RU2521973C1 - Method for preventing and treating arthropathies and methods for using same - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to agents for preventing and treating arthropathies containing a mixture of peptides H-Ala-Glu-Asp-OH, Lys-Glu-Asp and H-Lys-Glu-OH and a mixture of chondroitin and/or its salts, and/or glucosamines and/or its salts; methods for using them. The group of inventions provides a positive variation of clinical-biochemical dynamics. What is also provided is normalising the morphological structure of joint tissues, including a cytoarchitectonic characteristic of cartilaginous tissue with decreasing apoptotic chondrocyte count, especially at the final stages.

EFFECT: provided favourable effect on the metabolic processes in chondrocytes, activated synthetic processes and normalised biopolymer composition of the cartilage matrix An evident analgesic effect is manifested.

20 cl, 5 ex, 5 tbl

Description

The group of inventions relates to the creation of tools for the prevention and treatment of diseases of the joints, in particular arthritis, osteoarthritis, as well as other conditions and diseases accompanied by joint damage and pain, and methods for their use.

The causes of joint diseases are extremely diverse and in many ways have not yet been studied. Only the reasons for the development of specific infectious arthritis (tuberculosis, gonorrhea, brucellosis, etc.) associated with a particular infection are precisely known. As for all the other numerous forms of joint damage, then, according to modern concepts, their development is associated with a complex of many internal and external factors.

Nevertheless, the endpoint of the application of this diverse group of diseases is the defeat and further destruction of the cartilage tissue, which is necessary for the normal functioning of the joint.

For example, joint diseases such as osteoarthritis are a heterogeneous group of diseases of various etiologies, but with similar biological, morphological and clinical manifestations and outcome, which are based on the defeat of all components of the joint, especially cartilage, as well as the subchondral part of the bone, synovial membranes, ligaments, capsules and periarticular muscles. Osteoarthrosis as a chronic degenerative joint disease is characterized by progressive destruction of articular cartilage, a proliferative reaction of cartilage and bone tissue and is accompanied by reactive synovitis (Kovalenko V.N., Bortkevich O.P. Osteoarthrosis. Practical guide. - 2nd ed., Revised. and additional - K .: Morion, 2005 .-- 592 p.).

Osteoarthritis is the most common joint disease that affects at least 20% of the world's population (it usually begins at the age of over 40), while almost 25% of patients cannot cope with basic daily necessities of life. The progression of the disease, accompanied by constant pain in the affected joints, is the cause of disability of such patients, thereby exerting a severe economic and psychological impact not only on the patient, but also on his relatives.

The development of osteoarthritis leads to significant costs in the economic, social and psychological fields, which are associated with the widespread, frequent disability of patients and high economic costs, including the treatment of both the underlying disease and the prevention and treatment of possible complications of pharmacotherapy.

The same can be said of other joint diseases.

Therefore, the development of new effective means and methods of treatment and prevention of joint diseases is an urgent problem of modern pharmacology and clinical medicine, as it will not only increase the level of social activity and the quality of life of patients, but also reduce the costs of the health system associated with their provision.

Known drug glucosamine for the treatment of joint diseases and a method of treating these diseases, based on its introduction, which are widely used, for example, in primary and secondary osteoarthritis to reduce joint pain and slow the progression of the disease. However, this method using glucosamine cannot be considered quite effective, since its effect is associated only with one of the mechanisms of protecting cartilage from damage, namely, stimulation of the biosynthesis of aminoglycans (I. Zupanets, Experimental substantiation of the use of glucosamine and its derivatives in medicine: Dis. In the form scientific report ... doctor of medical sciences. - Kupavna, 1993. - 90 p.)

At the same time, these diseases lead to degenerative-dystrophic lesions of the joints with progressive destruction of the articular cartilage, proliferative reaction of cartilage and bone tissue and the development of synovitis. In addition, one of the important reasons for their development is the presence of concomitant diseases that cause circulatory disorders (atherosclerosis, diabetes mellitus, etc.), which can contribute to damage to cartilage tissue and the development of joint diseases.

Based on the foregoing, there is a need to find new peptide agents and methods of treatment based on their introduction to patients with joint diseases with chondroprotective activity, painkillers, repairers, and vasoactive properties, which will allow them to be successfully used to treat joint diseases of various etiologies, since activity of these funds will be aimed at correcting the main manifestations of the disease and the causes of its occurrence.

The prior art pharmaceutical composition for the prevention and treatment of diseases of the musculoskeletal system, including the peptide H-Ala-Glu-Asp-OH and its use for the treatment of the above diseases. This peptide, which is part of the known composition, has a pronounced chondroprotective activity against bone and cartilage. The dosage of the peptide is 0.01-100 mg / kg body weight (RF patent 2299741 C1 from 2007).

However, this composition and, accordingly, the method of its use cannot be considered effective, since they are based on the introduction of only one peptide in the form of an active principle, albeit with a pronounced chondroprotective effect.

At the same time, it is obvious that an increase in efficiency can be obtained by introducing different peptides with multidirectional activity, which will enhance the effectiveness of the therapy.

