RU2211035C1 - Anti-tuberculosis preparation - Google Patents

Abstract

FIELD: medicine, phthisiology, pharmacy. SUBSTANCE: invention relates to medicinal agents used in treatment of tuberculosis patients. An anti-tuberculosis preparation represents a pharmaceutical composition containing isoniazide (isonicotinic acid hydrazide) taken in the dose 5-15 mg/kg; 2,4-dioxo-5-fluoropyrimidine (fluorouracil) taken in the dose 0.1-2.5 mg/kg and pharmaceutically acceptable carrier. Ratio of isoniazide to fluorouracil can be equal (2:1)-(150:1) by the weight. Composition can be made as solution and administrated by intramuscular or intravenous route. Preparation shows high an anti-tuberculosis activity with combination of reduced toxic effect. EFFECT: enhanced and valuable medicinal properties of preparation. 4 cl, 2 tbl

Description

 The invention relates to medicine, in particular to anti-TB drugs and can be used to obtain drugs based on isoniazid and in chemotherapy for tuberculosis.

 The main problems of treating tuberculosis include the limited arsenal of anti-TB drugs, the rapid development of resistant forms of mycobacterium tuberculosis (MBT) during treatment and, as a result, the increase in the number of patients infected with primary resistant MBT to the main anti-TB drugs (11111). Therefore, there is a constant search for new anti-TB drugs (RF patents 2153339, 07.27.2000; 2099056, 12.20.1997; 2080114, 05.27.1997), as well as ways to increase the effectiveness of known anti-TB drugs: (RF patents 2169565, 06.27.2001; 2168994, 20.06. 2001; 2136276, 09/10/1999; 2045278, 10/10/95; 2008902, 03/15/1994).

 In the standard combination chemotherapy for tuberculosis, isoniazid - isonicotinic acid hydrazide is used as the main and most effective anti-tuberculosis drug (M.D. Mashkovsky. Medicines. - M.: Medicine, 1988. - V. 2. - S.315-316). Therefore, for the closest analogue, this pharmaceutical composition having an anti-tuberculosis effect, including isoniazid and a pharmaceutically acceptable carrier (prototype), was selected. An effective and safe dose of isoniazid is in the range of 5-15 mg / kg. The pharmaceutically acceptable carrier may be in the form of a solvent (isoniazid for injection) or a vehicle (isoniazid tablets).

 Characterized by high therapeutic activity, isoniazid also has side effects in the form of hematopoiesis disorders, liver function, and neurotoxic manifestations. But its main drawback is the rapid development of drug resistance of Mycobacterium tuberculosis, which reduces its effectiveness. Resistance of MBT to isoniazid in 70% of patients develops three months after the start of treatment, and 30% of patients pass into the category of chronic patients.

 The objective of the invention is the expansion of the range of anti-TB drugs.

 The technical result is an increase in the anti-tuberculosis activity of the drug, a bacteriostatic effect on the isoniazid-resistant MBT strain, and a decrease in the toxic effect of isoniazid.

 To achieve this technical result, a pharmaceutical composition having an anti-tuberculosis effect, comprising isoniazid - isonicotinic acid hydrazide and a pharmaceutically acceptable carrier, further comprises an effective and safe amount of 2,4-dioxo-5-fluoropyrimidine (fluorouracil). The ratio of isoniazid and fluorouracil is 2: 1-150: 1 by weight. The composition can be performed, in particular, in the form of a solution and administered both intramuscularly and intravenously.

 It was found experimentally in vivo and in vitro that the addition of a composition comprising isoniazid and a pharmaceutically acceptable carrier with fluorouracil increases its anti-tuberculosis activity, while the composition also has a bacteriostatic effect on the isoniazid-resistant MBT strain. This allows the use of minimal doses of isoniazid, which, of course, reduces its toxic effect. In an experiment on laboratory animals, an effective and safe amount of fluorouracil in the composition for treating tuberculosis was established, while the ratio of isoniazid and fluorouracil was 2: 1-150: 1 by weight. Pharmaceutically acceptable carriers in the composition may be, in particular, water, 0.9-5% sodium chloride solutions, etc. The composition in the form of a solution is convenient for intramuscular or intravenous administration of the drug.

