Drug targeting Mycobacterium tuberculosis cell wall synthesis: genetics of dTDP-rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-rhamnose.

An L-rhamnosyl residue plays an essential structural role in the cell wall of Mycobacterium tuberculosis. Therefore, the four enzymes (RmlA to RmlD) that form dTDP-rhamnose from dTTP and glucose-1-phosphate are important targets for the development of new tuberculosis therapeutics. M. tuberculosis genes encoding RmlA, RmlC, and RmlD have been identified and expressed in Escherichia coli. It is shown here that genes for only one isotype each of RmlA to RmlD are present in the M. tuberculosis genome. The gene for RmlB is Rv3464. Rv3264c was shown to encode ManB, not a second isotype of RmlA. Using recombinant RmlB, -C, and -D enzymes, a microtiter plate assay was developed to screen for inhibitors of the formation of dTDP-rhamnose. The three enzymes were incubated with dTDP-glucose and NADPH to form dTDP-rhamnose and NADP(+) with a concomitant decrease in optical density at 340 nm (OD(340)). Inhibitor candidates were monitored for their ability to lower the rate of OD(340) change. To test the robustness and practicality of the assay, a chemical library of 8,000 compounds was screened. Eleven inhibitors active at 10 microM were identified; four of these showed activities against whole M. tuberculosis cells, with MICs from 128 to 16 microg/ml. A rhodanine structural motif was present in three of the enzyme inhibitors, and two of these showed activity against whole M. tuberculosis cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth of M. tuberculosis in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 microg/ml.

Effective treatment of acute and chronic murine tuberculosis with liposome-encapsulated clofazimine.

The therapeutic efficacy of liposomal clofazimine (L-CLF) was studied in mice infected with Mycobacterium tuberculosis Erdman. Groups of mice were treated with either free clofazimine (F-CLF), L-CLF, or empty liposomes twice a week for five treatments beginning on day 1 (acute), day 21 (established), or day 90 (chronic) postinfection. One day after the last treatment, the numbers of CFU of M. tuberculosis in the spleen, liver, and lungs were determined. F-CLF at the maximum tolerated dose of 5 mg/kg of body weight was ineffective; however, 10-fold-higher doses of L-CLF demonstrated a dose response with significant CFU reduction in all tissues without any toxic effects. In acutely infected mice, 50 mg of L-CLF/kg reduced CFU 2 to 3 log units in all three organs. In established or chronic infection, treated mice showed no detectable CFU in the spleen or liver and 1- to 2-log-unit reduction in the lungs. A second series of L-CLF treatments cleared M. tuberculosis in all three tissues. L-CLF appears to be bactericidal in the liver and spleen, which remained negative for M. tuberculosis growth for 2 months. Thus, L-CLF could be useful in the treatment of tuberculosis.

Minocycline in lepromatous leprosy.

Twelve patients were treated with three dose levels of minocycline for 30 days, primarily to detect the dose-related effects on Mycobacterium leprae viability, followed by another 5 months of daily minocycline for overall efficacy and persistence of clinical and antibacterial effects. Subsequently, the patients were given standard WHO/MDT chemotherapy for multibacillary leprosy. Clinical improvement was recognizable during the first month, occurring much earlier among those on minocycline 200 mg daily than those who received minocycline 100 mg daily. A similar change also was observed in one patient 11 days after three daily doses of 100 mg of minocycline. At the end of 6 months, all patients were clinically improved with a slight reduction in the average bacterial index (BI) and logarithmic index of bacilli in biopsy (LIB). The effects of minocycline on viability by mouse foot pad inoculation and palmitic acid oxidation assays were noted beginning at 10 to 14 days of daily dosing and becoming more definite after 30 days of treatment. Both tests correlated fairly well. Doses of 200 mg daily did not appear to be more efficient than minocycline 100 daily. Phenolic glycolipid-I (PGL-I) antigen determinations done on some patients during the first month remained positive and did not correlate with changes in viability results. At the end of 6 months, after 5 months of 100 mg of minocycline monotherapy, no viable organisms could be demonstrated by mouse foot pad inoculation and palmitic acid oxidation assays; assays for PGL-I antigen were all negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical trial of fusidic acid for lepromatous leprosy.