The prior art known peptide Lys-Glu-Asp (RF patent 2295970 C1 from 2007) representing:

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

2. Structural formula:

H-Lys-Glu-Asp-OH

3. Gross formula without counterion: C ₁₅ H ₂₆ N ₄ O ₈ .

4. Molecular weight without counterion: 390.39.

5. Counterion: acetate.

6. Appearance: odorless white amorphous powder.

The peptide is used to correct metabolic vascular syndrome and diseases accompanied by impaired permeability of the vascular wall and fragility of capillaries. The effective dosage of the peptide is 0.01-100 mg / kg body weight. Moreover, the peptide has never been used to treat joint diseases.

Also known is the peptide H-Lys-Glu-OH (RF patent 2139085 C1), which has the ability to stimulate reparative processes. The effective dosage of the peptide is 0.01-100 mg / kg body weight. This peptide has also not been used to treat joint diseases.

From the document - Radar Handbook of 2009, it is known to use a combination of chondroitin sulfate and glucosamine as a means of stimulating the regeneration of cartilage tissue. The Teraflex preparation contains 500 mg of glucosamine hydrochloride and 400 mg of chondroitin sodium sulfate. The Kondronova capsule preparation contains 250 mg of glucosamine sulfate and 200 mg of chondroitin sulfate. The drug "Kondronova ointment for external use" contains 25 mg of glucosamine sulfate and 50 mg of chondroitin sulfate. The drug "Artra" contains 500 mg of glucosamine hydrochloride and 500 mg of chondroitin sodium sulfate.

Thus, the objective of this work was the development of pharmaceutical compositions, as well as methods for the prevention and treatment of joint diseases, aimed at using directly three peptides in the composition, which are selected in the optimal ratio, with multidirectional activity, which will enhance the effectiveness of the therapy and their combination with glucosamine and / or chondroitin sulfate.

Another objective of this work was to study the pharmacological properties (condoprotective, analgesic) of a new mixture of peptides, as well as a mixture of peptides with glucosamine sulfate (hydrochloride) and a mixture of peptides with chondroitin sulfate and a mixture of peptides with glucosamine sulfate (hydrochloride) and chondroitin sulfate.

The task is achieved by the group of the invention, aimed at solving one problem and achieving one result.

The technical result of the claimed group of inventions is the creation of a single pharmaceutical composition that combines different peptides in its composition, used in a specific weight ratio developed by us, with the possibility of including other compounds that affect the etiopathogenesis of joint diseases in the composition, with the achievement of a synergistic effect from such inclusion.

In addition, the use of the composition positively changes the dynamics of clinical and biochemical parameters, helps to normalize the morphostructure of articular tissues (including the cytoarchitectonics of cartilaginous tissue with a decrease in the number of chondrocytes prone to apoptosis, especially at its final stages) and favorably affects metabolic processes in chondrocytes (activation of synthetic processes and normalization of the biopolymer composition of the cartilage matrix), while a pronounced analgesic effect is manifested.

A pharmaceutical composition is proposed for the prevention and treatment of joint diseases, which is a powder, or a solution, or a gel, or a suspension of a mixture of the peptides H-Ala-Glu-Asp-OH, Lys-Glu-Asp and H-Lys-Glu-OH, taken the following mass ratio:

H-Ala-Glu-Asp-OH Lys-glu-asp H-Lys-Glu-OH 0,1-10 0,1-10 0,1-10

in a pharmaceutically acceptable carrier.

Peptides obtained by fine organic synthesis, based on optically pure L-amino acids, according to the above patents:

The pharmaceutical composition can be in the form of a tablet, a nasal (metered) spray, a sublingual (metered) aerosol, a capsule, a powder, a solution, a lyophilisate for the preparation of a solution, cream, ointment, liniment, gel, drops for oral administration, while the tablets can be sublingual, prolonged action and coated, and the solution can be for iv and / m administration. Three options are also proposed.

An agent for the prophylaxis and treatment of joint diseases, including the pharmaceutical composition described above, containing peptides and chondroitin and / or its salts in the following ratio of the indicated ingredients in the agent in wt.%:

peptide containing pharmaceutical composition 0.01-1.00 chondroitin and / or its salts rest

An agent for the prophylaxis and treatment of joint diseases, including the pharmaceutical composition described above, containing peptides and glucosamine and / or its salts in the following ratio of the indicated ingredients in the agent in wt.%:

peptide containing pharmaceutical composition 0.01-1.00 glucosamine and / or its salts rest

An agent for the prophylaxis and treatment of joint diseases, including the pharmaceutical composition described above, containing peptides, a mixture of chondroitin and / or its salts and glucosamine and / or its salts in the following ratio of these ingredients in the agent in wt.%:

peptide containing pharmaceutical composition 0.01-1.00 a mixture of chondroitin and / or its salts and glucosamine and / or its salts rest

the mass ratio of chondroitin and / or its salts: glucosamine and / or its salts in the tool is (1-9) :( 1-9).

The tool for all three options can be made in the form of a tablet, a nasal (dosed) spray, a sublingual (dosed) aerosol, a capsule, powder, solution, lyophilisate for the preparation of a solution, cream, ointment, liniment, gel, drops for oral administration, while tablets can be sublingual, prolonged action and coated, and the solution can be for iv and / m administration.

