Search for newer antileprosy drugs.

In 1991 World Health Organization proclaimed the goal of global elimination of leprosy as a public health problem by year 2000 by implementing multidrug therapy (MDT). Since then the prevalence rate has declined by 85%. However, during the same period the incidence rate of leprosy has remained constant or even has been increasing. This suggests that it will take a long time for the eradication of leprosy and that without in-vitro cultivation of M. leprae, eradication of leprosy is not likely to be achieved. While in-vitro cultivation is a long-term goal, as an immediate measure, there is an urgent need for the development of newer drugs and newer multidrug therapy regimens. Using the in-vitro system for screening potential antileprosy drugs and also using the mouse foot-pad system we have evaluated several compounds in four classes of drugs--dihydrofolate reductase inhibitors, fluoroquinolones, rifampicin analogues and phenazines--and identified at least two compounds that appear to be more potent than dapsone, rifampicin and clofazimine. Newer combinations of rifampicin analogues and fluoroquinolones have also been identified that seem to be better than the combination of rifampicin and ofloxacin.

ATP content of Mycobacterium tuberculosis grown in vivo and in vitro.

In order to determine the reason for the slow growth of Mycobacterium leprae either in a host or in vitro, the growth characteristics of Mycobacterium tuberculosis were studied. The ATP content of in vitro-grown M. tuberculosis was about 520 pg/10(6) viable organisms. The ATP levels from in vivo-derived organisms obtained from liver and spleen of mice was about 130 pg (in cases of chronic infection) and about 270 pg (in cases of acute infection). When the in vivo-derived organisms were inoculated into culture medium, the growth rates for both types of organisms, acute as well as chronic infection, were the same and the maximum growth was reached during the fifth subculture. Although the maximum ATP content for both types of organism was the same, it was attained during the 4th subculture for organisms obtained during acute infection and during the 6th subculture for those obtained during chronic infection. The comparison between the ATP content of M. leprae and of M. tuberculosis indicates the reason for the slow growth of M. leprae.

In vitro activity of epiroprim, a dihydrofolate reductase inhibitor, singly and in combination with brodimoprim and dapsone, against Mycobacterium leprae.

The antimicrobial effects of a new dihydrofolate reductase inhibitor, epiroprim, alone and in combination with dapsone and brodimoprim against Mycobacterium leprae were evaluated in vitro in cell-free culture system. Two biochemical parameters were used to measure metabolic activity (and growth) of the organism. The minimal inhibitory activity of epiroprim against M. leprae was 10 mg/l and the action was bactericidal. When combined with dapsone, epiroprim exhibited a strong synergism; on the other hand, combination of epiroprim and brodimoprim provided only additive effects. The results suggest that epiroprim can be a component in multidrug therapy regimen in leprosy.

In vitro and in vivo activity of K-130, a dihydrofolate reductase inhibitor, against Mycobacterium leprae.

The antimicrobial effects of a new dihydrofolate reductase inhibitor, K-130 (2,4-diaminodiphenyl sulfone substituted 2,4-diamino-5-benzylpyrimidine), alone and in combination with dapsone (CAS 80-08-0) against both dapsone-sensitive and dapsone-resistant strains of Mycobacterium leprae were evaluated in vitro, in cell-free culture system, and in vivo, in mouse foot pads. The minimal inhibitory concentration of K-130 against dapsone-sensitive as well as dapsone resistant strains of M, leprae was 0.03 microgram/ml, and the activity was bactericidal in both cases. However, when combined with dapsone, K-130 exhibited synergism in case of dapsone-sensitive M. leprae, while in case of dapsone-resistant M. leprae, the effect was merely additive. Similar synergistic effects were also observed in the mouse foot pad system for both types of M. leprae strains.

In vitro activities of phenothiazine-type calmodulin antagonists against Mycobacterium leprae.

