RU2235550C2 - Medicinal agent with bionormalizing effect and method for its preparing - Google Patents

Abstract

FIELD: medicine, medicinal industry.

SUBSTANCE: invention relates to preparing medicinal agents with bionormalizing effect based on humans or agricultural animals placental tissues. Placental tissue is milled, washed out from blood, chlorous acid solution is added to suspension in the ratio tissue/chlorous acid = 1:(0.1-1.5) by mass and modifying additives are added also that provide selectivity of process and oxidative-hydrolytic modification of placental tissues is carried out at temperature 18-45^oC. Medicinal agent comprises polypeptides in the amount 3.5-7.0 wt.-%, amino acids in the amount 50-60 wt.-%, aminosaccharides in the amount 4-5 wt.-% and hexuronic acids in the amount 8-9 wt.-% wherein indicated products are on oxidized water-soluble form, and polysaccharides and inorganic acids, the balance. Invention provides enhancing bionormalizing effect due to development of medicinal agent with high degree of oxidation and stability of properties.

EFFECT: improved preparing method, valuable medicinal properties of agent.

4 cl, 11 ex

Description

The invention relates to medicine and the medical industry, in particular to the field of obtaining drugs with bio-normalizing action based on placental tissues.

The bio-normalizing effect of a drug is understood to mean its ability, when used in medicine for therapeutic and prophylactic purposes, to eliminate or prevent the causes of the pathological state of the human and animal body and thereby provide a solution to the following problems:

- achieving a positive therapeutic effect (complete recovery) or persistent remission, regardless of the type and cause of the pathology without undesirable side effects;

- reducing treatment time and ensuring guaranteed improvement in health status and prolonging an active lifestyle;

- prevention of premature development of diseases associated with work in harmful working conditions, stress, physical, thermal and other overloads due to the preventive use of the drug;

- solution to the gerontological problem.

Almost all currently known drugs belong to the category of symptomatic drugs, i.e. to one degree or another, they eliminate individual symptoms of the disease, such as fever, arterial hypertension, runny nose, cough, etc. This is most characteristic in the treatment of chronic and especially chronically advanced pathologies, such as diseases of the musculoskeletal system (osteoarthritis, polyarthritis, Ankylosing spondylitis), as well as various diseases resulting from traumatic effects on the fetus during childbirth (cerebral palsy), past injuries of the skull, fractures of the extremities and the spine.

Of particular importance is the problem of treating diseases such as strokes, heart attacks, gunshot wounds. In the same category should be attributed diseases associated with immunodeficiency (chronic inflammatory processes of various organs and tissues, especially urogenital), oncological processes, etc.

The use of known drugs in the above and many other pathological processes provides, at best, the elimination of certain symptoms of the disease, which, as a rule, is temporary. It is known that all drugs belonging to the class of xenobiotics, exhibiting a therapeutic effect, simultaneously have a toxic effect on other systems or organs. The final balance of such treatment is zero or negative. This is due to the fact that with the help of well-known drugs in most cases it is impossible to eliminate the cause of the disease - a pathological condition that is the result of metabolic disturbance for one reason or another (metabolism and energy between the cell and the environment).

No less important is the issue of prevention of such diseases, because with the help of well-known drugs, it is not yet possible to develop a radical system for prolonging a person’s life in an active form.

There are known drugs of a protein nature such as interleukins, neuropeptides and others, obtained by genetic engineering or isolated from various substrates of the human body using affinity chromatography and clonal antibodies, which to one degree or another can be attributed to drugs with bio-normalizing effects (Status and prospects of drug development using biotechnology methods in capitalist countries: Overview in the collection "Economics in the medical and microbiological industry . Con- cern - M .: VNIISENTI NGO "Medbioekonomika", 1989, №1 / 2, 84) However, these drugs have a very significant drawbacks, namely:..

- laborious and very complex production technology and, accordingly, unreasonably high costs;

- rapid exo-and especially endogenous inactivation of the drug, as a result of which there is a short-term pharmacological effect and the appearance of undesirable side effects.

Closest to the claimed drug is a bio-normalizing effect, obtained on the basis of placental tissues and described in the patent of the Russian Federation No. 2036650 from 06/09/1995,

However, the drug according to the indicated copyright certificates, called Amniocene, is characterized by undesirable side effects, as a result of which, for example, it was excluded from the register of medicines of the Russian Federation in 2000.

The basis of the present invention is, in particular, the task of creating a medicinal product with bio-normalizing action containing products obtained from placental tissues, with a high degree of oxidation and stability of properties.

