RU2058786C1 - Antiteratogenic agent - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves the use of biopreparation of the known culture of plant tissue Poliscias filicifolia (plant adaptogene of Aralium family) as an antiteratogenic agent. Preparation shows antioxidant properties that normalize enzyme activity of antioxidant protection: catalase, superoxide dismutase, ceruloplasmin, the level of lipid peroxidation and also - enzymes of energetic metabolism: creatine kinase and enolase isoform in embryo brain in the process of alcoholic syndrome development in fetus. Proposed preparation ensures to provide necessary antiembryotoxic level of antiteratogenic agent. EFFECT: increased effectiveness of agent proposed. 2 cl, 6 tbl

Description

 The invention relates to medicine, namely to the production of medicines from plant materials, and can be used as an anti-teratogenic pharmacological agent that reduces or prevents the risk of developmental anomalies induced by toxic effects, in particular, alcohol intoxication during pregnancy.

 From publicly available literature, there is no evidence of a targeted search for anti-teratogenic agents, although with the complication of environmental problems, there is an increase in various developmental anomalies, including various congenital disorders of the higher nervous activity of a person. The frequency of congenital pathology and early perinatal death in our country has reached unprecedented limits. In this regard, it is necessary to develop pharmacological agents of anti-teratogens that protect the antenatal (intrauterine) development of a person from teratogenic factors of the environment, including industrial and domestic hazards.

 From the literature, isolated evidence of the antiterratogenic effect of various chemical compounds is known. In particular, cystine and glutathione significantly reduce the teratogenic effect of such an alkylating compound as cyclophosphamide.

 However, cystine prevents mainly anomalies of the extremities, while malformations of the central nervous system (CNS) persisted. Glutathione prevented both malformations of the extremities and CNS abnormalities, however, its protective effect was effective only at high doses of glutathione 400 mg / kg, which in principle excludes the possibility of its use as an anti-teratogenic agent for treating humans.

 It is also known to use sodium selenite, which is more effective than cystine and glutathione as an anti-teratogen with respect to the same model teratogen cyclophosphamide.

 Sodium selenite also effectively prevents the teratogenic effect of salts of certain heavy metals. Being an irreplaceable microelement, selenium is really necessary as a protective agent against a number of teratogenic effects, which is why an adequate content of this microelement in the diet of pregnant women is necessary.

 However, using it as a drug to prevent deformities is unsafe, since the toxic and therapeutic doses of this element are very close, which is why toxic effects of selenium from an overdose of this compound are very likely.

 As you know, significant success in reducing the frequency of congenital malformations of the central nervous system was achieved using a complex of vitamins in the first trimester of pregnancy. Efficiency in this case is achieved by removing vitamin deficiencies, which are a risk factor in the development of the nervous system. Obviously, the assertion that the corresponding complex of vitamins should become a routine preventive measure during pregnancy, especially in the early stages, does not cause objections. However, as is the prevention of any other nutritional deficiency.

 However, experimental and epidemiological data show that an adequate diet, being one of the factors for increasing the nonspecific resistance of an organism, still does not prevent embryogenesis from the pathogenic action of a significant number of chemical compounds with a wide spectrum of damaging effects: alkylating compounds, nucleic acid synthesis inhibitors, and compounds inducing lipid peroxidation, etc. One of these compounds, leading to the development of a characteristic deformity syndrome, called "fetal alcohol syndrome", is alcohol (ethyl alcohol). Ethyl alcohol metabolizes to toxic (acetaldehyde, superoxide radicals, etc.) and pharmacologically active (β-carbolines, tetrahydroisoquinolines, etc.) compounds, each of which has significant teratogenic activity, causing not only morphological malformations, but also deep lesions of higher nervous activity, up to moronity. Therefore, reducing the teratogenic potential of ethyl alcohol, being a socially significant task, is also important from a general biological standpoint as the search for pharmacological agents that prevent the teratogenic effect of various xenobiotics (unnatural synthetic agents), including industrial hazards and drugs. At the same time, the use of chemotherapeutic agents to reduce the toxic effects of alcohol during pregnancy is excluded, since synthetic agents can only enhance the teratogenic potential of ethyl alcohol. An example of this is the use of disulfiram in pregnant women with alcoholism, leading to an increase in teratogenic risk.

