GRIGORIEV, Evgeny Iosifovich (Zagrebsky bulvar, 5/1-105St.Petersburg, 4, 19228, RU)
MALININ, Vladimir Victorovich (Serebristy bulvar, 16/1-92St.Petersburg, 7, 19722, RU)
RYZHAK, Galina Anatolievna (ul. Partizana Germana 8/2-91, St.Petersburg, 5, 19820, RU)
KHAVINSON, Vladimir Khatskelevich (Bolshaya Raznochinnaya 3-21, St.Petersburg, 19711, RU)
GRIGORIEV, Evgeny Iosifovich (Zagrebsky bulvar, 5/1-105St.Petersburg, 4, 19228, RU)
MALININ, Vladimir Victorovich (Serebristy bulvar, 16/1-92St.Petersburg, 7, 19722, RU)
RYZHAK, Galina Anatolievna (ul. Partizana Germana 8/2-91, St.Petersburg, 5, 19820, RU)
| CLAIMS
1. Peptide alanyl-glutamyl-asparaginic acid with general formula: H-Ala-Glu-Asp-OH sequence 1 [SEQ ID NO:1] revealing biological activity, which consists in the normalization of metabolism in osseous and cartilaginous tissues.
2. Application of the peptide according to Claim 1 for manufacturing a pharmaceutical composition, normalizing the metabolism in osseous and cartilaginous tissues .
3. Pharmaceutical composition normalizing the metabolism in osseous and cartilaginous tissues, characterized by the fact, that it contains an effective amount of peptide alanyl-glutamyl-asparaginic acid with general formula H-Ala-Glu-Asp- OH sequence 1 [SEQ ID NO:1] as its active base, as well as a pharmaceutically acceptable carrier.
4. Pharmaceutical composition according to Claim 3, characterized in that it exists in the form intended for parenteral administration.
5. Method of prevention and treatment for locomotor apparatus diseases by means of normalizing the metabolism in osseous and cartilaginous tissues, such method consisting in the administration to the patient of a pharmaceutical composition, containing peptide alanyl-glutamyl-asparaginic acid with general formula: H-AIa- Glu-Asp-OH sequence 1 [SEQ ID NO:1] in the dose of 0,01 - 100 μg/kg of body weight as its active base, at least once a day during a period necessary for attaining the therapeutic effect.
6. Method according to Claim 5, characterized in that such administration is performed intramuscularly. |
Peptide substance normalizing the metabolism in osseous and cartilaginous tissues, pharmaceutical composition on its base and the method of its application
The invention is related to medicinal means of prevention and treatment for locomotor apparatus diseases, in particular degenerative-dystrophic diseases of the joints and the spine, and may be used as a means of normalizing the metabolism in osseous and cartilaginous tissues.
It is known, that degenerative-dystrophic diseases of the locomotor apparatus, as well as osseous tissue osteoporosis are among the most frequently occurring pathologies. A wide range of medications is used for the treatment of these diseases, depending on their clinical manifestations, as well as on the patient's age and the method of administration.
Different calcium preparations, D group vitamins and hormonal preparations (for women) are used for the treatment of osteoporosis. Drug therapy of degenerative-dystrophic diseases of the joints and the spine includes different medications, exerting a symptomatic and pathogenetic effect: analgesics and anti-inflammatory medications (Analgin, Novocain blockades, Reopyrin, Indometacine, Brufen); antihistaniinic medications (Dimedrole, Pipolphen); peripheral blood circulation improving medications (Pachicarpin, Platiphyllin); biostimulators (Rumalon, aloe, vitreous body, ATP); enzyme preparations (Lydase, Ronidase); anabolic steroids (Nerabol, Retabolyl).
Besides that, there are known medications, which are effective on cartilaginous tissue metabolism: Glucosamine, Chondroitin, sodium sulfate, (The Comprehensive Russian Encyclopaedia of Medicinal Means, Moscow, Remedium publishing house, V.I, 2002 (rus.), PP- 230-232).
However, the majority of the above medication groups is incompatible with other medications and has negative side effects, which narrows the range of indications and limits their field of application.
