The means with erythropoeisis stimulating, immunomodulatory and antibacterial effect

 

(57) Abstract:

A preparation containing as active principle kobazol formula I, has erythropoeisis stimulating, immunomodulatory and antibacterial properties, is an effective stimulator of hematopoiesis wide spectrum of action. 5 Il., 10 table.

The present invention relates to medicine and the application of new medicines Tetra-(1-vinylimidazole)cobalticyanide (kobazol) in medical practice as hemodilator a wide spectrum of action. The name "kobazol" assigned according VFS on substance (N 42-28-86-97) and lectora (N 42-28-87-97) of the drug.

Known blood stimulant of natural origin - cyanocobalamin (vitamin B12), folic acid (vitamin BC) (B. Belikov, "Pharmaceutical chemistry", M., 1985), as well as various preparations on their basis - vitogepat, kobamamid, hemocyanin (M. D. Mashkovsky "Drugs", Vilnius, 1993). However, the retrieval of cyanocobalamin from the liver of animals consuming and uneconomical. At the same time, the chemical synthesis of folic acid and microbiological method of obtaining vitamin B12are multistage expensive the relief of anemia. It is first of all preparations containing iron, which are used in the form of salts and complexes of organic and inorganic acids (M. D. Mashkovsky "Drugs", Vilnius, 1993). Among the drugs, which include cobalt, the most famous Hamid (ibid.). The main disadvantages of synthetic Antianemic drugs are adverse reactions, poor solubility in water, high toxicity, biologically inactive ligands (in the case of complex compounds), as well as multi-stage, time-consuming and uneconomical synthesis.

The aim of the invention is the creation and application of the drug, which is an effective stimulator of hematopoiesis wide spectrum of action, to increase nonspecific resistance of the organism of man and animals.

This goal is achieved by taking into account the fact that kobazol at a dose of 1-2 ml of 2% aqueous solution, having good portability, is an effective stimulator of blood.

The essence of the invention is that the new drug kobazol - dose 20-40 mg (1-2 ml of 2% aqueous solution) has a high activity for copyrow the iron, and leukopenia different Genesis with a total radiation and chemical lesions. In addition, kobazol has immunomodulatory effects and antibacterial properties.

As a substance for medicinal products proposed to use kobazol Tetra-(1-vinylimidazole)cobaltichloride the following formula:

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where four molecules of 1-vinylimidazole connected with one molecule of cobalt dichloride donor-acceptor bonds. Kobazol easily soluble in water, moderately toxic. The method of producing kobazol (with yields up to 99%) simple, one-step and based on the interaction of 1-vinylimidazole with cobalt dichloride in the environment of an organic solvent at room temperature

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In the IR spectrum of Tetra-(1-vinylimidazole)cobalticyanide observed shift of the frequencies of stretching vibrations of the heterocycle in the shortwave region of the spectrum: 1465 ---> 1500, 1511 ---> 1522 cm-1. Saved the absorption of the vinyl groups with a frequency of 1650 cm-1.

Example 1. The method of producing Tetra-(1-vinylimidazole)cobalt dichloride (kobazol).

To a solution of 14.3 g (0.11 mol) of cobalt dichloride was added 4 g (0.44 mol) of 1-vinylimidazole. Allocate are 5.36 g (99%) lilac proscar CLASS="ptx2">

Calculated, %: C 47,44; H 4,78; Cl 14,02; N 22,14.

Dosage form of kobazol (2% aqueous solution, ampoule 1 ml) made according to the technology of sterile filtration and aseptic bottling. We have developed projects temporary pharmacopoeial articles on substance (VFS-42-28-86-97) and lectora (VFS-42-28-87-97) of the drug.

In the scientific literature contains no information about gemostimuliruyuschee, immunomodulating and antibacterial effect of the medicinal product kobazol, as well as any information about its effect on people. The preparation according to the structure, efficiency and range of actions has no analogues in domestic and foreign medicine.

According to the complete preclinical safety study of kobazol medical institutions of Moscow (NGOs "Biotechnology" MC "Potential"), Ufa (Bashkir medical Institute) established that the drug is moderately toxic compound (LD50215 mg/kg). Chronic experiment (6 months) toxicity of kobazol on dogs indicates the absence of its influence on the function of internal organs (liver, kidney, spleen, heart, lungs, pancreas and thyroid gland), morphological and histological studies kuortane, not qualitaets in the body.

Pharmacokinetics study of kobazol showed that kobazol, having good solubility in water, quickly penetrates into tissues and organs, passes through the blood-brain barrier, is rapidly eliminated from the body, its bioavailability is 100%. After a single intramuscular injection at a dose of 10 mg/kg of the drug rapidly enters the blood, liver, kidney, heart, spleen, lungs and bone marrow, while the maximum concentration in these organs is observed in the 0.4 to 1.3 h after injection. The highest concentration in the bone marrow (1.42 times higher than in the blood). In the brain, kidneys, lungs, heart, concentration kobazol 1.3-1.8 times lower than in blood. The half-life of kobazol of organs and tissues is 3,1-4,4 h, which is close to the half-life of blood (~3,9 h).

