LV14468B - Antiinflammatory agent - Google Patents

Abstract

Meldonium acetylsalicylate salt, its solvates or polymorphs for use as medicament. Combination medicinal products comprising meldonium acetylsalicylate and another active agent selected from the group of niacin, statins, clopidogrel and dipyridamole.

Description

DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates to a novel medical product having anti-inflammatory, analgesic, antipyretic, antirheumatic, antihyperlipidemic, anti-atherosclerotic, anti-platelet and anti-thrombotic properties.

More particularly, the invention relates to a novel medical product containing meldonium acetylsalicylate. The invention also relates to a pharmaceutical composition comprising meldonium acetylsalicylate.

Meldonium acetylsalicylate was described in our patent application P-09-117. relating to new salts of acetylsalicylic acid. It is intended that the novel substance described in that application may have different polymorphic crystal types and solvates, preferably hydrates, which have similar biological properties and are therefore included in this application as variants of the compound described.

Because betaine-type compounds included in the new acetylsalicylic acid salts themselves have different pharmacological activities, the new acetylsalicylic acid salts may have beneficial activities in addition to those of the acetylsalicylic acid or betaines, including new pharmacological activities. We have already found that Meldonium acetylsalicylate slows down and significantly reduces nicotinic acid-induced dilation of skin blood vessels. Further experiments showed surprisingly pronounced pharmacological activities of meldonium acetylsalicylate.

ABBREVIATIONS USED

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

AdA - Adjuvant Arthritis

ASS - acetylsalicylic acid

H - cholesterol

CHD - Coronary heart disease

CIK - circulating immune complexes

KL - Clopidogrel

CRP - C-reactive protein

Di - dipyridamole

HDL - high density lipoproteins

LA - Laropiprant

LDL - Low-density lipoproteins

MDAS - Meldonium Acetyl Salicylate (Chemically: 3- (trimethylammonioamino) propanoate acetyl salicylate)

MD - Meldonium (SNN)

NS - Nicotinic acid RA - Rheumatoid arthritis TG - Triglycerides TR - Triton WR1339 SI - Simvastatin

TECHNICAL LEVEL

Platelets play an important role in the development of atherosclerosis and the formation of fatal thrombi in coronary heart disease. Antiplatelet agents have become very important in the prevention and treatment of various diseases, including various cardiovascular, cerebrovascular and peripheral arterial diseases (Meadows TA et al, Circ Res 2007; 100 (9): 1261-75). Acetylsalicylic acid is a very widely used drug known mainly for its anti-inflammatory, analgesic, antipyretic and anti-rheumatic properties. It is also used in low daily doses as an antiplatelet agent in cardiovascular patients (Eidelman RS et al., Arch Intern Med. 2003; 163: 2006-2010). Although antiplatelet agents have been known for many years, anti-inflammatory properties of ASA in cardiology have only recently begun to be more focused (Ridker PM et al, N Engl J Med 1997; 336: 973979). Consequently, clinical measurements of inflammatory markers such as C-reactive protein (CRP) may partially reflect atherosclerosis rates (Buckley DI et al, Ann Intern Med 2009; 151: 483-495). Current experience suggests that lowering CRP levels prevents CHD events (Ridker PM et al, Lancet 2009; 373: 1175-82). Ross suggested treating atherosclerosis as an inflammatory disease (Ross R, N Engl J Med 1999; 340: 115-126). Aspirin may not only affect the inflammatory aspect of atherosclerosis but also act directly to induce hypolipidemia (Kourounakis AP et al., Experimental and Molecular Pathology 2002, 73: 135-138).

NS is an effective lipid-sparing agent that prevents atherosclerosis and reduces the likelihood of cardiovascular events. NS has diverse effects on lipoproteins and arterial thrombosis, improving endothelial function, reducing inflammation, increasing plaque stability, and reducing thrombus formation (Rosenson RS, Atherosclerosis 2003; 171: 87-96).

NS almost completely prevents the formation of clots in the blood vessels caused by thromboplastin and pituitrin, suggesting its thrombolytic activity (Baluda VP, Cardiology 1974; 14 (11): 105-7 (Rus). The properties of NS anticoagulants have been described by several authors (Shestakov VA, Probl Gematol Pereliv Krovi, 1977; 22 (8): 29-35. Chekalina SI, Sov Med 1982 (5): 105-8. NS reduces blood clot formation (Chesney CM et al., Am Heart J, 2000; 140: 631-36).

NS inhibits platelet adhesion (Lakin KM, Farmakol Toksikol, 1980; 43 (5): 581-5). NS affects platelet activity in vitro, slightly inhibiting adhesion and promoting the release of significant prostaglandins, essentially without affecting the expression of major platelet receptors. The effect of NS is unique, unlike other anti-caking agents, indicating and potential use in combination therapy (Serebruany VL et al., Thrombosis and Haemostasis, 2010 (in press).

NS is an effective lipid-mediating agent that prevents atherosclerosis and reduces the likelihood of cardiovascular events (Drexel H, European Heart Journal Supplements 2006; Vol 8, Suppl F: F23-F29. Savel'ev AA, Shershevskii MG, Klin Med (Rus) 1996; 74: 48-52).

NS is available in 3 forms (fast release, sustained release and sustained release). Rapid release NS is associated with redness and increased blood glucose. Slow-release NS is associated with reduced redness but with the potential risk of hepatotoxic effects. Prolonged-acting NS is associated with reduced redness and a low risk of hepatotoxicity (McKenney J, Arch Intern Med 2004; 164 (7): 697-705).

The clinical application of NS was limited by skin redness. Slow-release nicotinic acid may help in the control of redness (Guyton JR et al., J Clin Lipidol, 2009; 3: 101-108).

ASA and other NSPLs have been proposed for the control of dermal redness in various pharmaceutical compositions providing NSPL administration prior to the NS dose (WO9632942, WO9906052, WO2009142731).

Recently, a specific prostaglandin D2 receptor subtype I antagonist laropiprant (Parhofer KG) has been proposed to limit NS-induced redness (Lai E et al, Clin Pharm Ther 2007; 81: 849-857. Davidson MH, AmJCardiol 2008; 101 [suppl]: 14B-19B). , Vascular Health and Risk Management 2009; 5: 901-908). Although the use of laropiprant reduces the incidence of redness, it cannot completely eliminate this side effect. Laropiprant does not alter the effects of nicotinic acid on lipids, nor does it alter the other side effects of nicotinic acid. Therefore, the combination of nicotinic acid and laropiprant may allow the use of higher doses of nicotinic acid and the full potential of this agent (Parhofer KG, Vascular Health and Risk Management 2009; 5: 901-908, Olsson AG, Expert Opinion on Pharmacotherapy 2010; 11 (10): 1715- 1726).

MD is a medicine with known beneficial effects on the heart and blood vessels. Some valid MD activity has been found in animal models of atherosclerosis (Veveris M, Smilsaraja B, White JLabAnimSci 2000; 10: 194-199. Veveris M et al, White JLabAnimSci 2002; 12: 116-122. Okunevich IV, Ryzhenkov VE, Patol Fiziol Eksp Ter (Rus) 2002; (2) 24-7). MD has also been reported to inhibit platelet aggregation (TsirkinVI, Ros Kardiol Zh 2002; 1: 45-52). Oral use of MD for two weeks in experimental arterial thrombosis therapy in rabbits and dogs has shown thrombolytic effects (Logunova L et al., Experim Clin Pharmacoter 1991; 19: 91-98 (Rus). No data are available on the prophylactic effect of MD on thrombosis control or prevention.

PROTOTYPES

The range of application of ASS is largely limited by the low water solubility (about 0.3%). In addition to the Li, Na, Mg and Ca salts, several islands with basic amino acids have been described [US-4,625,888]. None of these salts has found widespread use in medical practice. Therefore, studies of novel salts of ASS with improved solubility, lower toxicity and new pharmacological profiles are required.

OBJECTS OF THE INVENTION

This invention relates to a medical product, namely MDAS with anti-inflammatory, analgesic, antipyretic, anti-rheumatic, antihyperlipidemic, anti-atherosclerotic, anti-platelet and anti-thrombotic properties. Another subject of the present invention is a method of treating a subject in need of anti-inflammatory, analgesic, antipyretic, anti-rheumatic, antihyperlipidemic, anti-atherosclerotic, anti-platelet and anti-thrombotic therapy. A further subject of the present invention is a pharmaceutical composition comprising MDAS for the above purpose. Other objects of this invention will become apparent later, and others will be apparent to the skilled artisan.

DESCRIPTION OF THE INVENTION

The invention encompasses a medical product containing MDAS, preferably in a single dose dosage form. The particular form in which the active ingredient is administered is not important, as long as the desired effect of the invention is achieved. The active ingredient may be in the form of capsules, suspensions, dispersions, elixirs, syrups and the like.

We have unexpectedly found that MDAS has excellent anti-inflammatory, analgesic, antipyretic, anti-rheumatic, antihyperlipidemic, anti-atherosclerotic, anti-platelet and anti-thrombotic properties.

Therefore, this substance may be expected to be a desirable agent for the treatment of inflammation, pain, fever, rheumatic conditions, hyperlipidemia and atherosclerosis, to prevent platelet aggregation and thrombus formation in patients at risk.

The pharmaceutical product of the present invention may be in the form of an oral dosage form such as tablets or capsules.

It is contemplated that the medical product of the present invention may also contain various MDAS polymorphs and solvates with the same result.

The medical product of the present invention is also intended to include sustained release and sustained release dosage forms.

The pharmaceutical composition of the invention may be prepared by conventional procedures employing conventional pharmaceutically acceptable excipients or carriers and technologies.

The following examples illustrate but do not limit the invention.

EXAMPLES

Substances. NS (Acros Chemicals), MD (Grindex), ASS (Acros Chemicals), LA (MK 0524, Cayman Chemicals), KL bisulfate for in vitro assays (Molcan Corporation), KL for in vivo assays as Plavix ™ (Sanofi-Aventis), DI (Sigma-Aldrich).

Example 1: Determination of acute toxicity of MDAS

Acute toxicity of MDAS was determined in Wistar rats and ICR mice by administration

p.o.

Methods. IRC male mice weighing 20-22 g and Wistar rats 200-230 g were used. For acute toxicity, each dose was administered to 6 animals, increasing each subsequent dose by 500 mg / kg. LD50 was calculated from Karber using the method described by Akhila JS et al, Current Sci 2007; 93: 917-920, modified for Dose Confidence Interval (Tumer R In Screening Methods in Pharmacology, Acad. Press, New York, 1965, 61-9). 63).

LD ₅ o was calculated as follows:

LD ₅ o = Lowest lethal dose in the whole group - £ (axb) / NN - Number of animals in each group a - Dose difference b - Mean mortality (2 lethal groups / 2)

MDAS was diluted ex tempore in 0.2% agar solution and administered orally via a catheter in the stomach. The volume of liquid administered in this manner did not exceed 0.5 ml in mice and 2 ml in rats. Animals were observed until day 10 after administration.

Results. The results of the MDAS acute toxicity assay are shown in Figures 1-3. in the table.

Table 1

Acute toxicity of MDAS in mice p.o.

Group Dose mg / kg p.o. Number of animals per group (N) Letalitate (n) Average mortality (b) Probiti (a x b) 1 1000 6th 0 2 1500 6th 1 0.5 250 3 2000 6th 2 1.5 750 4 2500 6th 4 3 1500 5 3000 6th 6th 5 2500

LD ₅₀ = 3000 - (5000/6) = 2167

The coefficient f at P = 0.05 for this experiment is 1.32, so the LD ₅ o confidence interval is 1642-2860 (mg / kg).

