A PHARMACEUTICAL COMPOSITION COMPRISING MELDONIUM SUCCINATE AND MEDICAL USES THEREOF

Technical Field Present invention relates to medical use of Meldonium acid addition salt alone or in combination with succinic acid. The present invention provides novel medicinal products for prevention and/or treatment of disorder, selected from the group, consisting of dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease as chosen from the group of angina pectoris and myocardial infarction, chronic heart failure, transient and permanent ischemic attack including cerebrovascular accident and stroke and peripheral arterial occlusive disease. Background Art

Meldonium succinate (MDS) or (3-(2,2,2-trimethylhydrazinium)propionate succinate had been disclosed recently WO 2005/012233 A (GRINDEKS JSC, KALVINSH IVARS, BIRMANIS ANATOLIJS) 10.02.2005. This substance is described as non-hygroscopic, thermally stable and long-acting derivative of Meldonium (MD). No specific pharmacological data for this substance are available. Meldonium is described as clinically useful anti-ischemic and stress-protective agent for treating some diseases of heart vessels and other pathologies caused by tissue ischemization KARPOV, R.S., et al. Safety of Mildronate in Patients With Ischemic Heart Disease and Chronic Heart Failure. Kardiologiya. 2000, vol.6, p.69-74. Meldonium was shown to reduce myocardial infarct size in an experimental model of acute myocardial ischemia, without any effect on hemodynamics SESTI, C, et al. Mildronate, a Novel Fatty Acid Oxidation Inhibitor and Antianginal Agent, Reduces Myocardial Infarct Size Without Affecting Hemodynamics. Journal of Cardiovascular Pharmacology. 2006, vol.47, no.3, p.493-499. Meldonium is also indicated for treating chronic cerebrovascular insufficiency DZIAK, LA, et al. Use of mildronate for the treatment of patients with circulatory encephalopathy against a background of stenosis of major arteries of the head. Lik Sprava. 2003, vol.5-6, p.98-101. Meldonium was shown to possess antioxidant activity and offer protection against lipid peroxidation in clinic MUKHIN , I. V., et al. Experimental systemic enzyme therapy of gouty and primary glomerulonephritis. Eksp Klin FarmakoL 2003, vol.66, p.32-35. Patent application RU2005125660, claimed various alleged pharmacological activities to pharmaceutical composition comprising Meldonium and succinates, however without any proof of experimental data. A certain useful activity of Meldonium was discovered in animal models of atherosclerosis OKUNEVICH , I. V., et al. Anti-atherosclerotic action of mildronate in experiment. Patol Fiziol Eksp Ten. 2002, no.2, p.24-27. and observed in clinics KARPOV, R. S., et al. The clinical instrumental evaluation of treatment efficacy in patients with concomitant atherosclerosis of the coronary, cerebral and peripheral arteries. Ter Arkh.. 1991 , vol.63, no.4, p.90-93. Disclosure of Invention

When comparing the pharmacological profile of Meldonium succinate (1 :1) acid (addition salt of Meldonium with succinic acid) with that of Meldonium itself, we have unexpectedly discovered that Meldonium succinate possesses advantageous beneficial effects, significantly surpassing those of Meldonium. These differences can not be attributed only to possible differences in pharmacodynamics/pharmacokinetics of the Meldonium in salt form, but point to different action mechanisms. The Meldonium succinate containing medicinal products of the present invention are anticipated to be useful in a method for treatment and/or prevention of disorders, including dyslipidemia, hyperlipidemia, atherosclerosis, coronary heart disease as chosen from the group of angina pectoris and myocardial infarction, chronic heart failure, transient and permanent ischemic attack including cerebrovascular accident and stroke and peripheral arterial occlusive disease. In mice hyperlipidemia model Meldonium did not essentially change total and HDL-C, but displayed a tendency to prevent the LDL-C increase that was expressed in significant improvement of atherosclerosis coefficient at the 150 mg/kg/day dose. Contrary to that Meldonium succinate at the same dose of 150 mg/kg/day demonstrated statistically significant improvement of both the index and the coefficient. Investigation of atherosclerotic damage of aorta showed that Meldonium succinate protected aorta from damage significantly better than Meldonium alone. In rat model Meldonium succinate not only displayed stronger tendency than Meldonium to lower total cholesterol and LDL-C, but also significantly lowered triglyceride level. These results indicate that Meldonium succinate has a better potential than Meldonium in treatment and/or prophylaxis of hypercholesterolemia and atherosclerotic conditions.

