is non-toxic but still active until repeated doses of 0.0625 mg / kg do not show significant biological activity. In our experiment, single (2 h prior to thrombosis induction) and repeated (7 d once daily) doses of MASA and WF were used, as delayed onset of the WF effect can be expected as experimentally determined (Sato K et al. Jap J Pharmacol 1998; 8: 191-97) and clinic (Weiss P et al. Thromb Res 1987; 45: 783-90; Hirsh J et al. Chest 1995; 108: 231S-46S).

Rats were anesthetized with pentobarbital sodium (50 mg / kg i.p. and 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 uncovered by a neck incision, separated from the surrounding tissue, vagus nerve, and a flow probe (electromagnetic blood flow meter MFV 1200, Nicon Kohden, Japan) was placed on the exposed total carotid artery. After 15 min. During a long period of stabilization, thrombosis was induced by topically applying two (2x1 mm) strips of Whatman paper impregnated with 15% FeCF solution for 10 min (in contact with vascular advent).

The carotid artery thrombosis time was defined as the time required to completely stop blood flow and is shown as the time to occlusion (TTO). If the blood flow in the groups receiving the test substances did not stop for 90 min. time, TTO was reported as> 90 min.

All animals were measured for rat tail bleeding time and extent of blood loss - by known method (Sato K et al., Jap J Pharmacol. 1998; 78: 191-97).

Animals from the repeated dose groups were used to determine coagulation parameters and factors after the thrombosis experiment. Blood samples were taken from the vein cava inferior. Platelet-free plasma was obtained by centrifugation at 2000 g for 15 minutes. PT and aPTT were determined immediately with Sysmex CA-1500 according to the manufacturer's instructions (Sysmex Corporation, Japan); the remaining plasma was frozen at -20 ° C for subsequent determination of fibrinogen and coagulation factors VII and XII (by the above method and reagent kit from Siemens Healthcare Diagnostics Products GmbH, Germany).

The FeClfl control animals induced arterial thrombosis and arrest of carotid blood flow after approximately 23.6 min (Table 3).

Table 3. Effect of a single prophylactic dose of test substance on carotid artery thrombosis induced by iron trichloride - time to occlusion (TTO);

duration of bleeding (TBT) and the volume of blood lost. Mean ± SEM; N = 7-8. Group Time to Occlusion (TTO) Tail bleeding duration (TBT) Blood volume min % min % ml Control 23.6 + 1.72 100 7.34 + 1.41 100 0.064 + 0.03 MASS (10 mg / kg) 40.7 + 3.28 ²³ 173 11.39 + 1.92 155 0.11 + 0.03 ASA (5 mg / kg) 34.4 + 2.99 * 146 12.57 + 1.73 * 171 0.24 + 0.06 * WF (0.25 mg / kg) 28.3 + 2.15 120 8.54 + 0.95 116 0.07 + 0.03 ” ^c MASS + WF (10 + 0.25 mg / kg) 42.3 ± 2.72 ^3a " 179 12.04 + 1.75 164 0.12 + 0.02 " ASA + WF (5 + 0.25 mg / kg) 34.3 +1.98 ² 145 13.83 ± 1.85 ^la 188 0.27 + 0.06 *

P <0.05 versus control ² P <0.005 versus control ³ P <0.0005 versus control ^a P <0.05 versus WF ^b P <0.05 versus AS A + WF ^c P <0.05 versus ASA [0058 ] A single prophylactic dose of MASA induced a very reliable increase in TTO time (P <0.005 versus control). ASA induced a less pronounced increase in TTO compared to MASS (146 versus 173%), although the volume of lost blood was twice as long and the duration of bleeding was also significantly longer. A single dose (0.25 mg / kg) of WF was administered over 2 hours. prior to the experiment, did not result in a reliable change in TTO, bleeding duration, and lost blood volume, apparently due to delayed drug exposure, which is typically observed up to 24 h. after administration of an anticoagulant (USP 20080221204 A1). MASA in combination with WF showed significantly better protection against thrombosis than ASA + WF (Table 3). Bleeding time in the MASA + WF group was significantly lower than in the ASA + WF group.

Repeated administration of test substances increased TTO. Repeated administration of MASA and WF, in contrast to ASA, did not increase TBT and lost blood volume (Table 4).

Table 4. Effect of repeated administration of test substances on carotid artery thrombosis induced by iron trichloride - time to occlusion (TTO); bleeding time (TBT) and volume of blood lost. Mean + SEM; N-6-8.

