RU2080114C1 - Preparation for tuberculosis treatment - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention proposes the preparation that is complex of isonicotinic acid hydrazine with divalent iron of the formula: $$$. Preparation shows lower toxicity and increased activity as compared with isoniazide, prolonged action, does not form toxic metabolites in organism, exhibits the expressed bacteriostatic effect on mycobacterium strains that are resistant to streptomycin isoniazide. EFFECT: enhanced effectiveness of preparation. 10 tbl

Description

 The invention relates to medicine, specifically to anti-TB drugs.

Currently, 2 groups of drugs are used to treat tuberculosis:
a) First-line drugs (the main antibacterial derivatives of isonicotinic acid, p-aminosalicylic acid, antibiotics - streptomycin, rifalepicin).

b) Second-line drugs (reserve ethionamide, protionamide, ethambutol, cycloserine, kanamycin, etc.). [Mashkovsky MD Medicines, M. Medicine, 1993, part II, p. 366 367]
The disadvantage of the first-line drugs is that with the use of these highly active drugs, resistant mycobacteria of tuberculosis develop quite quickly, and the second-line drugs for independent use are not very effective.

 The closest in technical essence to the invention is the preparation of isoniazid hydrazide of isonicotinic acid (GINC) [Mashkovsky MD Medicines, M. Medicine, 1993, p. 367 -369] Being the most effective drug for the treatment of tuberculosis, GINC has the disadvantage that when it is used, mycobacterium tuberculosis resistant to GINC develops quite quickly, in addition, when it is used, some fairly toxic metabolites are formed in the body, which leads to an undesirable side the action of the drug.

 The purpose of the invention is the creation of a drug that is not inferior to GINC in anti-tuberculosis activity, but which has a bacteriostatic effect on strains of tuberculosis mycobacteria that are resistant to isoniazid and streptomycin and which do not form toxic metabolites in the body.

условно названным нами "Феназидом".The goal is achieved by using as an anti-tuberculosis drug a previously undescribed GINC complex with divalent iron structure

conditionally named by us "Phenazidum".

Phenazide is prepared by adding a 0.2 M solution of iron sulfate in distilled water to a 0.2 M solution of GINC in distilled water. The orange precipitate formed is filtered off, washed with distilled water, and dried in an oven at 40 ^° -50 ^° C. to constant weight. The yield of phenazide is 76% of theoretical.

C ₆ H ₁₁ FeN ₃ O ₇ S
Calculated, C 22.15; H 3.88; N, 12.92; Fe 17.23.

 Found, C, 23.04; H 4.13; N 13.08; Fe 16.62.

The water content in Phenazide was found (determined by the Fisher method) - 11.6%; calculated 11.07%
Upon emission of spectral characteristics in water and 0.2 M HCl, the following data were obtained (Fig. 1 4).

2. Комплекс ГИНК с сульфатом железа (II) полностью диссоциирует в 0,2 M HCl на ГИНК и ион железа.Spectral analysis data show that in water the Phenazide spectrum has two absorption maximums of 200 nm and 262 nm, the GINC spectrum under the same conditions - 212 and 262 nm. The absorption spectra of Phenazide and GINC in 0.2 M HCl completely coincide with the absorption maxima of 215 and 265 nm. This indicates two circumstances:
1. The complex of GINC with iron sulfate (II) is an intramuscular compound of the following structure:

2. The GINC complex with iron (II) sulfate completely dissociates in 0.2 M HCl into GINC and iron ion.

 The results obtained by studying the data of UV spectroscopy, indicating that the complex of GINC with iron sulfate is an intracomplex compound of this structure, is confirmed by the data of IR spectroscopy (Fig. 5). IR spectra were obtained in liquid paraffin.

The study of Phenazide in comparison with GINC by high pressure liquid chromatography (sorbent Nucleosil C ₁₈ , solvent 32% methanol, pH 6.25) shows that Phenazide is an individual (100%) substance - an intracomplex compound of GINC with iron (II) sulfate (Fig. .6, 7).

 Chromatographic studies were performed on a Millipore Pat. N 52507 chromatograph (USA).