Biological (pre-clinical) testing kobazol as hemodilator were carried out on outbred albino male rats weighing 170-180 g with anemia caused twice daily by intraperitoneal injection of 1% solution of phenylhydrazine in a dose of 30 mg/kg In 8 series of experiments 18 rats were divided into 3 groups of 6 animals. The treatment was carried out in the 1st group kobazol dose of 10 µg/kg, which wwwsexo 6 times during the experience; in the second group - ciankobalamin in the same dose 10 mcg/kg, administered 2 times per week during the same period. The third group was the control.

On the 4th day of observation the number of red blood cells in the control group of animals significantly decreased (P < 0,001). In the group of animals treated with vitamin B12also, there was a decline in the number of red blood cells (P < 0,001). In the group of animals treated with kobazol, the number of red blood cells remained similar to the initial data (P > 0.5), and when compared to control significantly higher (P < 0,05). Hemoglobin levels compared with the original data decreased in the control group animals (P < 0,01). In the animals treated with vitamin B12hemoglobin also decreased (P < 0,05). In the group of animals treated with kobazol, hemoglobin remained within the source, significantly higher than in the control group (P < 0,01), and higher than in the group treated with vitamin B12.

On the 7th day of experience in all groups there was a decline in the amount of hemoglobin and red blood cell counts compared with the original data. The number of leukocytes was similar benchmarks. In the group of animals treated with kobazol, the amount of hemoglobin decreased by 28% compared to the original, and the number of erythrocythemia 35 and 49% in relation to the original. In the control group 36 and 47%, respectively. Thus, the decrease in the number of erythrocytes in the group of animals treated with kobazol was lower than in the group of animals treated with vitamin B12(P < 0.0001) and that of the control animals (P < 0,05). The decrease in the number of erythrocytes in the group of animals treated with vitamin B12similarly the control group animals (P > 0,5).

On the 12th day of observations in all groups showed increased levels of hemoglobin and red blood cell counts. In the group of animals treated with kobazol, the hemoglobin level was significantly higher than in the control group (P < 0.05), in group of animals treated with vitamin B12(P < 0,05). In the group of animals treated with kobazol, the amount of hemoglobin is 5% lower than the original, and in the group of animals treated with vitamin B12, - 15.4%. The number of erythrocytes in the blood of rats treated with kobazol was significantly higher than in the control group (P < 0,001), and in the group treated with vitamin B12(P < 0,001), and was below the original 10.5%. In these terms the number of red blood cells in the groups of animals treated with vitamin B12and the control was lower than the initial 30%.

On the 21st day of observation in the group of animals treated with kobazol, the hemoglobin level increased significant is lobina also almost reached the initial level and was lower by 5.6%. The number of erythrocytes in the blood of rats treated with kobazol, on the 21st day reached the source of the data was higher than the number of red blood cells in the group of animals treated with vitamin B12(P < 0.002) and in the control (P < 0,001). In the group of animals treated with vitamin B12the number of erythrocytes was also higher than in control (P < 0,05), but has not reached the source data by 10%. The influence of kobazol and vitamin B12doses of 10 µg/kg for anemia, the amount of hemoglobin and the number of erythrocytes in the peripheral blood of rats are presented in table. 1 and in Fig. 1 and 2.

Study of the effect of kobazol on for anemia caused by irradiation was carried out in experiments on white rats in a single exposure at a dose of 800 P. Animals were divided into 3 groups. The first group of control animals of the 2nd group on a weekly basis, starting from the 1st day of radiation was administered intramuscularly kobazol at a dose of 15 mg/kg, 5 times. The third group of animals were administered a solution of vitamin B12in the dose of 10 mg/kg of body weight.