Group Time to Occlusion (TTO) Tail bleeding duration (TBT) Blood volume min % min % ml Control 24.8 + 1.98 100 7.7 + 1.25 100 0.076 + 0.02 MASS (10 mg / kg / d) 47.9 ± 3.65 ^3c 193 12.6 + 1.97 " 164 0.26 + 0.10 " ASA (5 mg / kg / d) 37.6 + 2.52 ² 153 14.9 + 1.91 * 200 0.34 + 0.10 * WF (0.25 mg / kg / d) 49.8 + 4.97 ² 201 11.3 + 1.61 " 159 0.26 + 0.09 " MASA + WF (10 + 0.25 mg / kg / d) 67.0 ± 5.93 ^4p 270 13.7 + 2.13 * " 205 0.30 + 0.11 " ASA + WF 56.4 ± 7.2 ^2c 227 22.2 + 3.01 ² 288 0.59 + 0.11 ²

(5 + 0.25 mg / kg / d) 1 1

P <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 MASS ^a P <0.05 versus WF ^b P <0, 05 versus ASA + WF ^c P <0.05 versus ASA MASA in combination with WF induced the most pronounced increase in TTO (270%) and had a significantly lower effect on blood loss and TBT compared to ASA + WF (Table 4).

A parallel experiment with a total control compared a significantly lower dose of WF (0.06 mg / kg) with MASA + WF given these preparations for 7 days. A dose of 0.0625 mg / kg / d is not known to reduce the amount of vitamin K-dependent clotting factors and does not exhibit anti-thrombotic effects (Smith G et al. Thromb Res 1988; 50: 163-74). Our data confirm that WF at a dose of 0.06 mg / kg / d, when administered for 7 days, does not prevent thrombosis, and does not alter TBT and lost blood volume (Table 5) compared to controls.

Table 5. Effect of repeated administration of WF, MASA and MASA + WF (10.0 + 0.06 mg / kg / d) on carotid artery thrombosis induced by iron trichloride - time to occlusion (TTO); bleeding time (TBT) and volume of blood lost.

Mean + SEM; N = 6-8.

Group Time to Occlusion (TTO) Tail bleeding duration (TBT) Blood volume min % min % ml Control 24.8 + 1.98 100 7.7 + 1.25 100 0.076 + 0.02 WF (0.06 mg / kg / d) 23.3 + 1.89 94 8.2 + 1.22 106 0.06 + 0.03 MASS (10 mg / kg / d) 47.9 + 3.65 ³⁸ 193 12.6 + 1.97 164 0.26 + 0.10 MASA + WF (10 + 0.06 mg / kg / d) 55.3 + 5.91 ²⁸ 223 12.5 + 2.08 162 0.27 + 0.09

² P <0.005 versus control ³ P <0.0005 versus control ^a P <0.0005 versus WF In this experiment, MASA showed significant protection against thrombosis as evidenced by increased TTO, although there was no reliable change in TBT and lost blood volume. (Table 5).

[0063] MASA in combination with WF caused a significant increase in TTO compared to MASA and WF (223 versus 193 or 94, respectively; see Table 5), but did not increase TBT and lost blood volume, indicating use of MASA or MASA + WF. safety in the clinic.

Ex vivo experiments Groups of animals to which test substances were repeatedly administered (7 days) were used to determine coagulation parameters and factors.

In contrast to ASA, administration of MASA at a dose of 10 mg / kg p.o., for 7 days resulted in a reliable increase in aPTT time and a decrease in factor XII (Fig. 6).

Table 1), which indicates the potential effect of MASA on the regulation of coagulation internal pathways (Hoffinan M, Monroe DM. Coagulation Hematol Oncol Clin North Am 2007; 21 (l): l-ll).

Table 6. Effects of test substances on PT, aPTT, fibrinogen and coagulation factors VII and XII after repeated administration. Mean ± SEM; N = 5-8.

Group PT % aPTT sec Fibrinogen mg / ml FVII % FXII % Control 95.7 + 6.7 29.8 + 0.84 3.10 + 0.08 126.5 + 12.8 101.5 + 5.2 MASS (10 mg / kg / d) 71.3 + 8.9 44.5 + 4.2 ' 3.25 + 0.21 123.4 + 12.0 67.6 + 10.3 ^lc ASA (5 mg / kg / d) 76.1 + 9.1 31.9 + 4.3 2.74 + 0.12 106.5 + 13.3 100.4 + 9.2 WF (0.25 mg / kg / d) 1 l, 6 ± 2.8 ³ * ^çc 46.8 + 5.3 " 3.16 + 0.16 13, l ± 3.9 ^3SScc 90.1 + 9.3 MASA + WF (10 + 0.25 mg / kg / d) 19.1 + 4.7 * " 79.2 ± 7.9 ^aabcc 3.20 + 0.17 12.3 ± 3.8 ^{3s $ cc} 41.6 ± 7.7 ^3ab " ASA + WF (5 + 0.25 mg / kg / d) 26.4 + 5.5 * " 53.2 + 6.4 ^lc 2.60 + 0.23 24.6 ± 9.7 ^3sec 86.6 + 11.9

