RU2778199C1 - Method for application of resveratrol to accelerate the regeneration of damaged nerves - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to neurology, and can be used to accelerate the recovery process of damaged somatic nerves. The method involves intramuscular administration of a suspension of resveratrol at a concentration of 0.1 M/l, which corresponds to a dose of 7 mg/kg of weight, for 30 days.

EFFECT: use of the invention makes it possible to accelerate the recovery process of damaged somatic nerves by increasing the amount of nerve growth factor (NGF) and restoring the functional activity of the damaged sciatic nerve.

1 cl, 2 dwg, 2 ex

Description

The invention relates to the field of regenerative medicine, namely to a method of using resveratrol to accelerate the process of recovery of damaged somatic nerves.

Analysis of the state of the problem of regeneration of damaged peripheral nerves shows a low level of efficiency in the restoration of the damaged nerve and its functional activity. The main causes of such injuries are road traffic accidents, professional and domestic injuries. The above leads to an increase in the number of people with disabilities and long-term disability.

In order to accelerate and stimulate the process of regeneration of damaged somatic nerves, various approaches are used, including the use of regeneration stimulants.

Today, stimulants of physical, chemical and biological nature are used as stimulators of regeneration processes. Laser, magnetic and electromagnetic radiation are used as physical stimulators, which requires rather complex and expensive hardware for their use. Among the stimulants of a chemical nature, one can distinguish various vitamin (thiamine chloride, pangamic and nicotinic acids), anticholinesterase drugs (prozerin, galantamine), as well as drugs with anti-inflammatory and analgesic effects, nootropic drugs such as Biotredin, Phenotropil, Neurobutal , their disadvantages include their synthetic nature and a rather low efficiency of regeneration of damaged nerves, and various side reactions can be observed when using them. Among the regeneration stimulators of a biological nature, one can single out growth factors and neurotrophic factors of exogenous and endogenous origin (nerve growth factor, insulin-like growth factor 1, brain growth factor), as well as natural biologically active substances-polyphenols. Neurotrophs and nerve growth factors have a huge disadvantage - high cost, besides, they have a short shelf life that requires certain conditions. At the same time, polyphenols exhibit their biological activity at low concentrations (milli- and micromoles/l) and have a lower cost compared to the use of neurotrophic factors. Secondly, they have a complex effect, they can simultaneously have anti-inflammatory, antioxidant and neuroprotective effects. Another advantage is their natural origin, which minimizes the side effects of treatment.

Known drug stefaglabrin sulfate 0.25% solution. Refers to drugs that act primarily on the peripheral nervous system. Has anticholinesterase activity; inhibits (suppresses the activity of enzymes) true and false cholinesterase. Stefaglabrin is an alkaloid isolated from tubers with roots of Stephanie glabra . Contraindications of this drug are: epilepsy, hyperkinesis (involuntary contractions of the muscles of the limbs), bronchial asthma, angina pectoris, bradycardia (rare pulse). The method consists in the fact that L-arginine is injected into the deposition medium based on the sodium salt of carboxymethyl cellulose, which fills the silicone tube, and the created conduit is implanted into an extended rupture of the peripheral nerve. The invention provides a pronounced stimulating effect on the restoration of the motor and sensory function of the nerve, effective maintenance of the survival of sensitive neurons and the germination of regenerating myelin fibers from the central to the peripheral segment of the nerve (RU 2369388, IPC A61K 31/00, A61K 36/59, A61K 9/08, publ. 10.10.2009).

The disadvantage of the known solution is that it is not a stimulator of reparative processes.

A known method of stimulating the regeneration of a damaged peripheral nerve in experimental animals after the operation of its cutting and stitching includes the introduction of drugs, which use B vitamins and anticholinesterase drugs. Additionally, in the postoperative period, animals are injected intramuscularly with neurotoxin of the variegated scorpion venom in doses of 40-50 μg/kg once every 2 days for 30-40 days, repeating the course 2-3 times with an interval of 20-30 days. The method provides an acceleration of the regeneration process and an increase in the volume of regenerating nerve fibers in the peripheral nerve (RU 2290187, IPC A61K 55/64, A61M 5/00, A61P 25/02, publ. 27.12.2006).