Taking into consideration the wide spread of locomotor apparatus diseases in different age groups, as well as their social significance, the development of new medications effective on the metabolism in osseous, cartilaginous and connective tissues is presently of significant importance.
Peptide alanyl-glutamyl-asparaginic acid with general formula: H-Ala-Glu-Asp- OH (Registration No. RN-85806-95-7; http://stnweb.fiz-karlsruhe.de) is presently known in the prior art.
The experimental study of peptide alanyl-glutamyl-asparaginic acid with general formula: H-Ala-Glu-Asp-OH revealed its previously unknown biological activity, consisting in the normalization of metabolism in osseous and cartilaginous tissues.
This invention has set and resolved the task of obtaining a peptide medication possessing a biological activity, which consists in the normalization of metabolism in osseous and cartilaginous tissues, as well as of a pharmaceutical composition containing the said peptide as an active base, and a method of its application.
The technical result of the invention consists in the peptide alanyl-glutamyl- asparaginic acid with general formula: H-Ala-Glu-Asp-OH revealing its biological activity, which consists in the normalization of metabolism in osseous and cartilaginous tissues, as well as in a pharmaceutical composition, containing said peptide as its active base, which, being administered into the organism, increases the quantity and enhances the functions of calcitonin producing cells of the thyroid gland, improves the trophies of osseous and cartilaginous tissue cells, thus normalizing the metabolism in osseous and cartilaginous tissues.
The possibility of objective attainment of the technical result by the use of the invention is substantiated by reliable data indicated in the examples, which contain experimental data obtained in the process of studies performed using the methods common for this field of knowledge.
To resolve the set task and to attain the indicated technical result, the authors have proposed a group of inventions combined by a common inventive concept. The invention is related to a peptide alanyl-glutamyl-asparaginic acid with general formula: H-Ala-Glu-Asp-OH sequence 1 [SEQ ID NO.l], possessing a biological activity, consisting in the normalization of metabolism in osseous and cartilaginous tissues.
Peptide alanyl-glutamyl-asparaginic acid with general formula: H-Ala-Glu-Asp- OH sequence 1 [SEQ ID NO:1] is proposed for manufacturing a pharmaceutical composition, normalizing the metabolism in osseous and cartilaginous tissues.
The other aspect of the invention is related to a pharmaceutical composition, normalizing the metabolism in osseous and cartilaginous tissues, characterized in that
such composition contains an effective amount of peptide alanyl-glutamyl-asparaginic acid with general formula; H-Ala-Ght-Asp-OH sequence 1 [SEQ ID NO:1] as its active base, as well as a pharmaceutically admissible carrier.
In this case such pharmaceutical composition exists in the foπn suitable for parenteral administration.
The next aspect of the invention is related to a method of prevention and treatment for locomotor apparatus diseases by means of normalizing the metabolism in osseous and cartilaginous tissues, such method consisting in the administration to the patient of a pharmaceutical composition, containing peptide alanyl-glutamyl-asparaginic acid with general formula: H-Ala-Glu-Asp-OH sequence 1 [SEQ ID NO: 1] in the dose of 0,01 - 100 μg/kg of body weight as an active base, at least once a day during a period necessary for attaining the therapeutic effect.
In this case the pharmaceutical composition is administered intramuscularly.
Peptide alanyl-glutamyl-asparaginic acid with general formula: H-Ala-Glu-Asp- OH is obtained using a classical method of peptide synthesis in a solution.
The peptide's biological activity was studied in the experiment with pinealectomized rats, as well as on cartilaginous tissue explants and in patients with degenerative-dystrophic diseases of the joints.
The notion "pharmaceutical composition" implies such different medicinal forms containing the peptide H-Ala-Glu-Asp-OH, which may find therapeutic usage in the medicine as a means of normalizing the metabolism in osseous and cartilaginous tissues.
To obtain pharmaceutical compositions covered by the invention, an effective amount of peptide H-Ala-Glu-Asp-OH as an active base is mixed with a pharmaceutically acceptable carrier according to the methods of compounding, which are universally accepted in pharmaceutics.
The notion "effective amount" implies the use of such amount of the active base, which, according to the quantitative indices of its activity and toxicity, as well as to the knowledge of a competent specialist, must be effective in this medicinal form.