 Natural remedies are also known that reduce the toxic effect of ethyl alcohol due to their inherent antioxidant properties: ginseng preparations (Panax ginseng), aralia and eleutherococcus (Eleuterococcus).

 The last representative of the Aralievs in the experiment significantly reduced the teratogenic potential of ethyl alcohol, and therefore is considered as the closest analogue that solves the same problem as the new object used.

 However, the raw material base for obtaining the drug from the roots of Eleutherococcus is very limited due to both the limited distribution area and the depletion of its natural resources. The aforementioned fundamental restrictions on the use of araliaevs as agents that reduce alcohol intoxication (and possibly also as anti-teratogenic agents with a wide spectrum of action due to the adaptive properties inherent in these plants) can be overcome by obtaining plant material in an industrial way by growing tissue cultures, as well as by finding other representatives of the Araliaceae with similar properties, but broader in distribution.

 It is known to use the biomass of ginseng, obtained by growing it in a tissue culture, to mobilize the body's defenses.

 The aim of the invention is to provide the required level of anti-embryotoxic effect of an anti-teratogenic agent based on natural compounds with respect to teratogenic effects while expanding its raw material base to obtain an anti-teratogen.

 This is achieved by using a biological product (known) of the tissue culture of the plant Poliscias filicifolia (plant adaptogen of the aralia family) as an anti-teratogenic agent.

 The plant adaptogen of the Araliaceae family Poliscias filicifolia (polysacias fern) is a tropical shrub that grows in southern Vietnam, Thailand, the Philippines, and New Zealand. Despite the fact that the tropics and subtropics are the habitat of the Poliscias genus, the biomass of Poliscias filicifolia can be obtained in significant quantities by the methods of plant tissue culture (like the ginseng biomass).

 In the Vietnamese Pharmacopoeia and folk medicine of the East, the use of the leaves and roots of Poliscias filicifolia as a moderate cardiotropic and psychostimulating agent, as well as a means of normalizing high and low blood pressure, is described.

 The biopreparation of plant tissue culture Poliscias filicifolia (poliscias) is currently being obtained at the Biopreparations Plant in Kirishi and at the Prenatal Safety Joint Venture in St. Petersburg, as well as at the Sanitas Experimental Production Plant in Lithuania. The passport of one of the poliscias strains obtained at the Chemical and Pharmaceutical Institute of St. Petersburg was transferred to the All-Union Collection of Higher Plant Cell Culture and approved by the All-Union Commission on Cell Cultures of the USSR Academy of Sciences.

 The use of a biopreparation of polyscias plant tissue culture as an anti-teratogenic agent was made possible thanks to the first anti-embryotoxic and antioxidant properties found in this preparation that normalize impaired activity of antioxidant protection enzymes: ceruloplasmin, superoxide dismutase and the level of lipid peroxidation (lipid peroxidation) and isoform enzymes: isoforms embryo in the development of fetal alcohol syndrome (ASP). This allows you to get a domestic anti-teratogenic drug with the necessary anti-embryotoxic level based on plant materials with the complete absence of a natural plant growing area in Russia.

 A comparison of the characteristics characterizing a biological product of a known culture of plant tissue polyscias in its new and known application shows that the proposed drug is not a simple derivative of the leaves or roots of an intact plant used in folk medicine for a known purpose, and therefore the new purpose of the biological product is not a direct consequence of the known properties of this plant or other representatives of the aralian family.

 As follows from the foregoing, the proposed object of the invention in comparison with the closest analogue of the same application of Eleutherococcus (namely: biomass tissue culture polyscias and extract from the roots of the plant Eleutherococcus) have a different set of essential features. The use of biologics with antiseratogenic properties in public patent and scientific literature is not described, and therefore, the claimed invention meets the criterion of "novelty."

 Based on the well-known use of the roots and leaves of the poliscias plant, it can be established that the provision of anti-embryotoxic and antioxidant protection of the fetus during pregnancy is a truly new purpose of tissue extracts of this plant along with their use to stimulate the central nervous and cardiovascular systems and, therefore, the known properties of tissues Plants do not clearly determine the possibility of using biomass polyscias obtained in tissue culture as an anti-teratogenic agent. Islands to protect the human fetus from toxic effects of alcohol and other toxic substances. Thus, the new purpose of the claimed object is not due to the known properties of tissues of an intact plant, and therefore, this invention does not follow explicitly from the prior art and meets the criterion of "inventive step".

