WO2017044002A1 - Active ingredient of a drug and a drug, a pharmaceutical composition and a method for treating demyelinating diseases of the human body, which includes prophylaxis - Google Patents

Abstract

The invention relates to the chemical and pharmaceutical industry and concerns the use of polyprenols of formula (1) to produce a drug, as well as the drug itself and a pharmaceutical composition containing compound (1) for the treatment and prophylaxis of demyelinating diseases of the human body. The invention also relates to methods for the treatment and prophylaxis of demyelinating diseases of the human body, in which an effective amount of polyprenols of formula (1) is introduced into a living organism. The invention makes it possible to minimize side effects when using a drug and a pharmaceutical composition of plant origin, which contain a compound of formula (1), in a method for the treatment and prophylaxis of demyelinating diseases of the human body.

Description

 The active ingredient of the drug, drug, pharmaceutical composition and method for the treatment of demyelinating diseases of a living organism, including the prevention of diseases

 Technical field

 The invention is a group of inventions and relates to the field of medicine, namely to means and methods for the treatment and prevention of demyelinating diseases of a living organism, including multiple sclerosis and conditions associated with demyelination, polyneuropathy and / or symptoms or signs of such conditions.

 State of the art

 Demyelinating diseases (hereinafter referred to as DZ) are among the autoimmune diseases, the main pathomorphological manifestation of which is the destruction of myelin in the white or gray matter of the central and / or peripheral nervous system (hereinafter CNS, PNS). In recent years, there has been a steady tendency to increase the incidence of DZ throughout the world, the number of cases with a clinical onset in children and adults over the age of 45 is growing, the nature of the course

DZ.

Currently, it is proved that the etiology of DZ is based on the interaction of environmental factors and the hereditary predisposition of the body. Among external factors, viruses (Epstein-Barr, herpes, measles, rubella, retroviruses), chronic bacterial and campylobacter infections, intoxications (solvents, gasoline, metals), nutritional features (predominance of animal fats and proteins), psychoemotional stress are of greatest importance. , environmental degradation [Zavalishin I.A., Zhuchenko TD, Peresedova A.V. Bulletin of the Russian Academy of Medical Sciences., N ° 7, 2001, S. 18-22]. The pathogenetic mechanisms of DZ are similar and consist in the development of autoimmune reactions to neuroantigens, which are a number of proteins and lipids of the myelin sheath. The initial mechanism for the development of these diseases is the activation of peripheral T-lymphocytes specific for the myelin basic protein (MBP, myelin basic protein). Several activation paths are possible: 1) due to molecular mimicry, when the antigenic structure of bacteria or viruses is similar to MBP, therefore T-cell receptors cease to distinguish between “their” and “foreign” antigens; 2) T cells can be activated by non-specific by due to the high local concentration of cytokines that accompanies any inflammatory reaction. The next stage of the pathological process is the penetration of autoreactive lymphocytes through the blood-brain barrier (BBB) by increasing the expression of adhesion molecules. Penetrated into the nervous system, T cells secrete pro-inflammatory cytokines (gamma interferon, lymphotoxin, TNF-a). Against this background, due to a sharp increase in the number of inflammatory mediators, the microenvironment of the brain is rapidly changing, and anti-myelin antibodies are formed in response. The final stage is the death of oligodendrocytes, the development of the demyelination process due to activation of the complement system, macrophages, microglia and phagocytosis of damaged structures.

One of the serious demyelinating diseases is multiple sclerosis (hereinafter referred to as PC), which is one of the most common. A feature of the disease is the simultaneous defeat of several parts of the central nervous system, which is manifested by a variety of neurological symptoms. For 85-90% of patients in the early stages, a remitting (undulating) course is characteristic, which subsequently, as a rule, is replaced by a progressive one. In addition, the specific causes of the disease are unclear, it is generally accepted that PC is a multifactorial disease that occurs in people who have a genetic predisposition, and who are also affected by some external factor. As a rule, the processes of demyelination and inflammation are preceded by biochemical changes, including nitrification and oxidative stress of the mitochondrial proteins of the respiratory complexes, which lead to a decrease in mitochondrial potential and apoptosis of neurons. Myelin-specific T lymphocytes cause damage to mitochondrial macromolecules, thereby breaking off the respiratory chain of mitochondria and reducing ATP synthesis, as well as causing apoptosis. [Bernadette Kalman et. all. The involvement of mitochondria in the pathogenesis of multiple sclerosis.// Journal of Neuroimmunology N 188, 2007, p. 1-12]. Nitric oxide (NO) is a free radical whose concentration increases during the inflammatory process during the development of multiple sclerosis and plays an important role in breaking the blood-brain barrier (BBB), as well as in the process of demyelination and death of oligodendrocytes and axon loss. Myelin is a white matter in the brain that covers the axons of neurons and contributes to the efficient conductivity of nerve impulses between the brain and others. body parts. The loss of myelin (i.e., demyelination) or the abnormal composition of myelin may be associated with a decrease in the speed or activity of processes in the nervous system and subsequent deterioration in cognitive function. The myelin sheath is a special type of cell membrane surrounding the processes of nerve cells, mainly axons in the central nervous system and the central nervous system. The main functions of myelin are: axon nutrition, isolation and acceleration of the nerve impulse and the preservation of ion fluxes by reducing the membrane capacity, as well as supporting and barrier functions. This is a lipoprotein membrane, consisting of a bimolecular lipid layer located between the monomolecular layers of proteins, spirally twisted around the nerve fiber. Myelins of the central nervous system and PNS differ in their chemical composition. In PNS, myelin is synthesized by Schwann cells. Myelin PNS is noticeably thicker than in the central nervous system. In the central nervous system, myelin is synthesized by oligodendrocytes, with one cell taking part in the myelination of several fibers [Huho F. Neurochemistry: fundamentals and principles: Translated from English. - M: Mir, 1990, 384s]. The destruction of myelin is a universal mechanism of reaction of nerve tissue to damage. From the point of view of pathogenesis, PC was considered as an inflammatory demyelinating disease in which the primary targets of the destructive process are the myelin sheath and oligodendrocytes.

Immunological changes in PC are manifested by deviations of cellular and humoral immunity. From the side of cellular reactivity, the following are determined: a decrease in the T-cell content of suppressors, a suppression of the T-cell response to mitogens, a change in the production of interferons, an increase in the cytotoxicity of mononuclear cells, and changes in the interleukin system. The involvement of multidirectional cytokines in the pathogenesis of PC has been repeatedly shown in experimental studies. Tumor necrosis factor-α (TNF-a) is considered a classic anti-inflammatory cytokine, but it has recently been shown that this cytokine has a significant anti-inflammatory and neuroprotective effect in demyelinating diseases [TNF neutralization in MS: results of a randomized, placebo-controlled multicenter study. The Lenercept Multiple Sclerosis Study Group and The University of British Columbia MS / MRI Analysis Group. // Neurology, 53, 1999, P. 457-465]. The clinical manifestations of PC are a consequence of the development of demyelinating foci of inflammation in the central nervous system. The autoimmune nature of this disease has both experimental and clinical evidence. At PC autoimmune aggression can be directed towards myelin basic protein (MBP), myelin basic protein, proteolipid protein (PLP, proteolipid protein), myelin glycoprotein (MAG, myelin-associated glycoprotein), myelin oligodendrocyte glycoprotein glycoprotein (MOG) ) and possibly other myelin antigens [Zhang J. T cell vaccination as an immunotherapy for autoimmune diseases. // Cell. Mol. Immunol. 2004; 1: P. 321-7]. Pathological foci formed in PC in the white matter of the brain and spinal cord are called plaques. The leading feature of these foci is demyelination. Activation of resident CNS cells — microglia and astroglia — stimulates their secretion of cytokines and chemokines (factors that attract cells to the focus of inflammation). Cells of hematogenous origin migrate to the foci - monocytes / macrophages, T - and B-lymphocytes. So the formation of plaque begins. In the focus of inflammation, cells secrete many active molecules: cytokines, antibodies, oxygen and nitrogen radicals, proteases. These molecules are major factors in damage to oligodendrocytes and myelin. Repeated exacerbation is manifested by inflammation along the periphery of the gliosis zone, the focus increases in size. Along with this, new foci appear, and some may regress. A histological examination of acute foci reveals partial or complete destruction and loss of myelin. Axonal endings generally remain intact. In addition to demyelination, plaques are characterized by a neuroglial reaction and infiltration by mononuclear cells (lymphocytes and macrophages). Chronic damage is represented mainly by gliosis [Homes J, Madgwick T, Bates D. The cost of multiple sclerosis.// Br. J. Med. Econ., 1995; 18: 181-93].

