WO2002000165A2 - Agent for reversal of drug resistance in mycobacterium tuberculosis - Google Patents

Agent for reversal of drug resistance in mycobacterium tuberculosis Download PDF

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WO2002000165A2
WO2002000165A2 PCT/IB2001/001136 IB0101136W WO0200165A2 WO 2002000165 A2 WO2002000165 A2 WO 2002000165A2 IB 0101136 W IB0101136 W IB 0101136W WO 0200165 A2 WO0200165 A2 WO 0200165A2
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inh
resistance
penicillin
tuberculosis
penicillins
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PCT/IB2001/001136
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WO2002000165A3 (en
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Bakulesh Mafatlal, Khamar
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Priority to EA200200290A priority Critical patent/EA004875B1/en
Priority to APAP/P/2002/002454A priority patent/AP2002002454A0/en
Priority to AU66257/01A priority patent/AU6625701A/en
Publication of WO2002000165A2 publication Critical patent/WO2002000165A2/en
Publication of WO2002000165A3 publication Critical patent/WO2002000165A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A61K31/431—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems containing further heterocyclic rings, e.g. ticarcillin, azlocillin, oxacillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis

Definitions

  • the present invention provides agents for reversal of INH resistance in Mycobacterium tuberculosis.
  • penicillins reverses resistances of Mycobacterium to INH.
  • 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 en . ocyl 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.
  • Ndh NADH dehydrogenase
  • INH resistant tuberculosis including multidrug resistant tuberculosis drugs like ciprofloxacin, Kanamycin, Pr ⁇ thionamide etc. are used along with other primary drugs.
  • 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,
  • beta-lactamase inhibitors The attempts have been made to overcome this problem by combining penicillins with beta-lactamase inhibitors.
  • MIC of ampicillin alone was 32 mcg/ml, but when combined with Clavulanic acid it was 4 meg /ml.
  • 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).
  • Sacchettini JC Blanchard JS. The structure and function of of the isoniazid target in M. tuberculosis.
  • Wilson TM Collins DM. ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex.
  • Banerjee A et al. inhA a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis.
  • Yuan Y et al. The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis.
  • the present invention overcomes the problem of INH resistance by penicillins.
  • 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.
  • 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 INH. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Amoxycillin was also evaluated.
  • the strength of amoxycillin evaluated were 1.0 and 2.0 mcg/ml
  • MIC appears to be 1 mcg/ml.
  • MIC ranges between 1 to 2 mcg/ml.
  • 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.

Abstract

Multidrug resistance to anti-tuberculous drugs poses threat in the treatment of tuberculosis. These strains are resistant to at least two first line anti-tuberculous drugs such as INH and rifampicin. Frequently, such MDR strains show resistance to all commonly used first-line agent i.e. INH, rifamcpicin, streptomycin, ethambutol and pyrazinamide. Isoniazid is the most widely used anti-tuberculous drug. Resistance to isoniazid can occur by increased expression in 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 AphC has been suggested as another mechanism of INH resistance in mycobacteria. The present invention overcomes INH resistance by the use of penicillins with INH. According to present invention, penicillins when used in effective amount, reduces the MIC of INH in multi-drug resistant strains of M. tuberculosis. The improved INH sensitivity, as brought by the present invention, falls within the range which can be exploited for effective therapeutic intervention.