 The use of this invention in obstetric practice for prenatal protection of the human fetus, as well as its wider use as a means of preventing alcohol intoxication, allows us to conclude that the invention meets the criterion of "industrial applicability".

 The present invention was tested in laboratory conditions of the Research Institute of Experimental Medicine RAMS and JV "Prenatal Safety".

The standard method of preparation of the biopreparation polyscias is the three-fold extraction of dried biomass with a 40% ethanol solution. Raw biomass 90% consists of parenchymal cells ranging in size from 30 to 90 microns. After drying at ⁶⁰ ° C, it is a pale yellow cell mass with a pleasant characteristic odor and flavor. The following biologically active substances were identified in biomass: 3-5% of triterpene glycosides, 1% β-sitosterol, 2% fructose, free amino acids, lysoglycerins and fatty acids. None of these compounds directly indicates the possibility of using poliscias as an anti-alcohol or anti-teratogenic drug, which was discovered experimentally.

 Table 1 shows the experimental data indicating a more effective prevention of acute alcohol intoxication compared with preparations of Eleutherococcus and bilberry.

In this series of experiments, ethanol was evaporated from polysacias biological preparations prepared in a standard way, the residue was lyophilized and dissolved in water. An aqueous solution of poliscias was administered to mice at a dose of 100 mg / kg at the same time as ethanol at a dose of 8.8 g / kg of body weight (in preliminary experiments, the lethal dose of ethanol was established as LD ₅₀ , causing 50% mortality of mice with a single (acute) injection) intraperitoneally. The death of animals was recorded during the day after administration of the drugs. In these experiments, poliscias prevented the death of animals in 100% of cases. At the same time, Eleutherococcus preparations (lyophilized pharmacy preparations of Eleutherococcus were used) and bioginseng, prepared in the same way as biopoliscias preparations, in the same dose and with the same method of administration had a significantly less pronounced protective effect. At the same time, the eleutherococcus preparation had the least protective effect. In this regard, in further comparative experiments, the unknown polysias analogue for the antiterratogenic effect of Eleutherococcus was used as a reference aralic drug, but even more similar in terms of the preparation method and anti-alcoholic activity in acute alcohol intoxication, but are less well-known from the literature are bilayer ginseng.

 A series of experiments was also conducted to prevent disturbances in energy metabolism in acute alcohol intoxication. At the same time, the activity of one of the most important enzymes in the energy metabolism of creatine kinase was investigated. Creatine kinase catalyzes the interconversion of creatine and phosphocreatine in a reaction involving ATP and ADR. In the brain tissue, creatine kinase maintains a stable level of the universal macro-energetic compound ATP, which is necessary, in particular, for the synthesis of neurotransmitters and bioelectric activity of the brain.

 Ethanol in the form of a 25% solution was administered to rats intraperitoneally at a dose of 3 g / kg 3 hours before the slaughter of animals. Phytopreparation was similarly administered at a dose of 20 mg / kg for 1 h immediately before slaughter. Creatine kinase activity was determined immediately after the slaughter of animals and expressed in ME (international units) per 1 mg of total soluble protein of brain tissue, or per 1 l of blood serum. The experimental results are given in table. 2.

 From table 2 it follows that the polyscias extract completely normalized the inhibition of creatine kinase activity in the brain caused by ethanol and its increase in blood serum. And in this regard, poliscias was more effective than ginseng extract at the same dose.

 In the table. Figure 3 also presents the results of experiments to prevent the death of rat fetuses during chronic consumption by females of alcohol during pregnancy and lactation.

The addition of female rats to the drinking solution (the only source of drinking a 20% ethanol solution) from the first day of pregnancy until the end of lactation of the polysacias ethanol extract to a final concentration of 0.8 (calculated as the extract volume per solution volume) led to the death rate newborns in the first 2 weeks of life decreased from 25% to 3%
The death toll in the case of a similar used ginseng extract in the same dosages was 14%
Thus, poliscias extract turned out to be significantly more effective than ginseng extract in preventing the death of fruits.

 An experimental analysis of the fetal growth retardation caused by chronic exposure to female alcohol during pregnancy and lactation was also conducted. The results of this analysis are given in table 4.

