WO2015118488A1

WO2015118488A1 - Agent for treating cardiovascular diseases

Info

Publication number: WO2015118488A1
Application number: PCT/IB2015/050897
Authority: WO
Inventors: Кенес Тагаевич ЕРИМБЕТОВ; Владимир Константинович ПОДГОРОДНИЧЕНКО; Виктор Владимирович ХОМИЧЕНОК; Анна Яковлевна ГОНЧАРОВА; Рахимджан Ахметджанович РОЗИЕВ; Алексей Викторович КЛЕЙМЕНОВ
Original assignee: Ооо "Диборнол Девелопмент"
Priority date: 2014-02-06
Filing date: 2015-02-06
Publication date: 2015-08-13
Also published as: EA201691465A1; RU2555335C9; RU2555335C1

Abstract

The invention relates to an agent for treating cardiovascular diseases consisting of a mixture of two structural isomers of 4-methyl-2,6-di-isobornyl phenol with an isomer ratio of from 60:40%wt. to 95:5%wt.

Description

Cardiovascular Treatment

The invention relates to medicine, specifically to pharmacology, and relates to agents having hemorheological, antiaggregatory, antithrombogenic, retinoprotective, endothelium protective, neuroprotective, antiarrhythmic and anti-ischemic activity, as well as increasing cerebral blood flow. An agent having antiradical, hemorheological cytoremic antithrombogenic activity [Patent N ° 2347561, A61K 31/05, 2008].

Known agent exhibiting anti-ischemic properties [Patent N ° 2499593, A61K 31/05, 2013].

Known agent exhibiting antiarrhythmic activity [Shchetinin P, P. Antiarrhythmic activity of dibornol under the conditions of the model of acute ischemia - myocardial reperfusion / Bulletin of Siberian Medicine, 2003, Volume 12, N ° 3, p.153-156].

Known agent with neuroprotective activity [Patent N ° 2406488, A61K 31/05, 2010].

Known tool with retinoprotective activity [Patent N ° 2406487, A61K 31/05, 2010].

A means is known that exhibits endothelium protective properties [Ivanov I. S. Neuroprotective and antithrombogenic activity of 4-methyl-2,6-diisobornylphenol / Abstract of the dissertation of the candidate of biological sciences, Tomsk, 2009, 24 pp.].

Known means that can increase cerebral blood flow [Patent N ° 2351321, A61K 31/05, 2009].

These agents are 4-methyl-2,6-diisobornylphenol.

It is known that this compound is a mixture of structural isomers [Patent N ° 2502719, С07С 39/17, 2013]; however, the ratio of these isomers to the mixture and the effect of their ratio on the pharmacological activity they exhibit are not known.

The objective of the invention is to expand the arsenal of drugs with simultaneous hemorheological, antiplatelet, antithrombogenic, retinoprotective, endothelium protective, neuroprotective, antiarrhythmic and anti-ischemic activity that increase cerebral blood flow. Also, the objective of the invention was to identify the pharmacological activity of the indicated agent exhibiting these properties. The problem is achieved using a mixture of structural isomers of 2,6-di (1, 7, 7-trimethylbicyclo [2.2.1] hept-2-yl) -4-methylphenol (isomer 1) and 2- (1,7,7-trimethylbicyclo [2.2.1] hept-2-yl), 6- (2,2, 1-trimethylbicyclo [2.2.1] hept-5-yl) -4-methylphenol (isomer 2) as a drug with hemorheological , antiplatelet, antithrombogenic, retinoprotective, endothelium protective, neuroprotective, antiarrhythmic and anti-ischemic properties, increasing cerebral blood flow with a high degree of activity in the wake appropriate ratio of isomers:

from 60 to 95% of the mass. for the first isomer and from 40 to 5% of the mass. for the second isomer.

There is no information in the literature on the use of a mixture of structural isomers of 2,6-di (1, 7, 7-trimethylbicyclo [2.2.1] hept-2-yl) -4-methylphenol and 2- (1,7,7-three - methylbicyclo [2.2.1] hept-2-yl), 6- (2,2, 1-trimethylbicyclo [2.2.1] hept-5-yl) -4-methylphenol with the indicated ratio of isomers as hemorheological, antiplatelet, antithrombogenic, retinoprotective, endothelium protective, neuroprotective, antiarrhythmic, anti-ischemic drugs that increase cerebral blood flow in diseases of the cardiovascular system. The literature describes the use of only one isomer - 2,6-di (1, 7, 7-trimethylbicyclo [2.2.1] hept-2-yl) -4-methylphenol, however, its isolation in pure form leads to large losses in output and cost of the drug.

New in the present invention is that as a hemorheological, antiaggregate, antithrombogenic, retinoprotective, endothelium protective, neuroprotective, antiarrhythmic, antiischemic, cerebral blood flow increasing agent, a mixture of these diastereomers of 2,6-di diastereomers is used (1, 7, 7 -trimethylbicyclo [2.2.1] hept-2-yl) -4-methylphenol and 2- (1,7,7-trimethylbicyclo [2.2.1] hept-2-yl), 6- (2,2, 1-trimethylbicyclo [2.2.1] hept-5-yl) -4-methylphenol with an isomer ratio of 60: 40% by mass ^ -95: 5% by mass, which can be used Xia for the treatment of cardiovascular disease. This mixture is obtained by selecting the conditions for the Friedel-Crafts alkylation reaction of para-cresol with Camphene and does not require expensive purification methods. The ratio of isomers was calculated from the NMR spectra (Fig. 1) and the ratio of peak areas on HPLC (Figs. 2 - 95: 5 and Fig. 3 - 60:40). For analysis, a SURVEYOR LC liquid chromatograph equipped with a UV detector with a photodiode array (Thermo, USA) was used.