P <0.05 versus control ² P <0.005 versus control ³ P <0.0005 versus control ^$ P <0.05 versus MASS ^{$ s} P <0.005 versus MAS A ^a P <0.05 versus WF ^ <0.05 versus ASA + WF ^c P <0.05 versus ASA ^cc P <0.005 versus ASA WF at 0.25 mg / kg / d significantly altered PT, increased aPTT time, and significantly lowered F VII levels. ASA did not cause reliable changes in PT, aPTT, and coagulation factors. MASA + WF, unlike ASA + WF, had a significantly stronger effect on the time course of aPTT and the decrease in factor XII activity (see Table 6). It should be noted that addition of WF to therapeutic doses at therapeutic doses further reduced Factor XII levels, whereas Factor VII levels were similar to those of WF in therapy. It should be noted that repeated administration of (7x) WF at a dose of 0.06 mg / kg did not result in a reliable change in PT and APTT, fibrinogen and coagulation factors VII and XII compared to controls (Table 7).

Table 7. Effect of repeated administration of (7x) WF (0.06 mg / kg / d), MASA and MASA + WF (10.0 + 0.06 mg / kg / d) on coagulation parameters and coagulation factors I, VII and XII; Mean ± SEM; N = 5-8.

Group PT,% aPTT, sec Fibrinogen (FI) mg / ml FVII % FXII % Control 95.7 + 6.7 29.8 + 0.84 3.10 + 0.08 126.5 + 12.8 101.5 + 5.2 WF (0.06 mg / kg / d) 92.3 + 9.0 30.7 + 0.89 3.26 + 0.18 112.5 + 10.3 104.3 + 5.7 MASS (10 mg / kg / d) 71.3 + 8.9 44.5 + 4.2 ¹³ 3.25 + 0.21 123.4 + 12.0 67.6 + 10.3 " MASA + WF (10 + 0.06 mg / kg / d) 71.9 + 7.3 ' 49.7 + 5.0 ¹³ 3.22 + 0.23 85.4 + 8.2 '* 62.4 + 13.3 "

* P <0.05 versus control ^a P <0.05 versus WF ^$ P <0.05 versus MASA [0068] Administration of MASA and MASA + WF (10 + 0.06 mg / kg / d) resulted in significantly higher aPTT and F XII changes than those induced by a sub-therapeutic WF dose of 0.06 mg / kg / d. Unexpectedly, combined administration of MASA + WF (10 + 0.06 mg / kg / d) resulted in a reliable decrease in F VII levels compared to MASA and control groups and was significantly greater than that induced by WF. This suggests that combinations of MASA and a sub-therapeutic dose of WF will produce beneficial effects in the prevention of thrombotic complications with a broader application than MASA alone.

III Effect of the Compounds Investigated on Thromboembolism Clinic WF is primarily used for the prevention of venous thrombosis and thromboembolism of various origins, including stroke prevention (Freeman WD, Aguilar ML Neurol Clin 2008; 26: 1129-1160).

We investigated the effects of MASA, WF and MASA + WF in an experimental model of thromboplastin-induced thromboembolism in mice (Sato K et al., Jap J Pharmacol. 1998; 78: 191-97). ICR male mice (26-30 g) were used in the experiments. The efficacy of repeated (7 days once daily) administration of MASA and WF was compared. In mice, 30 mg / kg of thromboplastin (dissolved in saline) was injected into the tail vein. Mouse survival at 15 minutes after thromboplastin injection was recorded and used as an index for antithrombotic effects.

[0071] The results were compared using χ ² test (Greenvvood PE, Nikulin MS. A guide to chi-squared testing. Wiley, Ν-Υ, 1996).

Table 8. Effect of repeated administration of test substances on animal survival in a thromboembolic experiment.

Group N Survival n % Control 10th 1 10th MASS 10 mg / kg 10th 8 * 80 WF 0.5 mg / kg 10th 9 * 90 MASS + WF (10 + 0.5 mg / kg) 10th 10 ² 100

'Ρ <0.05 versus control ² P <0.005 versus control [0072] Repeated (7x) administration of MASA and WF significantly increased the number of surviving animals, whereas combined administration of MASA + WF completely prevented thromboembolism and its induced animal death (Table 8). .

[0073] It is necessary for the clinically applicable drug to have good activity and a wide safe dose range. Therefore, we also considered this aspect in the case of WF and MASA. Doubling the MASS dose (from 5 to 10 mg / kg / d) resulted in a known (although not statistically significant) decrease in factor XII, XI and X and a comparatively longer bleeding time with slightly higher blood loss (Tables 1 and 2), although the differences were not statistically significant (P> 0.05). This indicates that in the case of MASA, the individual response of the patient to this compound would not lead to complications of bleeding and significant changes in efficacy. MASA reliably lowers intrinsic factor XII, with virtually no effect on extrinsic factor VII.

