RU2093177C1 - Preparation for treatment and prophylaxis of sickness in mammals, birds and reptiles - Google Patents

Abstract

FIELD: biotechnology, immunology. SUBSTANCE: preparation is organogermanium derivative of picolinic acid of the formula C₄₂H₂₄ N₆O_2IGe₃. Preparation shows immunomodulating (the main effect), antiviral, antitumor, radioprotective and antibacterial effects. Preparation shows an excellent antimutagenic and sedative effect with respect to domestic and industrial animals. Preparation can be used as repair agent for skin and tissue damages and as an accelerating agent of hair recovery after postinfections and metabolic alopecia damages. EFFECT: enhanced effectiveness of preparation. 14 ex, 4 tbl

Description

 The invention relates to biotechnology and relates to the production and use of biological products, in particular immunomodulators, for the correction of immune system disorders and used for the treatment and prevention of infectious, invasive and non-infectious diseases of mammals, birds and reptiles.

 Every living creature (be it a mammal, bird, or reptile) is given an excellent immune system protector from birth. The qualitative indicators of each species, its resistance to diseases and survival depend on its condition.

 Recently, a direct relationship between many infectious and non-infectious diseases with a violation of the immune system has been proved. In this regard, immunomodulators are increasingly being used for treatment, the purpose of which is to restore the body's own defenses.

 Known and well-established in clinical practice are new-generation drugs derived from biotechnology: natural and recombinant interferons, interleukins, phospholipids, etc. ("The current state and development prospects of the foreign market of immunomodulators" (review). NPO "Medbioeconomics", NIITEKHIM, 1990). However, the exogenous use of these expensive drugs requires large doses, since they are rapidly excreted from the body, which can lead to allergic reactions.

 The intensive development of organoelement chemistry created the prerequisites for the synthesis of new drugs based on organosilicon and organosilicon compounds, which differ favorably in their toxicological characteristics (Ignatenko M.A. "Antitumor activity of organosilicon and organosilicon compounds" (review). Chemical Pharmaceutical Journal , 1987, No. 4, pp. 402-408).

 An increased interest in the synthesis and study of the biological activity of organo-germanium compounds is associated with their wide spectrum of action. They have antitumor activity, have a beneficial effect in cardiovascular diseases, chronic respiratory diseases, neuropsychiatric disorders, metabolic disorders, some forms of allergies, diseases of the kidneys, liver, digestive organs, hypertension and cataracts.

The closest analogue is a drug for the treatment and prevention of diseases of vertebrates, including humans, based on an organo germanium compound of the formula (GeCH ₂ CH ₂ COX) ₂ O ₃ , where X = OH, NH ₂ or O-alkyl. The mechanism of action of the drug is based on its ability to stimulate the formation in a living organism of an immunomodulator of gamma-interferon, the most important factor of nonspecific resistance of the body (US Patent No. 4279892, A 61 K 39/00, 1981). However, the synthesis technology of the compound is associated with great difficulties and costs.

 An object of the invention is to expand the spectrum of action of an immunomodulator based on an organo-germanium compound.

 The technical result of the invention consists in screening a non-toxic immunomodulator that is able to suppress the reproduction of a number of viruses (herpes, arboviruses) exhibiting antistaphylococcal, antitumor, radioprotective and anthelmintic activity, as well as being able to restore and improve the quality of hairline in animals.

The inventive drug "DINA" for the treatment and prevention of mammals, birds and reptiles belongs to the group of immunocorreters of synthetic origin based on organogermanium compounds, namely, it is an organomagnesium derivative of picolinic acid of the following formula C ₄₂ H ₂₄ N ₆ O ₂₁ Ge ₃ .

 The drug "DINA" is able to correct the immune system to a normal state on all its links: it has an immunomodulating effect on B- and T-systems of immunity, stimulates immunoreactivity to specific antigens, the functional activity of macrophages, causes the production of alpha, beta and gamma interferons and lymphokines.

