RU2324492C1 - Agent for treatment and prevention of parkinson's disease - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: extract of the birch bark containing the specific amount of betulin is used in prevention and treatment of Parkinson's disease.

EFFECT: extract of birch bark is efficient for prevention and treatment of Parkinson's disease.

19 dwg, 7 tbl

Description

Technical field

The invention relates to the field of medicine. More specifically, it relates to finding new tools for the prevention and treatment of Parkinson's disease.

State of the art

Parkinson's disease is a chronic progressive degenerative disease of the central nervous system, clinically manifested by a violation of voluntary movements.

Parkinson's disease refers to primary, or idiopathic, Parkinsonism. Secondary parkinsonism is also distinguished, which may be due to various reasons, for example, encephalitis, excessive drug intake, cerebral arteriosclerosis. Parkinsonism applies to any syndrome in which neurological disorders characteristic of Parkinson's disease are observed.

In Russia, according to various sources, there are from 117,000 to 338,000 patients with Parkinson's disease. After dementia, epilepsy and cerebrovascular diseases, Parkinson's disease is the most common problem of older people, as evidenced by its prevalence in Russia (1996):

- 1.8: 1000 in the general population

- 1.0: 100 in the population of those over 70

- 1.0: 50 in the population of those over 80

The origin of Parkinson's disease remains unclear, however, a combination of several factors is considered as the cause of the disease:

- aging

- heredity

- some toxins and substances.

The average age at onset of Parkinson's disease is 55 years. At the same time, in 10% of patients, the disease occurs at a young age, up to 40 years. The incidence of Parkinsonism does not depend on gender and race, social status and place of residence. It is suggested that with an increase in the average age of the population in the coming years, the prevalence of Parkinson's disease in the population will increase.

Drug therapy for Parkinson's disease is aimed at filling the dopamine deficiency that develops in Parkinson's disease as a result of brain cell death, as well as inhibiting the progression of the disease, or the death of brain neurons - neuroprotective therapy.

Cholinolytic agents, aminoadamantane derivatives, DOPA-containing agents, type B monoamine oxidase (MAO) inhibitors, catechol-O-methyltransferase (COMT) inhibitors and dopamine receptor agonists are used for pharmacotherapy of parkinsonism.

Anticholinergics

This group of drugs began to be used to treat Parkinson's disease more than 100 years ago, but even now it has not completely lost its significance. In clinical practice, anticholinergic drugs are used both in isolation and in combination with other anti-Parkinsonian drugs, however, in recent years, a rather restrained attitude towards their use has prevailed due to relatively low effectiveness and a wide range of side effects. Cholinolytic drugs do not make up for dopamine deficiency and do not stop the progression of the disease, but only inhibit the increase in the activity of cholinergic systems. The use of these drugs is indicated in the initial stages of the disease and with individual clinically detectable positive dynamics. Drugs of this group are contraindicated in glaucoma, prostate adenoma, intellectual and mnestic disorders.

Cholinolytic drugs have the greatest impact on rigidity and autonomic manifestations of parkinsonism. Only a few are effective against tremors.

Commonly used anticholinergic drugs include cyclodol, biperiden.

Amantadine derivatives

There are several suggestions regarding the mechanism of action of amantadine and its analogues. The drugs of this group have a mild anticholinergic effect and inhibit the reuptake of dopamine, and also give an amphetamine-like effect, contributing to the release of dopamine from presynaptic terminals. In recent years, it has been established that amantadine acts as an antagonist of NMDA receptors, which, taking into account the proven impairment of glutamatergic neurotransmission in Parkinson's disease and its significance in changing corticostrial, thalamocortical and other relationships, is also one of the possible mechanisms of action of drugs of this group.

Amantadines are quite effective in rigid and akinetic forms, they have less effect on tremor. Their distinctive feature is low toxicity, rare side effects, and practically no contraindications, however, in general, the effectiveness of these drugs is low and no more than half of the patients improve (Walker et al., 1972). Side effects usually include dizziness, dry mouth, anxiety, and visual hallucinations. Drugs of this group should not be used in elderly patients with intellectual and mnestic disorders.

Amantadine hydrochloride and sulfate are among the drugs of this group.

Dofa - containing drugs

The role of dopamine, as a possible primary therapeutic agent in the treatment of Parkinson's disease, has been studied since the 50s, when Carlsson noted that symptoms of parkinsonism are caused by reserpine. In subsequent years, a decrease in the dopamine content in the striatum in Parkinson's disease was found (Ehringer, Hornykiewicz, 1960), which led to the emergence of a significant amount of dopamine-containing drugs, which are now the "gold standard" of treatment for Parkinson's disease.

Dopamine does not cross the blood-brain barrier. Therefore, the metabolic precursor of dopamine levodopa (L-dioxiphenylalanine), which passes through the blood-brain barrier and is converted into dopamine in the dopaminergic neurons by the action of cerebral DOPA decarboxylase (DDC), is used for replacement therapy (N.Quin, 1987).

One tablet of “pure” levodopa contains 250 or 500 mg of the active substance; the maximum daily dose is 3 g. However, due to the peculiarities of the metabolism of L-DOPA, which undergoes "premature" biochemical transformations outside the brain through decarboxylation, transamination and methylation, pure levodopa is practically not currently used.

The combination of levodopa with peripheral L-DDK inhibitors carbidopa or benserazide is pathogenetically justified and therefore widespread in the world. This allows you to reduce the dose and increase the duration of the drug (V.N. Shtok, 1995; V.N. Golubev et al., 1999, N.Quinn, C.Olanow, 1996). The most commonly used drugs of this group are madopar (levodopa / benserazide in a ratio of 4: 1), nakom (levodopa / carbidopa - 10: 1), cinematography (levodopa / carbidopa - 10: 1).

Side effects of levodopa are manifested in three areas: in the autonomic nervous system (nausea, vomiting, orthostatic hypotension, tachycardia), the motor system (dyskinesia, clinical fluctuations), the mental sphere (anxiety, agitation, hallucinations, depression with the danger of suicide), in addition, teratogenic effect of the drug was noted. Care must be taken while using other drugs that can enhance the therapeutic and side effects of levodopa (adrenergic agonists, tricyclic antidepressants) or, conversely, weaken them (vitamin B6, antipsychotics, reserpine, methyldopa).

MAO Inhibitors (B)

MAO inhibitors (B) by their pharmacological action interfere with the metabolism of dopamine formed by oxidative deamination with its conversion ultimately to homovanilic acid. Selegiline has also been shown to inhibit dopamine reuptake. MAO inhibitors thus increase the dopamine content in the brain tissue, preventing its breakdown. In addition, there is evidence of the antioxidant effect of MAO inhibitors (B), their neuroprotective ability, which justifies the use of selegiline in the early stages of parkinsonism (J. Knoll, 1996; Myllyla et al., 1992).