The carrier may have different forms, depending on the medicinal form of the preparation, which is desirable for the administration into the organism.
In case of parenteral administration the carrier normally includes a physiological solution or sterile water, although other ingredients, improving the stability or preserving sterility, may also be included.
The subject matter of the invention is explained by tables and a figure. Table 1 shows the effect of peptide H-Ala-Glu-Asp-OH on morphological and biochemical indices of guinea pig peripheral blood in the study of toxicity.
Table 2 illustrates the effect of peptide H-Ala-Glu-Asp-OH on the quantitative characteristics of studied thyroid gland parameters basing on the data of computer analysis of microscopic images.
The Figure shows the effect of peptide H-Ala-Glu-Asp-OH on the development of cartilaginous tissue explants.
The invention is illustrated by an example of synthesis of peptide alanyl-glutamyl- asparaginic acid with general formula: H-Ala-Glu-Asp-OH (Example 1), as well as by examples of the study of toxicity and biological activity of the peptide (Examples 2, 3, 4), and an example of the results of the peptide's clinical administration, confirming its pharmacological properties and the possibility of attaining the prophylactic and/or therapeutic effect (Example 5).
Example 1. Synthesis of peptide H-Ala-Glu-Asp-OH
1. Compound name: alanyl-glutamyl-asparaginic acid.
2. Structural formula: H-Ala-Glu-Asp-OH
3. General formula without ion pair: C 12 H 19 N 3 O 8 .
4. Molecular mass without ion pair: 333,29. 5. Ion pair: acetate.
6. Appearance: white amorphous powder without smell.
7. Method of synthesis: the peptide is obtained using a classical method of synthesis in the solution by the following scheme:
H-Asp(0Bzl)-0H
B0C-Glu(0Bzl)-Asp(0Bzl)-0H I) TFA
2) Z-AIa-OSu Z- AI - Asp(0Bzl)-0H
BOC - tert.butyloxycarbonyl group,
Z - benzyloxycarbonyl group,
OSu - N-oxysuccinimide ester,
DCC - NjN'-dicyclohexylcarbodiimide,
OBzI - benzyl ester, TFA - trifluoracetic acid.
Characteristics of the ready substance:
• Base substance content: 98,75 % (by HPLC, 220 nm),
• TLC - individual, R f =0,75 (acetonitrile-water 1:3),
• Humidity content: 6 %, • pH of 0,01% solution: 4,17,
• Specific rotary power: [α] D 22 : -20° (c=l, H 2 O), "Polamat A", Carl Zeiss Jena.
Example of synthesis:
1 ) BOC-GIU(OBZI)-OSU, N-oxy succinimide ester of N- tert.butyloxycarbonyl-(γ-benzyl)glutamic acid (I).
N-tert.butyloxycarbonyl-(γ-benzyl)glutamic acid BOC-GIU(OBZI)-OH (33,7 g, 0,1 mole) is dissolved in 50 ml of N,N'-dimethylformamide, cooled up to the temperature of -1O 0 C; cooled (4-6 0 C) solutions ofNjN'-dicyclohexylcarbodiimide (23,0 g, 0,11 mole) in
30 ml of N,N'-dimethylformamide and of N-hydroxysuccinimide (13,0 g, O 5 H mole) in 20 ml N,N'-dimethylformamide are added while stirring. The reactive mixture is stirred for 12 hours while cooled by ice, and for 24 hours more at the room temperature. The fallout N,N'-dicyclohexylurea is filtered out, and the obtained activated ester solution is used during the next stage without extraction.
2) BOC-GIU(OBZI)-ASP(OBZI)-OH, N-tert.butyIoxycarbonyl-(γ- benzyl)glutamyl-(β-benzyl)aspartate (II).