Fusidic acid was assessed for antileprosy activity in nine lepromatous leprosy patients. Patients received fusidic acid at either 500 mg/day for 12 weeks or 750 mg/day for 4 weeks followed by 500 mg/day for 8 weeks. All patients showed time-dependent clinical improvement and decreases in bacillary morphological index, radiorespirometric activity and PCR signal, and in serum phenolic glycolipid I. Fusidic acid appears to be a weakly bactericidal antileprosy agent which may have a role in the multidrug treatment of leprosy pending an evaluation of lepra-reaction-suppressive activity.

Clinical trial of clarithromycin for lepromatous leprosy.

Clarithromycin was administered to nine previously untreated lepromatous leprosy patients. Patients received two 1,500-mg doses on the first day, followed by 7 days of no treatment, in order to evaluate the potential efficacy of intermittent therapy. Patients then received 1,000 mg daily for 2 weeks followed by 500 mg daily for 9 weeks. The efficacy of therapy was monitored clinically, by changes in morphological index, mouse footpad infectivity, and radiorespirometric activity of Mycobacterium leprae obtained from serial biopsies and by serum levels of phenolic glycolipid I. Clarithromycin was well tolerated, with only minor side effects noted in two patients. Most patients showed reductions in morphological index and radiorespirometry 1 week after the first two doses. Within 3 weeks of starting treatment (total of 17 g of clarithromycin), biopsy-derived M. leprae specimens from all patients had a morphological index of zero, were noninfectious for mice, and had less than 1% of the radiorespirometric activity of pretreatment specimens. Reductions in serum phenolic glycolipid I levels were observed for most patients at 3 weeks. Significant clinical improvement was evident after 4 weeks of treatment. All analyses indicate that clarithromycin is rapidly bactericidal for M. leprae in humans.

Clinical trial of sparfloxacin for lepromatous leprosy.

Nine previously untreated patients with lepromatous leprosy were treated with 200 mg of sparfloxacin daily for 12 weeks to determine whether this drug is bactericidal for Mycobacterium leprae in humans. The efficacy of therapy was monitored both clinically and by measuring changes in morphological index, mouse footpad infectivity, and the radiorespirometric activity of M. leprae organisms obtained from serial biopsy specimens and also by determining titers of phenolic glycolipid-I in serum. Most patients showed clinical improvement within 2 weeks of treatment; this was accompanied by significant reductions in the morphological index, mouse footpad infectivity, and bacillary radiorespirometric activity. After 4 weeks of treatment, all patients had a morphological index of zero and specimens from most patients were noninfectious for mice, while the median decrease in radiorespirometric activity was > 99%. Overall results by the rapid radiorespirometric assay paralleled those of the mouse footpad and morphological index assays. Sparfloxacin given at 200 mg once daily appears to be rapidly bactericidal in humans, with activity similar to that observed in a previous clinical trial with 400 mg of ofloxacin.

Sparfloxacin is more bactericidal than ofloxacin against Mycobacterium leprae in mice.

The comparative bactericidal activities of sparfloxacin and ofloxacin against Mycobacterium leprae in mice were determined using the proportional bactericidal test at doses of 12.5 mg/kg-100 mg/kg. Significant bactericidal activity was found at 12.5 mg/kg sparfloxacin and 25 mg/kg ofloxacin. Sparfloxacin was significantly more bactericidal than ofloxacin at all doses, and the results with 25 mg/kg sparfloxacin were nearly identical to those obtained with 100 mg/kg ofloxacin. These results, together with pharmacokinetic and toxicological data in mice and man, suggest that sparfloxacin may have a higher therapeutic index than ofloxacin in leprosy, and that the tentative standard dosage of 200 mg sparfloxacin daily should be appropriate for a clinical trial.

Double-blind evaluation of BACTEC and Buddemeyer-type radiorespirometric assays for in vitro screening of antileprosy agents.