The problem posed above is solved by the fact that the composition of the drug bio-normalizing action containing products obtained from placental tissues includes polypeptides in an amount of 3.5-7.0 wt.%, Amino acids in an amount of 50-60 wt.%, Aminosaccharides in an amount 4-5 wt.% And hexuronic acids in an amount of 8-9 wt.%, Moreover, these products are in oxidized water-soluble form, the rest are polysaccharides and inorganic salts.

These products are water-soluble macromolecule precursors obtained by oxidative-hydrolytic modification of placenta biopolymers with a higher degree of oxidation than the natural products of biochemical reactions taking place in the human body. Oxidative-hydrolytic modification of placenta biopolymers means nothing more than their transfer to a metabolic state. The degree of oxidation of the products of modification of placental tissue biopolymers by oxygen, determined by calculation and analytical method, is 2 times higher than that of natural endoregulators formed in the human body due to oxidation of connective tissue proteins by prolingidoxylase or lysine hydroxylase enzymes and subsequent hydrolysis. And taking into account the formation of nitrogen-chlorine bonds, the oxidation state increases by more than 3 times. As a result, the claimed drug has a high bio-normalizing effect or, in other words, is an effective bio-normalizing agent.

A further improvement of the medicinal product in accordance with the invention is that the placenta tissues use the afterbirths of humans and farm animals. This allows you to significantly expand the scope of this drug and its volume of release.

High pharmacotherapeutic efficacy of the claimed drug as a bionormalizer has been established in studies on laboratory animals (mice, rats, rabbits, dogs), in the course of clinical trials in humans and as a result of postclinical use. The following are the results before the clinical, clinical and postclinical use of the claimed drug.

Immunotropic effect

Evaluation of the immunotropic activity of the claimed drug is determined by standard methods recommended by the World Health Organization (WHO). Specific pharmacological (immunological) activity is tested in model systems that quantitatively characterize the effect of the test drug on the main links of immunity. The experiments were performed on guinea pigs, Wistar rats, outbred mice, and CBA, C57B1 (CBAXC57B1) G ₁ mice. The effect of the claimed preparation on the cellular and humoral immune response (phagocytic activity, effect on the outcome of a generalized Pseudomonas infection, on the complement system, on the cooperation of T and B lymphocytes, on the transplant versus host reaction, on the delayed-type hypersensitivity reaction, on the humoral answer). As reference drugs, well-known drugs were used, such as levamisole, T-activin, cyclophosphamide, sodium nucleate, amniocene, and pyrogenal.

According to the test results, the inventive object significantly exceeds the above drugs in activity and only in some tests shows the same activity as T-activin. The most characteristic indicator characterizing the immunotropic (immunomoduliruyus) activity of any drug is the assessment of its effect on the delayed-type hypersensitivity reaction (HRT). In experiments on the induction of HRT in sublethally irradiated mice (ionizing radiation significantly violates the immunological competence of the body, inhibiting the manifestation of HRT), the claimed drug was administered subcutaneously 5 times in a total dose of 4.0 and 6.0 mg / kg, starting from the day after irradiation. As can be seen from the table below, treatment of animals at a dose of 4 mg / kg increases the hypersensitivity of the reaction by 30% with respect to the irradiated mice of the control group. At a dose of 6.0 mg / kg, the reaction index almost reaches the level of a group of unirradiated animals, which proves the possibility of protecting the body under radiation exposure.

Chondroprotective effect

Known chondroprotective drugs, such as rumalon, mucartrin and arteparone, were chosen as comparison preparations for assessing the activity of the claimed preparation by the ability to restore cartilage. Chondroprotective properties were evaluated using a standard model of post-traumatic arthrosis of the hip joint in rats, which develops after applying a defect in the form of a round hole with a diameter of about 2 mm passing through the articular cartilage into the subchondral region. The application of such a defect leads to the development of post-traumatic arthrosis. To test the activity of the claimed drug and comparison drugs, the following doses were selected: 0.01; 0.03; 0.06; 0.1; 0.3; 0.5; 1.0 ml / kg of animal weight. Evaluation of the effectiveness of chondroprotective action was carried out on a specially developed scale (in points). The maximum score is 12 points, which corresponds to the complete resorption of connective tissue and restoration of cartilage. Scoring results:

Rumalon 7.6

Mukartrin 8.4

Arteparon 8.9

The inventive drug 11.8

Anti-inflammatory effect

This type of action was tested on a model of experimental granuloma in rats. As comparison drugs, drugs obtained on the basis of human placenta — PDS (denatured suspended placenta) and amniocene — were used. Drugs were administered subcutaneously at the same time as the operation. The test results are presented in table.2.

