THE METHOD OF TREATING DRUG RESISTANT MYCOBACTERIUM TUBERCULOSIS INFECTION

Abstract: Multidrug resistance to anti-tuberculous drugs poses threat in the treatment of tuberculosis. These strains are resistant to at least tow first line anti-tuberculous drugs such as INH and rifampicin. Frequently, such MDR strains show resistance to all commonly used first-line agents i.e INH, rifampicin, streptomycin, ethambutol and pyrazinamide. Isoniazid is the most widely used anti-tuberculous drug. Resistance to isoniazid can occur by increased expression on inhA or by mutations that lower the enzyme's affinity to NADH. Mutations in katG, which encodes catalase peroxidase, is the most common source of resistance. Another mechanism of isoniazid resistance occurs by defects in NADH dehydrogenase (Ndh) of the respiratory chain. Increases expression of AhpC has been suggested as another mechanism of INH resistance in mycobacteria. The present invention provides method of treating drug resistance for Mycobacterium tuberculosis infection. According to present invention, a method is provided to employ penicillin with INH. This reduces the MIC of INH and makes resistant organisms sensitive. The level of penicillins required for reversal of INH resistance is known to be achieved and maintained by therapeutic doses of penicillins.

Description: THE PATENTS ACT, 1970 THE COMPLETE SPECIFICATION 1. THE METHOD OF TREATING DRUG RESISTANT MYCOBACTERIUM TUBERCULOSIS INFECTION. 2. Dr. Bakulesh Mafatlal Khamar, residing at 201 "Ashadha", Vasundhara Colony, Gulbai Tekra, Ellisbridge, Ahmedabad 380 006, Gujarat, India, Nationality: Indian 3. The following specification particularly describes the nature of this invention and the manner in which it is to be performed. FIELD OF THE INVENTION The present invention provides method of treating drug resistance for Mycobacterium tuberculosis infection. According to present invention, a method is provided to employ penicillin with INH. This reduces the MIC of INH and makes resistant organisms sensitive. THE BACKGROUND Isoniazid is the most widely used anti-tuberculous drug. It is one of the main therapeutic agent. The strains of mycobacterium are usually inhibited at the concentration of 0.2 mcg/ml or less. When a particular strain is not inhibited by INH concentration of 1.0 mcg/ml, it is labeled as resistant strain. Inh-A, NADH-dependent enocyl acyl carrier protein reductase is the primary target for this drug (Miesel L et al., 1998). A reactive form of isoniazid inhibits inh-A by reacting with the NADH cofactor to the enzyme active site forming a covalent adduct (isonicotinic acyl NADH) that is apt to bind with high affinity. There are more than one mechanisms by which mycobacterium becomes resistant to INH. Resistance to isoniazid can occur by increases expression of inh-A or by mutations that lower the enzyme's affinity to NADH (Miesel Let al., 1998). Both of these resistance mechanisms are observed in 30% of clinical tuberculosis isolates. Mutations in katG, which encodes catalase peroxidase, is the most common source for resistance (Miesel L et al., 1998). Another mechanism of isoniazid resistance occurs by defects in NADH dehydrogenase (Ndh). of the respiratory chain. Genetic, studies indicate that Ndh mutations confer resistance by lowering the rate of NADH oxidation and increasing the intracellular NADH/NAD+ ratio. Increased expression of AhpC has been suggested as another mechanism of INH resistance in mycobacteria (Telenti A et al., 1997; Dhandayuthapani S et al., 1996; Wilson TM et al., 1996). For the treatment of INH resistant tuberculosis, including multidrug resistant tuberculosis drugs like, ciprofloxacin, Kanamycin, Prothionamide. etc. are used along with other primary drugs. Infection caused by resistant organisms is on increase and is becoming a major problem. Efforts are made to discover new regimens, new drugs to minimize the incidence of the problem as well as treat it effectively. The management of resistant infection involve use of drugs to which organisms are sensitive. Penicillin is the oldest antibiotic available and in use even to-day. It has been evaluated for management of various infections including