Among the disadvantages of this method is that the introduction of neurotoxin venom of the motley scorpion at a dose of less than 40 mcg/kg and less than 30 days is ineffective, and more than 50 mcg/kg and more than 40 days increases the risk of side effects. .

There is a known method of accelerating regeneration processes in damaged peripheral nerves, in which intramuscular administration of a solution of clobetasol at a concentration of 0.5 mg/kg for 30 days is carried out (RU2745868, IPC A61K 31/573, A61P 25/02, publ. 04/02/2021).

The disadvantage of the known solution is that the introduction may cause local reactions: burning, itching, acne, dry skin, stretch marks; with prolonged use - atrophy of the skin and telangiectasia, hypertrichosis, pigmentation disorders, weakening of the barrier function of the skin, development of the pustular form of psoriasis.

Closest to the claimed invention is a method for evaluating the effectiveness of the chemoprophylactic activity of resveratrol in conditions of experimental carcinogenesis of liver cancer (RU 2736238, IPC, G09B23/28, A61P35/00, A61K31/05, publ. 11/12/2020).

The disadvantage of the prototype is that resveratrol is used in higher concentrations and for a longer period (100 mg/kg animal for 6 months), and besides, this method does not accelerate the recovery of damaged peripheral nerves.

The technical result consists in accelerating the process of recovery of damaged somatic nerves through the use of resveratrol, a polyphenol of the stilbene series, which increases the amount of NGF and restores the functional activity of the damaged sciatic nerve.

The essence of the invention lies in the fact that in a method for accelerating the regeneration of damaged somatic nerves, a suspension of natural, biologically active substance resveratrol at a concentration of 0.7 mg/kg of body weight is intramuscularly injected into the medial thigh muscle of the injured nerve for 30 days. The suspension contains in its composition: resveratrol - 0.7 mg; physiological NaCl solution (pH=7.4) - 900 µl, ethyl alcohol (70%) - 100 µl. The polyphenol resveratrol has a wide spectrum of action, regulates the activity of antioxidant enzymes, reduces the intensity of lipid peroxidation processes, increases the synthesis of nerve growth factors and restores the functional activity of the damaged nerve.

On FIG. 1 shows the change in the functional index of the damaged nerve with the introduction of resveratrol at a concentration of 0.1 mol/l and 0.001 mol/l (* - the significance of the difference in relation to the control, p≤0.05), Fig. Figure 2 shows the concentration of nerve growth factor NGF in the proximal area on the 30th day (* - significance of the difference in relation to the intact nerve, p≤0.01, ** - significance of the difference in relation to the control, p≤0.01).

Examples of the invention.

Example 1

The study of the function of the extensor muscles of the toes in trauma and the influence of resveratrol was carried out by determining the functional sciatic index (FSI).

On average, the FSI value in the intact group of animals was 8.8 conventional units (Fig. 1).

On day 1, there was a sharp decrease in the FSI index to - 26.6, that is, the functional loss according to the measurements of this parameter was 17.8%. At the end of the first week after the sciatic nerve compression operation, the animals practically did not use the injured limb, were inactive, the interdigital distance (MR) and the intermediate distance between the 2nd and 4th fingers (IR) on the injured limb were sharply reduced, while the length of the footprints of the injured limb increased .

The FSI value at the end of the first week after the operation was 40.16, that is, the functional loss according to the measurements of this parameter was 32%. The dynamics of changes in FSI showed that, starting from the third week after the nerve injury, there was a noticeable acceleration in the process of restoring the lost function of the extensor toes. It was at this time that a significant increase in the average value of FSI was observed up to - 19.87 on the third week (Fig. 1).

The data obtained are explained by the fact that with partial damage to the PNS at any level, the restoration of the original pattern of innervation occurs due to the remaining axons, and the latter begin to actively grow and branch (“sprouting”), giving numerous fibers heading to the denervated muscle fibers or skin areas; thus, under favorable conditions, the restoration of "own or primary" innervation occurs.

In order to identify possible differences in the characteristics of the restoration of the sciatic nerve, an analysis was made of the effect of injections of resveratrol at a concentration of 0.1 mol/l and 0.001 mol/l on the degree of recovery of the muscles of these groups at various times after injury (1, 7, 28 days). On day 1, a decrease in the FSI index was observed with the introduction of resveratrol at a concentration of 0.1 mol / l to - 17.34, a concentration of 0.001 mol / l to - 21.13, functional loss according to the measurements of this parameter, at a concentration of 0.1 mol / l was equal to 9%, at a concentration of 0.001 mol/l - 12.33% (Fig. 1).