Two radiorespirometric assays, the BACTEC 460 and Buddemeyer-type 14CO2 detection systems, were evaluated in a double-blind manner for their ability to discriminate between authentic antileprosy agents and inactive compounds. Freshly harvested, nude-mouse derived Mycobacterium leprae were incubated in axenic media in the presence of coded test solutions prepared in a remote laboratory. Activity was assessed by comparing the rate of 14CO2 evolution from [1-14C]palmitic acid to controls. Breaking the code revealed that both systems demonstrated a dose response to ethionamide, pefloxacin and rifampicin as well as sensitivity to dapsone. Most of the water, ethanol, sucrose, dabsyl chloride and riboflavin negative-control samples failed to effect a significant reduction in radiorespirometric activity. This study confirms the ability of the radiorespirometric assays to function as a primary drug screening system in leprosy.

Fusidic acid is highly active against extracellular and intracellular Mycobacterium leprae.

The activity of fusidic acid against Mycobacterium leprae was studied in axenic medium and in bacilli residing within mouse peritoneal macrophages. Activity was assessed by subsequent quantitation of bacillary radiorespirometric activity. Significant inhibition in both systems was observed at 0.156 micrograms/ml, and an approximately 50% reduction in activity occurred after exposure to 1.25 to 2.5 micrograms/ml. The excellent human pharmacokinetics and in vitro activity of fusidic acid against the leprosy bacillus warrant a clinical trial of this drug for leprosy.

Effects of activated macrophages on Mycobacterium leprae.

Five alternative methods were used to explore in vitro the effects of normal and activated murine macrophages on the metabolic well-being of intracellular Mycobacterium leprae: fluorescein diacetate-ethidium bromide staining, ATP content, synthesis of phenolic glycolipid 1, and two techniques to quantitate oxidation of palmitic acid. In relatively short-term experiments (7 to 10 days), each of these procedures provided strong evidence that activated macrophages exerted a deleterious effect on the leprosy bacillus. These findings appear to confirm the contention that activated macrophages underlie host resistance to clinical leprosy and limitation of M. leprae growth in paucibacillary leprosy.

L-arginine-dependent macrophage effector functions inhibit metabolic activity of Mycobacterium leprae.

Recently, L-arginine has been shown to be a necessary substrate for murine-activated macrophage-mediated tumor cytostasis and microbiostasis of certain fungi, bacteria, and intracellular protozoa. We report here the effects of the L-arginine-dependent pathway of activated mouse macrophages (MO) on the obligate intracellular prokaryote, Mycobacterium leprae. Due to the inability to culture M. leprae in vitro, a simple, quantitative assay was employed to measure the metabolism/viability of M. leprae released from MO: the metabolic capacity of M. leprae to oxidize 14C-palmitic acid to 14CO2. Murine normal MO or MO activated in vitro with IFN-gamma or in vivo by injection with Corynebacterium parvum were infected with viable M. leprae freshly harvested from the footpads of nu/nu mice. Activated MO strikingly inhibited the metabolism of M. leprae; however, in L-arginine-free medium or in medium containing L-arginase, the inhibitory effects of activated MO on M. leprae metabolism were abolished. The competitive inhibitor of L-arginine, NG-monomethyl-L-arginine, also blocked the inhibitory effects of activated MO for M. leprae, but the addition of supplemental L-arginine overcame the NG-monomethyl-L-arginine-induced block. Furthermore, in the culture supernatants, the levels of NO2-, an end product of L-arginine degradation, were directly proportional to the ability of the activated MO to inhibit M. leprae metabolism. These data present five lines of evidence that suggest that activated MO utilize the L-arginine-dependent pathway to cope with M. leprae.

In vitro activities of aminoglycosides, lincosamides, and rifamycins against Mycobacterium leprae.

The in vitro activities of a variety of aminoglycosides, lincosamides, and rifamycins against Mycobacterium leprae were evaluated with the BACTEC 460 system. At 20 micrograms/ml, gentamicin, kanamycin, tobramycin, streptomycin, and amikacin were inactive. Lincomycin was active at 20 micrograms/ml, and clindamycin was active at 0.31 micrograms/ml. Rifamycin SV, rifabutin, and rifampin were active at 3.1, 3.1 to 12.5, and 200 ng/ml, respectively. The in vitro assay correlates well with the in vivo response of M. leprae to antimicrobial agents, with the exception of the aminoglycosides.