Separation of the mixture of diastereoisomers was carried out on a Hyper carb column, 100 x 2.1 mm, particle size 5 μm (Thermo, USA). Mobile phase - acetonitrile + 0.1% ant - other acid, the elution mode is isocratic, the flow rate is 600 μl / min (Fig 2) or the acetonitrile + Н ₂ 0 + 0.1% formic acid eluent, the elution mode is gradient, the flow rate is 300 μl / min (Fig 3) . The structure of 2,6-di (1, 7, 7-trimethylbicyclo [2.2.1] hept-2-yl) -4-methylphenol was confirmed by X-ray analysis (Fig. 4).

For a specialist, these properties are not explicitly derived from the prior art.

Thus, the following compounds were the subject of research:

Compound 1 - 4-methyl-2,6-diisobornylphenol.

Compound 2 is a mixture of structural isomers and their diastereomers: 2,6-di (1, 7, 7-trimethylbicyclo [2.2.1] hept2-yl) -4-methylphenol and 2- (1,7,7-trimethylbicyclo [2.2. 1] hept-2-yl), 6- (2,2, 1-trimethylbicyclo [2.2.1] hept5 ~ yl) -4-methylphenol with a ratio of isomers from 60% to 95% of the mass. for the first isomer and from 40% to 5% of the mass. for the second isomer.

The efficacy of compound 2 compared with control and compound 1 was demonstrated in model experiments on Wistar rats with intragastric administration at a dose of 100 mg / kg.

In all examples, statistical processing of the results was carried out using the BioStat software packages for Windows using the Student t-test and the criterion. The non-parametric Mann-Whitney test was used to assess the significance of differences when comparing mean values.

The invention is illustrated by examples of specific performance.

Example 1. The experiments were performed on 90 female Wistar rats weighing 190-210 g. Rats were divided into 3 groups of 30 animals each. On the model of total transient cerebral ischemia using the method of WA Pulsinelli, JB Brierley [1979], neuroprotective effects were evaluated. For this, 1 day before the ischemia simulation in anesthetized rats (thiopental sodium 60 mg / kg, intraperitoneally), thermocoagulation of both vertebral arteries was performed at the level of the first cervical vertebra. 24 hours later, under ether anesthesia, occluders were applied to both carotid arteries for 30 minutes. The consistency of the model was assessed by blanching of the visible part of the choroid, expansion of the pupils, and development of hyperventilation. Reperfusion was carried out by the removal of occluders. 1 ml of 1% starch gel was administered intragastrically to rats of the control group, and compounds 1 and 2 were administered intragastrically to the animals of the experimental group at a dose of 100 mg / kg in 1 ml of starch gel once a day for 7 days. The first administration was carried out 1 hour after model of ischemia, the last injection - 1 hour before blood sampling. In the first 2-5 hours after the reproduction of the model of total transient cerebral ischemia in rats, the most severe symptoms of damage to the central nervous system were observed in the form of areflexia, seizures, spastic paralysis of the limbs, tonic tension of the muscles of the trunk, and lateral position. The functional state of higher nervous activity was evaluated on the 1st, 4th, 5th and 7th days after the creation of the model with total transient cerebral ischemia using the Stroke-index scale [McGraw CP., 1977] according to the following indicators: spontaneous engine - Naya activity (normal, increased or decreased, absence), gait disorders (stiffness, shakiness, slow movement, disorientation), reflexes of tail jerking, both fore and hind legs, reaction to sound, tremor, cramps, muscle tone of the trunk and extremities (normal, rep Weighted, absence), signs of ptosis (no, one-sided, two-sided). Each indicator was evaluated in points: 0 points - normal; 1 point - moderate changes; 2 points - pronounced changes. The neurological deficit of the animal was assessed by the sum of points for all indicators. In addition, in each group of rats, the proportion of animals with severe neurological disorders (6 points or more), with moderate impairment (3-5 points) and with mild changes (up to 2 points) was determined. Animal survival was recorded on the 1st, 4th, 5th and 7th day after total transient cerebral ischemia. At the end of the experiments, animal euthanasia was performed.

Research results show that 30% of the rats died in the control during the first days after total transient cerebral ischemia. At the same time, in treated animals with compounds 1 and 2, 15 and 11% of rats died on the first day, respectively, which was approximately 2 times lower than the control value (Table 1). Despite the fact that the average score of neurological deficit in embroidered rats of all groups did not significantly differ (Table 2), there was a clear tendency to decrease under the influence of compounds 1 and 2 the number of animals with severe neurological impairment and a corresponding significant increase in the number animals with an average degree of neurological deficit (table. 2). When treated with compound 2, there was a significant decrease in the mortality rate of rats and neurological impairment compared with both control and compound 1.

In the control, mortality of rats reached the maximum values (45%) by the fourth day after total transient cerebral ischemia (45%), during the next observation period, mortality did not increase. In the experimental groups, the death of animals by this time was significantly lower and did not change by 5-7 days (Table 1). The severity structure of neurological disorders was similar in surviving animals treated with compound 1 and control by 4 days. In rats treated with compound 2, an improvement in neurological disorders was noted (Table 2). Starting from the 4th day, in the animals of the control group there was a decrease in the severity of neurological symptoms. However, the restoration of neurological status in rats treated with compounds 1 and 2 occurred at a faster pace. So, by the 5th day, the average score of neurological deficit in the groups treated with compounds 1 and 2 was 1.6 and 2 times lower than the control, respectively, and by 7 days the tendency to complete restoration of central nervous system function continued.

Thus, the use of compound 2 in the treatment of ischemic brain damage to the greatest extent compared with compound 1 reduces the death of animals and accelerates the restoration of neurological status in embroidered animals.

