RU2157813C2 - Agent showing leukopoiesis stimulating, immunomodulating and antibacterial effect - Google Patents

Abstract

FIELD: organic chemistry, medicine. SUBSTANCE: invention proposes cobazol of the formula (I)

Description

 The present invention relates to medicine and relates to the use of a new drug tetra- (1-vinylimidazole) cobalt dichloride (cobazole) in medical practice as a hemostatic stimulator of a wide spectrum of action. The name "cobazole" is assigned in accordance with the VFS for the substance (N 42-28-86-97) and the drug form (N 42-28-87-97).

Known hemopoietic stimulants of natural origin - cyanocobalamin (vitamin B ₁₂ ), folic acid (vitamin B _C ) (V.G. Belikov "Pharmaceutical chemistry", M., 1985), as well as various drugs based on them - vitohepat, cobamamide, hemostimulin (M.D. Mashkovsky "Medicines", Vilnius, 1993). However, obtaining cyanocobalamin from the liver of animals is laborious and uneconomical. At the same time, the chemical synthesis of folic acid and the microbiological method for producing vitamin B ₁₂ are multi-stage expensive processes.

 Synthetic drugs that help relieve anemia are widely used. These are primarily iron-containing preparations, which are used in the form of salts and complexes of organic and inorganic acids (MD Mashkovsky "Medicines", Vilnius, 1993). Among the preparations, which include cobalt, the most famous is coamide (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 economically disadvantageous synthesis.

 The aim of the invention is the creation and use of a medicinal product, which is an effective stimulator of hematopoiesis of a wide spectrum of action, increasing the overall nonspecific resistance of the human body and animals.

 This goal is achieved taking into account the fact that cobazole in a dose of 1-2 ml of a 2% aqueous solution, having good tolerance, is an effective stimulator of hematopoiesis.

 The essence of the invention is that a new drug - cobazole - at a dose of 20-40 mg (1-2 ml of a 2% aqueous solution) has high activity for the relief of posthemorrhagic, hypochromic, hypotactic anemia, including anemia resistant to iron preparations, as well as leukopenia of various origins with general radiation and chemical lesions. In addition, cobazole has an immunomodulatory effect and pronounced antibacterial properties.

где четыре молекулы 1-винилимидазола соединены с одной молекулой дихлорида кобальта донорно-акцепторными связями. Кобазол легко растворяется в воде, умеренно токсичен. Способ получения кобазола (с выходом до 99%) прост, одностадиен и основан на взаимодействии 1-винилимидазола с дихлоридом кобальта в среде органического растворителя при комнатной температуре

В ИК спектре тетра-(1-винилимидазол)кобальтдихлорида наблюдается смещение частот валентных колебаний гетероцикла в коротковолновую область спектра: 1465 ---> 1500, 1511 ---> 1522 см^-1. Сохраняется поглощение винильной группы с частотой 1650 см^-1.It is proposed to use cobazole tetra- (1-vinylimidazole) cobalt dichloride of the following formula as a drug substance:

where four molecules of 1-vinylimidazole are connected to one cobalt dichloride molecule by donor-acceptor bonds. Cobazole is readily soluble in water, moderately toxic. The method of producing cobazole (with a yield of up to 99%) is simple, one-stage and is based on the interaction of 1-vinylimidazole with cobalt dichloride in an organic solvent at room temperature

In the IR spectrum of tetra- (1-vinylimidazole) cobalt dichloride, a shift in the frequencies of stretching vibrations of the heterocycle to the short-wave region of the spectrum is observed: 1465 ---> 1500, 1511 ---> 1522 cm ^-1 . The absorption of the vinyl group with a frequency of 1650 cm ^{-1 is maintained} .

 Example 1. A method of producing tetra- (1-vinylimidazole) cobalt dichloride (cobazole).

To a solution of 14.3 g (0.11 mol) of cobalt dichloride, 4 g (0.44 mol) of 1-vinylimidazole are added. Allocate 5.36 g (99%) of lilac powder with so pl. 164 ^o C.

 Found,%: C 47.75; H 4.97; Cl 14.10; N, 21.93.

C ₂₀ H ₂₄ Cl ₂ CoN ₈ .

 Calculated,%: C 47.44; H 4.78; Cl 14.02; N, 22.14.