 Studies of the last decade have shown that with PC, damage to axons and neurons occurs, the severity of which varies in intensity at different lesions in one patient, as well as when comparing lesions in different patients with PC. [Neurodegenerative process in multiple sclerosis and the possible neuroprotective effect of therapy with β-interferon 1a (avonex). M.V. Davydovskaya, A.N. Boyko, A.E. Podoprigora, I.N. Pronin, V.N. Kornienko, E.I. Gusev.// Journal of Neurology and Psychiatry. Ν ° 9. Vol. 2, 2012, S. 129-135].

The course of PC is very individual - there are no two patients with the same clinical manifestations of the pathological process. In most cases, after a certain period of time, individual for each patient, the remitting course of the PC becomes secondary-progressive with a gradual increase in neurological deficit, a reduction in the periods of clinical stabilization, and disability of patients.

 In general, the clinical manifestations of PC can be divided into 7 main groups:

 1) damage to the pyramidal system with hemi -, steam - and tetraparesis with a corresponding increase in tendon reflexes and pathological pyramidal symptoms;

 2) damage to the cerebellum and its pathways with the development of static and dynamic ataxia, muscle hypotension;

 3) sensory impairment, at the beginning of a deep one, with the development of sensory ataxia and sensory paresis, and then pain and temperature along the conductor type;

 4) damage to the white matter of the brain stem with various disorders of the cranial innervation, most often with the development of oculomotor symptoms, damage to the facial nerve (peripheral paresis of the muscles of the face);

 5) optic neuritis (including retrobulbar neuritis) with a decrease in visual acuity and the appearance of cattle;

 6) dysfunctions of the pelvic organs, often by the type of imperative urges, urinary retention, and subsequently until urinary incontinence;

 7) neuropsychological changes, weakening of memory, euphoria or depression, characterized by chronic fatigue syndrome.

In most cases, patients have symptoms of damage to both the brain and the spinal cord (cerebrospinal form). In some cases, the symptoms of spinal cord injury (spinal form) or cerebellum (cerebellar or hyperkinetic form) predominate in the clinical picture. In the latter case, intentional trembling can be so pronounced that it reaches a degree of hyperkinesis and makes any targeted movement impossible. Dysmetria, adiadochokinesis, chanted speech, severe ataxia occur. Magnetic resonance imaging (MRI) of the brain and spinal cord is currently one of the most informative research methods. Characteristic for DZ, the detection in T2 and proton modes of foci of increased intensity of circular or oval shape, of various sizes (from 3 mm to 3 cm) located in any brain area is considered.

 Other demyelinating diseases also include peripheral nervous system DZ - an acute onset (Guillain-Barré syndrome (GBS) occurs at any age (2 times more often in men). Chronic inflammatory demyelinating polyneuropathy (CID) has two peaks of disease onset: in 40 50 years and after 70 years (less pronounced), 3 times more often in men.CVDI is diagnosed in the presence of two obligate clinical signs:

 1) progressive or recurrent weakness in the limbs lasting more than 6 months;

 2) hypo - or areflexia in all four limbs.

 The distinctive clinical signs of CVD include:

 - mainly distal or mixed (distal and proximal) character of paresis of limbs;

 -expressed muscle hypotension and hypotrophy of the arms and legs;

 - more frequent and persistent sensitive and autonomic disorders in the limbs;

 - extremely rarely - the addition of respiratory and pelvic disorders.

 HDV prognosis is less favorable and depends on the type of course

(progressive, progressive-remitting, regressing, stationary), as well as the possible involvement of the central nervous system structures in the process. After a year, 30-60% of patients lose their ability to work [M. Piradov. // Nevrol. Journal, N ° 2, 2001, S. 4 - 9].

 Hereditary motor-sensory polyneuropathy type I (Charcot's disease -

Marie) is pathomorphologically a demyelinating disease. Polyneuropathy associated with anti-MAG (myelin-associated glycoprotein) monoclonal gammopathy has recently been isolated into an independent nosological form and is clinically manifested in isolation by sensory disturbances in the limbs. [Leger J.-M., Salachas F. // Eur. J. Neurol. 2001, N 8, P. 201-209].

Electroneuromyography reveals signs of myelin destruction and axonal degeneration. The most characteristic changes are significant a decrease in the speed of the pulse along peripheral nerves, an increase in distal latency, a change in the shape of the M-response, and F-wave delay. Using a special technique of sympathetic cutaneous evoked potential allows you to quantify the degree of autonomic disorders in the limbs.

 Despite numerous studies, the etiology of the disease is still unclear. Therapy of multiple sclerosis is the timely adequate relief of exacerbations, the appointment of drugs that slow the progression of the disease by preventing exacerbations.

 It is known that treatment of PC was reduced to relieving exacerbations of the disease using drugs of the corticosteroid group in order to suppress the activity of the immune system and conduct symptomatic therapy. The most commonly used is methylprednisolone, which is administered intravenously in a dose of 1000 - 2000 mg per day (per 400-500 ml of physiological saline) for 3-10 days, depending on the severity of the exacerbation [F. Barkhof et al. Comparison of MRI criteria at first presentation to predict conversion to clinically definite multiple sclerosis.// Brain. 1997, V.120, P.2059-2069].

 However, hormonal drugs have a number of side effects [Bashir K., Buchward L., Coyle R. K. Optimizing immunomodulatory therapy for MS patients // Int. JMCS Care. 2002, Vol. 4., P. 3-7]. Drugs with a cytostatic and antiproliferative effect, as well as general irradiation of lymphocytes, are used, as a rule, in malignant, steadily progressive MS in patients with corticosteroid intolerance. The largest number of studies on the effectiveness of cytostatics for the treatment of multiple sclerosis was carried out using azathioprine. Analysis of controlled studies showed a slight, but statistically significant decrease in the frequency of exacerbations after 2-3 years of therapy, a slight slowdown in patient disability compared with the control placebo group (about 0.2 points on the EDSS scale for 2 years) [A.A. Poryvaeva, On the use of cytostatics in the treatment of multiple sclerosis. // Modern high technology. N ° l, 2005, S. 72-73].

However, the use of such cytostatics is associated with the presence of serious side effects of this group of drugs [Bashir K., Buchward L., Coyle R. K. Optimizing immunomodulatory therapy for MS patients. // Int. JMCS Care. 2002, Vol. 4, P. 3-7]. In the 90s, preventive drugs of a pathogenetic orientation appeared, which significantly reduce the number of exacerbations of PC and slow its development, but, unfortunately, do not lead to a complete recovery. These are immunomodulatory agents that have proven themselves in clinical practice.

