RU2322991C1 - Method for treating experimental leprous infection - Google Patents

Abstract

FIELD: medicine, leprology.

SUBSTANCE: mice should be infected intraplantarly by Shephard's model. Moreover, it is necessary to apply fodder-medicinal mixture consisting of combined feedstuff, at 300 mg horseradish root/kg combined feedstuff. Peroxidase activity in horseradish root corresponds to 100 U/g. The mixture should be introduced in combination with the solutions of potassium iodide at the dosage of 0.2-0.4 mg/kg and potassium bromide at the dosage of 0.1-0.2 mg/kg murine body weight. The intake should be fulfilled daily perorally. The innovation increases antimicrobial action.

EFFECT: higher efficiency of therapy.

5 ex, 5 tbl

Description

The invention relates to medicine, namely to leprology, and can be used, in particular, for the treatment of leprosy.

From the practice of medicine, a method for treating experimental leprosy infection is known, which consists in using a preparation of diaminodiphenylsulfone (DDS) at a dose of 100 mg / kg of feed from the day of infection until the end of the experiment (Shepard CC Effect of DDS on established infectoins with Mycobacterium leprae // Int. J. Lepr. - 1967. - Vol. 35. - P.52-57). However, the known method has the following disadvantages: cases of drug resistance to the drug; it was experimentally established that treatment with this drug is not effective enough, since there are side effects associated with the phenomena of anemia, the toxic effect of the drug on the parenchyma and stroma of the liver of mice; prolonged use of the drug may have a carcinogenic effect on experimentally infected animals. These disadvantages do not allow to obtain a specific technical result - increasing the therapeutic effectiveness of the method.

There is also a method of treating leprosy in an experiment, which consists in using purified peroxidase enzyme at a dose of 100-200 mg / kg of feed (Maslov A.K., Kalyanina O.V. Influence of peroxidase on the development of experimental leprosy in mice infected with intraplantation // Bull. Experimental Biol. - 2000. - T.129, No. 5. - P.571-573; Maslov AK, Luzhnova SA Effect of experimental leprosy peroxidase therapy on the functional state of phagocytes, liver and blood picture in mice / / Bull.experimental biol. - 2000. - T.130, No. 7. - S.83-87). However, the known method has the following disadvantages: the high cost of the drug limits its use in practice; in the process of treatment there are side effects in the form of anemia, which does not allow to obtain a specific technical result - increasing the therapeutic effectiveness of the method.

The closest to the proposed method is a method of treating experimental leprosy infection with a horseradish root preparation containing peroxidase (Maslov A.K., Luzhnova S.A., Kalyanina O.V. Influence of horseradish root on the functional abilities of phagocytes, blood count and liver condition of mice with experimental leprosy // Bull. experimental biol. - 2002. - T.134, No. 8. - S.181-183).

The disadvantages of this method include: insufficient antimicrobial effect. This disadvantage does not allow to obtain a specific technical result - increasing the therapeutic effectiveness of the method.

This method is adopted by the authors as a prototype.

The present invention solves the main problem - increasing the therapeutic effectiveness of the method.

The problem is solved in the invention by the fact that as a therapeutic agent use horseradish root containing peroxidase with an activity of 100 U / g in combination with potassium iodide at a dose of 0.2-0.4 mg / kg of animal body weight and potassium bromide at a dose of 0 , 1-0.2 mg / kg of body weight of mice administered in solution daily.

A distinctive feature of the proposed method is that for the first time, the authors used peroxidase as a component of horseradish in combination with halogens, which increase the activity of one of the main antimicrobial systems, the myeloperoxidase system of phagocytes, for treating experimental leprosy infection. Improving the therapeutic effectiveness of the method makes it possible to recommend it for implementation in the laboratories of the health system, studying the effects of enzyme preparations on an experimental infection. Based on the proposed method, a method for the complex treatment of patients with leprosy using the components of the myeloperoxidase system, which represents one of the main bactericidal systems: peroxidase and cofactors (iodide and bromide), can be developed.

Currently, many studies are devoted to the study of myeloperoxidase of phagocytic cells, especially in case of chronic infectious and granulomatous diseases, since it has been found that acquired or hereditary deficiency of enzyme activity can contribute to the development of chronic infectious diseases, infectious complications in chronic diseases, or granulomatous disease, which includes leprosy.