Four variants of a method for the prevention and treatment of joint diseases using a pharmaceutical composition and three drug options are also proposed.

The chondroprotective properties of the described new mixture of peptides were studied experimentally by known methods.

RESEARCH OF SOME PHARMACOLOGICAL PROPERTIES

MIXTURES OF PEPTIDES IN THE COMPOSITION (EXAMPLE-AT THE MASS RELATIONSHIP OF COMPONENTS IN THE MIXTURE 1: 1: 1)

Carrageenin paw edema in rats (Winter et al., 1962). An acute inflammatory reaction (edema) was reproduced by subplanetary administration of 0.1 ml of a 1% carrageenin solution. The severity of the inflammatory reaction was assessed 3 hours (h) after the induction of inflammation by the change in the volume of the paw (oncometric method using a plethysmometer). The studied mixture of peptides in the form of a composition in various doses was administered 10-60 minutes (min) before the administration of carrageenin. The effect, estimated by reducing edema, was expressed as a percentage of control.

Peritonitis in rats. An acute exudative reaction - peritonitis - was caused by intraperitoneal (iv) administration of a 1% solution of acetic acid (1 ml per 100 g of mass). After 3 hours, the animals were killed, open the abdominal cavity and collect exudate. The studied mixture of peptides in the form of a composition in various doses was administered 10-60 minutes before the introduction of acetic acid. The antiexudative effect was evaluated by reducing the volume of exudate as a percentage of control.

Cramps caused by acetic acid in mice (V.M. Bulaev, N.V. Korobov, 2005). In experiments on mice, a specific pain reaction “cramp” (characteristic movements of animals, including contractions of the abdominal muscles, alternating with their relaxation, stretching of the hind limbs and arching of the back) was induced by the administration of a 0.75% solution of acetic acid (0.1 ml / 10 mg mass). Over the next 15 minutes after the introduction of the peptide mixture in the form of a composition in various doses (for 10-60 minutes), the number of writhing was calculated for each animal (8-10 animals in the group). The effect was evaluated by reducing the number of "writhing" as a percentage of control.

Acute toxicity (LN Sernov, VV Gatsura, 2000) of a mixture of peptides in the form of a composition was studied in mice. In this case, the death of animals was recorded 24 hours after the intravenous administration of a new mixture of peptides at a dose of 2 mg / kg. Since a mixture of peptides in doses of 1-800 μg / kg with a single intraperitoneal or intramuscular injection did not significantly change the studied parameters (unlike the comparison drugs indomethacin and diclofenac, the data on which are presented below in Tables 1-3), it was decided to study the effect of this mixtures with double intramuscular administration - 4 hours and 30 minutes before the introduction of phlogogenic agents. The results of this series of experiments are presented below in Table 1-3.

As can be seen from table 1, a mixture of peptides in doses of 2, 10, 50, 150, 300 and 600 μg / kg / day did not significantly exert antiexudative effect. In contrast, the indomethacin comparison drug with a single injection (at a dose of 10 mg / kg) gave a pronounced antiexudative effect.

As can be seen from table 2, a mixture of peptides in doses of 2, 10, 50, 150, 300 and 600 μg / kg / day did not significantly exert antiexudative effect. In contrast, the diclofenac comparison drug, when administered once (at a dose of 10 mg / kg), gave a pronounced ayatoexudative effect.

As can be seen from Table 3, a mixture of peptides at doses of 2, 10, 150, and 300 μg / kg / day did not significantly affect the number of writhing, and at doses of 50 and 600 μg / kg / day significantly reduced their number from 28 ± 1 ( control) to 23 ± 1 (p <0.01) and 22 ± 1 (p <0.001), respectively (or 18-21%). Comparison drug diclofenac with a single injection (at a dose of 10 mg / kg) gave a pronounced effect, significantly reducing the number of writhing to 17 ± 1 (p <0.001), or by 39%.

In a separate series of experiments, acute toxicity was evaluated in 9 mice weighing 22-27 g. A mixture of peptides was administered ip in a dose of 2 mg / kg. It was found that 24 hours after a single i / v administration, the mixture of peptides does not cause death of animals or significant toxic reactions.

FINDINGS

1. A new mixture of peptides in doses of 1-800 mcg / kg with a single intraperitoneal or intramuscular injection does not significantly change the studied parameters (in contrast to the drugs comparing indomethacin and diclofenac).

2. A new mixture of peptides in doses of 2, 10, 50, 150, 300, and 600 μg / kg / day with a double intramuscular injection does not significantly exert an antiexudative effect on rat carrageenin paw edema and peritonitis. Comparison preparations of indomethacin and diclofenac with a single administration give a pronounced antiexudative effect.

3. A new mixture of peptides in doses of 2, 10, 150 and 300 μg / kg / day does not significantly change the number of writhing in mice, and in doses of 50 and 600 μg / kg / day significantly reduces their number. Comparison drug diclofenac with a single injection gives a pronounced effect.

4. A new mixture of peptides at a dose of 2 mg / kg (once intraperitoneally) does not cause significant toxic reactions in mice.

Next, the level of chondroprotective activity of the mixture of peptides was studied under the conditions of experimental arthrosis development during treatment of animals with glucosamine hydrochloride.