Toxic effects occurred within the first few hours after administration of MDAS and some animals died within the first 2 days. In the surviving animals, the symptoms of toxicity gradually disappeared and after 5 to 8 days these animals did not differ from the same age control group. So the MDSA LD50 mice found p.o. and 2167 (1642-2860) mg / kg.

Results of the MDAS Acute Toxicity Test in the rat p.o. Table 2 is shown.

Table 2

Acute toxicity of MDAS in rats p.o.

Group Deva mg / kg Number of animals per group (N) Fatalities (n) Medium mortality (b) Probiti (axb) 1 1500 6th 0 2 2000 6th 2 1 500 3 2500 6th 4 3 1500 4 3000 6th 6th 5 2500

LD ₅₀ = 3000 - (4500/6) = 2250

The coefficient f at P = 0.05 for this experiment is 1.308, so the LD ₅₀ confidence interval is 1720- ^ 2944 (mg / kg).

Administration of MDAS in rats p.o. at a dose of 1500 mg / kg induced transient feeding and movement disorders, but all animals survived. Toxic symptoms began to disappear on day 3 after administration. Thus, the MDSA LD50 found in rats p.o. and 2250 (1720-2944) mg / kg.

Summary. Acute toxicity studies showed that MDAS is a low toxicity substance (LD50> 2000 mg / kg p.o. in mice and rats). The acute toxicity of ASA according to Boehringer Ingelheim Pharmaceuticals, Inc., Acros Chemicals and Sigma-Aldrich is 250 mg / kg in mice and 200 mg / kg in rats p.o., while Bayer AG gives LD50 in rats p.o. as> 1100 mg / kg, thus MDAS is less toxic than ASA.

Table 3

Acute toxicity of MDAS in mice and rats; N = 6.

Animals LD ₅₀ mg / kg po (confidence interval) Mice 2167 (1642-2860) The Rat 2250 (1720-2944)

Example 2: Comparison of MDAS, ASS, and MD analgesic, antipyretic, and anti-inflammatory activities in experiments

The analgesic, anti-inflammatory, and antipyretic effects of MDAS have been investigated using methods commonly used in the evaluation of NSAIDs. Insensitive laboratory mice and Wistar rats were used in the experiments. The animals were housed in groups of 7 to 8 in appropriate cages at 22 ± 1 ° C, 60 ± 5% relative humidity, and a 12/12 hour light / dark cycle with free access to food and water.

For comparing the effects of MDAS, ASS, and MD by oral administration, the following groups were formed:

GROUP PROCEDURE

The ASS50 received the ASS 50 mg / kg

ASS 100 received ASS 100 mg / kg

MD 100 received MD 100 mg / kg

MDAS75 received MDAS 75 mg / kg

MDAS 15 0 received MDAS 150 mg / kg

MDAS300 received MDAS 300 mg / kg

Aqueous solutions of the test substances were prepared ex tempore. In each series of experiments, a control group was used that received an identical volume of water p.o.

Statistics. Data were analyzed with Microsoft Excel 2007 software and expressed as Mean ± Standard Mean Error (SEM). The mean scores of the different groups were compared using one-way ANOVA with repeated comparison (Tukey's test). P <0.05 was considered significant.

2.1. Investigation of analgesic activity

2.1.1. Evaluation of analgesic activity using the mouse ..bend ”test

Method, The pain response was assessed by the chemical-irritation-bending assay (Charaborty A et al, Indian J of Pharmacology 2004; 36 (3): 148-150). Animals received i.p. 0.25 ml of 0.75% aqueous acetic acid solution. After injection, the animals were housed separately in specific boxes and observed for 10 minutes. Abdominal contractions were recorded. The analgesic activity consisted of a reduction in the number of cases of abdominal contraction over a 10-minute period. Test substances were administered for 30 min. before the irritant. The analgesic effect was expressed as the analgesic index calculated as follows:

A = (Cc-Ct) / Cc · 100% where

A - index of analgesia

Cc - number of contractions in the control group,

Ct - number of contractions in the test group.

The results are given in Table 4.

Table 4

The analgesic effect of the test substances in the "flexion" test model; N = 8; Mean + SEM

Group Animals with positive reactions / total Number of breaks Analgesia index Control 8/8 22.00+ 1.60 - MD100 8/8 22.10 + 1.47 -0.4 ASS50 6/8 5.75 + 1.32 *** " 7.1 MDAS75 8/8 12.13 + 0.61 ** " 4.5 MDAS 150 7/8 10.63 ± 1.10 ** " 5.2 MDAS300 5/8 6.00+ 1.39 *** " 7.3

** P <0.005 vs. Control *** P <0.0005 vs. Control ”P <0.005 vs. MD ^{I $ S} P <0.0005 vs. MD

MDAS showed a dose-dependent positive effect. The best results were observed in the ASS50 and MDAS300 (P <0.0005) groups while the MD was inactive. The analgesia index in the MDAS300 group was 7.3 (only 5 of 8 animals had a pain response).

2.1.2. Evaluation of analgesic activity in hot plate test mice

The method. The hot plate test was performed on 52 mice weighing 1726 g, according to the literature (Belyakov VA, Solov'ev IK. Narcotic analgesics, Nizhny Novgorod, 2001 (in Russian). The hot plate test is used for screening of central analgesics (Osterberg A et al, J Pharmacol Exper Ther 1958; 122: 59) Test solutions of aqueous solutions were administered 30 or 60 min prior to testing Time to paw bite was recorded The analgesic activity measure was the delay in response to the thermal irritant.

The results are given in Table 5.

Table 5

Response time of mice hot plate test; N = 8-10; Mean + SEM

Group Latent period, s 30 min after administration of the test substance 60 min after administration of the test substance Control 4.5 ± 0.42 5.0 ± 0.27 MD100 9.5 ± 0.68 *** ^&& 8.3 ± 0.53 *** ASS50 5.4 ± 0.46 9.7 ± 1.05 ** MDAS75 5.4 ± 0.38 4.6 ± 0.26 MDAS150 9.5 ± 0.53 *** ^{& ti} 7, l ± 0.55 ** MDAS300 9.6 ± l, 12 ** ⁱ 8.6 ± 0.60 ***

** P <0.005 vs. Control *** P <0.0005 vs. Control ^& P <0.05 vs. ASS (30 min) “P <0.005 vs. ASS (30 min) ^&&& P <0.0005 vs. ASS (30 min) )

Experimental data show that the MDAS150 and MDAS300 as well as the MD group show significant analgesic effect at 30 and 60 minutes. ASA significantly increased the pain threshold only after 60 minutes, indicating a slower onset of action (Table 5).

3. Comparative evaluation of test substances in fever test

3.1. Evaluation of Preventive Fever Activity in Pyrogenically Injected Rats

The method. Experiments were performed on 48 Wistar rats weighing 165182 g by intramuscular injection of 100 pg (Shwarz GY, Syubaev RD, Vedomosti NCEG lekarstvennyh sredstv MZ RF 2000; 1: 44-) into the muscle (Gamalei State Research Institutes, Moscow, Russia); 50 (Rus) Test substances were administered one hour before pyrogen injection, rectal temperature was measured with an electric thermometer TERMO before pyrogen injection (baseline) and 3 hours after injection, and the antipyretic activity was evaluated after 2 hours after pyrogen injection, which correlates well with published reaction peak data (Table 6) Room temperature was maintained at 20-21 ° C.

As shown by the data, the body temperature of the control animals gradually increased, reaching a peak after 2 hours and above the normal for another 1 hour.

Table 6

Changes in rectal temperature of the control group under the influence of pyrogen; N = 8; Mean ± SEM

Group Bazhnaya (° C) Rectal temperature (° C) after 30 min after 60 min pec 120 min in 180 min Control M-zv - 36.16 ± 0.16 36.25 ± 0.21 36.75 ± 0.12 ** 36.90 ± 0.10 # * ^# 36.70 ± 0.17 ^#

* P <0.05 versus baseline ## P <0.005 versus baseline ^W P <0.0005 versus baseline

Petamates did not significantly affect the normal body temperature of the animals, but significantly reduced the hyperthermia induced by pyrogen (Table 7).

Table 7

Effect of investigated compounds on hyperthermia induced by intramuscular pyrogen injection, N = 8; Mean + SEM

Group Baselines rectal temperature (° C) Rectal temperature (° C) 120 minutes after injection Control 36.2 + 0.12 36.9 + 0.16 MD100 35.9 + 0.18 36.2 + 0.16 * ASS50 35.8 + 0.17 35.6 + 0.19 *** ^s MDAS75 36.1 + 0.18 36.3 + 0.20 * MDAS150 35.8 + 0.15 36 + 0.21 ** MDAS300 35.9 + 0.14 35.8 + 0.16 *** '

* P <0.05 versus control ** P <0.005 versus control *** p <0.0005 versus control ** P <0.005 versus baseline ^s P <0.05 versus MD

In the hyperthermic model, the increase in body temperature induced by pyrogenic injection was completely reversed in the ASS50 and MDAS300 groups (Table 7). The anti-pyretic effect was less pronounced in MD100, MDAS75 and MDAS150 groups.

3.2. Evaluation of Preventive Fever Activity in Pyrogenically Injected Rats (Treatment Regimen)

The method. The anti-fever effect of the test substances in the therapeutic (treatment) regimen was investigated in 48 rats weighing 182-205 g by inducing hyperthermia with a pyrogenic injection at 100 pg (Shwarz GY, Syubaev RD, Vedomosti NCEG lekarstvennyh sredstv MZ RF 2000; 1: 44 -50 (Rus) The test substances were administered 2 hours after pyrogen administration, immediately after the elevated body temperature was measured Rectal temperature was measured with an electric thermometer TERMO before pyrogen

i.m. injections (baseline temperature), peak of hyperthermia (pyrogenic control), and 30 min. after test substance administration, i.e. 2½ hours after pyrogen administration. The temperature in the laboratory was maintained at 20-22 ° C.

The results are given in Table 8.

Table 8

Effects of test substances on hyperthermia induced by intramuscular pyrogen injection (treatment regimen); N = 8; Mean + SEM

Group Basal Rectal Temperature (° C) Rectal temperature (° C) after pyrogen injection (pyrogenic control) Rectal temperature (° C) 30 minutes after the procedure Control 36.10 + 0.15 36.90 + 0.20 ** 37.00 + 0.18 ** MD100 36.41 + 0.14 37.06 + 0.13 ** 37.10 + 0.14 ** ASS50 36.39 + 0.13 37.04 + 0.25 * 36.51 ± 0.15 * ^s MDAS75 36.23 + 0.12 37.01 + 0.11 ** 36.90 + 0.07 ^ MDAS150 36.25 + 0.20 36.96 + 0.15 * 36.75 + 0 {09 ^ MDAS300 36.11 + 0.14 36.76 + 0.11 ** 36.40 + 0.11 * " ^%

* P <0.05 versus control * P <0.05 versus baseline ^w P <0.005 versus baseline ^% P <0.05 versus pyrogenic control ^s P <0.05 versus MD ^K P <0.005 versus MD

Pyrogenic caused a significant and similar increase in body temperature in all the animals used in the experiment (cf. Pyrogenic control against the baseline, Table 8). The administration of test substances other than MD resulted in a decrease in body temperature against baseline and pyrogen control. Relatively higher hypothermia was observed in the MDAS300 and ASS50 groups, where body temperature reduction was significant compared to control and MD100. It should be noted that in the MDAS300 group, as opposed to ASS50, body temperature reductions were also significant against pyrogenic control. This points to the remarkable and rapid anti-pyretic effect of MDSA, which can be valuable in the clinic.