(β-benzyl)asparaginic acid H-Asρ(OBzl)-OH (28,0 g, 0,12 mole) and 36 ml (0,12 mole) of triethyl amine are suspended in 50 ml of N,N'-dimethylformarnide and stirred for 1 hour. Then activated ester solution BOC-GIU(OBZI)-OSU (I), which has been obtained during the previous stage, is added in portions. The reactive mixture is stirred at the room temperature for 48 hours. Then it is acidified with 0,5 N of sulfur acid up to pH 2-3 and extracted with ethyl acetate 4x50 ml. The extracts are put together and successively washed with 0,5 N H 2 SO 4 3x50 ml, water 2x50 ml, 5% NaHCO 3 solution 2x50 ml, water 2x50 ml, saturated NaCl solution 2x50 ml. The organic layer is dried over Na 2 SO 4 , solvent is removed in vacuum, residue is crystallized under hexane. 50 g of product are obtained (92%). R f = 0,34 (benzene-acetone 2:1).
3) TFA H-GIu(OBzI)- Asp(OBzl)-OH ( III ), trifluoracetate of (γ-benzyl)- glutamyl-(β-benzyl)aspartate.
N-tert.butyloxycarbonyl-(γ-benzyl)glutamyl-(β-benzyl)aspar tate ( I ) 5,68 g (« 0,01 mole) is dissolved in 20 ml of dichloromethane — trifluoracetic acid mixture (3:1). In 2 hours the solvent is removed in vacuum at the temperature of 4O 0 C, the removal is repeated with a new portion of dichloromethane (2x10 ml), residue is dried in vacuum over NaOH. 5,80 g of oil («100%) is obtained.
R f = 0,63 (n-buthanol-pyridine-acetic acid-water, 15:10:3:12).
4) Z-Ala-Glu(OBzl)-Asp(OBzl)-OH (IV), N-benzyloxycarbonyl-aIanyl-(γ- benzyl) glutamyl-(β-benzyl)aspartate. Trifluoracetate of (γ-benzyl) glutamyl-(β-benzyl)aspartate (II) 5,65 g (0,01 mole) is dissolved in 10 ml of dimethylformamide, added with triethyl amine 2,80 ml (0,02 mole)
and N-oxysuccinimide ester of N-carbobenzoxyalanine 4,14 g (0,013 mole). The mixture is stirred for 24 hours at the room temperature.
The product is sedentarized with 0,5 N sulfur acid solution (150 ml), extracted into ethyl acetate (3x30 ml), washed with 0,5 N sulfur acid solution (2x20 ml), water, 5% sodium bicarbonate solution (1x20 ml), water, 0,5 N sulfur acid solution (2x20 ml), water and dried over waterless sodium sulfate. Ethyl acetate is filtered out, removed in vacuum at the temperature of 40°C, the residue is crystallized in ethyl acetate/hexane system. The product is filtered out and dried in vacuum over P 2 O 5 . The output is 4,10 g (66 %). T me i t = 154 0 C. R f = 0,48 (benzene-acetone, 1:1), R f = 0,72 (n-buthanol-pyridine-acetic acid- water,
15:10:3:12).
5) H-Ala-Glu-Asp-OH, alanyl-glutamyl-aspartate.
Protected tripeptide (IV) 1,1 g is dissolved in the mixture of methyl spirit and water (4:1) and hydrated over catalyst Pd/C (5%) for 4 hours. The catalyst is filtered out, the solvent is removed in vacuum, the residue is dried in vacuum over KOH and P 2 O 5 . Crystallization is performed in water-methanol system.
Ultimately the residue is dissolved in 20 ml of deionized water and lyophilized. 105 mg of purified substance in the form of amorphous white powder without smell is obtained.
6) Analysis of the ready substance.
• The content of base substance was identified by HPLC on Phenomenex C 18 LUNA column 4,6x150 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 volume
20μl. Base substance content 98,75%.
• TLC: individual, R f = 0,75 (acetonitrile- water 1:3, Sorbfil plates, silicagel 8-12 μm, developing with chlorine/benzidine).
• Humidity content: 6% (gravimetrically, judging by weight loss by drying 20 mg at 100°C).
• pH 0,01% solution: 4,17 (potentiometrically).
• Specific rotary power: [α] D 22 : -20° (c=l, H 2 O), "Polamat A", Carl Zeiss Jena.