 The drug "DINA" enhances the protective mechanisms of the body of mammals, birds and reptiles and contributes to the treatment and prevention of a number of infectious and viral diseases, for example, salmonellosis, colibacteriosis, pyroplasmosis, otitis media, pneumonia, adenovirus, infectious hepatitis, viral enteritis, panleukopenia, viral rhinotracheitis, plague carnivores etc. skin diseases of various etiologies, eye diseases, for example, blepharitis, conjunctivitis, keratitis, invasive diseases.

 In addition, the drug "DINA" can be successfully used as a repair agent for skin and tissue lesions, as well as an accelerator of hair restoration in places of post-infection and metabolic alopecia.

 The drug "DINA" also enhances the effect of antibiotics on pathogenic microorganisms, showing a synergistic effect, which allows to reduce their dose by 2 times, and in most cases to exclude the use of hormonal drugs.

 The drug "DINA" is compatible with vaccines, eliminates post-vaccination complications and provides lasting immunity for a year. The use of the drug "DINA" for young animals (2-3 months) allows you to overcome the "immunological hole" without vaccination for 3-6 months.

 It was also revealed (according to the results of caprological studies) that in animals with impaired functions of the organs of the gastrointestinal tract and organs of internal secretion, a noticeable improvement in their functioning was noted.

 In addition, the drug "DINA" allows you to synchronize the reproductive cycle and helps preserve the fetus.

 For domestic and industrial animals, DINA is also an excellent antimutagenic and sedative drug.

The drug "DINA" is not toxic: LD ₅₀ is more than 2000 mg / kg. It is rapidly excreted from the body, disintegrating into carbon dioxide, water and non-toxic substances.

The drug "DINA" is a water-soluble, white powder with a maximum solubility up to a concentration of 0.5%
The main method of administration of the drug "DINA" is subcutaneous injection. The duration of the therapeutic course is determined by the severity and neglect of the disease. On average, the duration of the course is 6-10 injections of 0.5 ml subcutaneously after 8-10 hours. As a preventive measure, with the risk of infection, a single injection of 1 ml of the drug. The drug can also be administered intramuscularly or orally.

 In the treatment of eye diseases, a 0.15% solution is used, 1 drop 2 times a day.

 For skin diseases and to improve the hairline, it is recommended to do 3 injections of 0.5 ml subcutaneously with an interval of 1 week. Hairline improvement occurs in 3-4 weeks.

 Contraindications to use have not been established.

The drug "DINA" is obtained on the basis of available raw materials: substituted pyridines and chloro- (alkoxy) germans in three stages at a temperature of 80-90 ^o C. The product is isolated by filtration and drying under vacuum.

 Example 1. The drug "DINA" is used in veterinary practice in the treatment and prevention of certain infectious, invasive and non-infectious diseases of dogs and cats.

In the treatment of infectious viral diseases: carnivore plague (catarrhal, skin, intestinal forms), parvovirus enteritis, infectious hepatitis and other dog diseases (more than 1,500 animals of different breeds and ages), DINA was used in complex symptomatic therapy from 3 to 6 doses per a course of treatment. Efficiency was 80-87% (percentage of cure). In the treatment of the nervous form of the plague (82 dogs), the effectiveness is 50-60%
In the treatment of feline panleukopenia and viral rhinotracheitis of cats (about 300 animals), the efficacy was 90% and 83%, respectively.

In the treatment of skin diseases (demodicosis, microsporia, trichophytosis, eczema, dermatitis, allopecia of various etiologies), the drug was used for more than 1000 animals (cats and dogs). The efficiency in this case was 90-98%
The use of the drug before vaccination significantly reduced the number of post-vaccination complications and provided a more reliable immunity.

The drug provided a fairly reliable immune defense of animals from infectious diseases and without the use of vaccines. So, from the group of unvaccinated dogs (15 animals), which according to a certain scheme were administered the drug from three months of age to a year, only 3 animals fell ill during the observation period (1 year). In the control group for this period, the incidence was 35-40%
Example 2. The drug "DINA" is used for various skin lesions in cats: dermatitis, eczema, allopecia, and others. Cats of various breeds and age groups were used in the experiment. Identified lesions were non-contagious and infectious pathologies. The drug had a stable high result in these diseases, as well as in metabolic disorders in the body of the animal.

