RU2375960C2 - Method of determining areactogenic action of probiotic preparation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and pharmacology and can be used in clinical trials of probiotic preparations. In order to determine areactogenic action of probiotic preparation in 20 sexually mature, healthy and intact male inbred mice BALB/c, kept in standard vivarium conditions - each individual isolated from the rest, after handling during 1 minute at room temperature, excluding additional sound and light irritating factors, daily, during 5 days, as criteria of areactogenic action of probiotic preparation, determined are: presence of statistically reliable reduction, in comparison with initial averaged values, of averaged values of number of crossed peripheral squares, crossed central squares, peepings into holes, stands with support, stands without support, grooming and boluses, received during 2 minutes in test "open field", equivalent in terms of mouse size, 60 minutes after peroral introduction of maximal single dose of probiotic preparation equivalent in terms of animal weight, without or with subjecting mice to mild immobilising stress during last 30 minutes. Presence of areactogenic action of probiotic preparation is determined in case if at least two criteria of areactogenic action of probiotic preparation are present after peropral introduction of maximal single dose of probiotic preparation equivalent in terms of animal weight, and testing of probiotic preparation as psychotropic adaptogen is stopped.

EFFECT: method ensures self-descriptiveness of pre-clinical determination of areactogenic action of probiotic preparations.

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Description

The invention relates to medicine and pharmacology and can be used in preclinical studies of probiotic preparations.

A known method for determining the orientational and conditioned reflex activity of rats in an open field. According to the known method, the studies were carried out on outbred rats of both sexes in an open field, which is a round platform with a diameter of 100 cm, circumferentially bounded by opaque sides 40 cm high. The entire open field was drawn into squares with a side of 20 cm. At the intersection of the lines were 16 holes 3 cm in diameter are evenly spaced. The exposure time of each animal in an open field was 2 minutes. There were 9 visually distinct elementary behavioral acts: 1) grooming, 2) sniffing, 3) moving in place, 4) immobility, 5) locomotion, 6) peeping into minks, 7) a vertical stand without resting on a side, 8) a vertical stand with relying on the side, 9) numbness (Kremnevskaya SI, Zatsepin EP, Korolev SM “Ethological and pharmacological characteristics of central M-anticholinergics” // Physiological Journal of the USSR named after IM Sechenov No. 4, 1991; Kremnevskaya S.I., Gelman V.Ya., Zatsepin EP, Korolev S.M. “Isolation of various components of the approximate reaction AI rats in an open field "// Physiological Journal of the USSR them. IMSechenov №2, 1991).

The main disadvantage of this method of studying the behavior of used laboratory animals (rats) in an open field is that it is not informative in the preclinical determination of the areactogenic effect of the probiotic preparation in vivo, due to:

firstly, the short duration and softness of the areactogenic effect of probiotic preparations;

secondly, the insufficient sensitivity of rats to the areactogenic effect of probiotic preparations;

thirdly, the lack of combined elimination of factors that destabilize the results of the study: age, health and gender of mice; the effects of circadian animal rhythms; joint keeping of animals in a common cage; annoying informational and emotional factors; inadequate indicative behavior of mice in an open field of standard sizes;

fourthly, the lack of the use of an adequate dosed stress factor in combination with the optimal interval of action of probiotic preparations.

The basis of the invention is the task of ensuring the information content of the preclinical determination of the areactogenic effect of probiotic preparations.

The problem is solved in that in 20 sexually mature, healthy and intact males of BALB / c inbred mice kept under standard vivarium conditions, each individual is isolated from the others, after handling for 1 minute at room temperature, excluding sound and light irritating factors, daily for 5 days as a criterion of the reactogenic effect of the probiotic preparation, the presence of a statistically significant decrease compared with the initial average values of the average values of the number of crossed per spherical squares, intersected central squares, peeping into minks, struts with support, struts without support, grooming and boluses obtained within 2 minutes in the "open field" test, equivalent to the size of the mouse, 60 minutes after oral administration of the maximum single the dose of the probiotic preparation, equivalent in terms of the weight of the animal, without or with mice during the last 30 minutes of mild immobilization stress, and establish the presence of the reactogenic effect of the probiotic preparation in the presence of at least 2 areaktogennogo criteria of the drug after oral administration of the probiotic maximum single dose of the probiotic preparation, equivalent, based on the weight of the animal, the test is stopped and the probiotic preparation as a psychotropic adaptogena; the absence of the reactogenic effect of the probiotic preparation in the absence of criteria for the reactogenic effect of the probiotic preparation after oral administration of the maximum single dose of the probiotic preparation equivalent to the weight of the animal, and the testing of the probiotic preparation as a psychotropic adaptogen is continued.