mycobacterial infections (US Public Health Service General Research Support, 1973). Use of penicillin alone had not been found to be therapeutically effective in the treatment of tuberculosis. MIC of penicillins against mycobacterium is significantly high. It is not possible to achieve therapeutic concentration of penicillins required to treat infection effectively, The attempts have been made to overcome this problem by combining penicillins with beta-lactamase inhibitors. One such study evaluated ampicillin in combination with a beta-lactamase inhibitor. (Clavulanic acid, sulbactam, BL-P2013 or BL-P2090) in a ratio 1 :1 in a study by Sorg TB et al., 1987. MIC of ampicillin alone was 32 mcg/ml, but when combined with Clavulanic acid it was -4 meg /ml. Clavulanic acid- ampicillin combination was the most active combination with MICgo of 11 μM. Use of Beta-lactamase inhibitors alone were ineffective at highest concentrations tested. Other studies showed ampicillin-sulbactam to have a bactericidal effect at very high concentrations (15 meg ampicillin per ml) in vitro on exponential phase cultures of M. tuberculosis, thereby indicating its usefulness only in early period of anti-tuberculous treatment (Herbert D et al., 1996). However, they are unlikely to be effective as sterilizing drugs helping to kill persisting lesional bacilli. Studies by Cynamon CH et al., 1983 and Wong CS, 1988 reveal that Amoxycillin-Clavulanic acid combination was bactericidal at concentrations of Amoxycillin 4 mcg/ml and Clavulanic acid 2 mcg/ml in 14 of 15 strains tested. Even for this, dose required is very high. All of the above way was done for strains sensitive to drugs. The above use of beta- lactamase inhibitor-penicillin combination did not focus on multi-drug resistant strains of M. tuberculosis. Penicillin has not been tried to reverse the resistance of mycobacterium to INH so far. It has also not been successfully used in management of resistant mycobacterial infection. The combination of Amoxycillin and a beta-lactamase iπbibitor, Clavulanic acid was evaluated in a clinical setting for the treatment of multidrug-resistant M. tuberculosis without success (Nadler JP et al, 1991 ). In another study by Nakagawa et. al.(1999), the combination of a beta-lactamase inhibitor and penicillin was evaluated clinically in 4 patients with MDR tuberculosis. 2 patients discontinued therapy on account of diarrhoea and in other two, treatment was continued, but MDR-TB remained positive during the remaining period of therapy. Addition of ethambutol significantly reduces the MIC of Amoxycillin for isolates from 16 mg/L to < 0.5 mg/L (Abate G et al., 1998). Alternative approaches have been use of alternative drugs like clarithromycin to overcome the problem of multi-drug resistance (Cavalieri SJ et al., 1995). Ciprofloxacin has been used in newly diagnosed cases of pulmonary tuberculosis (Frederick A et al., 1997). However, emergence of resistance to ciprofloxacin against MDR strains has aroused concern (Fattorini L ae tal., 1999). One attempt was made to increase the susceptibility of multi-drug resistant M. tuberculosis to anti-tuberculous drugs (Jagannath C etal., 1995). The study involved used of ethambutol and dimethyl sulfoxide. Pre-exposure to these compounds enhanced the activity of anti-tuberculous drugs against multi-drug resistant strains of M. tuberculosis. REFERENCES 1. Sacchettini JC, Blanchard JS. The structure and function of of the isoniazid target in M. tuberculosis. Res Microbiol 1996; 147: 36-43. 2. Zhang Y, Young D. Strain variation in the katG region of M. tuberculosis. Mol Microbiol 1994; 14: 301-8. 3. 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J Antimicrob Chemother 1995; 35(3): 381-90. 26. Cavalieri SJ et al. Synergistic activities of clarithromycin and anti-tuberculous drug against multi-drug resistant Mycobacterium tuberculosis. Antimicrob Ag Chemother 1995; 39(7): 1542-5. 27. Wong CS et al. In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium kansasii to Amoxycillin and ticarcillin in combination with Clavulanic acid. J Antimicrb Chemother 1988; 22(6): 863-6. 