More effective in action, especially in the later stages, is the combination of MAO (B) inhibitors with DOPA-containing drugs, which can significantly reduce the dose of L-DOPA, and hence the severity of side effects.

Dopamine receptor agonists

An important and promising direction was the search for drugs that can "bypass" the degenerating neuron, bypassing its presynaptic part, directly on dopamine receptors. So a new class of antiparkinsonian drugs appeared - dopamine receptor agonists.

Dopamine receptor agonists are indicated as monotherapy in the early stages of the disease and at a later date in combination with levodopa drugs. In the early stages of the disease, the effectiveness of dopamine agonists is comparable to that of levodopa drugs. However, after several years, the effectiveness of treatment with dopamine agonists decreases and there is a need for the addition of levodopa drugs. The literature widely discusses the extent to which the use of dopamine receptor agonists can delay the development of complications when using L-DOPA preparations.

In patients with moderate or significantly pronounced signs of the disease, the addition of dopamine agonists to levodopa drugs can in many cases increase the effectiveness of treatment and reduce the severity of motor fluctuations. (A.Lieberman et al., 1983; C. Goetz et al., 1985; R. McDonald and R. Horowski, 1983; Y. Mizuno et al., 1995).

Side effects of dopamine agonists are nausea, vomiting, orthostatic hypotension, which is caused by stimulation of peripheral dopaminergic receptors. Among the side effects due to the effect on central dopaminergic receptors include hallucinations, nightmares, changes in higher mental functions. Some patients have constipation.

Currently, considerable experience has been gained with the use of dopamine receptor ergoline agonists (bromocriptine, pergolide, lisuride).

COMT Inhibitors

One of the promising and significant achievements in recent years has been the discovery of a fundamentally new, highly effective class of antiparkinsonian drugs - COMT inhibitors. COMT is one of the main enzymes that ensure the metabolism of levodopa, dopamine, other catecholamines and their metabolites. COMT is a fairly common enzyme; it is present in various parts of the body, including neurons and glia, with the exception of nigrostriatal dopaminergic neurons (A.Kastner et al., 1994). Peripheral COMT inhibitors reduce the metabolism of levodopa and thereby increase its content in the striatum.

It should be noted that drugs that are both central and peripheral COMT inhibitors (tolcapone), according to most authors, give significantly more pronounced side effects (K.Holm, C.Spencer, 1999). The feasibility of using tolcapone is currently under discussion.

SUMMARY OF THE INVENTION

Despite a fairly wide range of drugs used to treat Parkinson's disease that are actually used and under study, the task of finding new drugs for this purpose remains urgent, which is due not only to the insufficient effectiveness of the known drugs, but also to their side effects. It is to solve this problem that the present invention is directed.

Its essence lies in the unexpectedly discovered by the inventors of the possibility of using for the prevention and treatment of Parkinson's disease and, accordingly, for the manufacture of medicines for this purpose, the birch bark extract of the upper white part of the birch bark. The extract is a combination of triterpene compounds, with a predominance (up to 70%) of betulin (betulinol).

Disclosure of invention

In previous studies of birch bark extract by the authors of the present invention (application for invention of the Russian Federation No. 2005138574), it was found that in behavioral tests of general and directed screening in mice, the drug showed a psychotropic effect, the profile of which can be characterized as a manifestation of dopamine-positive activity, in particular, due to the fact that:

1. the extract at a dose of 50 mg / kg increases horizontal motor activity in the Ugo Basile actometer;

2. the extract enhances the timoleptic effect of the antidepressant Pyrazidol with the combined introduction of “despair” in the test;

3. The extract causes a nootropic effect in the tests “cross-shaped labyrinth” and “conditioned reflex of passive avoidance”.

In addition, other researchers have obtained experimental evidence in favor of the antioxidant properties of birch bark extract.

The totality of the above experimental data suggested that the combination of the described properties meets the requirements that can be presented to a drug with antiparkinsonian potential (Tillerson JL, Caudle WM, Reveron ME, Miller GW Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate temperature 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Exp. Neurol., 2002, 178: 80-90).

Further studies, a set of experiments using standard behavioral and pharmacological tests recommended for the detection of antiparkinsonian activity (Voronina T.A., Valdman E.A., Nerobkova L.N. Methodological guidelines for the study of antiparkinsonian activity of pharmacological substances. In the book: "Guidelines for the experimental (preclinical) study of new pharmacological substances", Moscow, 2000, 147-152), the essence and results of which are presented below, confirmed this assumption.

The studies were carried out using Dry Birch Bark Extract (BES) manufactured by Birch World LLC, Moscow, Russia and used for the manufacture of dietary supplements. The experiments were conducted on 196 male mice of the C57 / BI line with a weight of 20-25 g contained in a laboratory vivarium on a standard diet.

BAS was administered to mice 1.5 hours prior to testing using a probe intragastrically in the form of a suspension in a 1% solution of potato starch at a dose of 50 mg / kg of animal weight. The control group mice were injected with equivalent volumes of 1% starch.

The research results are reflected in the drawings, which depict:

figure 1 - Effect of BAS on the total time of catalepsy caused by haloperidol.

figure 2 - The effect of BAS on the latent period of tremor caused by Arecolin, 90 minutes after ingestion.

figure 3 - Dynamics of the influence of BAS on the latent period of the onset of tremor in mice.

figure 4 - The effect of BAS on the duration of arecoline tremor in mice.

figure 5 - Effect of BAS on the duration in mice caused by Arecolin, 90 minutes after ingestion.

6 - The effect of a 5-fold preliminary introduction of BAS (50 mg / kg / day) on the oligokinetic effect of MPTP (24 mg / kg) in the actometer (** / * - significant difference from column 2).

Fig.7 - The effect of a single injection of BAS (50 mg / kg, by mouth), Midantan (20 mg / kg, c, b) and their combination on the horizontal locomotor activity of C57BI / 6 mice in an open field test (arithmetic mean ± error standard deviation).

FIG. 8 - The effect of a single administration of BAS (50 mg / kg, by mouth), Midantan (20 mg / kg, c, b) and their combination on the motor activity of C57BI / 6 mice in an Ugo Basile actometer (arithmetic mean ± standard deviation).