 These include interferons beta (interferon beta-lb, interferon beta-1a) and preparations based on them (betaferon, rebif, avonex) and glatiramer acetate. All these drugs are first-line drugs recommended for prescribing to patients with a remitting form of the course of PC with mild to moderate disability. These drugs, having completely different mechanisms of action, lead to a reduction in the frequency of exacerbations by 30-35% [Freedman M, Hughes B., Mikol D. et al. Efficacy of disease-modifying therapies in relapsing-remitting multiple sclerosis: a systematic comparison. // Eur.Neurol., 2008, v.60, p. 1-1 1]. All these immunomodulators should be used continuously, injected and have a certain spectrum of adverse events.

 However, the most significant adverse event that occurs with the use of interferon beta is a flu-like syndrome with fever, headaches and muscle pain, which occurs after administration of the drug and sometimes lasts quite a long time.

When using glatiramer (copaxone), side effects also occur in the form of systemic post-injection reactions, manifested in the form of autonomic crises, and atrophy of adipose tissue at the injection site. The use of first-line immunomodulators in not all patients leads to the desired result. This is due to a number of reasons, and above all, the genetic characteristics of various patients. In addition, the use of interferon beta in some cases is accompanied by the formation of neutralizing antibodies that reduce the biological activity of drugs [Vartanian T., Sorensen P., Rice G. Impact of neutralizing antibodies on the clinical effect of interferon beta in multiple sclerosis. // J. Neurol., 2004, 251, suppl. 2, 25-30]. A significant factor in the lack of response to therapy is the incomplete adherence to treatment due to fear of injections, "fatigue" from the constant long-term injection of the drug, or intolerable side effects [Gold R. Adherence to treatment: value of patient support. // Cl. Trends in Neurol., 2005, vl, p. l 1-14]. It is known to use the drug fingolimod (Gilenia) manufactured by Novartis, which is used relatively recently. It has been approved for use in the United States (since 2010), Europe (since 2011) and in Russia (since 2010). The drug is a derivative of myriocin and is isolated from the fungus Saria sinclairii, used in traditional herbal treatment. Using sphingosine kinase, it in the liver turns into an active metabolite - fingolimod phosphate. Fingolimod is a modulator of 4 of the 5 known sphingosine phosphate receptors [Dominguez M., Casals D., Sabate M. Et al. Prediction of pharmacokinetic / pharmacodynamic behavior in humans of LAS 189913, a SI PI receptors agonist, based on preclinical studies in three animal species./ Mult.Scl, 2010, V.16, suppl. 10, S 150, P 453], which are present in different tissues, but especially widely in lymphocytes, cells of the central and peripheral nervous system.

 The number of "autoaggressive" T cells circulating in the blood decreases and the degree of inflammatory changes in the central nervous system decreases. In addition, it was shown that fingolimod, being lipophilic in nature, penetrates the blood-brain barrier into the central nervous system, where phosphate receptors of glia and neurons act on sphingosine-1, having a neuroprotective effect [Osinde M., Mullershausen F., Dev K. Phosphorylated FTY720 stimulates ERK phosphorylation in astrocytes via SIP receptors. // Neuropharm., 2007, v. 52, p. 1210-1218]. However, when using it, adverse side effects are often observed - headache, dyspnea, diarrhea, nausea.

For the first time, 3-4 months of treatment, the level of liver enzymes may increase. Its normalization occurs after about 2 months. after stopping the drug. Fingolimod has the effect of a “first dose”, resulting in a dose of 0.5 mg to symptomatic bradycardia in approximately 0.5% of patients, therefore, the use of fingolimod requires monitoring the patient for 6 hours after applying the first dose of the drug. Before prescribing the drug and 3-4 months after the start of its use, an oculist examination is necessary, because taking fingolimod at a dose of 0.5 mg led to the development of edema of the macula of the eye in 0.3% of patients. Patients with diabetes and a history of uveitis require special attention. [Recommendations on the use of new drugs (tisabri, guilenia, movectro) for the pathogenetic treatment of multiple sclerosis. All-Russian Society of Neurologists. Demyelinating Diseases Section. Moscow, 2011]. It is known that bone marrow stem cell transplantation has been proposed for the treatment of PC, but this is associated with the risk of re-autoimmunization of the body, and on the other hand, a higher mortality rate for allograft (15-35%) than for autoimmunization (3-10%). Granulocyte macrophage colony stimulating factor is also proposed for the treatment of PC [Application 201 1145434, PCT publication WO 2010/128035, 2010.1 1.1 1]. For the treatment of recurrent multiple sclerosis, drugs based on monoclonal antibodies, Lemtrada (Lemtrad, Alemtuzum), are proposed [Coles AJ, et al. Alemtuzumab vs interferon beta- la in early multiple sclerosis. // New England Journal of Medicine 2008; 359: 1786-1801]. However, significant side effects appear in the clinic - this is idiopathic thrombocytopenic purpura - a disease that disrupts blood coagulation, it was fatal in one case and was noted in 1-3% of participants, and dysfunction of the thyroid gland in 20-30% of patients.

 However, given that the PC has been going on for quite some time, these tools and treatments can be very expensive and not available to all patients. So, today the costs of treating patients in a moderate stage in European countries range from € 28,524 to € 43,948 per year. [Russia, Medical News. 2002, b, S. 3-7].

 The closest analogue to the claimed invention is the patent US N °

2444356, which proposes the use of polyunsaturated fatty acids for the treatment of dementia. An embodiment of this invention relates to a method for delaying demyelination or reducing the severity of demyelination in an individual. The method comprises administering to the individual a combination of polyunsaturated fatty acids (PUFAs) or their precursors, or sources to delay demyelination, or to reduce the severity of demyelination.

However, polyunsaturated fatty acids can be considered as adjuvants mainly for the prevention of demyelinating conditions, but not for treatment. In addition, polyunsaturated fatty acids and oils in which they are dissolved are usually unstable and transform into a transformation that is already harmful to humans, in particular, they increase intraocular pressure in people predisposed to glaucoma. Diet with a higher content of polyunsaturated fats than saturated (animals) more often leads to gallstone disease and can cause spasms of the gallbladder associated with thickening of bile. [Mary Dan Eijs. Vitamins and Minerals: A Complete Medical Guide. 2 ed. - St. Petersburg, 1996, 503c]. Therefore, prolonged administration of polyunsaturated fatty acids for the prevention and treatment of demyelinating diseases can be considered as a preventive measure.

 Disclosure of invention

 The tasks to which the invention is directed are to create a new drug, a new pharmaceutical composition and a new method of treatment, including the prevention of demyelinating diseases.

 The technical result of the invention is to obtain a minimal side effect when using a new medicinal product and a new pharmaceutical composition of plant origin, created on the basis of a fundamentally new class of compounds - polyprenols (long chain polyisoprenoid alcohols), in a method of treating demyelinating diseases of a living organism, including the prevention of diseases, including the number of multiple sclerosis and conditions associated with demyelination, and / or symptoms or signs of such Toyan.

 The tasks are solved as follows.

 Polyprenols of the formula (1)

CH ₃ CH ₃

H - (CH ₂ - C = CH - CH ₂ ) _P - CH ₂ - C = CH - CH ₂ OH, where n = 8-20

 were used as an active ingredient in the manufacture of a medicament for the treatment of demyelinating diseases of a living organism.

 Polyprenols of the formula (1)

CH ₃ CH ₃

 I I

H - (CH ₂ - C = CH - CH ₂ ) „- CH ₂ - C = CH - CH ₂ OH, where n = 8-20

have been used as an active ingredient in the manufacture of a medicament for the prevention of demyelinating diseases 0459

 - 12 - living organism.

 The drug for the treatment of demyelinating diseases of a living organism, including the prevention of the disease, is characterized in that it is a polyprenol of the formula (1)

CH ₃ CH ₃

H - (CH ₂ - C = CH - CH ₂ ) _P - CH ₂ - C = CH - CH ₂ OH, where n = 8-20.