28. Abate G and Miorner H. Susceptibility of multi-drug resistant strains of Mycobacterium tuberculosis to Amoxycillin in combination with Clavulanic acid and ethambutol. J Antimicrob Chemother 1998; 42: 735-40. 29. Herbert D et al. bactericidal action of ofloxacin, sulbactam-ampicillin, rifampicin and isoniazid on logarithmic- and stationary-phase cultures of Mycobacterium tuberculosis. Antimicrob Ag Chemother 1996; 40(10): 2296-99. 30. Zhang Y et al. 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SUMMARY OF THE INVENTION The present invention overcomes the problem of INH resistance by penicillins. According to present invention, penicillins are found to cause reduction in MIC of INH against multi-drug resistant strains of Mycobacterium tuberculosis, and making them sensitive to INH. The strains of mycobacterium not inhibited by INH at concentration of 1.0 mcg/ml by agar plate method gets inhibited by INH when 1.0 to 2.0 mcg/ml of penicillins is added to it. This concentration of penicillins can be achieved by using therapeutic dose of penicillin. DESCRIPTION OF THE INVENTION The present invention uses penicillins to overcome the problem of INH resistance. This has been done by using penicillins in combination with INH. Penicillins on its own, does not have activity against M. tuberculosis at a dose used or can be used. But when it is combined with INH, it improves sensitivity profile (MIC) of INH. The strains which are resistant to INH becomes sensitive to INK. The strain is known as resistant if it is not inhibited at 1.0 mcg/ml. For the purpose of demonstrating reversal of mycobacterial resistance to INH, an agar plate method has been used. EXAMPLE : I. Preparation of LJ media with combination drugs ( using penicillin and INH): Stock solution of Isoniazid and penicillin was prepared so as to obtain a strength of 10,000/ml. Three concentrations of INH were used (0.1 , 0.2 and 1.0 mcg/ml) along with penicillin. Appropriate solution was mixed with LJ medium so as to give the desired final concentrations as given below. All the flasks and tubes were marked before use, with the drug symbol and final concentration they obtain. Note: Figures with underline are for Isoniazid Figures with bold are for penicillin INH 0.1 + Penicillin: INH 0.2 + Penicillin: II. Evaluation of various penicillins for reversal of INH resistance of mycobacterium. II A. Cloxacillin: Ten strains (clinical isolates) of M. tuberculosis with various sensitive and resistant patterns to first-line anti-tuberculous drugs were evaluated using varying concentrations of INH (0.1 , 0.2 andl .O) and Cloxacillin ( 0.25, 0.5, 1.0 and 2.0) combination and MIC were determined for each, group. Three groups were made: One group had INH 0.2 and Cloxacillin and in this, 4 concentrations using 0.25, 0.5, 1.0 and 2.0 were used to determine MIC. In another group, INH 0.2 and Cloxacillin was used and this as well, 4 concentrations (0.25, 0.5, 1.0 and 2.0) were used to determine MIC. In the third group as well, INH 1.0 and Cloxacillin were taken and again similar 4 concentrations were used. So in each group, 4 LJ bottles for each M. tuberculosis strain was used. For one strain, in all, 12 bottles were used to determine MIC. The experiments were repeated more than once for consistency. At the end of period, each bottles were observed for presence or absence of growth. The results of the 10 strains are shown in the table below: Notes: NG: No growth • All the drug concentrations are in μg/ml • INH-lsoniazid, RIFA-Rifampicin, E B-Ethambutol, SM-Streptomycin The findings reveal that strains resistant to INH at more than 1.0 mcg/ml becomes sensitive to INH at 0.1 mcg/ml, when Cloxacillin 2.0 mcg/ml is used along with it. All strains become sensitive to INH 1.0 mcg/ml in presence of 1.0 mcg/ml of Cloxacillin. The change in sensitivity profile is dependent on amount of INH + Cloxacillin. Findings also suggests that amount of INH and Cloxacillin required is more if organism is resistant to more drugs. Findings also suggests that Cloxacillin concentration is more important than INH concentration, since 2.0 mcg/ml of Cloxacillin is effective for all strains irrespective of INH concentration. Example II B. Amoxycillin: Similarly, Amoxycillin was also evaluated. The strength of amoxycillin - evaluated were 1.0 and 2.0 mcg/ml Notes: • NG: No growth • All the drug concentrations are in μg/ml • INH-lsoniazid, RIFA-Rifampicin, EMB-Ethambutol, SM-Streptomycin The data reveals that findings obtained with Amoxycillin are identical to that seen with Cloxacillin. 