In rat hyperlipidemia model sodium succinate (SS) did not substantially influence lipid levels. Surprisingly, a combination of Meldonium succinate with sodium succinate significantly improved all relevant indices - lowered total cholesterol, low density lipoprotein cholesterol and triglyceride levels, as well as lowered both the atherosclerosis index and coefficient. These results indicate that a combination medicinal product Meldonium succinate+sodium succinate may have an even better beneficial effect in atherosclerosis conditions than Meldonium and Meldonium succinate alone. In rat myocardial ischemia and reperfusion test Meldonium succinate showed better protection against lethality and necrosis of ischemic zone, compared to

Meldonium. The antioxidative activity of Meldonium succinate in this test based on the lipid peroxidation levels in heart tissue and total antioxidant status (TAS) in serum was better than that of Meldonium. These results indicate that Meldonium succinate in clinic as cardioprotectant/antioxidant may be better than Meldonium. In rat experimental chronic heart failure model, induced by untreated infarction, Meldonium succinate almost completely restored the functional reserve of myocardial contraction, significantly reduced morphological changes - hypertrophy and dilatation of the left ventricle and surpassed Meldonium in all indices. These results indicate that Meldonium succinate would be better than Meldonium as agent for treating cardiac insufficiency.

In mice circulatory hypoxia test Meldonium succinct had higher activity than Meldonium. In mouse MCA occlusion test the prophylactic activity of Meldonium succinct was higher than that of Meldonium in protection from neural damage. The ischemic damage area in Meldonium succinct treated group was smaller than that in Meldonium group. These results indicate that in cerebral ischemia conditions Meldonium succinct has substantially better beneficial effect than Meldonium. In mice cerebral ischemia-reperfusion model Meldonium succinct displayed surprisingly high serum TAS levels, surpassing those of Meldonium. These results indicate that Meldonium succinate may be most beneficial in cerebral ischemia and post-stroke conditions. Best Mode for Carrying Out the Invention

The pharmacological activity of the product of present invention separately and in combination with another active agent was investigated by standard methods used in the art. All experiments were carried out in accordance with the European Community Council's Directive of 24 November 1986 (86/609/EEC) relative to experimental animal care. All efforts were made to minimize animal suffering and to reduce the number of animals used. Substances: Cholesterol (Across Organics), Meldonium (Grindeks), sodium succinct (LIOS), animal nutrition (R 70 Lactating), butter (commercial), Na Choate (Across Organics). Meldonium succinct was prepared by modification of WO 2005/012233 method and obtained as colourless crystalline powder, stable and non-hygroscopic at ambient conditions. Other substances were obtained from commercial sources. Influence on lipid levels Example 1. Rat hyperlipidemia model

Method. Male Wistar rats weighing 220-250 g were used. Rats received fat- cholesterol mixture (10% cholesterol and 1 % cholic acid p.o. in dose 10 ml/kg) for 10 days. Solutions of test substances or water for control groups were introduced one hour after the fat mixture. Blood for analysis was obtained on 11th day by cardiac puncture under ether anaesthesia.