Research objectives

To determine the effect of co-administration of a mixture of peptides and glucosamine hydrochloride on the course of systemic steroid arthrosis in rats.

The studied drug:

A mixture of peptides - lyophilized powder for injection for intramuscular injection.

Reference objects.

The substance is glucosamine hydrochloride manufactured by Protein Chemicals (Japan).

Animals:

View - rats

Sex - males

Mass - 250-300 g

Total number - 90 animals

Route of administration of drugs: Intramuscularly (i / m), orally (n / a). The distribution of animals in groups:

Group 1 - intact control (n = 10);

Group 2 - control pathology (n = 20);

Group 3 - rats with arthrosis receiving an i / m mixture of peptides at a dose of 100 μg / kg n = 12);

Group 4 — rats with arthrosis receiving an i / m mixture of peptides at a dose of 150 mcg / kg (n = 12);

Group 5 — rats with arthrosis receiving an i / m mixture of peptides at a dose of 100 μg / kg and p / o GA g / x at a dose of 50 mg / kg (n = 12);

Group 6 — rats with arthrosis receiving an i / m mixture of peptides at a dose of 150 μg / kg and p / o GA g / x at a dose of 50 mg / kg (n = 12);

7 group - rats with arthrosis receiving p / o GA g / x in a dose of 50 mg / kg (n = 12).

Study design:

The study is conducted on white outbred male rats weighing 250-300 g, obtained from one litter (of identical age and weight, with normal basic indicators of clinical and biochemical analyzes). We preliminary divide the animals into groups, weigh them, apply individual labels. Next, we simulate steroid arthrosis by injecting dexamethasone three times with an interval of 1 week in a single dose of 7 mg / kg. The first manifestations of arthropathy occur already at 4 weeks of the experiment. The degree of activity of the pathological process is evaluated in the control group by biochemical parameters of a blood test, for which some of the animals are removed from the experiment (10 rats) and collect blood. Starting from the 28th day of the experiment and for 4 weeks, all animals receive daily preparations in appropriate doses using a specific route of administration. The test mixture of peptides is injected intramuscularly, dissolving in the required amount of saline, and HA hydrochloride is administered p / o in saline using an intragastric probe. Throughout the study, clinical observation of animals is carried out, the functional state of the joints of rats (the degree of mobility, resistance to physical activity, swelling, hyperemia) is monitored. At the end of drug administration (on the 56th day of the experiment), the animals are decapitated under ether anesthesia in order to obtain biomaterials for biochemical and histomorphological studies. The studied parameters are taken into account in the form of initial data, for which we use intact animals, as well as on day 28 (for the control pathology group) and day 56 of the study.

The studied indicators and the timing of their observation

INTEGRAL INDICATORS Options Duration of observation Body mass 1, 14, 28, 56 days Eating food daily Animal behavior daily Functional condition of the joints daily BIOCHEMICAL INDICATORS Blood sialic acid 28, 56 days Fractions and GAG blood count 28, 56 days Blood glycoproteins 28, 56 days Chondroitin Sulfates 28, 56 days Total blood N-acetylglucosamine 28, 56 days Blood N-Acetylglucosamine Free 28, 56 days Associated Blood N-Acetylglucosamine 28, 56 days Cartilage N-Acetylglucosamine 28, 56 days PATHOMORPHOLOGICAL RESEARCH Articular cartilage morphostructure 56 days Articular Cartilage Ultrastructure 56 days

The duration of the study is 6 months.

Studies of the level of chondroprotective activity of a mixture of peptides under the conditions of development of experimental arthrosis showed an increase in the effectiveness of the use of a mixture of peptides in comparison with the control glucosamine hydrochloride.

In addition, the combined use of a mixture of peptides and glucosamine hydrochloride (the amount of a mixture of peptides is 0.2-0.3% by weight of glucosamine hydrochloride) exceeded the effect of the mixture of peptides and glucosamine hydrochloride separately (in terms of the individual components), which allows us to conclude synergistic effect of joint use, and an increase in the amount of a mixture of peptides in the composition causes a slow nonlinear increase in the efficiency of use with a pronounced analgesic effect.

Preclinical study of the effect of a mixture of peptides on the processes of apoptosis of chondrocytes in the development of experimental arthrosis

1. The purpose of the study

To study the effect of a mixture of peptides on the processes of apoptosis of chondrocytes in the development of experimental arthrosis.

2. Objectives of the study

To determine the effect of a mixture of peptides upon intramuscular administration on the intensity of the processes of apoptosis of chondrocytes in the articular cartilage of rats against the background of the development of systemic steroid arthrosis.

3. The study drug.

A mixture of peptides - lyophilized powder for injection for intramuscular injection.

4. Reference objects.

The substance is glucosamine hydrochloride, manufactured by Protein Chemicals (Japan).

5. Justification of the chosen research scheme.

During preliminary studies, it was found that the original substance containing a mixture of short-chain peptides is an agent with chondroprotective activity. At the stage of electron microscopic examination of the effect of this substance on the structure of articular cartilage, a decrease in the number of chondrocytes susceptible to apoptosis (chondroptosis), especially at its final stages, was detected. This suggests the presence of an antiapoptotic effect in this substance. The highest activity was observed in the dose range of 50-150 μg / kg.

