RU2299741C1 - Peptide normalizing metabolism in osseous and cartilage tissue, pharmaceutical composition based on thereof and method for its using - Google Patents

Abstract

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to drugs used in prophylaxis and treatment of the locomotor system, in particular, degenerative-dystrophic joint and backbone diseases. Invention proposes a pharmaceutical composition normalizing metabolism in osseous and cartilage tissues and comprising the effective amount of peptide alanyl-glutamyl-aspartic acid of the general formula: H-Ala-Glu-Asp-OH of the sequence 1 [SEQ ID NO:1] as an active component, and pharmaceutically acceptable carrier. Invention proposes peptide alanyl-glutamyl-aspartic acid of the general formula: H-Ala-Glu-Asp-OH of the sequence 1 [SEQ ID NO:1] possessing the biological activity manifesting as normalization of metabolism in osseous and cartilage tissues. Invention proposes a method for prophylaxis and treatment of locomotor system by normalization of metabolism in osseous and cartilage tissues involving administration in a patient of a pharmaceutical composition containing as an active component peptide alanyl-glutamyl-aspartic acid of the general formula: H-Ala-Glu-Asp-OH of the sequence 1 [SEQ ID NO:1] in the dose 0.01-100 mcg/kg of the body mass for at least once per a day for time necessary for achievement of the therapeutic effect. Invention can be used as agent normalizing metabolism in osseous and cartilage tissues.

EFFECT: valuable medicinal properties and high effectiveness of peptide and pharmaceutical composition.

6 cl, 2 tbl, 1 dwg, 1 ex

Description

The invention relates to medicines for the prevention and treatment of diseases of the musculoskeletal system, in particular degenerative diseases of the joints and spine, and can be used as a means of normalizing metabolism in bone and cartilage tissues.

It is known that degenerative diseases of the musculoskeletal system, as well as osteoporosis of bone tissue are the most common pathology.

A wide range of drugs is used to treat these diseases, depending on the clinical manifestations of the disease, the age of the patient, and the method of application.

So, for the treatment of osteoporosis, various calcium preparations, group D vitamins and hormonal drugs (for women) are used.

Drug therapy for degenerative diseases of the joints and spine includes the use of various medications of symptomatic and pathogenetic action: analgesics and anti-inflammatory drugs (analgin, novocaine blockade, rheopyrin, indomethacin, brufen); antihistamines (diphenhydramine, pipolfen); drugs that improve peripheral blood circulation (pachycarpine, platifillin); biostimulants (rumalon, aloe, vitreous, ATP); enzyme preparations (lidase, ronidase); anabolic steroids (nerabol, retabolil).

In addition, drugs are known that affect the metabolism in cartilage: glucosamine, chondroitin sodium sulfate (Big Russian Encyclopedia of Medicines, Remedium, M .: 2002, v. 1, p. 230-232).

Moreover, most of the listed groups of drugs are incompatible with other drugs, cause negative side effects, which narrows the range of indications and limits their use.

Given the prevalence of diseases of the musculoskeletal system in different age groups, as well as their social significance, the development of new drugs that affect metabolism in bone, cartilage and connective tissues is relevant at the present time.

The peptide known in the art is the alanyl-glutamyl-aspartic acid peptide of the general formula H-Ala-Glu-Asp-OH (Registration number RN-85806-95-7; http://stnweb.fiz-karlsruhe.de).

In an experimental study of the peptide alanil-glutamyl-aspartic acid of the general formula H-Ala-Glu-Asp-OH, its previously unknown biological activity was revealed, which manifests itself in the normalization of metabolism in bone and cartilage tissues.

The present invention has set and solved the problem of obtaining a peptide agent with biological activity, which is manifested in the normalization of metabolism in bone and cartilage tissues and a pharmaceutical composition containing this peptide as an active principle, as well as a method for its use.

The technical result of the invention is the manifestation of the peptide alanyl-glutamyl-aspartic acid of the general formula H-Ala-Glu-Asp-OH of biological activity, manifested in the normalization of metabolism in bone and cartilage, as well as containing the pharmaceutical composition as an active principle, using which helps to increase the number and function of calcitonin-producing thyroid cells, improves trophism of bone and cartilage tissue cells and, thus, normalizes metabolism in bone and cartilage tissues.