 A pharmaceutical composition for treating demyelinating diseases of a living organism, including disease prevention, is characterized in that it comprises an effective amount of polyprenols of the formula (1)

CH ₃ CH ₃

H - (CH ₂ - C = CH - CH ₂ ) _P - CH ₂ - C = CH - CH ₂ OH, where n = 8-20

and pharmaceutically acceptable excipients, including carriers, and / or solvents, additives and / or lubricants.

 The pharmaceutical composition may be in the form of a solution, suspension, capsule, tablet, granule, suppository.

 A method of treating demyelinating diseases of a living organism, including disease prevention, is characterized in that an effective amount of polyprenols of the formula (1) is introduced into a living organism

CH ₃ CH ₃

H - (CH ₂ - C = CH - CH ₂ ) _P - CH ₂ - C = CH - CH ₂ OH, where n = 8-20

in the form of a stand-alone preparation or in the form of a pharmaceutical composition further comprising auxiliary ingredients.

 The above polyprenols are isolated from conifers, including pine and spruce, fir, gingko biloba needles from woody green.

 The use of polyprenols according to the formula (1) is possible in complex therapy together with a remyelinating agent and anti-inflammatory agent, which is an adrenocorticotropic hormone, corticosteroid, interferon, glatiramer acetate or non-steroidal anti-inflammatory drug.

Brief Description of the Drawings The invention is illustrated by drawings. Figure 1 shows a diagram of the demyelination of the brain of mice under the action of cuprizone, figure 2 is a diagram of the restoration of the area of the corpus callosum under the influence of Ropren after demyelination, Fig. 3 is a diagram of the effect of Ropren on demyelination of the brain of mice according to the results of immunohistochemical studies, Fig. . 4 is a diagram of the effect of Ropren on neurogenesis in the subventricular zone of the brain of mice in a demyelination model; FIG. 5 is a diagram of the effect of Ropren on neurogenesis in the dentate gyrus of the hippocame of the brain of mice in a demyelination model.

 The implementation of the invention

 The inventors have identified the previously unknown possibility of using polyprenols of the formula (1) for the treatment and prevention in patients of conditions associated with demyelination and / or symptoms or signs of such conditions with impaired passage of a nerve impulse through the myelin sheath, including patients suffering from multiple sclerosis

CH ₃ CH ₃

H - (CH ₂ - C = CH - CH ₂ ) p - CH ₂ - C = CH - CH ₂ OH, where p = 8-20. (1) Polyprenols of the formula (1) is a natural mixture of oligomers (isoprenoids) behaves as a single substance (Ropren), but can be divided into individual isoprenoids on reverse phase sorbents. Ropren is obtained from coniferous herbs (pine, spruce, fir) (patent RU2017782). This polyprenol showed antiulcer action [N.A. Skuya et al. Products of woody greens processing - prospects for use in gastroenterology. / In the book: Functional diagnostics and treatment of digestive diseases. Vilnius, 1988, part 4, p. 675-676], hepatoprotective (patent US5731357), immunomodulating (patent RU2137479). The purest fraction of isoprenols - Ropren obtained by the method described in patent PCT / RU 2004/000162. The drug stimulates the processes of natural regeneration of the liver (RU 2252026), is used as a tool for the treatment of alcoholism and complications associated with it (PCT / RU 2008/000298, RU 2327448), as a tool for the treatment of patients with dementia syndrome (PCT / RU 2008/000297 )

However, currently drugs to speed recovery myelination of nerve endings and acceleration of neurogenesis are not used. The use of herbal preparations based on polyprenols is a promising direction.

 These include the drug Ropren, which can serve as an exogenous source of isoprenoid units, from which the endogenous product in the body is formed - dolichol. The isoprene units that make up Ropren can be used by the body, both for building cholesterol and for building ubiquinone - vital metabolites for the body. Polyisoprenode alcohols (prenols / dolichols) are metabolites in a living organism that are important, they are present in any living organism - from bacteria to every cell in the human body. They participate in the mevalonate pathway of cholesterol biosynthesis and in violation of this biosynthesis, which can take place both with dolichols and without them, which can lead to a violation of lipid metabolism in the body. Another way of biosynthesis, in which polyprenols in the form of phosphorylated derivatives are involved, is the biosynthesis of glycoproteins, where they participate in the processes of glycosylation of proteins and recognition of glucoprotein on the membrane (for example, as a cofactor in the formation of phosphatidylionisitol (GPI), which, as an anchor, is attached to the membrane and in transmission of a nerve impulse). They also participate in the recognition of antigens on the membrane, regulate the fluidity and viscosity of the membranes, and, consequently, the opening of ion channels, as well as the passage of a nerve impulse through the membrane. All the functions of polyprenols are not completely open, but based on studies, these compounds are of great interest for pharmacology [Surmacz L, Swiezewska E. Polyisoprenoids - Secondary metabolites or physiologically important superlipids, Biochem Biophys Res Commun. 201 1 Apr 22; 407 (4): 627-32. doi: 10.1016 / j.bbrc.201 1.03.059. Epub 201 1 Mar 16].

There is evidence that polyprenol (dolichol) is found in the black substance of the brain in neuromelanin, and its content is quite high - up to 15%, but its functions are still unknown. [Joshida K., Engel J., Liljequst S. Naunyn. Investigation of lipid component of neuromelani (NM). // Schmiedebergs Arch. Pharmacol 1982, Oct., 321 (1), 3-74,]. Ropren is a promising drug for the treatment of neurodegenerative diseases such as dementia and Alzheimer's disease (AD) due to its effect on the activity of mitochondrial enzymes: butyrylcholinesterase (BChE) and monoamine oxidase (MAO).

 As you know, the white matter of the brain in higher organisms consists of more than 50% of myelin, therefore, disorders in the formation of myelin during ontogenesis or changes in the structure of myelin in the developed nervous system during pathology lead to demyelinating diseases and severe neuropathies.

 Typically, proteins predominate in membranes, but in the case of the myelin sheath of nerves, three quarters of the mass is lipids, with cholesterol of the total lipid mass being 28% and located on the outside of the membrane. The total lipid content in myelin is 2 times higher - they are approximately 70-85%, and proteins make up to 20% of the dry mass of myelin compared to other plasma membranes. Myelin lipids are represented by phospholipids, glycolipids and steroids. Myelin does not metabolize completely, the half-life of lipids ranges from 5 weeks to 2-4 months (in phosphatidylserine and phosphotidylcholine), up to 1 year (in cholesterol). For proteins, this period ranges from 2 to 6 weeks (basic protein and proteolipid). However, it is interesting that the half-life is different for each component of the membrane and they are updated independently of each other, i.e. exchange occurs between all layers of the membrane [Huho F. Neurochemistry: fundamentals and principles: Trans. from English M: Mir, 1990.91-109s].