 Traditionally, fluorouracil is classified as an antimetabolic group of cytostatic agents and is used in the treatment of cancer in daily doses of 10-15 mg / kg in short courses for up to 10 days (M.D. Mashkovsky. Medicines. - M .: Medicine, 1988, v. 2, p. .455). The tuberculostatic properties of fluorouracil were first discovered by us in experiments in vitro (RF patent 2153339, 2000), and its antituberculous activity in the treatment of experimental tuberculosis (RF patent 2165761, 2001).

It can be assumed that a composition comprising two drugs with anti-tuberculosis activity will also have an anti-tuberculosis effect. However, it is known that the combination of several tuberculostatic agents is not always advisable because of the mutual increase in toxicity, as well as because of the possible incompatibility of the drugs and the characteristics of their effect on the biological object. In vitro experiments by vertical diffusion showed that isoniazid and fluorouracil are compatible when mixed, while the tuberculostatic activity of the composition increases dramatically. The acute toxicity of LD _{50 of the} claimed composition, which we determined by the method of Kerber (GN Pershin. Methods of experimental chemotherapy. - M .: Medicine, 1971, p. 526-529) on 80 white mice, amounted to 267.75 mg / kg. The LD _{50 of} isoniazid is 188.4 mg / kg (RF patent 2153878, 2000), i.e. The claimed composition has better tolerance.

The anti-tuberculosis activity of the claimed composition, studied by us in vivo, was higher than in the prototype. In this experiment, an effective and safe amount of fluorouracil was determined with long-term treatment of experimental tuberculosis in laboratory animals infected with laboratory H ₃₇ Rv (example 1) or clinical isoniazid-resistant strains of the office (example 2). It was found that the administration to animals of a composition containing 5-15 mg / kg of isoniazid and 0.1 ÷ 2.5 mg / kg of fluorouracil (which corresponds to a ratio of isoniazid and fluorouracil from 2: 1 to 150: 1 by weight) is effective and safe. The content in the composition of fluorouracil less than 0.1 mg / kg does not lead to significant differences from the prototype, i.e. not effective, and more than 2.5 mg / kg - is accompanied by toxic lesions of the body. High anti-tuberculosis activity of the claimed composition is also confirmed in a clinical trial (example 3).

 The inventive composition can be prepared as follows.

 Option 1. By mixing the dry substances "isoniazid" and "fluorouracil" in the indicated proportions, followed by dissolution in 5% sodium chloride solution and sterilization. The dose depends on the individual, body weight and the nature of the tuberculosis process. A composition containing from 5 to 10 mg / kg of isoniazid and from 1 to 1.5 mg / kg of fluorouracil, i.e. a single dose for a person weighing 50 kg may contain 250–500 mg of isoniazid and 50–75 mg of fluorouracil.

 Option 2. By adding to the finished solution of isoniazid the required amount of the finished solution of fluorouracil. For example, to 10 ml of a 5% official solution of isoniazid add 1.5 ml of a 5% official solution of fluorouracil. The result is a single dose of 11.5 ml for intravenous administration to a person weighing 50 kg, which contains 500 mg (10 mg / kg) of isoniazid and 75 mg (1.5 mg / kg) of fluorouracil. A composition for treating experimental animals weighing 200 g, containing 10 mg / kg of isoniazid and 1.5 mg / kg of fluorouracil, was prepared by mixing 4 ml of a 5% solution of isoniazid and 0.6 ml of a 5% solution of fluorouracil with 45.4 ml of physiological saline. 0.5 ml (single dose for intramuscular injection) of the resulting solution contains 2 mg of isoniazid and 0.3 mg of fluorouracil. Similarly, you can prepare all of the claimed composition.

The study of the anti-tuberculosis activity of the composition
Example 1. Two series of experiments (a and b) were conducted for 2 months each on guinea pigs, males and females, weighing from 200 to 400 g with the formation of homogeneous groups by weight and sex. Each group consisted of 7 animals, a total of 77 animals. The disease was simulated by subcutaneous injection of an MBT culture (laboratory strain H ₃₇ Rv) at a dose of 0.025 mg / kg in a volume of 0.5 ml in the right inguinal region. Treatment began 2 weeks after infection.