Table 1

The effect of intragastric administration of compounds 1 and 2 at a dose of 100 mg / kg on mortality to patients after total t ansitic cerebral ischemia,%

Note: - p <0.05 compared with the control;

^# - p <0.05 when comparing the group of animals treated with compound 2 with the group of rats treated with compound 1.

table 2

The effect of intragastric administration of compounds 1 and 2 at a dose of 100 mg / kg on the severity of neurological deficiency in rats after total cerebral ischemia

Table 2 Advice

- p <0.05 compared with the control;

- p <0.05 when comparing a group of animals treated with compound 2 with a group of rats treated with compound 1.

Example 2. A study of the endothelial protective activity of compounds 1 and 2 was carried out on models of pathological conditions (incomplete cerebral ischemia and diabetes mellitus).

The model of incomplete cerebral ischemia in anesthetized (ether anesthesia) Wistar female rats was reproduced by complete occlusion of the left carotid artery and restriction of blood flow in the right carotid artery to 50% of the initial one under the control of an electromagnetic flowmeter. False-operated animals underwent a similar surgical intervention, but without vascular ligation. The wounds were treated with antiseptics and sutured.

To assess endothelial dysfunction, the endothelium-dependent and endothelium-independent vascular response in rats was studied. For this, anesthetized animals continuously recorded systemic blood pressure (SBP). To measure SBP, animals were implanted with a catheter filled with a mixture of saline and heparin in the right carotid artery. Endothelium-dependent vasodilation was recorded with bolus intravenous administration of acetylcholine (5 μg / kg), endothelium-independent vasodilation with bolus intravenous administration of sodium nitroprusside (30 μg / kg). Acetylcholine and sodium nitroprusside were injected into the femoral vein. The functional state of the endothelium was assessed using the coefficient of endothelial dysfunction (QED), calculated as the ratio of the areas under the blood pressure reduction curves in response to the intravenous administration of sodium nitroprusside and in response to the intravenous administration of acetylcholine [Tyurenkov I.Yu., Voronov A.V. , 2008].

Experiments with the reproduction of a model of incomplete cerebral ischemia were carried out on female Wistar rats weighing 260-280 g. 5 groups of animals were formed, 10 each. Rat groups: intact, control, pseudo-operated and treated with compounds 1 and compound 2. Animals of the control, pseudo-operated and intact groups were administered intragastrically 1 ml of starch gel. Rats treated with compounds 1 and 2 were administered drugs at a dose of 100 mg / kg. The starch gel and suspension of compounds 1 and 2 in the starch gel were administered to the animals once a day for 5 days. The first introduction was through 1 an hour after creating a model of incomplete cerebral ischemia. On the 5th day, 1 hour after the last administration of compounds 1 and 2 or starch gel, the rats were anesthetized (thiopental sodium 60 mg / kg, ip) and the endothelial function was evaluated.

The results of the studies indicate that in the group of false-operated rats, changes in endothelium-independent and endothelium-dependent vasodilation compared with intact animals were not detected, QED was 2.1 ± 0.2 (Table 3). Therefore, all the observed changes in the control and groups treated with compounds 1 and 2 are not associated with the influence of surgical intervention. In ischemic rats of the control group, the QED was 3.4 ± 0.4, which is 60% higher than the value in the group of false-operated rats (Table 3). Thus, incomplete cerebral ischemia in rats was accompanied by endothelial dysfunction. The described changes in endothelial function with a predominant impairment of endothelium-dependent vasodilation are consistent with data on the formation of endothelial dysfunction during ischemic and reperfusion damage to rat brain tissue. In the group of rats with cerebral ischemia treated with Compound 2, the QED was 1.9 ± 0.2, which was significantly different from the control and the group treated with Compound 1 and was similar to falsely operated animals.

Thus, with a course intragastric administration, Compound 2 exhibits higher endothelial protective activity compared to Compound 1 under the conditions of the model of incomplete cerebral ischemia in rats.

Table 3

The effect of intragastric administration of compounds 1 and 2 at a dose of 100 mg / kg on function

endothelium to ys with incomplete cerebral ischemia

Note: - p <0.05 compared with the control;

To create a model of diabetes mellitus, female rats of the Wistar strain were injected with intraperitoneal injection of streptozotocin at a dose of 60 mg / kg in citrate buffer. Animals with a blood glucose level of 19 to 26 mmol / L were selected for the experiment. Whole blood glucose was measured using a glucometer (Hand- guidelines for preclinical studies of drugs. Part one. - M.: Grif and K, 2012 .-- 944 s).

Evaluation of endothelial dysfunction was carried out in accordance with the methodology described in the previous section.

Experiments with the reproduction of a model of diabetes mellitus were carried out on female Wistar rats weighing 240-260 g. In 30 animals, a diabetes mellitus model was reproduced and 1 month after injection of streptozotocin, rats were selected into the groups treated with compound 1, compound 2 and control with a close level of glucose in the blood, ranging from 19 to 26 mmol / l. Over the next month, starch gel was administered intragastrically to animals of the control and intact groups, and compounds 1 and 2 were administered to rats of the experimental groups at a dose of 100 mg / kg in starch gel. 1 hour after the last administration of compounds 1 and 2, the rats were anesthetized (thiopental sodium 60 mg / kg, ip) and the endothelial function was evaluated.

The research results showed that endothelial dysfunction was revealed in rats of the control group. This was evidenced by a significant increase in QED by 1.5 times in comparison with intact animals. The described changes in the control group of animals are consistent with data on the presence of endothelial dysfunction in patients with diabetes mellitus and in animals with models of this disease. In animals treated with compound 2, QED was significantly significantly 1.6 and 1.2 lower, respectively, than in the control and when using compound 1 in the treatment of diabetes mellitus.

Table 4

The effect of course intragastric administration of compounds 1 and 2 at a dose of 100 mg / kg on

Endothelial function for diabetes mellitus

Note: - p <0.05 compared with the control;

Thus, in the course of intragastric administration, compound 2 exhibits higher endothelial protective activity in diabetes mellitus in rats compared to compound 1.