Calmodulin-like protein has been established as the primary receptor for calcium in eukaryotic as well as prokaryotic cells. The calmodulin-calcium complex regulates a variety of enzymes including nucleotide phosphodiesterase. Recently, the presence of this protein in Mycobacterium leprae has been demonstrated and the effects of phenothiazine-type calmodulin antagonists on in vitro growth of M. leprae in a cell-free culture system were investigated. Two biochemical parameters were used to measure metabolic activity and growth of the organism. Among the six phenothiazine derivatives tested, trifluoperazine appeared to be the most potent in inhibiting the in vitro growth of M. leprae, with an MIC of 10 micrograms/ml. Chlorpromazine, triflupromazine and thioridazine were less active than trifluoperazine, with an MIC of 20 micrograms/ml each, while the other two, acetopromazine and fluphenazine, were totally ineffective even at 80 micrograms/ml. All four compounds inhibited the uptake of labelled acetate, glycine and thymidine by whole cells of M. leprae. This suggests that these phenothiazine derivatives have multiple sites of action and probably affect the synthesis of lipids, proteins and DNA.

Factors influencing the in vitro growth of Mycobacterium leprae: effect of inoculum.

Factors responsible for the in vitro growth of Mycobacterium leprae in Dhople-Hanks (DH) medium, and also to improve the technique devised earlier, and the source of the M. leprae used as inoculum, were investigated. M. leprae were obtained from armadillos and nude mice, both inoculated earlier with human- or armadillo-derived M. leprae. The growth of M. leprae in DH medium was monitored using two biochemical indicators. Normal growth was obtained when inocula were from livers and spleens of M. leprae-infected armadillos. The M. leprae harvested from the footpads of nude mice failed to multiply in the same medium. Using inocula from livers and spleens of infected armadillos, a gradual decrease in inoculum size resulted in a proportionately slower multiplication of M. leprae. When the DH medium was supplemented with whole M. leprae, or cell-free extracts of M. leprae, from irradiated livers and spleens of infected armadillos, nude mouse-derived M. leprae exhibited growth in the DH medium in accord with that obtained using armadillo-derived M. leprae. Similar results were obtained with cell-free extracts of M. leprae harvested from non-irradiated livers and spleens of infected armadillos, but no growth was obtained when the medium was supplemented with extracts from livers or spleens of normal armadillos. These results indicate the possible existence of a growth factor in armadillo-derived M. leprae.

The activity of rifabutin against Mycobacterium leprae in armadillos.

The activity of rifabutin (LM 427) against Mycobacterium leprae was evaluated in armadillos inoculated earlier with human-derived M. leprae. Rifabutin was administered daily at a dose of 6 mg/kg body weight/day. The effect of rifabutin on M. leprae harvested from armadillos was determined by measuring the intracellular levels of ATP (an indicator of metabolic activity) of M. leprae and also their ability to multiply in the mouse footpads and in vitro in DH medium. Within 2 weeks of initiating the treatment, ATP levels declined to 21% of the original (pre-treatment level) and these M. leprae failed to multiply in the footpads of mice as well as in the in vitro culture system. This suggests that rifabutin was able to kill all M. leprae within 2 weeks. After 8 weeks the treatment was terminated and results showed that M. leprae from the treated armadillos remained non-viable in the mouse footpad system as well as in the in vitro system, indicating bactericidal action of rifabutin. The results suggest that rifabutin can be a substitute for rifampin in the leprosy multi-drug therapy regimen.

Comparative in vitro activities of rifamycin analogues against Mycobacterium leprae.

Comparative activities of various rifamycin analogues against leprosy were studied by evaluating their effects on in vitro growth of Mycobacterium leprae in DH medium as described earlier. Among the seven analogues studied, KRM-1648 was found to be the most potent in inhibiting the growth of rifampicin-sensitive strains of M. leprae, MIC being 0.05 microgram/ml. This was followed by KRM-2312 and T9 (MIC of each being 0.1 microgram/ml) and rifabutin (MIC, 0.2 microgram/ml). Rifampicin, along with KRM-1657 and KRM-1668, were least effective, with MIC for each being 0.4 microgram/ml. The effects of each at their respective MICs were bactericidal. The results were similar for rifampicin-resistant strains of M. leprae, but the MICs were higher than those obtained with rifampicin-sensitive strains of M. leprae. Thus, even though rifampicin has been the first-line drug in the treatment of leprosy, the results in present studies suggest that other rifamycin analogues are available that are more potent than rifampicin against both rifampicin-sensitive as well as rifampicin-restraint strains of M. leprae.

In vitro activity of a new rifamycin derivative against Mycobacterium leprae.