4. Comparative anti-inflammatory activity of test substances

4.1. Model of acute inflammatory edema

The method. Experiments were performed using the carrageenan assay (Winter C et al, Proc Soc Exptl Biol and Med 1962; III (3): 544-547. Wei Jia et al., Journal of Ethnopharmacology 2003 (89): 139-141; Sutharson Lingadurai et al. al, African Journal of Traditional, Complementary and Alternative Medicine, 2007, 4 (4): 411-416) with 42 rats weighing between 226 and 274 g. One (1%) solution of carrageenan (Sigma) in saline (0.1 ml) was injected into the hind paw. Test substances were administered p.o. (via catheter in rat's stomach) 30 min. after injection of carrageenan. Paw volume was measured with an oncometer at baseline and 4 hours after carrageenan injection.

The percent protection index (suppression of edema) was calculated using the formula:

Protection index (%) = (1 - Vo / Vc) x 100 where

I - Protection Index% (suppression of edema)

Vo - difference between paw volume at baseline and experimental conditions;

Vc - analogous difference in control group.

The results are given in Table 9.

Table 9

Antiinflammatory activity of test substances in carrageenan inflammatory model; N = 7, Mean ± SEM

Group Volume of paw (ml) at baseline Paw volume (ml) after carrageenan injection ml Index,% Control 1.6 ± 0.30 2.70 ± 0.13 0 MD100 1.5 ± 0.34 2.13 ± 0.12 * 43 ASAI 00 l, 6 ± 0.32 l, 97 ± 0.10 ** 66 MDAS75 1.6 ± 0.44 2.51 ± 0.1 l 18th MDAS150 1.6 ± 0.31 l, 54 ± 0.15 *** ^{s &} 93 MDAS300 1.7 ± 0.25 l, 79 ± 0.15 ** 91

* P <0.05 versus control ** P <0.005 versus control *** P <0.0005 versus control ^& P <0.05 versus ASS 100 ^s P <0.05 versus MD

In the model of acute inflammatory edema, the volume of the affected limb increases approximately

1.6 times. The most pronounced effect of the inflammatory process was prevented in the MDAS150 group, where the prevention index was 93% versus the control group. In the MDAS300 group, activity was slightly lower - swelling was reduced by 91%. Decreased edema was also observed in the MD100 and ASS50 groups.

4.2. The anti-inflammatory activity of the test substances against carrageenan edema in the prophylactic regimen

The method. Caregenin edema was studied by a known method (Okunevich IV, Ryzhenkov VE, Patol Fiziol Eksp Ter, 2002 (2): 24-7 (Rus) in 42 rats weighing 178-220 g. Within 5 days: Immediately after administration of the test substances on day 6, rats were injected with 0.1 ml of 1% carrageenan solution in the hind paw The volume of the paw was measured with an oncometer at baseline and 4 hours after injection of carrageenan. the prophylactic administration of 6-fold agents resulted in a reduction in edema compared to untreated animals (Table 10).

Table 10

Preventive anti-edema effect of test substances on carrageenan; N = 7, Mean ± SEM

Group Volume of paw (ml) at baseline Volume of paw (ml) after carrageenan injection ml % Reduction Control 1.43 ± 0.12 l, 93 ± 0, l 1 0 MD100 1.33 ± 0.09 1.66 ± 0.10 34 ASS50 1.40 ± 0.06 1.55 ± 0.04 * 70 MDAS 150 l, 37 ± 0, ll l, 40 ± 0.09 ** ^J 94 MDAS300 1.34 ± 0.07 l, 41 ± 0.04 ** ^s 86

* P <0.05 versus control ** P <0.005 versus control ⁵ Ρ <0.05 versus MD

In this inflammatory model, significant protective activity was observed in the MDAS 150 and MDAS300 groups (94%), which was higher than in the MD100 (34%) and ASS50 (70%) groups (Table 10).

To measure the intensity of the inflammatory process, a CRP level was determined on the INTEGRA 400+ analyzer at the end of the experiment (5 hours after carrageenan injection) using a standard method.

Table 11 shows the results of blood CRP determination.

Table 11

CRP levels in rat blood in a carrageenan model of inflammation in rats; N = 7, Mean ± SEM

Group CRP mg / L CRP increase% Control 0.17 ± 0.015 0 Carrageenan control 0.23 ± 0.016 ** 100 MD100 0.22 ± 0.014 * 83 ASS100 0.20 ± 0.018 50 MDAS 150 0.19 ± 0.009 ^{# $} 33 MDAS300 0.21 ± 0.014 * 67

* P <0.05 versus control ** P <0.005 versus control * P <0.05 versus carrageenan control ^s P <0.05 versus MD

As can be seen from these data, carrageenan causes an increase in CRP levels in the blood. In the ASS 100 group, the increase in CRP level was reduced by 50% (Table 11). Unexpectedly, in the MDAS150 group, CRC elevation was significantly less pronounced (only 33% versus control). This supports the conclusion that MDAS may have a positive effect on the inflammatory process in the clinic.

Example 5. Study of MDAS anti-rheumatic activity versus ASA and MD

Clinical experience has shown that rheumatoid arthritis (RA) patients are predisposed to atherosclerosis and cardiovascular disease (Nasonov EL, West Ross Akad Nauk 2003 (7): 6-10). Patients with long-term RA have atherosclerosis more often than patients of the same age with a more recent onset. Systemic inflammation may increase the age-related risk of cardiovascular disease (Del Rincon I et al., Atherosclerosis 2007; 196 (2): 3 54-360).

Rheumatoid arthritis has the first place among rheumatoid disorders. The most appropriate experimental animal model of human rheumatoid arthritis is the adjuvant arthritis model induced by Freind's adjuvant injection in the hind foot of a rat. It is widely used in the selection of anti-arthritis agents (Wei Jia et al., Journal of Ethnopharmacology 2003 (89): 139-141; Sutharson Lingadurai et al., African Journal of Tradition.1, 2007 ;, 4 (4): 411416).

Methods. Our experiments were designed to test the effect of MDAS on the development of adjuvant arthritis relative to MD and ASS. Experiments were performed on Wistar rats with an initial body weight of 153-185 g. Rats were housed in air-conditioned rooms at 22 ± 1 ° C, 60 ± 5% relative humidity, and 12/12 light / dark cycles. Each standard cage housed 7 rats with unrestricted access to drinking water and pelleted standard food. All experiments were carried out in accordance with Council Directive 86/609 / EEC of 24 November 1986 on the use of animals for scientific purposes. Every effort was made to minimize animal suffering and reduce the number of animals used.

A modified standard procedure for the induction and progression of chronic adjuvant arthritis was used (Bellavite P, Ortolani R, Conforti A, Immunology and Homeopathy. 3. Experimental Studies on Animal Modeh, Advance Access Publication 2.05, 2006, 171-186). Rats were injected with complete Freind's adjuvant in the hind foot 0.1 ml and intraperitoneally 0.05 ml.

Test substance solutions were prepared ex tempore. AS was used as 0.1% and 1%, MD as 1% and MDAS as 0.25%, 1% and 2% aqueous solution. The test solutions in the animals were administered p.o. with a catheter in the stomach.

The following groups of animals were created (N = 7):

-GROUP PROCEDURE

Group 1 intact animals used as controls (Control);

Group 2 animals with induced adjuvant arthritis (AdA);

Group 3 animals receiving ASA at a dose of 10 mg / kg / day for 28 days following initiation of adjuvant arthritis (ASA 10);

Group 4 animals receiving ASA at a dose of 100 mg / kg / day for 28 days following initiation of adjuvant arthritis (ASA 100);

Group 5 animals receiving MD at a dose of 100 mg / kg / day for 28 days following the initiation of adjuvant arthritis (MD100);

Group 6 animals receiving MDAS at a dose of 25 mg / kg / day for 28 days following the initiation of adjuvant arthritis (MDAS25);

Group 7 animals receiving MDAS at a dose of 100 mg / kg / day for 28 days following the initiation of adjuvant arthritis (MDAS 100);

Group 8 animals receiving MDAS at a dose of 200 mg / kg / day for 28 days following the initiation of adjuvant arthritis (MDAS200);

Animals in the control group and the AdA group received water p.o. instead of test substances. following the same plan as the study group.

Statistics. Data were analyzed with Microsoft Excel 2007 software and expressed as Mean ± Standard Mean Error (SEM). The mean scores of the different groups were compared using one-way ANOVA with repeated comparison (Tukey's test). P <0.05 was considered significant.

Results. The dynamics of clinical manifestations of arthritis were investigated on day 14 and day 28. The effects of the test substances were assessed using the following criteria:

1. Evaluation of local manifestations of arthritis - volume of paw and ankle joint.

2. Blood Score Assessment (WBC).

3. Biochemical Assay Evaluation (CRP).

4. Evaluation of immunological parameters (CIC level).

Edema, i.e., the volume of the hind paw, was measured with an oncometer. The protection percentage (suppression of edema) was calculated using the formula:

A (%) = (Vc - Vt) / Vc x 100 where

Vc - paw volume in the control group.

Vt - volume of paw in test group

A -% Protection (suppression of edema).

Hematology was determined by standard methods using the PENTRA 120 hematology analyzer, CRP was determined by INTEGRA 400+.

Serum CIC was determined spectrometically using ethylene glycol.

After injection of Freind's adjuvant, all animals in the test groups developed local inflammation, rats became fatigued, aggressive to insult, and swollen. However, dietary habits did not differ from control in all groups. The body weight gain in all groups did not differ significantly from the control group.

Localized manifestations of arthritis are shown in Tables 12 and 13: paw volume (representing soft tissue edema) and ankle joint volume / size (representing arthritis-like organic lesions in connective tissue) at day 14 and day 28.

Table 12

Effect of test substances on paw volume (ml) 14 and 28 days after Freind's adjuvant injection; N ~ 7; Mean ± SEM

Group Volume of paw Day 14 Volume of paw Day 28 ml Protection% ml Protection% Control 1.03 ± 0, Ι2 ** - 1.08 ± 0.17 * " - AdA 2.42 ± 0.17 0 2.23 ± 0.24 0 ASS10 2.53 ± 0.10 -7 2.08 ± 0.12 7th ASS100 2.35 ± 0.14 3 l, 52 ± 0.07 * 32 MD100 2.60 ± 0.21 -7 1.93 ± 0.13 13th MDAS25 2.20 ± 0.21 9th l, 32 ± 0.12 41 MDAS 100 2.07 ± 0.17 ^& 15th 1.22 ± 0 _; 08 ^{&&&<s! I} 45 MDAS200 2.13 ± 0.1, ^& 12th l, 7 ± 0.09 ^{# &} ® 24th

^e P <0.05 vs. AdA *** P <0.0005 versus AdA ^& P <0.05 versus ASS 10 ^ <0.005 versus ASS 10 ^{&& Λ} Ρ <0.0005 versus ASS 10 @P <0.05 versus ASS 100 ^J P <0.05 vs. MD

As shown in Table 12, on day 14 all animals in the study group developed pronounced soft tissue edema. Treatment for 14 days had relatively little effect on the development of edema. However, animals in the MDAS100 and MDAS200 groups had significantly less pronounced edema than the animals in the AS10 group. MD and ASS 10 at Day 14 did not prevent the development of edema but even increased volume compared to the AdA group (negative protection -7%). MDAS significantly protected against edema development at all doses and ASS 100 after 28 days (% protection, 41, 45, 24 and 32%, respectively). It should be noted that the MDAS 100 showed significantly better protection than the ASS 10, ASS 100 and MD.