Example 3. On the model of diabetic retinopathy, the retinoprotective effects of compounds 1 and 2 were evaluated. The experiments were performed on 40 female rats Wistar strain, weight 220-250 g. The model of diabetic retinopathy in rats was reproduced by a single intraperitoneal administration of streptozotocin at a dose of 60 mg / kg body weight. The level of hyperglycemia (over 18 mmol / L), weight loss, the severity of polyuria and polydipsia were the criteria for the severity of diabetes. 30 days after the administration of streptozotocin, rats were divided into 3 groups. Compounds 1 and 2 were administered intragastrically to rats of experimental groups with a glucose concentration of 21.8 ± 2.5 (compound 1) and 22.0 ± 1 ₅ 9 (compound 2) mmol / l daily for 30 days at a dose of 100 mg / kg body weight in suspension in 1 ml of starch gel. Animals of the control group with a glucose concentration of 22.3 ± 2.2 mmol / L were injected with the same amount of starch gel according to the same scheme. A group of intact animals in similar conditions. The central sections of the retina of rats were the material of the study, which were prepared (60 days after the start of streptozotocin injection) immediately after euthanasia by ether anesthesia of animals. The central parts of the posterior wall of the eye were fixed in a 2.5% solution of glutaraldehyde in 0.2 M cacodilate buffer (pH 7.4), after which they were fixed in a 2% solution of osmium tetroxide and poured into a mixture of epon-araldite resins. Semi-thin sections were stained with toluidine blue, ultra-thin sections were contrasted with uranyl acetate and lead citrate, viewed and photographed using an electron microscope. Semi-thin sections counted neurosensory cells with pycnosis of the nucleus per 1000 cells from each retina, hyperchromic and pycnomorphic associative neurons and radial gliocytes, ganglion neurons with total, focal chromatolysis and pycnomorphic 200 cells from each retina. Using Avtandilov's ocular measuring grid, the numerical density of nuclei in the outer nuclear layer over an area of 900 μm, the specific area of vessels with stasis, sludge, and thrombosis, and the gleoneuronal index were calculated.

The results of the studies indicate that after the development of streptozotocin diabetes in the retinas of animals, morphological signs of degeneration of all structural components of the retina are noted. Destructive changes in sensor cells are expressed in the appearance of pericarions of various sizes, deformations of the nuclei between which hypertrophic scleral processes of radial glia grow. A shift of single nuclei of neurosensory cells to the photosensor and outer reticular layers is observed. Quantitative analysis showed a threefold increase in the percentage of neurosensory cells with pycnosis of the nucleus, characterized by an increase in osmiophilia of the nucleus and cytoplasm, and a 1.56-fold decrease in the density distribution of the nuclei of neurosensory cells in the outer nuclear layer in relation to intact control (table 5). Changes in associative neurons are reactive in nature. In streptozotocin diabetes, rats in the control group showed an increase in the percentage of hyperchromic neurons as compared with intact control (Table 6). Degeneration of multipolar neurons with prolonged hyperglycemia in the presence of streptozotocin diabetes is characterized mainly by varying degrees of chromatolysis. In the control group, chromatolytic changes in ganglionic neurons were first noted (Table 7): an increase in the level of neurons with focal and total chromatolysis by 6–7 times with respect to the intact control, while the number of pycnomorphic neurons increased by 3.8 times. With prolonged hyperglycemia in rats of the control group, destructive processes in radial gliocytes were revealed, which was expressed by an increase in the electron density of the cytoplasm and nucleus. The number of pycnomorphic radial gliocytes relative to the values of intact control increases, which is accompanied by a decrease in the glyoneuronal index (Table 8). In the control group, pronounced microvascularization disorders were noted, characterized by a significant increase compared with the intact control of the specific area of choroid vessels with sludge and stasis of shaped elements (Table 9).

Table 5

The effect of intragastric administration of compounds 1 and 2 at a dose of 100 mg / kg on indicators of destruction of the outer nuclear layer of rat retinas with streptozotocin diabetes

Note: - p <0.05 compared with the control;

^# - p <0.05 when comparing the group of animals treated with compound 2 with the group of rats treated with compound 1. Table 6

The effect of intragastric administration of compounds 1 and 2 at a dose of 100 mg / kg on indicators of the destruction of associative neurons in the inner nuclear layer of rat retinas

Note: - p <0.05 compared with the control;

Table 7

The effect of intragastric administration of compounds 1 and 2 at a dose of 100 mg / kg

Note: - p <0.05 compared with the control;

Table 8

Note: - p <0.05 compared with the control;

^# - p <0.05 when comparing the group of animals treated with compound 2 with the group of rats treated with compound 1. Table 9

The effect of intragastric administration of compounds 1 and 2 at a dose of 100 mg / kg on the specific area indicator (%) of choroid vessels with stasis, sludge and thrombosis of the retina kys with stptozotocin diabetes

Note: - p <0.05 compared with the control;