Biochemistry: Total C, HDL-C, LDL-C and TG were detected by COBAS INTEGRA 400 (Roche). Atherosclerosis index (reflecting coronary atherosclerosis and discriminator for peripheral atherosclerosis) was calculated as follows: Index = LDL-C/HDL-C Atherosclerosis coefficient (reflecting coronary atherosclerosis) was calculated as follows: Coefficient =Total C/HDL-C. Statistics. The data obtained were mathematically processed using Microsoft Excel program and the results were expressed as mean ± standard error (SEM). Mean results of different groups were compared using single-factor analysis according to ANOVA and Student's t-test. Difference of the results were considered significant at P<0.05. Results. Rats receiving nutrition with fats and cholesterol mixture developed pronounced hyperlipidemia and hypercholesterolemia within 10 days. Main indices like total C, LDL-C and TG were significantly different from those of intact control animals. During the short treatment period (10 days) MDS contrary to MD significantly reduced TG levels (P<0.05 vs C control), significantly improved the atherosclerotic index and rate of total C/HDL-C in this model (Table 1). This indicates positive influence on lipid metabolism and allows positioning MDS as potential agent for prophylaxis and/or treating of atherosclerosis.

Table 1 Influence of MD and MDS on lipid levels; Mean±SEM, n=6

^* P<0.05 vs C control * ^* P<0.005 vs C control

In another series of tests the effects of MDS combination with SS was compared to those of MDS alone. The experiment was conducted as described above. The results are given in Table 2. Table 2 Influence of MDS, SS and MDS+SS on lipid levels; Mean±SEM, n=6

^* P<0.05 vs C control * ^* P<0.005 vs C control ^& P<0.05 vs MDS @P<0.05 vs SS

Succinic acid salts (sodium, potassium, calcium or magnesium) themselves did not have substantial influence on lipid levels in this rat hypercholesterolemia model. Surprisingly, the combined use of MDS with succinates augmented the effects of MDS or SS. In particular, the combined use of MDS with SS significantly lowered the total C, LDL-C and TG levels, as well as the atherosclerosis index and the rate of total C/HDL-C (Table 2). Surprisingly, combined use of MDS with SS more significantly lowered the rate of total C/HDL-C than MD or SS alone, indicating the synergy effect of combination and indicated potential use of

MDS+SS combination in prophylaxis and/or treating of hypercholesterolemia and hyperlipidemia.

Example 2. Mice atherosclerosis model

Method. Mice atherosclerosis model proposed in literature was used [Smith JD, Breslow JL, J Intern Med 1997;242: 99-109]. Cholesterol and fat were added to standard nutrition of experimental C57BL/6 female mice. Within 22 weeks the test group animals developed pronounced atherosclerotic changes. During this time the evaluation of anti-atherosclerotic activity of test substances was made. The studies employed the following experimental protocol: Cholesterol (1.25%), butter (15%) and sodium cholate (0.5%) were added to nutrition of all animals (excluding intact control). The experiment continued for 22 weeks according to the following scheme:

1) Intact Controls group (animals received only standard nutrition).

2) Cholesterol control group (atherosclerotic diet, without treatment); 3) Two MD groups (animals additionally were given MD, 50 mg/kg/day or

150 mg/kg/day);

4) Two MDS groups (animals additionally were given MDS, 50 mg/kg/day or 150 mg/kg/day).

MD and MDS were added to drinking water. The doses of the preparations were corrected according to the actually consumed nutrition/water during the experiment. As an average, during a day mice consumed the amount of food that made up to 10% of their body weight, and drank water about 12% of their body weight. The doses of MD and MDS were selected on the basis of preliminary experiments. A positive effect on atherosclerotic changes was observed with doses as low as 20 mg/kg/day of MDS. Higher doses of MDS (up to 250 mg/kg/day) lead to more pronounced effects. The dosage used in Examples serves as illustration and does not limit the scope of invention.

At the end of the experiment the animals were anaesthetized (pentobarbital, 60 mg/kg, i.p). After laparotomy the blood was collected from the abdominal vein (with Vacutainer™). The blood sample was centrifuged (2000 x min- ¹ for15 min) and serum obtained was stored at -2O ⁰ C before biochemical tests. Aorta arches, descending and abdominal aorta were separated and preserved for further investigation in 4% buffered paraformaldehyde solution. Then determination of lipids in aorta root was based on the technique developed by Paigen et al. [Paigen B et al, Atherosclerosis 1987;68:231-240]. The total area of atherosclerotic changes in the internal surface of aorta was determined by ,,en face" technique. The longitudinally opened aorta was stained by Sudan IV or Oil red-O for estimation of lipids. The specimens were photographed and the images analyzed by planimetric application using Image-Pro Plus 4.5.1. The atherosclerotic damage was expressed as percentage of the area of lipid infiltrate from the total area of aorta. Six to eight specimens were analyzed for each group of animals.