Example 2. Study of peptide H-Ala-Glu-Asp-OH toxicity
Common toxicity of peptide H-Ala-Glu-Asp-OH was studied in accordance with the requirements stated in the "Manual for experimental (pre-clinical) study of new pharmacological substances" (2000): acute toxicity in case of a single administration of the substance, as well as sub-acute and chronic toxicity in case of long-term administration of the peptide.
The study of acute toxicity was performed on 66 white mongrel male mice with body weight of 19-22 g. The animals were randomly subdivided into 6 equal groups. The substance was administered to the animals once, intramuscularly, in the 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. The control animals received 0,9% NaCl solution in the same volume.
The study of sub-acute toxicity was performed on 64 white mongrel male rats with body weight of 180-220 g. The substance was administered to the experimental animals daily, once a day, for 90 days, intramuscularly in the doses of 1 μg/kg, 0,1 mg/kg, 1 mg/kg in 0,5 ml of sterile 0,9% NaCl solution. Control animals received sterile 0,9% NaCl solution in the same volume. Morphological composition and properties of the animals' peripheral blood were evaluated before the administration of the substance, as well as on the 30 th , 60 th and 90 th day since the beginning of the substance administration. Upon completion of the experiment biochemical and coagulologic indices of the animals' blood were studied.
The study of chronic toxicity lasted for 6 months, basing on the duration of the recommended clinical indication of the substance. It was performed on 90 male guinea pigs with body weight of 270-300 g. Experimental animals received the peptide daily, once a day, for 6 months, intramuscularly in the doses of 1 μg/kg, 0,1 mg/kg, 1 mg/kg in 0,5 ml of sterile 0,9% NaCl solution. The control animals received sterile 0,9% NaCl solution in the same volume and by the same schedule.
The following indices were identified in the peripheral blood of the animals by using common methods: quantity of erythrocytes, hemoglobin, reticulocytes, thrombocytes, leukocytes, leukocyte formula, erythrocyte sedimentation rate (ESR), erythrocyte resistance. Alongside with that, total protein level in the blood serum was identified using Lowry's method, as well as the content of potassium and sodium using the method of plasma spectrophotometry. After the completion of the experiment a
pathomorphologic study of the brain, spinal cord, spinal cord ganglia, thyroid gland, parathyroid glands, adrenal glands, testis, pituitary gland, heart, lungs, aorta, liver, kidneys, urinary bladder, pancreas, stomach, small intestine, large intestine, thymus, spleen, lymph nodes and bone marrow was performed. The study of acute toxicity showed, that a single administration of the studied peptide to the animals in the dose exceeding the therapeutic one, recommended for the clinical administration, by more than 5000 times, does not cause any toxic reactions, which points out the wide therapeutic diapason of the substance.
The studies of sub-acute and chronic toxicity confirm the absence of side effects in case of a long-term administration of the substance in the doses, which exceed the therapeutic one by 100-1000 times. The study of peptide effect on the morphological composition and biochemical indices of guinea pig peripheral blood did not reveal any statistically significant change in the indices after 3 and 6 months of peptide administration (Table 1). The evaluation of general status of the animals, as well as of morphological and biochemical indices of their peripheral blood, morphological status of the internal organs, of the status of cardiovascular and respiratory systems, of liver and kidney functions did not reveal any pathologic alterations in the organism.
The absence of common toxicity allows to recommend the pharmaceutical composition, containing the peptide H-Ala-Glu-Asp-OH as its active base, for the clinical studies.
Example 3. Effect of peptide H-Ala-Glu-Asp-OH on the structural and functional organization of calcitonin producing cells of the thyroid gland in pinealectomized rats
The study was performed on 23 male Wistar rats with body weight of 130-140 g. The animals had their pineal gland removed. Pinealectomy was performed under ester narcosis according to the special method. Beginning 3 weeks after the operation (on the 21 st day) and throughout the successive 10 days peptide H-Ala-Glu-Asp-OH was administered to the animals subcutaneously in the dose of 0,5 μg per rat in 0,5 ml of sterile 0,9% NaCl solution.
Control pinealectomized rats and healthy animals received sterile saline solution in the same volume and by the same schedule.