On the 7th day, a decrease in MR and PR parameters was observed (Fig. 1), the FSI index with the introduction of resveratrol at a concentration of 0.1 mol / l was equal to - 35.06, the concentration of 0.001 mol / l was up to - 37.51, functional loss according to measurements of this parameter, at a concentration of 0.1 mol/l was equal to 26%, at a concentration of 0.001 mol/l - 28%.

Starting from the third week, the animals already freely used the injured limb. The FSI value on the 28th day at a concentration of 0.1 mol/l was 17.01, at a concentration of 0.001 mol/l it was 17.61.

The studied FSI reflects functional changes in the work of the damaged nerve. Resveratrol can activate signaling between the vascular endothelial growth factor (VEGF) family and other nerve growth factors and their receptors in the motor neuron and thus promote nerve regeneration.

Several studies have shown that various polyphenols, including flavonoids such as genistein ( Bondonno CP et al., 2012), quercetin, liquiritin (Schroeter H. et al. 2006), al. 2016), and acetylated flavonoid glycosides from Scopariadulcis (Zheng XX et al. 2013), as well as the stilbene compound resveratrol (a polyphenol present in grapes and red wine) (Ras, RT et al. 2011) cause a significant increase in the synthesis neurotrophins (nerve growth factor [NGF] and brain-derived neurotrophic factor [BDNF]) mediate neurite outgrowth in PC12 cells.

Some polyphenolic compounds have been reported to increase BDNF levels; various flavonoids such as puerarin (Wightman E. L. et al. 2015) and quercetin (Wong R. H. X. et al. 2016), as well as other polyphenols such as curcumin and resveratrol (Ras, R. T. et al. 2011; Bowtell J. L. et al. 2017 ) are also reported to have this property.

Example 2

Since resveratrol contributes to an increase in the number of growth factors, and their increase directly affects the survival of neurons and their further regeneration. We have studied the effect of resveratrol (0.1M/l and 10mkM/l) on the content of nerve growth factor in the proximal and distal parts of the damaged sciatic nerve for 30 days (Fig. 2).

When studying the protein composition after damage to the peripheral nerve on day 30, with the introduction of resveratrol concentrations of 10 μM / l and 0.1 M / l, an increase in the amount of nerve growth factor NGF in the proximal section by 12 and 21% was found, compared with the undamaged one, with a concentration of 0, 1 M/l had a more pronounced effect.

Tracing the dynamics of the results of this experiment, it can be seen that the concentration of nerve growth factor decreases on day 30 without the administration of the substance, and the use of resveratrol contributes to an increase in the concentration of NGF. According to I. N. Shchanitsyn, this is due to the fact that Schwann cells produce a number of neurotrophic and neurotropic molecules (BDNF, neurotrophin, NGF and IGF), which stimulate the growth of axons.

Resveratrol, as a polyphenol with a positive effect on the nervous system, may be a resource for the discovery of new neurotrophic agents in the future. The neurotrophic actions of various polyphenols are based on Trk receptor agonist activity, activation of the ERK, PI3Kinase/Akt and CREB pathways, activation of the Nrf2 pathway, and upregulation of antioxidant and detoxification enzymes. An in-depth study of the molecular mechanisms of action of these compounds in the future will expand the horizon of research for the search for new drugs for the treatment of neurodegenerative diseases and other disorders of the nervous system (Moosavi F. Modulation of neurotrophic signaling pathways by polyphenols / F. Moosavi et. al. //Drug design , development and therapy. - 2016. - Vol. 10. - P.23).

Compared with the prototype, the claimed invention is capable of stimulating the process of regeneration of the damaged nerve, which is confirmed by data on an increase in the amount of nerve growth factor and restoration of the functional activity of the sciatic nerve when resveratrol polyphenol is administered against the background of injury.

Claims (1)

A method of using resveratrol to accelerate the regeneration of damaged nerves by intramuscular injection of a suspension of the natural biologically active substance resveratrol at a concentration of 0.1 M/l, which corresponds to a dose of 7 mg/kg of body weight, for 30 days.

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