Results. After 22 weeks all Control animals had a lipid infiltration in aorta that comprised 5.41 % from the total inner surface of aorta for untreated cholesterol controls. In the groups receiving MD a dose-related tendency of reduction of atherosclerotic area was observed. Surprisingly the same doses of MDS (50 or 150 mg/kg/day) demonstrated a more pronounced and statistically significant reduction of damaged area versus Intact control and MD group both in aorta root and total aorta (Table 3). Table 3

Influence of MD and MDS on the rate of atherosclerotic damage in aorta root (A) and internal surface of aorta (B); M±SEM, n=6-8

^* P<0.05 vs C control * ^* P<0.005 vs C control

^& P<0.05 vs MD in the same dose

After feeding atherosclerosis-inducing diet experimental animals show increase in total and low density (LDL-C) cholesterol with only a slight decline in high density cholesterol. Atherosclerosis index increased 12.8 times (from 0.17 to 2.18, Table 4). MD at a dose of 50 mg/kg/day or 150 mg/kg/day did not essentially change total and HDL-C, but displayed a tendency to prevent the LDL-C increase that was expressed in significant improvement of atherosclerosis index at the 150 mg/kg/day dose (Table 4). Contrary to MD, MDS at the same dose (50 or 150 mg/kg/day) demonstrated more expressed and statistically significant improvement of both the index and the coefficient and lowered the LDL-C levels.

Table 4 Influence of MD and MDS on cholesterol (C) fractions; M±SEM, n=7-8

^* P<0.05 vs C control ^** P<0.05 vs C control * ^** P<0.0005 vs C control &P<0.05 vs the same dose of MD

Influence on heart and circulation

Example 3. Cardioprotective and antioxidant properties in rat infarction model Method. The rat infarction model was used. Male Wistar rats weighing 320 -360 g were randomized into 4 groups:

1) I/R Control (n=16), animals received 0.9% NaCI solution 2 ml/kg p.o. for 7 days

2) Sham control animals underwent the surgery without occlusion of artery (n=8) received 0.9% NaCI solution 2 ml/kg p.o. for 7 days.

3) MD (n=12), animals received 150 mg/kg MD p.o. for 7 days

4) MDS (n=12), animals received 150 mg/kg MDS p.o. for 7 days. After the experiment ended blood was sampled from abdominal vena, serum separated by centrifugation. A 100 mg tissue sample was collected from the left ventricle for biochemical analyses. Tissues were homogenized and a 10% homogenate sample prepared. Heart homogenate and serum MDA level was measured by the modified method of [Andreeva Ll, Kozhemyakin LA, KishkunAA, Lab delo (Rus) 1988;11 :41-43]. TAS was measured by a colorimetric method using TAS Randox® kit (Cat No. NX2332, Randox laboratories, Ltd, UK). Statistics. Data are presented as means ± SEM of 7 to 11 separate experiments. Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). P<0.05 was considered as significant.

Results. Myocardial ischemia with following reperfusion caused serious heart rhythm disturbances with 6 lethalities in the control group (Table 5).

Table 5

Effect of test substances on heart rhythm disturbance and lethality

Test groups had less pronounced life-threatening rhythm disturbances (VF and VT) and lethality. Morphological analysis of the myocardium confirmed that all test substances significantly reduced the area of necrosis zone (Table 6).

Table 6 Effect of investigated substances on heart morphology; n= 8-11 ; Mean±SEM

^* P<0.05 vs I/R Control * ^* P<0.005 vs I/R Control &P<0.05 vs MD

¹ Necrotic index = Necrotic zone/lschemic zone x 100

The most pronounced cardioprotective activity in this model was displayed by

MDS that significantly (P<0.005) reduced the size of necrosis zone relative to the left ventricle and ischemic zone. MD was significantly less active in necrotic index test (Table 6).

