RU2704815C2 - Method of treating muscular dystonies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns a method of treating muscular dystonias in a human being consisting in administering to the patient the therapeutically effective amounts of peptide DNWWPKPPHQGPRPPRPRPKP.

EFFECT: invention provides a local therapy of muscular dystonia with no side effects.

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Description

The technical field to which the invention relates.

The present invention relates to medicine, namely to a method for treating muscular dystonia. More specifically, this invention relates to a composition and method for administering a drug based on the Azemiopsin peptide.

State of the art

Muscular dystonia - “a syndrome of constant muscle contractions, often causing repeated,“ twisting ”movements or pathological postures of the trunk, neck, arms, legs and muscle spasms of the face” (B.S. Oppenheimer). Examples of muscular dystonia: blepharospasm (involuntary squeezing), cervical dystonia (torticollis), spasticity (hypertonicity) of skeletal muscles, cramping spasm, foot dystonia, etc. The problem of constant increased tone of certain muscle groups also exists with a spastic form of cerebral palsy (Cerebral Palsy). According to the European and American dystonia societies [1], the number of patients with various forms of dystonia is 500,000 in Europe and 300,000 in the USA. According to the Research Foundation for the Association of Cerebral Palsy Patients [2] in the United States, there are approximately 760 thousand patients with this disease. In Russia, the number of patients with muscular dystonia is estimated at 80,000-140,000 people, and cerebral palsy - 150,000-200,000 people. A number of departmental and regional target programs have been directed to the treatment and rehabilitation of patients with cerebral palsy in the Russian Federation. The main treatment for muscle dystonia and the spastic form of cerebral palsy is the injection of botulinum toxin into the muscles involved in hyperkinesis. In 1989, Botox (one of the drugs based on botulinum toxin) was approved by the FDA for the treatment of blepharospasm, in 2000 - cervical dystonia, and in 2010 muscle spasticity in the elbow, wrist and fingers. The clinical effect is achieved in 85-90% of cases and lasts for 2-3 months. As a rule, patients need repeated injections of the botulinum toxin preparation: with spastic crankshaft - 2 injections per year, with blepharospasm - 3-4, with cerebral palsy - 2 times a year .

The type A botulinum toxin molecule consists of heavy (with a molecular weight of 100,000 Da) and light (with a molecular weight of 50,000 Da) chains linked by a disulfide bridge. The heavy chain has a high binding affinity for specific receptors located on the surface of target neurons. The light chain has a Zn2 + -dependent protease activity specific for the cytoplasmic regions of a synaptosomal-bound protein having a molecular weight of 25,000 Da (SNAP-25) and participating in exocytosis processes. The first stage of the action of botulinum toxin type A is the specific binding of the molecule to the presynaptic membrane. This process takes 30 minutes. The second stage is the penetration of the bound toxin into the cytosol of the nerve through endocytosis. The intracellular light chain acts as a Zn2 + -dependent cytosol protease, selectively cleaving SNAP-25, which in the third stage leads to blockade of the release of acetylcholine from presynaptic terminals of cholinergic neurons. The final effect of this process is persistent chemo-denervation.

With the intramuscular administration of Botox®, 2 effects develop: direct inhibition of extrafusal muscle fibers by inhibiting alpha-motor neurons at the level of the neuromuscular synapse and inhibition of muscle spindle activity by inhibiting the gamma-motor neuron cholinergic synapse on the intrafusal fiber. A decrease in gamma activity leads to a relaxation of the intrafusal fibers of the muscle spindle and reduces the activity of 1a afferents. This leads to a decrease in the activity of muscle stretch receptors, as well as to the efferent activity of alpha and gamma motor neurons. Clinically, this is manifested by pronounced relaxation of the injected muscles and a significant reduction in pain in them. Along with the denervation process, reinnervation proceeds in these muscles by the appearance of lateral processes of nerve endings, which leads to the restoration of muscle contractions 4-6 months after injection.

When administered locally in therapeutic doses, Botox® does not penetrate the BBB and does not cause significant systemic effects. Apparently, there is minimal presynaptic capture and reverse axonal transport from the site of its introduction.

Antibodies to the complex of botulinum toxin type A with hemagglutinin are formed in 1-5% of patients after repeated injections of Botox®. The formation of antibodies is facilitated by the administration of the drug in high doses (> 250 units) and repeated injections in small doses at short intervals. In the case of the formation of antibodies to botulinum toxin type A, the effect of the subsequent reaction can be reduced. With the obvious effectiveness of Botox, there are a number of disadvantages associated with side effects: itching, burning, swelling at the injection site, some patients have general muscle weakness during the first two weeks after using the drug, antibody formation is observed in 3-10% of patients , which reduces the effectiveness of "Botox" in repeated applications. Even the duration of its effect is a drawback, because it does not allow to promptly adjust the dose of the drug in accordance with individual tolerance. A significant drawback of the drug is associated with the mechanism of action of botulinum toxin at the molecular level.