As a result of our preclinical studies of the properties of probiotic preparations in vivo on the basis of establishing the short duration of the peak (60 minutes) and the softness of the reactogenic effect of probiotic preparations, we selected the animals most sensitive to the action of probiotic preparations - BALB / c mice, introduced informative parameters of the reactogenic effect of probiotic preparations and the conditions for their determination are regulated (including: use of the “open field” test, equivalent in terms of mouse size, selection of sex and sexuality healthy, intact male BALB / c inbred mice, isolation of each individual from others, taking into account the influence of circadian animal rhythms, dosage of probiotic preparations, time intervals for determining indicators, conducting studies at room temperature, eliminating sound and light irritating factors, using adequate dosed stress factor in combination with the optimal interval of action of probiotic preparations), which together provide an increase in the information content of the proposed method.

The inventive method for determining the areactogenic effect of the drug probiotic is new and not described in the literature.

The technical result of the claimed invention is to ensure informativeness of the preclinical determination of the areactogenic effect of probiotic preparations by establishing the short duration and softness of the areactogenic effect of probiotic preparations, selecting the most sensitive to the action of probiotic preparations of animals - BALB / c mice, introducing informative parameters of the reactogenic effect of probiotic preparations and regulating conditions their definitions.

The invention is illustrated by the following examples, showing the information content of the preclinical determination of the areactogenic effect of probiotic preparations when using the claimed invention.

Example 1. A group of 20 male adult, healthy and intact inbred BALB / c mice was used. Each individual was kept under standard vivarium conditions in isolation from others. During the experiment, each mouse was handled for 1 minute before each placement in the “open field”. Initially, with the exclusion of irritating informational and emotional factors, within 2 minutes each mouse determined daily baseline values for 5 days of the number of crossed peripheral squares, crossed central squares, peeping into minks, struts with support, struts without support, grooming and boluses in the test " open field ”, equivalent to the size of the mouse. Then, 60 minutes after oral administration of the maximum single dose of the drug acylact, equivalent in terms of animal weight, with 10 mice during the last 30 minutes of immobilization stress, at room temperature, with the exception of additional sound and light irritating factors for 2 minutes indicators of the number of crossed peripheral squares, crossed central squares, peeping in minks, struts with a support, struts without a support, grooming and bo Yusov in the test "open field" equivalent in terms of the size of a mouse. We calculated and compared the averaged values of the number of crossed peripheral squares, crossed central squares, peeping into minks, struts with support, struts without support, grooming and boluses, initial and obtained 60 minutes after oral administration of acylact. It was found that there are no defined criteria for the areactogenic effect of acylact after oral administration of the maximum single dose of a probiotic preparation, equivalent to the weight of the animal, therefore, the absence of a reactogenic effect of a probiotic preparation was established, and preclinical trials of acylact in tablets as a psychotropic adaptogen were continued.

