RU2497540C1 - Cerebroprotective agent - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, more specifically to pharmacology, neurology and cell engineering. What is described is using Napelline as a cerebroprotective agent. The mode of action of the agent is activation of cerebral neural stem cells.

EFFECT: invention achieves extending the range of high-effective cereroprotective agents.

3 tbl, 2 ex

Description

The invention relates to medicine, specifically to pharmacology, neurology and cellular technology.

A large number of cerebroprotective agents and methods for the treatment and prevention of brain damage are known [1].

The disadvantage of these funds is often their low efficiency [2].

The objective of the present invention is to expand the arsenal of highly effective cerebroprotective agents.

The task is achieved by the use of napellin as a cerebroprotective agent.

New in the invention is the use of napellin as a cerebroprotective agent.

The original napellin product used was developed and obtained by the FSBI Research Institute of Pharmacology SB RAMS (Tomsk) together with the National Research Irkutsk State Technical University (Irkutsk) and was a 0.0005% aqueous solution of this alkaloid. Napellin was extracted from plant materials (grasses of plants of the buttercup family) in the form of free bases by extraction by the standard method [3].

To date, for cerebroprotection for the treatment and prevention of the development of brain lesions, a wide range of drug and non-drug treatment methods are used [1, 2]. Moreover, the pharmacological effect of existing cerebroprotectors is mainly to protect or stimulate the preserved cellular elements. However, this concept of pharmacological intervention in some cases is completely untenable. Existing cerebroprotectors are often unable to not only completely restore the morphofunctional state of the organ, but also prevent the development of the progressive nature of the pathological process [1].

In this regard, the development of fundamentally new cerebroprotective agents with qualitatively new mechanisms of action seems to be relevant.

The information obtained in recent years on the properties and patterns of life of multipotent progenitor cells of an adult organism has opened the possibility of developing a new direction in the treatment of many diseases - using cell therapy. Moreover, the most physiological and promising approach to solving the problems of regenerative medicine is the pharmacological strategy of cell therapy, based on the principle of stimulation of endogenous stem cells (SC) by imitating the activity of natural regulatory systems of their functioning in the body [4].

The FSBI Research Institute of Pharmacology SB RAMS has shown the fundamental possibility and high efficiency of the pharmacological strategy of cell therapy for different models of diseases, including pathological conditions of the central nervous system [4]. At the same time, using the thin layer chromatography method, we determined the content of napellin (C22H33NO3) in the alkaloid fraction of plants of the buttercup family and revealed its pronounced stimulating effect with respect to stromal progenitor skin cells and wound healing processes of surface tissues when used externally [5].

At the same time, the effect of napellin on the processes of tissue regeneration of internal organs, including those affected by the pathological process of the central nervous system, and the possibility of cerebroprotection due to the activation of regeneration mechanisms associated with stem cells (SC) when used in vivo. The experiment showed unpredictable results.

The fact of the use of napellin with the achievement of a new technical result, which consists in obtaining pronounced cerebroprotective effects, is not obvious for a specialist.

New properties do not follow explicitly from the prior art in this field and are not found in the patent and scientific literature.

The present invention can be used in medicine.

Based on the foregoing, the claimed technical solution should be considered relevant criteria. “Novelty”, “Inventive step”, “Industrial applicability”.

The experiments were conducted on 68 outbred mice. The animals were obtained from the nursery of the experimental biomedical modeling department of the Research Institute of Pharmacology SB RAMS.

The studies were carried out in accordance with the rules of laboratory practice (GLP), Order of the Ministry of Health and Social Development of the Russian Federation No. 708n dated 08/23/2010 “On approval of the rules of laboratory practice”, Federal Law of April 12, 2010 No. 61-FZ “On the circulation of medicines”, “Guide on preclinical studies of drugs ”(Moscow, 2012).

Example 1

The proposed tool was obtained from the grass of Baikal aconite and high liveliness. The aerial part of plants collected during the flowering period in the Irkutsk Region was ground to a particle size of less than 5 mm, treated with a solution of sodium carbonate and subjected to continuous extraction with chloroform for 5 days. The chloroform extract was evaporated to a small volume and carefully extracted with 5% sulfuric acid. The acid extract was made alkaline with sodium carbonate to pH 9-10 and sequentially extracted first with ether and then with chloroform. The ether extract was evaporated to dryness, dissolved in a small amount of ether and chromatographed on deactivated alumina in a hexane-acetone system (90 → 50%). The ester-soluble fraction was subjected to fractional extraction with buffer solutions with increasing pH values. The ether solution remaining after extraction with the most alkaline buffer was evaporated to dryness and chromatographed on alumina in a hexane-methanol system eluting with napellin. The substance was dissolved in distilled water to a final concentration of 0.0005%.