The antimicrobial effects of T9 (3-(4-cinamyl-1-piperazinyl)iminomethyl rifamycin SV) alone and in combination with ofloxacin, against strains of M. leprae were evaluated, using an in vitro cell-free culture system. The minimum inhibitory concentrations (MICs) of T9 against rifampin-sensitive and rifampin-resistant strains of M. leprae were 0.1 microgram/ml and 0.4 microgram/ml, respectively. Furthermore, in common with rifabutin, but not with rifamycin, T9 demonstrated synergy with ofloxacin against both rifampin-sensitive rifampin-resistant strains of M. leprae. The results suggest that T9, in combination with ofloxacin as part of multidrug regimens, warrants further evaluation as treatment for patients with leprosy.

In vitro susceptibility of Mycobacterium leprae to oxygen-mediated damage.

In order to evaluate factors responsible for the failure of Mycobacterium leprae to multiply in cell-free cultures in vitro studies were undertaken to determine the possible poisoning of the organism by hydroxide and superoxide radicals produced in the growth medium. The superoxide dismutase activity was very low, 10% of the levels found in armadillo cells, while measured activity of catalase and glutathione peroxidase was negligible. Susceptibility of M. leprae to hydrogen peroxide was enhanced by potassium iodide but not by lactoperoxidase. The addition of high amounts of catalase completely prevented hydrogen peroxide-mediated killing of M. leprae. Superoxide generated by the action of xanthine oxidase on xanthine was lethal to M. leprae, but superoxide dismutase added to the reaction mixture gave significant protection. Thus superoxide radicals may be a major cause for the sudden termination of growth of M. leprae in primary cultures and also for failure of subcultures.

In vivo activities of novel benzoxazinorifamycins against Mycobacterium leprae.

Among the four newly synthesized benzoxazinorifamycin derivatives, KRM-1648 and KRM-2312 completely inhibited the multiplication of rifampicin-sensitive as well as rifampicin-resistant strains of M. leprae in the foot-pads of mice. Both were found to be more potent than rifampicin and were bactericidal. In combination with ofloxacin, another potent bactericidal drug against M. leprae, both KRM-1648 and KRM-2312 exhibited synergism. Thus, combination of one of these benzoxazinorifamycin derivatives and ofloxacin in multidrug regimens is worth evaluating in clinical trials.

In vitro activity of levofloxacin, singly and in combination with rifamycin analogs, against Mycobacterium leprae.

The in vitro susceptibility of Mycobacterium leprae to levofloxacin was studied by using two biochemical parameters to measure the metabolic activity of the organism. Levofloxacin consistently exhibited twofold greater bactericidal activity than ofloxacin, with the MIC being 0.75 microgram/ml. When combined with one of the three rifamycin analogs, synergism was obtained with KRM-1648 and rifabutin but not with rifampin.

The in-vitro activities of novel benzoxazinorifamycins against Mycobacterium leprae.

The activities of four newly synthesized benzoxazinorifamycin derivatives, either alone or in combination with ofloxacin, against strains of Mycobacterium leprae were determined by assessing their effects on two biochemical parameters of metabolic activity which served as surrogate markers for growth in vitro. KRM-1648 and KRM-2312 were the most active agents tested against both a rifampicin-susceptible isolate (MICs of 0.05 and 0.1 mg/L respectively) and a rifampicin-resistant isolate (MICs of 0.2 and 0.3 mg/L respectively); both compounds were more active than either rifampicin or rifabutin. The activities of the two other derivatives, KRM-1657 and KRM-1668, against a rifampicin- susceptible strain (MICs of 0.3 mg/L) were similar to that of rifampicin, while the MIC of each of these agents for the rifampicin-resistant strain was 1.0mg/L. In common with rifabutin, both of the more active derivatives demonstrated synergy with ofloxacin against the rifampicin-susceptible isolates. The results of this study suggest that these compounds, in combination with ofloxacin as part of multidrug regimens, warrant further evaluation as treatment for patients with leprosy.

In vitro activities of 2,2'-bipyridyl analogs against Mycobacterium leprae.

In vitro susceptibility of Mycobacterium leprae to two bipyridyl analogs was studied by using two biochemical parameters to measure the metabolic activity of the organism. VUF-8514 at 0.16 micrograms/ml, but not VUF-8842, completely inhibited the metabolic activity of M. leprae, and the action was bactericidal. When compared to rifampin (MIC 0.3 micrograms/ml), VUF-8514 was equally bactericidal against M. leprae.