According to modern concepts, acid hydrolases are capable of destroying only those bacteria that were killed by the myeloperoxidase system, non-enzymatic cationic proteins, lysozyme and lactoferrin in the phagolysosomes of phagocytes. With severe deficiency of phagocyte myeloperoxidase, the latter are not able to kill Candida, staphylococci and other pathogenic microorganisms.

Analyzing the literature data, we can with a high degree of reliability believe that such an indicator of the bactericidal system of phagocytes as the activity of myeloperoxidase is a reliable criterion for assessing their phagocytic ability.

Previously, the authors revealed that lyophilized horseradish peroxidase, as well as horseradish root, in experimentally selected effective dosages increase phagocyte activity due to the stimulation of the myeloperoxidase system of phagocytes (during treatment, myeloperoxidase activity of neutrophilic granulocytes increases). Myeloperoxidase is known to have antibacterial properties, acting in conjunction with hydrogen peroxide and halogens, of which iodide and less effective bromide are considered the most effective. The concentration of iodide in serum is much lower than that necessary for the effective action of the myeloperoxidase system. With the transition of iodine to a positive monovalent form, it exhibits the highest antibacterial activity, and myeloperoxidase in the presence of even small amounts of hydrogen peroxide transfers iodine to this highly active state. The myeloperoxidase-H ₂ O ₂ iodide system iodinates the molecules of bacterial poisons when they are neutralized. The dependence of iodide fixation by phagocytes that absorb bacterial cells on the content of myeloperoxidase in them (Klebanoff SJ lodination of bacteria, a bactericidal mechanism // J. Exper. Med. 1967. Vol.126. P.1063-1069) is proved.

Early observations of researchers related to the 40s and 50s of the last century (Malov G.A., Translators I.N., Zaks A.S. On the hypersensitivity of some patients to iodides // Transactions of the AGMA. T.10. Astrakhan, 1952. P.217-218), indicate that iodine has a fairly good therapeutic effect in leprosy, but often undesirable side effects are noted. However, in the described cases, potassium iodide was prescribed as monotherapy and the doses were quite large (75-100 times higher than that used by us). Patients then did not respond to potassium iodide when they simultaneously took sedatives or hypnotics (including bromides). In our study, KI was used in conjunction with peroxidase contained in KX, with which it forms a joint myeloperoxidase system, and potassium bromide, which, in addition to the smoothing effect of taking KI, is a cofactor in the myeloperoxidase system.

The method is as follows: experimental CBA mice with an initial weight of 25-30 g were infected with an intraplant (in the paw of the right hind paw) suspension of M. leprae isolated from patients and passaged on experimental animals (Shepard leprosy model). The dose of infection was 1 × 10 ⁴ microbial bodies in 0.03 ml of suspension.

Due to the fact that the experiments used mycobacteria leprosy isolated from different patients (i.e. different strains), the intensity of reproduction of mycobacteria in the paws of mice can vary depending on the strain. In one example, a single strain of mycobacterium leprosy was used.

2 months after infection, the mice were given a feed-drug mixture consisting of animal feed, 1 kg of which contained a previously effective dose of horseradish root (300 mg), which we detected earlier, whose peroxidase activity was 100 U / g, as well as potassium iodide and bromide aqueous solution. Horseradish root (KX) was dried at room temperature, cut into small pieces and ground in a conventional electric coffee grinder. The feed-drug mixture was prepared in a Rotomixer mixer (England).

For control, mice of the same line and with the same initial weight were used. After infection with the same strain and the same dose of mycobacterium leprosy, one group of control mice was given a feed-drug mixture consisting of feed in 1 kg of which contained 100 mg of diaminodiphenyl sulfone powder (DDS) (the main drug used to treat leprosy). Another group of control mice did not receive treatment after infection. The method allows to increase the phagocytic activity of macrophages in the paws of animals, which leads to an increase in therapeutic efficacy compared to the known method.

The proposed method was successfully tested at the Federal State Institution "Research Institute for the Study of Leprosy of Roszdrav" on 600 mice of the CBA line with an initial weight of 25-30 g.