 The course of treatment was 2-3 injections of the drug. After treatment, a positive result in 85-87% of cases was observed on days 17-24. The lesion sites were covered with wool and the general condition of the animal improved.

 Example 3. The drug "DINA" is used on various types of animals (calves, dogs, cats) for the treatment of such eye diseases as surface-purulent keratitis, conflicticulitis keratitis, catarrhal conjunctivitis, blepharitis.

The drug is administered subcutaneously and in the form of eye drops. Improvement usually occurs 3-4 days after the start of administration; the cure rate is, on average, 90%
Example 4. The drug "DINA" is used to treat reptiles of turtles, lizards, snakes.

 The drug increases the natural resistance of the organism of reptiles, has antiviral and antimicrobial effects.

 DINA is used to treat the following diseases: pneumonia, parvovirus enteritis, intestinal infections, staphylococcal infections, dermatitis of various etiologies, stomatitis and purulent abscesses.

 The drug is administered to reptiles subcutaneously or orally in 0.2 ml (3 drops) 1 time per day for 3-5 days.

 In case of a threat of stressful situations (during transportation, changing the conditions of detention, etc.), the drug is given 3 drops 1 time per day.

 To accelerate the healing of postoperative sutures, various skin lesions and dermatitis, lotions are made with the drug 2 times a day.

 The drug "DINA" showed a high therapeutic effect in the treatment of these diseases of reptiles.

 Example 5. The drug "DINA" is used to treat domestic (chickens, ducks, geese, etc.) and decorative (parrots, canaries, etc.) birds.

 The drug is used to treat the following bird diseases: colds, intestinal infections, metabolic disorders, plumage disorders, as well as for the prevention of poultry diseases, mainly young animals.

 The drug is given orally: poultry 3 drops, chickens and decorative birds 1-2 drops 2 times a day for 3-5 days.

 The drug can be used to prevent disease and reduce mortality in the event of an epidemic. For this purpose, produce a drink with a diluted solution of the drug: for 100 chickens (chicks) a solution of 5 ml of the drug in 0.5 l of water.

 Using the drug makes it possible to reduce the percentage of mortality of young animals by 1.5-2 times compared with the control. A large increase in the weight of young animals receiving the drug was also noted.

 Example 6. The determination of the antiviral activity of the drug "DINA" in relation to the herpes virus and cytomegalovirus is carried out on diploid cells of a human lung embryo (LEC).

LEC cells are cultured in 96-well plates in Eagle's medium with 10% fetal calf serum (seeded dose of 4 x 10 ⁶ cells per well). On the third day after the formation of the monolayer, the cells are treated with DINA at a concentration of 3 to 50 μg / ml, incubated for 1 h at 37 ^° C in the presence of 5% CO ₂ , after which the preparation is removed, the cells are washed with Hanks solution and added to each well 100 μl of 10-fold dilutions of either herpes simplex virus type 1 (HSV-1), for example, strain SG-16 in the initial titer of 10 ^5.5 -10 ^6.0 TCD ₅₀ , or cytomegalovirus (CMV), strain AD 169 initial titer 10 ⁸ -10 ^8.5 TCD ₅₀ .

On the 4th day for HSV-1 or on the 7-11th day for CMV, the titer of the virus is determined by titration according to the cytopathic effect (CPD ₅₀ ) on the monolayer of LEC cells; the wells with the virus infected, but not treated with the DYNA drug, are LEC cells.

Optimum results were obtained with 50 μg / ml of the drug (Table 1), while there was a decrease in HSV titer by 0.3 3.2 lg TCD _{50 / 0.1 ml} or CMV by 3.5-3.75 _lg TCD _{50/0, 1 ml} Thus, the DINA drug is able to inhibit the development of these viruses in cells, inhibiting their reproduction by 100-8000 times.