Myocardial ischemia with a following reperfusion caused substantial increase in the MDA levels in serum and almost doubled the levels in heart tissue (Table 7).

Table 7 Indices of the antioxidative activity in rats

^* P<0.05 vs I/R Control * ^* P<0.005 vs I/R Control $P<0.05 vs Sham &P<0.05 vs MD MDS contrary to MD significantly protected from the increase of MDA in heart tissue. The decrease in total antioxidant status (TAS) in serum was significantly prevented only by MDS. Example 4. Rat infarction model Method. Male Wistar rats weighing 330 - 360 g were randomized into 4 groups:

1) I/R control, animals received 0.9% NaCI solution 2 ml/kg for 7 days

2) MDS group, animals received 150 mg/kg MDS p.o. for 7 days

3) SS group, animals received 50 mg/kg SS p.o. for 7 days

4) MDS+SS group, animals received 150 mg/kg MDS and 50 mg/kg SS p.o. for 7 days.

One hour after introduction of test substance the animals were narcotized (pentobarbital sodium 50 mg/kg i.p.) and under mechanical ventilation prepared for blocking of left coronary artery. Experimental infarction was induced by 45 min. long occlusion of coronary artery with following 2 h long reperfusion. The following data were registered: the number of animals with VT, VF and lethality. After the experiment the ischemic and necrotic area were detected by triphenyltetrazolium- Evans blue perfusion-staining method. Left ventricle was dissected and weighed and the morphological criteria calculated: percentage of the ischemic zone of the left ventricle, percentage of the necrotic zone of the left ventricle and ratio of the necrosis zone to ischemic zone (necrosis index). The results are presented as means ± SEM for each group.

Results. Myocardial ischemia with following reperfusion caused serious heart rhythm disturbances with lethalities in the control group (Table 8). Although SS itself was essentially inactive, its combination with MDS augmented the activity of the latter, particularly reducing the life-threatening rhythm disturbances and lethality.

Table 8

Effect of test substances on heart rhythm disturbance and lethality

Table 9

Effect of test substances on heart morphology of rats; n= 6-10; Mean±SEM

^* P<0.05 vs I/R Control * ^* P<0.005 vs I/R Control

¹ Necrotic index = Necrotic zone/lschemic zone x 100

Heart morphology showed that the rate of necrotic zone vs left ventricle area and the necrotic index was significantly lower in the MDS and MDS + SS group versus control (Table 9). Thus we have surprisingly discovered that MDS and MDS+SS significantly reduced necrotic damage caused by myocardial ischemia. Example 5. Rat chronic heart failure model

Method. Male Wistar rats weighing 300 - 330 g were used. Chronic heart failure was initiated by a known method in which the heart failure developed after the experimental myocardial infarction caused by blocking of the left coronary artery [Sasaki H et al, J MoI Cell Cardiol 2001 ;33:283-294. Batista M et al, J Appl Physiol 2007; 102:2033-2039].

Animals were anesthetized by sodium pentobarbital solution (50 mg/kg i.p.). Surgery field was prepared after the inset of anesthesia and switching the animal to mechanical ventilation. Under sterile conditions the chest and pericardium was opened and the left coronary artery occluded by filament snare at the level of the left atrial appendage. After 45 min of ischemia, the snare was released and the heart was allowed to reperfuse. Reperfusion was readily confirmed by hyperaemia over the surface of the previously ischemic-cyanotic segment. The chest wall was closed in layers, s.c. analgesic was injected and animal was placed on a heating pad while recovering from anesthesia. Control animals (Sham control) underwent the same manipulations except the occlusion of the artery. Animals that underwent the infarction were randomized in 3 groups on the next day:

1) Myocardial infarction control (Ml control, N = 10), received water p.o. one time each day 2) MD group (MD; N = 8), received MD 150 mg/kg p.o. once each day