Polyprenols (dolichol) also work on the membrane, they exhibit membrane-active properties that affect the fluidity and viscosity of the membranes. Apparently, due to the binding of the polyisoprenoid fragment at its a-terminus to glycoproteins, and then to the protein, the transport of the protein or peptide and the transmission of the nerve impulse are facilitated. The main myelin protein and myelin glycoproteins are the main oligodendrocyte macromolecules. It can be assumed that while suppressing protein synthesis in the myelin sheath, polyprenols (Ropren) promote and / or participate in the transfer of myelin proteins, including the myelin basic protein (MBP, myelin basic protein) and secrete them through the membrane and thereby facilitate the transmission of nerve impulses and protect them from autoimmune aggression. In addition, in each cell in the vesicles, along with cholesterol, dolichol is always contained, which is involved in the biosynthesis of cholesterol. It can be assumed that when under the influence of various factors (for example, oxidative stress) the myelin sheath is damaged, then polyprenols come to the rescue and compensate for the lack of cholesterol, thereby preventing the loss of myelin (i.e. demyelination), as well as restoring the speed or activity of processes in the nervous system, as evidenced by new data. Namely, sterols and other lipids (dolichol) involved in cholesterol biosynthesis are the main protein conductors through the membrane and are involved in the regulation of the nerve signal [Friedhelm Schroeder, Barbara P. Atshaves at al-Sterol Carrier Protein-New roles in regulating lipid rafts andsignaling.// Biochem. Biophys. Acta, 2007, June, 1771 (6), p. 700-718]. Long-chain polyprenols and polyprenyl phosphates are ubiquitous and the main components of cell membranes in all areas of life. [Hartley MD, Imperiali B. At the membrane frontier: a prospectus on the remarkable evolutionary conservation of polyprenols and polyprenyl-phosphates. // Arch Biochem Biophys. 2012, Jan.l5; 517 (2): 83-97.doi: 10.1016 / j. abb.201 10.10.018. Epub 2011, Nov. 10.]. In addition, polyprenols in membranes can cause intracellular resonance and appear to be involved in signal transmission [Knudsen MJI, Troy FA Nuclear magnetic resonance studies of polyisoprenols in model membranes.// Chem. Phys. Lipids 1989, Nov; 51 (3-4): 205-12].

In an experimental study on animals with cerebral ischemia, it was shown that Ropren has a cerebroprotective effect. [Report on the topic: "Study of the effect of the drug Ropren on energy metabolism in cerebral ischemia in rats." // Russian Military Medical Academy named after SM. Kirova, Department of Pharmacology, St. Petersburg]. Other demyelinating diseases also include peripheral nervous system DZ. The use of Ropren at a dose of 4.3 mg / kg and 11.3 mg / kg for 7 days after occlusion of the common carotid arteries reduces the severity of neurological disorders in rats and increases their survival in the postischemic period. Ropren in these doses protects the brain from the development of metabolic acidosis and prevents the development of metabolic disorders. Against the background of cerebral ischemia and hypoxia, piracetam only by 46.5%, and ropren at a dose of 1 1, 6 mg / kg by 62% (i.e. almost two-thirds) reduces lactic acidosis. At the same dose, roprene increases the content of creatine phosphate (piracetam by 60%) and by 173% the content of adenosine triphosphate (ATP) in the brain tissue by 73%. The levels of adenosine diphosphate (ADP) ropren and the piracetam comparison drug are approximately the same, reducing their level by 40-43%. In this case, and piracetam and Ropren restored to normal the energy charge characterizing the general state of energy metabolism in the brain. The energy-stabilizing effects of roprene are comparable to the effects of piracetam. [Shabanov P. D., Zarubina I.V., Sultanov BC. To the mechanism of action of polyprenols in cerebral ischemia. // Medical academic journal 201 1, t. 11, N 2., S.25-32]. Polyprenols have immunomodulatory and antiviral effects [Safatov AS at all, A prototype prophylactic anti-influenza preparation in aerosol form on the basis of Abies sibirica polyprenols // J. Aerosol Med. 2005, Spring; 18 (1): 55-62]. Ropren is able to increase the level of interferon (INF) in the blood and, unlike other drugs (Cycloferon), it has a prolonged effect [Zarubaev V.V., Sultanov VS, Sukhinin V.P., Roshchin V.I., Nikitina T.V. Study of the dynamics of interferonogenic activity of Ropren preparations and its modified analogue in experiments on white mice. // Materials of the 4th international conference “Pasteur’s ideas in the fight against infections”, St. Petersburg, June 2-4, 2008, p.12]. In addition, when studying the effect of Ropren on the level of TNF-α in mice with ascitic and solid Ehrlich carcinoma in the experiment, it was found that in this case, a decrease in the concentration of TNF-a (TNF-α) in the blood serum is observed (for Ropren by 38% with a solid tumors and by 78% for ascites, and for the reference drug Glutoxim - by 29 and 75%, respectively [Report on the topic: "Study of the antitumor activity and mechanisms of action of the Ropren drug in models of solid and ascites forms of Ehrlich carcinoma in white mice", Research Institute of Influenza Russian Academy Medical Sciences, St. Petersburg, 2007]. Perhaps this Ropren property (reduction of TNF-alpha serum concentration) will help prevent, reduce inflammation and slow down PC development.

It is known that with autoimmune aggression and the development of multiple sclerosis, a decrease in the level of TNF-α is a good prognostic factor, reducing inflammation. A significant increase in the production of TNF-α in PC patients to the active stage of a remitting or primary progressive disease has been established compared to control parameters or data for stabilization of the disease, an increase in production of TNF-a is considered a harbinger of activation of the immunopathological process in PC. The maximum increase in the production of this cytokine is observed 5-14 days before the clinical exacerbation of the disease, dropping into the subacute stage [Evtushenko S. K., Derevyanko I.N. Modern approaches to the treatment of multiple sclerosis: achievements, disappointments, hopes. // International Neurological Journal. 2006, No 2 (6), S. 23-35].

 Embodiments of the invention

 The inventors conducted additional studies on the drug

Ropren revealed a previously unknown property of this compound, namely, a polyprenol-based composition has a positive effect in demyelinating conditions, including a remyelinating (restoring) effect, accelerates (increases) the process of neurogenesis, and also has an immunomodulatory and metabolic effect. This new property of Ropren is confirmed by experimental data.

 The basis for the use of the compounds of formula (1) for the declared purpose was the identification of a number of new activities previously unknown for this compound.

In an animal experiment, the effect of Ropren on myelination of white and gray matter was studied on a model of multiple sclerosis by cuprizone intoxication. Cuprizone intoxication is an excellent model used worldwide to study pathologies such as multiple sclerosis, epilepsy, and schizophrenia. [Preisner A1, Albrecht S, Cui QL, Hucke S. Non-steroidal anti-inflammatory drug indometacin enhances endogenous remyelination.// Acta Neuropathol. 2015, May 6. (Epub ahead of print)]. It has been observed that a three-week exposure to cuprizone is sufficient to cause deep demyelination. [Dewar, D., Underbill, SM, Goldberg, M. P. Oligodendrocytes and ischemic brain injury. // Cereb. Blood Flow Metab. 23., 2003, P. 263-274]. The introduction of cuprizone causes axonal edema, which occurs due to the redistribution of Na + channels and mitochondrial deficiency, which leads to disruption of sodium and calcium homeostasis. However, there are differences - there are no inflamed vessels in the Cuprizon model, and T cells are in a minimal amount [Rossi, L., Lombardo, MF, Ciriolo, MR, Rotilio, G. Mitochondrial disfunction neurodegenerative diseases associated with copper imbalance. // Neurochem. Res. 29. 2004. H. 493-504]. Under the action of cuprizone, there is massive apoptosis of oligodendrocytes in the fourth week of exposure with the participation of the immune system, and by 6 weeks cells of oligodendrocyte precursors differentiate into adult cells. Early changes in neurons occur at the synapse level, where the mediator concentration is impaired due to changes in the activity of synthesizing enzymes. In addition, inhibition of transport protein activity can be observed in demyelinated axon regions, which ultimately leads to axon loss. A certain number of neurons survive during cuprizone intoxication, despite the fact that they are equally or often more vulnerable to metabolic disorders compared with oligodendrocytes. A possible explanation is the metabolic relationship of neurons with astrocytes, which protect them from oxidative stress. During oxidative stress, astrocytes can trigger the glycolysis process, thereby creating a buffer that protects neurons from oxidative stress [Biancotti, J. C, Kumar, S., de Vellis. Activation of inflammatory response by a combination of growth factors in cuprizone-induced demyelinated brain leads to myelin repair. // J. Neurochem. Res. 33, 2008, P. 2615-2628].