The possibility of an objective manifestation of a technical result when using the invention is confirmed by reliable data given in the examples containing experimental information obtained in the process of conducting research using methods adopted in this field.

To solve the problem and achieve the technical result, a group of inventions is proposed, united by a common inventive concept.

The present invention relates to the alanyl-glutamyl-aspartic acid peptide of the general formula H-Ala-Glu-Asp-OH of sequence 1 [SEQ ID NO: 1], which has biological activity that manifests itself in the normalization of metabolism in bone and cartilage tissues.

Another aspect of the present invention relates to a pharmaceutical composition that normalizes bone and cartilage metabolism, containing as an active principle an effective amount of an alanyl-glutamyl-aspartic acid peptide of the general formula H-Ala-Glu-Asp-OH sequence 1 [SEQ ID NO: 1] and a pharmaceutically acceptable carrier.

In this case, the pharmaceutical composition is in a form suitable for parenteral administration.

It is also proposed the use of the peptide alanyl-glutamyl-aspartic acid of the General formula H-Ala-Glu-Asp-OH sequence 1 [SEQ ID NO: 1] for the preparation of a drug that normalizes metabolism in bone and cartilage

A further aspect of the present invention relates to a method for the prevention and treatment of diseases of the musculoskeletal system by normalizing metabolism in the bone and cartilage tissues, which comprises administering to the patient a pharmaceutical composition containing, as an active principle, an alanyl-glutamyl-aspartic acid peptide of the general formula H-Ala-Glu- Asp-OH sequence 1 [SEQ ID NO: 1] at a dose of 0.01-100 μg / kg body weight at least once a day for the period necessary to achieve a therapeutic effect.

In this case, the administration is carried out intramuscularly.

The alanyl-glutamyl-aspartic acid peptide of the general formula H-Ala-Glu-Asp-OH is obtained by the classical method of peptide synthesis in solution.

The study of biological activity was carried out in an experiment on epiphysectomized rats, on cartilage tissue explants, and in patients with degenerative joint diseases.

The term "pharmaceutical composition" refers to various dosage forms containing the peptide H-Ala-Glu-Asp-OH, which can find therapeutic applications in medicine as a means of normalizing metabolism in bone and cartilage tissues.

To obtain pharmaceutical compositions of the invention, an effective amount of the H-Ala-Glu-Asp-OH peptide, as an active principle, is mixed with a pharmaceutically acceptable carrier according to pharmaceutically acceptable compounding methods.

The term "effective amount" means the use of such an amount of active principle, which, in accordance with its quantitative indicators of activity and toxicity, as well as on the basis of specialist knowledge should be effective in this dosage form.

The carrier may take various forms, which depend on the dosage form of the drug desired for administration to the body.

For parenteral administration, the carrier usually includes saline or sterile water, although other ingredients that promote stability or to maintain sterility may be included.

The invention is illustrated in table 1 and 2 by drawing.

Table 1 shows the effect of the peptide H-Ala-Glu-Asp-OH on the morphological and biochemical parameters of the peripheral blood of guinea pigs in the study of toxicity.

Table 2 shows the effect of the peptide H-Ala-Glu-Asp-OH on the quantitative characteristics of the studied parameters in the thyroid gland according to computer analysis of microscopic images.

The drawing shows the effect of the peptide H-Ala-Glu-Asp-OH on the development of cartilage explants.

The invention is illustrated by an example of the synthesis of the alanyl-glutamyl-aspartic acid peptide of the general formula H-Ala-Glu-Asp-OH (example 1), examples of the toxicity and biological activity of the peptide (examples 2, 3, 4), as well as an example of the results of the clinical use of the peptide confirming its pharmacological properties and the possibility of achieving a preventive and / or therapeutic effect (example 5).

