Lepr Rev (2011) 82, 253–258

The activity of several newer antimicrobials against logarithmically multiplying M. leprae in mice

JASMIN BURGOS, EDUARDO DE LA CRUZ, ROSE PAREDES, CORA REVELYN ANDAYA & ROBERT H. GELBER Leonard Wood Memorial Center for Leprosy Research, Cebu, Philippines

Accepted for publication 01 December 2010

Summary Introduction: Moxifloxacin, rifampicin, rifapentine, linezolid, and PA 824, alone and in combination, have been previously administered, as single doses and five times daily doses, to M. leprae infected mice during lag phase multiplication and were each found to have some bactericidal activity. Design: The fluroquinolones, ofloxacin, moxifloxacin and gatifloxacin, (50 mg/kg, 150 mg/kg and 300 mg/kg) and the rifamycins (5 mg/kg, 10 mg/kg, and 20 mg/kg), rifampicin and rifapentine, were evaluated alone and in combination for bactericidal activity against M. leprae using the mouse footpad model during logarithmic multiplication. Linezolid and PA 824 were similarly evaluated alone and linezolid in combination with rifampicin, minocycline and ofloxacin. Results: The three fluroquinolones and rifamycins were found alone and in combination to be bactericidal at all dosage schedules. PA 824 had no activity against M. leprae, while linezolid at a dose of 25 mg/kg was bacteriostatic, and progressively more bactericidal at doses of 50 mg/kg and 100 mg/kg. No antagonisms were detected between any of these drugs when used in combinations. Conclusion: Moxifloxacin, gatifloxacin, rifapentine and linezolid were found bactericidal against rapidly multiplying M. leprae.

Introduction

Antimicrobial therapy is the major medical intervention that favourably alters the course of disease in leprosy patients and the only means currently available to check its dissemination to others. Combinations of dapsone, clofazimine and rifampicin, are currently recommended by the WHO1 for treating leprosy patients. Rifampicin2–5 is the only component that is bactericidal for M. leprae. The WHO treatment regimens are used to treat nearly all leprosy patients and are provided by Novartis free of charge. Unfortunately, patients may

Correspondence to: Robert H. Gelber, 220 Scenic Avenue, San Anselmo, CA 94960 USA (Tel: þ415.454.8765; Fax: þ415.454.8191; e-mail: [email protected])

0305-7518/11/064053+06 $1.00 q Lepra 253 254 J. Burgos et al. experience intolerance, side effects and toxicity from each of the regimen’s components. The development of resistance in M. leprae6–8 to dapsone and rifampicin, and treatment failure9–12 is of concern. Therefore, alternative agents and more bactericidal combinations need to be tested, particularly if the duration of chemotherapy, can be shortened. In addition to the components of MDT, the number of available antimicrobials to treat leprosy is currently limited. Though pefloxacin/ofloxacin, minocycline and clarithromycin have been found in clinical trials13 –17 to be more active than dapsone2 and clofazimine,2 clinical application of these has been largely confined to the use of single dose rifampicin, ofloxacin and minocycline (ROM) for single lesion PB leprosy.1 We18 and others19 have found several fluroquinolones, including moxifloxacin, to be more active against M. leprae in mice than pefloxacin and ofloxacin, while moxifloxacin has proven more bactericidal in leprosy patients.20,21 Though there are studies utilising moxifloxacin,22,23 gatifloxacin,24,25 linezolid26 and PA 82427,28 in murine tuberculosis, these agents have only undergone limited evaluation19,29 in the mouse leprosy model and primarily in ‘stationary phase’ multiplication. Moxifloxacin,22,23 gatifloxacin,24,25 and linezolid26 are licensed for human use and are being used to treat several bacterial infections, while PA 824, a nitromidazofuran compound, is in drug development, is uniquely and substantially active against M. tuberculosis at low concentrations and is being developed primarily to treat the emerging problem of multidrug resistance tuberculosis. In order to confirm and expand upon the work on the aforementioned antimicrobials, herein we evaluated the activity of these agents alone and in combination against rapidly dividing M. leprae in the murine model.

