WO2008069691A1

WO2008069691A1 - Agent for preventing and treating a parkinson's disease

Info

Publication number: WO2008069691A1
Application number: PCT/RU2007/000017
Authority: WO
Inventors: Georgy Ivanovich Kovalev; Denis Alexandrovich Abaimov; Julia Jurievna Firstova; Vladimir Vladimirovich Balakshin; Aleksey Nikolaevich Chistyakov
Original assignee: Obschestvo S Ogranichennoi Otvetstvennostju 'berezovy Mir'
Priority date: 2006-12-04
Filing date: 2007-01-18
Publication date: 2008-06-12
Also published as: RU2324492C1

Abstract

The invention relates to the use of a birchbark extract for preventing and treating a Parkinson's disease.

Description

MEANS FOR PREVENTION AND TREATMENT OF PARKINSON'S DISEASE

The technical field The invention relates to the field of medicine. More specifically, it relates to the search for new drugs 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 • certain 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. Suggest that with increasing 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.

For the pharmacotherapy of parkinsonism, cholinolytic drugs, ^' aminoadamantane derivatives, DOPA-containing drugs, monoamine oxidase inhibitors (MAO) type are used. B, catechol-O-methyltransferase (KOMT) inhibitors and dopamine receptor agonists. 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 the 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. Preparations of this group are contraindicated in glaucoma, prostate adenoma, and intellectual-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, as well as give an amphetamine amino acid effect, promoting 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 disorders of glutamatergic neurotransmission in Parkinson's disease and its significance in changing corticostria, thalamocortical and other 5 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, almost no 1.0 contraindications, however, in general, the effectiveness of these drugs is low and ^' improvement is observed in no more than half of patients. (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 intellectually-15 mnestic disorders.

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

Dofa - containing drugs

The role of dopamine, as a possible main therapeutic agent in the treatment of Parkinson's disease, has been studied since the 1950s, when Srlssop noted that symptoms of parkinsonism are caused by reserpine. In subsequent years, a decrease in the dopamine content in the striatum in the disease was found

Parkinson's (Ехriпгер, Номукиевісz, 1960), which led to the emergence of a significant number of dopamine-containing drugs, which constitute 5 now the "gold standard" of therapy 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 under the action of cerebral DOPA-0 decarboxylase (DDC) in dopaminergic neurons, is used for replacement therapy (N.Qшп, 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 currently practically not 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. Quiši, S. Olapow, 1996). The most commonly used drugs in this group - Madopar (levodopa / benserazide in the ratio 4: 1) Nacom (levodopa / carbidopa - ^'10: 1), Sinemet (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. Koll, 1996; Mullula 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. Liebermap et al., 1983; C. Goetz et al., 1985;

R. McDonald, R. Horowski, 1983; Y. Mizupo 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. AT . considerable experience has been accumulated on the use of dopamine receptor ergoline agonists (bromocriptine, pergolide, lisuride).

KOMT Inhibitors

One of the promising and significant achievements in recent years has been the discovery of a fundamentally new, highly effective class of anti-Parkinsonian drugs - KOMT inhibitors. KOMT is one of the main enzymes that provide the metabolism of levodopa, dopamine, other catecholamines and their metabolites. KOMT is a fairly common enzyme that is present in various parts of the body, including neurons and glia, with the exception of nigrostriatal dopaminergic neurons (A. Caster et al., 1994). Peripheral KOMT 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 KOMT inhibitors (tolcapone), according to most authors, give significantly more pronounced side effects (K.Holm,

S. Strepser, 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).

SUMMARY OF THE INVENTION In previous studies of the birch bark extract by the inventors of the present invention (RF patent application N ° 2005138574), it was found that in behavioral tests of general and directed screening in mice, the drug exhibits 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 Vasile actometer; 2. the extract enhances the timoleptic effect of the antidepressant Pyrazidol when combined with the introduction of the “despair” test

3. the extract causes a nootropic effect in the tests “perfect labyrinth” and “the conditioned reflex of passive avoidance”.

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

The combination of the above experimental data suggested that a combination of the described properties meets the requirements that can be presented to a drug with antiparkinsonian potential (Tillersop J.L., Caudle W.M., Reverop

M.E., Miller G. W. Detective of bеhavioral imrairmepts correlated to mеtre détеs оf l-methyl-4-phenyl-tеr-2,3, 2,3. Exp Nеurol., 2002, 178: 80-90).

Further studies, a set of experiments using standard behavioral and pharmacological tests recommended for the detection of antiparkinsonian activity (Voronina TA., Waldman EA, Nerobkova LN Methodological guidelines for the study of antiparkinsonian activity of pharmacological substances. In book: {(Manual (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 “Cyxogo Extract Samples” (BES), manufactured by Berezovy Mir LLC, Moscow, Russia and used for the manufacture of biologically active additives. The experiments were conducted on 196 male mice of the C57 / B1 line with a weight of 20-25 g. Contained in a laboratory vivarium on a standard diet.

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

The research results are reflected in the figures, which depict: Fig.l The effect of BAS on the total time of catalepsy caused by haloperidol.

Fig. 2 The effect of BAS on the latent period of tremor caused by Arecolin, 90 minutes after oral administration.