It is known that apoptosis occurs in articular cartilage in response to compression, mechanical damage, trophic disturbance, experimental exposure and is the main mechanism of articular cartilage cell death in osteoarthritis. Moreover, apoptosis of chondrocytes has a number of morphological features, which makes it possible to use even a separate germin, chondroptosis, in experimental practice.

In this regard, with the aim of an in-depth study of the mechanisms of chondroprotective action of this substance, of great scientific and practical interest is the study of apoptosis in pathologically altered articular cartilage under its influence at doses of 100 and 150 μg / kg. For this, it is possible to use the immunohistochemical method of TUNEL reactions (TdT-mediated X-dUTP nick end labeling) on semi-thin paraffin sections of articular cartilage using In Situ Cell Death Detection Kit (Roche, Germany) as a marker of apoptotic cells. The main advantage of this method is its high sensitivity and selectivity, which allows highly accurate cell identification in the early stages of cell death. Another important point is the possibility of conducting a morphometric assessment on large areas of cartilage tissue, which is not allowed to do electron microscopy.

It is advisable to use the following biochemical indicators as parameters for assessing the intensity of the pathological process in the experimental groups: the content of sialic acids, glycoproteins, chondroitin sulfates, glycosaminoglycans, N-acetylglucosamine in the blood serum, since these indicators informatively reflect the intensity of the course of degenerative-dystrophic processes in the connective tissue. As a comparison drug, it is advisable to use glucosamine hydrochloride as one of the most common and studied means of chondroprotective action, at a dose of 50 mg / kg, corresponding to an average effective dose for anti-inflammatory activity

6. Animals:

View - rats

Sex - males

Mass - 250-300 g

Total number - 66 animals

7. The route of administration of drugs.

Intramuscularly (i / m), orally (n / a)

8. The distribution of animals in groups.

Group 1 - intact control (n = 10)

Group 2 - control pathology (n = 20);

Group 3 - rats with arthrosis receiving an i / m mixture of peptides at a dose of 100 μg / kg (n = 12);

Group 4 — rats with arthrosis receiving an i / m mixture of peptides at a dose of 150 mcg / kg (n = 12);

Group 5 - rats with arthrosis receiving a p / o drug for comparison of glucosamine hydrochloride at a dose of 50 mg / kg (n = 12).

9. Study design

The study was conducted on white outbred male rats weighing 250-300 g, obtained from one litter (of identical age and weight, with normal basic indicators of clinical and biochemical analyzes). We preliminary divide the animals into groups, weigh them, apply individual labels. Next, we simulate steroid arthrosis by injecting dexamethasone three times with an interval of 1 week in a single dose of 7 mg / kg. The first manifestations of arthropathy occur already at 4 weeks of the experiment. The degree of activity of the pathological process is evaluated in the control group by biochemical parameters of a blood test, for which some of the animals are removed from the experiment (10 rats) and collect blood. Starting from the 28th day of the experiment and for 4 weeks, all animals daily receive IM peptides in appropriate doses. Animals of group 5 receive daily glucosamine hydrochloride at a dose of 50 mg / kg, which is administered orally in solution using an intragastric tube. Throughout the study, clinical observation of animals is carried out, the functional state of the joints of rats (the degree of mobility, resistance to physical activity, swelling, hyperemia) is monitored. At the end of drug administration (on the 56th day of the experiment), the animals are decapitated under ether anesthesia in order to obtain biomaterials for biochemical and immunohistochemical studies. Identification of the apoptiated cells in the tissues of the articular cartilage is carried out immunohistochemically on semi-thin paraffin sections using the TUNEL reaction using the In Situ Cell Death Detection Kit manufactured by Roche (Germany).

10. The studied indicators and terms of their observation

INTEGRAL INDICATORS Options Duration of observation Body mass 1, 14, 28, 56 days Eating food daily Animal behavior daily Functional condition of the joints daily BIOCHEMICAL INDICATORS Blood sialic acid 28, 56 days Fractions and GAG blood count 28, 56 days Blood glycoproteins 28, 56 days Chondroitin Sulfates 28, 56 days N-acetylglucosamine 28, 56 days PATHOMORPHOLOGICAL RESEARCH Chondrocyte Apoptosis 56 days

The duration of the study is 6 months.

As a result of the experimental data, it is recommended to prescribe these agents for osteoarthritis of the peripheral joints and spine (osteoarthritis; arthropathy; intervertebral osteoarthritis, as a treatment and prevention of osteoarthrosis.

In order to study the breadth of variation in the ratio of peptides in the mixture, we conducted additional studies, which are shown in table 1 and 2.

Table 1 shows the dynamics of rat body mass under the influence of the studied mixture of peptides (0.1-1-10) against the background of the development of systemic steroid arthrosis.

Table 2 shows the dynamics of rat body mass under the influence of the studied mixture of peptides (10-2-0.1) against the background of the development of systemic steroid arthrosis.

