New drugs for the treatment of tuberculosis: hope and reality.

The objective of this review is to report evidence about the efficacy and potential of currently licensed drugs and new molecules beyond pre-clinical development for improving the chemotherapy of tuberculosis (TB). Rifapentine, a rifamycin with low minimum inhibitory concentration, long half-life and potent sterilizing activity in mice did not confirm its potential in a recent short-term clinical trial and is being extensively re-evaluated. Moxifloxacin, a fluoroquinolone, improved the activity of the standard drug regimen when substituted for ethambutol (EMB). It is being studied to shorten the duration of treatment for fully drug-susceptible TB (Remox study). Clofazimine, a fat-soluble dye with experimental activity against TB, but used only for leprosy in the last 50 years, requires further study because it has been included in a successful short 9-month combined drug regimen for the treatment of multidrug-resistant TB. The diarylquinoline TMC207 is the most promising among the new TB drugs because of its experimental and clinical rate of culture conversion. Also exciting, 200 mg daily doses in humans of the nitroimidazo-oxazine PA-824 and the nitro-dihydro-imidazooxazole OPC-67683 were safe and induced a bactericidal effect of respectively 0.098 ± 0.072 log(10) and 0.040 ± 0.056 log(10) per day. The new oxazolidinones PNU-100480 and AZD-5847 might be at least as active as linezolid and much less toxic. SQ109 is an EMB analogue that does not have cross-resistance with EMB and might have synergistic activity in combined regimens. Benzothiazinones and dinitrobenzamides show exciting in vitro anti-microbial activity and deserve careful attention.

Promising antituberculosis activity of the oxazolidinone PNU-100480 relative to that of linezolid in a murine model.

Oxazolidinone antibiotics have activity against Mycobacterium tuberculosis. Linezolid, the only marketed oxazolidinone, has been used off-label in combination regimens to treat multidrug-resistant tuberculosis, but its precise contribution to the efficacy of such combinations is unclear. Another oxazolidinone, PNU-100480, has been demonstrated to have more potent activity in vitro and in a murine model of tuberculosis. In this study, we compared the pharmacokinetics and the antituberculosis activities of these two oxazolidinones over a range of doses and found that linezolid has limited activity at clinically relevant doses in the murine model compared to that of PNU-100480, which has potent bactericidal activity, even at lower drug exposures. These findings were unexpected, given the similar in vitro activities of PNU-100480, its major metabolites, and linezolid. Moreover, the incorporation of PNU-100480 dramatically improved the bactericidal activities of regimens containing current first-line antituberculosis drugs and moxifloxacin. For example, the addition of PNU-100480 (100 mg/kg of body weight/day) to the standard daily regimen of rifampin (rifampicin), isoniazid, and pyrazinamide resulted in an additional 2.0-log(10)-unit reduction in lung CFU counts during the first 2 months of treatment. The combination of PNU-100480, moxifloxacin, and pyrazinamide, which does not contain either rifampin or isoniazid, was also more active than rifampin, isoniazid, and pyrazinamide. These results suggest that PNU-100480 may have the potential to significantly shorten the duration of therapy for drug-susceptible as well as multidrug-resistant tuberculosis.

Paucibacillary tuberculosis in mice after prior aerosol immunization with Mycobacterium bovis BCG.