 Example 7. Determination of the antitumor effect of the drug "DINA" is carried out in vitro is carried out on a model of herpes-induced tumors on Syrian hamsters weighing 100 g, also examining the interferon status of the animal organism.

 The drug "DINA" is administered three times in 0.2 ml at a concentration of 5 mg / kg body weight 24 hours before the introduction of tumor cells, simultaneously with tumor cells and 6 days after inoculation of tumor cells.

 An antitumor and immunomodulating effect was found in a tumor induced by HSV-1 DNA in Syrian hamsters. Blood taken from hamsters after treatment with DINA showed a high titer of immune interferon.

 The duration of the latent period of the onset of tumors in the experimental group reached 37 days from the moment of injection of tumor cells compared with 12 days in the control. There was a difference in the size of the tumors compared to the control (the size of the tumors was reduced by a factor of 2–3) under the influence of the DYNA preparation.

 Under the influence of the DINA drug, the lifespan of tumor carrier hamsters has significantly increased. So by the 42nd day in the control only 25% of hamsters were alive, in the experimental group of animals, death by this time was not observed. By the 71st day after the introduction of tumor cells at 100% death of animals in the control, 50% of the animals survived in the experimental. In 25% of the animals of the experimental group, tumors did not occur until the 113th day after the implantation of tumors. In 15% of the animals treated with DINA, the tumors did not develop during the observation period (about 1 year) (Table 2).

 Thus, the drug "DINA" has inhibitory activity against viruses of the herpes groups both in vitro and in animals. In its tumor-inhibiting activity, it is not inferior to phage RNA and yeast RNA.

 The drug "DINA" can be recommended in complex therapy for cancer, the etiological factor of which may be the viruses of the herpes group.

Example 8. To determine the effectiveness of the drug "DINA" in a particularly dangerous infection of arboviruses of the virus of Western encephalomyelitis in horses (alphavirus infection), outbred mice are injected subcutaneously with 10-12 g of the drug in a volume of 0.1 ml in a single dose of 50 μg / kg of weight according to the scheme: 24 hours, simultaneously and 24 hours after infection with 10 LD _{50 of the} virus. The course dose of DYNA is 150 mcg / kg of body weight. The virus is administered intramuscularly in a volume of 0.1 ml. The results are presented in table. 3.

 The effectiveness of the drug "DINA" is not inferior to the well-known inducer of interferon ridostin. The drug "DINA" has a wide spectrum of action, is active in acute and physiological routes of administration, is clinically promising for chemotherapy of neuroviral infections caused by alphaviruses (ZEL), which is especially important, since there are no specific prophylaxis against infections caused by ZEL and treatment.

 Example 9. The interferon-inducing activity of the drug "DINA" is determined on CBA mice with oral, intraperitoneal, intramuscular and subcutaneous injections in an optimal dose of 0.5 mg / kg The level of synthesis of interferon (IFN) is determined in blood serum at various time intervals after the administration of the drug "DINA" (from 5 to 72 hours). IFN peak) is observed 48 hours after drug administration. IFN is most actively synthesized by oral and subcutaneous administration. With the oral administration of the drug "DINA", the peak of IFN activity is shifted to 24 hours.

 A study of the degree of involvement of immunocytes in the production of IFN allowed us to establish that thymocytes are the least active in synthesizing IFN for all routes of administration. With the oral and intraperitoneal route of administration, IFN cells of bone marrow and splenocytes are synthesized, with intramuscular splenocytes, with subcutaneous bone marrow cells and thymocytes.

 Example 10. The determination of the antiviral activity of the drug "DINA" in relation to bunyavirus infections: Rift Valley fever (LDR), Issyk-Kul fever and Crimean hemorrhagic fever (CHF) is carried out on outbred white mice weighing 15-20 g.

 The drug "DINA" has a pronounced protective effect against experimental infections of white mice LDR (80%), CHF (70%) and Issyk-Kul fever (85%).

 The drug is active in the oral, subcutaneous and intramuscular routes of administration at a concentration of 50 μg / mouse. The greatest efficiency is observed with preventive or preventive regimens.