Example 1. The synthesis of the peptide H-Ala-Glu-Asp-OH

1. Name of the compound: alanyl-glutamyl-aspartic acid.

2. Structural formula: H-Ala-Glu-Asp-OH

3. Gross formula without counterion: C ₁₂ H ₁₉ N ₃ O ₈ .

4. Molecular weight without counterion: 333.29.

5. Counterion: acetate.

6. Appearance: odorless white amorphous powder.

7. Method of synthesis: the peptide obtained by the classical method of synthesis in solution according to the scheme

BOC - tert-butyloxycarbonyl group,

Z is benzyloxycarbonyl group,

OSu - N-oxysuccinimide ester,

DCC - N, N'-Dicyclohexylcarbodiimide,

OBzl - benzyl ether,

TFA is trifluoroacetic acid.

Characteristics of the finished product:

- content of the main substance: 98.75% (by HPLC, 220 nm),

- TLC - individual, R _f = 0.75 (acetonitrile-water 1: 3),

- moisture content: 6%,

- pH 0.01% solution: 4.17,

- Specific optical rotation: [α] _D ²² : -20 ° (c = 1, H ₂ O), "Polamat A", Carl Zeiss Jena.

Synthesis Example:

1) BOC-Glu (OBzl) -OSu, N-tert-butyloxycarbonyl- (γ-benzyl) glutamic acid N-oxysuccinimide ester (I).

N-tert-butyloxycarbonyl- (γ-benzyl) glutamic acid BOC-Glu (OBzl) -OH (33.7 g, 0.1 mol) was dissolved in 50 ml of N, N'-dimethylformamide, cooled to a temperature of -10 ° С , cooled (4-6 ° C) solutions of N, N'-dicyclohexylcarbodiimide (23.0 g, 0.11 mol) in 30 ml of N, N'-dimethylformamide and N-hydroxysuccinimide (13.0 g, 0) were added with stirring , 11 mol) in 20 ml of N, N'-dimethylformamide. The reaction mixture was stirred for 12 hours while cooling with ice and then for 1 day at room temperature. The precipitated N, N'-dicyclohexylurea was filtered off and the resulting activated ester solution was used without isolation in the next step.

2) BOC-Glu (OBzl) -Asp (OBzl) -OH, N-t-butyloxycarbonyl- (γ-benzyl) glutamyl- (β-benzyl) aspartate (II).

(β-Benzyl) aspartic acid H-Asp (OBzl) -OH (28.0 g, 0.12 mol) and 36 ml (0.12 mol) of triethylamine were suspended in 50 ml of N, N'-dimethylformamide and stirred for 1 h. Then, a solution of the activated BOC-Glu (OBzl) -OSu (I) ester obtained in the previous step was added in portions. The reaction mixture was stirred at room temperature for 2 days. Then acidified with 0.5 N. sulfuric acid to pH 2-3 and was extracted with ethyl acetate 4 × 50 ml. Hoods were combined and washed sequentially with 0.5 N. H ₂ SO ₄ 3 × 50 ml, water 2 × 50 ml, 5% NaHCO ₃ solution 2 × 50 ml, water 2 × 50 ml, saturated NaCl solution 2 × 50 ml. The organic layer was dried over Na ₂ SO ₄ , the solvent was evaporated in vacuo, the residue was crystallized under hexane. Received 50 g of the product (92%). R _f = 0.34 (benzene-acetone 2: 1).

3) TFA H-Glu (OBzl) -Asp (OBzl) -OH (III), trifluoroacetate (γ-benzyl) -glutamyl- (β-benzyl) aspartate.

N-tert-butyloxycarbonyl- (γ-benzyl) glutamyl- (β-benzyl) aspartate (I) 5.68 g (≈0.01 mol) was dissolved in 20 ml of a mixture of dichloromethane-trifluoroacetic acid (3: 1). After 2 hours, the solvent was evaporated in vacuo at 40 ° С, the evaporation was repeated with a new portion of dichloromethane (2 × 10 ml), and the residue was dried in vacuo over NaOH. An oil of 5.80 g (≈100%) was obtained.

R _f = 0.63 (n-butanol-pyridine-acetic acid-water, 15: 10: 3: 12).