In addition, experimental studies have shown the following possibility of creating different dosage forms and recommendations for their use:

Example 1

Solid forms (tablets, including for resorption, and capsules):

Capsules are obtained in a well-known manner (Chueshov V.I. et al. Industrial technology of drugs, T.2, MTK - Book; Publishing House of NFAU, 2002, pp. 400-413).

To prepare the capsules, the ingredients are mixed based on the composition of one capsule: a mixture of peptides in the form of a powder, for example, in a ratio of 1: 1: 1 - 150 μg; glucosamine (and its salts) - 750 mg; excipients: magnesium stearate 5 mg, manganese sulfate 1 mg, stearic acid 10 mg.

One of the methods for producing capsules is the granulation method: the capsule mass is prepared as follows: powders of a mixture of petids and / or glucosamine (and its salts), and / or chondroitin (and its salts), lactose and calcium or magnesium stearate, or any other pharmaceutically acceptable carriers are sieved and weighed. Then the powders are mixed in mass ratios determined according to the technical regulations of the manufacturer, the obtained intermediate mixture of powders is moistened with 70% ethanol, the wet intermediate mixture is sieved through a sieve with a hole diameter of 3 mm to obtain wet granules (granules). The granules are dried, dusted, and then filled with gelatin capsules.

Tablets were prepared in a well-known manner (ibid., Pp. 353-368).

For the preparation of tablets, the ingredients are mixed based on the composition of one tablet: a mixture of peptides (which in powder form, for example, in a ratio of 1: 2: 1) - 150 μg; chondroitin (and its salts) - 200 mg; excipients: calcium stearate 4.8 mg, crospovidone 12.39 mg, magnesium carbonate, povidone 9.312 mg, microcrystalline cellulose 96 mg).

The stage of preparation of the tablets includes the following operations: starting materials → preparatory stage + preparation of the working recipe → weighing → grinding → sieving → (possibly wet granulation) → mixing powders → moisturizing → granulation → drying → dusting → pressing → packing

The resulting dosage forms may be used as follows.

Adults and children over 12 years of age per 1 unit (the content of the mixture of peptides is 150 μg, the content of glucosamine sulfate is 500 mg) 2 times a day for the first three weeks; 1 unit once a day for the following weeks and months. A stable therapeutic effect is achieved when taking the drug for at least 6 months.

Example 2

Injection solutions were obtained by a well-known method (ibid., Pp. 510-543).

To prepare a solution for intravenous and intramuscular administration, the ingredients are mixed based on the composition of one ampoule (2 ml): a mixture of peptides in the form of a powder, for example, in a ratio of 1: 3: 1 - 100 μg; chondroitin (and its salts) - 100 mg; glucosamine (and its salts) - 100 mg; lidocaine hydrochloride 10 mg, trometamol to pH 2-3, water for injection - up to 2 ml.

The stage of preparation of the solution includes the following operations: starting materials → preparatory stage + preparation of the working recipe → weighing → dissolution → isotonization → stabilization → the introduction of preservatives → filtering → ampoule filling → packing. Depending on the properties of medicinal substances, some of the operations may be excluded, for example, isotonation, stabilization, the introduction of preservatives)

The solution is transparent, colorless or with a slightly yellowish tint, odorless.

The resulting dosage forms must be used as follows.

Injections: The drug is prescribed IM 50 mg (1 amp. The content of the mixture of peptides 50 μg, glucosamine hydrochloride 50 mg) daily. With good tolerance, the dose is increased to 100 mg (2 amp.), Starting with the fourth injection. The course of treatment is 25-30 injections. If necessary, repeated courses of treatment are possible.

Example 2a

To prepare a solution for intravenous and intramuscular administration, the ingredients are mixed based on the composition of one ampoule (2 ml): a mixture of peptides in the form of a powder, for example, in a ratio of 1: 2: 1 - 150 μg; lidocaine hydrochloride 10 mg, trometamol to pH 2-3, nipagin 10 mg, water for injection - up to 2 ml.

The solution is transparent, colorless or with a slightly yellowish tint, odorless.

Injection: The drug is prescribed in / m 2 ml (1 amp. The content of the mixture of peptides 150 μg) daily. With good tolerance, the dose is increased to 300 μg (2 amp. Daily), starting with the fourth injection. The course of treatment is 25-30 injections. If necessary, repeated courses of treatment are possible.

Example 3

Nasal form: Drops in the nose 5 ml (peptide mixture content of 75 μg, glucosamine sulfate 800 mg). Adults and children over 15 years: apply drops of 1-2 drops in each nasal passage 2-3 times a day for the first three weeks, 1-2 drops 1 time per day for the next 24 weeks. For injection forms, the ratio of active substances is similar.

Example 4

The stage of preparation of aerosol release forms includes the following operations:

1. starting materials → preparatory stage + preparation of a working recipe → weighing → grinding → sieving → mixing of powders → (preparation of a eutectic mixture of active substances)

2. preparation of a eutectic mixture of excipients

3. the introduction of a eutectic mixture in an emulsion of excipients

4. The finished product is pumped from the reactor or transferred to collectors, from where it is fed by gravity or under pressure to the filling line to the drug dosage unit.

5. Preparation of a mixture of propellants. Technological operations associated with the preparation of propellants vary depending on how the propellant is transported to the filling line. Transportation can be carried out either using a pump, or under pressure created by an inert gas - nitrogen or vapors of the propellants themselves.