Below are the results of testing.

Example 1. 50 mice were infected as described above with a suspension of mycobacterium leprosy isolated from patient K. (source. Bol. No. 2662) and passaged twice in mice.

2 months after infection, the mice were given a feed preparation with KX. At the same time, mice received in a solution daily potassium iodide at a dose of 0.2 mg / kg mass and potassium bromide at a dose of 0.1 mg / kg mass. As a control, a group of mice (50 animals) was given a fodder mixture containing 100 mg of DDS powder in 1 kg of feed. The third group of mice (50 animals) infected intraplantally with the same suspension and the same dose of mycobacterium leprosy did not receive treatment (control).

Mice were sacrificed by decapitation in experimental control groups of 5-6 animals from the group at 3, 6, and 9 months after treatment. The number of leprosy mycobacteria in the paws of mice was calculated according to the method of Shepard and MacRe. The activity of myeloperoxidase in neutrophilic granulocytes (NG) of the peripheral blood of mice is a semi-quantitative method of Astaldi and Verg.

The results of counting the number of mycobacteria leprosy in the paws of mice and the level of myeloperoxidase (MP) are presented in table 1.

Table 1.
The reproduction dynamics of M.leprae and the level of MP in the peripheral blood neutrophils of mice in the treatment of KX + KI (at a dose of 0.2 mg / kg) + KBr (at a dose of 0.1 mg / kg) (M ± m). Indicator; treatment period (months) Control (without treatment) DDS treatment KX + KI + KBr treatment Mycobacteria (10 ⁶ ) 3 20.05 ± 2.9 0.32 ± 0.06 * 0.12 ± 0.03 * 6 41.2 ± 12.06 0.38 ± 0.13 * 0.084 ± 0.04 * 9 54.5 ± 15.31 0.51 ± 0.15 * 0.013 ± 0.006 ** MP level (conventional units) 3 1.79 ± 0.07 2.01 ± 0.06 ** 2.16 ± 0.09 ** 6 1.69 ± 0.04 2.08 ± 0.11 * 2.1 + 0.15 *** 9 1.6 ± 0.032 2.25 ± 0.06 ** 2.39 ± 0.07 *** Note. * P <0.001, ** P <0.01, *** P <0.05 compared with the control.

Combination therapy with KX + KI + KBr (group I) after 3 months of treatment led to a significant suppression of the growth of M. leprae in the paw pads of mice in relation to the control and in relation to monotherapy DDS 2.7 times. After 6 and 9 months of treatment, the combination of drugs had an antimicrobial effect, statistically exceeding the effect of DDS monotherapy (p <0.001).

In a cytochemical study of NG blood of mice treated with a complex of drugs for a long time, an increase in the activity of intracellular MP was revealed in comparison with the control (table).

Thus, the treatment of mouse experimentally infected mycobacterium leprosy with KX + KI + KBr complex at a dose of KI of 0.2 mg / kg and KBr of 0.1 mg / kg of mouse weight leads to a significant decrease in the number of leprosy pathogen in the paw pads of mice compared to the control, which is an indicator of the activation of completed phagocytosis of microorganisms. In this case, the predominance of the pharmacological effect compared with the prototype and DDS.

Example 2. The experimental conditions are similar to example 1. 150 mice of the CBA line (3 groups of 50 animals each) were infected with a suspension of mycobacterium leprosy isolated from patient B. (source. Bol. No. 3865) and passaged three times in mice. After infection, the 1st group of mice was given a feed-drug mixture containing preparations as shown in Example 1, but potassium iodide and bromide were given at doses of 0.3 mg / kg and 0.15 mg / kg of animal weight per day, respectively. The control was carried out analogously to example 1. Animals were slaughtered 3, 6 and 9 months after treatment. The number of mycobacteria in the paws of mice and the level of MP activity in NG peripheral blood of mice were calculated by the method specified in example 1.

The results are presented in table.2.