 The dosage form of cobazole (2% aqueous solution, 1 ml ampoule) was made using sterile filtration and aseptic spill technology. We have developed projects of temporary pharmacopoeial articles on the substance (VFS-42-28-86-97) and the drug form (VFS-42-28-87-97).

 In the scientific and technical literature there is no information about the hemostimulating, immunomodulating and antibacterial effect of the drug cobazole, as well as any information about its effect on humans. The drug has no analogues in domestic and foreign medicine in structure, effectiveness and spectrum of action.

According to a complete preclinical study of the safety of cobazole in medical institutions in Moscow (NPO Biotechnology, MC Potential), Ufa (Bashkir Medical Institute), it was found that the drug is a moderately toxic compound (LD ₅₀ 215 mg / kg). A chronic experiment (6 months) of the toxic effect of cobazole on dogs indicates the absence of its effect on the functions of internal organs (liver, kidney, spleen, heart, lungs, pancreas and thyroid gland), morphological and histological studies of which did not reveal differences from animal organs in the control . Cobazole does not have teratogenic and mutagenic effects, does not cumulate in the body.

 A study of the pharmacokinetics of cobazole showed that cobazole, having good solubility in water, quickly penetrates into organs and tissues, 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, the drug quickly penetrates the blood, liver, kidneys, heart, spleen, lungs and bone marrow, while the maximum concentration in these organs is observed 0.4-1.3 hours after administration. The highest concentration is in the bone marrow (1.42 times higher than in the blood). In the brain, kidneys, lungs, heart, the concentration of cobazole is 1.3-1.8 times lower than in the blood. The half-life of cobazole from organs and tissues is 3.1-4.4 hours, which is close to the half-life of blood (~ 3.9 hours).

 Biological (preclinical) tests of cobazole as a hemostatic stimulator were carried out on white outbred male rats weighing 170-180 g with anemia caused by daily double intraperitoneal administration of a 1% solution of phenylhydrazine at a dose of 30 mg / kg. In the 8th series of experiments, 18 rats were divided into 3 groups of 6 animals. The treatment was carried out in the 1st group with cobazole at a dose of 10 μg / kg of body weight, which was administered intramuscularly twice a week, starting from the day after the last phenylhydrazine administration, only 6 times during the experiment; in the second group - cyancobalamin in the same dose of 10 μg / kg, also administered 2 times a week at the same time. The third group was a 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 B ₁₂ , a decrease in the number of red blood cells was also observed (P <0.001). In the group of animals treated with cobazole, the number of red blood cells remained similar to the initial data (P> 0.5), and when compared with the control, it was significantly higher (P <0.05). The hemoglobin level compared to the initial data decreased in the control group of animals (P <0.01). In animals treated with vitamin B ₁₂ , hemoglobin levels also decreased (P <0.05). In the group of animals treated with cobazole, hemoglobin remained within the original range, significantly higher than in the control group (P <0.01), and higher than in the group treated with vitamin B ₁₂ .

On the 7th day of the experiment, in all groups there was a decrease in the number of hemoglobin and the number of red blood cells compared with the initial data. The number of leukocytes was similar to baseline. In the group of animals treated with cobazole, the amount of hemoglobin decreased by 28% compared to the original, and the number of red blood cells - by 37%. The decrease in the number of hemoglobin and erythrocytes in the group of animals that received vitamin B ₁₂ was 35 and 49% in relation to the initial one. In the control group, 36 and 47%, respectively. Thus, the decrease in the number of red blood cells in the group of animals treated with cobazole was less than in the group of animals that received vitamin B ₁₂ (P <0.0001) and in the control animals (P <0.05). The decrease in the number of red blood cells in the group of animals treated with vitamin B ₁₂ is similar to the control group of animals (P> 0.5).

On the 12th day of observation in all groups, an increase in hemoglobin level and the number of red blood cells was noted. In the group of animals treated with cobazole, the hemoglobin level was significantly higher than in the control group (P <0.05), and in the group of animals treated with vitamin B ₁₂ (P <0.05). In the group of animals treated with cobazole, the amount of hemoglobin is 5% lower than the original, and in the group of animals that received vitamin B ₁₂ , by 15.4%. The number of red blood cells in the blood of rats treated with cobazole was significantly higher than in the control group (P <0.001) and in the group treated with vitamin B ₁₂ (P <0.001), and was 10.5% lower than the initial one. During these periods, the number of red blood cells in groups of animals treated with vitamin B ₁₂ and the control was 30% lower than the initial one.