Absorbable action

The absorbable effect has been studied on the model of an adhesion process that occurs in non-pregnant female rats as a result of a dosed injury to the uterine horn. The results of the experiments are presented in table.3.

Reparative action

This type of pharmacological action is established on a standard model with a full-layer skin defect in mice, which were administered the claimed drug in doses of 5-10 mg / kg. Under the influence of the drug, the healing time compared with the control is reduced from 17 days to 10. A similar effect is exerted by the drug from the placenta amniocene only at a dose of 75 mg / kg. In this case, a significant advantage of the use of the claimed drug is the absence of scars in the healing process.

Antioxidant effect

The antioxidant effect of the claimed drug was established during lipid peroxidation, the effectiveness of which exceeds almost 10 times ascorbic acid. This fact is essential in the practical use of the claimed drug in oncology.

Fertile action

In the process of studying the inventive preparation for mutagenic properties (on the model of dominant lethal mutations), an increase in the fertility and fecundity of experimental animals was detected compared to the control. At the stage of mature sperm, a significant decrease in post-implantation death is observed, which is in good agreement with published data on the antimutagenic effect of preparations from the placenta.

Adaptogenic action

This type of action in the process of creating new medicines acquires one of the most relevant areas of search, because this can make it possible to influence physiological processes during physical, thermal, chemical overloads, especially to prevent poisoning, burns, increase body endurance, etc. In the process of studying the inventive preparation, it was found that preliminary treatment of animals (mice) for 7 days in a conditionally therapeutic dose (0.1 ml / kg) prevents the death of animals from a toxic dose of toxic substances (strychnine), lethal exposure, Pseudomonas aeruginosa infection.

Anti-stress action

The anti-stress effect of the claimed drug was studied on the basis of multidimensional methods for assessing the state of various components of resistance to emotional stress in rats (behavioral and visceral components). As a result of these studies, it was found that the claimed drug has the ability to increase resistance to emotional stress, i.e. to prevent the development of functional disorders of higher nervous activity, general immunity and metabolic disorders of cardiac activity. The most effective dose was 30 μl per 100 g of rat mass, which corresponds to a dose of 2 ml per 1 injection in humans.

Lactogenic action

In the process of practical use of the inventive preparation in veterinary medicine, it has been established that during agalactia in sows, the inventive object leads to the restoration of the lactation process.

Anticoagulant effect

In the process of studying the degree of influence of the claimed drug on blood coagulability, it was found that at a dose of 1.5 mg / kg with a monthly injection, an anticoagulant effect appears, which disappears 30 days after the end of the drug administration. This fact can be of significant practical importance in the treatment of preinfarction and postinfarction conditions.

The claimed drug is fully investigated for acute and chronic toxicity in accordance with the methods recommended by WHO.

Acute toxicity

Testing of the claimed drug for acute toxicity was carried out in mice by a gradual (double) increase in the dose of the drug, starting from 10 mg / kg of animal weight to 100 g / kg. At the maximum permissible dose (100 g per 1 kg of weight), the animals do not die, which proves the absolute harmlessness of the drug. At high doses exceeding the conditionally therapeutic by 5,000 times, of course, animals exhibit physical inactivity, apathy, lethargy, but restore their activity after 3-4 days without signs of poisoning.

Chronic toxicity

A study of the chronic toxicity of the claimed drug was carried out in accordance with WHO recommendations in three groups of animals (rats, rabbits and dogs) during a 6-month daily administration of the drug in doses exceeding the conditionally therapeutic 10, 20, 50 and 100 times. After a 6-month administration of the drug, 50% of the animals were killed to assess the degree of influence of the claimed drug on organs and tissues, and the rest of the animals were rehabilitated for 1 month and then again killed for a comparative assessment of the pathological effect of the drug.

As a result of a study of the internal organs of animals, after a 6-month administration of drugs in the above doses, none of the animals died. The detected changes in the lungs, brain and kidneys after a month of rehabilitation were fully restored, which confirms the safety of the claimed drug (high bioavailability and biocompatibility).

Clinical trial results

Clinical trials of the claimed drug were carried out in three stages. The first phase is volunteers (30 people) who were intramuscularly injected with a 2% solution of the claimed drug in water for 10 days. Toxic effects and other side effects have not been established. The second phase (90 people) - determination of the therapeutic dose when using the drug as an anti-inflammatory and analgesic for osteoarthritis (as one of the most promising areas of the biological effect of the drug).