Example 2. A group of 20 male adult, healthy and intact inbred BALB / c mice was used. Each individual was kept under standard vivarium conditions in isolation from others. During the experiment, each mouse was handled for 1 minute before each placement in the “open field”. Initially, with the exclusion of irritating informational and emotional factors, within 2 minutes each mouse determined daily baseline values for 5 days of the number of crossed peripheral squares, crossed central squares, peeping into minks, struts with support, struts without support, grooming and boluses in the test " open field ”, equivalent to the size of the mouse. Then, 60 minutes after oral administration of the maximum single dose of the drug containing Bifidobacterium bifidum 1C, equivalent in terms of the weight of the animal, with 10 mice during the last 30 minutes of immobilization stress, at room temperature, with the exception of additional sound and light irritating factors in within 2 minutes, the indicators of the number of crossed peripheral squares, crossed central squares, peeping in minks, racks with support, racks without Ora, grooming and boluses in the test "open field" equivalent in terms of the size of a mouse. We calculated and compared the average values of the number of crossed peripheral squares, crossed central squares, peeping into minks, struts with support, struts without support, grooming and boluses, initial and obtained 60 minutes after oral administration of a preparation containing Bifidobacterium bifidum 1C. The presence of all determined criteria for the reactogenic effect of a preparation containing Bifidobacterium bifidum 1C was established after oral administration of the maximum single dose of a probiotic preparation equivalent to the weight of the animal; therefore, the presence of a reactogenic effect of a probiotic preparation was established and preclinical trials of a preparation containing Bifidobacterium bifum 1 were discontinued as a psychotropic adaptogen.

Example 3. A group of 20 male adult, healthy and intact inbred BALB / c mice was used. Each individual was kept under standard vivarium conditions in isolation from others. During the experiment, each mouse was handled for 1 minute before each placement in the “open field”. Initially, with the exclusion of additional annoying informational and emotional factors, within 2 minutes each mouse determined daily baseline values for 5 days of the number of crossed peripheral squares, crossed central squares, peeping into minks, struts with support, struts without support, grooming and boluses in the test "Open field" equivalent in terms of mouse size. Then, 60 minutes after oral administration of the maximum single dose of the drug containing Bifidobacterium bifidum 1C and Bifidobacterium bifidum 791, equivalent in terms of the weight of the animal, without mice undergoing immobilization stress during the last 30 minutes at room temperature, with the exception of additional sound and light irritating factors for 2 minutes in each mouse re-determined indicators of the number of crossed peripheral squares, crossed central squares, peeping into minks, racks with op swarm racks without support, grooming and boluses in the test "open field" equivalent in terms of the size of a mouse. We calculated and compared the average values of the number of crossed peripheral squares, crossed central squares, peeping into minks, struts with support, struts without support, grooming and boluses, initial and obtained 60 minutes after oral administration of a preparation containing Bifidobacterium bifidum 1C and Bifidobacterium 79 Bifidobacterum bifidum 1C and Bifidobacterum 2. The presence of 2 defined criteria for the reactogenic effect of a preparation containing Bifidobacterium bifidum 1C and Bifidobacterium bifidum 791 was established after oral administration of the maximum single dose of the probiotic preparation, equivalent in terms of animal weight, therefore, the presence of the reactogenic effect of the probiotic preparation was established, and preclinical trials of the preparation containing Bifidobacterium bifidum 1C and Bifidobacterium bifidum 791 as a psychotropic adaptogen were discontinued.

Claims (1)

A method for determining the areactogenic effect of a probiotic preparation, characterized in that in 20 sexually mature, healthy and intact male BALB / c inbred mice kept under standard vivarium conditions, each individual is isolated from the others, after handling for 1 min, at room temperature, with the exception additional sound and light irritating factors are determined daily for 5 days as a criterion of the reactogenic effect of the probiotic preparation, the presence of a statistically significant decrease compared with and the running average values of the average values of the number of crossed peripheral squares, crossed central squares, peeping into minks, struts with support, struts without support, grooming and boluses obtained for 2 min in the open field test, equivalent in terms of mouse size, through 60 min after oral administration of the maximum single dose of the probiotic preparation, equivalent in terms of the weight of the animal, without or with mice during the last 30 minutes of mild immobilization stress, and anavlivayut: the presence of the drug areaktogennogo probiotic with at least two criteria areaktogennogo probiotic action of the drug after oral administration of the maximum single dose of the probiotic preparation, equivalent, based on the weight of the animal, the test is stopped and the probiotic preparation as a psychotropic adaptogena; the absence of the reactogenic effect of the probiotic preparation in the absence of criteria for the reactogenic effect of the probiotic preparation after oral administration of the maximum single dose of the probiotic preparation equivalent to the weight of the animal, and the testing of the probiotic preparation as a psychotropic adaptogen is continued.