Table 2.
The reproduction dynamics of M.leprae and the level of MP in the peripheral blood neutrophils of mice in the treatment of KX + KI (at a dose of 0.3 mg 7 kg) + KBr (at a dose of 0.15 mg / kg) (M ± m). Indicator; treatment period (months) Control (without treatment) DDS treatment KX + KI + KBr treatment The number of mycobacteria (10 ⁵ ) 3 106.2 ± 31.8 9.12 ± 1.44 ** 5.42 ± 0.92 * 6 182.6 ± 48.0 5.72 ± 1.1 * 1.47 ± 0.08 * 9 284.2 ± 101.0 4.12 ± 1.71 * 0.12 ± 0.03 ** MPO level (conventional units) 3 1.75 ± 0.03 1.96 ± 0.07 * 2.19 ± 0.22 *** 6 1.66 ± 0.04 2.12 ± 0.08 ** 2.23 ± 0.12 * 9 1.56 ± 0.06 2.23 ± 0.12 * 2.3410.12 * Note. * P <0.001, ** P <0.01, *** P <0.05 compared with the control.

After 3 months of treatment, combination therapy with KX + KI + KBr (group I) led to a significant inhibition of M.leprae growth in the paw pads of mice in relation to untreated control and in relation to DDS monotherapy by 3.9 times. After 6 and 9 months of treatment, the combination of drugs had an antimicrobial effect, statistically exceeding the effect of DDS monotherapy (p <0.05).

In a cytochemical study of NG blood of mice treated with a complex of drugs for a long time, an increase in the activity of intracellular MP was revealed in comparison with the control (table).

Thus, the treatment of mouse experimentally infected mycobacterium leprosy with KX + KI + KBr complex at a dose of KI of 0.3 mg / kg and KBr of 0.15 mg / kg of mouse weight leads to a significant decrease in the number of leprosy pathogen in the paws of mice compared to the control, which is an indicator of the activation of completed phagocytosis of microorganisms. In this case, the predominance of the pharmacological effect compared with the prototype and DDS.

Example 3. The experimental conditions are similar to example 1. 150 mice of the CBA line (3 groups of 50 animals each) were infected with a suspension of mycobacterium leprosy isolated from patient B. (source. Bol. No. 3865) and passaged three times in mice. After infection, the 1st group of mice was given a feed-drug mixture containing the preparations as shown in Example 1, but potassium iodide and bromide were given at doses of 0.4 mg / kg and 0.2 mg / kg of animal weight per day, respectively. The control was carried out analogously to example 1. Animals were slaughtered 3, 6 and 9 months after treatment. The number of mycobacteria in the paws of mice and the level of activity of myeloperoxidase in NG peripheral blood of mice were calculated by the method described in example 1.

The results are presented in table.3.

Table 3.
The reproduction dynamics of M.leprae and the level of MP in the peripheral blood neutrophils of mice in the treatment of KX + KI (at a dose of 0.05 mg / kg) + KBr (at a dose of 0.05 mg / kg) (M ± m). Indicator; treatment period (months) Control (without treatment) DDS treatment KX + KI + KBr treatment The number of mycobacteria (10 ⁶ ) 3 40.56 ± 6.5 0.81 ± 0.25 ** 0.23 ± 0.09 ** 6 68.52 ± 7.05 0.83 ± 0.41 * 0.15 ± 0.07 * 9 111.6 ± 14.8 1.19 ± 0.05 * 0.028 ± 0.01 ** MPO level (conventional units) 3 1.74 ± 0.02 2.21 ± 0.14 ** 2.24 ± 0.1 ** 6 1.72 ± 0.03 2.25 ± 0.06 ** 2.38 ± 0.12 9 1.61 ± 0.03 2.3 ± 0.12 ** 2.43 ± 0.12 ** Note. * P <0.001, ** P <0.01 compared with the control.

After 3 months of treatment, combination therapy with KX + KI + KBr (group I) led to a significant inhibition of M.leprae growth in the paw pads of mice in relation to control and in relation to DDS monotherapy by 3.5 times. After 3 and 9 months of treatment, the combination of drugs had an antimicrobial effect, statistically exceeding the effect of DDS monotherapy (P <0.05).

In a cytochemical study of NG blood of mice treated with a complex of drugs for a long time, an increase in the activity of intracellular MP was revealed in comparison with the control (table).