 A mass spectrometric study of Phenazide on an MI-1201 E instrument (USSR) showed that three mass peaks identified as iron (M = 56), GINC (M = 137) and phenazide ( M = 289). Mass 289 corresponds to the molecular weight of Phenazide excluding two water molecules (325 36 = 289).

 Using potentiometric titration, the stability constant of Phenazide in aqueous solutions at physiological pH values of 6.9522 was determined. This suggests that Phenazide will be absorbed in the gastrointestinal tract of animals and humans without decomposition and dissociation into components.

 The oil-water phenazide partition coefficient was determined to be 1.48. This allows us to conclude that Phenazide should penetrate well through the lipid membrane of mycobacterium tuberculosis.

образуя бактериостатическое соединение изоникотиновую кислоту и нетоксичные продукты (азот, протоны, ионы железа).When studying the kinetics of the oxidation of Phenazide in an aqueous solution at room temperature with atmospheric oxygen (taking into account the data of thermogravimetric analysis), it was shown that the preparation is oxidized according to the scheme:

forming a bacteriostatic compound isonicotinic acid and non-toxic products (nitrogen, protons, iron ions).

Pharmacological study of phenazide
The experimental study of the bacteriostatic and chemotherapeutic activity of Phenazide was carried out in in vitro and in vivo experiments on cultures of human and bovine tuberculosis microbacteria freshly isolated from pulmonary tuberculosis patients resistant to known anti-tuberculosis drugs GINC and streptomycin, as well as on disease models.

The study of bacteriostatic activity of Phenazide in the experiment "in vitro"
A culture of Mycobacterium tuberculosis was seeded with 0.2 ml of V optical (casein-salt). The concentration of mycobacteria in this case was 1 • 10 ⁷ million cells in 1 ml of culture medium.

A solution of Phenazide in various concentrations (from 10 to 100 μg / ml) was introduced into mycobacterial cultures and kept under sterile conditions for 10 days at 37 ^o C. After this period, the supernatant was removed, fixed smears were prepared from the precipitate, stained with Tsil -Nielsen and in 100 fields of view considered the number of formed "braids". The results of the study were processed statically.

 It was found that Phenazide at a concentration of 40 μg / ml has a bactericidal, and at a concentration of 10 and 20 μg / ml bacteriostatic effect against laboratory strains of tuberculosis-sensitive mycobacterium GINC. Especially valuable is its antibacterial effect against strains freshly isolated from patients with pulmonary tuberculosis, resistant to the effects of highly active tuberculostatics GINK and the antibiotic streptomycin. In this case, Phenazide has the same activity of 40 μg / ml, as in relation to sensitive mycobacterium tuberculosis, table. 1-3.

Study of the chemotherapeutic activity of Phenazide in the long-term treatment of tuberculosis in white mice
Outbred white male mice weighing 20-22 g were used for the chemotherapeutic experiment. Animals were infected intravenously (in the tail lateral vein) with a culture of human type Mycobacterium at a dose of 0.025 mg in 0.5 ml.

 Phenazide was administered orally at doses of 5.0 mg / kg and 10.0 mg / kg, each dose was studied in 10 animals; 10 mice served as control.

 As a result of the experiment, it was established: control animals died within 1 month after the start of the experiment. The cause of death is generalized hematogenous-desiminated tuberculosis.

 In the 1st experimental group, which received 5 mg / kg Phenazid daily, out of 10 mice, there were 2 individuals per month, in the remaining 8 animals, the tuberculosis lesion index ranged from 0.5 to 2 units, which corresponds to the presence of single tuberculous foci ( table 4).

 Microbiological examination of the organs of animals after 1 month of treatment with Phenazide showed that in 6 examined mice in the homogenate of the internal organs it was not possible to detect mycobacterium tuberculosis both by seeding and by bacterioscopy; in 2 animals, mycobacterium tuberculosis was found in the homogenate of the spleen and lungs. When sowing the material, the growth of mycobacteria was not revealed.

 In the 2nd experimental group of white mice that received Phenazide orally at a dose of 10 mg / kg daily for a month, all animals remained alive.