On the 21st day of observation in the group of animals treated with cobazole, the hemoglobin level increased significantly more than in the control (P <0.05). At the same time in animals treated with vitamin B ₁₂ , the amount of hemoglobin also almost reached the initial level and was lower by 5.6%. The number of red blood cells in the blood of rats treated with cobazole on the 21st day reached baseline data and was higher than the number of red blood cells in the group of animals that received vitamin B ₁₂ (P <0.002) and in the control (P <0.001). In the group of animals treated with vitamin B ₁₂ , the number of red blood cells was also higher than in the control (P <0.05), but did not reach the initial data by 10%. The effect of cobazole and vitamin B ₁₂ in doses of 10 μg / kg on the course of anemia, the amount of hemoglobin and the number of red blood cells in the peripheral blood of rats is presented in table. 1 and in FIG. 1 and 2.

The effect of cobazole on the course of anemia caused by radiation was studied in experiments on white rats with a single exposure at a dose of 800 P. The animals were divided into 3 groups. The first group is the control, animals of the 2nd group weekly, starting from the 1st day of exposure, was injected intramuscularly with a dose of 15 mg / kg cobazole, only 5 times. The third group of animals was injected with a solution of vitamin B ₁₂ at a dose of 10 μg / kg of body weight.

The study of the main hematological parameters of the blood of animals (Table 2) showed that in animals treated with cobazole, they fell to lower numbers and over a shorter time. The increase in indicators began earlier and was more intense, i.e. their recovery occurred in a shorter time. When analyzing changes in the number of leukocytes (Fig. 3), it was found that their greatest decrease occurs on the 6th day, and in animals treated with cobazole, this decrease was significantly lower than in animals treated with vitamin B ₁₂ and in the control group . The increase in the number of leukocytes began on the 12th day, and on the 18th day in the group treated with cobazole, was significantly higher than in other groups. By the end of the month, it was 19% lower than the initial level, while the leukocyte counts in the blood of animals from the control group and treated with vitamin B ₁₂ remained comparatively lower (30-26%, respectively) by the same period. The hemoglobin level (Fig. 4) in irradiated animals treated with cobazole dropped to 12 days, and in other groups to 18 days after irradiation. Moreover, at all times, the amount of hemoglobin in animals treated with cobazole remained significantly higher, and the restoration of hemoglobin indices went more intensively and at all times than in animals of other groups. In the group of animals treated with vitamin B ₁₂ , restoration of the hemoglobin level began from the 24th day of treatment and by the 31st day was 15% lower than the initial level. By this observation period, the hemoglobin level in the control group was 18% lower than the initial one. (Cobazole in the same period - 8%).

Similar data were obtained by analyzing changes in the number of red blood cells after irradiation (Fig. 5). The fall in the number of red blood cells lasted up to 18 days. But in animals treated with cobazole, this drop was relatively small, and on the 24th day it was restored to almost the initial level, while in the other two groups the recovery was very slow, and after a month in the group of animals that received vitamin B ₁₂ , the number of red blood cells was lower than the original by 13.6%.

 The leukocyte formula (Table 3) did not change dramatically, and its changes in all groups were almost similar, although in the control group the number of neutrophils increased slightly (and lymphocytes decreased accordingly), and by the end of the month it did not decrease to the initial level.

 The data obtained suggest that, in radiation sickness, cobazole is more effective as a stimulator of erythro- and leukopoiesis.

 The antibacterial properties of cobazole were determined by studying the effect of the drug on bacterial growth: Proteus, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa (15 cups with 6 discs each). The effects of cobazole were compared with the activity of tetracycline and streptomycin. It was shown that cobazole at a dose of 10 μg inhibits staphylococcus growth similar to tetracycline and is 2 times more active than streptomycin (Table 4). At a dose of 30 μg, cobazole inhibits the growth of Proteus, Klebsiella, Pseudomonas aeruginosa like streptomycin, second only to it in relation to Escherichia coli. According to the action on staphylococcus, cobazole has 3 times greater activity than streptomycin. At the same dose (30 μg), cobazole is superior in its action to tetracycline in relation to protein, Klebsiella, Pseudomonas aeruginosa and 1.5 times in action on staphylococcus (Table 5).

