RU2554500C2 - Method of treating ischemic stroke - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely to neurology, and deals with the treatment of ischemic stroke. For this purpose the introduction of ubidecarenone by injection, mainly intravenous, is carried out.

EFFECT: introduction of the medication provides the reduction of the zone of brain tissue injury and reduction of neurologic deficiency expression due to the accumulation of ubidecarenone, possessing an expressed neuroprotective action, in brain tissues.

7 cl, 4 dwg, 3 ex

Description

FIELD OF THE INVENTION

The invention relates to medicine, specifically to the treatment of dysfunctions of the central nervous system caused by ischemic lesions, namely ischemic stroke.

State of the art

Ischemic stroke occupies a significant place in the structure of mortality, leads to frequent disability of the working population. One of the promising areas in neurology and clinical pharmacology is the study of drugs with neuroprotective activity aimed at improving the survival of neurons and the normalization of neurometabolic processes in brain ischemia. So, for example, many drugs with neuroprotective properties (noopept, gliatilin) are presented in oral form. However, the use of neuroprotective agents in oral forms is ineffective in acute conditions. Injection administration maximally meets the goals and conditions of emergency therapy in acute severe central nervous system lesions such as stroke. The most widespread in medical practice are injectable peptide neuroprotective drugs - cortexin, cerebrolysin, the injectable form of the drug noopept is known (RF patent No. 2330680, December 9, 2005). In addition, with ischemic stroke, the use of antihypoxants and antioxidants is justified and shown - drugs that improve the utilization of oxygen circulating in the body, inhibit the processes of free radical damage to cells and thus increase resistance to hypoxia. The antihypoxant used in the clinic for the treatment of acute stroke is the drug 2-ethyl-6-methyl-3-hydroxypyridine succinate - Mexidol (Russian Medicines Register. Encyclopedia of Medicines. M.: RLS-2006, issue 14, p. 489-490). It is a membrane protector with nootropic, antioxidant and anxiolytic effects. However, it has several disadvantages: it is not always effective, it has a set of side effects, and is also quickly excreted from the body. Coenzyme Q ₁₀ is an endogenous fat-soluble coenzyme that is part of the respiratory chain of mitochondria and shunts electron transfer from the 1st and 2nd complex of the respiratory chain to cytochrome C. It has powerful antioxidant properties and, like ubidecarenone (the international non-proprietary name for coenzyme Q ₁₀ ), is drugs and dietary supplements used in the treatment of cardiovascular diseases. The principal disadvantage of the dosage forms of ubidecarenone for oral administration is their low bioavailability (less than 2%). As a result, even the use of high doses does not provide a blood concentration sufficient to overcome the blood-brain barrier and the entry of ubidecarenone into the brain. An injection dosage form of ubidecarenone does not currently exist.

Disclosure of invention

The invention is directed to the development of a new method for the treatment of ischemic stroke using intravenous administration of ubidecarenone to ensure its neuroprotective effect.

The authors of the present invention first suggested and showed that the injection of ubidecarenone is effective in the treatment of ischemic stroke. The method proposed by the authors ensures the effectiveness of treatment by ensuring maximum bioavailability of ubidecarenone and allows you to create high concentrations in blood plasma, which in turn ensures overcoming the blood-brain barrier and the entry of ubidecarenone into the brain. The technical result of the invention is the expansion of the indications for the use of ubidecarenone, the expansion of the arsenal of drugs for the treatment of ischemic stroke.

Ubidecarenone is 2- (3,7,11,15,19,23,27,31,35,39-decamethyl-2,6,10,14,18,22,26,30,34,38-tetracontadecaenyl) -5,6-dimethoxy-3-methyl-2,5-cyclohexadiene-1,4-dione and is characterized by the structural formula below:

In the experiments conducted by the authors, the pharmacokinetics of ubidecarenone (as part of the Kudesan preparation, ZAO Akvion, Russia) was studied after intravenous administration to male rats of the Wistar strain at a dose of 30 mg / kg. The drug Kudesan (ZAO Akvion, Russia) is an oral liquid containing the active substance ubidecarenone (30 mg in 1 ml). Of the excipients, the preparation includes: alpha-tocopherol acetate - 4.5 mg, ascorbyl palmitate - 1 mg, macrogol glyceryl hydroxystearate (Cremophor RH-40) - 105 mg, sodium benzoate - 2 mg, (food grade citric acid) - 1, 6 mg, purified water - up to 1 ml. The drug Kudesan is presented in the form of a 3% and 6% solution (the dose of the active substance is 30 mg / ml and 60 mg / ml, respectively).