The third phase is a study conducted in three clinics in Moscow with a total of 156 patients (men and women aged 18 to 78 years) with a diagnosis of osteoarthritis of various etiologies and severity. The main clinical manifestation of osteoarthritis is pain, the assessment of which was carried out on a specially developed scale in points on a 5-point system, both before treatment and after treatment. The inventive drug was administered parenterally and intraarticularly, a dose of 2 ml, first every other day (10 injections), then daily (20 injections). Each patient received 60 ml of the drug. A subjective assessment of the effectiveness of the use showed that in 58 people the pains significantly decreased (37%), in 42 people (27%) they completely disappeared, in 33 people (21%) the pains completely disappeared in the knee joints and significantly decreased in the hip joints. All patients increased range of motion, including when walking up the stairs. In 12 patients admitted to the hospital on crutches, it became possible to move independently. Side effects during the trial have not been established.

Postclinical trials of the claimed drug showed a high effect in many pathological conditions, which proves its bio-normalizing effect. The following are examples of such tests.

Example 1

Patient L. 33 years. The diagnosis is the consequences of cerebral palsy (spasticity of the right hand and entire arm, physical inactivity). After treatment with the claimed drug for one year, in combination with physiotherapeutic methods, also using the claimed drug, almost complete recovery has been achieved. The patient began to write, peel potatoes and perform other small movements with high accuracy. He started to run, swim. The use of known drugs over the past 30 years has not yielded positive results.

Example 2. The use of the claimed drug in children and adolescent neuropsychiatric practice

The therapeutic correction of mental and neurological syndromes formed as a result of an early organic lesion of the central nervous system, closed craniocerebral trauma and neuroinfection in children and adolescents remained a difficult to resolve problem before using the claimed drug. When using the claimed preparation for these purposes within 25-30 days in combination with physiotherapeutic methods, the Ukrainian Research Institute for the Protection of the Health of Children and Adolescents achieved a high persistent therapeutic effect (95.3%). The remaining patients showed a significant improvement in their health status.

Example 3. The use of the claimed drug for toxoplasmosis

Toxoplasmosis is a zootropic parasitic disease characterized by a polymorphic clinical picture, latent, acute and chronic course of an acquired or congenital infection. Toxoplasmosis, as a rule, is not amenable to effective treatment, and the disease becomes protracted chronic. The use of the claimed drug in the treatment of toxoplasmosis according to the company "Bacteria" at the Dnepropetrovsk Medical Academy has proven high pharmacotherapeutic efficacy, characterized in most cases by complete recovery.

Example 4. The use of the claimed drug for chronic cerebrovascular accidents

According to the results of the treatment of patients with a diagnosis of stroke, regardless of the prescription, received at Polyclinic No. 3, Kharkov, a complete resorption of the brain hematoma, the resulting adhesions and other pathological defects was established. Ultimately, the restoration of lost motor function and speech. The duration of treatment depended on the severity and prescription of the pathological condition.

Example 5. The effectiveness of the treatment of urogenital infections

The use of the claimed drug in urological practice in the treatment of diseases such as chlamydia, uroplasmosis and others, especially when combined, gives a high therapeutic effect. According to the results obtained in the urological clinic at the Kharkov Medical University, a positive effect was achieved in 94.7% of cases.

Example 6. The use of the claimed drug in cardiology

More than 200 patients with cardiovascular insufficiency (post-infarction condition, angina pectoris, myocarditis, etc.) were treated at the clinic number 6 in Kharkov. In the vast majority of patients, a stable therapeutic effect was obtained, especially with angina pectoris and post-infarction state.

Example 7. The use of the claimed drug in the treatment of injuries and diseases of the musculoskeletal system

According to the results of the treatment of patients with diseases of the musculoskeletal system of various etiologies conducted on the basis of the clinic number 6 in Kharkov, a positive effect was achieved in almost 100% of cases.

Example 8. The use of the claimed drug in gynecological practice

The high effectiveness of the treatment of gynecological diseases such as endometritis, diffusion fibromyomas, adnexitis, polycystosis, inflammation of the appendages, etc. is shown on the results of its use in gynecological institutions of Kharkov.

The high efficiency of the claimed drug is also established in oncology, ophthalmology, surgery and other branches of medicine. The drug is approved for medical use.

Known methods for producing drugs based on placental products in an oxidized state. By the method described in A.S. USSR No. 948001, the placenta is treated with chlorine. By the method described in A.S. USSR No. 1392684, processing is carried out with chlorine dioxide.

Closest to the claimed method for producing a medicinal product with bio-normalizing action is the method according to the patent of the Russian Federation No. 2036650, publ. 06/09/95, bull. No. 16, according to which the oxidation of ground placenta is performed with sodium chlorite in a hydrochloric acid medium.