Thus, the treatment of mouse experimentally infected mycobacterium leprosy with KX + KI + KBr complex at a dose of KI of 0.4 mg / kg and KBr of 0.2 mg / kg of mouse weight leads to a significant decrease in the number of leprosy pathogen in the pads of mice compared to the control, which is an indicator of the activation of completed phagocytosis of microorganisms. In this case, the predominance of the pharmacological effect compared with the prototype and DDS.

Example 4. The experimental conditions are similar to Example 1. 150 mice of the CBA line (3 groups of 50 animals each) were infected with a suspension of mycobacterium leprosy isolated from patient K. (source. Bol. No. 3866) and passaged twice in mice. After infection, the 1st group of mice was given a feed-drug mixture containing the preparations as shown in Example 1, but potassium iodide and bromide were given at doses of 0.1 mg / kg and 0.05 mg / kg of animal weight per day, respectively. The control was carried out analogously to example 1. Animals were slaughtered 3, 6 and 9 months after treatment. The number of mycobacteria in the paws of mice and the level of MP activity in NG peripheral blood of mice were calculated by the method specified in example 1.

The results are presented in table 4.

Table 4.
The reproduction dynamics of M.leprae and the MPO level in the peripheral blood neutrophils of mice in the treatment of KX + KI (at a dose of 0.4 mg / kg) + KBr (at a dose of 0.2 mg / kg) (M ± m). Indicator; treatment period (months) Control (without treatment) DDS treatment Treatment KX + KI + KBr The number of mycobacteria (10 ⁶ ) 3 15.2 ± 2.9 0.76 ± 0.08 * 0.82 ± 0.17 ** 6 30.8 ± 12.2 0.42 ± 0.13 * 0.48 ± 0.09 * 9 42.6 ± 14.8 0.48 ± 0.05 * 0.32 ± 0.04 ** MPO level (conventional units) 3 1.87 ± 0.05 2.0 ± 0.07 *** 2.2 ± 0.1 * 6 1.7 ± 0.04 2.2 ± 0.08 * 2.22 ± 0.09 * 9 1.64 ± 0.03 2.21 ± 0.1 ** 2.4 ± 0.07 * Note. * P <0.001, ** P <0.01, *** P <0.05 compared with the control.

From table 4 it is seen that after 3 and 6 months of treatment, the number of M.leprae in the paws of mice treated with the complex KX + KI + KBr and DDS was almost the same. After 9 months of treatment, less M.leprae was found in the paws of mice treated with the complex of drugs compared to untreated mice and 1.5 times lower than those treated with DDS.

Thus, the treatment of experimentally infected M.leprae mice with horseradish root peroxidase in combination with potassium iodide and bromide in the given doses leads to a decrease in the number of leprosy pathogen in the pads of mice compared to DDS treatment and control, which is an indication of the activation of completed phagocytosis of microorganisms . In this case, there was a slight predominance of the pharmacological effect compared to the prototype (treatment only with KH) and no predominance was noted compared to the main drug for the treatment of leprosy - DDS.

Example 5. The experimental conditions are similar to example 1. 150 mice of the CBA line (3 groups of 50 animals) were infected with a suspension of mycobacterium leprosy isolated from patient B. (source. Bol. No. 3865) and passaged twice in mice. After infection, the 1st group of mice was given preparations, as shown in Example 1, but potassium iodide and bromide were given at doses of 3 mg / kg and 1.5 mg / kg of animal weight per day, respectively (i.e. 15 times more than in example 1). Control and indicators were determined similarly to the previous examples.

The results are presented in table.5.

Table 5.
The reproduction dynamics of M.leprae and the level of MP in the peripheral blood neutrophils of mice in the treatment of KX + KI (at a dose of 3 mg / kg) + KBr (at a dose of 1.5 mg / kg) (M ± m). Indicator; treatment period (months) Control (without treatment) DDS treatment KX + KI + KBr treatment The number of mycobacteria (10 ⁶ ) 3 32.2 ± 5.9 0.44 ± 0.21 0.26 ± 0.06 * 6 48.7 ± 4.01 0.83 ± 0.18 ** 0.082 ± 0.03 * 9 37.8 ± 7.27 0.38 ± 0.12 * 0.01 ± 0.004 ** MPO level (conventional units) 3 1.76 + 0.06 1.9 + 0.16 *** 2.2 ± 0.06 ** 6 1.68 ± 0.02 2.1 ± 0.12 ** 2.39 ± 0.16 ** 9 1.62 ± 0.04 2.29 ± 0.13 ** 2.48 ± 0.12 ** Note. * P <0.001, ** P <0.01, ** P <0.05 compared with the control.