Description

1. AGENTS FOR REVERSAL OF DRUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS.
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 agents for reversal of INH resistance in Mycobacterium tuberculosis. According to present invention, penicillins reverses resistances of Mycobacterium to INH.
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 en.ocyl 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 increased 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, Prόthionamide 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 ith MIC90 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 inhibitor, 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.
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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. Telenti A et al. Genotypic assessment of isoniazid and rifampicin resistance in Mycobacterium tuberculosis: A blind study at reference laboratory level.
J Clin Microbiol 1997; 35: 719-23. 4. Ddhandayuthapani S et al. Oxidative stress response and its role in sensitivity to isoniazid in mycobacteria: Characterization and indicibility of ahpC by peroxidases in Mycobacterium smegmatis and lack of expression in M. aurum and M. tuberculosis.
J Bacteriol 1996; 178: 3641-49.
5. Wilson TM, Collins DM. ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex.
Mol Microbiol 1996; 19: 1025-34.
6. Banerjee A et al. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis.
Science 1994; 263: 227-30.
7. Phetsurksih B et al. Antimycobacterial activities odf isoxyl and new derivatives through the inhibition of mycolic acid sysnthesis.
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8. Miesel L et al. Mechanism for isoniazid action and resistance. Novartis Found Symp 1998; 217: 209-20.
9. Yuan Y et al. The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis.
Mol Microbiol 1998; 29(6): 1449-58.
10. Mdluli K et al. Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid.
Science 1998; 280(5369): 1607-10. 11. Mdluli K et al. Mechanisms involved in the intrinsic resistance of Mycobacterium avium.
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12. Rozwarski DA et al. Modification of the NADH of the isoniazid target (inhA) from Mycobacterium tuberculosis.
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13. Barry CE. New horizons in the treatment of tuberculosis. Biochem Pharmacol 1997; 54(11): 1165-72.
14. Bardou F et al. effect of isoniazid on ultrastructure of Mycobacterium aurum and Mycobacterium tuberculosis and on production of secreted proteins. Antimicrob Ag Chemother 1996; 40(11): 2459-67.
15. Wheeler PR and Anderson PM. Determination of the primary target for isoniazid in mycobacterial mycolic acid biosynthesis with Mycobacterium aurum A+.
Biochem J 1996; 318 (Pt 2): 451-7.
16. Quemard A et al. Certain properties of isoniazid inhibition of mycolic acid synthesis in cell-free systems of M. aurum and M. avium.
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17. Quemard A et al. Mycolic acid synthesis: a target for ethionamide in mycobacteria?
Antimicrob Ag Chemother 1992; 36(6): 1316-21. 18. Quemard A et al. Isoniazid inhibition of mycolic acid synthesis by cell extracts of sensitive and resistant strains of M. aurum.
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19. Tomiyasu I and Yano I. Isonicotinic acid hydrazide induced changes and inhibition in mycolic acid synthesis in Nocardia and related taxa.
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Amer Rev Resp Dis 1973; 105: 632-7.
21. Sorg TB and Cynamon MH. Comparison of four beta-lactamase inhibitors in combination with ampicillin against Mycobacterium tuberculosis.
J Antimicrob Chemother 1987; 19(1 ): 59-64.
22. Cynamon MH and Palmer GS. In vitro activity of.Amoxycillin in combination with Clavulanic acid against Mycobacterium tuberculosis.
Antimicrob Ag Chemother 1983; 24(3): 429-31.
23. Kirk SM et al. Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid and rifampin in 2 hours.
J Clin Microbiol 1998; 36(6): 1568-73.
24. Cooper CE et al. Effect of low concentrations of Clavulanic acid on the in vitro activity of Amoxycillin against beta-lactamase producing Branhamella catarrhalis and Haemophilus influenzae.
J Antimicrob Chemother 1990; 26(3): 371-80. 25. Jagannath C et al. Enhancement of drug susceptibility of multi-drug resistant strains of Mycobacterium tuberculosis by ethambutol and dimethyl sulfoxide.
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.
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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. Beta-lactamase inhibitors and the inducibility of the beta- lactamase of Mycobacterium tuberculosis.
Amer Rev Resp Dis 1992; 145(3): 657-60. 31. Segura C et al. Contribution of beta-lactamases to beta-lactam susceptibilities of susceptible and multi-drug resistant Mycobacterium tuberculosis clinical isolates.
Antimicrob Ag Chemother 1998; 42(6): 1524-26.
32. Randhawa B et al. Bactericidal action of oral ampicillin-sulbactam against Mycobacterium leprae.
J Antimicrob Chemother 1999; 44: 279-81.
33. Nakagawa Y et al. A study of beta-lactamase activity of mycobacteria and clinical trial of penicillin/beta-lactamase inhibitor combinations in the treatment of drug-resistant Mycobacterium tuberculosis.
Kekkaku 1999; 74(5): 447-52.
34. Nadler JP et al. Amoxycillin-clavulanic acid for treating drug-resistant Mycobacterium tuberculosis.
Chest 1991 ; 99(4): 1025-6.
35. Abate G et al. Drug resistance in Mycobacterium tuberculosis strains isolated from re-treatment cases of pulmonary tuberculosis in Ethiopia and susceptibility to first line and alternative drugs.
Int J Tuberc Lung Dis 1998; 2(7): 580-4.
36. Frederick A et al. The early bactericidal activity of Ciprofloxacin in patients with pulmonary tuberculosis.
Am J Resp Crit Care Med 1997; 156: 901-5.
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Microb Drug Resist 1999; 5(4): 265-70. 38. Goodman and Gilman's The Pharmacological Basis of Therpaeutics, 1992; 8th Ed., pp 1158.
39. Prabhakaran K. et al. Postantibiotic effect of ampicillin/sumbactam against mycobacteria.
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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.
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 INH. 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 sp 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:
Figure imgf000013_0001
INH 0.2 + Penicillin:
Figure imgf000014_0001
INH 1.0 + Penicillin:
Figure imgf000015_0001
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:
Figure imgf000017_0001
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
Figure imgf000018_0001
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.
Figure imgf000020_0001
Notes:
• NG: No growth
• All the drug concentrations are in μg/ml
• INH-lsoniazid, RIFA-Rifampicin, EMB-Etham.butol, SM-$treptomycin
Figure imgf000021_0001
Notes:
• NG: No growth
• All the drug concentrations are in μg/ml
• INH-lsoniazid, RIFA-Rifampicin, EMB-Ethambutol, SM-Streptomycin
Figure imgf000022_0001
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.

Claims

I claim:
1. Penicillin reverses resistance of mycobacterium to INH.
2. Penicillins, as claimed in claim 1 can be a broad spectrum penicillin or a narrow spectrum penicillin.
3. Penicillin, as claimed in claim 1 and 2 can be any single penicillin or combination of more than one penicillins.
4. Penicillin, as claimed in claim 1 to 3 can have activity against beta-lactamase producing organism.
5. Penicillin, as claimed in claim 1 to 3 can have activity against penicillinase producing organism.
6. Penicillins, as claimed in claim 1 to 3 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.
7. Mycobacterium, as claimed in claim 1 can be Mycobacterium tuberculosis.
PCT/IB2001/001136 2000-06-28 2001-06-26 Agent for reversal of drug resistance in mycobacterium tuberculosis WO2002000165A2 (en)

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