 In accordance with the international classification, 4 main types of PC flow are distinguished. Lucchinetti C, describes 4 main types of multiple sclerosis: types 1 and 2 are considered autoimmune, while in types 3 and 4 oligodendropathy (apoptosis of oligodendrocytes) is considered the primary cause. [Lucchinetti, C, Bruck, W., at al ,. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelization.// Ann. Neurol. 2000, 47, 707-717]. In particular, the third type of multiple sclerosis has many similarities with cuprizone intoxication: actively demyelizing sites with a small participation of lymphocytes, but a large participation of microglia and macrophages, hypoxic tissue damage and signs of oxidative stress, as well as mitochondrial deficiency, on this model the authors studied the effect of the drug Ropren. The work was performed on 24 mice of the males of the CD-I line (age - 8 weeks at the beginning of the experiment) obtained from the nursery of the E.D. Research Institute of Pharmacology and Regenerative Medicine Goldberg (Tomsk).

The studies were carried out in accordance with the rules adopted by the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes. The experimental procedures were approved by the Bioethics Commission of the Institute of Cytology and Genetics SB RAS (Novosibirsk). Animals were divided into 3 experimental groups: "Control", "Demyelination" and "Ropren + demyelination". Animals of the Demyelination and Ropren + demyelination groups received standard food with 0.5% cuprizone for 10 weeks, and Control animals received standard food for 10 weeks. The animals of the Ropren + demyelination group ”were injected with the Ropren drug intraperitoneally at a dose of 12 mg / kg starting from the 6th week of the experiment, the animals of the Control and Demyelination groups were injected intraperitoneally with the same dose of a solvent - vegetable olive oil ( containing a mixture of polyunsaturated and saturated fatty acids).

 After 10 weeks, a brain scan of the mice was performed on a Broker BioSpec 1 17 / 16USR magnetic resonance imaging scanner under gas anesthesia using 2% isoflurane.

 At the end of the experiment, the animals were killed under ether anesthesia by transcardial perfusion in a large circle of blood circulation with 4% paraformaldehyde. The rat brain was recovered and placed in a 4% formaldehyde solution for 24 hours. After 24 hours, the brain was removed from formalin and placed in a 10% sucrose solution for a day, then it was transferred to a 20% sucrose solution, then it was frozen in liquid nitrogen vapor and placed in a freezer at -80 ° C for further immunohistochemical analysis.

 The study was conducted using modern methods of magnetic resonance imaging (MRI) of the brain and immunohistochemical analysis of sections of the brain of mice.

 Research methods

MRI studies. 10 weeks after the start of the experiment, a mouse brain scan was performed using several sequences (Turbo Rare, T2-weighted image, Study on three-dimensional (3D) protocol for mapping the macromolecular proton fraction (MPF) in the rodent brain with the following parameters: MT- w sequence: TR = 22 ms, TE = 2.5 ms, rotation angle 9 °, spectral bandwidth 125 kHz; for saturation of macromolecular protons an extra-resonant radio frequency pulse of a Gaussian shape with a duration of Strongly 10ms detuning frequency 6 kHz and a nominal angle of rotation of 900 °), as well as the Tl-w sequence: TR = 16 ms, TE = 2.5 ms, rotation angle 16 °, spectral bandwidth 125 kHz, and also with PD-w sequence: TR = 16 ms, TE = 2.5 ms, rotation angle 3 °, spectral bandwidth 125 kHz, as well as with FieldMap sequence for mapping the VO field (double echo method): TR = 20 ms, TE 1 = 2.4 ms, TE 1 = 4.1 ms, rotation angle 8 °, spectral band width 200 kHz., And finally with the AFI sequence for mapping the B1 field (double TR method): TR1 = 12 ms, TR2 = 60 ms, TE = 3.6 ms, rotation angle 60 °, spectral bandwidth 59.5 kHz, increment phase angle of 39 °.

 In the last decade, the MPF (molecular proton fraction) parameter, also known as the “bound pool fraction” or “restricted proton fraction” parameter, has been widely used as a marker of myelin due to strong correlations between MPF and myelin content in nerve tissue. Pulse sequences and the method of mapping the macromolecular proton fraction (MPF) were developed by Dr. V.L. Yarnykh [Yarnykh VL. Fast macromolecular proton fraction mapping from a single off-resonance magnetization transfer measurement./ Magn. Reson Med 2012; 68 (1): 166-178]. A number of independent groups and Dr. V.L. Yarnykh showed a high correlation of the MPF parameter with the quantitative content of myelin in various areas of the brain [Underhill HR, Rostomily RC, Mikheev AM, Yuan C, Yarnykh VL .. Fast bound pool fraction imaging of the in vivo rat brain: Association with myelin content and validation in the C6 glioma model./ Neuroimage 201 1; 54 (3): 2052-2065 .; Samsonov D Alexander AL, Mossahebi P, Wu YC, Duncan ID, Field AS. Quantitative MR imaging of two-pool magnetization transfer model parameters in myelin mutant shaking pup. / Neuroimage 2012; 62: 1390-1398].

 Scanning was carried out in axial projection. The geometry parameters, spatial resolution and the number of signal averages were set for the mouse: field of view 20x20x24 mm, matrix 200x200x80, resolution 100x100x300 μm, 4 signal averages, total scan time 48 minutes. For the sequences of mapping the fields BO and B1, the resolution in the plane was set half as high as for the remaining sequences due to the smoothness of the spatial changes in the field. Accordingly, a 100x100x80 matrix was used.

For groups of mice with demyelination and the control group were reconstructed MPF maps of the brain using a single-point algorithm using a synthetic reference image [Yarnykh VL. Fast macromolecular proton fraction mapping from a single off-resonance magnetization transfer measurement./ Magn. Reson Med 2012; 68 (1): 166–178] and studied the degree of demyelination in the structures of white matter (corpus callosum, arch, commissure, internal capsule) and gray (cortex, thalamus, hippocampal fissure, dentate gyrus and field CA1 of the hippocampus).

 The average MPF values in the studied structures of the brain substance in demyelinated and control mice were calculated using Image J software. To do this, on a MPF map, a region of interest (ROI) of a fixed size was placed in the corresponding anatomical structure, and the average value of the parameter in the region of interest was measured. The obtained parameter values were used to assess myelin loss during demyelination.

 The area of the structure of the white matter of the brain - Corpus callosum (corpus callosum) for control groups, "Demyelination",

“Demyelination + Ropren” was measured in pixels using Image J software for the sum of the areas of 12-14 slices from the front edge of the olfactory bulb using T2-weighted images.

 The contouring of the corpus callosum and the measurement of MPF were carried out by the same operator for all groups of animals blindly, without informing the operator about the animal's membership in a particular group.

 Immunohistochemical studies

Coronary sections of the brain of mice with a thickness of 10 μm were obtained using the NM525 cryotome. Slices were obtained in zones 0.74-S.26 and -1.34 - ^ - 1.70 from Bregma according to the atlas (G.Paxinos, K.V. J.Franklin, 2001). The sections obtained were immunohistochemically stained for basic myelin protein (MBP) and doublecortin (DCX, a marker of young neurons) using primary (Goat anti MBP (sc-13914), Goat anti Doublecortin (C-18): sc-8066, Santa Cruz) and secondary (Donkey anti goat AlexaFlour 488 (705-545-147), Jackson Immunoresearch) antibodies. Photographing was carried out using an Axio Imager Z2 microscope (Carl Zeiss, Germany) and Axio Vision 4.8 software (Carl Zeiss, Germany) with a MozaiX module, which allows obtaining images of ~ 1 1 mm2 in size by combining 20-28 smaller images, depending on size cut. Microphotographs of whole sections of the brain stained with MBP were evaluated by a method similar to processing MPF cards: the average fluorescence intensity over the area of the studied brain structures was calculated (corpus callosum, internal capsule, cortex, thalamus, hippocampal CA1 field, hippocampal fissure, dentate gyrus, commissure, arch ) The obtained intensity was normalized by the background and average intensity of the whole slice, the obtained values were included in further statistical processing. For each animal, photographs of 10-14 sections of the brain were analyzed.