After 3 months of treatment, combination therapy with KX + KI + KBr (group I) led to a significant suppression of the growth of M.leprae in the paw pads of mice with respect to untreated control and 1.7 times with respect to DDS monotherapy. After 6 and 9 months of treatment, the combination of drugs had an antimicrobial effect, statistically exceeding the effect of DDS monotherapy (p <0.05).

In a cytochemical study of NG blood of mice treated with a complex of drugs for a long time, an increase in the activity of intracellular MP was revealed in comparison with the control (table).

Thus, the treatment of mouse experimentally infected mycobacterium leprosy with KX + KI + KBr complex at a dose of KI of 3 mg / kg and KBr of 1.5 mg / kg of mouse weight leads to a significant decrease in the number of leprosy pathogen in the paws of mice compared to the control, which is an indicator of the activation of completed phagocytosis of microorganisms. In this case, the predominance of the pharmacological effect compared with the prototype and DDS. However, in this case, a slightly worse antibacterial effect was noted compared with the first three examples and, according to some authors, high doses of potassium iodide can cause poisoning.

Thus, the analysis of the given examples shows that the best results with respect to suppressing the growth of mycobacterium leprosy in the paw pads of mice (antimicrobial effect) are achieved in the treatment of KX mouse experimentally infected with mycobacterium leprosy in the form of a dietary supplement containing peroxidase 100 U / g in combination with iodide and potassium bromide in doses of 0.2-0.4 and 0.1-0.2 mg / kg of animal weight, respectively, administered in solution daily, which leads to a decrease in the number of leprosy pathogen in the paw pads of mice compared to the control (we shea without treatment) and compared with treatment. When reducing the dose of iodide and potassium bromide in 2 times, the worst therapeutic effect compared with the prototype.

With an increase in the doses of potassium iodide and bromide, a slightly better, but statistically subtle antibacterial effect was noted compared to the dose given at the beginning.

An analysis of the patent and scientific literature showed that previously enzyme therapy with peroxidase contained in KX, in combination with cofactors of the myeloperoxidase bactericidal system (iodide and bromide) of the experimental leprosy infection, was not carried out. The proposed method achieves an increase in the therapeutic effectiveness of the method in comparison with the prototype (treatment only KH).

Used in the claimed method, the therapeutic effect of peroxidase contained in the horseradish root, in combination with iodide and bromide (according to the studies conducted by the authors, increases the phagocytic ability of the macrophage) is a significant difference in the approach to the treatment of experimental leprosy infection.

This difference allowed us to obtain a positive result in the form:

- a significant suppression of the growth of mycobacterium leprosy in the paw pads of mice with experimental leprosy infection in comparison with the prototype;

- the absence of side effects, since the horseradish root is a plant product and is widely used in everyday life.

The proposed method allows to increase the activity of completed phagocytosis of M.leprae in animal macrophages, which leads to an increase in therapeutic efficacy compared to the known method.

The proposed method can be recommended for implementation in the laboratories of the health system involved in the study of the effects of enzyme preparations on an experimental infection.

Based on the proposed method, a method for the complex treatment of patients with leprosy using the components of the myeloperoxidase system, which represents one of the main bactericidal systems: peroxidase and cofactors (iodide and bromide), can be developed.

Claims (1)

A method of treating experimental leprosy infection, which consists in suppressing the multiplication of leprosy mycobacteria in the paws of mice infected intraplantally according to the Shepard model, characterized in that as therapeutic agents a feed-drug mixture consisting of compound feed containing 1 mg of horseradish root in 1 kg is used, with activity peroxidase 100 U / g, in combination with potassium iodide solutions at a dose of 0.2-0.4 mg / kg and potassium bromide at a dose of 0.1-0.2 mg / kg of body weight of mice, the introduction is carried out orally daily.