 At autopsy, isolated tuberculous lesions were noted in the spleen, bronchial lymph nodes were enlarged in the lung tissue. No tuberculous changes were detected in the liver. Mycobiological examination of the internal organs of white mice treated with Phenazide at a dose of 10 mg / kg showed that no tuberculosis mycobacteria were found in the homogenates of the internal organs of animals (Table 4).

Изучение химиотерапевтической активности Феназида при длительном лечении туберкулеза у морских свинок.Thus, the efficacy index of chemotherapy with phenazide in this experiment was:

The study of the chemotherapeutic activity of Phenazide in the long-term treatment of tuberculosis in guinea pigs.

The study of the chemotherapeutic activity of Phenazide in doses of 5 and 10 mg / kg
To conduct this experiment, 30 male guinea pigs weighing 200-220 g were used. Animals were infected with a culture of human mycobacteria at a dose of 0.025 mg in 0.5 ml, which was injected subcutaneously into the right inguinal region. 10 animals served as a control. Experimental groups: 1st, 10 animals that were injected daily with 5.0 mg / kg of phenazide daily, and 2nd, 10 animals that were daily administered with phenazide at a dose of 10.0 mg / kg.

 Control animals died during the first month from the start of the experiment.

 In the first experimental group (Phenazide, 5 mg / kg), 4 out of 10 guinea pigs fell during the experiment (3 months). Large translucent foci of gray were detected in the lungs of dead animals, the liver and spleen also contained lesions. Diagnosis: hematogenous disseminated tuberculosis of internal organs. In 6 guinea pigs, the tuberculosis lesion index ranged from 1 to 1.5 units, which corresponds to the presence of single foci of tuberculosis in the spleen. Tracheo-bronchial lymph nodes were enlarged in the lung tissue. No tuberculous changes were detected in the liver (Table 5). A microbiological study of the internal organs of animals after 3 months of treatment with Phenazide at a dose of 5 mg / kg showed that of the 6 examined guinea pigs in five animals, mycobacterium tuberculosis in the homogenate of the internal organs were not detected by both inoculation and bacterioscopy. Mycobacterium tuberculosis was found in homogenates of the liver, lungs and spleen in only one guinea pig. Sowing this material did not allow to obtain a positive result, that is, the growth of mycobacteria was not detected.

 Thus, in 50% of the animals in the first experimental group, foci were sterilized after parenteral administration of tuberculosis microbacteria with phenazide chemotherapy at a dose of 5 mg / kg for three months.

 The 2nd group of experimental animals received finazide at a dose of 10 mg / 0 kg. All 10 animals remained alive during the three-month experiment. At the end of the experiment, an autopsy revealed single tuberculosis lesions in the spleen. In the homogenates of the internal organs during microbiological examination of mycobacteria were not found.

Изучение химиотерапевтической активности Феназида в дозах 20 и 40 мг/кг.The chemotherapy efficacy index in this guinea pig experiment was:

The study of the chemotherapeutic activity of Phenazide in doses of 20 and 40 mg / kg.

 To assess the chemotherapeutic activity of Phenazide at doses of 20.0 and 40.0 mg / kg, 30 male guinea pigs weighing 200-220 g were also used. The animals were infected with a culture of human mycobacterium tuberculosis at a dose of 0.025 mg in 0.5 ml, which injected subcutaneously into the right groove area. 10 animals served as a control. Experimental groups: 1st, 10 animals that were daily injected with phenazide inside at a dose of 20 mg / kg; 2 nd, 10 animals that were daily injected with phenazide orally at a dose of 40 mg / kg. The duration of the experiment is three months.

 Control animals died between two weeks and 1.5 months from the start of the experiment.

 In the first experimental group, by the end of the experiment, all animals are alive. The tuberculosis lesion index ranged from 0 to 0.5. Internal organs were without pathological changes. In six animals, foci of inflammation replaced by connective tissue were found in the lungs (Table 6). A microbiological examination of the internal organs of experimental animals after three months of treatment with Phenazide at a dose of 20 mg / kg showed that in all animals of this group mycobacterium tuberculosis were not detected by both seeding and bacterioscopy.

 When examining the 2nd experimental group, it was shown that the tuberculosis lesion index was from 0 to 0.5. However, in this group of animals in only two individuals, small allowed foci of inflammation replaced by connective tissue were found in the lungs. The remaining internal organs were found without pathological changes. Microbiological examination of homogenates of the internal organs of culture methods and bacterioscopic did not reveal the presence of mycobacterium tuberculosis.