Example 2

The pharmacological properties of the drug were studied on a model of hypoxic damage to the central nervous system. The hypoxic effect was hermetic volume hypoxia and was modeled using a heat chamber [6]. The heat chamber was a glass vessel with a calibrated volume of 500 ml (± <1.0%) and a hermetically sealed lid. The animals were placed in a heat chamber, after which its lid was tightly closed. The mice were there until the death of a convulsive seizure or the first agonal inhalation, after which they were removed and allowed to "catch their breath." To obtain pronounced encephalopathy, hypoxic exposure was modeled twice with an interval of 48 hours. Moreover, when using a 500 ml heat chamber in animals, immediately after a hypoxic injury, a quick (within 2-3 hours) restoration of all functions is observed. Functional disorders from the side of the central nervous system caused by hypoxic exposure were assessed by changes in orientational research behavior in the open field and the safety of passive avoidance reaction [7].

With this formulation of the technique, within 10-15 days after the last hypoxia of the mouse, the mice exposed to it practically do not differ from normal animals, however, from 14-21 days on, mnemonic activity begins to appear in them, and by the end of the fourth week there is a partial death. Assessment of the condition of the animals by the safety of passive avoidance reaction was carried out 48 hours later, at 7, 14, 21, and 28 days after the development of the reflex. The death of animals was taken into account by the 35th day of the experiment. The results were evaluated by the proportion of animals with a preserved reflex and by the proportion of animals that died by the end of the fifth week after a hypoxic injury. Approximate research behavior in the open field and the production of passive avoidance reaction was carried out 1 hour after repeated hypoxic injury.

The experimental setup “open field” was a camera 40 × 40 × 20 cm in size with a square floor and white walls [7]. Its floor, divided into 16 squares, had a circular hole in each square with a diameter of 3 cm. From above, the camera was illuminated by an electric incandescent lamp with a power of 100 watts, located at a height of 1 m from the floor. The mouse was placed in one of its corners and for 3 minutes, separately in the first and two subsequent minutes, the number of movements from square to square (horizontal activity), the number of hind legs rising (vertical activity), and the number of hole examinations (mink) were recorded reflex), the number of washings (grooming) and the number of bowel movements according to the number of fecal balls (boluses) and the coefficient of asymmetry of behavior was calculated in the form of the ratio of the number of horizontal movements to total motor activity and expressed as a percentage. The results of the first and two subsequent minutes of testing were evaluated separately and in total.

The passive avoidance reaction method is based on the suppression of the innate reflex of preference for the dark space available in rodents [7]. The experimental setup was a camera consisting of two compartments: large - lighted and small - dark. The animal was placed in the light compartment and soon (after 10-20 seconds), due to the innate reflex of preference for dark space, it passed into the small compartment, after which the door connecting both compartments was closed and fed to the floor of the dark compartment, consisting of parallel alternating electrodes, electric current pulses with a duration of 50 MS, a frequency of 5 Hz and an amplitude of 50 mA. After 10 seconds, the door was opened and the animal could jump out into the bright compartment with the usual floor. As a result of the described procedure, a conditioned reflex of avoiding dark space was developed in animals. When checking the reproducibility of the reflex, the animals were placed in the light compartment at an angle opposite from the entrance to the dark compartment and observed for 3 minutes. The time of the first entry into the dark compartment (latent time of entry) and the total time spent in the dark compartment were recorded. The developed reflex was considered if during all 3 minutes of observation the animal had never visited the dark compartment or the latent time of approach exceeded 150 s. The quality of the reflex was judged by the proportion of animals with the presence of a reflex. Additional indicators characterizing conditioned reflex activity and behavioral status were the number of bowel movements, grooming, the number of examinations of the entrance to the dark compartment, the time spent in the light compartment, the number of visits to the dark compartment and the time spent in the dark compartment. Animals that, after being placed in the light compartment, remained motionless, and did not approach the entrance to the dark compartment, were not taken into account when calculating the results.

In order to study the mechanisms of action of the drug using the in vitro cloning method, its effect on the neural SC of the paraventricular region of the brain of animals undergoing hypoxia was studied [8].

To study the cerebroprotective properties of the drug in a dose of 0.2 ml was administered orally in the form of a 0.0005% solution in two ways: 1) daily, starting 4 days before hypoxic exposure and 5th time 1 hour before hypoxia; 2) 1 time per day for 5 days after modeling the pathological condition. These administration schemes allow us to evaluate the cerebroprotective, antihypoxic and nootropic effects of pharmacological substances [9].

G-CSF (Neupogen, Hoffman-la Roche, Switzerland) was used as a comparison drug, which was administered subcutaneously at a dose of 125 μg / kg according to similar schemes. This tool is the closest in technical essence of the achieved result. It has a pronounced cerebroprotective effect, determined by the stimulation of the “deep reserve” regeneration mechanisms associated with SC [8], but it is quite toxic [4], which makes it unlikely to be used in effective conditions in the clinic according to these indications.