Comparative in vitro activities of 20 fluoroquinolones against Mycobacterium leprae.

The in vitro activities of 20 fluoroquinolones against Mycobacterium leprae were evaluated by using the BACTEC 460 system. M. leprae was incubated in BACTEC 12B medium at 33 degrees C under reduced oxygen for 2 to 3 weeks in the presence of fluoroquinolones at 0.31 to 5 micrograms/ml. Activity was determined by a reduction in 14CO2 evolution compared with that of drug-free controls. Of the commercially available agents, ofloxacin was most active, while enoxacin and norfloxacin were inactive. However, a number of newer fluoroquinolones (AT-4140, OPC-17100, OPC-17066, PD-117596, PD-124816, PD-127391, and WIN-57273), all containing a cyclopropyl group at R-1 and, with the exception of WIN-57273, either a halogen or methyl group at R-8, were more active than ofloxacin in vitro. Further in vivo evaluations of these agents should help determine their potential for use against leprosy.

Drug susceptibility testing of Mycobacterium leprae in the BACTEC 460 system.

The susceptibility of Mycobacterium leprae to clinical and experimental antileprosy agents was assessed in the BACTEC 460 system. Nude-mouse-derived M. leprae (10(7) cells), incubated in BACTEC 12B medium at 33 degrees C under reduced oxygen, maintained a fairly constant growth index (14CO2 evolution) for 2 to 3 weeks. At concentrations ranging from 0.031 to 2.0 micrograms/ml, dapsone, rifampin, clofazimine, ethionamide, ofloxacin, clarithromycin, and minocycline all effected reductions in the growth index within 1 to 2 weeks, the extent of inhibition increasing with the incubation time. An in vivo rifampin-resistant isolate displayed markedly reduced susceptibility to rifampin compared with an in vivo-susceptible strain. This system appears to be highly suitable for in vitro drug susceptibility testing of M. leprae.

Structure-activity relationships of tetramethylpiperidine-substituted phenazines against Mycobacterium leprae in vitro.

In a previous study of structure-activity relationships of selected phenazines against Mycobacterium leprae in vitro, compounds containing a 2,2,6,6-tetramethylpiperidine substitution at the imino nitrogen were most active. Therefore, the effect of substitution at the para positions of the phenyl and anilino groups in tetramethylpiperidine-substituted phenazines was assessed. As determined by radiorespirometry, activity in ascending order was observed in compounds substituted with hydrogens or fluorines, ethoxy groups, methyl groups, chlorines, and bromines and correlated with partition coefficients in octanol-water.

In vitro and in vivo activities of macrolides against Mycobacterium leprae.

We previously demonstrated the potent in vitro activity of erythromycin against Mycobacterium leprae as determined by its effect on ATP pools and rates of palmitate oxidation and phenolic glycolipid I synthesis. In the present study, the relative in vitro activities of a number of new macrolides with superior pharmacokinetic properties were assessed. In addition, for the most active compounds, concentrations in serum were determined by bioassay during continuous administration in the feed of mice, and in vivo activity against M. leprae was assessed by the kinetic mouse footpad technique. Both clarithromycin and roxithromycin were more potent than erythromycin in vitro, with the former showing the highest activity in accelerating rates of ATP decay and reducing rates of palmitate oxidation. In mice, concentrations of clarithromycin in serum were higher than those of roxithromycin and erythromycin, with the latter undetectable even when administered at 0.1% (wt/wt) in the diet. When administered at 0.01% (wt/wt) in the diet, erythromycin and roxithromycin were unable to inhibit growth of M. leprae in mouse footpads whereas clarithromycin demonstrated bactericidal-type activity. On the basis of these data and other properties of macrolides, a clinical trial of clarithromycin in leprosy is warranted.

Inhibition of phenolic glycolipid-I synthesis in extracellular Mycobacterium leprae as an indicator of antimicrobial activity.