 Thus, in all animals of this group, after a three-month treatment with Phenazide at a dose of 40 mg / kg, the foci completely sterilized after parenteral administration of mycobacterium tuberculosis.

Изучение метаболизма и фармакокинетики Феназида
1. Изучение метаболизма Феназида
ГИНК, являясь субстратом N-ацетилтрансферазы, при длительном применении может оказывать и не оказывать побочное действие на организм больного в зависимости от фенотипа ацетилирования. Это обстоятельство предполагает проведение комплекса мероприятий в клинике, направленного на обеспечение индивидуализации разовых и курсовых доз этого препарата для каждого больного. Реализация же побочных эффектов изониазида связана с хелатирующими свойствами его молекулы.The chemotherapy efficacy index in this experiment was:

The study of metabolism and pharmacokinetics of phenazide
1. The study of the metabolism of phenazide
GINC, being a substrate of N-acetyltransferase, with prolonged use may and may not have a side effect on the patient's body, depending on the acetylation phenotype. This circumstance involves a set of measures in the clinic aimed at ensuring the individualization of single and course doses of this drug for each patient. The realization of the side effects of isoniazid is associated with the chelating properties of its molecule.

 Modification of the GINC molecule by means of complexation with iron (P) pursued, first of all, the goal, the achievement of which would ensure greater safety of tuberculosis chemotherapy, since the chelate site of the GINC molecule blocked by iron (P) loses its ability to interact with the active centers of metal-containing enzymes, and the inclusion of the primary amino group of hydrazine in the chelate cycle of the complex deprives it of the properties of the substrate of N-acetyltransferase.

 In this regard, we conducted a study of the metabolism of Phenazide in order to directly verify the theoretical and experimental results.

 White male rats weighing 200-220 g were injected with 0.1 g of Phenazide and GINK. The animals were placed for a day in Kassel exchange cells (Germany). During the experiment, the animals did not receive food, water was received as needed.

Twenty-four hours later, daily urine was collected and examined using the Defrancini, Zumboui method described by J. Hirts (1975). To obtain chromatograms, Whatman-dense paper and an isopropanol-water solvent system in a ratio of 85: 15 were used. Vertical chromatography was performed for 5 hours. After the process was completed, the chromatograms were dried for 16 hours at room temperature in the dark, then developed with iodine vapor at 25 ^o C for 45 minutes Chromatograms were identified using the results obtained by the authors of the method under similar conditions (Table 7).

 The results shown in Table 7 show that when Phenazide is introduced into the body of an animal (unlike GINCs) in the daily urine, isoniazid and acetylisoniazide are absent, the main compounds responsible for the development of side effects during long-term treatment of patients with GINC preparations. Therefore, Phenazide is not a substrate of N-acetyltransferase and when it is prescribed to patients, there is no need to individualize the dosage of the drug. In addition, our data confirm that the main pathway of phenazide metabolism is oxidation.

Pharmacokinetics of Phenazide
The pharmacokinetics of phenazide was studied on white outbred male rats with an average weight of 300 g. The drug was administered once through a probe intragastrically in the form of an aqueous suspension at a dose of 30 mg / kg.

 Blood for analysis was taken after decapitation after 1.5; 3.0; 6.0; 9.0; 12.0; 18.0; 24.0 h. For each time point of the pharmacokinetic curve, 6 animals were taken into the experiment.

 The determination of the drug content in the blood serum of experimental animals was carried out spectrophotometrically.

 Calculation of the pharmacokinetic curve and parameters was carried out according to the "farm" program on a personal computer HP-85 by Hewlett-Packard Company (USA).

 Averaged rat serum phenazide concentrations are shown in Table 8.

 According to the data obtained, the change in the concentration of Phenazide in the blood serum of rats is well described by the exponential equation in the single-particle pharmacokinetic model with absorption (Soloviev V.N. et al. 1980). A slight difference between the experimental and calculated values of the concentrations of Phenazide testifies in favor of the correctness of the selected model (table. 9, Fig. 8).