Compound 2 treatment more effectively prevents the destruction of neurosensory cells, which is reflected in a decrease in the percentage of pyknotic nuclei of neurons to intact control values and an increase in the number of rows of nuclei relative to the control group and the group of rats treated with compound 1 (Table 5). At the same time, the density distribution of the nuclei of neurosensory cells in the outer nuclear layer remains reduced, which is probably due to the proliferative-progressive reaction of radial glia in the form of proliferation and hypertrophy of scleral processes. The administration of compound 2, unlike compound 1, contributed to the complete preservation of the neurons of the inner nuclear layer from the damaging effects of persistent prolonged hyperglycemia, which was expressed in the absence of significant differences in all indicators of destruction of associative neurons in rats treated with compound 2 and the intact group ( table 6). Intragastric administration of compound 2 had a positive effect on the state of retinal ganglion neurons, and the percentage of neurons with total and focal chromatolysis decreased relative to the values of the control group and the group of animals treated with compound 1, while remaining significantly higher than the values of intact control (Table 7 ) Also, when rats were administered with compound 2, complete restoration of all indicators of radial gliocyte deformation was observed (Table 8). Thus, the percentage of pycnomorphic radial gliocytes decreased while the gloneuronal index increased in relation to both the control values and the values of the group of rats treated with compound 1. When animals were administered with compound 2, there was a significant improvement in hemodynamics, which was expressed in a significant decrease in the specific area of vessels with stasis , sludge and thrombosis in comparison with the same indices of control rats and treated with compound 1 (Table 9). Thus, the course treatment with compound 2 at a dose of 100 mg / kg compared with compound 1 for 30 days in rats with diabetic retinopathy that developed against streptozotocin diabetes had a more pronounced retinoprotective effect, leading to a decrease in the severity of destruction of all structures - tour components of the retina. Compound 2, unlike compound 1, has a significantly higher retinoprotective activity.

Example 4. The effect of compounds 1 and 2 upon single parenteral and triple intragastric administration to rats on cerebral blood flow was studied by recording the velocity of blood flow in the carotid artery using an electromagnetic flow meter.

Experiments to study the effect of a single parenteral administration of compounds 1 and 2 on the dynamics of cerebral blood flow (MK) and systemic blood pressure (CAD) were performed on 26 male Wistar rats weighing 340-360 g. 3 groups of animals were formed. Animals of the control group (n = 8) were injected subcutaneously with 1 ml of cremophor. Compounds 1 and 2 were administered subcutaneously to rats of the experimental groups (n = 18) at a dose of 100 mg / kg as a solution in 1 ml of cremophor.

In anesthetized rats (thiopental sodium 60 mg / kg, ip), the right femoral artery was dissected and a catheter was implanted to measure systemic blood pressure. The left common carotid artery was repaired and the external carotid artery was ligated. A cuff sensor was placed on the common carotid artery, and cerebral blood flow was recorded using an electromagnetic blood flow meter.

Experiments on the effect of triple intragastric administration of compounds 1 and 2 on cerebral blood flow were carried out on 18 male Wistar rats weighing 250-280 g. 3 groups of animals were formed. The rats of the control group (n = 6) were injected intragastrically once daily for 3 days with an equivalent volume of starch gel. Compounds 1 (n = 6) and 2 (n = 6) were administered intragastrically to the animals of the experimental groups once daily for 3 days at a dose of 100 mg / kg in starch gel. On the 3rd day of the experiment, 1 hour after the last injection, rat cerebral blood flow measurements were taken.

The results of the studies indicate that in the control, with a single parenteral (subcutaneous) administration of cremaphore to rats, the MK level was stable for 1 hour, a slight tendency to a decrease in cerebral blood flow was revealed in the next 30 minutes of measurement. At the same time, a tendency was noted. to a decrease in SBP during the measurement, which is obviously due to the conditions of the experiment (the presence of surgical trauma and the use of anesthesia) (Table 10). With a three-day intragastric administration of starch gel in animals of the control group, the value of cerebral blood flow was 5.9 ± 0.5 ml / min (Table 11).

Subcutaneous administration of an oil solution of compounds 1 and 2 to rats contributed, from 5 minutes of measurement to the end of the observation, a significant increase in MK. In the period from 45 to 90 min of the experiment, the MK values in rats treated with compound 2 were significantly higher than the values of this indicator in the control groups and treated with compound 1. Moreover, according to the level of SBP during the entire observation period, significant differences between groups of animals not detected (table 10).

In the course of intragastric administration to rats of compound 2 at a dose of 100 mg / kg, the value of cerebral blood flow was 8.9 ± 0.4 ml / min, which was, respectively, 47 and 22% higher than that of the control group and animals treated with compound 1 (tab. 11).

Thus, subcutaneous administration of an oil solution of compound 2 at a dose of 100 mg / kg increases cerebral blood flow more significantly compared to the control and animals treated with compound 1, without causing a decrease in systemic blood pressure. Three intragastric administration of compound 2 at a dose of 100 mg / kg causes a significant increase in cerebral blood flow and, in terms of the severity of the effect, exceeds compound 1. The present invention expands the arsenal of drugs that can increase cerebral blood flow.

Table 10

The effect of subcutaneous administration of compounds 1 and 2 in cremophor (100 mg / kg) on the dynamics

MK, ml / min and CA, mm t.st.

Note: - p <0.05 compared with the control;

^# - p <0.05 when comparing the group of animals treated with compound 2 with the group of rats treated with compound 1. Table 11

The effect of three intragastric administration of compound 1 and 2 (100 mg / kg) on

MK

Note: - p <0.05 compared with the control;

Example 5. A study of hemorheological, antitrobocytic and antithrombogenic activities of compounds 1 and 2 was carried out on ex vivo models of blood hyperviscosity, intravascular thrombosis in rats.

Experiments on the hemorheological properties of compounds 1 and 2 were carried out on 20 male Wistar rats weighing 270-290 g. Rats were randomized into 4 groups of 5 animals each. The rats of the control group were injected with starch gel, the animals of the experimental groups - pentoxifylline (400 mg / kg) or compounds 1 and 2 (100 mg / kg). The preparations and the equivolume amount of starch gel (2 ml) were administered intragastrically through a probe one hour before blood sampling.

Blood viscosity and erythrocyte aggregation were evaluated in blood samples immediately after collection and 1 hour after blood incubation at a temperature of 20.0 ± 0.4 ° C. Blood was drawn from the common carotid artery under ether anesthesia. A 3.8% sodium citrate solution in a ratio of 1: 9 blood was used as an anticoagulant. Blood viscosity was measured on a rotational viscometer in the range of shear rates from 5 to 300 s ^"1 before and after incubation of the samples at a temperature of 20.0 ± 0.4 ° C for 60 minutes.