6. emulsion filtration → filling vials with an emulsion → installing a dispenser and rolling up a vial → packaging. The filling line can be either a series of separate semiautomatic devices or automatic equipment compactly combined into one line according to the sequence of technological operations.

Aerosol form 100 ml (the content of the mixture of peptides is 1.2 mg, chondroitin sulfate 2.5 g, glucosamine sulfate 3.5 g). The drug is used topically. Before use, the protective cap is removed from the spray gun; when using, the balloon should be held vertically so that the spray gun is on top; Do not use the cylinder upside down. After applying the drug, a protective cap should be put on the sprayer. Adults and children over 12 years of age: spray 1-2 sprayings 2-3 times a day for the first three weeks, 1-2 sprayings 1 time a day for the next 24 weeks.

Example 5

Oral form 10 mg (drops containing a mixture of peptides 400 μg, glucosamine hydrochloride 1.8 g.): Administer to adults and children. older than 15 years, 1-2 drops of the solution 2-3 times a day for the first three weeks, 1-2 drops 1 time per day for the next 24 weeks. A similar ratio for oral-aerosol complexes.

The analysis of the obtained data showed that in the treatment of experimental osteoarthritis in rats, a mixture of peptides and a mixture of peptides with glucosamine sulfate (hydrochloride) and a mixture of peptides with chondroitin sulfate and a mixture of peptides with glucosamine sulfate (hydrochloride) and chondroitin sulfate positively changes the dynamics of clinical and biochemical parameters, contributes to the normalization of the morphostructure of articular tissues (including the cytoarchitectonics of cartilaginous tissue with a decrease in the number of chondrocytes prone to apoptosis, especially on a horse GOVERNMENTAL stages) and a positive effect on metabolic processes in chondrocytes (activation processes and synthetic biopolymer composition normalization of cartilage matrix), thus manifests a pronounced analgesic effect.

Table 1 The effect of a new mixture of peptides with intramuscular injection (2 times) and indomethacin comparison drug (1 time) on the development of carrageenin edema in rats Substance (dose, mcg / kg / day) The number of rats Antiexudative effect, increase in paw volume,% The control 10 139 ± 6 Mixture (2) 6 159 ± 10 Mixture (10) 7 148 ± 8 Mixture (50) 8 125 ± 4 Mixture (150) 7 129 ± 5 Mixture (300) 8 135 ± 4 Mixture (600) 8 126 ± 4 Indomethacin (10 mg / kg once) 8 36 ± 2 ∗∗∗ Note. The differences are statistically significant compared with the control (student criterion): ∗∗∗ - p <0.001

table 2 The effect of a new mixture of peptides with intramuscular injection (2 times) and a diclofenac comparison drug (1 time) on rat peritonitis model Substance (dose, mcg / kg / day) The number of rats Antiexudative effect (decrease in exudate volume), in% of control Mixture (2) 6 125 ± 13 Mixture (10) 6 117 ± 11 Mixture (50) 8 89 ± 6 Mixture (150) 7 98 ± 6 Mixture (300) 7 101 ± 7 Mixture (600) 8 92 ± 5 Diclofenac (10 mg / kg once) 8 54 ± 2 ∗∗∗ Note. The differences are statistically significant compared with the control (student criterion): ∗∗∗ - p <0.001

Table 3 The effect of a new mixture of peptides with intramuscular injection (2 times) and the drug comparison diclofenac (1 time) on the model of "vinegar cramps" Substance (dose, mcg / kg / day) The number of mice The number of cramps (M ± m) Effect, % The control 12 28 ± 1 - Mixture (2) 10 30 ± 1 107 (+7) Mixture (10) 10 29 ± 1 104 (+4) Mixture (50) 10 23 ± 1 ∗∗ 82 (-18) Mixture (150) 8 25 ± 2 89 (-11) Mixture (300) 10 26 ± 1 93 (-7) Mixture (600) 10 22 ± 1 ∗∗∗ 79 (-21) Diclofenac (10 mg / kg once) 10 17 ± 1 ∗∗∗ 61 (-39) Note. The differences are statistically significant compared with the control (student criterion): ∗∗ - p <0.01, ∗∗∗ - p <0.001

Table 4 Experience Conditions Dose of the drug Body weight g Initial data 14 days 28 days 56 days Control pathology (n = 40) 262.0 ± 4.0 273.5 ± 3.7 287.9 ± 3.3 ∗ 320.4 ± 4.5 ∗ A mixture of peptides (0.1-1-10) (n = 12) 10 mcg / kg 260.3 ± 3.4 270.7 ± 3.6 282.2 ± 3.2 ∗ 316.7 ± 4.0 ∗ 50 mcg / kg 261.3 ± 4.2 272.4 ± 3.2 280.1 ± 3.4 ∗ 307.7 ± 3.9 ∗ 150 mcg / kg 263.0 ± 4.7 274.1 ± 3.6 285.3 ± 3.2 ∗ 302.4 ± 3.8 ∗ / ∗∗ 600mkg / kg 260.9 ± 3.4 269.3 ± 3.5 280.0 ± 3.4 ∗ 300.6 ± 3.8 ∗ / ∗∗ Glucosamine hydrochloride (n = 12) 50 mg / kg 260.8 ± 3.2 269.4 ± 3.5 ∗ 287.8 ± 3.2 ∗ 302.2 ± 4.0 ∗ Notes: 1) ∗ - p≤0.05 relative to the source data 2) ∗∗ - p≤, 05 relative to the control pathology group