Today it has been established that the mechanism of action of botulinum toxin consists of four phases: binding to the membranes of the nerve endings of motor neurons, internalization (incorporation into the neuronal cell through endocytosis), translocation into the cytosol and proteolysis of a specific site of one of the proteins included in the so-called SNARE synapse protein complex, which participates in the release of a mediator (acetylcholine) into the synaptic cleft. As a result, botulinum toxin breaks down the proteins of the presynaptic membrane that are part of the SNARE complexes, leading to the cessation of acetylcholine release. Activation of nAChR does not occur and neuromuscular transmission is interrupted.

The negative aspects of this mechanism of action of botulinum toxin (and especially its duration) are progressive atrophy of the muscle fiber with a decrease in the average fiber diameter, scattering of nAChR from the synapse site and a decrease in the activity of synaptic acetylcholinesterase. In rabbits, it was shown that with botulinum toxin injections continued for six months, muscle mass reduction can reach 76%, and contractile fibers can be replaced by adipose tissue elements [3]. So far, more than 10 years have passed since the FDA approved the use of Botox in the treatment of muscle spasm. The accumulated experience suggests that Botox has a number of significant side effects. In the absence of alternative effective muscle blockers of the company, Botox manufacturers are trying to improve drugs based on botulinum toxin, producing essentially the same drug under other commercial names (Botox, Xeomin, Lantox, Dysport, etc.).

The task of creating new effective drugs for the local treatment of muscular dystonia without side effects is relevant.

Disclosure of the invention.

A comprehensive study of the specific activity and selectivity of Azemiopsin SEQ ID NO: 1 in in vitro experiments (in electrophysiological tests and in calcium imaging) revealed a high selectivity of the drug with respect to the target target - muscle nAChR. The affinity of azemiopsin for the most similar pharmacological profile of α7 nAChR was reduced by almost two orders of magnitude and was IC50 = 2.7 μM. To other representatives of Cys-loop receptors, which are widely represented in the body on both nerve and immune cells, GABA-A receptors and heteromeric nAChR, azemiopsin did not show affinity, which speaks in favor of the absence of side effects due to the action on other molecular structures similar the target. Studies of specific activity in mice in vivo showed that the introduction of the drug Azemiopsin into the muscles of the forelimbs in doses of 0.03; 0.1 and 0.3 mg / kg causes a decrease in muscle strength of the limbs. The action of the drug is observed from the 5th minute after administration for doses of 0.1 and 0.3 mg / kg and from the 10th minute after administration for doses of 0.03 mg / kg. The longest duration of the drug was shown for the highest dose of 0.3 mg / kg, it was 55 minutes and lasted from the 5th to 60 minutes after administration. The maximum muscle relaxant effect of Azemiopsin for all studied doses occurred 10 minutes after administration. The average effective dose, when calculating the effect at the maximum response point, is 0.09 mg / kg. A pharmacokinetic study in mice and rats showed that with intravenous and intramuscular methods of administration, the drug is almost completely eliminated from free blood flow during the day. With the intravenous route of administration, a higher maximum concentration of the drug (Cmax) was observed than with the intramuscular route. The result of the acute toxicity study showed that a single intramuscular injection of the studied drug Azemiopsin with its single parenteral administration to male and female SD rats in doses of 1.0; 1.5; 2.0; 2.3 and 2.5 mg / kg revealed a dose-dependent death of animals and allowed to calculate LD50 (males: 1.83 ± 0.16 mg / kg, females: 1.87 ± 0.18 mg / kg). Intoxications were characterized by neurotoxic manifestations, and had a dose-dependent character. It was noted that the identified violations are associated with a specific pharmacological muscle relaxant effect of the drug and are complications caused by the use of toxic doses of the substance. Compensation of neurotoxic effects occurred one day after administration. No disturbances caused by the toxic effect of large doses of the drug in comparison with the control were recorded 14 days after administration. In a study of the subchronic toxicity of the drug Azemiopsin with 14-day intramuscular injection in two doses to female and male SD rats with a two-week withdrawal period, the drug showed no signs of toxicity. Based on the conducted study of reproductive toxicity, it can be concluded that repeated parenteral administration of the test drug Azemiopsin to male and female Wistar outbred runoff rats for 48 days and 15 days, respectively, does not adversely affect the generative function of males and females. As a result of the direct mutation test on mammalian cells in vitro in order to determine the mutagenic properties of the drug Azemiopsin, when it was once introduced into cell cultures in doses from 2000 to 2.8 μg / ml of growth medium, no mutagenic effect of the test drug was revealed. After analysis, the results of a micronucleus test on polychromatophilic erythrocytes of the bone marrow of mice in order to determine the mutagenic properties in vivo of the studied preparation Azemiopsin SEQ ID NO: 1 (DNWWPKPPHQGPRPPRPRPKP) with its single intramuscular administration to male BALB / c mice in two doses (0.15 mg) 0.5 mg / kg) can be considered negative. However, at a dose of 0.5 mg / kg of Azemiopsin, a weak tendency toward an increase in the frequency of polychromatophilic red blood cells with micronuclei was revealed relative to the negative control. The results of a micronuclear test for polychromatophilic bone marrow erythrocytes in order to determine the carcinogenic properties of the studied drug Azemiopsin (DNWWPKPPHQGPRPPRPRPKP) with its 5-day intramuscular injection into BALB / c mice of both sexes in two doses (0.15 and 0.5 mg / kg) negative. Immunotoxic studies showed that none of the tests revealed toxic effects of the study drug on the immune system. Based on the conducted allergenicity studies in two animal species, no allergenic effect of the studied preparation Azemiopsin was revealed. Thus, a 0.9% sodium chloride solution for injection containing the active ingredient Azemiopsin peptide is suitable for the treatment of muscular dystonia.