4) Z-Ala-Glu (OBzl) -Asp (OBzl) -OH (IV), N-benzyloxycarbonyl-alanyl- (γ-benzyl) glutamyl- (β-benzyl) aspartate.

Trifluoroacetate (γ-benzyl) glutamyl- (β-benzyl) aspartate (II) 5.65 g (0.01 mol) was dissolved in 10 ml of dimethylformamide, triethylamine 2.80 ml (0.02 mol) and N-oxysuccinimide ether were added N-carbobenzoxyalanine 4.14 g (0.013 mol). The mixture was stirred for 24 hours at room temperature.

The product was planted with 0.5 N. a solution of sulfuric acid (150 ml), was extracted into ethyl acetate (3 × 30 ml), washed with 0.5 N. sulfuric acid solution (2 × 20 ml), water, 5% sodium bicarbonate solution (1 × 20 ml), water, 0.5 N. a solution of sulfuric acid (2 × 20 ml), water and dried over anhydrous sodium sulfate. Ethyl acetate was filtered, evaporated in vacuo at 40 ° C, and the residue was crystallized in ethyl acetate / hexane. The product was filtered and dried in vacuo over P ₂ O ₅ . Yield 4.10 g (66%). Mp = 154 ° C.

R _f = 0.48 (benzene-acetone, 1: 1), R _f = 0.72 (n-butanol-pyridine-acetic acid-water, 15: 10: 3: 12).

5) H-Ala-Glu-Asp-OH, alanyl-glutamyl-aspartate.

Protected tripeptide (IV) 1.1 g was dissolved in methyl alcohol-water (4: 1) and hydrogenated over a Pd / C catalyst (5%) for 4 hours. The catalyst was filtered off, the solvent was evaporated in vacuo, the residue was dried in vacuo over KOH and P ₂ O ₅ . Crystallization from a water-methanol system.

Finally, the residue was dissolved in 20 ml of deionized water and lyophilized.

Received 105 mg of pure preparation in the form of an odorless amorphous white powder.

6) Analysis of the finished product.

- The content of the basic substance was determined by HPLC on a Phenomenex C 18 LUNA column 4.6 × 150 mm. A: 0.1% TFA; B: MeCN; grad. B 0-100% in 10 min. Flow rate 1 ml / min. Detection at 220 nm, scanning 190-600 nm, sample 20 μl. The content of the main substance is 98.75%.

- TLC: individual, R _f = 0.75 (acetonitrile-water 1: 3, PTCX-P-B-UV Sorbfil plates, CTX-1VE silica gel 8-12 μm, manifestation of chlorine / benzidine).

- Moisture content: 6% (gravimetrically by weight loss during drying 20 mg at 100 ° C).

- pH 0.01% solution: 4.17 (potentiometric).

- Specific optical rotation: [α] _D ²² : -20 ° (c = 1, H ₂ O), "Polamat A", Carl Zeiss Jena.

Example 2. The study of the toxicity of the peptide H-Ala-Glu-Asp-OH

The general toxic effect of the peptide H-Ala-Glu-Asp-OH was studied in accordance with the requirements of the Guidelines for the experimental (preclinical) study of new pharmacological substances (2000): acute toxicity with a single administration of the drug, as well as subacute and chronic toxicity with prolonged administration of the peptide .

A study of acute toxicity was conducted on 66 outbred white male mice weighing 19-22 g. Animals were randomly divided into 6 equal groups. The drug was administered to animals once intramuscularly at doses of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg in 0.25 ml of sterile 0.9% NaCl solution. Animals of the control group were injected with the same volume of 0.9% NaCl solution.

Studies on the study of subacute toxicity were carried out on 64 white mongrel male rats weighing 180-220 g. Once daily, the animals of the experimental groups were injected intramuscularly for 90 days at doses of 1 μg / kg, 0.1 mg / kg, 1 mg / kg 0.5 ml of a sterile 0.9% NaCl solution. The animals of the control group were injected in the same volume with a sterile 0.9% NaCl solution. Before administration of the drug on the 30, 60 and 90 days after the start of drug administration, the morphological composition and properties of peripheral blood were studied in animals. At the end of the experiment, biochemical and coagulological blood parameters were examined.