Table 13

Effect of test substances on ankle joint changes on days 14 and 28 following Freind's adjuvant injection. N = 7; Mean * SEM

Group Dimensions of ankle joint Day 14 mm Dimensions of ankle joint Day 28 mm Control 5.6 * 0.09 ** 5.8 ± 0.12 ** AdA 7.2 * 0.14 7.3 ± 0.16 ASS 10 7.2 ± 0.12 6.8 * 0.14 * ASS 100 6.9 * 0.15 7, I ± 0.16 MD100 7.0 ± 0.13 7.0 * 0.13 MDAS25 7.0 ± 0.14 6.7 * 0.15 * MDAS100 7.0 ± 0.13 6.6 * 0.16 * ® * MDAS200 6.8 * 0.10 6.9 * 0.11

* P <0.05 versus AdA ** P <0.005 versus AdA ^& P <0.05 versus ASS 10 ®P <0.05 versus ASS 100 ^s P <0.05 versus MD

Analysis of ankle joint data (Table 13) shows that on day 14 only MDAS200 significantly protected against arthritis damage. On day 28, significant protection was demonstrated by MDAS25, MDAS100, and ASS 100. Comparatively better protection was demonstrated by MDAS100 in this experiment. We found that at day 28 the level of edema (Table 12) and arthritic changes in the ankle joint (Table 13) were reduced. MDAS100 had a significantly greater effect than ASS or MD.

Blood Scene (WBC) assessment showed an increase in leukocyte counts under Freind's adjuvant (Table 14). Leukocytosis is a characteristic feature of the inflammatory process.

Table 14

Changes in blood levels caused by test substances on days 14 and 28 following Freind's adjuvant injection. N = 7; Mean ± SEM

Group Leukocytes 10 ³ / mm ³ Day 14 Day 28 Control 15.17 * 0.76 ** 16.50 * 0.52 ** AdA 21.48 * 1.47 ** 20.32 * 0.90 ** ASS10 15.83 * 1.23 * 16.68 * 1.22 * ASS10 17.07 * 1.43 16.91 * 1.17 * MD100 14.42 * 1.41 * 15.55 * 1.26 * MDAS25, 14.72 * 1.63 * 16.77 * 1.78 MDAS100 14.13 * 0.53 ** 15.52 * 0.57 ** MDAS200 15.42 * 1.40 * 14.35 * 0.84 **

** P <0.005 versus Control * P <0.05 versus AdA * P <0.005 versus AdA

The use of test substances resulted in a decrease in white blood cell counts compared to the AdA group, which indicates anti-inflammatory activity. Although there was no statistically significant difference in the effect of the test substances on the increase in white blood cell count, on day 14 comparatively higher activity was observed in MDAS100 and on day 28 in the MDAS200 group (Table 14).

CRP levels at days 14 and 28 were determined to assess the development of the inflammatory process. CRP levels are known to increase during the inflammatory process.

Table 15

Changes in CRP levels in rats at 14 and 28 days after exposure to test substances

after Freind ad uvanta injections. N = 7, MeaniSEM Group CRP, mg / l Day 14 Day 28 Control 0.19 ± 0.01 ^e "" 0,16 ± 0,02 ^## AdA 0.30 ± 0.02 *** 0.24 ± 0.02 ** ASS 10 0.26 ± 0.01 ** 0.19 ± 0.01 # ASS 100 0.29 ± 0.01 *** 0.18 ± 0.02 # MD100 0.26 ± 0.02 ** 0.17 ± 0.02 * MDAS25 0.29 ± 0.02 ** 0.15 ± 0.02 MDAS100 0.29 ± 0.02 ** 0, I6 ± 0.01 MDAS200 0.25 ± 0.01 * # ^fe 0.19 ± 0.0l "

* P <0.05 vs. Control ** P <0.005 vs. Control *** p <0.0005 vs. Control * P <0.05 vs. AdA ** P <0.005 vs. AdA ** P <0.0005 vs. AdA ^& P <0.05 versus ASS 10 @P <0.05 versus ASS 100

As shown in Table 15, an increase in CRP levels was observed on day 14 in all study groups, suggesting an inflammatory process. In our experiment, only MDAS200 showed significant protection against CRP increase on day 14. It should be noted that on day 14, MDAS200 showed significantly better results than ASS 100. On day 28, MDAS25 and MDAS100 showed significantly better protection than ASA 10 (Table 15).

CIC levels were determined by a standard spectrophotometric method (Baranovskii PV, Rudyk Bl, Laboratornoe delo 1982; 12: 35-39 (Rus). The dynamics of immunological factors were assessed on days 14 and 28. The changes in CIC level are shown in Table 16.

Table 16

Amount of CIC (in units) 14 and 28 days after injection of Freind's adjuvant. N = 7; Mean ± SEM

Group CIC units, Day 14 CIC units, Day 28 Control 9.4 ± 1.05 * 7.8 ± 0.49 ^## AdA 17.4 ± 1.29 ** 13.4 ± 1.25 ** ASS 10 10.8 ± 0.74 ^## 8.2 ± 0.53 * ASS 100 15.0 ± 1.23 * 14.4 ± 1.66 * MD100 11.8 ± 2.22 8.6 ± 0.93 # MDAS25 12.6 ± 0.68 * 11.8 ± 1.53 MDAS100 20.8 ± 2.99 * 5.8 ± 0.74 ^ ® ' ⁵ ' ⁵ MDAS200 21.6 ± 3.26 * 6.2 ± l, 02 ** ®

* P <0.05 versus Control ** P <0.005 versus Control * P <0.05 versus AdA * # P <0.005 versus AdA ^s P <0.05 versus MD ^& P <0.05 versus ASS 10 @P < 0.05 vs. ASS 100 @@ P <0.005 vs. ASS 100

On days 14 and 28, CIC levels were higher in the study groups than in the control group. At day 14, only the ASS10 and MDAS25 groups had lower CIC levels than the AdA group. At day 28, the CIC levels in the groups receiving test substances, except for the ASS 100 group, were close to Control.

During the experiment, an increase in CIC on day 14 was observed in the ASS 100, MDAS100 and MDAS200 groups. At day 28, CIC levels in the MDAS100 and MDAS200 groups had normalized. Elevated serum CIC levels may be observed in a variety of abnormal immune conditions. Significant increases in CIC are observed in inflammatory processes, including systemic states, where CIC levels characterize the intensity of the pathological process (Bier 0 et al., New York, Heidelberg, Berlin, p. 442). Prolonged treatment with MDAS at various doses lowered CIC levels to normal. In the case of sufficiently high immunity, the major part of the CIC is removed by Kupfer cells and the decrease in CIC is seen as a positive effect. The fact that administration of MDAS in the MDAS 100 and MDAS200 groups showed a normalizing effect on CIC level at day 28 suggests that prolonged administration of multiple doses of MDAS in the treatment of arthritis may be more clinically viable than elevated doses of ASA.

Example 6. Investigation of antihyperlipidemic properties

Atherosclerosis is a multifunctional process (Berliner JA et al, Circulation 1995; 91: 2488-2495) whose clinical effects increase with increasing symptoms of coronary heart disease. Inflammation and the body's response to it play an important role in atherosclerosis (Ross R, Am Heart J 1999; 138: S419-S420). Clinical observations indicate that anti-inflammatory therapy reduces the manifestations of atherosclerosis (Stoller DK et al, J Surg Res 1993; 54: 7-11). Experimental data for at least COX-1 inhibitors confirm a significant correlation between anti-inflammatory activity and hypolipidemic activity (Kourounakis AP et al., Exper Mol Pathol 2002; 73: 135138). We compared the hypolipidemic activity of ASS and MDAS at equivalent doses.

6.1. Comparative activity of test substances on lipid levels in a rat model of acute hyperlipidemia

Methods. Wistar male males weighing 250-270 g were used. Animals were housed in groups of 6-8 in appropriate cages at 22 ± 1 ° C, 60 ± 5% relative humidity, and a 12/12 hour light / dark cycle with free access to food and water. Acute experimental hyperlipidemia / hypercholesterolemia was induced by Triton WR1339 as described in Kourounakis AP et al, Exper Mol Pathol 2002; 73: 135-138). Rats were given i.p. administered Triton WR1339 (TR), dissolved in isotonic saline at a dose of 250 mg / kg. The test substance solution or water was administered to TR and control animals p.o. one hour before and 20 hours after administration of TR as described below.

Blood for biochemical analysis was collected the next day (24 hours after TR injection) by cardiac puncture under ether narcosis. Serum was removed by centrifugation and total H, HDL, LDL and TG levels were determined with commercial assay kits.

Three series of experiments were performed.

Statistics. Data were analyzed with Microsoft Excel software and expressed as mean +/- Mean ± SEM. The mean scores of the different groups were compared using one-way analysis by ANOVA and t-Student tests. P <0.05 was considered significant.

Series I - Comparison of ASS, MD and MDAS

GROUP PROCEDURE Number of animals

Controls 6

TR TR 250 mg / kg 8

ASS45 TR 250 mg / kg + ASS 45 mg / mg 6

ASS90 TR 250 mg / kg + ASS 90 mg / mg 8

MD150 TR 250 mg / kg + MD 150 mg / kg 8

MDAS75 TR 250 mg / kg + MDAS 75 mg / kg 6

MDAS 150 TR 250 mg / kg + MDAS 150 mg / kg 8

MDAS300 TR 250 mg / kg + MDAS 300 mg / kg 8

Results. Rats receiving TR exhibited marked hypercholesterolemia and hyperlipidemia with levels of total cholesterol, LDL, and TG significantly different from the control group (6-7 fold increase in total H, 30 fold greater in TG, see Table 17). ASA therapy, particularly at 90 mg / kg, limited total H, LDL and TG but did not significantly alter HDL levels. MD in our experiment did not significantly protect against TR-induced changes in lipid levels. Treatment with MDAS provided dose-dependent protection against TR-induced hyperlipidemia / hypercholesterolemia. MDAS at 75 mg / kg is no different from ASS45, but MDAS150 is significantly more effective than ASS45 and MD150 at protecting against TR effects. The MDAS300 significantly lowered total H, LDL and TG levels than the ASS45 and ASS90. This indicates that MDAS may be useful in the prevention and / or treatment of hypercholesterolemic and hyperlipidemic states and, in view of its anti-inflammatory activity, may be useful in the prevention and / or treatment of atherosclerosis and other medical conditions mediated by disorders of lipid metabolism and inflammation.

Table 17

Comparative effects of MD, ASA and MDAS on lipid levels in a rat hyperlipidemia model; n = 6-8; Mean ± SEM

Group Hmg / dl HDL mg / dL LDL mg / dL TG mg / dl Control 80.7 ± 4.7 *** 56.5 ± 2.8 21.1 ± 2.6 *** 44.0 ± 6.9 *** TR 453.6 ± 40.0 60.3 ± 9.6 386.0 ± 36.5 1399 ± 129.7 ASS45 381.0 ± 30.3 62.8 ± 10.9 307.4 ± 37.8 973 ± 82.7 * ASS90 288.1 ± 23.5 * 60.0 ± 7.4 219.1 ± 23.9 * 791 ± 73.9 ** MD150 345.6 ± 34.1 63.8 ± 9.3 273.9 ± 31.6 * 1022 ± 80.7 * MDAS75 341.9 ± 16.3 * 68.1 ± 8.7 270.1 ± 12.8 * 861 ± 105.7 * MDAS 150 249.6 ± 22.2 ** ' ^a 58.2 ± 8.1 182.4 ± 19.6 ** ¹⁴ · "668I004A ** ⁵ ® MDAS300 219.0 ± 16.7 ** ^Wi 56.3 ± 8.6 158.6 ± 19.4 ** ^lsi 548 ± 73.2 *** ^{& w}

* P <0.05 versus TR ** P <0.005 versus TR *** P <0.0005 versus TR ^s P <0.05 versus ASS45 * P <0.05 versus ASS90 ^& P <0.05 versus MD150

HT Series - ASS. MDAS. Comparison of NS and ASS + NS, MDAS + NS

GROUP PROCEDURE Number of animals Control 6th TR TR 250 mg / kg 8th ASS45 TR 250 mg / kg + ASS 45 mg / mg 6th MDAS TR 250 mg / kg + MDAS 150 mg / kg 6th

NS TR 250 mg / kg + NS 50 mg / kg 7

ASS + NS TR 250 mg / kg + ASS 45 mg / mg + NS 50 mg / kg 7

MDAS + NS TR 250 mg / kg + MDAS 150 mg / mg + NS 50 mg / kg 7

Results. In our experiment, NS provided significant protection against changes in lipid (H, LDL and TG) levels induced by TR (see Table 18). The combination of ASA with NS did not significantly alter the effect of NS on lipid levels. Surprisingly, MDAS and NS significantly potentiated the NA50 effect and exceeded the protective effect of MDAS on TG elevation induced by TR (Table 18). Combined use of MDAS and NS was also significantly more effective than ASS45 + NS50 in normalizing LDL and TG levels.