Materials and Methods

The kinetic technique of Shepard was used.30 Untreated control and treated mice (CBA/j) were infected in both hind feet with 5,000 M. leprae derived from a skin biopsy taken from an untreated lepromatous leprosy patient, and treated from Day 60 to 150 after footpad infection. At Day 150 and at intervals thereafter mice (both hind feet) were harvested, and M. leprae therein enumerated. Growthwas considered to have occurredif $ 105 bacilli/footpad were found. Drugs were considered inactive on Day 150 if M. leprae growth occurred at the same rate as in untreated control mice, bacteriostatic if growth begins immediately upon drug discontinuation, and by previously utilised criteria,18 partially bactericidal if growth is further delayed and fully bactericidal if growth of M. leprae is not detected 9 months after the completion of therapy. In the first study we administered by gastric gavage moxifloxacin (Bayer Pharmaceuticals), gatifloxacin (Bristol-Meyers Squibb Company) and ofloxacin each at doses of 50 mg/kg, 150 mg/kg and 300 mg/kg five times weekly, as well as rifampicin and rifapentine (Aventis Incorporated) at 5 mg/kg, 10 mg/kg and 20 mg/kg once weekly. Also, to assess the potential for synergism, the three fluoroquinolones at doses of 50 mg/kg five times weekly were administered to groups of mice together with rifampicin or rifapentene 5 mg/kg once weekly. In the second study groups of mice were treated by gastric gavage with linezolid (Upjohn Company) at 25 mg/kg, 50 mg/kg and 100 mg/kg five times weekly, as well as rifampicin 5 mg/kg once weekly by gavage, dapsone 0·001% in diet, minocycline 0·01% in diet, and ofloxacin 50 mg/kg five times weekly by gavage. In order to assess the potential that linezolid provides additive activity when combined with other active agents, groups of mice were treated with linezolid at the lowest tested dose (25 mg/kg), together with other study Newer Antimicrobials against M. leprae 255 antimicrobial agents at the dosage schedules which had previously been established active but not fully bactericidal. In the third study, PA 824 was evaluated in groups of mice treated by gastric gavage five times weekly at dosages of 3·2 mg/kg, 6·3 mg/kg, 12·5 mg/kg, 25 mg/kg, 50 mg/kg and 100 mg/kg.

Results

M. leprae did not grow in any mice treated with the three dosage levels of oxfloxacin, gatifloxacin, moxifloxacin, rifampicin and rifapentine which were harvested at intervals up to nine or more months after the completion of therapy (Table 1). No M. leprae grew in mice treated with combinations of each of the three fluroquinolones and two rifamycins. Thus, by the criteria employed, the three regimens of all three fluroquinolones and the two rifamycins, as well as the combination, were fully bactericidal against logarithmically multiplying M. leprae. The findings of the second study are presented in Table 2. Multiplication of M. leprae in untreated control mice was detected 133 days after footpad infection. In all treated groups of mice M. leprae growth was found undetectable in seven or more mice at varying intervals beginning 120 days after footpad infection and prior to when growth was first detected or last found undetectable. Linezolid at a dosage of 25 mg/kg administered to mice five times weekly was found to be bacteriostatic, at 50 mg/kg found partially bactericidal and at 100 mg/kg fully bactericidal. The low dose of linezolid, rifampicin, minocycline and ofloxacin alone all permitted M. leprae multiplication, while in

Table 1. Study 1: fluoroquinolones and rifampycins

Mice demonstrating M. leprae multiplication/total number Days after M. leprae Treatment of mice studied Infection (Range)

No therapy 50/54 152–624 Moxifloxacin 50 mg/kg 0/10 161–425 Moxifloxacin 150 mg/kg 0/11 161–420 Moxifloxacin 300 mg/kg 0/10 161–440 Gatifloxacin 50 mg/kg 0/17 161–407 Gatifloxacin 150 mg/kg 0/11 161–561 Gatifloxacin 300 mg/kg 0/8 161–550 Ofloxacin 50 mg/kg 0/8 161–550 Ofloxacin 150 mg/kg 0/9 161–617 Ofloxacin 300 mg/kg 0/10 161–642 Rifampicin 5 mg/kg 0/8 161–481 Rifampicin 10 mg/kg 0/11 161–425 Rifampicin 20 mg/kg 0/11 161–624 Rifapentene 5 mg/kg 0/11 161–329 Rifapentene 10 mg/kg 0/11 161–379 Rifapentene 20 mg/kg 0/12 161–334 Moxifloxacin 50 mg/kg þ Rifampicin 5 mg/kg 0/11 161–621 Gatifloxacin 50 mg/kg þ Rifampicin 5 mg/kg 0/11 161–567 Ofloxacin 50 mg/kg þ Rifampicin 5 mg/kg 0/11 161–411 Moxifloxacin 50 mg/kg þ Rifapentene 5 mg/kg 0/11 161–494 Gatifloxacin 50 mg/kg þ Rifapentene 5 mg/kg 0/8 161–312 Ofloxacin 50 mg/kg þ Rifapentene 5 mg/kg 0/12 161–652 256 J. Burgos et al.