The killing of the animals and the extraction of their thyroid gland was performed in the morning, from 10 till 12 o'clock in the conditions of daylight and under Nembutal narcosis (50 mg/kg). Some pinealectomized rats of both experimental and control groups was killed 3 days after the completion of injections (on the 33 rd day since the surgery and the beginning of the experiment), and some - 12 days after the completion of the injections (on the 42 nd day since the pinealectomy).
Fragments of the thyroid gland were fixed for 24 hours in the sour Buen's liquid for light microscopy studies and by Karnovsky's method for electronic microscopy. The analysis of calcitonin-irnrnune positive cells (C-cells) in the thyroid gland was performed basing on the index of volume proportion (pc a ), as well as of quantitative (Nca/lmm 2 ) and optic density (OpDca).
It was shown, that the thyroid gland of control animals largely retains its structural peculiarities after 1 month since the pinealectomy. However, there is a reduced amount of colloid in the follicles and somewhat increased mitotic activity in the follicular epithelium. The quantity of C-cells is increased by 24% as compared to the intact animals and makes 3012±172 per 1 mm 2 . The quantitative density of C-cells after 1,5 months since the pinealectomy is increased by more than 50% as compared to the intact animals, making up to 3668+158 cells per 1 mm 2 (Table 2). After 1 month since the pinealectomy the content of C-cells in the thyroid gland of experimental animals treated with peptide H-Ala-Glu-Asp-OH also has its peculiarities, consisting mainly in a statistically significant decrease of their quantity by 35%. The content of C-cells in the thyroid gland after 1,5 months since the pinealectomy (12 days after the completion of peptide H-AIa- Glu-Asp-OH administration) is ultimately normalized and largely does not differ from the control, making 2346+162 per 1 mm 2 . The quantitative density of C-cells on this stage is significantly different from the results of the control group, where this index made 3668+158 per 1 mm 2 , i.e. it exceeds the control values by more than 50% (Table
2). The studies of functional morphology of experimental pinealectomized rats' thyroid glands under conditions of peptide H-Ala-Glu-Asp-OH effect demonstrates the substance's stimulating influence on tissue and cellular metabolism. The administration of peptide H-Ala-Glu-Asp-OH exerts a compensatory effect on the structural and
functional organization of thyroid gland cells in pinealectomized animals. In 3 days after the completion of peptide administration its effect manifests itself by completely compensated consequences of pinealectomy, said effect being preserved after 12 days, i.e. until the completion of the experiment. The increased quantity and enhanced functions of thyroid gland C-cells under the effect of peptide H-Ala-Glu-Asp-OH limelight the important compensatory role of the peptide in calcium metabolism regulation and in the increase of calcium resorption by the osseous tissue, which is valuable for the prevention and treatment of osteoporoses of different etiologies.
Example 4. Effect of peptide H-Ala-Glu-Asp-OH on the development of cartilaginous tissue explants
The experiments were conducted on 28 fragments of cartilaginous tissue from femoral bone proximal head of Wistar rats with body weight of 160-200 g. Nutritional medium for explants cultivation consisted of 35% Eagle's solution, 25% calf fetal serum, 35% Hanx solution, 5% chicken embryonic extract, with the addition of glucose (0,6%), insulin (0,5 units/ml), penicillin (100 units/ml), glutamine (2mM). Cartilaginous tissue fragments were placed into this medium and cultivated in Petri dishes in the thermostat at the temperature of 36,7°C for 48 hours. Peptide H-Ala-Glu-Asp-OH was added into the medium, reaching ultimate concentration of 1, 10, 100, 200 and 400 ng/ml.
Area index (AI), i.e. the ratio of total explant area together with the growth zone to the initial area of cartilaginous tissue fragment served as the criterion of biologic activity. AI values were expressed in per cent, control AI values being considered as 100%. The Figure displays the effect of peptide H-Ala-Glu-Asp-OH on the development of cartilaginous tissue explants.
It was found, that after 24 hours of cultivating the explants sprawled upon the collagen substrate, and proliferating and migrating cells began disseminating around the area of the explant. By the 3 r day of the cultivation in case of peptide H-Ala-Glu-Asp- OH dose making 100 ng/ml a statistically significant AI growth by 26% was observed as compared to the control AI indices. The study of cartilaginous tissue explants after longer periods of cultivating (7 days) revealed the same stimulating effect in case of the same concentration of peptide H-Ala-Glu-Asp-OH.