 The pharmacokinetic parameters of phenazide are given in table.9.

An analysis of the results indicates that Phenazide is characterized by slow absorption from the gastrointestinal tract and even slower excretion. So, the level of the maximum concentration of phenazide in the blood is reached after 9, 18 hours, while the absorption rate is 2.6 μg ml ^-1 h ^-1 . At the same time, the half-life of the drug is 7.21 hours, and the elimination rate constant is 0.0961 h ^-1 . The apparent volume of distribution of Phenazide is 1.09 L kg ^-1 , total clearance 0.1047 L kg ^-1 h ^-1 .

According to the data given in Table 8, the equations describing the curve of the dependence of the concentration of Phenazide on time have the form:
C _(t) = 27.0 • (1st ^-0.09661 • ^T ),
if t <T _abc , i.e. for the ascending branch of the curve
C _(t) = 17.2 • e ^{-0.0961 (t-9.18)} ,
if t> T _abc , i.e., for the descending branch of the curve
Where
T = tT _lag

 It should be noted that for Phenazide, the time to reach the maximum level of the drug in the blood is determined by the ratio between the absorption and elimination constants and does not depend on the administered dose of the drug. Moreover, since the values of the absorption and elimination constants differ slightly, each of the branches of the pharmacokinetic curve characterizes both processes simultaneously (Soloviev V.N. et al. 1980).

 Thus, Phenazide is a drug of prolonged action in contrast to GINK, which is of great importance for phthisiology. A single appointment of Phenazide ensures the presence of a bacteriostatic concentration of the drug in the blood for 24 hours (GINC 4 hours).

Phenazide Acute Toxicity Results
The acute toxicity study of Phenazide was performed on non-linear white male mice weighing 18 to 20 g and non-linear white male rats weighing 18 to 20 g and non-linear white male rats weighing 200 to 220 g. Phenazide was administered intragastrically and intraperitoneally in the form of an aqueous suspension. Five doses of the drug were administered to each animal species. For each dose, 10 animals of each species were used. Observation of experimental animals after drug administration was carried out for 10 days. In rats, death was not observed at very high doses of Phenazide (Table 10). In mice, the death of individual individuals was observed only on the next day after administration of the drug. LD _{50 was} calculated by the Litchfield-Wilcoxon method.

From published data it is known that LD _{50 of} GINC for white mice with intraperitoneal administration is 211.2 mg / kg (Vitolia MA 1957), and the death of animals occurs in the first hour after the introduction of GINC with a characteristic pattern of intoxication.

Comparing the data of Table 10 and the literature, we can conclude that the blockade of the chelate node of the GINC molecule by the iron (II) ion sharply reduces the toxic properties of the drug, moreover, due to the different pathways of GINC and Phenazide metabolism, the molecular mechanism of intoxication and death of experimental animals changes. So, if with the introduction of GINC, the death of animals occurs in 5 10 minutes 1 1.5 hours (depending on the method of administration of the drug), then with the load of experimental animals (mice) with Phenazide, death is observed only on the next day after administration of the drug, and in rats, it was not at all possible to establish LD _{50 of} Phenazide with intragastric administration of the drug.

 Thus, a previously undescribed GINC complex with divalent iron under the conditional name Phenazide was proposed as a drug for treating tuberculosis, a production method was developed, the physicochemical properties were studied, and the structure of Phenazide was established.

 The study of the bacterioscopic and chemotherapeutic activity of Phenazide in the in vitrpo and in vivo experiments showed that Phenazide is superior to GINC in terms of chemotherapeutic activity and has a pronounced bacterioscopic effect on tuberculosis microbacterial strains resistant to isoniazid and streptomycin.

 The study of metabolism and pharmacokinetics Phenazide, unlike GINC, does not form toxic metabolites in the body of experimental animals. A single administration of Phenazide ensures the maintenance of the bacteriostatic concentration of the drug in the blood for 24 hours. The effect of prolongation does not depend on the dose of Phenazide, while Phenazide in acute experiments showed much less toxicity than GINK. 10 tab.

Claims (1)

A drug for the treatment of tuberculosis, which is a complex of isonicotinic acid hydrazide with ferrous iron of the general formula

in

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