 The number of DCX + (newly formed young neurons) was visually calculated in two main neurogenic zones - the subventricular zone near the lateral ventricles (SVZ) and the subgranular zone of the dentate gyrus of the hippocampus (DG).

 The differences between the groups were evaluated using Statistica 6.0 software by analysis of variance (ANOVA) and subsequent post-hoc analysis using the T-test or the non-parametric Mann-Whitney test in case the distribution differs from the normal one. Differences were considered statistically significant at p <0.05.

 Example 1. The effect of Ropren on myelination of the brain according to the results of MRI studies. The study of the white and gray matter of the brain using the mapping of the macromolecular proton fraction of MPF. Dispersion analysis of MPF maps showed the significance of the effect of cuprizone administration factor on mouse brain myelination (F (8, 40) = 3.9490, p = 0.00161). Post-hoc analysis confirmed the demyelination under the action of cuprizone for the main brain structures using the T-test and showed the presence of significant demyelination for the white matter of the brain: a decrease in the MPF parameter by 1, 9 times in the corpus callosum and 1.5 times in the commissure, and also for gray matter: a 1.3-fold decrease in the MPF parameter in the cortex and thalamus. Thus, a 10-week oral administration of cuprizone led to significant demyelination of the structures of both white and gray matter, as shown in the diagram of Fig. 1, where the MPF parameter of mice with cuprizone demyelination and control animals is visible ** - p <0.01, * - p <0.05, T-test.

T2-weighted images of mice from the control groups, “Demyelination”, “Demyelination + Ropren” were obtained, where the corpus callosum demyelinated in the mouse with the introduction of cuprizone and without demyelination was visualized for the Control and Demyelination + Ropren groups. A quantitative assessment of the area of the corpus callosum in mice of different groups was carried out, which showed a significant decrease in the area of the corpus callosum in mice from the introduction of cuprizone, compared with the control group. At the same time, with the introduction of Ropren, the area of the corpus callosum practically did not differ from the control group, despite the introduction of cuprizone, which indicates a protective effect of Ropren on the brain during intoxication with cuprizone, which causes its demyelination. This effect is shown in the diagram of figure 2, where the area of the corpus callosum in mice of the Demyelination group is significantly (P <0.05, T-test) 21.5% less than the same indicator for the Control and Demyelination + Ropren groups, at the same time, there are no significant differences between the Control and Demyelination + Ropren groups.

 Thus, a 10-week oral administration of cuprizone according to the results of MRI studies leads to significant demyelination of the brain. When demyelination detected a significant decrease in the area of the corpus callosum. The introduction of Ropren has a protective effect on the white matter of the brain, namely, restoring the area of the corpus callosum to control values.

 Example 2. The effect of Ropren on myelination of the brain according to the results of immunohistochemical studies. Microphotographs of brain sections stained for basic myelin protein (MBP), mice of the Control, Demyelination, Demyelination + Ropren groups clearly indicate a decrease in fluorescence intensity and contrast between the white and gray matter of the brain. While in microphotographs of brain sections stained for basic myelin protein (MBP), the fluorescence intensity in mice of the Demyelination + Ropren group does not decrease and visually the slices look the same as in the control group of mice.

A quantitative analysis of the fluorescence intensity showed a significant decrease in the intensity, reflecting the content of the main MBP myelin protein, for the Demyelination group compared with the control from 10 to 54% in all the studied brain structures, except for commissure, where * - p <0.05, ** - ρθ .01, T-test, as well as differences between the groups "Demyelination" and "Control" were - p <0.01, T-test. The fluorescence intensity indices reflecting the content of the MBP myelin basic protein in the Demyelination + Ropren group were higher than in the Demyelination group, by 20.0 -75.0%, and also exceeded control values in various brain structures (diagram in Fig. 3).

 The introduction of Ropren statistically significantly increased myelination in these structures, in terms of structures, myelination was higher than in the control group, which indicates the restoring (remyelinating) effect of Ropren,

 Example 3. The effect of Ropren on neurogenesis in neurogenic zones of the brain according to the results of immunohistochemical studies.

 The effect of Ropren on neurogenesis in mice was analyzed. The obtained micrographs of sections of the brain of the subventricular zone near the lateral ventricles for animals of different groups stained with doublecortin, which is a marker of young neurons and visualized using specific antibodies conjugated with green fluorochrome (AF488), show a decrease in young neurons in the Demyelination group of mice compared to control. Micrographs of brain sections of the same zone from the Demyelination + Ropren group show an increase in the number of young neurons compared to the Demyelination and Control groups.

 It was found that with cuprizone demyelination, the number of young neurons in all major neurogenic zones of the brain decreases compared with the control group (p <0.001). In the subventricular zone (SVZ) in mice treated with a preparation based on polyprenols (Ropren), there is a tendency to increase the level of neurogenesis compared with the control group (p = 0.05) and a statistically significant increase in the number of young neurons compared to the Demyelination group (diagram in Fig. 4).

 In the granular layer of cells of the dentate gyrus of the hippocampus (DG), there are no statistically significant differences between the control group and the group of mice that received the drug based on polyprenols (p> 0.05), while for this group Demyelination + Ropren, the number of young neurons is significantly higher compared with the Demyelination group (p <0.001) (diagram in Fig. 5).

The results obtained in experiments on animals on the model of demyelination with cuprizone made it possible to establish with confidence the new properties of Ropren, namely, the drug restores myelination in the structures of the white and gray matter of the brain to the control level, which is confirmed by the results of MRI and immunohistochemical studies. On the cuprizone scattered model sclerosis there was a significant demyelination of structures, both white and gray matter, confirmed by the results of MRI studies using mapping of the macromolecular proton fraction and immunohistochemical studies. A quantitative assessment of the intensity of staining of brain sections on the main myelin protein showed that treatment with Ropren protects the brain from demyelination.

 The study showed a significant increase under the action of Ropren, the level of neurogenesis in the main neurogenic zones of the brain in mice with demyelination, while the level of neurogenesis during demyelination decreases by 3.6 times.

 In the granular layer of the cells of the dentate gyrus of the hippocampus (DG), Ropren significantly increases - the number of young neurons is 4 times greater than demyelination, they become even more than in the control by 15% (Diagram 5). The same tendency is observed in another zone - the subventricular zone in mice (SVZ), the number of neurons in this region increases 2.2 times compared with demyelination, and 37.5% compared with the control.

 These new, previously unknown properties of Ropren were first clearly demonstrated and proven using MRI and immunohistochemical studies, which makes it possible to use this drug in the treatment of multiple sclerosis and demyelinating diseases and conditions.

 Clinical Experience with Ropren

 (with toxic encephalopolneuropathy with severe demyelination, impaired conduction of both limbs)

 Chronic alcoholic polyneuropathy (PNP) is a primary degenerative process with the breakdown of myelin, the destruction of axial cylinders and the replacement of nervous tissue with connective tissue rich in blood vessels. First of all, the motor nerves are affected: first of all, the radial nerve in the upper limbs and the perineal nerves in the lower extremities. The pectoral and phrenic nerves are involved in the pathological process. The optic nerve and retina are often affected. [Trinitatsky Yu.V., Trinitatsky I.Yu. Treatment of chronic demyelinating polyneuropathies., State Medical University, Rostov-on-Don, 2003, p.176].