Determination of the ability of birch bark extract (BAS) to alter the catalepsy caused by haloperidol in mice

Haloperidol is a typical antipsychotic derived from butyrophenone. Its action comes quickly and lasts a long time. The mechanism of action of haloperidol is associated with blockade of dopamine receptors, a central alpha-adrenergic blocking effect, and disruption of the process of reverse neuronal uptake and deposition of catecholamines. The most common side effect of haloperidol is extrapyramidal disorders.

In the experiment, 48 male C57BL / 6 mice were used at the age of about 2 months (body weight 18-23 g) randomly distributed into 4 experimental groups:

1. Oral - 1% starch; in / b - saline solution (FR);

2. Oral - BES; v / b - FR;

3. Oral - 1% starch; iv - 1 mg / kg haloperidol;

4. Oral - BES; in / b - 1 mg / kg haloperidol.

The duration of the cataleptic state of each mouse was recorded for 2 min sessions 30, 60, and 90 min after haloperidol administration. A suspension of BAS was administered using an intragastric probe inside 1.5 hours before the injection of an antipsychotic. The results of statistical processing of data by groups are presented in figure 1.

The statistically significant effect of catalepsy developed by 90 minutes after the administration of an antipsychotic (chart on the right), taking 112 seconds from a 120-second session (p <0.02). The effect of BAS itself after oral administration at a dose of 50 mg / kg did not differ from the placebo effect, however, it significantly (more than doubled, p <0.05) reduced the duration of catalepsy induced by haloperidol.

Therefore, BAS under these experimental conditions exhibits dopamine-positive activity, counteracting the effect of the blocker of DA receptors of haloperidol.

Determination of the ability of birch bark extract (BAS) to influence tremor caused by arecoline in mice.

Acetylcholine (AX) is the neurotransmitter of numerous interneurons of the caudate nucleus, and it is in this structure that the highest concentration of AX is observed (Guttman M., Kish SJ, Furukawa Y. Current concepts in the diagnosis and management of Parkinson's disease. CMAJ, 2003, 168 (3) , 293-301). The cholinergic model of Parkinson's syndrome is reproduced by the primary activation of the caudate nucleus cholinergic neurons by systemic administration of arecoline or oxotremorine. In this experiment, an Arequolin m-cholinergic agonist was used in a standard tremorogenic dose of 25 mg / kg, subcutaneously.

In the experiment, 24 male C57BL / 6 mice were used at the age of about 2 months (body weight 18-23 g), randomly distributed into 2 experimental groups:

1. Oral - 1% starch; s / c - 25 mg / kg arecoline;

2. Oral - BES; s / c - 25 mg / kg arecoline.

A suspension of BAS (50 mg / kg) was administered in accordance with the protocol presented above. 30, 60, 90, 120 and 150 minutes after the administration of arecoline in mice, the following were recorded: a) the latent period of the onset of tremor (sec); b) duration of tremor (sec).

The results of statistical processing of the data are presented in Figs. 2, 3, 4 and 5. It was found that the administration of arecoline on the background of placebo leads to the onset of tremor by 33 seconds, while the same on the background of 50 mg / kg BES delays the beginning of tremor to 73 seconds ( Figure 2; p <0.00000).

Moreover, the latent period under the influence of BAS increases within 2 hours after the introduction of arecoline, and then begins to decrease (figure 3).

The dynamics of the duration of arecoline tremor after administration of BES is presented in Fig. 4, from which it follows that the action of the extract leads to a decrease in duration up to one and a half hours after administration of arecoline. In later periods of observation (2 and 2.5 hours), the effectiveness of BES disappears.

Thus, the maximum effectiveness of the action on the duration of tremor caused by arecoline occurs in 90 minutes (F (1, 22) = 4.9959 at p = 0.03588) (Fig. 5).

Thus, BAS demonstrates the presence of cholinolytic properties, manifested in a decrease in the latent period and the duration of arecoline tremor.

Determination of the ability of birch bark extract (BAS) to influence the decrease in motor activity of mice in the Ugo Basils actometer and “open field”, on a model of parkinsonism syndrome caused by systemic administration of MPTP neurotoxin in the mode of prophylactic use of BES

The model of parkinsonism syndrome caused by the administration of MPTP neurotoxin (4-phenyl-1,2,3,6-tetrahydropyridine) is the most appropriate model of parkinsonism, since MPTP selectively damages dopaminergic neurons of the substantia nigra. The toxin under the influence of MAO-B in astrocytes turns into 1-methyl-4-phenylpyridinium (MFP +), which is captured by DA-ergic neurons using the dopamine transporter. The effects of MPTP are due to the toxic effect of MPP +, which causes energy deficiency due to inhibition of complex-1 of the mitochondrial respiratory chain. This effect is most pronounced in primates, and in mice in the C57BI / 6 line (Feldman RS, Meyer JS, Quenzer LF Parkinson's disease and Alzheimer's disease. In: Principles of Neuropsychopharmacology, Sinauer Associates, Inc., Publishers, Massachusetts, 1997, 861 -909).

The present experiment was aimed at detecting the alleged antitoxin activity of BAS, when MPTP was administered once against the background of a preliminary 5-fold application of an extract suspension. In this experiment, it was supposed to evaluate the possible “preventive” significance of BAS.

In the experiment, 36 male C57BI / 6 mice were used. A suspension of BAS was administered orally daily at a dose of 50 mg / kg / day for 5 days. A toxin at a dose of 25 mg / kg or a placebo was administered 30 minutes before the start of a 10-minute observation of motor activity in the Ugo Basile actometer.

Animals were randomly assigned to 3 experimental groups:

1. Inside - 1% starch 5 times; in / b - saline (RF) 1-fold;

2. Inside - 1% starch 5 times; v / b - MPTP 1-fold;

3. Inside - BES 5 times; v / b - MPTP 1-fold.

The general behavior of animals after the introduction of BAS or placebo did not reveal significant differences from the usual. However, after the introduction of the toxin in mice, deviations from normal vegetative and behavioral signs were observed: piloerection, salivation, retro-pulsation, respiratory failure.

Statistically processed indicators of horizontal motor activity, automatically measured in the Ugo Basile actometer for a 10-minute session, are presented in Fig.6.

The data obtained indicate that administration of BES to C57BI / 6 mice for 5 days at 50 mg / kg per day leads to a restoration of the total number of horizontal movements in the Ugo Basile actometer in a 10-minute session by 50% (p <0.01), reduced by a single injection MPTP on the 5th day of the experiment.

The result indicates that the extract has protective properties against oligokinesia caused by the toxic effect of MPTP on DA-ergic neurons.