Alcohol intoxication of rats led to a decrease in the average number of newborns in the litter by 25% and body weight of newborns at the time of birth by 16% and also to a decrease in the rate of increase in body weight during postnatal development by 26% (during the first 15 days of life) and postnatal death of animals by 25%
At the same time, there was a delay in the formation of motor skills according to testing of 16-day-old pups according to the generally accepted open field technique. This delay is expressed, firstly, in a 42% decrease in the number of visited squares of the field and in maintaining the pendulum reflex, which by this time has almost disappeared in control animals (norm 1.7; alcoholization of females 6.2).

 Addition of poliscias extract to drinking ethanol led to normalization of female fetal growth retardation caused by alcoholization in all of the above parameters.

 As follows from Table 4, ginseng extract has a similar effect. However, its effect is less effective, since it does not at all provide an increase in the number of newborns per 1 litter compared to the group of females that received only ethanol solution and prevents postnatal death less effectively than poliscias (Table 3).

 Next, a series of experiments was carried out to prove that polysacias preparations have high antioxidant activity. The experiments, the results of which are presented in Table 5, were carried out on 14-day-old embryos and newborn rat pups at the age of 21 days, as well as 1 month after alcohol withdrawal.

 The level of lipid peroxidation was determined by measuring the products of lipid peroxidation, reacting with 2-thiobarbituric acid (2 TBA). To determine the activity of superoxide dismutase, a modified method was used, based on the inhibition of oxidation of NADH mediated by superoxide radicals with a SOD (tissue extract, blood serum) sample. To determine the activity of ceruloplasmin in the experiments, the Raven method was used.

 The addition of poliscias extract to the ethanol drinking solution led to a decrease in the level of lipid peroxidation to the range of normal values and to a similar change in the activity of superoxide dismutase and ceruloplasmin antioxidant enzymes in both pre and postnatal exposure to alcohol (14-day-old embryos and newborn rats in 21 days old), and for animals 1 month after the withdrawal of alcohol. Thus, for the first time, the presence of high antioxidant activity in polysacias drugs was proved.

 A comparative study of the normalization of the above biochemical changes using extracts of poliscias and ginseng shows that, according to this indicator, the preparations of poliscias are not inferior to the preparations of ginseng and surpass the latter in a number of other tests.

 In addition to studying the antioxidant properties of polysacias and ginseng, an analysis was made of the effect of these drugs on the isoenzyme spectrum of brain enolase. Enolase is an enzyme of the glycolytic pathway through which the main energy substrate of the brain enters glucose metabolism of the central nervous system tissues. In animal tissues, enolase is represented by 3 dimeric isoenzymes: 13 -αα-, αγ-, γγ-, and the γγ-isoform is neurospecific.

 The experimental results of this series of experiments are shown in table.6. It is seen that chronic alcoholization leads to a decrease in the proportion of activity of the γγ-isoenzyme, and this decrease persists after a considerable time after ethanol withdrawal to 5 months. We can assume that alcohol causes irreversible changes in the isoenzyme spectrum of enolase.

 The addition of poliscias extract to the drinking ethanol solution led to the complete restoration of both the specific enolase activity and the dynamics of the growth of the γγ isoenzyme in ontogenesis. At the same time, the use of ginseng extract did not cause such effects, and the ratio of the activity of enolase isoenzymes almost coincided with the enolase spectrum of alcoholized animals.

Based on the results of the experiments, it can be concluded that the proposed anti-teratogenic agent has several advantages compared to the selected analogues, namely:
the drug poliscias significantly more than the ginseng drug reduces the proportion of dead fruit in chronic alcohol poisoning of pregnant and lactating females;
the drug poliscias normalizes the delayed fetal development caused by alcoholization of pregnant and lactating females, both in terms of growth and behavior. Ginseng is less effective in this regard;
the poliscias drug is effective in preventing the death of animals from acute alcohol intoxication, and its effect is much more pronounced than the effect of the preparations Eleutherococcus and Ginseng under similar experimental conditions. In addition, the polysacias drug normalizes the activity of creatine kinase in the brain tissue and blood serum, superior in this ability to ginseng preparations;
Poliscias has a high antioxidant activity, comparable to the activity of such a powerful antioxidant as ginseng.