Studies on the study of chronic toxicity were carried out for 6 months, based on the duration of the recommended clinical prescription of the drug in 90 male guinea pigs weighing 270-300 g. Animals of the experimental groups received a single daily intramuscular peptide for 6 months. in doses of 1 μg / kg, 0.1 mg / kg, 1 mg / kg in 0.5 ml of a sterile 0.9% NaCl solution. In the control group, animals were injected in a similar fashion with a sterile 0.9% NaCl solution in the same volume.

In animals, peripheral blood was determined by conventional methods: the number of red blood cells, hemoglobin, reticulocytes, platelets, white blood cells, white blood cell count, erythrocyte sedimentation rate (ESR), and erythrocyte resistance. Along with this, the content of total protein in blood serum was determined by the method of Lowry, potassium and sodium by plasma spectrophotometry. After the experiment was completed, a pathomorphological study of the brain and spinal cord, spinal ganglia, thyroid gland, parathyroid glands, adrenal glands, testes, pituitary gland, heart, lungs, aorta, liver, kidney, bladder, pancreas, stomach, small intestine, colon, thymus, spleen, lymph nodes, bone marrow.

When studying acute toxicity, it was found that a single administration of the studied peptide to animals at a dose exceeding the therapeutic recommended for clinical use by more than 5000 times does not cause toxic reactions, which indicates a large therapeutic breadth of the drug.

The study of subacute and chronic peptide toxicity indicates the absence of side effects with prolonged use of the drug in doses exceeding the therapeutic by 100-1000 times. When studying the effect of the peptide on the morphological composition and biochemical parameters of the peripheral blood of guinea pigs, no significant changes in the parameters were revealed 3 and 6 months after the administration of the peptide (Table 1).

When assessing the general condition of animals, morphological and biochemical parameters of peripheral blood, the morphological state of internal organs, the state of the cardiovascular and respiratory systems, and liver and kidney function, pathological changes in the body were not detected.

The absence of a general toxic effect makes it possible to recommend a pharmaceutical composition containing the peptide H-Ala-Glu-Asp-OH as an active principle for conducting clinical trials.

Example 3. The effect of the peptide H-Ala-Glu-Asp-OH on the structural and functional organization of calcitonin-producing thyroid cells of epiphysectomized rats.

The work was performed on 23 male Wistar rats weighing 130-140 g. The pineal gland was removed from animals. The operation of epiphysectomy was performed under ether anesthesia according to the developed technique.

The animals of the experimental group three weeks after surgery (for 21 days) for the next 10 days were subcutaneously injected with the peptide H-Ala-Glu-Asp-OH at a dose of 0.5 μg per rat in 0.5 ml of sterile physiological 0.9% solution NaCl. Epiphysectomized rats of the control group and healthy animals were injected with a similar volume of sterile physiological saline in the same way.

The killing of animals and the isolation of the thyroid gland was carried out in the morning from 10 to 12 hours under natural light under nembutal anesthesia (50 mg / kg). Part of the epiphysectomized rats of the experimental and control groups were killed 3 days after the end of the injections (33 days after the operation and the start of the experiment), and part of the animals - 12 days (42 days after the epiphysectomy).

Pieces of the thyroid gland were fixed for 24 hours with Buen's acidic liquid for light microscopy and Karnowski's for electron microscopy. To analyze calcitonin-immunopositive cells (C-cells) in the thyroid gland, the volume fraction (ρ _Ca ), quantitative (N _Ca / 1 mm ² ) and optical density (OpD _Ca ) were used.