Determination of the ability of birch bark extract (BAS) to influence the reduction of the motor activity of mice in the actometer and “open field”, on the model of the parkinsonism syndrome caused by the systemic administration of MPTP neurotoxin in the regimen of therapeutic effect of BAS

The present experiment was aimed at detecting the alleged antitoxin activity of BAS against the background of a double preliminary use of MPTP. In this version of the experiment, it was supposed: a) to evaluate the intrinsic antitoxin activity of BAS; b) evaluate the ability of the extract to modulate the effect of the well-known anti-Parkinsonian drug Midantan (Morozov I.S., Petrov V.I., Sergeeva S.A. Pharmacology of adamantanes, Volgograd, 2001).

In the experiment, 88 male C57BI / 6 mice were used. A toxin at a dose of 30 mg / kg or a placebo was administered intraperitoneally once a day for 2 consecutive days. BES was administered on the second day once in a dose of 50 mg / kg 1.5 hours before MPTP injection. Midantan was administered once at a dose of 20 mg / kg, ip 30 minutes before the introduction of the toxin. Previously, animals were randomly assigned to 5 experimental groups:

Table 1 Experiment Log Group number 1st day 2nd day 1 (n = 12) Saline solution, in / b Placebo inside
Saline solution, in / b 2 (n = 19) MPTP, v / b Placebo inside
MPTP, v / b 3 (n = 19) MPTP, v / b Midantan, w / w
MPTP, v / b 4 (n = 19) MPTP, v / b BES, inside
MPTP, v / b 5 (n = 19) MPTP, v / b BES, inside
Midantan, w / w
MPTP, v / b

30 minutes after the second injection of MPTP in mice, the behavior in the actometer (5 min) and in the “open field” (3 min) were sequentially evaluated.

30-40 minutes after the administration of MPTP on the second day, the horizontal motor activity (the number of crossed squares) significantly decreased from 101.17 ± 6.74 in group 1 to 4.58 ± 1.21 in group 2 (p = 0.000000, Student t-test).

The results of the introduction of BAS, midantan and their combination on horizontal activity are presented in Fig.7. It is seen that the acute effect of 50 mg / kg BES is almost equipotential to the action of the Midantan comparison drug (20 mg / kg): the first increased the number of movements to 331% (p = 0.021, Student's t-test), and the second to 327% (p = 0.008, Student's t-test) relative to this indicator in the group with MPTP.

The effectiveness of using their combination also differed from the toxin effect - 413% (p = 0.007, Student t-test), but statistical significance from the results of the influence of drugs separately on noted.

Similar results were obtained in the case of measuring the motor activity of the same groups of mice using an actometer (Fig. 8):

Twofold administration of the toxin led to a decrease in the number of movements from 234.8 ± 32.2 in group 1 to 11.2 ± 3.2 in group 2 (p = 0,000; Student's t-test). In turn, the acute use of BAS, Midantan and their combination partially restored the induced motor deficit to 265% (p = 0.013; student t-test), 275% (p = 0.033; student t-test) and 311% (p = 0.040 ; t-student test), respectively, relative to the values of group 2, taken as 100%.

It should be noted that not only the number of movements, but also the qualitative composition of motor activity changed under the influence of MPTP and drugs: the introduction of the toxin also led to a decrease in vertical activity (stance) and search activity (peeping into holes) in the “open field” test from 31: 1: 1.7 in group 1 to 0.11 ± 0.11 in group 2 (p = 0.000; Student t-test) and from 5.3 ± 0.1 to 0.6 ± 0.2 (p = 0.000 ; t-student test), respectively.

The introduction of BES and its combination with Midantan partially, but statistically significantly restored these parameters of locomotor activity selectively. Thus, the extract and combination increased the reduced rate of “vertical activity” with a statistical reliability of 0.04 and 0.03 according to the Mann-Whitney test, respectively.

Thus, experimentally selected conditions made it possible to detect in two test variants (“open field” and automatic actometer Ugo Basile) that birch bark extract at a dose of 50 mg / kg, once administered orally, has the ability to partially but statistically significantly compensate for locomotor deficiency in male mice C57BI / 6 caused by double administration of MPTP toxin.

This effect was quantitatively comparable with the acute effect of the clinically common anti-Parkinsonian drug Midantan (Amantadine) used in an intraperitoneal dose of 20 mg / kg.

The combination of these substances also led to an increase in the number and improvement of the structure of motor activity in mice with MPTP-induced parkinsonism syndrome, somewhat better in comparison with the effects of both drugs separately.

The purpose of the further study was to conduct a further set of in vitro and ex vivo experiments aimed at studying the alleged dopamine-positive properties of BAS using generally accepted neurochemical approaches characterizing the pharmacodynamics of the specific effects of BAS.

To achieve the goal, the following tasks were set:

1. To evaluate the possibility of interaction of BAS with receptors of dopamine, serotonin, glutamate and acetylcholine using membrane preparations and tritium-labeled ligands of these receptors.

2. To study the effect of BAS on the functional indicators of presynaptic activity: synaptosomal uptake, dopamine biosynthesis and secretion, as well as dopamine and serotonin metabolism in homogenates of competent brain structures of mice and rats.

Materials and animals

The experiments were conducted on male mice of the C57 / BI strain with a mass of 20-25 g and male Wistar rats with a weight of 180-200 g contained in a laboratory vivarium on a standard diet.

In ex vivo experiments, BAS was administered to animals 1.5-2 hours before decapitation using a probe inside in the form of a suspension in a 1% solution of potato starch at a dose of 50 mg / kg. Individuals of control groups were injected with equivalent volumes of 1% starch.

Research results

Experiment 1. Study of the influence of BAS on specific binding to neurotransmitter receptors in rat brain structures

The setting of this series of experiments pursued 2 goals: (1) to study whether the receptors of the main brain neurotransmitters are possible molecular targets for the primary (direct) pharmacological action of BAS - in vitro; and (2) to examine whether systemic administration of BAS does not have a significant indirect effect on receptor characteristics after 2 and 24 hours - in ex vivo conditions.

The degree of possible affinity of the extract to receptors in in vitro experiments was estimated by the IC50 value equal to the concentration of BAS inhibiting radioligand binding by 50%.

Possible dynamic changes in the receptors under the influence of BAS in ex vivo experiments were expressed in Bmax and Kd values, reflecting the number of binding sites and the degree of affinity for the receptor, respectively.