 The animals were divided into groups as follows: two control groups — without treatment; two comparison groups - animals that were daily injected intramuscularly with a composition containing an official solution of isoniazid in a dose of 10 mg / kg; seven experimental groups - animals that received a daily intramuscular injection of a composition containing isoniazid and fluorouracil in a ratio of 2: 1-150: 1 by weight (the minimum number of preparations in the compositions was: isoniazid - 5 mg / kg and fluorouracil - 0.1 mg / kg; maximum amount: isoniazid - 15 mg / kg and fluorouracil - 2.5 mg / kg). Compositions prepared according to the above option 2.

 Research methods: intravital assessment of the course of tuberculosis in animals (change in body weight at the end of the experiment); biochemical and clinical blood tests; pathomorphological studies of parenchymal organs and lymph nodes; microbiological examination of homogenates of parenchymal organs for the presence of MBT by culture.

 The results of the study. In the control groups, by the end of the experiment, more than 50% of the animals had fallen; all animals had revealed generalized tuberculosis with characteristic laboratory and pathomorphological changes, MBT (+).

 All animals receiving treatment survived. Changes in laboratory parameters in them did not have significant differences and did not go beyond the age norm, with the exception of animal comparison groups and the group receiving the composition with 2.5 mg / kg of fluorouracil, in which moderate anemia was detected.

The inventive composition was more effective than the prototype, as a result of treatment compared with the prototype:
- the number of positive MBT cultures was reduced (Table 1), specific pathological changes in parenchymal organs in groups receiving the composition with the least amount of fluorouracil (0.1 and 0.5 mg / kg) were reduced;
- there is no growth of MBT in homogenates of parenchymal organs when using compositions with high concentrations of fluorouracil (up to 2.5 mg / kg); pathomorphological examination revealed only residual changes in the transferred generalized tuberculosis.

 The increase in body weight of animals of both experimental groups and comparison groups significantly exceeded this indicator in animals of control groups, which confirms the antituberculous activity of the composition used (Table 1).

 Example 2. The experiment was conducted on guinea pigs, males and females, weighing from 300 to 400 g with the formation of homogeneous groups by weight and gender. Each group consisted of 7 animals, a total of 49 animals. The duration of the experiment is three months. The disease was simulated in animals of all groups by subcutaneous injection of an MBT culture (clinical isoniazid-resistant strain isolated from the patient’s sputum) at a dose of 0.025 mg / kg in a volume of 0.5 ml into the right inguinal region. The strain was identified and its drug resistance to isoniazid at a concentration of 100 μg / ml was established. Treatment began 2 weeks after infection. The groups of animals were divided as follows. The control group - without treatment; comparison group - animals that were daily injected intramuscularly with a composition comprising isoniazid at a dose of 10 mg / kg; five experimental groups - animals that received a daily intramuscular injection of a composition comprising isoniazid and fluorouracil in a ratio of 4: 1-100: 1 by weight (the amount of isoniazid in the compositions was 10 mg / kg, fluorouracil - from 0.1 to 2.5 mg / kg ) Compositions prepared according to the above option 2.

 Research methods are similar to the first example. All animals in all groups survived. The clinical picture of the disease in all cases was erased. Changes in laboratory parameters in the studied animals had no significant differences and did not go beyond the age norm. The results of inoculation of homogenates of internal organs for the detection of MBT in all animals of all groups were negative. Animals of the control group that did not receive treatment, in terms of weight gain, significantly lagged behind the animals of the other groups (Table 2). In a pathomorphological study, all of these animals revealed multiple tuberculous lesions of the internal organs with numerous foci of caseous necrosis. Thus, despite the erased clinical picture due to the weak clinical strain of MBT, generalized tuberculosis was detected in all animals of the control group.

