ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, p. 229-231

Vol. 34, No. 2

0066-4804/90/020229-03$02.00/0 Copyright © 1990, American Society for Microbiology

Comparative In Vitro Activities of 20 Fluoroquinolones against Mycobacterium leprae SCOTT G. FRANZBLAU* AND KENNETH E. WHITE Laboratory Research Branch, Gillis W. Long Hansen's Disease Center, Carville, Louisiana 70721 Received 7 August 1989/Accepted 13 November 1989 The in vitro activities of 20 fluoroquinolones against Mycobacterium kprae were evaluated by using the BACTEC 460 system. M. keprae was incubated in BACTEC 12B medium at 33°C under reduced oxygen for 2 to 3 weeks in the presence of fluoroquinolones at 0.31 to 5 Lg/ml. Activity was determined by a reduction in "4CO2 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.

Recent laboratory and clinical studies have demonstrated the potential of the fluoroquinolones in treating mycobacterial infections (12). While the number of new compounds continues to increase, only a few of these have been tested against Mycobacterium leprae in mice (1, 10, 14, 15; K. Kohsaka, Int. J. Lepr. 57[Suppl.]:316-317, 1989; S. R. Pattyn, Int. J. Lepr. 57[Suppl.]:381, 1989; S. Tsutsumi and M. Gidoh, Int. J. Lepr. 57[Suppl.]:383, 1989). Ciprofloxacin was found to be inactive, probably due to poor pharmacokinetics in mice (1, 10), while pefloxacin and ofloxacin demonstrated bactericidal activity when administered with sufficient frequency (10, 14, 15). Pefloxacin and ofloxacin were therefore selected for clinical trials against leprosy, with encouraging preliminary results (J. Grosset, C. C. Guelpa-Lauras, L. N'Deli, and E. Pasani, Int. J. Lepr. 57[Suppl.]:316, 1989; L. N'Deli, C. C. Guelpa-Lauras, and J. Grosset, Int. J. Lepr. 57[Suppl.]:317, 1989). Considering the significant associated toxicities of fluoroquinolones (17), it seems highly advisable to evaluate the numerous new derivatives which continue to be produced worldwide in order to identify those compounds with the best therapeutic indices for treating leprosy. However, until recently the only available drug-screening method for antileprosy agents was the lengthy and costly mouse footpad model. The cost and time required to evaluate most of the currently available compounds in the mouse footpad system might be prohibitive, especially since the fluoroquinolones seem to require administration by gavage. In addition, compounds might be eliminated from further consideration only because of poor pharmacokinetics in the (nonhuman) mouse model. Our recent development of in vitro radiorespirometric drug screening allows for screening of large numbers of compounds (4). We have employed radiorespirometry to evaluate a variety of macrolides (6) and clofazimine derivatives (7, 8). In addition, we have recently demonstrated that the BACTEC 460 system can also be used in the radiorespirometric assessment of drug activity against M. Ieprae in the absence of increases in cell numbers (5). Here, we used the BACTEC system to evaluate the comparative activities of 20 fluoroquinolones. *

MATERIALS AND METHODS Inoculum preparation. M. leprae was harvested from the footpads of nude mice as previously described (6, 7). Briefly, footpads containing approximately 1010 acid-fast bacilli were surface decontaminated with iodine and ethanol, minced with scissors, and homogenized in Middlebrook 7H9 broth containing 1% (wt/vol) bovine serum albumin. Tissue debris was removed by centrifugation (108 x g, 5 min, 10°C), and the bacilli were pelleted (2,710 x g, 45 min, 10°C) and suspended to a density of approximately 109/ml. Suspensions were treated with 50 ,ug of ampicillin per ml and 2.5 ,ug of amphotericin B per ml for contamination control, held at 4°C until used, and then diluted with Middlebrook 7H9 broth to 108/ml in a serum vial. Drugs. Temafloxacin, A-62412, A-63345, A-67937, and A-69137 (Abbott Laboratories, Abbott Park, Ill.); AT-4140 (Dainippon Seiyaku, Osaka, Japan); fleroxacin (HoffmannLa Roche, Nutley, N.J.); norfloxacin (Merck Sharp & Dohme, Rahway, N.J.); ciprofloxacin (Miles Laboratories, Inc., West Haven, Conn.); ofloxacin (Ortho Diagnostics, Inc., Raritan, N.J.); OPC-17100 and OPC-17066 (Otsuka Pharmaceutical, Tokushima, Japan); enoxacin, PD-117596, PD-124816, and PD-127391 (Parke, Davis & Co., Ann Arbor,

Mich.); pefloxacin (Rhone-Poulenc, Monmouth Junction, N.J.); amifloxacin and WIN-57273 (Sterling-Winthrop, Rensselaer, N.Y.); and T-3262 (Toyama, Tokyo, Japan) were gifts from the manufacturers and were solubilized at 2 mg/ml in NaOH or deionized water, according to the instructions of the manufacturers. The structures of these compounds are shown in Fig. 1. In some cases, concentrated solutions were stored at -20°C for 1 or 2 days before use. Drug solutions were filter sterilized and then diluted in Middlebrook 7H9 broth (Difco Laboratories, Detroit, Mich.). Susceptibility testing. BACTEC 12B medium (4 ml of 7H12 medium plus 4 p.Ci of [1-'4C]palmitic acid in a 20-ml serum vial; Becton Dickinson and Co., Towson, Md.) was inoculated with 107 M. leprae in 0.1 ml of Middlebrook 7H9 medium, and 0.1 ml of diluted drugs was added. Ampicillin and amphotericin B were added in a volume of 0.1 ml for control of occasional contaminants. Vials were flushed with 2.5% 02-10% CO2 (balance, N2) and incubated at 33°C, and the growth index ("'CO2 evolution) was measured at appropriate intervals with a BACTEC 460 instrument. Drugs at

Corresponding author. 229

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ANTIMICROB. AGENTS CHEMOTHER.

FRANZBLAU AND WHITE

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various concentrations were considered to be active if they effected a significant (Student's t test) reduction in the growth index during the reading interval of days 11 to 18 compared with that of drug-free controls. RESULTS With most of the fluoroquinolones, a clear dose response observed; the higher drug concentrations resulted in lower '4CO2 evolution (growth index). AT-4140, PD-127391, was

PD-124816, PD-117596, OPC-17100, OPC-17066, and WIN57273 were the most active compounds, all showing significant reduction (P