In the same series of experiments, spontaneous aggregation of erythrocytes was studied using the sillectometric method. The criterion for the aggregation activity of erythrocytes was the half-period of aggregation Tm — the time during which the magnitude of the photometric signal is halved.

The initial values of the blood viscosity of animals of the control group at shear rates from 5 to 300 s ^"1 were in the range from 8.3 ± 0.1 to 4.5 ± 0.1 MPa s. Incubation of these blood samples for 60 minutes at a temperature of 20.0 ± 0.4 ° C led to a significant (16–31%) increase in blood viscosity. Pentoxifylline caused a significant decrease in blood viscosity at low shear rates prior to incubation compared to control. After incubation, an increase in blood viscosity was observed throughout the investigated range of shear rates, however, the values of this indicator would whether significantly lower compared with the values in the control group. Consequently, the intragastric administration of pentoxifylline to rats improved the initial hemorheological parameters at low speed and slowed down the development of “hyperviscosity” in vitro. Compound 2 compared with control and compound 1 had a pronounced effect on blood viscosity. In animals treated with compound 2 compared with the control and the group of animals with the introduction of compound 1, immediately after blood collection, a significant decrease in blood viscosity was revealed both at low shear rates and after incubation at high shear rates (Table 12).

The initial values of red blood cell aggregation between groups did not differ significantly. Incubation of the blood of rats in the control group led to an increase in red blood cell aggregation by 37%. The introduction of pentoxifylline prevented the acceleration of red blood cell aggregation during incubation. Intragastric administration of Compound 2 to rats slowed down the aggregation of red blood cells during blood incubation, which resulted in a significant increase in the half-life of red blood cell aggregation compared to control and Compound 1 by 55% and 22%, respectively (Table 13).

Thus, compound 2 is able to limit the development of blood hyperviscosity and increase ex vivo red blood cell aggregation. The effect of compound 2 is more pronounced than the effect of compound 1 and is comparable to pentoxifylline.

Table 12

The effect of a single intragastric administration of pentoxifylline (400 mg / kg) and compounds 1 and 2 (100 mg / kg) on blood viscosity up to (1) and

after 1 h after incubation at room temperature at 20.0 ± 0.4 ° C 2

Note: - p <0.05 compared with the initial values;

- p <0.05 compared with the control group;

* - p <0.05 when comparing the group of animals treated with compound 2 with

a group of rats treated with compound 1.

Table 13

The effect of a single intragastric administration of pentoxifylline (400 mg / kg) and compounds 1 and 2 (100 mg / kg) on the floor during the period of red blood cell aggregation before (1) and 1 hour after

incubation at room temperature 20.0 ± 0.4 ° C 2

Note: - p <0.05 compared with the initial values;

⁺ - p <0.05 compared with the control group;

^# - p <0.05 when comparing the group of animals treated with compound 2 with

a group of rats treated with compound 1.

Experiments to study the antiplatelet activity of compounds 1 and 2 were carried out on 20 male Wistar rats weighing 300-320 g, which Rats were divided into 4 groups (5 animals each). An hour before blood collection, the animals of the experimental groups were injected with pentoxifylline (400 mg / kg) or compounds 1 and 2 (100 mg / kg). Equivolume amounts of starch gel were administered to rats of the control group. Blood was taken from the common carotid artery under ether anesthesia. A 3.8% sodium citrate solution in the ratio of 1: 9 blood was used as an anticoagulant. Platelet aggregation was determined by the nephelometric method. According to the standard method, rich (BTP) and poor (BeTP) plasma were obtained by platelets and the platelet count was calculated. After determining the platelet count in BTP, platelet count was standardized, for which BTP was diluted with the required amount of BeTP to 400 ± 27 thousand platelets in 1 mm ³ in the sample. To evaluate the antiplatelet effect of the vascular wall, BTP and BeTP of intact donor rats were used. Platelet aggregation was evaluated in a standardized plasma on the instrument, aggregatograms were recorded using a recorder. After the addition of the aggregation inducer, a change in the optical density level of BTP was recorded. An indicator of the degree of aggregation (in%) was used as a criterion for platelet aggregation activity, characterized by a change in the optical density of BTP after adding an aggregation inducer to the cuvette. At the same time, the optical density of BeTP was taken as 100%, and the optical density of BTP was taken as 0%. ADP was used as an aggregation inducer at a final concentration of 4 · 10 ^"6 M.

The results of the studies showed that in animals of the control group the amplitude of platelet aggregation induced by ADP at a final concentration of 4-10 ^" ° M was 29 ± 2%. The amplitude of ADP-induced platelet aggregation after administration of pentoxifylline was significantly lower than the same indicator at control group. After administration of compound 2, the amplitude of platelet aggregation was also significantly significantly lower than in the control group and the group administered with compound 1 (Table 14).

Thus, compound 2 in comparison with compound 1 has a pronounced effect on vascular platelet hemostasis. Antithrombogenic activity of compound 2 may be due to both antiplatelet and endothelium protective properties.

Table 14

The effect of a single intragastric administration of pentoxifylline (400 mg / kg) and compounds 1 and 2 of 100 mg / kg on A ph-indi ovanny aggregation of t platelets

Note: - p <0.05 compared with the control group;

^# - p <0.05 when comparing the group of animals treated with compound 2 with

a group of rats treated with compound 1.