Table 18

Effects of MD, ASS, and MDAS, alone and in combination, on lipid levels in a rat hyperlipidemia model; n = 6-8; Mean + SEM_

Group H mg / dl HDL mg / dL LDL mg / dL TG mg / dl Control 80.7 + 4.7 *** 56.5 + 2.8 21.1 + 2.6 *** 44 + 6.9 *** TR 453.6 + 40.0 60.3 + 9.6 386.0 + 36.5 1399 + 129.7 ASS45 381.0 + 30.3 62.8 + 10.9 307.4 + 37.8 973 + 82.7 * MDAS 150 249.6 + $ 22.2 ** $ 58.2 + 8.1 182.4 + $ 19.6 ** 668 + 70.9 ** $ NS50 327.5 + 38.4 * 66.5 + 14.6 246.3 + 32.5 * 591 + 43.3 ** ASS45 + NS50 316.0 + 43.1 * 57.9 + 14.3 251.2 + 33.8 * 618 + 42.8 ** MDAS150 + NS50 226.3 + 24.9 *** $ 63.1 + 10.2 163.2 + 19.3 ** ^m 468 + 34.7 * * $ ^#% ®

* P <0.05 versus TR ** P <0.005 versus TR *** P <0.0005 versus TR $ P <0.05 versusASS45 ®P <0.05 versus MDAS 150 P <0.05 versus NS50 ^% P < 0.05 vs. ASS45 + NS50

Series III - ST MD, MDAS and SI + MD combination. Comparison of SI + MDAS

GROUP PROCEDURE Number of animals

Controls 6

TR TR 250 mg / kg 8

Sl TR 250 mg / kg + SI 5 mg / kg 7

MD TR 250 mg / kg + MD 150 mg / kg 6

MDAS TR 250 mg / kg + MDAS 150 mg / kg 6

SI + MD TR 250 mg / kg + SI 5 mg / kg + MD 150 mg / kg 7

SI + MDAS TR 250 mg / kg + SI 5 mg / kg + MDAS 150 mg / kg 7

Results, In our experiment, Sl at 5 mg / kg provided significant protection against changes in lipid (H, LDL and TG) levels induced by TR (see Table 19). The combination of SL with test substances increased the normalizing effect on lipid levels. MDAS in combination with Sl was significantly more effective than MD + SI in preventing TR-induced increases in LDL and TG (Table 19). The MD combination with strut is described in WO2006099244 without specific data. Combination of statins with ASA requires a special pharmaceutical composition as these substances are pharmacologically and chemically incompatible (US Pat. No. 6,235,311), so synergy is not possible.

Table 19

MD. Effects of MDAS and SI, individually and in combination, on lipid levels in a rat hyperlipidemia model, n = 6-8, Mean ± SEM

Group H mg / dl HDL mg / dL LDL mg / dL TG mg / dl Control 80.7 ± 4.7 *** 56.5 ± 2.8 21.1 + 2.6 *** 44 + 6.9 *** TR 453.6 ± 40.0 60.3 ± 9.6 386.0 + 36.5 1399 + 129.7 THIS 321.3 ± 32.3 * 53.7 ± 12.4 259.3 + 30.1 * 826 + 44.1 ** MD 345.6 ± 34.1 63.8 + 9.3 273.9 + 31.6 * 1022 + 80.7 * MDAS 249.6 ± 22.2 ** ^& 58.2 + 8.1 182.4 + 19.6 *** 668 + 70.9 *** SI + MD 281.5 ± 31.2 ** ^& 59.5 + 14.9 217.1 ± 26.2 * 690 + 32.6 ** ^{& l} SI + MDAS 230.7 ± 28.6 ** ^{& i} 62.8 + 14.6 153.4 + 28.0 *** ^{1 #} 512 + 40.2 **

* P <0.05 versus TR * * P <0.005 versus TR *** P <0.0005 versus TR ^& P <0.05 versus MD ^s P <0.05 versus SI #P <0.05 versus MD + THIS

Summary. These results indicate the potential of MDAS for the prevention and / or treatment of hypercholesterolemia and hyperlipidemia. Given the anti-inflammatory activity of MDAS, it may be more effective than AS or MD in preventing and / or treating atherosclerosis and other conditions contributing to inflammation. Combined use of MDAS and NS enhances the positive effects of the individual substances on experimentally elevated lipid levels more than ASS + NS. Not only was MDAS in combination with SI more effective than SI alone, it was also significantly more effective than MD + SI in preventing TRL elevated LDL and TG.

6.2. NS and MDAS. alone and in combination, effects on lipid levels in a rat model of chronic hyperlipidemia

Methods. Wistar male rats were used. Animals were housed in air-conditioned rooms at 22 ± 1 ° C, 60 ± 5% relative humidity, and a 12/12 hour light / dark cycle with free access to water and food. The initial weight of the animals was 220-240 g. Experimental chronic (subchronic) hyperlipidemia / hypercholesterolemia was induced by TR using the method described by Levine and Saltzman (Levine S. Saltzman A.J. Pharmacol ToxicolMeth 2007; 55: 224-226). The animals received 250 mg / kg TR solution in the tail vein three times a week for 3 weeks. Test substance solutions in experimental groups or water were administered p.o. to the Control and TR groups. once daily for one hour prior to injection of TR solution or blood sampling according to the following scheme:

GROUP PROCEDURE Number of animals

Control 10th TR TR 250 mg / kg 14th TR + NS TR 250 mg / kg + NS 50 mg / kg / d 14th TR + MDAS TR 250 mg / kg + MDAS 150 mg / kg / d 14th TR + NS + MDAS TR 250 mg / kg + NS 50 mg / kg + MDAS 150 mg / kg / d 14th

Blood for biochemical analyzes was taken at 1, 2 and 3 weeks (the day after TR injection) with cardiac puncture under ether narcosis. Serum was removed by centrifugation and total H, HDL, LDL and TG levels were determined with commercial assay kits.

Statistics. Data were analyzed with Microsoft Excel software and expressed as mean +/- Mean ± SEM. The mean scores of the different groups were compared using one-way analysis by ANOVA and t-Student tests. P <0.05 was considered significant.

Results. Repeated TR injections resulted in pronounced and stable hypercholesterolemia and hyperlipidemia characterized by significant increases in total H, LDL and TG compared to Control (total H increased 6-7 fold, TG 30 fold and greater, see Table 20). NS treatment, more significantly in the first week, limited the increase in total H, LDL and TG but significantly increased HDL levels at weeks 2 and 3 only. MDAS almost completely reduced NS and HL and elevated HDL levels, but less than NS prevented TR-induced TG increase (see Table 20).

Unexpectedly, the combined administration of NS + MDAS after 3 weeks significantly lowered total H, LDL and TG levels and increased HDL levels than NS or MDAS alone. In this way, the combination NS + MDAS can be expected to be useful in the prevention and / or treatment of hypercholesterolemia and hyperlipidemia.

Table 20

Effects of NS and MDAS, alone and in combination, on lipid levels in a rat model of chronic hyperlipidemia; n = 9-14; Mean + SEM

Group Total H pec for weeks 1, 2 and 3, mg / dl Cl C2 C3 Control 77.6 + 4.9 * 75.1 + 5.1 * 72.7 + 2.5 * TR 487.6 + 25.4 501 + 16.7 513 + 41.1 TR + NS 345 + 15.7 * 401.1 + 25.1 ** 405.5 + 25.9 * TR + MDAS 375.9 + 25.8 * 406.7 + 19.8 ** 409 + 39.4 TR + NS + MDAS 331.7 + 28.4 ** 379.1 + 24.7 ** 375.8 + 31 *

Table 20 (cont'd)

Group HDL after 1.2 and 3 dances, mg / dl ABL1 ABL2 ABL3 Control 54.6 + 1.9 * 54.1 + 1.3 53.7 + 1.0 * TR 76.3 + 6.9 76.2 + 11.4 77 + 10.2 TR + NS 111.3 + 9.1 * 144.7 + 13.5 * 127.3 + 10.9 * TR + MDAS 100.1 + 9.4 113.8 + 13.1 128.3 + 18.5 * TR + NS + MDAS 112.3 + 10.9 * 129.2 + 13.1 * 154.1 + 19 *

Table 20 (cont'd)

Group LDL at 1, 2 and 3 weeks, mg / dl ZBL1 ZBL2 ZBL3 Control 18.7 + 3.8 * 19.7 + 4.4 * 16.3 + 2.0 * TR 388.7 + 26.7 402.1 + 19.2 405.1 + 41.7 TR + NS 216.3 + 14.0 250.3 + 20.6 * 265.1 + 18.4 * TR + MDAS 263.3 + 19.4 287.6 + 18.5 * 261.9 + 12.6 ** TR + NS + MDAS 205.7 + 18.5 ** 222.4 + 16.5 ** 214.4 + 15.1 "*

Group TG at 1, 2, and 3 weeks, m g / dl TG1 TG2 TG3 Control 38 + 2.9 * 37 + 3.2 * 38 + 4.4 * TR 1240 + 80.1 1297 + 78.3 1234 + 114.1 TR + NS 734 + 81.6 * 860 + 73.8 ** 828 + 44.7 * TR + MDAS 964 + 94.9 * 1079 + 84 982 + 72.7 TR + NS + MDAS 721.6 + 52.4 ** 807.6 + 80.1 ** 714 + 27.3 ***

* P <0.05 versus TR

Table 20 (cont.) ** P <0.005 vs. TR * <0.0005 vs. TR ^s P <0.05 vs. NS ^& P <0.05 vs. MDAS

Summary

Combined use of MDAS and NS is significantly more effective than separate administration of these agents.

Example 7. Effect of test substances on platelet adhesion and thrombus formation

7.1, Platelet adhesion

ASA is one of the most widely used prophylactic antiplatelet agents (Miner J et al, Tex Heart Inst J 2007; 34 (2): 179-186). ASA is combined with NS as an anti-inflammatory (US 3,312,593) and antiplatelet agent (WO 9632942). Many other agents and combinations thereof are known. MD has been found to normalize vascular tone, inhibit platelet adhesion and fatty acid oxidation, and optimize oxygen consumption in myocardial ischemia (Tsirkin VI, Ros Kardiol Zh 2002; 1: 45-52). NS also slightly inhibits platelet aggregation (Lakin KM, Farmakol Toksikol, 1980.43; 43 (5): 581-5 (Rus). The typical anti-caking agent clopidogrel is used alone (US 4,529,596, US 4,847,265, US 5,576328) or in combination) with ray (WO9804259) or ASA (WO9729753) Dipyridamole, an anti-caking agent, can also be combined with ASA (Halkes PH et al, Lancet 2006,367 (9523): 1665-73) Clinical experience has shown that combinations of different agents have a more versatile use .