The composition, including isoniazid and fluorouracil, was more effective than the prototype, as a result of treatment compared with the prototype:
- increased weight gain of animals (table. 2);
- in groups receiving compositions with the least amount of fluorouracil (0.1 and 0.5 mg / kg), specific pathological changes are less pronounced, there are no foci of caseous necrosis in the lymph nodes;
- in the groups receiving the compositions with the highest amount of fluorouracil, there are no pathological changes in the parenchymal organs, i.e. after the treatment, no pathomorphological changes were found either in the lungs, or in the liver, or in the spleen, or in the lymph nodes, which indicates the recovery of the animals.

 The results of the studies (examples 1 and 2) showed a high anti-tuberculosis activity of the claimed composition, regardless of the nature of the pathogen. The advantages of using this composition for the treatment of tuberculosis (in contrast to the prototype) are both in accelerating the elimination of MBT from the animal organism (within 2 months from the start of treatment) against the background of normalization of homeostasis and in the reduction or absence of specific pathological changes in parenchymal organs. It has been experimentally shown that the use of a pharmaceutical composition containing isoniazid and fluorouracil in the treatment of tuberculosis leads to the cure of animals in the experiment.

 Example 3. A clinical trial of the anti-tuberculosis effect of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, isoniazid at a dose of 10 mg / kg and fluorouracil at a dose of 1-1.5 mg / kg was carried out on 4 patients with progressive infiltrative pulmonary tuberculosis in the decay phase. The extent of the lesion in two cases was limited to one lobe of the lung. Two other patients had foci of seeding on the opposite side. In all cases, the resistance of the office to two or more major anti-TB drugs has been proven. The duration of combination chemotherapy in patients before admission to the clinic ranged from 12 to 14 months. In the clinic, the patient continued chemotherapy with an additional prescription of the above composition, which was administered in the form of a solution daily intravenously for up to 3 months. Assessment of the condition of patients was carried out on the basis of changes in the clinical picture of the disease and indicators of homeostasis, the dynamics of the x-ray picture and the timing of the cessation of bacterial excretion.

 As a result of a comprehensive anti-tuberculosis therapy supplemented with a composition including isoniazid with the addition of fluorouracil, positive dynamics of the process was observed in all patients, which was expressed in the rapid, up to 2-3 weeks, relief of symptoms of intoxication and normalization of blood counts. A month after the start of treatment, all patients experienced a regression of radiological changes in the form of resorption of infiltrates, reduction in size or closure of decay cavities, complete or partial resorption of foci of seeding. In all cases, abacillation was achieved within up to 2 months.

 Two patients were operated on 2 months after the start of treatment - lobectomy was performed. In both cases, pulmonary tissue fibrosis was detected in the surgical material within the resected areas. Postoperative anti-relapse therapy was carried out using only a composition comprising isoniazid and fluorouracil and lasted for one month, after which the patients were discharged under the supervision of a TB doctor. A follow-up examination after 12 months showed no signs of tuberculosis reactivation in these patients.

 In two other cases, treatment was limited to the chemotherapy described above. Three months after the start of treatment in patients with x-ray examination in the lungs at the site of the existing decay cavities, cicatricial changes were detected with complete resorption of the infiltration. During the follow-up examination of these patients after 6 and 12 months, new pathological changes in the lungs were not found in them.

 Thus, the claimed composition expands the range of anti-TB drugs, as it has high anti-TB activity, has an effect on the isoniazid-resistant MBT strain, and reduces the toxic effect of isoniazid.

Claims (4)

 1. A pharmaceutical composition having an anti-tuberculosis effect, comprising isoniazid - isonicotinic acid hydrazide in an amount of 5-15 mg / kg and a pharmaceutically acceptable carrier, characterized in that it additionally contains 2,4-dioxo-5-fluoropyrimidine (fluorouracil) in an amount of 0, 1-2.5 mg / kg.  2. The composition according to p. 1, characterized in that the ratio of isoniazid and fluorouracil is 2: 1 ÷ 150: 1 by weight.  3. The composition according to p. 1 or 2, characterized in that it is made in the form of a solution.  4. The composition according to p. 3, characterized in that it is administered intramuscularly or intravenously.

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