However, all these methods of processing the placenta do not provide a bio-normalizing effect of the resulting preparation due to the relatively low oxidation state of its constituent products and the constantly changing composition of the finished product, because the oxidation process is uncontrollable.

The basis of the present invention regarding the method of obtaining the described drug with a bio-normalizing effect is the task of improving the known method so that by introducing new reagents and new conditions for its implementation to achieve a higher degree of oxidation of the finished product with a stable composition and thereby obtain a drug with bio-normalizing action.

The problem is solved in that the known method, including the oxidative-hydrolytic modification of placental tissues with oxygen compounds of chlorine, is improved as follows: the process of oxidative-hydrolytic modification is carried out with chloride acid in the presence of modifying additives.

These modifying additives provide the necessary selectivity of the oxidation-hydrolytic modification process. This makes it possible to obtain a drug with the required degree of oxidation of the ingredients due to the formation of carbon – oxygen, nitrogen – oxygen, sulfur – oxygen, and nitrogen – chlorine bonds by substituting a hydrogen atom in the amide group (peptide bond) or in the free amino group.

In a specific embodiment of the method, the modification process is carried out at a tissue / chloride acid ratio of 1: (0.1-1.5) at a temperature of 18-45 ° C.

In yet another specific embodiment of the process according to the invention, organic compounds of nickel or cobalt are used as modifying additives.

The duration of the processing process to the desired degree of oxidation (in terms of oxygen and chlorine) depends on temperature and ranges from 2 to 40-45 days.

The following are examples of specific performance of the proposed method.

Example 9

The placenta, prepared in maternity hospitals in frozen form, is crushed, washed from blood, the resulting suspension is loaded into a glass reactor equipped with a stirrer and a coil for heating or cooling, add the pre-prepared mixture in equivalent amounts of the sodium salt of an amino acid (preferably lysine) and a chloride modifier cobalt or nickel - in a tissue / modifier mass ratio of 1: (0.001-0.1), then a 5% chloride solution is loaded at a tissue / acid mass ratio of 1: (0.1-1.5) and incubated in t chenie 1-45 days prior to the desired degree of oxidation. The oxidation state is determined by the percentage mass content of oxygen and chlorine in the reaction products and compared with the same indicators in the initial product. The content by weight of oxygen in the finished product compared to the feedstock should be 1.8-2.1 times more. The content of total chlorine - free and organically bound - should be in the range of 8.0-20 wt.% In terms of dry matter.

The polypeptide fraction in the form of an aqueous solution is separated from the precipitate, subjected to purification by filtration, neutralized with an alkaline agent and transferred to an injectable dosage form by known methods.

Example 10

Under the conditions of Example 9, sodium chlorite is used instead of hydrochloric acid in the presence of an equivalent amount of hydrochloric acid. The main active substance (oxidizing agent) in this case is chloride, which is released during the processing of sodium chloride with hydrochloric acid. The ratio of the oxidizing agent / tissue of the placenta by weight in terms of sodium chloride 1: (0.15-1.8). The target product is isolated analogously to example 9. In this case, just cobalt chloride in the same proportions is used as a modifying additive.

Example 11

In the conditions of example 9, the processing of crushed placental tissues is carried out with chlorine dioxide at a tissue / chlorine dioxide ratio of 1: (0.1-0.9) by weight. The active substance in this case is also chloride, which is formed by the interaction of ClO ₂ with water. As a modifying additive, in this case, a mixture of the sodium salt of lysine and cobalt chloride is preferable. The selection of the target product is carried out in a similar manner.

Claims (4)

1. The drug has a bio-normalizing effect, characterized in that it contains polypeptides in an amount of 3.5-7.0 wt.%, Amino acids in an amount of 50-60 wt.%, Amino saccharides in an amount of 4-5 wt.% And hexuronic acids in an amount of 8-9 wt.%, and these products are in oxidized water-soluble form, the rest are polysaccharides and inorganic acids. 2. A method of obtaining a medicinal product with a bio-normalizing effect, characterized in that the placental tissue is crushed, washed from the blood, a solution of hydrochloric acid is added to the suspension at a tissue / chloride acid weight ratio of 1: (0.1-1.5) and modifying additives, ensuring the selectivity of the process, and the oxidative-hydrolytic modification of placental tissues is carried out at a temperature of 18-45 ° C. 3. The method according to claim 2, in which, as modifying additives, cobalt or nickel compounds are used in a tissue / modifier mass ratio of 1: (0.001-0.1). 4. The method according to claim 2 or 3, in which the placenta tissue uses the afterbirth of a person or farm animals.