In the study of receptor binding, the following ligands were used: for dopamine D1-, D2- and D3-subtypes in the striatum - [G- ³ H] -SCH-23390, [G- ³ H] -spiperone, 7-OH- [G- ³ H] -DPAT, respectively; for serotonin 5-HT2 subtype in the frontal cortex - [G- ³ H] -ketanserin; for the glutamate NMDA subtype in the hippocampus - [3H-G] (+) MK-801; for cholinergic receptors of the nicotine subtype in the whole brain - [3H-G] - (±) Nicotine. All labeled ligands were obtained by solid-phase catalysis at OKHVA IMG RAS (Head - Academician of the Russian Academy of Sciences NF Myasoedov). The selection of specific ligands was carried out in accordance with the recommendations of IUPHAR ("Guide to Receptors and Channels", SPHAIexander, A.Mathie and JAPeters, BJP, v.147, Suppl.3, 2006).

1.1. In vitro binding to dopamine receptors.

a) Methodology

The study of radioreceptor binding to DA receptors was carried out according to the method of Sun et al. (2003) with modifications. Rat striatums were homogenized in 10 ml of ice-cold (0-4 ° C) 50 mM Tris-HCl buffer (pH 7.4 at 4 ° C) using a Teflon-glass homogenizer. The resulting suspension was centrifuged at 40,000 g for 20 min in an Optima L-70K ultracentrifuge (Beckman Coulter). After centrifugation, the supernatant was discharged, the precipitate was resuspended by repeated homogenization in the same volume of the buffer, then centrifuged again. The washing procedure was carried out three times, the precipitate was resuspended in 10 ml of 50 mM Tris-HCl incubation buffer (pH 7.4 at 37 ° C) with the addition of salts (120 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ -2H ₂ O, 1 mM MgCl ₂ -6H ₂ O) [11] and used 250 μl each in the binding procedure of [G- ³ H] ligands of dopamine receptors. The obtained membrane fraction was incubated with [G- ³ H] -SCH23390 (specific activity 60 Ci / mmol), [G- ³ H] -spiperone (95 Ci / mmol), 7-OH- [G- ³ H] -DPAT ( 120 Ci / mmol). Upon binding of 7-OH- [G- ³ H] -DPAT, in order to increase the selectivity to D3 receptors, divalent cations were excluded from the reaction mixture and 1 mM Na ₄ EDTA was added.

The [G- ³ H] ligand was added to the incubation mixture in a volume of 50 μl at a final concentration of 0.1 nM and incubated for 20 min at 37 ° C using a solid-state thermostat “Termite” (NPO “DNA-technology”). Nonspecific binding was determined in the presence of 50 μl of unlabeled ligand (20 μM), which was 12-14% of the total. Specific binding was calculated as the difference between total and non-specific binding.

IC ₅₀ with respect to the binding of [G- ³ H] ligands was determined by adding 50 μl of the test compounds to the incubation medium at final concentrations in the range of 10 ^-3 -10 ^-8 M. The volume of the incubation mixture was 500 μl. The binding process was stopped by adding 2 ml of ice-cold 50 mM Tris-HCl buffer and rapid filtration through GF / B type glass filters (Whatman), followed by washing with ice-cold buffer with a total volume of 14 ml. The filters were pre-wetted before the experiment in ice-cold 50 mM Tris-HCl buffer for 3 hours.

The filters were dried for 12 hours at room temperature, then placed in a 4 ml scintillation fluid (Bray reagent) and used for scintillation counting. The radioactivity of each sample was measured for 2 min on a Wallac 1411 scintillation counter. The counting efficiency was 40-45%.

b) Results

D1-dopamine receptors. The concentration dependence of the influence of BAS on dopamine D1-type receptors is presented in Fig.9.

Conclusion: BES does not have an affinity for D1-type dopamine receptors.

D2 dopamine receptors. The concentration dependence of the influence of BAS on dopamine D2-type receptors is presented in Fig.10.

It was shown that the influence of the generally accepted antagonist of this type of Haloperidol receptor is described by the classical curve (dashed line) and is characterized by IC50 = 0.57 μmol / L. The introduction of BAS does not reveal the effect of competition for receptor binding sites up to a maximum used concentration of 1 mM (solid curve).

Conclusion: BES does not have an affinity for D2-type dopamine receptors.

D3 dopamine receptors. The concentration dependence of the influence of BAS (Betulin) on dopamine DS-type receptors is presented in Fig. 11.

The influence of the standard antagonist of this type of receptor (+) - 7-OH-DPAT is described by the classical curve (dashed line) and is characterized by an IC50 value of 14.6 nmol / L. The introduction of BAS did not reveal the effect of competition for receptor binding sites up to a maximum used concentration of 1 mM (solid curve).

Conclusion: BES does not have an affinity for D3-type dopamine receptors.

1.2. In vitro serotonin receptor binding

a) Methodology

The study of radioreceptor binding to CT receptors was carried out according to the method of Sun et al. (2003), described for the study of dopamine D2 receptors (see 1.1.) With the exception that the membrane preparations of the frontal cortex were taken into the study, and [G-3H] -ketanserin with a specific radioactivity of 60 curie / mmol was used as a specific ligand .

b) Results

Serotonin 5-HT2 receptors

The concentration dependence of the influence of BAS on serotonin receptors of 5-HT2 type is presented in Fig. 12.

It can be seen that the influence of a standard antagonist of this type of ketanserin receptor is described by the classical curve (dashed line) and is characterized by an IC50 value of 0.70 μmol / L. The introduction of BAS did not reveal the effect of competition for receptor binding sites up to a maximum used concentration of 1 mM (solid curve).

Conclusion: BES does not have an affinity for serotonin receptors of 5-HT ₂ type.

1.3. In vitro binding to NMDA receptors.

a) Methodology

The study of binding to NMDA subtypes of receptors was carried out according to the method of L.-M. Zhou et al. (1996) with modifications. Tritium-labeled MK-801 (disocylpin) with a specific activity of 210 Curie / mol was used. The hippocampal tissue or cerebellum was homogenized in a Potter homogenizer (Teflon glass) in 10 volumes of 5 mM HEPES / 4.5 mM Tris buffer (pH 7.6) containing 0.32 M sucrose (buffer No. 1). The homogenate was diluted to 50 volumes of assay buffer No. 2 (5 mM HEPES / 4.5 mM Tris buffer (pH 7.6) and centrifuged for 10 min at 1000 g. The supernatant was collected and centrifuged again for 20 min at 25000 g. The pellet was homogenized in 50 volumes of buffer no. 2 and centrifuged for 20 min at 8000 g. The supernatant and its soft, unsteady layer were collected and centrifuged for 20 min at 25000 g. The resulting pellet was suspended in buffer No. 3 (5 mM HEPES / 4.5 mM Tris buffer containing 1 mM Na ₄ EDTA (pH 7.6 ), and the suspension was again centrifuged. This washing procedure was carried out four times, and when EDTA was removed by ice-cold washing. The final precipitate was resuspended in 5 volumes of buffer No. 2 and stored in liquid nitrogen. The reaction mixture (final volume 0.5 ml) contained 200 μl of buffer No. 2, 50 μl of labeled ligand (50 nM pp) and 250 μl protein suspension. Non-specific binding was determined in the presence of 50 μl of unlabeled ligand.