Table 2. Linezolid therapy

M. leprae growth detected (day after infection when growth first detected Treatment or last day growth found still undetectable)

No therapy þ (133) Linezolid 25 mg/kg þ (196) Linezolid 50 mg/kg þ (226) Linezolid 100 mg/kg 2 (225) Dapsone 0·001% in diet 2 (279) Rifampicin 5 mg/kg þ (201) Minocycline 0·01% in diet þ (257) Ofloxacin 50 mg/kg þ (238) Linezolid 25 mg/kg þ Dapsone 0·001% in diet 2 (336) Linezolid 25 mg/kg þ Rifampicin 5 mg/kg 2 (196) Linezolid 25 mg/kg þ Minocycline 0·01% in diet þ (371) Linezolid 25 mg/kg þ Ofloxacin 50 mg/kg 2 (267)

In all treated groups of mice M. leprae growth was found undetectable in seven or more mice at varying intervals beginning 120 days after footpad infection and prior to when growth was first detected or last found undetectable. mice treated with dapsone growth of M. leprae was not detected even in the final harvest at 279 days after footpad infection. In all instances the combination of linezolid and rifampicin, minocycline and ofloxacin resulted in no detectable M. leprae growth, while the combination of linezolid and dapsone resulted in detectable growth of M. leprae at a later time than dapsone alone. Thus, no antagonism between linezolid when combined with either dapsone, rifampicin, minocycline and ofloxacin was observed, and, in fact, for all combinations additive activity was suggested by our findings. In the third study at the completion of therapy (Day 150), M. leprae grew in control mice and all mice treated with PA 824 five times weekly at all dosage schedules (3·5 mg/kg to 100 mg/kg) – this demonstrates that PA 824 is inactive against M. leprae.

Discussion

We have demonstrated that moxifloxacin, gatifloxacin and linezolid are active against rapidly multiplying M. leprae in mice and bactericidal, gatifloxacin having not been evaluated previously for its activity against M. leprae. Also, during logarithmic multiplication we have confirmed that both rifampicin and rifapentene are bactericidal for M. leprae and that no antagonism between the three tested fluoroquinolones and the two rifamycins was observed. Though previously we18 had found by the criteria employed herein that ofloxacin 50 mg/kg was only partially bactericidal, while fully bactericidal at 150 mg/kg and 300 mg/kg, unfortunately in this study ofloxacin at 50 mg/kg was fully bactericidal, therefore we could not establish if equivalent doses of moxifloxacin and gatifloxacin were superior. In these studies a dose- response curve for linezolid activity against M. leprae was observed. While 25 mg/kg five times weekly was bacteriostatic, 50 mg/kg five times weekly was partially bactericidal and 100 mg/kg was fully bactericidal. Previously, Ji et al.29 had found in M. leprae infected mice treated with a single dose or five daily doses of linezolid 100 mg/kg one day after footpad infection that linezolid had very modest bactericidal activity, less than found previously for the bacteriostatic agent, dapsone. These current studies Newer Antimicrobials against M. leprae 257 demonstrate that linezolid at the highest dose studied, 100 mg/kg during active multiplication, results in activity which is fully bactericidal and that linezolid was not found antagonistic with established agents-dapsone, rifampicin, minocycline and ofloxacin, and in fact was found additive with each of these agents. It is noteworthy that a newer oxazolidinone congener of linezolid, PNU 100480, has been found to be significantly more potent against murine tuberculosis than linezolid itself,31 was well-tolerated in human volunteers and resulted in serum levels which were significantly more active against M. tuberculosis than those obtained by linezolid.32 In view of these findings we would recommend that PNU 100480 should be tested in M. leprae-infected mice. In these studies PA 824 was found inactive against rapidly multiplying M. leprae. Previously, Ji et al.29 found it modestly bactericidal in ‘stationary phase’ multiplication. Our results in rapidly multiplying M. leprae parallel those of Manjunatha et al.33 who found it inactive against M. leprae in the mouse model, which accords with their findings that M. leprae lacks the genes necessary to convert PA 824 to its active moiety. Combinations of antimicrobials found to be individually active against M. leprae when studied previously in combination have been regularly observed not to be antagonistic and generally additive34 – 36 in their antimicrobial activity in both mice and an immunosuppressed rat model, the neonatally thymectomized Lewis rat (NTLR). As prospects that a rifamycin and fluroquinolone might well in combination improve the treatment of leprosy has been proposed, 20-21 it is particularly noteworthy in our previous studies that when rifampicin and sparfloxacin were combined in the treatment of mouse leprosy additive antimicrobial activity was observed35 and in the NTLR ofloxacin added to the activity of rifampicin.36 Both moxifloxacin and gatifloxacin, both representative of a newer unique class of fluoroquinolones, 8-methoxyquinolones, have proved more active against M. tuberculosis in mice than other quinolones; furthermore their bactericidal activity approximates that of rifampicin. Consequently both moxifloxacin and gatifloxacin are currently being evaluated as components of the initial treatment for active pulmonary tuberculosis, with the goal to even further shorten the duration required for treatment and, also in regimens to treat multi-drug resistant tuberculosis. Herein, we have confirmed the profound bactericidal activity of moxifloxacin in mice against rapidly multiplying M. leprae that Consigny et al.19 had found against stationary M. leprae. Thus, it is not surprising that in clinical trials of moxifloxacin in lepromatous leprosy M. leprae viability was lost very rapidly and at a rate only previously found by rifampicin,20,21 while in those studies leprosy infiltration, also resolved most rapidly. These findings in clinical trials suggest that a rifamycin/moxifloxacin based regimen presents the promise of a superior regimen to treat leprosy.

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