To develop a murine model of paucibacillary tuberculosis for experimental chemotherapy of latent tuberculosis infection, mice were immunized with viable Mycobacterium bovis BCG by the aerosol or intravenous route and then challenged six weeks later with virulent Mycobacterium tuberculosis. The day after immunization, the counts were 3.71 +/- 0.10 log(10) CFU in the lungs of aerosol-immunized mice and 3.65 +/- 0.11 and 4.93 +/- 0.07 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Six weeks later, the lungs of all BCG-immunized mice had many gross lung lesions and splenomegaly; the counts were 5.97 +/- 0.14 and 3.54 +/- 0.07 log(10) CFU in the lungs and spleens of aerosol-immunized mice, respectively, and 4.36 +/- 0.28 and 5.12 +/- 0.23 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Mice were then aerosol challenged with M. tuberculosis by implanting 2.37 +/- 0.13 log(10) CFU in the lungs. Six weeks after challenge, M. tuberculosis had multiplied so that the counts were 6.41 +/- 0.27 and 4.44 +/- 0.14 log(10) CFU in the lungs and spleens of control mice, respectively. Multiplication of M. tuberculosis was greatly limited in BCG-immunized mice. Six weeks after challenge, the counts were 4.76 +/- 0.24 and 3.73 +/- 0.34 log(10) CFU in the lungs of intravenously immunized and aerosol-immunized mice, respectively. In contrast to intravenously immunized mice, there was no detectable dissemination to the spleen in aerosol-immunized mice. Therefore, immunization of mice with BCG by the aerosol route prior to challenge with a low dose of M. tuberculosis resulted in improved containment of infection and a stable paucibacillary infection. This model may prove to be useful for evaluation of new treatments for latent tuberculosis infection in humans.

Genomics and the chemotherapy of leprosy.

The information deduced from the genome sequence of Mycobacterium leprae is of immense value for the chemotherapy of leprosy. Knowing the complete set of genes, enzymes and proteins allows us to understand why some drugs are without effect whereas others are fully active. It may also enable better use to be made of existing drugs, such as beta-lactams, and opens new avenues for the development of novel compounds. M. leprae is relatively susceptible to a wide range of drugs, unlike the highly related tubercle bacillus, and several new multidrug regimens are in clinical trials. Genomics provides a number of possible explanations for this broader susceptibility as some of the genes encoding enzymes involved in antibiotic inactivation have decayed whereas the number of transporters available to contribute to drug efflux is considerably lower than in Mycobacterium tuberculosis. Several leads for new drug targets have been uncovered.

A method for rapid detection of rifampicin-resistant isolates of Mycobacterium leprae.

A genotypic method for predicting rifampicin resistance in Mycobacterium leprae has been developed and rigorously tested on mouse footpad-derived and clinical specimens. A series of immobilized oligonucleotide capture probes can discriminate between wild type and mutant rpoB alleles, and positive controls are available for the most frequent mutation affecting Ser425. Two different non-radioactive detection formats have been tested with comparable success in both an industrialized and a developing country. The standardized procedure could now be used in a prospective study of potential rifampicin resistance among multibacillary patients.

Newer drugs in leprosy.

During the last 15 years, new drugs active against Mycobacterium leprae have been identified. All of them belong to the fluoroquinolone, cycline and macrolide drug families. In the mouse model and in humans, minocycline, ofloxacin, and clarithromycin have demonstrated, individually or in combination, antileprosy activities much superior to those of the standard drugs dapsone and clofazimine. In humans, a single dose of the combination ofloxacin 400 mg + minocycline 100 mg was able to kill 68% to 98% of viable M. leprae and a single dose of ROM, a three-drug combination of rifampin 600 mg + ofloxacin 400 mg + minocycline 100 mg, was killing more than 99% of viable M. leprae. As a result of a double-blind, control, clinical trial, the Seventh Report of the WHO Expert Committee on Leprosy recommended in 1997 the use of single-dose ROM for the treatment of patients with single-lesion paucibacillary leprosy. Recently moxifloxacin, a new fluoroquinolone, and rifapentine, a long-lasting rifamycin derivative, have demonstrated in the mouse model highly promising antileprosy activities, justifying their assessment in humans.

Chemoprophylaxis of leprosy in the southern Marquesas with a single 25 mg/kg dose of rifampicin. Results after 10 years.