The method. Platelet adherence was studied using full-blood impedance aggregometry with Multiplate (Multiple Platelet Function Analyzer, Dynabyte Medical, Germany) (Toth O et al, Thromb Haemost 2006; 96: 781-788. Velik-Salchner C et al, Anesth Analg 2008; 107 : 1798-1806). Blood samples for the in vitro experiment were taken from healthy donor B. (37 years), who had not used ASA or other anti-caking agents, in hirudin-coated plastic tubes (Dynabyte Medical, Germany) and used for measurement within 30 min. up to 4 hours after collection. For the ex vivo experiment, blood was collected in plastic hirudin-coated tubes (Dynabyte Medical, Germany) from anesthetized rats that had received the test substance for the previous 3 days. Measurements were made according to a modified Dynabyte Medical protocol. Isotonic sodium chloride solution (0.3 ml, or saline with test substance at a final concentration of 10 ⁴ mmol / ml each) was warmed to 37 ° C and pipetted into measuring cells with 0.3 ml of whole blood sample supplemented with hirudin as an anticoagulant. . After 5 minutes of incubation and mixing at 37 ° C, a solution of the appropriate agonist (from Dynabyte Medical, Germany) was added and measured as follows:

1) adenosine diphosphate (ADF) - ADF test. ADF stimulates platelet activation by ADF receptors (Ρ2Υ12, etc.).

2) arachidonic acid (AS) - ASPI test: activation with AS - a substance that produces cyclooxygenase to thromboxane A2 (ΤΧΑ2), a potent platelet agonist.

3) ADF HS test (prostaglandin Ej with ADF). Addition of an endogenous inhibitor such as PGEj makes the ADF HS assay more sensitive to the effects of clopidogrel and related substances compared to the ADF assay.

Aggregation curves were recorded for 6 minutes and analyzed with Dynabyte Medical software. The following platelet adhesion parameters were calculated:

1) Amax, maximum value of platelet adhesion in units of relative adhesion (AU);

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

Statistics. Results were expressed as mean and mean standard error (Mean ± SEM). A one-way ANOVA test was used to assess the reliability of the differences. If the null hypothesis was rejected, a post-hoc Student-Newman-Keuls test was used.

Results. As shown in Table 21, MDAS concentration ΚΓ ⁴ mol significant protection against the ADF, and especially against platelet aggregation and ADP + AS PGEi affect (AUC and a significant decrease in Amax, Table 21). NS (10 ⁴ mmol / ml) also reduced ADF-induced adhesion (see Amax, Table 21). Combined action of both agents resulted in significantly higher and more pronounced reduction in platelet adhesion in the ADF or ADF + PGEi assay, which appears in both AUC and Amax data (Table 21).

Table 21

Effect of MD, NS and their combination on ADF, AS and ADF + PGE, induced platelet adhesion; N = 5-8; Mean ± SEM.

Group ADF AUC (AU * min) Amax (AU) Control 942 + 43.7 169.3 + 6.4 MDAS 10 ' ⁵ 828 + 63.5 153.5 + 8.8 MDAS 10 798 + 38.9 ' 140.5 + 7.2 ' MD 10 869 ± 36.3 153.2 + 6.1 ASS ΙΟ ⁴ 883 + 50.3 151.7 + 9.3 NS 10- ⁴ 859 + 62.5 148.0 + 5.2 ' MD 10 + NS 10 474+ 34.9 ^{34 # SS3} 81.0 ± 5.7 ^444IIa MDAS 10 + NS IC ⁴ 403+ 75.3 + 6.8 ² ^ ^3552244 ASS + NS 805 + 47.3 ^lb 137 + 7, l ' ^b

Table 21 (cont'd)

Group AS AUC (AU * min) Amax (AU) Control 1023 + 46.3 178.8 + 6.9 MDAS 10 ' ⁵ 832 + 54.1 ¹ 148.4 + 5.7 ' MDAS 10 298 + 25.3 ^34AS 66.1 ± 6.8 ^{34 & I} MD 10 1050 + 37.6 ^& 179.5 + 7.3 * ASS 10 450 + 24.8 ^{2i |} ® 103, l ± 5.9- ^e + NS 10 1010 + 41.7 161.1 + 9.4 MD 10 + NS 10 1106 + 55.4 ² 173.3 + 12.1 ' MDAS 10 + NS 10 216i 18 48 5 ± 6 ASS + NS 463 + 35.2 ^2bS4 99.4 + 8.2 ²⁶⁵⁴

Table 21 (cont'd)

Group ADF + PGE, AUC (AU * min) Amax (AU) Control 1005 + 46.5 175.3 + 8.9 MDAS 10 595 + 45.3 ' 99.3 + 4.8 ² MDAS 10 533 + 20.3 ³ 87.7 + 4.6 ³ MD 10- ⁴ 587 + 37.4 ² 101.4 + 2.2 ² ASS ΙΟ ^-4 - - NS 10- ⁴ 862 + 51.9 146.7 + 8.6 MD 10 + NS 10 306 + 35.5 ^34Sa 54.5 + 5.8 ³⁴⁵³ MDAS 10 + NS 10 50.2 + 7.3 ASS + NS 603 + 42.5 ^1Jb 98.0 + 7.2 ²¹⁶

'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 Control * P <0.005 versus MD 10 ** P <0 , 0005 versus MD 10 ^s P <0.005 versus NS 10 ^JI P <0.0005 versus NS 10 ®P <0.05 versus MDAS 10 ′ P <0.05 versus ASS + NA Τ <0.05 versus MD + NS

MDAS was significantly better than ASA or MD in protecting platelets from AS-induced adhesion (Table 21). The combination of MDAS + NS showed significantly higher anti-platelet adhesion activity in AS, exceeding the activity of individual substances, as well as that of ASS + NS and MD + NS (Table 21).

Parallel experiments were conducted with dipyridamole (DI) and the combination of DI with ASA or MDAS on ADF or AS platelet adhesion (Table 22). DI exhibits anti-thrombotic and anti-adherent activity (Mammen EF, Thrombosis Research Supplement 1990 XII, 1-3). Dipyridamole with aspirin is more active than aspirin after arterial cerebral ischemia (Halkes PH et al. Lancet 2006, 367 (9523): 1665-73).

Table 22

Effects of test substances, individually and in combination, on ADF or AS-induced platelet adhesion. Mean ± SEM; N = 5-8.

Group ADF AS AUC (AU * min) Amax (AU) AUC (AU * min) Amax (AU) Control 942 + 43.7 169.3 + 6.4 1023 + 46.3 178.8 + 6.9 MDAS KD 798 + 38.9 ' 140.5 + 7.2 ' 298 + 25.3 ^{3aa &} 66, l ± 6.8 ^{Share &} ASS lCfi 883 + 50.3 151.7 + 9.3 450 + 24.8 ³ '® 103, l ± 5.9 ^Za ® DI 3Χ10- ⁴ 665 + 44.1 " 111.1 + 6.9 " 1104 + 45.5 * ®'® 173.9 + 5, MDAS IOADI 3x1- ⁴ 465 + 27.θ " ^{! Ϊ́} " 69 7 + 3 2 ^J ® ^S ” 38.5 ± 3.6 ⁴ ®® ' ^ilaaS ASS IOADI 3Χ10- ⁴ 667 + 39.4 " 105.2x6.7 "'- 207.9 + 27.5 "" 62.8 + 6.8 "

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 ASS ®P <0.05 versus MDAS @@ P <0.005 versus MDAS ^a P <0.05 versus DI “P <0.005 versus DI” +><0.0005 versus DI ^s P <0.05 versus ASS + DI ^si P <0.005 versus ASS + DI

The highest activity in this series was MDAS + DI, which was significantly higher than that of ASS + DI (Table 22).

7.2. Thrombosis

Two weeks of oral treatment in experimental arterial thrombosis therapy in rabbits and dogs showed MD thrombolytic effects (Logunova L et al., Experim Clin Pharmacoter 1991; 19: 91-98 (Rus). No data are available on the prophylactic effect of MD on thrombosis control. NS reduces thrombosis by various mechanisms (Rosenson RS et al., Atherosclerosis 1998; 140: 271-80).

The method. We chose an experimental thrombosis model based on FeCl2-induced rat arterial thrombosis (Kurz K et al, Thromb Res 1990, 60: 269-280. Wang X, Xu L, Thromb Res 2005, 115: 95-100). Oxidative damage to tissues with iron promotes adhesion of platelets to the damaged surface and adhesion, followed by activation of coagulation and fibrin deposition. Wistar male males weighing 350-420 g were used in the experiments. The animals were housed in groups of 7 to 8 in appropriate cages at 22 ± 1 ° C, 60 ± 5% relative humidity, 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 use of animals for scientific purposes. Every effort was made to minimize animal suffering and reduce the number of animals used. Rats were randomized to several study groups with at least 7 animals each. Carrier or test substance MD (25 mg / kg), NS (25 mg / kg),

MDAS (10 mg / kg), ASS (5 mg / kg) and combinations MD + NS (25 + 25 mg / kg), MDAS + NS (10 + 25 mg / kg) and ASS + NA (5 + 25 mg / kg) kg) were administered orally 2 hours before thrombosis was induced. Parallel experiments were conducted to compare the effects of a single dose of the test substance (administered 2 hours prior to thrombosis) and repeated doses (once daily for 3 days). Animal groups (7-8) received the following substances: MDAS (10 mg / kg), clopidogrel (CL) (5 mg / kg), ASS (5 mg / kg) and combination MDAS + CL (10 + 5 mg / kg). or ASS + CL (5 + 5 mg / kg). Rats were anesthetized with pentobarbital sodium (50 mg / kg, i.p. and 10 mg / kg / h) and placed on a heated operating table during the experiment to maintain a body temperature of 37 ° C. One of the carotid arteries was detected by a neck incision, separated from the surrounding tissue, the vagus nerve, and a flow probe (electromagnetic blood flow meter MFV 1200, Nicon Kohden, Japan) applied to the open total carotid artery. After 15 min. after a long period of stabilization, two pieces of Whatman filter paper (2x1 mm) soaked in 15% FeCf solution were induced by thrombosis upon application to the artery in contact with the vascular surrounding tissue. The time of carotid thrombosis was recorded as the time at which the blood flow stopped completely and is shown as the time to occlusion (LLO). If in the active treatment group 90 min. blood flow did not stop, LLO was recorded as> 90 min.

The duration of the rat tail bleeding was further measured during the thrombosis experiment. The tail was cut with a scalpel 5 mm from the tip and immediately immersed in 37 ° C warm isotonic saline until the bleeding stopped. Bleeding stop was defined as the time to complete bleeding, when the bleeding did not recur within the next 30 s.

Following the thrombosis experiment, narcotic animals receiving test substances for 3 days were used for the platelet adhesion test ex vivo. An abdominal cavity was opened and blood from a venous cava inferior was collected in plastic hirudin-coated tubes (Dynabyte Medical, Germany).

Blood samples were used for measurements within 30 min. up to 4 hours after collection. Measurements were made according to the modified Dynabyte Medical protocol (see Platelet Adherence above).

Statistics. The results were analyzed with Microsoft Excel 2007 software. Data are shown as Mean ± Mean Standard Error (Mean ± SEM). Differences between experimental groups were compared using one-way ANOVA with repeated comparison (Tukeja test). P <0.05 was considered significant.

Results. The mean time to FeCf-induced vascular thrombosis and the time to stop induced arterial flow in the control group was 24.4 min. (Table 23).

Table 23

Effect of investigated substances on FeCl ₃ induced carotid thrombosis. Mean ± SEM; N = 7-8.