After 15 minutes, 30 minutes, 1, 2, 6, 8, 12, 24, 48 hours and 8 days after administration of the drug, the animals were decapitated, the brain was seized and the tissue content of ubidecarenone was determined (5 animals in the group). For this, brain tissue was homogenized, and extraction was performed twice with a mixture of ethanol 95%: hexane (2: 5). The selected upper hexane layer was evaporated to dryness. The dry residue was dissolved in an aliquot of ethyl alcohol. The determination was carried out by high performance liquid chromatography (HPLC) with coulometric detection.

Data processing was performed using the STATISTICA 6.0 software package and Microsoft Excel. The data are presented as cf. value ± stand. deviation. The differences were considered significant at p <0.05.

It was shown that the level of coenzyme Q ₁₀ in the brain tissue after injection was increased (p <0.05) relative to control animals by 54% after 15 minutes, by 72% after 30 minutes, by 71% after 60 minutes, by 75% after 2 hours , 53% after 6 hours, 36% after 8 hours and 32% after 12 hours. After 24 hours, 48 hours and 8 days there were no significant differences in the level of coenzyme Q ₁₀ compared with intact animals (figure 1).

Thus, a single intravenous administration of ubidecarenone at a dose of 30 mg / kg provides an increase in its content in the brain by more than 50% within 15 minutes after administration and maintain elevated levels of coenzyme Q ₁₀ for at least 12 hours.

The determination of doses and modes of administration of the drug can be selected individually in each specific clinical case and is obvious to a specialist. The selection of the optimal dosage of the drug for use in clinical practice was carried out by the authors of the present invention using the table of dosage correlations between humans and animals given in the manual Yanlin Wang-Fisher "Manual of stroke models in rats", 2008. According to the table for conversion of dose to average weight an adult (70 kg), it is necessary to multiply the dose of ubidecarenone proposed by the authors (30 mg / kg) by the average rat weight (0.2 kg) and by a factor of 56. The resulting dose, normalized to 70 kg, is 5 mg / kg, which corresponds to 10 ml of 3% solution "K hung up "or 5 ml of a 6% solution. In accordance with the data obtained, the inventors propose the use of a sterile composition containing ubidecarenone intravenously as a single injection at a dose of 5-10 mg / kg in the acute period of ischemic stroke or a double intravenous injection in the acute period and 6 hours after the first injection. Also, according to the standards for the treatment of acute ischemic stroke, after a single injection of the drug at a dose of 5-10 mg / kg intravenously to maintain a constant concentration of ubidecarenone in the blood, an intravenous drip of a solution of ubidecarenone at a dose of 5-10 mg / kg dissolved in 200 ml of physiological solution is possible through 2 hours after the first bolus injection. In addition, to ensure the prolonged effect of ubidecarenone, intravenous drip of a solution at a dose of 5-10 mg / kg daily for 10 days after the first bolus injection can be recommended.

The composition for the treatment of ischemic stroke proposed by the authors contains ubidecarenone as the main active ingredient, and may also additionally contain auxiliary substances and additives, in particular solubilizers and / or emulsifiers, preservatives, for example, macrogolglyceryl hydroxystearate (solubilizer), ascorbyl palmitate and / or sodium benzo , and / or citric acid (preservatives), purified water.

A brief description of the drawings (Fig. 1-4)

Figure 1. The content of coenzyme Q ₁₀ (CoQ ₁₀ ) in the brain tissue of rats after various time intervals after intravenous administration of a solution of ubidecarenone at a dose of 30 mg / kg * - p <0.05 compared with intact animals.

Figure 2. The content of coenzyme Q ₁₀ (CoQ ₁₀ ) in the ipsilateral cerebral hemisphere of rats of various experimental groups: intact; intact + physical solution; falsely operated; rats undergoing occlusion of the middle cerebral artery + phys. solution (OSMA + physical solution); rats undergoing occlusion of the middle cerebral artery + solution of ubidecarenone at a dose of 30 mg / kg (OCMA + CoQ ₁₀ ). * - p <0.05 - relative to other experimental groups.