 Thus, the obtained preliminary test results were a prerequisite for further study of the drug. In accordance with the decision of the Pharmacological Committee of the Ministry of Health of the Russian Federation (protocol No. 2 dated 01/27/94), clinical trials were conducted on the tolerability and effectiveness of the drug cobazole at the bases of the gynecological departments of the Angarsk Oncology Center of the Ministry of Health of the Russian Federation and the Irkutsk City Medical and Diagnostic Center of Obstetrics, Gynecology and Perinatology. The drug was investigated in the dosage form for injection - 2% aqueous solution, ampoules of 1 ml.

 Example 2. The manufacture of a dosage form of cobazole.

The drug’s cobazole form, the substance of which was obtained at the Institute of IIH SB RAS in accordance with VFS-42-28-86-97, was made using sterile filtration and aseptic spill technology. A sample of the drug substance cobazole in the form of a lilac powder is dissolved in distilled water to obtain a 2% solution. The prepared solution of the drug is pink and transparent according to the VFS project for the cobazole leform (VFS-42-28-87-97). The sterilizing filtration of the cobazole solution is carried out through the Millipor filter (0.22 μm pore diameter) mounted in a special coil connected to the filtration unit under the pressure of purified nitrogen. After thorough filtration (the solution is filtered 4 times), the hose system with the filter is disconnected from the solution bottle and attached to a sterile burette for bottling. The drug is bottled in sterile and thoroughly tested 3 ml ampoules, which are then sealed. Then the ampoules with leform are viewed for the content of solids and burns on the control table GF-42 for the purpose of rejection. The certification of cobazole leform in accordance with VFS-42-28-87-97 was performed by the control department of the Irkutsk enterprise of immunopreparations. Storage of the dosage form of the drug at 20-30 ^o C showed that for two years it retains all its physicochemical properties according to the VFS for lexform.

 An open study of the effectiveness of cobazole was carried out in 50 oncological and gynecological patients with mild to moderate anemia and leukopenia. The study included patients who reached the age of 18 years, who gave oral consent to participate in clinical trials, who did not receive any stimulants of erythro- and leukopoiesis 7 days before the study. After the patient is included in the study, hemostatic stimulants, iron preparations, prednisone, and blood preparations are completely excluded from the treatment regimen.

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

 The study of the effectiveness of cobazole was carried out in the following groups: 1st group - gynecological patients with posthemorrhagic and iron deficiency anemia, 20 people; 2nd group - cancer patients with leukopenia of various origins, 30 people. To compare the efficacy and duration of treatment, the control groups were studied: the 3rd group using placebo, 8 people, and the 4th group of retrospective data in the treatment of anemia with iron preparations (ferroplex peroral, ferrumlek parenteral route), 30 people.

 In the 1st group, according to the severity of anemia (WHO classification), patients had: I Art. - in 10 (50%); II Art. - in 9 (45%); III art. - in 1 (5%). Anemia as a complication of hemorrhagic syndrome was observed: with uterine myoma in 60% of patients, with genital endometriosis in 20%, after dysfunctional uterine bleeding in 10%, and bleeding in ectopic pregnancy in 10%.

 In the 2nd group according to the severity of leukopenia (WHO classification), patients had: I Art. - in 17 (56.6%); II Art. - in 10 (33.3%); III art. - in 3 (10%). Leukopenia as a complication of chemotherapy was observed: in ovarian cancer, in 50% (I stage of the main disease had 4 patients (13.3%); II stage - 2 (6.6%); III stage - 5 (16.6 %); IV st. - 4 (13.3%); with breast cancer - 13.3% (10% of patients had II stage of disease, 3.3% - III stage), with lymphogranulomatosis - 3 , 3%. Leukopenia as a complication of radiation therapy was found in cervical cancer in 13.3% (6.6% of patients had stage I diseases, stage II and IV - 3.3% each), and endometrial cancer of stage I. - in 3.3%. In 6.6% of patients, cobazole was used for uterine fibroids in combination with sarcoma, in 3.3% for colon cancer, in 3.3% for tumors of serous histology.