It was established that in animals of the control group, 1 month after epiphysectomy, the thyroid gland mainly retains structural features. At the same time, the amount of colloid in the follicles decreases, and the mitotic activity in the follicular epithelium increases. The number of C cells compared with intact animals increases by 24% and amounts to 3012 ± 172 per 1 mm ² . The quantitative density of C cells after 1.5 months after epiphysectomy increases by more than 50% in relation to intact animals, reaching 3668 ± 158 cells per 1 mm ² (table 2). 1 month after epiphysectomy, the content of C cells in the thyroid gland of animals of the experimental group treated with the peptide H-Ala-Glu-Asp-OH also has features that are reduced to a significant reduction in their number in relation to the control by 35%. The content of C cells in the thyroid gland 1.5 months after epiphysectomy (12 days after the end of the administration of the peptide H-Ala-Glu-Asp-OH) is completely normalized and practically does not differ from the control, amounting to 2346 ± 162 per 1 mm ² . The quantitative density of C cells at this stage is significantly different from the results obtained in the control group, where this indicator was 3668 ± 158 per 1 mm ² , i.e. more than 50% exceeds the control values (table 2).

The results of studies of the functional morphology of the thyroid gland of epiphysectomized rats of the experimental group under the action of the peptide H-Ala-Glu-Asp-OH demonstrate its stimulating effect on tissue and cell metabolism. The introduction of the peptide H-Ala-Glu-Asp-OH has a compensatory effect on the structural and functional organization of thyroid cells in epiphysectomized animals. Its effect is manifested 3 days after the end of the administration of the peptide by completely leveling the effect of epiphysectomy and persists after 12 days, i.e. until the end of the experiment.

An increase in the number and enhancement of the function of thyroid C-cells under the action of the peptide H-Ala-Glu-Asp-OH indicates an important compensatory role of the peptide in the regulation of calcium metabolism and increased bone resorption, which is of great importance in the prevention and treatment of osteoporosis of various etiologies .

Example 4. The effect of the peptide H-Ala-Glu-Asp-OH on the development of cartilage explants

The experiments were performed on 28 fragments of cartilaginous tissue of the proximal femur of the Wistar rats weighing 160-200 g. The culture medium for explant cultivation consisted of 35% Eagle's solution, 25% fetal calf serum, 35% Hanks' solution, 5% chicken embryonic extract , glucose (0.6%), insulin (0.5 units / ml), penicillin (100 units / ml), glutamine (2 mM) were added to the medium. Cartilage fragments were placed in this medium and cultured in Petri dishes in an incubator at a temperature of 36.7 ° C for 2 days. The peptide H-Ala-Glu-Asp-OH was added to the experimental medium to final concentrations of 1, 10, 100, 200, and 400 ng / ml. The criterion of biological activity was the area index (PI) - the ratio of the area of the entire explant along with the growth zone to the outgoing area of the cartilage tissue fragment. The IP values were expressed as a percentage, the control IP value was taken as 100%.

The drawing shows the effect of the peptide H-Ala-Glu-Asp-OH on the development of cartilage explants.

It was found that after 1 day of cultivation, the explants spread on a collagen substrate and the proliferation of proliferating and migrating cells began on the periphery of the explant. On the 3rd day of cultivation, at a concentration of the H-Ala-Glu-Asp-OH peptide of 100 ng / ml, a significant increase in explant PIs was observed by 26% compared with the reference PI values. When studying cartilage tissue explants for longer cultivation periods (7 days), a similar stimulating effect of the H-Ala-Glu-Asp-OH peptide at the same concentration was revealed.

Thus, with respect to cartilage, the H-Ala-Glu-Asp-OH peptide had a tissue-specific effect, which manifests itself in stimulating the growth of explants.

Example 5. The effectiveness of the peptide H-Ala-Glu-Asp-OH in patients with osteoarthritis of the knee

The study was conducted on 29 patients aged 52-72 years with osteoarthrosis of the knee joints. Patients complained of pain and restriction of flexion and extension in the joints when walking. In persons of the older age group, the characteristic signs were joint deformity, atrophy of the femoral muscles and weakening of the ligament apparatus of the joints. The duration of the disease ranged from 5 to 20 years, there was a progressive dynamics of the development of the pathological process. All patients previously received analgesics and anti-inflammatory drugs for a long time, the use of which caused a short-term therapeutic effect, requiring an increase in the dose of drugs for the course of treatment and their long-term use.

Patients were randomly divided into 2 groups.