Figure 3. The content of coenzyme Q10 (CoQ10) in the contralateral hemisphere of the brain of rats of various experimental groups: intact; intact + physical solution; falsely operated; rats undergoing occlusion of the middle cerebral artery + phys. solution (OSMA + physical solution); rats undergoing occlusion of the middle cerebral artery + solution of ubidecarenone at a dose of 30 mg / kg (OCMA + CoQ ₁₀ ). * - p <0.05 - relative to other experimental groups.

Figure 4. The content of coenzyme Q10 (CoQ10) in the cerebral hemispheres of rats after occlusion of the middle cerebral artery: in the group that received an intravenous injection of physical. solution (OSMA + physical solution), and in the group that received an intravenous injection of ubidecarenone at a dose of 30 mg / kg (OSMA + CoQ ₁₀ ). # - p <0.05 - between the ipsilateral and contralateral hemispheres.

The implementation of the invention

The study of the neuroprotective role of coenzyme Q ₁₀ was carried out on a model of ischemic stroke with reperfusion, which allows the most complete imitation of ischemic stroke in humans.

The study was conducted on male Wistar rats weighing 270-370 g. Animals were divided into 5 groups: intact animals (n = 3), intact animals that were injected with a solution of ubidecarenone at a dose of 30 mg / kg (n = 4), falsely operated animals subjected to all manipulations, except for setting the occluder (n = 9), animals that underwent occlusion of the middle cerebral artery and were injected with an equivalent amount of physiological saline (OSMA + physical solution, n = 12) and a group of animals that underwent surgery , which was administered a solution of ubidecarenone at a dose of 30 g / kg (OCMA + CoQ _10, n = 11). Under intraperitoneal anesthesia (chloral hydrate at a dose of 400 mg / kg), the operation for occlusion of the middle cerebral artery, described by Longa EZ (1989), was performed under sterile conditions. The common carotid, external carotid and internal carotid arteries were isolated. The common carotid and internal carotid arteries were squeezed using microvascular clips, and the external carotid artery was ligated. Through a small incision in the external carotid artery, an occluder filament was introduced into its lumen, which is a 30 mm nylon thread with a tip coated with silicone (manufactured by DOCCOL, USA). The filament was carefully guided through the external carotid artery into the lumen of the internal carotid artery and advanced until resistance was felt. In this way, it was possible to completely block the mouth of the middle cerebral artery and ensure the development of brain ischemia in its pool. Occlusion was performed for an hour. 45 minutes after the onset of ischemia, a test group of animals was injected with a solution of ubidecarenone at a dose of 30 mg / kg through a catheter in the femoral vein. Animals from the control group were injected with the same volume of NaCl solution. An hour after the onset of ischemia, the filament was removed, providing reperfusion. 24 hours after ischemia, neuroprotective efficacy was determined by the severity of neurological deficit (Example 1) and by the volume of the lesion (Example 2). Then, the tissue content of coenzyme Q10 was evaluated in tissues of the ipsilateral and contralateral hemispheres of rat brain by HPLC (Example 3).

Example 1

Neurological deficit was determined blindly using the modified Neuropathy Symptom Score (mNSS) scale, which uses a score of 6 parameters. Each parameter was assigned a value from 1 to 3: the higher the score, the more preserved the function.

1. Spontaneous activity of the animal in the cell for 5 minutes

The activity of the animal was recorded by the number of cell corners visited in 5 minutes. In animals treated with the drug, greater physical activity was noted compared with the control group of animals (p <0.01).

2. The symmetry of the movements of the limbs of the affected and unaffected sides of the body

In animals treated with the drug, less pronounced paralysis of the limbs developed on the side of the body opposite the affected hemisphere of the brain. They also noted a less pronounced difference in the movements of the limbs of the affected and unaffected sides of the bodies compared with animals from the control group (p <0.01).

3. The symmetry of the movements of the limbs when hanging the animal by the tail

Animals that did not receive the drug, compared with animals that received the drug, were unable to pull up to the tail and showed a more pronounced difference in the use of the right and left limbs (p <0.01).

4. The ability to climb up when sitting on a vertical grid

Animals that did not receive an injection of ubidecarenone, compared with animals that received the drug, were unable to fit on the net, fell; there was also a more pronounced difference in the use of left and right limbs (p <0.01).

5. Assessment of sensitivity when touching the affected side of the body

In animals that received an injection of ubidecarenone, a less pronounced loss of sensitivity to touch was observed compared with animals in the control group (p <0.01).