The reaction mixture was incubated at room temperature for 2 hours. At the end of the incubation, the samples were filtered through GF / B glass fiber filters (Whatman), pre-soaked in 0.3% polyethyleneimine for 2 hours at 4 ° C. Each tube was washed once with cold buffer No. 2, then the filters were washed three times with the same volume of buffer. The filters were air dried until completely dry and transferred to scintillation vials. Filters were filled with 5 ml of scintillation mixture containing 4 g of PPO, 0.2 g of POPOP per 1 liter of toluene. Radioactivity was determined on a Wallac 1411 scintillation counter, with a counting efficiency of about 45%.

b) Results

Glutamate NMDA receptors

The concentration dependence of the effect of BAS (Betulin) on glutamate receptors of the NMDA type is presented in Fig.13.

It can be seen that the influence of the standard antagonist of this type of receptor MK-801 (disocilpin) is described by the classical curve (solid) and is characterized by an IC50 value of 7.3 nmol / L. The introduction of BAS (Betulin) reveals the effect of competition for receptor binding sites with IC50 = 8.5 μmol / L.

Conclusion: BES has a micromolar affinity for glutamate NMDA receptors.

1.4. In vitro binding to n-cholinergic receptors

a) Methodology

The study of radioreceptor binding to nicotinic receptors was carried out according to the method (Michael et al., 1986) with modifications. The final volume of the incubation mixture contained 50 μl of [3H-G] (±) Nicotine, 250 μl of buffer and 200 μl of protein suspension of the membranes. To determine non-specific binding, unlabelled nicotine was added in a concentration range from 10 ^-10 to 10 ^-4 M. The tubes were thermostated in a shaker-incubator at 37 ° C for 40 minutes (Charles et al., 1997). The tubes were then cooled in ice for 20 minutes (Regina et al., 2002). Filtration was carried out through GF / C filters, previously moistened for a day in a 0.3% solution of polyethyleneimine at 4 ° C, followed by washing with cold buffer. The filters were transferred to scintillation vials and 5 ml of dioxane-based scintillation fluid was added. Radioactivity was determined on a Wallac 1411 scintillation counter with a counting efficiency of about 42%.

b) Results

Nicotinic Acetylcholine Receptors

The concentration dependence of the influence of BAS on n-cholinergic receptors is presented in Fig. 14.

It can be seen that the influence of the standard antagonist of this type of receptor, Nicotine, is described by the classical curve (solid) and is characterized by an IC50 value of 13.4 nmol / L. The introduction of BAS (Betulin) does not show the effect of competition for receptor binding sites up to 10 ^-4 mol / L.

Conclusion: BES does not show affinity for nicotinic choline receptors.

2.1. Ex vivo dopamine receptor binding

The results of a study of the effect of a single administration of a suspension of BAS (50 mg / kg, by mouth) on the receptor binding constants of labeled spiroperidol after 2 and 24 hours in comparison with the placebo effect are presented in Fig. 15.

From the data in the drawing it follows that the introduction of BAS leads to a decrease in the binding of the labeled ligand in the striatum both after 2 hours and one day after injection. Moreover, these changes do not occur due to a decrease in affinity for dopamine receptors (Cd), but due to BES-induced decrease in the number of binding sites (Bmax): after two hours, their density drops from 904 fmol / mg protein in the control to 670 fmol / mg protein, continuing decrease after 24 hours to 625 fmol / mg protein.

Conclusion: acute administration of BAS at a dose of 50 mg / kg induces a gradual decrease in the density of D2-dopamine receptors in the striatum of rats.

2.2 Ex vivo binding to serotonin receptors

The results of a study of the effect of a single administration of a suspension of BAS (50 mg / kg, by mouth) on the receptor binding constants of labeled ketanserin after 2 and 24 hours in comparison with the placebo effect are presented in Fig. 16.

From the data in the drawing it follows that the introduction of BAS leads to an increase in the density of serotonin receptors in the frontal cortex after 2 hours from 106 fmol / mg protein to 164 fmol / mg protein, while after a day there is a slight decrease in this indicator (130 fmol / mg protein) , however, not reaching values with placebo. Noteworthy is the decrease in the affinity of the ligand for the receptor from 5 to 8 nM observed 2 hours after the introduction of BAS, which disappears after 24 hours (5.9 nM).

Conclusion: acute administration of BAS at a dose of 50 mg / kg induces a transient increase in binding to serotonin receptors in the cerebral cortex.

2.3 binding to ex vivo glutamate NMDA receptors

The results of a study of the effect of a single administration of a suspension of BAS (50 mg / kg, by mouth) on the receptor binding constants of labeled MK-801 after 2 and 24 hours in comparison with the placebo effect are presented in Fig. 17.

From the presented data it follows that the introduction of 50 mg / kg BAS leads to a significant increase in the density of NMDA receptors in the rat hippocampus after 24 hours from 3154 fmol / mg protein to 8276 fmol / mg protein without changing affinity. At the same time, 2 hours after the introduction of BAS, a transient deterioration in affinity for receptors by 22% is also noted.

Conclusion: acute administration of BAS at a dose of 50 mg / kg induces an increase in binding to NMDA receptors in the hippocampus of rat brain after 24 hours.

2.4 Ex vivo binding to n-cholinergic receptors

The results of a study of the effect of a single administration of a suspension of BAS (50 mg / kg, by mouth) on the receptor binding constants of labeled nicotine after 2 and 24 hours in comparison with the placebo effect are presented in Fig. 18.

From the presented data it follows that the introduction of 50 mg / kg BAS leads to a significant increase in the density of nicotinic receptors in the frontal cortex of rats after 24 hours from 232 protein to 334 fmol / mg protein without changing affinity. 2 hours after the introduction of BAS, significant changes in the receptor characteristics were not observed.

Conclusion: acute administration of BAS at a dose of 50 mg / kg induces a significant increase in binding to n-cholinergic receptors in the cerebral cortex of rats.

Experiment 2. Study of the influence of BAS on the functional system of dopamine reuptake in rat brain synaptosomes

The system of reuptake (presynaptic transport) of CNS neurotransmitters is an effective way to regulate synaptic neurotransmission, therefore it is the target of many psychotropic drugs: psychostimulant, antiparkinsonian, antidepressant.