Patients of the main group were divided into 3 subgroups according to age and severity of joint deformity. Patients of the main group over 65 years of age with the most pronounced joint deformation and restricted movement were administered a pharmaceutical composition containing the H-Ala-Glu-Asp-OH peptide, once daily intramuscularly at a dose of 5.0 mg in 1 ml of sterile physiological 0.9% NaCl solution for 20 days. Patients of the main group aged 60-65 years with moderate joint deformation were injected with a pharmaceutical composition containing the peptide H-Ala-Glu-Asp-OH, once daily intramuscularly at a dose of 10.0 μg in 1 ml of sterile physiological 0.9% NaCl solution within 20 days. Patients of the main group aged 52 to 60 years with the initial stage of the disease, expressed in joint pain and some limited movement in the joints during the exacerbation of the disease, were administered a pharmaceutical composition containing the H-Ala-Glu-Asp-OH peptide once daily intramuscularly at a dose of 1.0 μg in 1 ml of sterile physiological 0.9% NaCl solution for 20 days.

The control group included 12 patients who received injections of sterile saline according to a similar scheme.

The effectiveness of the use of a pharmaceutical composition containing the peptide H-Ala-Glu-Asp-OH in patients was evaluated by the dynamics of clinical indicators and X-ray data.

It should be noted that the radiological symptoms of degenerative-dystrophic joint diseases are not only objective diagnostic criteria for the stage of development of the pathological process, but also have great prognostic significance in the course of drug therapy.

It was found that the use of the H-Ala-Glu-Asp-OH peptide in patients with osteoarthritis of the knee joints of all subgroups contributed to a decrease in pain and increased joint mobility in 54.5-62.7% of cases, depending on the severity of the disease. Moreover, the pain symptomatology most completely disappeared at the radiologically determined initial stages of the disease: narrowing of the joint gap between the patella and hip, lateral osteophytes of the patella and femoral condyle. Significant dynamics of radiological symptoms during this period were not observed.

Patients in the advanced stage of arthrosis also observed a similar dynamics of subjective indicators, but less pronounced, since a long complex treatment is required at this stage of the disease. Since this stage of the disease was diagnosed in people of an older age group, the positive dynamics of subjective indicators was characterized as very favorable.

The results of the study indicate that the therapeutic efficacy of the pharmaceutical composition containing the peptide H-Ala-Glu-Asp-OH is based on normalizing metabolism and slowing down involutional changes in the bone and cartilage tissues of the knee joints by improving trophism of their cells.

Thus, it is advisable to use a pharmaceutical composition containing an effective amount of the H-Ala-Glu-Asp-OH peptide as an active principle for therapeutic or prophylactic purposes, including in combination with any means of symptomatic and pathogenetic therapy used to treat degenerative degenerative diseases of the joints and spine, using various dosages of the pharmaceutical composition from 5 mg to 1 μg in 1 ml of sterile physiological 0.9% NaCl solution, depending on the patient's age and degree the severity of the pathological process in the joints and spine.