Examples of carrying out the invention.

Example 1. The study of the specific activity of Azemiopsin in experiments in vivo.

The study examined the specific activity of azemiopsin. The study was conducted on male ICR mice (CD-1) at the age of 11-12 weeks. The drug was administered once into the muscles of the forelimbs in doses of 0.03; 0.1 and 0.3 mg / kg. The effect was recorded by a change in the muscle strength of the retention behind the bars of the forelimbs. The measurements were carried out using a Grip strength meter (Columbus, USA) before the start of the experiment and at 5, 10, 15, 20, 30, 60, 90 minutes after administration. The effect was evaluated relative to the control group of animals, which were injected with saline. It was found that intramuscular administration of a drug based on the azemiopsin polypeptide to mice caused a muscle relaxant effect, which was characterized by a decrease in the holding power of animals. The effect in varying degrees of severity was observed with the introduction of all doses of 0.03; 0.1 and 0.3 mg / kg. The onset of the drug was observed from the 5th minute after administration (doses of 0.1 and 0.3 mg / kg) and from the 10th minute (dose of 0.03 mg / kg). The duration of action was up to the 30th minute after administration (doses of 0.03 and 0.1 mg / kg) and up to the 60th minute (dose of 0.3 mg / kg). The maximum muscle relaxant effect of Azemiopsin occurred 10 minutes after administration and lasted up to the 30th minute. The average effective dose, when calculating the effect at the 10th minute, is 0.09 mg / kg. Thus, the introduction of the drug Azemiopsin into the muscles of the forelimbs in doses of 0.03; 0.1 and 0.3 mg / kg caused a decrease in limb muscle strength. The onset of action of the drug was observed from the 5th minute after administration for doses of 0.1 and 0.3 mg / kg and from the 10th minute after administration for doses of 0.03 mg / kg. The longest duration of the drug was shown for the highest dose of 0.3 mg / kg, it was 55 minutes and lasted from the 5th to 60 minutes after administration. The maximum muscle relaxant effect of Azemiopsin for all studied doses occurred 10 minutes after administration. The average effective dose, when calculating the effect at the maximum response point, is 0.09 mg / kg.

Example 2. The study of pharmacokinetics.

A study of the pharmacokinetic parameters of the drug Azemiopsin with the introduction of mice ICR (CD-1). Doses and route of administration: intravenously and intramuscularly once in doses of 0.25 and 0.50 mg / kg. In each of the four studied groups, there were 5 animals (males). Blood samples were taken from the orbital sinus before administration of the drug, after 1.5, 15, 30 minutes, 1, 2, 4 and 24 hours after drug administration. The concentration of the drug (125I-labeled Azemiopsin) in blood samples was measured using a Wallac 1470 WIZARD® Gamma Counter (PerkinElmer) gamma counter taking into account the specific radioactivity of the derivative obtained at the time of measurement. Descriptive statistics are calculated for all quantitative source data. The main pharmacokinetic parameters were determined, allowing to evaluate the processes of excretion and elimination of the drug.