A clinical trial involved patients with chronic alcohol polyneuropathy, which were divided into two groups according to the severity of clinical symptoms: with moderate PNP without signs of demyelination and with severe and severe forms of PNP, taking alcohol substitutes, which led to more persistent disorders in neurological symptoms and to a demyelinating state as a result of myelin degeneration .

 In the control group of patients with a diagnosis of polyneuropathy (PUP), there were 25 people, 14 of them were diagnosed with toxic encephalopolneuropathy. In the experimental group - 57 people, of which 23 patients were diagnosed with toxic encephalopolneuropathy. Toxic encephalopolneuropathy (demyelinating state - DS) was clinically characterized by the addition of limb polyneuritis, mainly lower extremities, expressed in paresthesia and numbness of the extremities, pain, burning, in a decrease in reflexes, pain threshold, vibrational, temperature, tactile sensitivity of the limbs of the legs and hands. The severity of PNP was evaluated on the basis of a study of the thresholds of 4 types of sensitivity (tactile, pain, temperature and vibration) and reflexes (Achilles and knees) based on standardized international tests on the Young scale (M. Washsh, 1993) based on the total points:

 - from 3-5 points - a moderate form of PUP,

 - from 6-8 points - a pronounced form of PUP,

 - from 9-10 points - a severe form of PUP.

In the experimental group of 23 patients with severe and severe form of DS within 1 month of treatment with Ropren, there was an improvement in the demyelinating state of patients, which consisted in the transition of PNP to the milder stage of the disease according to the Young scale, and only in 2 patients these manifestations were less expressed. Three patients with demyelinating condition recovered, and in the control group no one recovered by the end of treatment. In the control group, a slightly pronounced effect in 3 patients occurs by the 15th day of treatment, while in the experimental group, by the same day of treatment, the effect occurred in the majority of patients. After 30 days of treatment with Ropren, out of 16 patients with severe PNP, 3 people (18.8%) have a slight improvement and transition to a pronounced form, in 1 1 people (68.8%) - a transition to a moderate form, i.e. more significant improvement, and 2 people recovered by the end of treatment. From 7 people with the expressed form of ANP improved in 6 people (85.7%), and 1 person recovered and had no signs of ANP. Thus, in the experimental group of 23 patients, 3 people recovered during the period of treatment with Ropren, the rest showed a positive dynamics. In the control group for the same period in patients who underwent treatment (they received nootropics, cerebroprotectors and B vitamins as injections), no significant improvement was observed in the condition. So out of 9 patients with severe form of PNP, there was a transition to the expressed form of PNP in 8 people (89.0%), and 1 (1 1%) the patient was without dynamics. The remaining 5 patients with a pronounced form of PNP, of which 2 people (40%) went into the moderate stage, and 3 (60%) were without dynamics. The effectiveness of treatment with Ropren is most visible with severe forms of PNP, when it is quite difficult to succeed in treating the disease. Such an effect in the control group of patients with severe forms of DS does not occur, even towards the end of treatment.

 According to electroencephalography (EEG), an improvement in the condition of patients with demyelinating disease was noted. Before treatment with Ropren, in the majority of the studied patients in the initial EEG, there was a clear decrease in the severity of alpha activity and an increase in the severity of beta activity, which often had an emphasis in the frontal areas. Of the 23 people in the experimental group after treatment, 13, that is, more than 50% of patients showed pronounced positive dynamics on the EEG. After treatment with Ropren, marked changes in bioelectric activity were noted, reflecting distinct positive neurophysiological changes. The increased severity of the rhythm, its more correct organization reflects the normalization of the general functional state of the central nervous system and the optimization of cortical-subcortical relationships.

Example 4. (Experience). Patient, 49 years old, disease duration 5 years. Diagnosis: Toxic encephalopolneuropathy (DS) with epileptic syndrome (intensification of seizures last 3 years), chronic alcoholism of the 2nd degree, convulsive symptom. Improvement occurred on the 15th day, a decrease in focal cerebral symptoms and polyneurotic prolapse. There were no attacks of epilepsy. By the 30th day of treatment - a marked improvement in focal cerebral symptoms. PNP has passed from a severe to a moderate stage. Regression of neurological status is significant. EEG data show positive dynamics - increased alpha rhythm.

 Example 5. (Experience). Patient, 39 years old, disease duration 10 years. Diagnosis: Toxic encephalopolneuropathy (DS), chronic alcoholism of the 2nd degree. On day 16, a significant regression of focal neurological symptoms: nystagmus disappeared, cerebellar disturbances decreased. By the 30th day, only micro-focal cerebral symptoms persist, a significant regression of PNP (from 10 points to 4 points on the Young scale, i.e., from a severe form of PNP went into a moderate form). According to the EEG data, after treatment, there is a positive dynamics in the form of a significant increase in the alpha rhythm, a decrease in the severity of the assimilation of photostimulation rhythms (RFS).

 Industrial applicability

 The results of a study of the effects of a drug and a pharmaceutical composition based on compound (1), Ropren in animals on a cuprizone model of multiple sclerosis, which causes demyelination in the brain of mice, as well as in the treatment of patients with toxic encephalopolineuropathy (demyelinating disease) in a clinic, led to the conclusion the effectiveness of the treatment of demyelinating diseases. Ropren stimulates the formation of new neurons in the neurogenic zones of the brain, significantly improves neurological disorders and leads to remyelination (restoration of the demyelinating state).

 The drug Ropren is an inexpensive tool, its preparation is based on the known method of extraction of coniferous woody green.

Claims

Claim  1. The use of polyprenols of the formula (1) СН ₃ СНз Н - (СН ₂ - С = СН - СН ₂ ) „- СН ₂ - С = СН - СН ₂ ОН, where n = 8-20 (1) as an active ingredient in the manufacture of a medicament for the treatment of demyelinating diseases of a living organism.  2. The use of polyprenols of the formula (1) CH ₃ CH ₃  I I H - (CH ₂ - C = CH - CH ₂ ) „- CH ₂ - C = CH - CH ₂ OH, where n = 8-20 (1) as an active ingredient in the manufacture of a medicament for the prevention of demyelinating diseases of a living organism.  3. A drug for the treatment of demyelinating diseases of a living organism, including the prevention of disease, characterized in that it is a polyprenol of the formula (1) CH ₃ CH ₃ H - (CH ₂ - C = CH - CH ₂ ) - - CH ₂ - C = CH - CH ₂ OH, where n = 8-20 (1).  4. The pharmaceutical composition for the treatment of demyelinating diseases of a living organism, including disease prevention, characterized in that it comprises an effective amount of polyprenols of the formula (1) CH ₃ CH ₃ H - (CH ₂ - C = CH - CH ₂ ) - - CH ₂ - C = CH - CH ₂ OH, where n = 8-20 (1). and pharmaceutically acceptable excipients, including carriers, and / or solvents, additives and / or lubricants.  5. The pharmaceutical composition according to paragraph 4, characterized in that it is made in the form of a solution, suspension, capsule, tablet, granule, suppository. 6. A method of treating demyelinating diseases of a living organism, including disease prevention, characterized in that an effective amount of polyprenols of the formula (1) is introduced into a living organism CH ₃ CH ₃ H - (CH ₂ - C = CH - CH ₂ ) „- CH ₂ - C = CH - CH ₂ OH, where n = 8-20 (1) in the form of an independent preparation or in the form of a pharmaceutical composition, additionally including auxiliary ingredients.