Table 1
A peptide that normalizes metabolism in bone and cartilage tissues, a pharmaceutical composition based on it and a method for its use Indicator The introduction of the peptide H-Ala-Glu-Asp-OH (1 μg / kg) 3 months 6 months Control (n = 24) Peptide (n = 24) Control (n = 24) Peptide (n = 24) Red blood cells, × 10 ¹² / l 5.3 ± 0.6 5.4 ± 0.1 5.4 ± 0.3 5.3 ± 0.5 Hemoglobin, g / l 14.2 ± 1.4 14.6 ± 1.1 14.5 ± 1.3 13.7 ± 0.8 Reticulocytes,% 1.3 ± 0.07 1.2 ± 0.04 1.1 ± 0.05 1.1 ± 0.02 Platelets × 10 ⁹ / L 143.7 ± 7.9 144.8 ± 6.1 144.5 ± 8.6 145.3 ± 7.2 White blood cells × 10 ⁹ / L 9.4 ± 0.5 9.7 ± 0.6 9.6 ± 0.5 10.5 ± 0.2 Band neutrons,% 0.31 ± 0.04 0.28 ± 0.05 0.33 ± 0.04 0.30 ± 0.03 Segmented neutrophils,% 45.8 ± 2.1 45.1 ± 1.9 46.2 ± 3.5 44.6 ± 2.8 Eosinophils,% 0.69 ± 0.05 0.74 ± 0.03 0.72 ± 0.04 0.68 ± 0.04 Basophils,% 0.61 ± 0.04 0.64 ± 0.06 0.72 ± 0.03 0.65 ± 0.02 Monocytes,% 2.5 ± 0.02 2.6 ± 0.05 2.6 ± 0.06 2.5 ± 0.07 Lymphocytes,% 48.9 ± 2.5 51.2 ± 2.1 51.3 ± 2.7 49.7 ± 2.1 ESR, mm / h 1.69 ± 0.05 1.73 ± 0.05 2.01 ± 0.05 1.85 ± 0.05 Erythrocyte resistance,% NaCl - maximum 0.41 ± 0.02 0.40 ± 0.06 0.42 ± 0.04 0.40 ± 0.02 - minimum 0.32 ± 0.05 0.30 ± 0.01 0.34 ± 0.04 0.30 ± 0.03 Total protein in blood serum, g / l 72.9 ± 3.1 71.9 ± 2.7 73.1 ± 3.4 7131 ± 2.6 Sodium in blood serum, mmol / l 153.9 ± 5.7 155.2 ± 4.9 155.5 ± 6.2 153.9 ± 5.9 Serum potassium, mmol / l 5.1 ± 2.3 5.4 ± 2.5 5.2 ± 2.1 5.3 ± 2.0

table 2
A peptide that normalizes metabolism in bone and cartilage tissues, a pharmaceutical composition based on it and a method for its use Group of animals ρ _Ca ,% N _Ca / 1 mm ² OpD _Ca , c.u. Intact animals 14.5 ± 1.9 2424 ± 169 0.241 ± 0.005 Control (epiphysectomized animals + saline solution) 3 days after the end of administration 17.8 ± 1.6 ^* 3012 ± 172 ^* 0.263 ± 0.003 Control (epiphysectomized animals + saline solution) 12 days after the end of administration 22.0 ± 2.3 ^* 3668 ± 158 ^* 0.258 ± 0.012 Epiphysectomized animals + H-Ala-Glu-Asp-OH peptide 3 days after the end of administration 9.5 ± 1.5 ^** 1575 ± 163 ^** 0.344 ± 0.008 ^** Epiphysectomized animals + H-Ala-Glu-Asp-OH peptide 12 days after the end of administration 14.1 ± 1.8 ^** 2346 ± 162 ^** 0.32810.011 ^{** *} - P <0.05 compared with that in intact animals;
^** - P <0.05 compared with the indicator in animals of the control group.

Claims (6)

1. The peptide alanyl-glutamyl-aspartic acid of the General formula H-Ala-Glu-Asp-OH sequence 1 [SEQ ID NO: 1], which has biological activity, manifested in the normalization of metabolism in bone and cartilage. 2. The peptide according to claim 1 for the preparation of a drug that normalizes metabolism in bone and cartilage. 3. A pharmaceutical composition that normalizes metabolism in bone and cartilage, characterized in that as an active principle contains an effective amount of the peptide alanyl-glutamyl-aspartic acid of the General formula H-Ala-Glu-Asp-OH sequence 1 [SEQ ID NO: 1 ] and a pharmaceutically acceptable carrier. 4. The pharmaceutical composition according to claim 3, characterized in that it is in a form suitable for parenteral administration. 5. A method for the prevention and treatment of diseases of the musculoskeletal system by normalizing metabolism in the bone and cartilage, comprising administering to the patient a pharmaceutical composition containing, as an active principle, an alanyl-glutamyl-aspartic acid peptide of the general formula H-Ala-Glu-Asp-OH sequence 1 [SEQ ID NO: 1] at a dose of 0.01-100 μg / kg body weight, at least once a day for the period necessary to achieve a therapeutic effect. 6. The method according to claim 5, characterized in that the introduction is carried out intramuscularly.