Group Time to Occlusion (LLO) Duration of tail bleeding min % min % Control 24.4 + 1.45 100 8.9 + 1.28 100 NS (25 mg / kg) 30.3 + 3.12 124 11.5 + 1.39 129 MD (25 mg / kg) 29.8 + 2.29 122 10.5 + 1.01 118 MD + NS (25 + 25 mg / kg) 34.0 + 2.78 ' 139 11.4 + 1.42 128, MDAS (10 mg / kg) 41.7 ± 3.95 ² * 171 12.1 + 2.20 136 MDAS + NS (10 + 25 mg / kg) 53, l ± 4.12 ^{2iabc #} 218 13.5 + 4.19 152 ASS (5 mg / kg) 35.2 + 3.02 ' 144 13.8 + 3.27 155 ASS + NS (5 + 25 mg / kg) 42.5 ± 4.24 ^{2S #} 174 15.2 + 2.12 ' 171

'P <0.05 versus Control ² P <0.005 versus Control #P <0.05 versus MD ^s P <0.05 versus NS ³ P <0.05 versus ASS ^b P <0.05 versus ASS + NS ^c P < 0.05 vs. MD + NS

Prophylactic administration of MDAS yielded a significantly longer LLO prolongation (P <0.005 vs. Control) but, unlike ASA, had less effect on the tail bleeding assay (136 vs. 155%). MDAS + NS (10 + 25 mg / kg) resulted in comparatively longer thrombosis inhibition than MD + NS or ASS + NS (Table 23). It should be noted that with the use of ASS and ASS + NS, the increase in LLO goes hand in hand with the increase in tail bleeding time, whereas for MDAS or MDAS + NS, the increase in LLO is significantly greater than the increase in tail bleeding time (Table 23).

The effect of ASS, MDAS, and CL, and combinations thereof, on thrombosis was studied in parallel experiments with a joint control. Test substances were administered as a single dose (2 hours before testing) or once daily for 3 days. ASA or CL, administered 2 hours prior to the thrombosis test, significantly prolonged LLO compared to the control group receiving water (Table 24).

Table 24

Effect of a single dose of test substance on LLO and tail bleeding duration in a FeCl-induced carotid artery thrombosis experiment. Mean ± SEM; N = 7-8.

Group Time to Occlusion (LLO) Duration of tail bleeding min % min % Control 24.4 + 1.45 100 8.9 + 1.28 100 MDAS (10 mg / kg) 41.7 ± 3.95 ^2b 171 12.1 + 2.20 136 ASS (5 mg / kg) 35.2 + 3.02 ' 144 13.8 + 3.27 155 CL (5 mg / kg) 31.7 + 2.40 ' 130 11.9 + 3.62 134 MDAS + CL (10 + 5 mg / kg) 61.5 + 4.3 L ^3Sabc 252 15.7 + 3.16 176 ASS + CL (5 + 5 mg / kg) 45.4 ± 4.80 ^2ab 186 16.3 ± 2.25 ' 183

P <0.05 vs Control ² P <0.005 versus control ³ P <0.0005 vs Control ^of P <0.05 versus MDAS ^a P <0.05 versus ASA ^b P <0.05 versus CL ^c P <0, 05 against ASS + CL

One dose of MDAS provided a more significant LLO extension than CL. MDAS in combination with CL significantly increased LLO but only slightly altered tail bleeding duration (Table 24). It should be noted that the combination of MDAS + CL resulted in a significant increase in LLO compared to the individual substances and the combination of ASS + CL.

Repeated administration of test substances resulted in a further prolongation of LLO and tail bleeding (Table 25).

Table 25

Effect of repeated administration of test substance on LLO and tail bleeding time in FeCl-induced carotid artery thrombosis experiment. Mean ± SEM; Ν = 7-8.

Group Time to Occlusion (LLO) Duration of tail bleeding min % min % Control 25.3 + 1.75 100 8.6 + 1.37 100 MDAS (10 mg / kg) 48.4 + 4.35 ² 191 13.1 + 2.63 152 ASS (5 mg / kg) 40.9 + 3.25 ' 162 17.8 + 3.17 ' 207 CL (5 mg / kg) 49.1 + 7.75 ' 194 18.3 + 3.78 ' 213 MDAS + CL (10 + 5 mg / kg) 64.6 + 8.01 " 255 20.7 + 3.88 ' 241 ASS + CL (5 + 5 mg / kg) 61.0 + 10.32 ²³ 241 27.4 + 4.98 ²¹³ 319

'P <0.05 versus Control ² P <0.005 versus Control * P <0.05 versus MDAS ^a P <0.05 versus ASS

Repeated administration of MDAS or CL resulted in significantly greater effects on LLO than ASA (191 and 194 vs. 162%, respectively), but MDAS, in contrast to CL or ASA, did not prolong tail bleeding (Table 25). Repeated administration of MDAS + CL or ASS + CL resulted in a significant and relatively similar increase in LLO, but MDAS + CL had a relatively smaller effect on the duration of tail bleeding compared to ASS + CL (241% versus 319%) (Table 25).

Experiment ex vivo

Following the thrombosis experiment, animal blood was collected and aggregation parameters determined. Three-day administration of MDAS at a dose of 10 mg / kg resulted in a significant reduction in platelet adhesion for all tested adhesion promoters (Table 26).

Table 26

Effect of MDAS, ASS, CL and combinations thereof on ex vivo platelet adhesion ', N = 5-7; Mean ± SEM.

Group ADF AUC (AU * min) Amax (AU) Control 794 + 33.5 149.7 + 9.4 MDAS (10 mg / kg / d) 528 + 46,5 ' 75.5 + 12.8 ² ASS (5mg / kg / d) 768 + 53.2 150.3 + 13.5 CL (5mg / kg / d) 423 + 39.4 " 68.2 + 15.1 " MDAS + CL (10 + 5 mg / kg / d) 140 + 16.8 ³ ® ^{& ab} 35.3 + 9.4 ASS + CL (5 + 5 mg / kg / d) 230 + 29.8 " 53.3 + 11.5 "

Table 26 (cont'd)

Group AS AUC (AU * inin) Amax (AU), Control 935 + 62.3 167.8 + 11.6 MDAS (10 mg / kg / d) 232 + 44, l " 48.2 + 9.7 " ASS (5mg / kg / d) 550 + 31.3 ² 86.1 + 6.8 ² CL (5mg / kg / d) 905 + 53.5 ^& 160.7 + 13.5 MDAS + CL (10 + 5 mg / kg / d) 187 + 29.4 "* 30.7 ± 9.8 ^{3 &} ASS + CL (5 + 5 mg / kg / d) 239 + 32.7 " 39.8 + 14.1 "

Table 26 (cont'd)

Group ADF + PGE, AUC (AU * min) Amax (AU) Control 855 + 40.5 159.5 + 12.2 MDAS (10 mg / kg / d) 195 + 35.3 ³ 41.4 + 14.8 ² ASS (5 mg / kg / d) - - CL (5 mg / kg / d) 259 + 49.3 ² 49.7 + 9.9 ³ MDAS + CL (10 + 5 mg / kg / d, 87 + 15.8 ³ ® ^ 15.5 ± 4.8 ³ ® ^ ASS + CL (5 + 5 mg / kg / d) 128 + 19.4 ² ® 24.5 ± 4.8 ³ ® ^b

'P <0.05 vs. Control ² P <0.005 vs. Control ³ P <0.0005 vs. Control®P <0.05 vs. MDAS ^& P <0.05 vs. ASS P <0.05 vs. CL ^b P <0, 05 against ASS + CL

Administration of CL at a dose of 5 mg / kg / d for 3 days resulted in significant anti-adhesion induced by ADF and ADF + PGEj but did not prevent AS-induced adhesion. ASA provided significant protection against AS-induced adhesion but was not effective against ADF (Table 26). The combination of MASA and CL (10 + 5 mg / kg / d x3) afforded comparatively superior protection against various substances induced adhesion, which was significantly better than that of ASS + CL in the ADF and ADF + PGE tests (Table 26).

SUMMARY

MDAS significantly better protects against AS-induced platelet adhesion than MD or ASA. MDAS + NS outperforms MD, NS and ASS, as well as the combination of ASS + NS against all adhesion initiators, and MD + NS against AS-induced adhesion.

Given the beneficial effects of MDAS and MDAS + NS on platelet adhesion and LLO prolongation in vivo, the combination of MDAS or MDAS + NS may find application in reducing the risk of platelet adhesion and thrombosis in patients with severe atherosclerosis, possible myocardial infarction and stroke, and peripheral blood flow. The fact that the combination of MDAS and MDAS + NS does not prolong the duration of tail bleeding suggests the potential use of this combination in patients at increased risk of pre- and postoperative bleeding.

MDAS + DI protects against ADF- and AS-induced platelet adhesion significantly better than ASS + DI.

In the thrombosis experiment, a single dose of MDAS + CL provides better protection against FeCl 2 induced thrombosis than ASS + CL. MDAS + CL prolongs the duration of tail bleeding comparatively less than ASS + CL. In an ex vivo experiment, MDAS + CL provides significantly better protection against platelet adhesion than CL, ASS, or MDAS. MDAS + CL provides better protection against ADF and ADF + PGEj-induced platelet adhesion than ASS + CL.

These facts suggest that MDAS + CL may find application in the clinic for immediate protection against an increased risk of platelet adhesion, imminent or onset thrombosis.

Example 8. Comparative Study of MDAS / NS, MD / NS and LA / NS Combined Applications for Reduction of NS-induced Redness

Nicotinic acid (niacin, NS) is effective in lowering serum cholesterol, LDL and triglycerides, increasing HDL levels. However, in patients receiving the immediate or sustained release form of nicotinic acid, there is a limiting side effect: rapid, significant warming of the skin and vasodilation, which is termed flush, which in severe cases leads to discontinuation (Gupta EK, Ito MK , Heart Dis 2002; 4: 124-137). Laropiprant (MK0524) (LA) has been proposed as one of the most active and promising means to reduce NS-induced redness (Cheng K et al, PNAS 2006; 103: 66826687). '

The aim of our study was to compare experimentally the effects of MDAS, MD and LA on NS-induced redness (changes in skin temperature).

Methods. Wistar male males weighing 280-330 g were used. The animals were housed in groups of 6 in an air-conditioned room at 22 + 1 ° C, 60 + 5% relative humidity and a 12/12 hour light / dark cycle with free access to water and food (Lactamin AB, Sweden). A contactless temperature measurement method was used to measure skin temperature changes in intact rats (Papaliodis D et al, Br J Pharmacol 2008; 153: 13821387). Temperature measurements were made with a manual infrared thermometer (Modei Proscan 510, TFA-Dostman). Animals were habituated to hand-picking and infrared probe 3 days prior to use. Temperature readings from the dorsal side of both ears were obtained without anesthesia at the beginning and during the experiment. Ear temperature was measured every 5 minutes for up to 60 minutes. over a long period. Between measurements, animals were returned to cages. NS, MD and MDAS were dissolved in saline and pH was adjusted just prior to administration. LA (MK 0524, Cayman Chemicals) was first dissolved in DMSO and then diluted with 0.9% NaCl daily before the experiment. The NS to LA ratio in combination is based on the Tredaptive ™ (nicotinic acid / laropiprant) 1000 mg / 20 mg controlled-release tablet.

Statistics. For each time point, the average of the six temperature measurements (for each ear) was recorded. Data were analyzed with Microsoft Excel software and expressed as mean +/- Mean ± SEM. The mean scores of the different groups were compared using one-way ANOVA and t-Student test. P <0.05 was considered significant.