6. Assessment of sensitivity when touching vibrissa

In animals that received an injection of ubidecarenone, a less pronounced loss of sensitivity to touch was observed compared with animals in the control group (p <0.01).

The total score for all six parameters in the group of control animals was 9.0 ± 1.0, and in the group of animals that received ubidecarenone, it was significantly higher - 14.8 ± 0.8 (p <0.01).

Example 2

After determining the neurological deficit, the animals were decapitated. Brain sections were prepared, treated with metabolic dye 2,3,5-triphenyltetrazolium chloride for 20 minutes, which stained the viable tissue in raspberry color, while the necrosis zone remained unpainted - pale yellow. The obtained sections were photographed and processed using the ImagJ program (USA) to determine the affected area and calculate the volume of the stroke. The area of necrosis was calculated using a formula that takes into account cerebral edema (Zhang Y, Pardridge WM, 2001):

S _ish = S _kont - (S _ipsa -S _necr ), where

S _ish - stroke area

S _kont - area of the affected hemisphere

S _ipsa - intact hemisphere area

S _necr is the area of necrosis.

The stroke volume was calculated by the formula:

V = ΣS _ish * d

where d is the thickness of the slice.

The stroke volume in animals that received an injection of ubidecarenone solution was 3.5 times less compared to animals in the control group (38.1 ± 27.8 mm ³ and 133.0 ± 64.6 mm ^3, respectively, p <0.01 )

Example 3

The level of coenzyme Q ₁₀ in intact animals, intact animals that received a solution of ubidecarenone, and in false-operated animals did not significantly differ and amounted to 27.3 ± 1.38 μg / g, 27.7 ± 1.16 μg / g and 26.6 ± 2.77 μg / g, respectively. This confirms the above data that in intact animals after intravenous administration of a solution of ubidecarenone, the tissue content of coenzyme Q ₁₀ after 24 hours does not differ from the norm.

In operated animals receiving physical. solution, the level of coenzyme Q ₁₀ was significantly lower (p <0.05) and amounted to 23.7 ± 2.8 μg / g and 21.1 ± 2.25 μg / g in the contralateral (figure 3) and ipsilateral (figure 2) hemispheres respectively. This confirms the authors' hypothesis that in ischemic brain tissue, the levels of endogenous coenzyme Q ₁₀ decrease in both cerebral hemispheres, while its level in the ipsilateral hemisphere is significantly lower than in the opposite contralateral one. In the group of animals that underwent surgery and received an injection, there was a significant increase (p <0.05) in tissue level of coenzyme Q ₁₀ relative to the group of control animals: 28.2 ± 2.76 μg / g and 29.9 ± 2.72 μg / g in the ipsi and contralateral hemisphere, respectively (figure 4). The data obtained show that the intravenous administration of a solution of ubidecarenone in the acute period of ischemic stroke provides an increase in the tissue content of coenzyme Q ₁₀ to at least normal levels within 24 hours, which allows for a prolonged neuroprotective effect.

Thus, to ensure the neuroprotective effect, ubidecarenone should be administered intravenously bolus with a possible supportive introduction of ischemic stroke into the acute phase or immediately before thrombolytic therapy.

Claims (7)

1. A method of treating ischemic stroke, comprising injecting the patient a pharmacologically effective amount of ubidecarenone. 2. The method according to claim 1, characterized in that the injection is an intravenous administration. 3. The method according to claim 1, characterized in that in acute ischemic stroke the patient is administered the composition according to claim 1 in the form of an intravenous single injection at a dose of 5-10 mg / kg 4. The method according to claim 1, characterized in that for acute ischemic stroke the patient is administered a composition containing ubidecarenone in the form of a double intravenous injection at a dose of 5-10 mg / kg in the acute period and 6 hours after the first injection. 5. The method according to claim 1, characterized in that for acute ischemic stroke the patient is administered a composition containing ubidecarenone in the form of a single bolus intravenous injection at a dose of 5-10 mg / kg, followed by intravenous drip at a dose of 5-10 mg / kg in 200 ml of saline 2 hours after the first injection. 6. The method according to claim 1, characterized in that for acute ischemic stroke the patient is administered a composition containing ubidecarenone in the form of a single intravenous injection at a dose of 5-10 mg / kg followed by daily intravenous drip at a dose of 5-10 mg / kg in 200 ml of saline for 10 days. 7. The use of ubidecarenone for the treatment of acute ischemic stroke.

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