The conducted studies showed (Tables 1, 2) that the transferred hypoxia caused a decrease in the orientational-research behavior in the open field with a shift in the coefficient of asymmetry of behavior. In the hypoxic control group, an increase in the latent time of entry into the dark chamber during the development of the reflex was noted, which indicates a violation of the orientational reflex. The use of the proposed drug in both schemes led to the normalization of orienting-exploratory behavior in the open field, while G-CSF was effective only with the 2nd administration scheme (Table 1).

Manifestations of posthypoxic encephalopathy in animals of hypoxic control during registration of mnestic functions (passive avoidance reaction) began from 14 days, progressively increasing by 28 days, while in this group 65% of the animals were observed to die. The use of napellin had a protective effect on animals. There was an increase in the level of reproducibility of the reflex and the prevention of death of animals. In this case, the use of the comparison drug was accompanied by the development of therapeutic effects only when it is administered after modeling the pathological condition (Table 2)

The study of the mechanisms of the cerebroprotective action of this agent revealed its significant stimulating effect against regional neural progenitor cells of the paraventricular region of the brain, which manifested itself in a significant increase in their number (Table 3).

The results obtained indicate a pronounced cerebroprotective activity of napellin associated with activation of the realization of the growth potential by resident neural SC of the brain.

Table 1 The effect of napellin in comparison with G-CSF on the indicators of the orientational research behavior of outbred male mice in the open field after a hypoxic injury (X ± m) Observation groups, dose Total motor activity Horizontal activity Vertical activity Mink reflex Grooming Defecation Asymmetry coefficient Intact control 65.9 ± 7.4 * 32.3 ± 3.8 * 4.2 ± 1.2 24.1 ± 2.8 1.0 ± 0.3 1.5 ± 0.2 * 51 ± 4 * Hypoxic control 35.7 ± 3.2 27.5 ± 2.8 1.0 ± 0.6 5.7 ± 1.4 1.2 ± 0.5 0.2 ± 0.1 73 ± 4 G-CSF according to the first scheme 37.8 ± 4.3 28.4 ± 1.9 0.9 ± 0.3 6.2 ± 1.1 0.4 ± 0.2 0.1 ± 0.1 71 ± 5 G-CSF according to the second scheme 61.7 ± 6.3 * 29.7 ± 4.2 * 3.5 ± 1.3 * 19.7 ± 3.8 * 0.9 ± 0.4 0.3 ± 0.3 53 ± 3 * The inventive tool according to the first scheme 59.9 ± 3.7 * 31.7 ± 5.8 * 3.7 ± 0.8 * 19.7 ± 2.4 * 0.9 ± 0.2 0.2 ± 0.1 57 ± 3 * The inventive tool according to the second scheme 64.3 ± 4.2 * 30.9 ± 3.7 * 4.1 ± 0.9 * 25.6 ± 1.5 * 0.8 ± 0.2 1.3 ± 0.4 * 52 ± 3 * * - the differences are significant in comparison with the hypoxic control at p <0.05

table 2 The effect of napellin in comparison with G-CSF on the safety of the conditioned reflex of passive avoidance of outbred male mice after suffering a hypoxic injury (X ± m) Observation groups, dose The proportion of animals with a preserved reflex when checked through (in%) The proportion of animals that died by the end of 35 days, in% 48 hours 7 days 14 days 21 days 28 days Intact control one hundred* one hundred* one hundred* 92 one hundred* 0 * Hypoxic control 83 83 33 17 17 65 G-CSF according to the first scheme 80 80 33 eighteen eighteen fifty* G-CSF according to the second scheme one hundred* one hundred* 83 * 92 * 90 * 0 * The inventive tool according to the first scheme one hundred* one hundred one hundred* 92 * 92 * 0 * The inventive tool according to the second scheme one hundred* one hundred one hundred* one hundred 90 * 0 * * - the differences are significant in comparison with the hypoxic control at p <0.05

Table 3 The effect of napellin compared with G-CSF on the dynamics of the content of neural stem cells (NSC) in the paraventricular region of the brain of outbred mice after modeling encephalopathy (X ± m) Study time, days NSC, at 5 × 10 ⁴ nuclears Intact control 5.87 ± 0.39 5th Hypoxic control 6.3 ± 0.62 G-CSF according to the first scheme 5.93 ± 0.36 G-CSF according to the second scheme 12.64 ± 0.63 * The inventive tool according to the first scheme 9.3 ± 0.57 * The inventive tool according to the second scheme 11.6 ± 0.45 * * - the differences are significant in comparison with the hypoxic control at p <0.05

Literature

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Claims (1)

The use of napellin as a cerebroprotective agent.