Experiments to study the antithrombogenic activity of compounds 1 and 2 were carried out on 24 male Wistar rats weighing 260-280 g. 4 groups of 6 animals each were formed. The animals in the control group were injected with starch gel. Pentoxifylline (400 mg / kg), compounds 1 and 2 (100 mg / kg) were respectively administered to rats of the experimental groups. Compounds and the equivolume amount of starch gel (2 ml) were administered intragastrically through a probe 1 time per day for 5 days. On the 5th day of the experiment, 1 hour after the last injection, the animals were anesthetized (sodium thiopental at a dose of 60 mg / kg, intraperitoneally), the left common carotid artery was isolated, and the model of intravascular thrombosis was reproduced. Cuff sensors were placed on the prepared carotid artery, and blood flow through the vessel was recorded using an electromagnetic blood flow meter. Next, strips of filter paper and plastic film were placed under the carotid artery. A strip of filter paper was superimposed on this artery from above, onto which 1 drop of a 10% solution of FeCl _{2 was} applied. Measurements of the amount of blood flow in the artery were carried out from the moment of application of iron chloride on the left carotid artery to the complete stop of the blood flow in it (time of thrombus formation). The calculation of the decrease in blood flow relative to the initial value of blood flow was expressed as a percentage. The mass of the thrombus was estimated by the gravimetric method.

In animals of the control group, after exposure to the vascular wall with a solution of FeCl ₂ , a complete cessation of blood flow through the vessel occurred. Average time full stopping blood flow to zero was 22 ± 1 minutes. The mass of the thrombus, determined one day after the application of iron chloride, was 1.40 ± 0.1 mg (Table 15). In the course of intragastric administration of pentoxifylline to rats, a decrease in blood flow by 28% from the initial value by 90 minutes of the experiment was recorded. The next day in the lumen of the carotid arteries in rats of this group of blood clots was not found. The use of pentoxifylline reduces thrombus formation in the stenosis zone. The course intragastric administration of compound 2 to animals prevented the cessation of blood flow after application with FeCl ₂ solution. By 90 minutes of the experiment, rats treated with compound 2 showed a decrease in blood flow by 9%. The introduction of compound 2 completely prevented thrombosis in the carotid artery the next day after application with a solution of FeCl ₂ . Compared to antithrombogenic activity, compound 2 significantly exceeded compound 1.

Thus, compound 2 in comparison with compound 1 has a pronounced antithrombogenic activity.

Table 15

The effect of intracranial exchange rate intramuscular administration of pentoxifylline (400 mg / kg) and compounds 1 and 2 (100 mg / kg) on the decrease in blood flow in the carotid artery for 90 minutes and weight

t omba through tk after application of iron chloride

Note: - p <0.05 compared with the control group;

^# - p <0.05 when comparing the group of animals treated with compound 2 with

a group of rats treated with compound 1.

Example 6. The study of anti-ischemic and anti-arrhythmic activity of compounds 1 and 2 was performed according to the Guidelines for conducting preclinical studies of drugs (Part One. - M.: Grif and K, 2012.- 944 p.) On the model of ischemia and rat myocardial reperfusion, which is recommended for the reproduction of ischemia and ventricular arrhythmias.

The experiments were performed on 66 Wistar male rats weighing 260-280 g. Rats were randomized into 3 groups. Compounds 1 and 2, respectively, were administered to rats of the experimental groups at a dose of 100 mg / kg in the form of a suspension on 1% starch gel (2 ml) intragastrically through a probe 1 time per day for 3 days before and 3 days after model creation. The animals of the control group were injected with an equivalent volume of starch gel according to a similar scheme. Compounds 1 and 2 were investigated using electrocardiographic (ECG) and morphological methods. To reproduce the model, the animals were anesthetized with sodium thiopental (60 mg / kg, intraperitoneally), intubated, and connected to a ventilator. After thoraco- and pericardotomy, occlusion of the left coronary artery was performed at the level of the lower edge of auricula sinistra without disturbing the topography of the heart in the chest by the method of Kogan [Kogan A.Kh. The surgical method for modeling coronary occlusion infarction and heart aneurysm in rats // Pathological physiology and experimental therapy. 1979. N ° 3. from. 79-81]. The duration of occlusion of the left coronary artery was 20 min, after which the ligature was untied and postischemic reperfusion was carried out. During the period of ischemia and 10 minutes of reperfusion, ECG monitoring was carried out in standard II lead using a computer electrocardiograph. Verification of the correctness of the ligature and the adequacy of the model was monitored by the degree of change in the height of the ST segment on the ECG. In false-operated animals, a similar surgical intervention was performed without ligature on the left coronary artery.

On the 3rd day after reperfusion, the rats were again anesthetized with sodium thiopental (60 mg / kg, intraperitoneally), intubated and connected to a ventilator, and a ligature was applied. To identify the hypoperfusion zone, 0.2 ml of a 5% patent blue violet dye solution was bolus injected into the femoral vein, and the heart was removed for the next 20-30 seconds. Parts of the myocardium with preserved perfusion were stained green, non-perfused remained unpainted.

Heart sections were prepared on a freezing microtome. Some of the sections were left in their native form to assess the hypoperfusion zone. Another part of the sections was stained with nitro-blue tetrazolium (HCT) in order to detect dehydrogenase activity in the tissue. Slices were enclosed in a glycerol-gelatin gel and scanned. The size of hypoperfusion zones and changes in dehydrogenase activity was estimated using Adobe Photoshop. Zones with a sharp decrease in dehydrogenase activity were taken as a heart attack zone. For this purpose, sections were identified on the myocardial sections, the total color intensity of which was reduced by 2.5 times or more with respect to the intact regions of the myocardium, which reflected the topography of the decrease in the same degree of dehydrogenase activity [Kogan A.Kh. The surgical method for modeling coronary occlusion infarction and heart aneurysm in rats // Pathological physiology and experimental therapy. 1979. N ° 3. from. 79-81].

The research results indicate that in the control group during the first hours after the restoration of blood supply to the left ventricular myocardium 11 animals out of 22 (50%). In animals treated with compound 2, out of 22 animals died during reperfusion 1. Mortality in the group of rats treated with compound 2 was 4%, which was significantly different from the control and the group of animals treated with intragastric compound 1 (Table 16).