The study of the basic hematological parameters of blood of animals (PL. 2) showed that animals treated with kobazol, they fell to less than low numbers and for a shorter time. The increase in same indicators were started earlier and went through Bakiyev (Fig. 3) found that the greatest reduction occurs on the 6th day, and animals treated kobazol, this reduction was significantly lower than in animals treated with vitamin B12and in the control group. The increase in the number of leukocytes was started on the 12th day, and on the 18th day in the group treated with kobazol was significantly higher than in the other groups. By the end of the month is 19% lower than the initial level, whereas the leukocyte count in the blood of the animals of the control group and treated with vitamin B12remained to the same period of relatively lower (30-26%, respectively). The level of hemoglobin (Fig. 4) in irradiated animals treated kobazol, fell to 12 days, while the other groups up to 18 days after exposure. At the same time, all the time quantity of hemoglobin in the animals treated with the kobazol remained significantly higher, and the recovery of hemoglobin was more intensely and in all periods than in other animal groups. In the group of animals treated with vitamin B12, restore haemoglobin levels began with a 24-day treatment and 31st days was lower than the initial level by 15%. By this time monitoring the level of hemoglobin in the control group was below baseline at 18%. (Cobasouli irradiation (Fig. 5). The fall in the number of erythrocytes continued for up to 18 days. But in the animals treated with the kobazol, this decline was relatively small, and 24-e day was restored almost to its original level, whereas in the other two groups, the recovery was very slow, and after a month in the group of animals treated with vitamin B12the number of erythrocytes was lower than the initial 13.6%.

WBC (PL. 3) has changed dramatically, and change it from all groups were almost similar, while in the control group more sharply increased the number of neutrophils (and, correspondingly, decreased lymphocytes), and by the end of the month to the initial level was not decreased.

Our data indicate that radiation sickness kobazol more effective as a stimulator of Erythro - and leucopoiesis.

Antibacterial properties of kobazol was determined in the study of the effect of the drug on the growth of bacteria: Proteus, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus and Pseudomonas aeruginosa (15 cups 6 disks). A comparison was made of the action of kobazol activity of tetracycline and streptomycin. It is shown that kobazol at a dose of 10 µg retards the growth of Staphylococcus similar to tetracycline and the sticks like streptomycin, giving him only in activity against E. coli. The action on Staphylococcus kobazol has 3 times more active than streptomycin. At the same dose (30 µg) kobazol superior in their effect tetracycline against Proteus, Klebsiella, Pseudomonas aeruginosa and 1.5 times in Staphylococcus (table. 5).

Thus, the preliminary results of the test was a prerequisite for further study of the drug. In accordance with the decision of the Pharmacological Committee of Ministry of health of Russia (minutes No. 2 dated 27.01.94, conducted clinical trials on the tolerability and efficacy of the drug kobazol on the bases of gynecological departments Angarsk oncologic dispensary MOH and Irkutsk city medical diagnostic center of obstetrics, gynecology and Perinatology. The investigated drug in the dosage form for injection 2% aqueous solution, ampoule 1 ml

Example 2. The manufacture of dosage forms of kobazol.

Legform drug kobazol, the substance of which is received in Erich SB RAS in accordance VFS-42-28-86-97 produced by technology of sterile filtration and aseptic bottling. A portion of the substance of the drug kobazol in Vidor drug pink and transparent according to the project VFS on lectora of kobazol (VFS-42-28-87-97). Sterilizing filtration of a solution of kobazol put through a filter Millipore (pore size 0.22 μm), mounted in a special coil connected to the installation of filtration under pressure of purified nitrogen. After careful filtering (solution 4 times filtered) system hose with filter cut off from the flask with the solution and attached to the sterile burette to spill. Spill preparation is carried out in a sterile and thoroughly tested for purity ampoules of 3 ml, which is then sealed. Then the ampoules with lekforma viewed on the content of mechanical impurities and burns on the control table GF-42 with the aim of rejection. Certification legform of kobazol in accordance VFS-42-28-87-97 performed by the control Department of the Irkutsk enterprise immunopreparatov. Storage dosage form of the drug at 20-30oC showed that within two years it retains all its physico-chemical properties according to VFS on lectora.

Open study of the effectiveness of kobazol conducted in 50 cancer and gynecological patients with symptoms of anemia and leukopenia light and medium severity. The study included patients aged 18 years, gave oral coz the th until the time of the study. After inclusion of the patient into the study of the regimen completely excluded hemodilator, iron preparations, prednisolon, blood.

Age patients were distributed as follows: from 28 to 30 years examined three patients (6%); from 31 to 40 years - 7 (14%); from 42 to 50 years old - 25 (50%); 51 to 60 years - 7 (14%); over 60 years of age - 8 (16%).

To study the efficacy of kobazol conducted in the following groups: 1st group - gynecologic patients with posthemorrhagic and iron-deficiency anemia, 20; 2nd group - cancer patients with radiation of various origins, 30 people. To compare the effectiveness and duration of treatment are investigated control group: 3rd group using a placebo, 8 people, and group 4 retrospective data in the treatment of anemia iron supplementation (veraplex pills, ferroelec - parenteral), 30 people.