3) MDS group (MDS; N = 8), received MDS 150 mg/kg p.o. one time each day

4) Sham control (N=6) animals received water p.o. one time each day. On the 28th day after the induction of Ml animals were repeatedly put under anesthesia and switched to mechanical ventilation. Catheter was insert into the left ventricle and left ventricular pressure and contractility parameters (LV dP/dt max and dP/dt min was obtained by a differentiator EQ-601 G) were recorded on Polygraph system RM 6000 (Nihon Kohden, Japan). After recording of basal contractility parameters (LV dP/dt max and dP/dt min), left ventricular contractile functional reserve was obtained by stress test as previously described [Kaga S et al, J MoI Cell Cardiol 2006;40: 138-147). At the end of experiment, an additional anesthesia was injected and saturated KCI was introduced via the femoral vein to stop the heart in diastole. Heart was isolated, left ventricle weight and maximum (diastolic) volume measured. Statistics. Results are expressed as mean from 6 to 8 measurements ± standard error of the mean (SEM). Differences between groups were tested for statistical significance by Student ^' s T-test (P<0.05).

Results. During 28 days after the experimental Ml the animals developed heart failure, characterized by drop in left ventricle systolic pressure (LVSP), left ventricle end diastolic pressure (LVEDP) increase and reduced left ventricle contractility (systolic +dP/dt and diastolic -dP/dt) (Table 10). MD reduced changes of LVEDP and +dP/dt that indicated a protective activity against the developing heart failure. Surprisingly the same dose of MDS provided a substantially better protection against the changes of LVSP and -dP/dt (P<0.05 in case of MI+MDS vs Ml control and non-significant in case of MI+MD vs Ml control). Table 10

Influence of test substances on hemodynamic indices and left ventricle contractility (+/-dP/dt); Mean ± SEM; N=6-9

^* P<0.05 vs Ml Control

^** P<0.005 vs Ml Control aHR - heart rate, beats per min bLVSP - left ventricular systolic pressure, mmHg;

⁰ LVEDP - left ventricular end-diastolic pressure, mmHg.

The stress test indicated that Ml group animals had substantially lower functional reserve of myocardium contractility compared to Sham control animals (Table 11). The average +dP/dt and -dP/dt increase during the test for Ml control was 13%, while in Sham control group it was 21 %, P<0.005. Surprisingly the MDS therapy statisticaly significantly better than MD terapy increased myocardium contractility (+ dP/dt and -dP/dt) during the test period, indicating that MI+MDS group animals had substantially higher functional reserve of myocardium contractility compared to MI+MD or Ml control group (Table 11).

Table 11

Influence of MD and MDS on contractility of left ventricle under load-stress test; Mean ± SEM; N=6-9

^* P < 0.05 vs Ml control #P < 0.005 vs Ml control Ψ < 0.0005 vs Ml control & P<0.05 vs MI+MD The analysis of morphometric data showed that animals within the 28 day period after the myocardial infarction had developed hypertrophy and dilatation of heart and left ventricle (Table 12). MDS significantly diminished the hypertrophy of the left ventricle (characterized by the rate LV/heart) and dilatation (characterized by significantly smaller increase of diastolic volume).

Table 12 Influence of test substances on heart morphometric indices

a - body weight *P<0.05 vs Ml Control * ^* P<0.005 vs Ml Control

These results indicate substantial cardioprotective and adaptogenic activity of

MDS in the rat chronic heart failure model and allow to propose using MDS in clinic to reduce and prevent complications after myocardial infarction, including treating cases of systolic and diastolic insufficiency [Dickstein K et al, European

Heart J 2008;29:2388-2442].

Influence on cerebral hypoxia and ischemia

Example 6. Mice cerebral hypoxia and ischemia models

Mice cerebral circulatory hypoxia model Metod. Male ICR mice weighing 21 - 25 g were randomized into 5 groups (Table

13). Animals received per os or by intragastric catheter the test substances as aqueous solutions either once or for 7 days. The control group received the same volume of water (10 ml/kg). The last dose was given 1 hour before the experiment.

Experimental circulatory hypoxia was induced by MgCb introduced during 3 s into the tail vein (2% MgCb, dose 200 mg/kg). Time till cessation of the last respiratory movements was registered and defined as survival time.