Fig. 3 Dynamics of the influence of BAS on the latent period of the onset of tremor in mice. Fig. 4 Effect of BAS on the duration of arecoline tremor in mice

Fig. 5 The effect of BAS on the duration of Arecolin-induced mice 90 minutes after oral administration

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

Fig. 7 The effect of a single administration of BAS (50 mg / kg, inside), Midantan (20 mg / kg, c, b) and their combination on the horizontal motor activity of C57B1 / 6 mice in the open-field test (arithmetic mean + standard deviation error) . Fig. 8 The effect of a single administration of BAS (50 mg / kg, by mouth), Midantan

(20 mg / kg, c, b) and their combinations on the motor activity of C57B1 / 6 mice in an Ugo Basile actometer (arithmetic mean + standard deviation error).

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 a 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; v / b - Saline solution (RF); 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 fixed for 2 minutes. sessions 30, 60 and 90 minutes after the administration of haloperidol. 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 Fig. 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 action 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-haloperidol receptors.

Determining the ability of birch bark extract (BAS) to influence tremor caused by arecoline in mice. Adetylcholine (AX) is the neurotransmitter of the numerous interneurons of the caudate nucleus, and it is in this structure that the highest concentration of AX is observed (Guttmap M., Kish SJ., Furukawa Y. Surprise iptapdp apd mapagamep оf Рarkiпеспс 168Меее 3ееесе,. ), 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 data processing are presented in Fig. 2, 3, 4, and 5. It was found that the administration of arecoline against the background of placebo leads to the onset of tremor by 33 seconds, while the same against the background of 50 mg / kg BES delays the onset of tremor to 73 seconds (Fig. 2; pθ.00000) .

Moreover, the latent period under the influence of BAS increases within 2 hours after the introduction of arecoline, after which it begins to decrease (Fig. C).

The dynamics of the duration of arecoline tremor after administration of BAS 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 the administration of arecoline. In later periods of observation (2 and 2.5 hours), the effectiveness of BES disappears.

Thus, the maximum effect on the duration of tremor caused by arecoline occurs in 90 minutes (F (I, 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 reduction of the motor activity of mice in the Ugo Vasile and Open Field actometer on a model of parkinson syndrome caused by systemic administration of MPTP neurotoxin in the mode of prophylactic use of BAS.

The model of Parkinson's syndrome caused by the administration of MPTP neurotoxin (4-phenyl-1.2.3.6-tetrahydropyridine) is the most an adequate 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 l-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 the mitochondrial respiratory chain complex-1. This effect is most pronounced in primates, and in mice in the line C57B1 / 6 (Feldmap RS, Meuer JS, Quepzer LF Parcelsop, Disp. Apt Alzheimers dissent, Incomp. 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 use of an extract suspension. In this version of the experiment, it was supposed to evaluate the possible "prophylactic" significance of BES.

In the experiment, 36 male C57B1 / 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 an Ugo Vasile actometer.

Animals were randomly assigned to 3 experimental groups:

.one. Inside - 1% starch 5-fold; 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 administration of the toxin in mice, deviations from normal autonomic and behavioral signs were observed: pilo erection, salivation, retro-pulsation, respiratory failure.

Statistically processed indices of horizontal locomotor activity, automatically measured in an Ugo Vasile actometer for a 10-minute session, are presented in Fig. 6: The data obtained indicate that administration of BES to C57B1 / 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 Vasile actometer for 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 DAergic neurons.

Determination of the ability of birch bark extract (BAS) to influence the decrease in the motor activity of mice in the actometer and open field on a model of parkinsonism syndrome caused by 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 Midantana (Morozov I.S., Petrov B.I., Sergeeva CA. Pharmacology of adamantanes, Volgograd, 2001).

In the experiment, 88 male C57B1 / 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

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, t-student test).

The results of the introduction of BAS, midantan and their combination for 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's t-test), but statistical significance from the results of the influence of drugs separately on was noted.

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

Twice 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 (ρ = 0,000; Student's t-test). In turn, acute use of BAS, Midantan and their combinations partially restored the induced motor deficit to 265% (p = 0.013; t-student criterion), 275% (p = 0.033; t-student 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 under the influence of MPTP and drugs, not only the number of movements changed, but also ica-iεctvepiii, i-th motor composition activity: the introduction of the toxin also led to a decrease in vertical activity (rack) and search activity (peering into holes) in the “open floor” test from 31 + 1.7 in group 1 to OD 1 + 0.11 in group 2 (p = 0.000 ; t-student test) and from 5.3 + 0 D to 0.6 + 0.2 (p = 0.000; t-student test), respectively.

^~ Introduction of BES and its combination with midantanom partially but statistically significantly restore these parameters of locomotor activity selectively. Thus, the extract and combination increased the decreased indicator “vertical activity)) with a statistical reliability of 0.04 and 0.03 according to the Mann-Whitney test, respectively.

Thus, the experimentally selected conditions made it possible to detect in two test variants (“open field” and the automatic actometer Ugo Vasile) that the birch bark extract at a dose of 50 mg / kg with a single oral administration has the ability to partially but statistically significantly compensate for locomotor deficiency in male mice C57B1 / 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 the aforementioned substances _ also led to an increase in the number and improvement of the structure of motor activity in mice with MPTP-induced parkinsonian syndrome, somewhat better in comparison with the effects of both drugs separately.

Claims

CLAIM

1. The use of birch bark extract for the prevention and treatment of Parkinson's disease.

2. The use of birch bark extract for the manufacture of medicines for the prevention and treatment of Parkinson's disease