In the 1st group on the severity of anemia (who classification) patients had: I tbsp. - in 10 (50%); II Art. - in 9 (45%); (III tbsp. in 1 (5%). Anemia as a complication of hemorrhagic syndrome was observed: uterine fibroid in 60% of patients with genital endometriosis 20%, after dysfunctional uterine bleeding in 10%, bleeding in ectopic pregnancy 10%- 10 (33,3%); III senior 3 (10%). Leukopenia as a complication of chemotherapy was observed: ovarian cancer - 50% (I tbsp. of the underlying disease had 4 patients (13.3 per cent); (II Art. - 2 (6,6%); Art. III - 5 (16.6 percent); Art. IV - 4 (13,3%); breast cancer - 13.3 per cent (10% of patients had II Art. disease, 3,3% - III century), with chlamydia - 3.3 percent. Leukopenia as a complication of radiation therapy is installed in cancer of the cervix in 13,3% (6.6% of the patients I had Art. diseases, II and IV century - 3.3%) and cancer of the endometrium Art. I - 3.3 percent. In 6.6% of patients kobazol was applied with uterine cancer in combination with sarcoma, 3.3 percent for cancer of the large intestine, at 3.3 percent for tumors serous gotostate.

Kobazol was applied in the form of a 2% aqueous solution by volume of 2 ml at the rate of 5-7 injections. Clinical evaluation of the effectiveness of kobazol as hemodilator was based on laboratory data for 72 h and weekly, adverse reactions - from the moment of injection up to 2-3 days. Laboratory parameters were evaluated on the following parameters: full hematological analysis (number of erythrocytes, leukocytes, platelets, reticulocytes, ESR, WBC, hemoglobin, hematocrit), biochemical analysis of blood (serum iron, total bilirubin, total protein, the levels to the of TATUS blood [T-lymphocytes (helper the suppressor), B-lymphocytes, phagocytic index, phagocytic number, the levels of CEC, Lg A, M, G].

Example 3. Study of the efficacy and tolerability of the drug kobazol.

The duration of drug was 1 less 2 weeks depending on the initial level of hemoglobin in group II and the number of leukocytes in the second. The use of kobazol during the week increased the level of hemoglobin of 5 g/l at 16.6 percent, 10 g/l - 44,4%, 15 g/l in 33.3% of patients with anemia. Weekly treatment kobazol patients with leukopenia increased the number of leukocytes in the following way: (1.5 to 2.0) 109- with 13.7% of patients; for (2.1 to 3.0) 109- 55,1%, (3,1-4,0) 109- 20,6%, (4,1-5,0%) 109- at 10.3 percent. Thus, the use of kobazol during the week led to full recovery 45% of patients with anemia and to 93.3% with radiation. To compare the effectiveness and duration of treatment anemia data of iron supplementation (table. 6). Clinical effect of kobazol obtained in 85% of patients with anemia and in 96.6% of patients with leukopenia. Evaluation of changes in mean values of hematological parameters of blood in the study and control groups are shown in table. 7.

When the weekly analysis of the dynamics of changes in the level of hemoglo what's preparations of iron, hemoglobin levels increased by 8 g/l In the group with placebo, the increase in hemoglobin is not identified (chart).

The data of biochemical analysis of blood and hemostatic parameters indicate the absence of pathological changes (fluctuations are within normal values). Changes in the level of serum iron is more pronounced in the control group (an increase 4,32 µmol/l) than in the study - an increase of 1.13 µmol/l (table. 8). When examining the state of the cardiovascular system pathological changes in blood pressure, heart rate was not detected (table. 9). Adverse reactions of moderate severity in the form of General weakness, nausea, dizziness, local hyperemia was observed in 12% of women. In one patient the drug was withdrawn due to individually intolerance.

In the process of studying the effectiveness of kobazol the group of patients (24) evaluated the immunological profile of blood (before and after treatment), as anemic syndrome and multiple effects on the body of the cytotoxic factors (chemotherapy, radiotherapy) lead to the development of immune deficiency, as evidenced by doscover the CLASS="ptx2">

The use of kobazol was achieved preservation of immune status within normal values, and in some cases, marked improvement (table. 10). This is confirmed by the persistent increase in the number of T-lymphocytes from 55 to 82-90% 45.8% of the patients, b-lymphocytes from 7.4% to 15% from 37%. In 45.8% of women observed increase in phagocytic activity of neutrophils (PHI) from 31 to 69%. The use of kobazol was accompanied by reduced levels of circulating immune complexes (CIC) 40% 50% of patients. 100% of women saw an increase in the production of immunoglobulins.

Thus, the test results indicate a pronounced effectiveness of kobazol as hemodilator and allow to prove the feasibility of its use in posthemorrhagic, hypochromic anemia, anemia resistant to iron preparations, and when leukopenia. Immunomodulatory effect and antibacterial properties of kobazol increase its clinical value in the treatment of anemia and leukopenia.

The means with erythropoeisis stimulating, immunomodulatory and antibacterial action, containing as active principle Tetra-(1-vinylimidazole)cobaltichloride (kobazol).

 

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