Statistics. Data are presented as means ± SEM of 6 to 10 separate experiments.

Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). P<0.05 was considered as significant.

Results. The cerebral hypoxia induced by stopping the blood flow resulted in loss of reflexes (death) already after 24.8 seconds in the control group (Table 13 below). The investigated substances displayed substantial antihypoxic activity as evidenced by the prolonged survival time of animals receiving the 7 day prophylactic therapy. MDS displayed substantially better antihypoxic activity than MD. The combined application of MDS and SS caused surprising increase in the protective activity surpassing the activity of each individual substance, indicating possible synergy. The results obtained in this test indicate a possible use of MDS and MDS+SS in treating hypoxic conditions in clinic.

Table 13

Influence of test substances on survival time; M±SEM, n=6-8

^* P<0.05 vs Intact control * ^* P<0.005 vs Intact control * ^** P<0.0005 vs Intact control $P<0.05 vs SS

⁶ PO.05 vs MD #P<0.05 vs MDS

Middle cerebral artery (MCA) occlusion model

Method. Male ICR mice weighing 21 to 26 g were randomized in 4 groups. Middle cerebral artery (MCA) origin was occluded by intraluminal filament technique according to known method [Longa EZ et al, Stroke 1989;20:84-91] adapted for mice by Iwai M. et al. [Circulation 2004; 110:843-848]. Preventive (once per day for

7 days) treatment protocol was used. Animals were anesthetized by chloral hydrate (400 mg/kg i.p.). The body temperature was supported at 37±0.3 ⁰ C by electronically controlled heating plate. Briefly, after a midline neck incision had been made, the right common and external carotid arteries were isolated and temporally ligated. Nylon monofilament suture 6/0 (Ethicon) , blunted at tip and coated with silicon resin (Xantopren; Bayer Dental), was inserted into the right internal carotid artery, and advanced approximately 10 mm distal to the carotid bifurcation to occlude the origin of the middle cerebral artery. Sham group underwent similar surgery without occlusion. Post-operative pain was alleviated by s. c. application of tramadol (20 mg/kg) and infection prevented by ampicillin (100 mg/kg). The neurological deficit scores that reflect overall sensorimotor dysfunction were determined 24 h after MCA occlusion [Wei EQ et al, Acta Physiol Sin 2003;55:742-747]: 0, no deficit; 1 , flexion of contralateral forelimb upon lifting of the whole animal by the tail; 2, circling to the contralateral side; 3, falling to contralateral side; 4, no spontaneous motor activity. After the evaluation of neurological status the animals received a lethal dose of sodium pentobarbital (100 mg/kg i.p.), brains removed, and cut into 6 slices (~ 1.5 mm). The slices were incubated for 15 mins at 37 ⁰ C in buffered 2% triphenyltetrazolium sodium solution for staining of living tissues. Staining slices were photographed (Nicon Coolpic 990, Japan) and necrotized area (non staining area) was determined. As the most adequate for calculating the brain ischemic damage the 3rd slice from cranial side on the chiazma opticum level was selected, since it was completely supplied by blood from the middle cerebral artery.

Statistics. Data are presented as means ± SEM of 6 to 10 separate experiments. Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). Differences of neurological scores between groups were analyzed by unpaired Student's t-test. P<0.05 was considered as significant.

Results. 24 hours after the MCA occlusion all 7 control group animals had neurological disturbances of various intensity (from 1 to 4 points) - average 2.71 point. In the sham group only one animal had slight disturbances (0.14 points, P<0.0005; Table 14). Prophylactic therapy by MD prevented from the deterioration of the neurological status, but was comparatively less active than MDS. MCA occlusion caused ischemic damage of brain tissue that for control group constituted 24.42 % of the brain surface area. MD did not prevent the ischemic damage of brain tissue. MDS displayed surprisingly good protection against the damage of brain tissue, caused by the MCA occlusion (Table 15). These results indicate the MDS has significant protective effect against morphological and functional damage of brain, caused by ischemia, substantially surpassing that of MD (Table 15).