 Cobazole was used as a 2% aqueous solution of 2 ml in the amount of 5-7 injections. A clinical evaluation of the effectiveness of cobazole as a hemostatic stimulator was carried out on the basis of laboratory data for 72 hours and weekly, adverse reactions - from the moment of injection to 2-3 days. Laboratory parameters were evaluated by the following parameters: complete hematological analysis (the number of red blood cells, white blood cells, platelets, reticulocytes, ESR, white blood cell count, hemoglobin level, hematocrit), biochemical blood test (serum iron level, total bilirubin, total protein, creatinine, urea, ALT, ACT), indicators of hemostasis (fibrinogen, prothrombin index), indicators of the immune status of blood [T-lymphocytes (helpers, suppressors), B-lymphocytes, phagocytic index, phagocytic number, CEC levels, Lg A, M, G].

 Example 3. The study of the effectiveness and tolerability of the drug cobazole.

The duration of the drug was 1 less than 2 weeks, depending on the initial hemoglobin level in group II and the number of leukocytes in group II. The use of cobazole during the week increased the hemoglobin level by 5 g / l in 16.6%, by 10 g / l in 44.4%, and by 15 g / l in 33.3% of patients with anemia. Weekly treatment with cobazole in patients with leukopenia increased the number of leukocytes as follows: by (1.5-2.0) • 10 ⁹ - in 13.7% of patients; by (2.1-3.0) • 10 ⁹ - by 55.1%; by (3.1-4.0) • 10 ⁹ - by 20.6%; by (4.1-5.0%) • 10 ⁹ - by 10.3%. Thus, the use of cobazole during the week led to the full recovery of 45% of patients with anemia and 93.3% with leukopenia. To compare the effectiveness and terms of treatment of anemic syndrome, data on the use of iron preparations are given (Table 6). The clinical effect of cobazole was obtained in 85% of patients with anemia and in 96.6% of patients with leukopenia. The assessment of changes in the average values of hematological blood parameters in the studied and control groups is shown in table. 7.

 An analysis of the weekly dynamics of hemoglobin level changes in the study group revealed an increase of this indicator by 13 g / l, while in the group of patients treated with iron preparations, the hemoglobin level increased by 8 g / l. In the placebo group, hemoglobin growth was not detected (chart).

 The data of biochemical analysis of blood parameters, as well as hemostasis indicators indicate the absence of pathological changes (fluctuations are within normal values). The change in the level of serum iron is more pronounced in the control group (an increase of 4.32 μmol / L) than in the study group - an increase of 1.13 μmol / L (Table 8). When studying indicators of the state of the cardiovascular system, pathological changes in blood pressure and heart rate were not detected (table. 9). Adverse reactions of moderate severity in the form of general weakness, nausea, dizziness, local hyperemia were noted in 12% of women. In one patient, the drug was canceled due to individual intolerance.

 In the process of studying the effectiveness of cobazole in a group of patients (24 people), the immunological profile of the blood was assessed (before and after treatment), since anemic syndrome and repeated exposure of the body to cytostatic factors (chemo-, radiation therapy) lead to the development of immune deficiency, which is confirmed a significant decrease in T and B lymphocytes.

 Example 4. The study of the immunomodulatory activity of cobazole.

 The use of cobazole achieved the maintenance of indicators of the immune status within normal values, and in some cases their improvement was noted (Table 10). This is confirmed by a persistent increase in the number of T-lymphocytes from 55 to 82-90% in 45.8% of patients, B-lymphocytes from 7.4 to 15% in 37%. 45.8% of women showed an increase in the phagocytic activity of neutrophils (PI) from 31 to 69%. The use of cobazole was accompanied by a decrease in the level of circulating immune complexes (CIC) by 40% in 50% of patients. In 100% of women, an increase in the production of immunoglobulins was noted.

 Thus, the test results indicate the pronounced effectiveness of cobazole as a hemostatic stimulator and make it possible to justify its use in posthemorrhagic, hypochromic anemia, anemia resistant to iron preparations, as well as in leukopenia. The immunomodulating effect and antibacterial properties of cobazole increase its clinical value in the treatment of anemia and leukopenia.

Claims (1)

 A tool with leukopoiesis-stimulating, immunomodulating and antibacterial action, containing as the active principle tetra- (1-vinylimidazole) cobalt dichloride (cobazole).

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