In the case of WF, a dose change from 0.5 to 0.25 and even lower to 0.06 mg / kg / d significantly reduced the effect on coagulation factors (see Tables 2, 6 and 7), and significantly changed the risk of bleeding (see changes in TBT and blood loss from WF 1,

Tables 3 and 4).

As such, we have found that the effects of WF and MASA on the coagulation cascade are different pathways (WF on the outer pathway, but MASA on the inner pathway) and a combination of both acts on factor VII as well as on factor XII. Surprisingly, the combination of MASA with a sub-therapeutic dose of WF resulted in a graded decrease in factor VII, indicating a synergy between MASA and WF.

Our observations reveal new beneficial dual effects of MASA in which the known anti-caking efficacy is complemented by anticoagulant activity. Synergistic effects of MASA and WF anticoagulants without increased risk of bleeding were observed. By varying the doses and proportions of the individual agents in the combination of MASA and WF, it would be possible for the clinician to purposefully alter the specific effect of this combination and to influence undesirable effects (such as the risk of bleeding). The new combination WF + MASA provides a significant reduction in the WF dose in the clinic, which may raise the WF safety threshold.

INDUSTRIAL APPLICATION

MASA is intended to be used as an oral anticoagulant and can be administered in the form of the usual oral dosage forms, such as tablets or capsules. Because of the beneficial effects of MASA over a wide dose range, it is possible to adjust the dose of MASA or its pharmaceutical composition according to clinical needs. The combination of MASA with WF has beneficial effects already when WF is administered at doses that are generally considered to be non-anticoagulant. Therefore, it is possible to use a minimal dose of WF in synergistic combination with MASA to achieve a beneficial effect. Clinical experience will show the best ratio and amount of components in a combined pharmaceutical product that can be produced as a combination of these formulations in a single dosage form.

Because MASA is highly water soluble, MASA solution may find use in intravenous administration in emergency situations.

Claims

Claims (13)

Claims Use of Meldonium acetylsalicylate as a coagulation factor XII inhibitor. A pharmaceutical composition having anticoagulant activity comprising Meldonium acetylsalicylate together with a pharmaceutically acceptable excipient, carrier or excipient. A method of providing anticoagulant activity to a patient in need of such treatment, comprising administering an effective amount of meldonium acetylsalicylate according to claim 1 or a pharmaceutical composition according to claim 2. The use of Meldonium acetylsalicylate according to claim 1 or the pharmaceutical composition of claim 2 for providing anticoagulant activity to a patient in need of such treatment. Use of Meldonium acetylsalicylate for the manufacture of a medicament with anticoagulant activity. A pharmaceutical combination comprising a therapeutically effective amount of Meldonium acetylsalicylate and a therapeutically effective amount of warfarin sodium. A method for the prevention and / or treatment of a thromboembolic disorder comprising administering to a patient in need thereof a therapeutically effective amount of meldonium acetylsalicylate and a therapeutically effective amount of warfarin sodium. The method of claim 7, wherein the meldonium acetyl salicylate and warfarin sodium are administered as separate dosage forms. The method of claim 7, wherein the meldonium acetylsalicylate and warfarin are administered as a single dosage form. The method of any one of claims 7, 8 and 9, wherein the thromboembolic disorder is selected from the group consisting of arterial cardiovascular thromboembolic disorder, venous cardiovascular thromboembolic disorder, arterial cerebrovascular thromboembolic disorder and venous cerebrovascular accident. health disorder. The method of any one of claims 7, 8 and 9, wherein the thromboembolic disorder is selected from the group consisting of unstable angina, acute coronary syndrome, atrial fibrillation, first myocardial infarction, recurrent myocardial infarction, sudden ischemic death , transient ischemic attacks, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary artery thrombosis, cerebral artery thrombosis, cerebral embolism, cerebral embolism, cerebral embolism, , where the blood comes in contact with an artificial surface that promotes thromboembolism. A pharmaceutical composition comprising the first therapeutic agent, meldonium acetylsalicylate, and the second therapeutic agent, warfarin sodium, together with a pharmaceutically acceptable excipient, carrier, or excipient. Use of a therapeutic combination according to claim 6 or a pharmaceutical composition according to claim 12 for the manufacture of a medicament for use in the treatment of arterial cardiovascular thromboembolic disorder, venous cardiovascular thromboembolic disorder, arterial cerebrovascular thromboembolic disorder and venous cerebrovascular accident / venous disorder / treatment.