 However, the effect of the polysacias drug in preventing biochemical changes in fetal tissues caused by chronic alcohol poisoning of pregnant and lactating females is not limited only to its antioxidant effects. Only poliscias reveals the ability to completely normalize such an important biochemical indicator of brain energy metabolism as the isoenzyme spectrum of enolase.

 The example below shows that poliscias drugs are effective not only in preventing the teratogenic effects of alcohol, but also protect the fetus from teratogens with a different spectrum of metabolic activity.

 To prove this point, the most studied mammalian teratogen was used: the anti-malarial drug chloridin (2,4-diamino-5-parachlorophenyl-6-ethylpyrimidine), which inhibits DNA synthesis and causes a characteristic complex of deformities in rat fetuses, the chloride deformity syndrome.

 According to I. Akimova, the maximum embryotoxic effect of chloridine is on the 1st and 13th day of rat embryogenesis. With intragastric administration of chloridine at a dose of 1 mg / kg in the early stages of rat embryogenesis (1, 2, 3 days of pregnancy), preimplantation death was 67.1% and postimplantation 54.8% Complex administration of polyscias (1 mg / kg) and chloridine (1 mg / kg) on the 1st, 2nd, 3rd day of pregnancy led to a decrease in preimplantation death by almost half by 39.5% (p <0.05). However, with this method of administration, post-implantation death of the embryos remained high (63.8%), apparently due to implantation of non-viable embryos.

 With intragastric administration of chloridine at a dose of 15 mg / kg on the 13th day of pregnancy (group 1), post-implantation death of the embryos was 7.5% of abnormal embryos 88.5% All abnormal embryos had pronounced characteristic for chloridine swelling, microcephaly, hypognathia, micromelia, shortening tail, abnormalities of the extremities, including brachidactyly, hemiomelia, hematomas in the places of missing fingers.

 The average body weight of the fetus after exposure to chloridine on the 13th day of pregnancy was 1.54 ± 0.21 g.

 On the contrary, with the prophylactic administration of the drug poliscias, its anti-teratogenic properties were manifested quite clearly. So, with the prophylactic administration of the drug intragastrically from 1 to 11 days of pregnancy (at a dose of 1 mg / kg), followed by the introduction of chloridine on the 13th day of pregnancy (15 mg / kg) (group II) and with the preliminary administration of polyscias on 11, 12 and 13 the day of pregnancy (1 mg / kg) with the combined introduction of chloridine on the 13th day of pregnancy at a dose of 15 mg / kg (group III) the following results were obtained.

 The number of abnormal embryos in group I was 88.5% in group II 57.4%. Thus, the most effective anti-teratogenic effect of poliscias is with prolonged prophylactic administration.

 The embryotoxic effect of chloridine with the same method of administration on the 13th day of pregnancy also reliably depended on the administration of polysias and was 96.5% 68% and 58.8% in groups I, II and III, respectively.

 It should be noted that the spectrum and severity of malformations changes with the introduction of chloridine against the background of the drug. Thus, the spectrum of anomalies in the group with preliminary administration of polyscias from the 1st to the 11th day of pregnancy (group II) does not differ from the complex of anomalies in the group of rats that were injected only with chloridine (group I). On the contrary, after administration of the drug directly against the background of teratogen, only isolated anomalies of the lower extremities (adactyly and brachidactyly) were noted in the absence of the characteristic chloride syndrome of deformities. A decrease in the embryotoxic effect in this experimental group was expressed not only in a decrease in the incidence of abnormal embryos and a narrowing of the spectrum of anomalies, but also in a significant increase in the average fetal mass to almost the control value of 2.20 ± 0.23 g compared to 1.55 ± 0. 25 g when chloridine was administered in isolation.

 Thus, the study of a biological product of a known tissue culture of the plant adaptogen of the aralia family Poliscias filicifolia as an anti-teratogenic agent with a wide spectrum of action allows, along with high antioxidant activity, to increase the efficiency of normalization of the activity of energy metabolism enzymes in comparison with the known anti-teratogenic agents. This, in turn, makes it possible to increase the level of the anti-embryotoxic effect of an anti-teratogenic agent based on plant materials against teratogenic effects while expanding its raw material base for the preparation.

Claims (2)

 1. ANTITERATOGENIC PRODUCT, characterized in that it is a biomass preparation of Poliscias filicifolia obtained by tissue culture.  2. The tool according to p. 1, characterized in that it is a biomass extract.

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