Given the pharmacological profile of the action of BAS, including these types of activity, in this series of experiments, the effect of BAS on the dopamine reuptake system in the isolated nerve endings of rat striatum was studied.

a) Methodology

Synaptosomal capture [ ³ N] -YES

Obtaining a crude synaptosome fraction: striatums dissected from the brain of rats were homogenized using a glass-teflon homogenizer in 10 volumes of a cooled solution of 0.32 M sucrose balanced with 10 mM Tris-HCl at 0-4 ° C. The resulting homogenate was centrifuged in a centrifuge at 1000 g for 10 minutes. Next, the supernatant (S1) was decanted and centrifuged again at 12000 g for 25 minutes. The resulting P2 pellet (“coarse synaptosome fraction”) was resuspended in modified Krebs buffer (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO ₄ , 1.2 mM CaCl ₂ , 10 mM HEPES, 10 mM D-glucose; pH 7.4), based 5 ml of buffer per 1 g of the original tissue.

The study of the capture of [ ³ N] -YES. The capture procedure was carried out according to the protocol [13]. For the capture reaction, an incubation medium containing 125 mM NaCl, 5 mM KCl, 1.2 mM MgSO ₄ , 1.2 mM CaCl ₂ , 10 mM HEPES, 10 mM D-glucose, 200 μM pargyline solution (MAO-B inhibitor) was used (incubation pH Wednesday = 7.4). To determine the proportion of non-specific processes, instead of the incubation medium, we used a 0.32 M sucrose solution containing 10 mM Tris-HCl (pH 7.4), in which synaptosomes were incubated at 0-4 ° C.

The capture reaction was started by introducing into the incubation medium containing the required concentration of [ ³ H] -DA, pargyline and the test substance, 50 μl of a suspension of synaptosomes. The tubes were incubated in the solid-state thermostat "Termite" (NPO "DNA-technology") for 3 min at 37 ° C. The reaction was stopped by rapid ultrafiltration through Whatman GF / C glass fiber filters. The filters were washed twice with 5 ml of ice-cold saline solution, dried at room temperature, transferred to scintillation vials and 7 ml of Bray scintillation liquid were added, after which their radioactivity was measured using a Wallac 1411 scintillation counter with a tritium counting efficiency of 40-45%.

b) Results

An in vitro study of the effect of BAS on synaptosomal reuptake of dopamine was started by introducing 50 μl of a synaptosome suspension into the incubation medium containing the required concentration of [ ³ H] -DA, pargyline (an MAO inhibitor) and the test substance. It was previously shown that PPS Midantan is able to inhibit dopamine reuptake with a half-inhibition concentration with an IC50 lying in a concentration range of ~ 200-250 μM. In this regard, dopamine reuptake was measured at a concentration of Midantan (250 μM) in the incubation mixture.

The influence of BAS on the reuptake of dopamine in rat brain tissue was determined in 4 groups: group 1 - physical. rr (0.9% NaCl); Group 2 - BES (Betulin) 500 μm; Group 3 - Midantan (250 μM); Group 4 - Midantan (250 μM) + BAS (Betulin) 500 μM.

A statistically significant inhibition of dopamine reuptake was found (40%). Co-administration of Midantan (250 μM) + BAS (Betulin 500 μM) revealed a more pronounced inhibition of reuptake compared to the administration of Midantan (250 μM) - about 60%, which indicates the ability of BAS to potentiate 30% of the inhibitory effect of Midantan on reuptake of dopamine (Fig. 19).

Conclusion: BES does not have a direct effect on the functioning of the dopamine transporter, but it is able to potentiate the inhibitory effect of Midantan by an additional 30%.

Experiment 3. The study of the influence of BAS on the regulation of the biosynthesis of dopamine and serotonin in the brain structures of mice in ex vivo

The biosynthesis of neurotransmitters in presynaptic nerve endings is the second of the functional units of neurotransmission, which is under the regulatory influence of membrane receptors and intracellular factors. Therefore, the possibility of the influence of BAS on the biosynthesis of dopamine and serotonin in the homogenates of the cerebral cortex, striatum and hippocampus of mice was studied.

a) Methodology

The content of monoamines was determined by high performance liquid chromatography with electrochemical detection (HPLC / ED).

The content of monoamines and their metabolites in the brain tissue of mice was determined in 2 groups. The test substances were administered intraperitoneally in a volume of 2 ml / kg NSD1015 for 30 minutes, BES 2 hours before decapitation of animals.

The first group of animals was injected phys. oral solution and NSD 1015 (50 mg / kg) intraperitoneally. The second group of animals was administered BAS (50 mg / kg) orally and NSD 1015 (50 mg / kg) intraperitoneally.

The need to use NSD1015 to assess biosynthesis is due to the high intensity of the processes of biosynthesis of monoamines, which results in a rather low content of their precursors in the brain. NSD1015 blocks the synthesis of dopamine from DOPA by inhibiting the enzyme L-aminodecarboxylase of aromatic acids (DAAA). In this case, there is an accumulation of DOPA and, accordingly, a decrease in the concentration of dopamine and its metabolic products, tyrosine also accumulates, which leads to an increase in the concentration of 5-GTP (serotonin precursor).

b) Results

The data presented in Tables 2-4 indicate that systemic administration of BAS at a dose of 50 mg / kg after two hours leads to a decrease in the activity of the biosynthesis of neurotransmitters in the striatum of the mouse brain: dopamine synthesis (concentration of dopamine precursor DOPA) is suppressed by 22%, and the synthesis of serotonin ( the concentration of the precursor of serotonin 5-GTP) - by 32% (Table 2).

table 2 The effect of BAS on the content of biogenic amines in the striatum of C57B1ask / 6 mice against the background of NSD 1015 (m ± S.E.M) DOFA ON DOFUK 5-gtr YES 5-GIUK GVK ST Fiz. rr + NSD1015 2.87 ± 0.31 1.95 ± 0.18 1.43 ± 0.18 1.06 ± 0.06 45.37 ± 2.75 2,30 ± 0,27 4.30 ± 0.62 3.23 ± 0.20 BES + NSD1015 2.24 ± 0.08 * 1.82 ± 0.14 1.26 ± 0.18 0.72 ± 0.07 * 52.98 ± 2.60 2.43 ± 0.32 3.85 ± 0.75 3.78 ± 0.19 * - statistically significant difference, Mann-Whitney U-test, p <0.05.