Table 5 Experience Conditions Dose of the drug Body weight g Initial data 14 days 28 days 56 days Control pathology (n = 40) 262.0 ± 4.0 273.5 ± 3.7 287.9 ± 3.3 ∗ 320.4 ± 4.5 ∗ A mixture of peptides (10-2-0.1) (n = 12) 10 mcg / kg 260.4 ± 2.5 269.6 ± 3.5 280.0 ± 3.1 ∗ 310.8 ± 3.3 ∗ 50 mcg / kg 261.8 ± 3.9 270.4 ± 2.2 275.1 ± 2.2 ∗ 305.4 ± 2.7 ∗ 150 mcg / kg 263.0 ± 2.7 273.2 ± 3.1 281.3 ± 3.9 ∗ 301.0 ± 3.8 ∗ / ∗∗ 600 mcg / kg 260.7 ± 1.7 267.2 ± 3.8 280.0 ± 1.2 * 289.6 ± 2.4 ∗ / ∗∗ Glucosamine hydrochloride (n = 12) 50 mg / kg 260.8 ± 3.2 269.4 ± 3.5 ∗ 287.8 ± 3.2 ∗ 302.2 ± 4.0 ∗ Notes: 1) ∗ - p≤0.05 relative to the source data 2) ∗∗ - p≤, 05 relative to the control pathology group

Claims (20)

1. The pharmaceutical composition for the prevention and treatment of diseases of the joints, comprising a mixture of peptides H-Ala-Glu-Asp-OH, Lys-Glu-Asp and H-Lys-Glu-OH, taken in the following weight ratio:
H-Ala-Glu-Asp-OH Lys-glu-asp H-Lys-Glu-OH 0,1-10 0,1-10 0,1-10
in a pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to claim 1, is made in the form of a tablet, a nasal (metered) spray, a sublingual (metered) aerosol, a capsule, a powder, a solution, a lyophilisate for preparing a solution, a gel, drops for oral administration. 3. The pharmaceutical composition according to claim 2, where the tablets can be sublingual, prolonged action and coated. 4. The pharmaceutical composition according to claim 2, where the solution may be for iv and / m administration. 5. The tool for the prevention and treatment of diseases of the joints, including the pharmaceutical composition according to claim 1 and chondroitin and / or its salts in the following ratio of these ingredients in the tool, in wt.%:
pharmaceutical composition according to claim 1 0.01-1.00 chondroitin and / or its salts rest 6. The tool according to claim 5, is made in the form of a tablet, a nasal (metered) spray, a sublingual (metered) aerosol, a capsule, a powder, a solution, a lyophilisate for preparing a solution, a gel, drops for oral administration. 7. The tool according to claim 6, where the tablets can be sublingual, prolonged action and coated. 8. The tool according to claim 6, where the solution can be for iv and / m administration. 9. The tool for the prevention and treatment of diseases of the joints, including the pharmaceutical composition according to claim 1 and glucosamine and / or its salts in the following ratio of these ingredients in the tool, in wt.%:
pharmaceutical composition according to claim 1 0.01-1.00 glucosamine and / or its salts rest 10. The tool according to claim 9, is made in the form of a tablet, a nasal (metered) spray, a sublingual (metered) aerosol, a capsule, a powder, a solution, a lyophilisate for preparing a solution, a gel, drops for oral administration. 11. The tool of claim 10, where the tablets can be sublingual, prolonged action and coated. 12. The tool of claim 10, where the solution may be for iv and / m administration. 13. The tool for the prevention and treatment of diseases of the joints, including the pharmaceutical composition according to claim 1, a mixture of chondroitin and / or its salts and glucosamine and / or its salts in the following ratio of these ingredients in the tool, in wt.%:
pharmaceutical composition according to claim 1 0.01-1.00 a mixture of chondroitin and / or its salts and glucosamine and / or its salts rest
the mass ratio of chondroitin and / or its salts: glucosamine and / or its salts in the tool is (1-9) :( 1-9). 14. The tool according to item 13, is made in the form of a tablet, a nasal (metered) spray, a sublingual (metered) aerosol, a capsule, a powder, a solution, a lyophilisate for preparing a solution, a gel, drops for oral administration. 15. The tool according to 14, where the tablets can be sublingual, prolonged action and coated. 16. The tool according to clause 15, where the solution can be for in / in and / m introduction. 17. A method for the prevention and treatment of diseases of the joints, characterized in that they use the pharmaceutical composition according to claim 1. 18. A method for the prevention and treatment of diseases of the joints, characterized in that they use the tool according to claim 5. 19. A method for the prevention and treatment of diseases of the joints, characterized in that they use the tool according to claim 9. 20. A method for the prevention and treatment of diseases of the joints, characterized in that they use the tool according to item 13.