In the rats of the control group, the appearance of a pathological Q wave, indicating the presence of necrotic changes in the myocardium, and a decrease in the amplitude of the T wave relative to the initial values were observed on the ECG in the first days and during the next 3 days after the resumption of perfusion of the left ventricular myocardium. After restoration of myocardial blood supply during the reperfusion period in rats of the control group, a significant decrease in the amplitude of the R wave relative to the initial values was observed, which characterizes the violation of the process of ventricular depolarization due to the shutdown of part of the myocardium from the excitation processes. In the rat group administered with Compound 2, compared with the control and the group of animals treated with Compound 1, a significant decrease in Q wave amplitude on the ECG after an ischemic / reperfusion episode was observed within 1-3 days after myocardial reperfusion. During the reperfusion period by the third day in the group of rats treated with compound 2, a significant significant increase in the amplitude of the R and T waves was revealed relative to the control and the group of animals with the introduction of compound 1 (Table 17).

As can be seen from table 19, the size of the hypoperfusion zone did not differ significantly from the total area of myocardial transverse sections. In animals treated with compound 2, compared with the control and group of rats with compound 1, there was a significant decrease in the zone of sharply reduced dehydrogenase activity from the total myocardial area and from the area of the hypoperfusion zone. Therefore, the therapeutic and prophylactic administration of compound 2 at a dose of 100 mg / kg reduces the mortality of animals after an episode of ischemia / reperfusion and significantly reduced the infarction area both from the total myocardial area and from the area of hypoperfusion compared to the same indices of the control group and the group of animals treated with the compound 1 (Table 18).

Table 16

The effect of repeated intragastric administration of compounds 1 and 2 (100 mg / kg) on the survival of animals during the first course after ischemia / myocardial epilepsy

Note: - p <0.05 compared with the control group;

^# - p <0.05 when comparing the group of animals treated with compound 2 with the group of rats treated with compound 1. Table 17

The effect of repeated intragastric administration of compounds 1 and 2 (100 mg / kg) per day

Note: - p <0.05 compared with the initial values;

⁺ - p <0.05 compared with the control group;

Table 18

The effect of multiple intragastric administration of compounds 1 and 2 (100 mg / kg) on the sizes of the hypoperfusion zone and the zone of sharply reduced dehydrogenase activity on the 3rd

Note: - p <0.05 compared with the control group;

In animals of the control and experimental groups during ischemic and reperfusion periods in all ratios, all types of Lown ventricular arrhythmias of high gradations were noted in different ratios: polytopic ventricular extrasystole, ventricular tachycardia, ventricular fibrillation. In control rats during the ischemic period, the main share in cardiac arrhythmias was ventricular fibrillation (50%) and ventricular tachycardia (27%) (Table 20). Mortality in the control group of rats was 50% (table. 16). Fatal arrhythmias, in particular left ventricular fibrillation followed by cardiac arrest, were the main cause of animal death. In animals of the experimental groups, the prophylactic administration of compounds 1 and 2 during an episode of ischemia did not significantly change the structure of the heart rhythm in comparison with the frequency and nature of arrhythmias in control animals (Table 19). Significant differences in the number and severity of arrhythmias in rats of the studied groups were revealed during the reperfusion period. In the group of animals treated with compound 2, compared with the control and the group administered with compound 1, the number of animals without ventricular arrhythmias significantly (6 times and 1.7 times, respectively) and the number of rats with fatal ventricular fibrillation significantly decreased (Table 20) . A decrease in pathological changes in the heart rhythm and a decrease in the number of rats with the most severe type of arrhythmia - ventricular fibrillation - were manifested by a decrease in the lethality of animals protected by compound 2 during the perfusion period.

Compound 2 at a dose of 100 mg / kg of body weight, in contrast to control and compound 1, when it was administered under the conditions of a model of myocardial ischemia and reperfusion, statistically significantly reduced the incidence and severity of arrhythmias, and reduced animal mortality due to fatal arrhythmias.

Thus, a significant decrease in the zone of myocardial infarction, a decrease in the frequency and severity of arrhythmias and mortality in animals protected by compound 2, is due to its anti-ischemic and anti-arrhythmic actions.

Table 19

The effect of compounds 1 and 2 on the frequency and nature of arrhythmias during acute myocardial ischemia

Note: - p <0.05 compared with the control group;

^# - p <0.05 when comparing the group of animals treated with compound 2 with

a group of rats treated with compound 1. Table 20

Note: - p <0.05 (%) compared with the control group;

^# - p <0.05 (χ ² ) when comparing the group of animals treated with compound 2 with the group of rats treated with compound 1.

The present invention expands the arsenal of agents that simultaneously have hemorheological, antiplatelet, antithrombogenic, retinoprotective, endothelial, neuroprotective, antiarrhythmic and anti-ischemic activity that increase cerebral blood flow.

Claims

Claim

1. An agent for the treatment of cardiovascular diseases, which is a mixture of 2,6-di (1, 7, 7-trimethylbicyclo [2.2.1] hept-2-yl) -4-methylphenol and its diastereomers, and 2- (1,7,7-trimethylbicyclo [2.2.1] hept-2-yl), 6- (2,2,1-trimethylbicyclo [2.2.1] hept-5-yl) -4-methylphenol and its diastereomers with the ratio 2,6-di (1, 7, 7-trimethylbicyclo [2.2.1] hept-2-yl) -4-methylphenol and 2- (1,7,7-trimethylbicyclo [2.2.1] hept-2-yl) , 6- (2,2, 1-trimethylbicyclo [2.2.1] hept-5-yl) -4-methylphenol from 60 to 95% of the mass. for the first isomer and from 40 to 5% of the mass. for the second isomer.