In the cerebral cortex, BES had no effect on the synthesis of DA or CT synthesis (Table 3), and only the synthesis of serotonin was suppressed in the hippocampus (Table 4):

Table 3 The effect of BAS on the content of biogenic amines in the cortex of C57Blask / 6 mice against the background of NSD 1015 (m ± S.E.M) DOFA ON DOFUK 5-gtr YES 5-GIUK GVK ST Fiz. rr + NSD1015 0.98 ± 0.09 3.45 ± 0.36 0.10 ± 0.04 1.00 ± 0.15 0.37 ± 0.09 1.86 ± 0.20 0.68 0.06 5.61 ± 0.24 BES + NSD1015 0.86 ± 0.11 4.21 ± 0.37 0.10 ± 0.03 0.82 ± 0.05 0.36 ± 0.07 1.79 ± 0.19 0.41 ± 0.07 * 7.98 ± 0.90 * * - statistically significant difference, Mann-Whitney U-test, p <0.05

Table 4 The effect of BAS on the content of biogenic amines in the hippocampus of C57Black / 6 mice against the background of NSD 1015 (m ± S.E.M) DOFA ON DOFUK 5-gtr YES 5-GIUK GVK ST Fiz. rr + NSD1015 0.51 ± 0.12 2.39 ± 0.36 0.12 ± 0.04 1.39 ± 0.27 1.58 ± 0.71 3.05 ± 0.46 0.48 ± 0.20 3.42 ± 0.58 B3C + NSD1015 0.33 ± 0.10 1.89 ± 0.37 0.06 ± 0.02 0.78 ± 0.14 * 1.28 ± 0.61 2.72 ± 0.60 0.28 ± 0.13 2.96 ± 0.59 * - statistically significant difference, Mann-Whitney U-test, p <0.05

Conclusion: a single administration of 50 mg / kg BAS leads to the suppression of DA biosynthesis in striatum tissue, as well as to inhibition of CT biosynthesis in striatum and hippocampal tissue.

Experiment 4. Study of the influence of BAS on intracellular metabolism and dopamine secretion in striatum tissues. mouse frontal cortex and hippocampus in ex vivo

The effect of BAS (50 mg / kg, by mouth, after 2 hours) on the metabolism of monoaminergic neurotransmitters and dopamine secretion was studied in the brain structures of C57BI / 6 mice.

a) Methodology

The content of monoamines was determined by high performance liquid chromatography with electrochemical detection (HPLC / ED).

The content of monoamines and their metabolites in the brain tissue of mice was determined in 2 groups. Injection of the substance was carried out 2 hours before decapitation of animals.

The first group of animals was given a placebo, and the second group was administered BAS (50 mg / kg) orally.

b) Results

Metabolism of catecholamines (dopamine and norepinephrine) and indolamines in presynaptic ends is carried out by monoamine oxidase enzymes of types A (serotonin) and B (catecholamines), as a result of which 5-hydroxyindoleacetic acid (HIAA) is formed from the first, and dioxiphenylacetic acid detectable intracellularly. Extracellular, synaptically active dopamine is exposed to the COMT enzyme (catecholamine-o-methyltransferase) and is metabolized to homovanilic acid (HVA), the extracellular dopamine metabolite.

Therefore, by the level of GVK it is possible to judge the intensity of secretory processes in the brain ex vivo. So, two hours after the administration of BAS, no changes in the intensity of dopamine secretion are observed in the studied brain structures (Tables 5, 6, and 7).

Table 5 The effect of BAS on the content of biogenic amines in the striatum of C57Black / 6 mice (m ± S.E.M) DOFA ON DOFUK 5-gtr YES 5-GIUK GVK ST 5-GIUK / ST The control 0.74 ± 0.15 3.01 ± 0.10 3.54 ± 0.33 0
0 64.57 ± 7.37 2.08 ± 0.20 6.20 ± 0.65 5.33 ± 0.23 0.39 ± 0.05 BES 0.74 ± 0.08 1.88 ± 0.43 * 4.78 ± 0.43 * 0
0 63.55 ± 2.84 3.77 ± 0.22 * 5.82 ± 0.40 4.59 ± 0.18 0.82 ± 0.04 * * - statistically significant difference, Mann-Whitney U-test, p <0.05

However, in the striatum there is a decrease in the level of norepinephrine (-37%), accompanied by a corresponding increase in the concentration of its intracellular metabolite DOPUK (+ 35%), which indicates activation of the metabolism of norepinephrine (Table 4). The effect of accelerating the intracellular metabolism of catecholamines is also observed in the cerebral cortex, where the level of DOPUK increases by 52% (Table 6).

Table 6 The effect of BAS on the content of biogenic amines in the cortex of C57Black / 6 mice. (m ± S.E.M) ON DOFUK 5-gtr YES 5-GIUK GVK ST 5-GIUK / ST The control 3.53 ± 0.32 0.29 ± 0.05 0.07 ± 0.01 0.74 ± 0.17 1.56 ± 0.18 0.75 ± 0.11 5.90 ± 0.09 0.27 ± 0.03 BES 4.15 ± 0.34 0.44 ± 0.07 * 0.07 ± 0.01 0.73 ± 0.08 3.21 ± 0.36 * 0.90 ± 0.16 6.46 ± 0.64 0.50 ± 0.02 * * - statistically significant difference, Mann-Whitney U-test, p <0.05

The effect of BAS on serotonin metabolism is noteworthy - in all three brain structures, a significant activation of the metabolism of this neurotransmitter is recorded, which is estimated by an increase in the concentration of the main metabolite of serotonin - 5-hydroxyindoleacetic acid.

Table 7 The influence of BAS on the content of biogenic amines in the hippocampus of C57Black / 6 mice. (m ± S.E.M) ON DOFUK 5-gtr YES 5-GIUK GVK ST DOFUK / YES 5GIUK / ST The control 4.27 ± 0.25 0.35 ± 0.04 0.05 ± 0.01 1.20 ± 0.55 3.05 ± 0.28 * 0.47 ± 0.06 4.44 ± 0.18 0.37 ± 0.06 * 0.68 ± 0.05 * BES 2.09 ± 0.47 0.51 ± 0.19 0.05 ± 0.01 1.05 ± 0.28 4.20 ± 0.73 * 0.44 ± 0.15 3.62 ± 0.54 1.18 ± 0.25 * 1.32 ± 0.10 *

Conclusion: The introduction of BAS does not lead to a significant change in dopamine secretion, but activates the metabolism of DA. At the same time, the previously studied ST-ergic component plays an essential role both as a functional dopamine antagonist and as an independent one. The spectrum of pharmacological activity of BAS confirms the importance of the latter statement.

Claims (1)

The use of birch bark extract with a predominant up to 70% betulin content for the prevention and treatment of Parkinson's disease.

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