The maximum concentration with intramuscular administration is achieved within five minutes after the injection, after which the concentration decreases within an hour with a half-life of about 15-20 minutes for 0.25 mg / kg and about 40 minutes for 0.50 mg / kg. During the observation period, from 1 hour to one day, the concentration decreased more slowly: the elimination half-life was about 4 hours. With the intravenous route of administration, the maximum concentration was observed 1 minute after the injection, after which the concentration of the drug decreased with a half-life of about 15-20 minutes for 1 hour. In the period from 1 hour to a day after the injection, the recorded half-life reached 5.5 hours.

With both methods of administration, the drug is almost completely eliminated from free blood flow during the day. With the intravenous route of administration, a higher maximum concentration of the drug (Cmax) was observed than with the intramuscular route.

Example 3. The study of acute toxicity.

The study studied the toxicity of the drug Azemiopsin with a single intramuscular injection to male and female SD rats in doses of 1.0; 1.5; 2.0; 2.3 and 2.5 mg / kg. The main indicator defined in the study was the mortality of animals with the introduction of various doses of the drug. During the intravital phase of the study, the clinical signs of possible intoxication, body weight, and feed intake were recorded in animals. Animals were euthanized on the 15th day of the study. At the end of the study, all animals were opened, organs examined for the presence of macrodamage and weighed.

After a single intramuscular administration of the drug Azemiopsin to male and female rats, a dose-dependent death of animals was observed, which occurred in the period from the 15th to the 48th minute after the injection. The introduction of Azemiopsin in the minimum dose did not lead to the death of animals. The maximum dose of 2.5 mg / kg caused the death of animals in 100% of cases. Based on the results obtained, the average lethal dose of LD50 was calculated with a single intramuscular injection. For male rats, the LD50 was 1.83 ± 0.16 mg / kg, for females - 1.87 ± 0.18 mg / kg. The likely cause of death was a violation of respiratory function due to muscle relaxation of skeletal muscles. Clinical signs of intoxication were detected 10, 30 and 60 minutes after administration. In general, it was shown that intramuscular administration of the drug Azemiopsin led to the occurrence of dose-dependent neurotoxic effects. The effects were characterized by impaired posture maintenance, impaired reflex responses to various stimuli, respiratory disorders, decreased muscle strength and motor activity. When using maximum doses, convulsions and paralysis occur. Full compensation for impaired signs of clinical health occurred after 24 hours. During the entire period of the study, animals did not differ from the control in terms of weight gain and feed intake. At the autopsy of the surviving animals 14 days after administration, no macroscopic deviations from the norm of the internal organs were found. The mass of organs did not statistically significantly differ between groups.

Thus, a single intramuscular injection of the studied medicinal product Azemiopsin with its single parenteral administration to male and female SD rats in doses of 1.0; 1.5; 2.0; 2.3 and 2.5 mg / kg revealed a dose-dependent death of animals and allowed to calculate LD50 (males: 1.83 ± 0.16 mg / kg, females: 1.87 ± 0.18 mg / kg). Clinical signs of intoxication were characterized by neurotoxic manifestations and had a dose-dependent character. Identified violations are associated with a specific pharmacological muscle relaxant effect of the drug and are complications caused by the use of toxic doses of the substance. Compensation of neurotoxic effects occurred one day after administration. 14 days after the introduction, there were no violations caused by the toxic effect of large doses of the drug in comparison with the control.

BIBLIOGRAPHY

1.http: //dystonia-europe.org, http://www.dystonia-foundation.org/

2. UCPA: http://www.ucp.org/

3. Herzog W, Longino D, Clark A. The role of muscles in joint adaptation and degeneration. Langenbecks Arch Surg. 2003 Oct; 388 (5): 305-15

4.http: //www.alomone.com/p/azemiopsin/sta-100/95/

5. Guidelines for preclinical studies of drugs. Part 1. M .: Grif and K, 2012. p. 944.

Claims (4)

1. A method of treating muscular dystonia in humans, comprising administering to the patient therapeutically effective amounts of the peptide SEQ ID NO: 1. 2. The method according to p. 1, characterized in that the peptide solution in 0.9% sodium chloride is administered intramuscularly. 3. The method according to p. 1, characterized in that the average effective dose of the peptide SEQ ID NO: 1 is 0.09 mg / kg 4. The method according to any one of paragraphs. 1-3, characterized in that muscle dystonia is focal or segmental muscle dystonia.

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