In 1988, a programme of leprosy chemoprophylaxis, employing a supervised, single 25 mg/kg dose of rifampicin, was implemented in the Southern Marquesas Islands. Of the 2786 inhabitants, 2751 (98.7%) were treated. In addition, 3144 South Marquesans living elsewhere in French Polynesia were administered the same chemoprophylaxis. During the following 10 years, seven leprosy patients were detected among those who had been administered chemoprophylaxis. Of these, two were very likely missed cases of leprosy, and cannot be considered a failure of chemoprophylaxis. The epidemiometric projection model, based on cases of leprosy observed in the Southern Marquesas during the 20 years preceding implementation of the programme, predicted that 17 leprosy cases could be expected in the South Marquesan population if no chemoprophylaxis were given. In fact, only five cases were detected in the treated population, a number significantly smaller than 17, suggesting that the chemoprophylaxis was 70% effective, assuming that no change of detection rate would have occurred without chemoprophylaxis. However, during the 10 years following implementation of the chemoprophylaxis programme, the detection rate in the Polynesian population that was not administered chemoprophylaxis declined by about 50%. Therefore, the effectiveness of the chemoprophylaxis was only 35-40%.

Clarithromycin resistance and susceptibility patterns of Mycobacterium avium strains isolated during prophylaxis for disseminated infection in patients with AIDS.

A randomized, placebo-controlled trial was conducted to evaluate the efficacy of clarithromycin in the prevention of disseminated Mycobacterium avium complex (MAC) infection in patients with AIDS; special attention was given to the development of clarithromycin resistance. The median time to documented MAC bacteremia was 199 days for placebo-treated patients, 217 days for clarithromycin-treated patients infected with clarithromycin-susceptible MAC, and 385 days for clarithromycin-treated patients infected with clarithromycin-resistant MAC. Most of the patients with clarithromycin-resistant isolates (91%) had a baseline CD4 T-cell count of < 20/microL, while these low counts occurred in only 25% of patients having clarithromycin-susceptible breakthrough isolates. The emergence of clarithromycin resistance did not affect the total period of survival. Resistance to clarithromycin in breakthrough MAC isolates emerges most likely when the patient is extremely immunodeficient at the time of initiation of the preventative therapy.

High relapse rate among lepromatous leprosy patients treated with rifampin plus ofloxacin daily for 4 weeks.

Fifty-one lepromatous leprosy patients, all of whom had relapsed after previous dapsone (DDS) monotherapy, were treated between 1990 and 1991 with 600 mg of rifampin (RMP) plus 400 mg of ofloxacin (OFLO) daily for 4 weeks, and the great majority of the patients were followed up at least once a year after completion of the treatment. After only 173 patient-years of follow-up, 5 relapses had been detected; the overall relapse rate was 10.0% (confidence limits, 1.7 and 18.3%), or 2.9 relapses (confidence limits, 0.4 and 5.4) per 100 patient-years. The unacceptably high relapse rate indicated that 4 weeks of treatment with daily RMP-OFLO was unable to reduce the number of viable Mycobacterium leprae organisms to a negligible level. In addition, the M. leprae from one of the relapses were proved to have multiple resistance to DDS, RMP, and OFLO. To avoid further relapses, the follow-up was terminated and the great majority of the patients were retreated with the standard 2-year multidrug therapy from 1994. No further relapse has been diagnosed since the beginning of retreatment.

Field trial on efficacy of supervised monthly dose of 600 mg rifampin, 400 mg ofloxacin and 100 mg minocycline for the treatment of leprosy; first results.