Example 11. The study of the drug "DINA" for resistance to staphylococcal infection is carried out on CBA mice with subcutaneous administration in the dose range from 0.1 to 10 μg per mouse. After administration of the DYNA preparation, animals are infected with 1.25 • 10 ⁹ Staph microbial cells. aureus strain Wood 46 and determine the survival rate of animals as a percentage of the survival rate of mice in the control group to which the drug is not administered. The results are presented in table. 4.

 The experimental results indicate that the drug "DINA" in the dose range from 0.1 to 10 μg per mouse cause an increase in non-specific resistance to staphylococcus. The stimulating effect is most pronounced when using the smallest of the tested doses of 0.1 μg per mouse (stimulation index 2.56).

 Example 12. The drug "DINA" affects the functional activity of macrophages in many ways: chemotaxis, severity of the lysosomal apparatus, secretion of protein products, phagocytic function.

Dynamic experimental studies on mice of CBA and C ₅₇ B1 / 6 lines show that this increase is observed already 15 minutes after intraperitoneal administration of the drug at a dose of 20 μg and lasts from 1 to 3 days.

 The urgent nature of the effect of the DYNA drug allows it to be used in cases where it is urgent to strengthen the nonspecific defense link of the body, for example, with some viral and other infectious diseases.

 Example 13. The drug "DINA" is used to treat infectious and viral diseases of foxes, minks and Arctic foxes, mainly orally, in severe cases, in the form of injections. The course of treatment is 3-6 doses of a 0.15% solution. For prevention, only 1 dose of the drug is enough.

 The use of the drug "DINA" increases the vitality of young animals and improves the quality of the fur.

 Example 14. The drug "DINA" is used to treat gastrointestinal and respiratory diseases of cattle.

 The test of the drug is carried out on groups of animals selected by type of analogues (with control setting).

 A group of animals (40 calves) where the drug is not used (control group) is taken as a base for comparison.

 The drug "DINA" is tested with medical (20 calves) and prophylactic purposes (60 calves) in cattle (age group 5 days 2 months). In sick animals, clinical signs characteristic of respiratory and gastrointestinal diseases were observed.

 Before using the drug, a clinical examination of the animals is carried out according to the scheme: temperature, pulse, respiration, condition of individual systems. A clinical examination according to the full scheme is carried out once every five days throughout the entire experiment. The duration of observations of the experimental and control groups is two months.

 When determining the therapeutic efficacy of the DINA preparation, the duration of clinical symptoms (in comparison with the control group), the number of required treatments, and the rate of recovery of sick animals are assessed.

 The therapeutic efficacy of the DINA preparation in animals with clinical symptoms characteristic of mixed respiratory infections was 74%. The treatment course was 5-6 injections.

In animals with clinical symptoms characteristic of gastrointestinal infections (colibacteriosis, salmonellosis), 84%
The therapeutic course depends on the severity of the course and form of the manifestation of the disease and is on average 5-6 days.

The drug "DINA" is recommended for the prevention and treatment of respiratory and gastrointestinal diseases of calves of different age groups in combination with other specific and symptomatic agents. Its use leads to recovery of animals on average by 5 days, reduces the use of antibiotics by 50%
In the group of animals that received a prophylactic dose of DINA (0.5 ml, twice) and for two months (observation period), no diseases were noted. The condition of the animals is good.

Claims (4)

1. A preparation for the treatment and prevention of diseases of mammals, birds and reptiles based on an organo-germanium compound, characterized in that it contains an organo-germanium compound, a picolinic acid derivative of the formula C ₄₂ H ₂₄ N ₆ O ₂₁ Ge ₃ , characterized by immunomodulating, antiviral, antibacterial, anthelmintic, radioprotective and antitumor activity.  2. The drug under item 1, characterized in that it is characterized by anti-herpes activity.  3. The drug according to claim 1, characterized in that it is characterized by antistaphylococcal activity.  4. The drug under item 1, characterized in that it is characterized by the ability to restore the hair of mammals.

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