Thus, peptide H-Ala-Glu-Asp-OH exerted a tissue specific effect on the cartilaginous tissue, consisting in the stimulation of explants growth.
Example 5. Efficacy of peptide H-Ala-Glu-Asp-OH in patients with knee joints osteoarthrosis
The study was conducted on 29 patients aged 52-72 with knee joints osteoarthrosis. The patients complained of pains and restricted flexion and extension in the joints while walking. Joints deformity, atrophy of femoral muscles and weakening of joints ligamentous apparatus were characteristic of elderly patients group. The duration of the disease was from 5 to 20 years, with progressive dynamics of pathological process development. Before participating in the study all patients had been long treated with analgesics and antiphlogistic drugs, attaining only a short-term therapeutic effect and being in need of increased doses of preparations their long-term intake. The patients were randomly divided into 2 groups.
Main group patients were subdivided into 3 groups depending on the age and severity of joints deformity. Main group patients older than 65 years with the most pronounced joint deformity and restriction of joint movement received the pharmaceutical composition containing peptide H-Ala-Glu-Asp-OH once a day, intramuscularly in the dose of 5,0 mg in 1 ml of sterile 0,9% NaCl solution for 20 days. Main group patients aged 60-65 with intermediate severity of joints deformity were treated with the pharmaceutical composition containing peptide H-Ala-Glu-Asp-OH once a day, intramuscularly, in the dose of 10,0 μg in 1 ml of sterile 0,9% NaCl solution for 20 days. Main group patients aged 52-60 with the initial stage of the disease, manifested by joint soreness and some restriction of joint movement in case of disease exacerbation, received the pharmaceutical composition containing peptide H-Ala-Glu-Asp-OH once a day, intramuscularly in the dose of 1,0 μg in 1 ml of sterile 0,9% NaCl solution for 20 days.
Control group consisted of 12 patients, who received sterile 0,9% NaCl solution by the same schedule.
The efficacy of the pharmaceutical composition containing H-Ala-Glu-Asp-OH peptide was estimated judging by the dynamics of the clinical indices and results of the rontgenologic assessment.
It is significant that rontgenologic symptoms of the degenerative dystrophic joints diseases are not only objective diagnostic criteria for pathologic process stage estimation, but also have prognostic significance in case of drug therapy.
It was found that peptide H-Ala-Glu-Asp-OH application in patients with lcnee- joints osteoarthrosis contributed to the reduction of the pain syndrome and increase in the joint movement in 54,5-62,7% of cases, depending on the severity of the disease. At the same time almost all pain symptoms (contraction of joint space between patella and thigh, lateral patella and condyle osteophytes) disappeared in case of the disease being at its initial stage, which was diagnosed rontgenologically. There was registered no significant dynamics of rontgenologic symptoms during this period.
Patients with intermediate severity of the arthrosis revealed the same dynamics of the subjective indices, but less pronounced, as on this stage of the disease the patient needs a long-term complex treatment. As this stage of the disease was diagnosed in patients of the older age group, positive dynamics of these subjective indices was considered to be very propitious.
The results of the conducted study show that the efficacy of the pharmaceutical composition, containing peptide H-Ala-Glu-Asp-OH, is based on the normalization of metabolism and slowing down the involution in osseous and cartilaginous tissues of knee joints by the improvement of their cells trophies.
Thus, it is expedient to use the pharmaceutical composition, containing an effective amount of peptide H-Ala-Glu-Asp-OH as its active base, for prevention and treatment, as well as in combination with any preparations of symptomatic or pathogenetic therapy, used for treatment of degenerative dystrophic joint and spinal column diseases, using different doses of pharmaceutical composition from 5 mg up to 1 μg in 1 ml of sterile 0,9% NaCl solution depending on the patient's age and severity of the pathologic process in the joints and spinal column.
Table 1
Table 2
* - P < 0,05 as compared to the index in intact animals; ** - P < 0,05 as compared to the index in control animals.
....•in.] πiβ (W
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