In 1995, a field trial was implemented in Senegal in order to evaluate the efficacy of a regimen based on the monthly supervised intake of rifampin 600 mg, ofloxacin 400 mg and minocycline 100 mg to treat leprosy. During the first year of the trial, 220 patients with active leprosy (newly detected or relapsing after dapsone monotherapy) were recruited: 102 paucibacillary (PB) (60 males and 42 females) and 118 multibacillary (MB) (71 males and 47 females). All of them accepted the new treatment (none requested to be preferably put under standard WHO/MDT), no clinical sign which could be considered as a toxic effect of the drug was noted, and none of the patients refused to continue treatment because of any clinical trouble. The compliance was excellent: the 113 patients (PB and MB) detected during the first 6 months of the trial have taken six monthly doses in 6 months, as planned. The rate of clearance and the progressive decrease of cutaneous lesions was satisfactory. Although it is too soon to give comprehensive results, it should be noted that no treatment failure was observed in the 56 PB patients who have completed treatment and have been followed up for 6 months. The long-term efficacy of the new regimen is to be evaluated on the rate of relapse during the years following the cessation of treatment. If that relapse rate is acceptable (similar to that observed in patients after treatment with current standard WHO/ MDT), the new regimen could be a solution to treat, for instance, patients very irregular and/or living in remote or inaccessible areas since no selection of rifampin-resistant Mycobacterium leprae should be possible (a monthly dose of ofloxacin and minocycline being as effective as a dose of dapsone and clofazimine taken daily for 1 month). Nevertheless, until longer term results of this and other trials become available, there is no justification for any change in the treatment strategy, and all leprosy patients should be put under standard WHO/MDT.

Bactericidal activity of single dose of clarithromycin plus minocycline, with or without ofloxacin, against Mycobacterium leprae in patients.

Fifty patients with newly diagnosed lepromatous leprosy were allocated randomly to one of five groups and treated with either a month-long standard regimen of multidrug therapy (MDT) for multibacillary leprosy, a single dose of 600 mg of rifampin, a month-long regimen with the dapsone (DDS) and clofazimine (CLO) components of the standard MDT, or a single dose of 2,000 mg of clarithromycin (CLARI) plus 200 mg of minocycline (MINO), with or without the addition of 800 mg of ofloxacin (OFLO). At the end of 1 month, clinical improvement accompanied by significant decreases of morphological indexes in skin smears was observed in about half of the patients of each group. A significant bactericidal effect was demonstrated in the great majority of patients in all five groups by inoculating the footpads of mice with organisms recovered from biopsy samples obtained before and after treatment. Rifampin proved to be a bactericidal drug against Mycobacterium leprae more potent than any combination of the other drugs. A single dose of CLARI-MINO, with or without OFLO, displayed a degree of bactericidal activity similar to that of a regimen daily of doses of DDS-CLO for 1 month, suggesting that it may be possible to replace the DDS and CLO components of the MDT with a monthly dose of CLARI-MINO, with or without OFLO. However, gastrointestinal adverse events were quite frequent among patients treated with CLARI-MINO, with or without OFLO, and may be attributed to the higher dosage of CLARI or MINO or to the combination of CLARI-MINO plus OFLO. In future trials, therefore, we propose to reduce the dosages of the drugs to 1,000 mg of CLARI, 100 mg of MINO, and 400 mg of OFLO.

Bactericidal activities of combinations of new drugs against Mycobacterium leprae in nude mice.

The bactericidal activities of 12 regimens with various combinations of new drugs (clarithromycin [CLARI], minocycline [MINO], and ofloxacin [OFLO]) and the standard antileprosy drugs, especially rifampin (RMP), were compared in nude mice with established Mycobacterium leprae infection. The longest duration of treatment was 24 weeks for intermittent (once every 4 weeks) therapy and 8 weeks for daily therapy. Bactericidal effects were monitored by titrating the proportion of viable M. leprae isolates by subinoculating the organisms into the footpads of immunocompetent and nude mice. The results indicate that RMP was more bactericidal than any combination of the new drugs. A single dose of CLARI-MINO, with or without OFLO, displayed bactericidal activity as great as that of 4 weeks of daily treatment with dapsone (DDS) plus clofazimine (CLO); thus, intermittent CLARI-MINO, with or without OFLO, may replace DDS and CLO of the standard multidrug regimen, and these will become regimens that can be administered monthly and under full supervision. Additional evidence that this may be the case is provided by the finding that intermittent RMP-CLARI-MINO or RMP-CLARI-MINO-OFLO administered for 12 or 24 weeks was as active as the standard multidrug regimen. While the intermittent treatment always displayed significantly greater bactericidal activity than the same number of doses of daily treatment, daily treatment with CLARI-MINO and CLARI-MINO-OFLO were more active than the drugs given as intermittent treatment for the same duration; therefore, unless these combinations are to be administered together with intermittent RMP, they should be given daily, especially for the treatment of RMP-resistant cases of infection. Finally, 12 weeks of daily treatment with DDS-CLO was more bactericidal than had been expected, suggesting that it may not be necessary to administer the standard multidrug regimen for multibacillary leprosy for as long as 24 months in order to minimize the risk of developing RMP resistance.