Table 14

Influence of test substances on neurological status 24 h after MCA occlusion; n= 7-8; Mean±SEM

^* P<0.05 vs Intact control #P<0.0005 vs Intact control

Table 15

Effect of 7 day application of test substances on size of brain ischemic-infarction damage zone

^* P<0.05 vs Intact control & PO.05 vs MD

In another series of tests the effects of MDS+SS were compared with those of MDS and SS separately. Table 16

Influence of test substances on neurological state 24 h after MCA occlusion , n= 7- 8; Mean±SEM

^* P<0.05 vs Intact control

# P<0.0005 vs Intact control $P<0.05 vs SS

Although prophylactic therapy with MDS prevented from the deterioration of the neurological status, it was comparatively less efficient than the combination of MDS and SS (Table 16).

Table 17

Effect of 7 day application of test substances on size of brain ischemic-infarction damage zone

P<0.05 vs Intact control *P<0.005 vs Intact control

Morphological investigation showed that SS did not display significant protection pret ischemic damage zone in this model (Table 17). Combination of MDS and SS displayed surprising increase in the protective activity that was higher than that of separate components (P<0.005 in case of MDS+SS vs Control, not significant in case of SS vs Control and only P<0.05 in case of MDS vs Control). These data indicate possible synergy in activity of MDS+SS against ischemic damage in MCA occlusion model.

Example 7. Mice common carotid artery occlusion model Method. Antioxidative properties of test substances were estimated by mice common carotid artery occlusion model [Hoyte LC, Buchan AM, Encyclopedia of Neuroscience 2009. p.465-472][ widely used in evaluation of antihypoxic activity and protective action against central ischemic damage [Lee H, Bae JH, Lee SR, J Neurosci Res 2004;77(6): 892-900]. ICR male mice weighing 21 - 25 g were randomized in 4 groups. The animals received p.o. for 7 days:

1) MD group, 150 mg/kg/d MD

2) MDS group, 150 mg/kg/d MDS

3) Intact control group, received only water

4) Sham (control) group, received only water. The last dose of substances was given 1 hour before the experiment.

Experimental central ischemia was induced by 30 min long occlusion of both carotid arteries (a. carotis comunis) and following 24 hours long reperfusion. Sham group underwent a similar surgery without occlusion. At the end of the experiment the animals were repeatedly anesthetized (sodium pentobarbital, 60 mg/kg i.p.) and blood for biochemical analyses sampled from vena cava. Serum was separated by centrifugation and analyzed to contents of MDA and TAS. From each of animals ex tempore removed brain, performed its homogenization (400 rpm) and determined in homogenate (10%) the activity of malondialdehyde (MDA) and total antioxidant status (TAS). Brain hemolyzate and serum MDA level was measured by the modified method of Andreeva et al [Andreeva LI et al, Lab delo (Rus) 1988; 11 :41-43]. TAS was measured by a colorimetric method using TAS Randox® kit (Cat No. NX2332, Randox laboratories, Ltd, UK). Statistics. Data are presented as means ± SEM of 6 to 10 separate experiments. Differences between each experimental group were compared using one-way ANOVA with repeated comparisons (Tukey's test). P<0.05 was considered as significant.

Results. Brain ischemia with following reperfusion caused doubling of MDA levels in blood serum and more than tripled increase in brain tissues (Table 18). Table 18

Antioxidative properties of test substances

^* P<0.05 vs Intact control * ^* P<0.005 vs Intact control * ^** P<0.0005 vs Intact control & PO.05 vs MD

MD did not provide a significant protection against the increase of MDA in serum and brain tissue. Contrary to MD, MDS significantly lowered the levels of MDA in brain tissue. Surprisingly the TAS level was increased only by MDS. MDS therapy provides for a significantly higher TAS levels in serum versus both control and MD group. High TAS level signifies potentially better outcome in central ischemia, including stroke cases [Kumar A et al, Drugs Today 2008; 44(10):757-766]. Augmentation of in vivo antioxidant defences (TAS) substantially improves brain functions, including depressive states [Zafir A, Ara A, Banu N, Prog Neuropsych 2008].