8.1. Evaluation of the effect of time and solvent on skin temperature

GROUP PROCEDURE Number of animals SolvLA LA solvent 6th SolvNS NS, MDAS and MD solvent 6th NS NS 15 mg / kg s.c. 6th

8.2. Effects of individual test substances on skin temperature at simultaneous Γ01 or 30 min, pre-emptive [301 s.c. administration

The effect of separate administration of LA, MD or MDAS on skin temperature was examined. Each test substance was administered s.c. with NS as LA + NS [0] or 30 min. before NS as LA + NS [30],

GROUP PROCEDURE Number of animals Control / Solvent 6th NS NS 15 mg / kg 6th LA LA 0.3 mg / kg 6th LA + NS LA 0.3 mg / kg + NS 15 mg / kg 6th MD MD 45 mg / kg 6th MD + NS MD 45 mg / kg + NS 15 mg / kg 6th MDAS MDAS 45 mg / kg 6th MDAS + NS MDAS 45mg / kg + NS 15mg / kg 6th

8.3. Effect of MDAS / NS and MD / NS combinations on skin temperature at simultaneous [01 or 45 min, pre-emptive [45 ul administration p. o.

GROUP PROCEDURE Number of animals

Control / Solvent 6

NS NS 40 mg / kg 8th MD MD 100 mg / kg 6th MD + NS MD 100 mg / kg + NS 40 mg / kg 6th MDAS 150 MDAS 150 mg / kg 6th MDAS75 + NS MDAS 75 mg / kg + NS 40 mg / kg 6th MDAS150 + NS MDAS 150 mg / kg + NS 40 mg / kg 6th

RESULTS.

R 8.1. Testing the effects of time and solvent on skin temperature

The mean baseline ear temperature was 28.4-30.6 ° C, recorded between 10am and 2pm. A study of the response to NS over time (15 mg / kg sc) showed a maximum temperature increase of 2.32 ± 0.37 ° C against the baseline and 2.57 ± 0.43 ° C against the solvent group (P <0.005). 10 minutes (Image). The effect of LA solvent on ear temperature was found to be significantly different from that of NS, MDAS and MD only in the first 5 minutes after injection, therefore only one control group was used in temperature calculations after 10 minutes.

R 8.2. Effects of individual test substances on skin temperature at simultaneous [0] or 30 min, pre-emptive [301 s.c. administration

Subcutaneous injection of MDAS, MD, or LA did not cause significant changes in rat ear skin temperature (Table 27). There was no temperature difference between MD + NS [0] when MD was co-administered with NS and MD + NS [30] when MD was administered for 30 min. ahead of NS.

Table 27

Effect of MDAS, LA, and MD on NS-induced changes in skin temperature; N = 6, Mean + SEM

Group Original temperature ° C Maximum temperature ° C An increase, % Control / Dissolve hey 29.5 + 0.29 $ 29.62 + $ 0.25 " - NS 29.61 + 0.40 32.2 + 0.42 *** 100 MDAS 29.3 + 0.35 29.2 + 0.38 ¹¹¹ - MDAS + NS Γ01 29.9 + 0.31 31.5 ± 0.40 ** 62 MDAS + NS Γ301 29.6 + 0.32 30.9 ± 0.32 * $ 50 LA 29.43 + 0.27 29.5 + 0.35 $$$ - LA + NSfO] 29.72 + 0.31 31.45 + 0.40 ** 67 LA + NS Γ301 29.51 + 0.32 30.73 + 0.34 * $ 47 MD45 29.42 + 0.38 29.6 + 0.31 $$$ - MD + NS roi 2953 + 0.29 31.33 + 0.48 * 69 MD + NS 1301 29.68 + 0.26 31.40 + 0.39 * 65

* P <0.05 versus Control ** P <0.005 versus Control *** p <0.0005 versus Control * P <0.05 versus Control * P <0.0005 versus Control

Concurrent NS and MDAS (NS + MDAS [0] group; pre-emptive time = 0) resulted in a decrease in NA redness similar to that co-administered with NS and LA or NS and MD, the NS-induced increase in temperature was reduced from 100% (NS), respectively. effect) to 62%, 67 and 69% (Table 27). In our MDAS + NS experiment and LA + NS, MDAS or LA administration s.c. 30 min before NS resulted in significant and similar protection against NS-induced skin temperature increase (Table 27).

R 8.3. Effect of NS / MDAS and NS / MD combination on skin temperature upon oral administration

Oral (p.o.) administration of NS at 40 mg / kg resulted in a significant and sustained (up to 60 min) increase in rat ear skin temperature with a maximum between 15 and 45 minutes (Table 28).

Table 28

Effects of MDAS or MD on NS-induced skin hyperthermia with simultaneous [0] or pretreatment [45]; N = 6-8, Mean + SEM

Group Initial temperature ° C Temperature, 15 min. Temperature, 30 min. Temperature, 45 min Max. An increase, % Control / Solvent 29.9 + 0.32 30.2 + 0.48 ** 30.0 + 0.33 *** 30.1 + 0.37 ** 9th NS 30.0 + 0.41 32.3 + 0.46 ** 33.1 + 0.41 *** 32.4 + 0.48 ** 100 MDAS 150 29.7 + 0.28 29.9 + 0.33 ** 29.5 + 0.35 " 29.6 + 0.39 ** 6th MDAS75 + NS [0] 29.7 + 0.33 31.9 + 0.51 * 32.3 + 0.37 *** 32.1 + 0.46 * 84 MDAS150 + NS [0] 29.8 + 0.37 30.9 + 0.47 * 32.0 + 0.42 ** 31.95 + 0.38 * 70 MD 29.9 + 0.35 30.1 + 0.39 ** 29.8 + 0.38 *** 30.0 + 0.34 ** 6th MD + NS [0] 29.6 ± 0.26 31.3 ± 0.35 ** 32.5 ± 0.42 ** 32.2 ± 0.40 * 96 MD100 + NS [45] 29.3 ± 0.35 31.2 + 0.33 ** 32.3 + 0.41 ** 32.1 + 0.44 * 95 MDAS75 + NS [451 29.4 + 0.30 31.3 + 0.53 * 31.9 + 0.45 * 31.5 + 0.42 ** 80 MDAS150 + NS [45] 29.7 + 0.38 30.7 + 0.41 * 31.2 + 0.44 ** 30.2 + 0.38 **** ® 48

* P <0.05 vs. Control ** P <0.005 vs. Control *** P <0.0005 vs. Control * P <0.05 vs. Control ** P <0.005 vs. Control *** P <0.0005 vs. Control ^& P < 0.05 vs. MDAS150 + NS [0] ®P <0.05 vs. MDAS 75 + NS [45] * P <0.05 vs. MD + NS [45]

Oral administration of MDAS or MD did not cause significant changes in skin temperature. Simultaneous p.o. Administration of MDAS and NS [0] at a dose of 75 mg / kg resulted in a minor protection and at a dose of 150 mg / kg a significant protection against NS-induced increase in skin temperature (Table 22). MD (100 mg / kg), co-administered with NS, prevented the rise in temperature for 15 minutes but did not provide significant protection against maximum NS-induced skin temperature increase (see 30 min, 45 min, Table 28). Analysis of the results showed that MDAS at 150 mg / kg, when co-administered with NS, reduced the skin temperature increase to 70%, which is significantly better than the decrease from NS + MD (96%). The increment caused by NS is considered 100%. Prophylactic administration - 45 min. before NS, the temperature-lowering effect of MDAS150 + NS (45) increased and was significantly better than that of MD + NS [45] or MDAS75 + NS [45] (Table 28).

MDAS by administering p.o. or s.c., reduces NS-induced increase in skin temperature. When administered s.c., MDAS, like laropiprant, reduced NS-induced increase in skin temperature when given concomitantly and prophylactically. Significant MDAS activity against redness when administered p.o. in combination with NS or prophylactically, indicates the potential utility of MDAS for reducing the adverse effects of NS on the skin (redness).

SUMMARY

Because MDAS has anti-inflammatory, antihyperlipidemic and antiplatelet activity, MDAS can be considered as a novel therapeutic agent for the treatment of various pathologies caused by platelet adhesion. The combination of MDAS with NS, strut, CL or DI can significantly expand the potential indications.

METHODS OF IMPLEMENTING THE INVENTION

The present invention provides a medical product comprising MDAS as an anti-inflammatory, analgesic, antipyretic, anti-rheumatic, antihyperlipidemic, anti-atherosclerotic, anti-stick and anti-thrombotic agent. The medical product of the invention may be administered in the form of a pharmaceutical composition. In this regard, the invention provides a pharmaceutical composition comprising MDAS in admixture with a pharmaceutically acceptable diluent or carrier.

Since the use of a medical product as an anti-inflammatory, analgesic, antipyretic, anti-rheumatic, antihyperlipidemic, antiatherosclerotic, anti-caking and antithrombotic agent requires sustained use of the medical product, the best embodiment of the invention is in the form of an oral capsule, e.g.

According to another aspect of the present invention there is provided the use of a medical product as described herein in the manufacture of a medicament for the treatment of inflammation, pain, fever, rheumatism, hyperlipidemic conditions, atherosclerotic conditions, antiplatelet and thrombus formation.

Another aspect of the present invention pertains to combination medical products comprising MDAS and another agent selected from the group consisting of NS, Stands, CL and DI. These products may be based on pharmaceutical compositions based on MDAS.

Claims (13)

Claims Addition salts of 3- (trimethylammonioamino) propanoate (meldonium) acetylsalicylic acid, solvates or polymorphs thereof for use as a medicament. A pharmaceutical composition comprising the addition salt of 3- (trimethylammonioamino) propanoate (meldonium) acetylsalicylic acid according to claim 1 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 2, as a quick release, sustained release, and sustained release dosage form. A pharmaceutical composition according to claim 2, suitable for oral administration. Meldonium acetylsalicylic acid addition salt according to claim 1 for the prevention and / or treatment of inflammation, pain, fever, rheumatic conditions, hyperlipidemic and atherosclerotic state, platelet adhesion pathology or thrombus formation, wherein the platelet such as myocardial infarction or stroke, or thrombosis and thromboembolism. A pharmaceutical composition according to claim 2 for the prophylaxis and / or treatment of inflammation, pain, fever, rheumatic conditions, hyperlipidemic and atherosclerotic state, platelet adhesion pathology or thrombus formation, wherein the platelet adhesion pathology, e.g. , myocardial infarction or stroke, or thrombosis and thromboembolism. A combination medical product comprising an effective amount of a meldonium acetylsalicylic acid addition salt according to claim 1 and an effective amount of dipyridamole for the prophylaxis and / or treatment of platelet adhesion pathology, particularly stroke. A combination medicinal product comprising an effective amount of a meldonium acetylsalicylic acid addition salt according to claim 1 and an effective amount of clopidogrel or a pharmaceutically acceptable salt thereof for the prevention and / or treatment of platelet adhesion-related pathology, wherein the platelet adhesion-related pathology comprises: such as myocardial infarction or stroke, or thrombosis and thromboembolism, acute coronary syndrome, sudden cardiac death, and complications following coronary angioplasty or coronary bypass grafting. A combination medical product comprising an effective amount of a meldonium acetylsalicylic acid addition salt according to claim 1 and an effective amount of nicotinic acid or a pharmaceutically acceptable salt thereof for the prevention and / or treatment of platelet adhesion-induced pathology, wherein such as myocardial infarction or stroke, or thrombosis and thromboembolism. The product of claim 9, wherein the meldonium acetylsalicylic acid addition salt is in the form of a quick release, sustained release, and sustained release dosage form. The product of claim 9, wherein the nicotinic acid or pharmaceutically acceptable salt thereof is in the form of a quick release, sustained release, and sustained release dosage form. A combination medical product according to claim 9 for the prophylaxis and / or treatment of dyslipidemia, hyperlipidemia and atherosclerosis. A combination medical product comprising an effective amount of a meldonium acetylsalicylic acid addition salt of claim 1 and a statin selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, mvastatin, pitavastatin, pravastatin, and rosuvastatin, intended for the prophylaxis and / or treatment of dyslipidemia, hyperlipidemia and atherosclerosis.