Clarithromycin is inactive against Mycobacterium tuberculosis.

When 10% oleic acid-albumin-dextrose-catalase-enriched Mueller-Hinton agar medium was employed, the MICs of clarithromycin (CLARI) at which 50 and 90% of 12 strains of Mycobacterium tuberculosis were inhibited were 64 and > 128 micrograms/ml, respectively, which are significantly greater than the achievable peak CLARI concentrations in serum and in lung tissue in humans. In two different mouse experiments, 4 to 6 weeks of treatment with CLARI at 200 mg/kg of body weight six times weekly produced neither bactericidal nor bacteriostatic effects against M. tuberculosis. Therefore, we conclude that CLARI as a single drug is inactive against M. tuberculosis.

Preventive therapy of tuberculosis with rifapentine in immunocompetent and nude mice.

The effectiveness of intermittent administration of rifapentine (RPT), with or without isoniazid (INH), for preventive therapy of tuberculosis was evaluated in immunocompetent (normal) and nude mice. After infection with a small inoculum of Mycobacterium tuberculosis H37Rv, normal mice developed a chronic and nonfatal infection, and the bacterial population became relatively stable after an initial period of limited multiplication. On the other hand, nude mice developed an acute and fatal infection, and all untreated mice died within 5 wk, with very high colony-forming-unit (CFU) counts in their organs. Various degrees of bactericidal activity were shown in normal mice after daily treatment with rifampin (RMP) plus pyrazinamide (PZA) for 13 wk, INH daily for 26 wk, or RPT once weekly for 13 or 26 wk or once fortnightly for 26 wk. The activity of RPT was significantly enhanced when INH was added at the same dosing frequency. In nude mice the response of M. tuberculosis infection to certain regimens was less favorable than that in normal mice, suggesting that preventive therapy may be less effective in severely immunodeficient hosts even during treatment. After chemotherapy was stopped, virtually all nude mice relapsed within 12 wk regardless of the regimen administered, whereas no or very few relapses were observed in normal mice that had been treated with RMP+PZA daily for 13 wk, or RPT alone or RPT+INH once weekly for 26 wk. The latter three regimens and RPT+INH once weekly for 13 wk may be applied for fixed-duration preventive therapy in human immunodeficiency virus (HIV)-negative subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Implications of multidrug resistance for the future of short-course chemotherapy of tuberculosis: a molecular study.

Tuberculosis-control programmes are compromised by the increased frequency of multidrug-resistant strains of Mycobacterium tuberculosis. We used the polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis techniques to establish the molecular basis of resistance in 37 drug-resistant isolates of M tuberculosis, and correlated these findings with clinical and antibiotic-sensitivity data. Resistance to isoniazid was found in 36 strains, 16 of which were also resistant to ethionamide. Of the 36 isoniazid-resistant strains, 23 had mutations in the katG gene, and 5 of these also had mutations in the inhA gene. A further 5 strains had alterations in the inhA locus without the katG gene being mutated. Rifampicin resistance was less frequent (13 strains) and usually associated with isoniazid resistance (11 of 13 strains). Mutations in the rpoB gene were detected for all these rifampicin-resistant isolates. Mutations in the rpsL and rrs genes, associated with streptomycin resistance, were found in 13 of 25 and 2 of 25 streptomycin-resistant strains, respectively. The same chromosomal mutations, or combinations of mutations, were found in strains displaying single or multidrug resistance, from cases of both primary and secondary resistance, and from patients infected with human immunodeficiency virus. Thus, multidrug resistance is not due to a novel mechanism and tuberculosis chemotherapy is not subject to a new threat.