1. For strains resistant to INH only, MIC appears to be 1 mcg/ml. 2. For strains resistant to INH, Rifampicin and ethambutol, MIC ranges between 1 to 2 mcg/ml. 3. For strains resistant to all 4 drugs, MIC of 2 and higher may be required. Example II C. Combination of penicillin: Cloxacillin is a narrow spectrum penicillin with more effect on organism producing beta-lactamase and/or penicillinase, while Amoxycillin is a broad spectrum penicillin but has no activity on organisms producing beta-lactamase or penicillinase. To find out whether one or the other is more useful, same clinical isolates against which Amoxycillin was evaluated were evaluated with various concentrations of combination of Amoxycillin and Cloxacillin. The combinations used has Amoxycillin and Cloxacillin in ratio of 1 :1 , 1 :2 and 1 :4 respectively. Notes: NG: No growth All the drug concentrations are in μg/ml INH-lsoniazid, RIFA-Rifampicin, EMB-Ethambutol, SM-Streptomycin Notes: • NG: No growth • All the drug concentrations are in μg/ml • INH-lsoniazid, RIFA-Rifampicin, EMB-Ethambutol, SM-Streptomycin Notes: • NG: No growth • All the drug concentrations are in μg/ml • INH-lsoniazid, RIFA-Rifampicin, EMB-Ethambutol, SM-Streptomycin The findings show that for reversal of resistance to INH, all penicillins have identical effect when used alone or in combination with each other. The above example clearly demonstrates that penicillins reverses the resistance of mycobacterium for INH. The examples also indicates the amount of penicillins required to lower the MIC of INH for INH resistant mycobacterium. The amount required is easily achievable in clinical settings without any adverse effects. The amount of penicillins required for reversal of INH resistance (1 to 2 mcg/ml for Cloxacillin, Amoxycillin or its combination) is known to be achieved and maintained by therapeutic doses of Amoxycillin and Cloxacillin. Accordingly for the first time it is demonstrated that multi-drug resistant mycobacterium can be treated by therapeutic amounts of two drugs e.g. Penicillins and INH. Both the drugs have been use since long time with established safety and efficacy even after long term use.

Claims: I claim: 1. The method of treating drug-resistant mycobacterial infection in an animal comprising administration to the said animal, Penicillin and INH. 2. The method of claim 1 wherein penicillin is administered simultaneously with INH. 3. The method as claimed in claim 1 wherein penicillin and INH are administered sequentially. 4. Penicillins, as claimed in claim 1 to 3 can be a broad spectrum penicillin or a narrow spectrum penicillin. 5. Penicillin, as claimed in claim 1 to 4 can be any single penicillin or combination of more than one penicillins. 6. Penicillin, as claimed in claim 1 to 5 can have activity against beta-lactamase producing organism. 7. Penicillin as claimed in claim 1 to 4 can be active against penicillinase producing organisms. 8. Penicillins, as claimed in claim 1 to 5 can be selected from the group of penicillin comprising Benzylpenicillin, Phenoxymethylpenicillin, Propicillin, Clemizole penicillin, Procaine penicillin, Phenethicillin, Hetacillin, Metampicillin, Pivampicillin, Cyclacillin, Epicillin, Pevmecillinam, Apalcillin, Aspoxicillin, Temocillin, Methicillin, Oxacillin, Nafcillin, Ampicillin, Amoxycillin, Carbenicillin, Ticarcillin, Azlocillin, Mezlocillin, Piperacillin, Cloxacillin, Dicloxacillin, Lenampicillin, Bacampicillin, Azidocillin, Talampicillin, Mecillinam, Flucloxacillin, Aztreonam, Imipenem, Meropenem and the like. 9. Penicillin in claim 1 can be given by oral, parenteral or rectal route. 10. Penicillin and INH as claimed in claim 1 can be given separately or as a single fixed dose combination.

Inventors: BAKULESH MAFATLAL KHAMAR, BAKULESH, MAFATLAL, KHAMAR
Applicants: BAKULESH MAFATLAL KHAMAR, BAKULESH, MAFATLAL, KHAMAR
Application year: 2001
Main classification: A61K
IPC codes: A61K31, A61P31