The effects of 22 antimicrobial agents on the incorporation of [U14C] palmitic acid ([U14C] PA) into the unique phenolic glycolipid-I (PGL-I) antigen of Mycobacterium leprae were studied. Nude-mouse-propagated M. leprae were incubated in a modified Dubos medium in the presence of antimicrobial agents for 4 days. [U14C] PA was then added and incubation was continued for 8 days. The antileprosy agents dapsone, rifampin, and clofazimine (2 micrograms/ml each) caused a significant reduction in [U14C] PA incorporation into PGL-I. Among other agents, the most active were erythromycin, chloramphenicol, and cerulenin. Low concentrations of ethionamide, tetracycline, and minocycline stimulated label incorporation. This system may prove useful in the evaluation of antileprosy agents.

Structure-activity relationships of selected phenazines against Mycobacterium leprae in vitro.

Structure-activity relationships of phenazines against Mycobacterium leprae were investigated by using an in vitro radiorespirometric assay. In general, activity in ascending order was observed in compounds containing no chlorine atoms, a monochlorinated phenazine nucleus, and chlorines in the para positions of both the anilino and phenyl rings. The most active compounds contained a 2,2,6,6-tetramethylpiperidine substitution at the imino nitrogen. Most of these chlorinated phenazines were considerably more active in vitro than clofazimine (B663).

Leprosy.

Leprosy affects over 10 million people in the world. The disease is a model of graded cell-mediated immunity, in this case to the causative organism, Mycobacterium leprae. The clinical manifestations are due to (i) bacterial progression, (ii) immunologic responses of the host, (iii) peripheral nerve damage due to either or both bacterial progression and immunologic responses of the host, and (iv) preventable secondary deformities following nerve damage, which account for most of the stigma of the disease. Treatment modalities are now available to control or minimize the effects of bacterial progression, harmful immunologic responses of the host, peripheral nerve damage, and secondary deformities. Unique biochemical characteristics of M. leprae reside in the cell wall and associated macromolecules. Some of these molecules are potent immunogens in humans, while others constitute the structural integrity of the bacillus. Proteins of M. leprae are currently under intensive investigation as a result of deoxyribonucleic acid cloning of M. leprae genes. Structure-function and antigenic relationships of M. leprae proteins should become available by using recombinant deoxyribonucleic acid procedures coupled with T- and B-cell cloning to advance our understanding of the immunologic reactions encountered in Hansen's disease. Until recently, the study of the immunology of leprosy has been stymied by the lack of immunologically specific M. leprae antigens. The definition of specific antigens and production of recombinant and synthetic immunologic reagents have fostered state-of-the-art research efforts into new immunodiagnostic procedures and development of a leprosy vaccine. Also discussed is progress in understanding of the mechanism(s) underlying the M. leprae-specific immunodeficiency associated with lepromatous leprosy, including the role of suppressor T cells and defective macrophage function. Metabolic studies of M. leprae suggest intact catabolic pathways and energy generation with purine bases and catalase as possible growth factors. Special attention may also need to be given to biophysical parameters for eventual in vitro cultivation. Rapid in vitro systems, using quantitation of bacillary metabolic activity, may soon replace the lengthy mouse footpad test for determining the viability and drug susceptibility of the leprosy bacillus.

Biophysical optima for metabolism of Mycobacterium leprae.

The metabolic response of freshly harvested, nude-mouse-derived Mycobacterium leprae to biophysical parameters was studied to facilitate an understanding of axenic culture requirements. Quantitation of intracellular ATP and the rate of [U-14C]palmitic acid incorporation into phenolic glycolipid I (PGL-I) were used as metabolic indicators after axenic incubation in modified Dubos medium under various biophysical conditions. PGL-I synthesis was optimal at 33 degrees C, whereas ATP was optimally maintained at less than or equal to 33 degrees C. Both metabolic indices showed sharp reductions at 37 degrees C. After 5 days of incubation, PGL-I synthesis and ATP maintenance showed pH optima of 5.1 to 5.6, with the higher value appearing optimal for ATP maintenance after extended incubation. Metabolic activity was negatively affected by strong reducing agents, and ATP maintenance was optimal when the gaseous environment was maintained at 2.5 to 10% oxygen. The results may partially explain the failure to cultivate the leprosy bacillus in vitro.