In vivo susceptibility of Mycobacterium leprae to ofloxacin either singly or in combination with rifampicin and rifabutin. Anti-leprosy activity of ofloxacin and ansamycins in mice.

The antimicrobial effects of ofloxacin against Mycobacterium leprae, either alone or in combination with rifampicin and rifabutin, were studied using mouse foot pad assay technique. When used singly, the minimum concentrations of the drugs needed to completely inhibit the growth of Mycobacterium leprae in foot pads of mice were 50 mg/kg body weight for ofloxacin and 0.003% and 0.0001%, respectively, for rifampicin and rifabutin. However, excellent synergistic effects were observed when mice were fed with 25 mg/kg body weight of ofloxacin along with 0.00003% rifabutin, but not rifampicin. Thus, incorporation of ofloxacin and rifabutin in the multiple drug therapy of leprosy patients is suggested.

In-vitro activity of three new fluoroquinolones and synergy with ansamycins against Mycobacterium leprae.

The efficacy of three fluorinated quinolones, clinafloxacin (PD 127391), sparfloxacin (PD 131501) and PD 131628, either alone or in combination with rifampicin/rifabutin, against Mycobacterium leprae was evaluated in vitro using two biochemical parameters to measure the metabolic activity of the organism. Clinafloxacin was found to be most effective with an MIC of 0.75 mg/L, followed by sparfloxacin (MIC 1.5 mg/L) and PD131628 (MIC 3.0 mg/L). When combined with rifampicin each of the three quinolones were additive to the activity. However, when combined with rifabutin, both clinafloxacin and sparfloxacin demonstrated pronounced synergic activity. Incorporation of clinafloxacin and rifabutin in a multi-drug therapy regimen is suggested.

In vitro synergistic activity between ofloxacin and ansamycins against Mycobacterium leprae.

The antimicrobial effects of ofloxacin, alone and in combination with either rifampicin or rifabutin, were evaluated against M. leprae, using in vitro cell-free culture system. The minimum inhibitory concentration (MIC) of ofloxacin against M. leprae was 1.5 micrograms/ml, while MICs of rifampicin and rifabutin were 0.4 and 0.2 microgram/ml, respectively. Combination of 0.375 microgram/ml ofloxacin and 0.05 microgram/ml of rifabutin exhibited synergistic bactericidal activity while the effect of combination of 0.75 microgram/ml ofloxacin and 0.2 microgram/ml rifampicin was additive bactericidal. Thus, combination of ofloxacin and rifabutin deserves further attention in multi-drug therapy of leprosy.

Utility of beige mouse in leprosy research.

Dissemination of M. leprae to visceral organs is seen by four months onwards only in beige (C57BL/6/bgj) but not BALB/c mice following intravenous or intraperitoneal infections. Inoculation of the beige mouse derived M. leprae showed all the characteristics of M. leprae, including growth pattern in the foot-pads of BALB/c mice. M. leprae inoculated into foot-pads of beige mice multiplied faster than those in the foot-pads of BALB/c mice. The possibility of using beige mouse in chemotherapeutic studies in leprosy is discussed.

Isolation of cultivable mycobacteria from feces and lungs of armadillos infected with Mycobacterium leprae.

In the past, no cultivable mycobacteria were isolated from armadillos captured in the state of Florida, U.S.A. But recent findings of acid-fast bacilli (AFB) in the lungs of armadillos infected with Mycobacterium leprae prompted us to undertake this study to determine the correlation between systemic leprosy infection and the occurrence of cultivable mycobacteria in the lungs and stools of these animals. No AFB could be isolated from noninfected animals. Seventy percent of the infected animals developed disseminated infection, but no cultivable mycobacteria were isolated from their livers and spleens. However, cultivable mycobacteria were isolated from the lungs and stools of a large number of armadillos showing disseminated infection. The most common among these were M. gordonae, M. fortuitum, and M. avium. There was a close correlation between the development of disseminated leprosy infection and the occurrence of cultivable mycobacteria in their lungs and stools, perhaps due to the decline in the immune system in these animals in the later stages of infection.