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

Abstract

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).Clofazimine [3-(p-chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-(isopropylimino)phenazine; B663] has been used effectively in treating leprosy for over 25 years (2). The World Health Organization recommends its use along with rifampin and dapsone in multidrug therapy of lepromatous leprosy (8). Major advantages of B663 include low host toxicity (16); suppression of erythema nodosum leprosum (10, 11), a reactive state in leprosy; and few reports of bacterial resistance. The single most common drawback of B663 is a dose-related reddish-black skin pigmentation, making its continued use objectionable to a number of patients. The most serious and dose-limiting toxicity is gastrointestinal (16).Recently, a series of phenazines were synthesized. They share a number of properties, including activity against clofazimine-resistant Mycobacterium smegmatis 607 and lack of uptake by body fat (and thus nonpigmentation) (13). We studied the relative activities of these compounds against Mycobacterium leprae in vitro to determine structure-activity relationships with a view to ultimately finding a less toxic substitute for clofazimine in treating leprosy.Unlike that for in vitro-cultivable microorganisms, drug susceptibility testing of M. leprae has, until recently, been possible only in vivo. The established mouse footpad model is expensive and time-consuming, taking 6 to 12 months to complete, requiring gram quantities of new compounds for maximum-tolerated-dose determinations, and relying upon favorable pharmacokinetics in a nonhuman system (8). Therefore, a number of in vitro systems have been devised which involve exposure of intracellular or extracellular M. leprae to test compounds followed by measurement of some bacillary metabolic activity (6-9, 12, 15). Although these systems appear to be useful, most are somewhat cumbersome for large-scale screening or clinical use. We therefore recently used a simpler, semiautomated, radiorespirometric assay for rapidly assessing the metabolic status of M. leprae (4). This assay is based on the oxidation of palmitic acid to carbon dioxide and is similar to that used for rapid drug * Corresponding author. susceptibility testing of cultivable mycobacteria in the BACTEC method (14). We describe here its use in investigating structure-activity relationships of a number of phenazine derivatives against M. leprae.Susceptibility testing was performed by a slight modification of the method of Franzblau (4). Briefly, footpads of M. leprae-infecte...

Comparative in vitro activities of 20 fluoroquinolones against Mycobacterium leprae

“…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).…”

“…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.…”

Drug susceptibility testing of Mycobacterium leprae in the BACTEC 460 system

“…Among these was the use of a Buddemeyer-type detection system to measure 14CO2 production from [1-14C]palmitic acid by M. leprae in an axenic medium (1). This system is very sensitive, and its simplicity has facilitated screening of a variety of macrolides (3), clofazimine derivatives (4,5), and fluoroquinolones (S. G. Franzblau ) consists of (final concentrations) polymyxin B (50 U/ml), amphotericin B (5 pg/ml), nalidixic acid (20 pug/ml), trimethoprim (5 jig/ml), and azlocillin (10 ,ug/ml). Susceptibility testing.…”

Fusidic acid is highly active against extracellular and intracellular Mycobacterium leprae

“…We have previously demonstrated the utility of radiorespirometry in evaluating the in vitro activities of a variety of macrolides (9), fluoroquinolones (11), phenazines (10,12), and aminoglycosides, lincosamides, and rifamycins (7) against M. leprae in axenic media with either a Buddemeyertype detection system (5) or the BACTEC 460 instrument (6) as a means of assessing drug activity in the absence of bacterial multiplication. We and our colleagues have also used assays quantitating phenolic glycolipid-1 synthesis and adenosine triphosphate concentration (8) as indices of drug activity against both extracellular (16) and intracellular (20) M. leprae.…”

“…In vitro screening for drugs active against the uncultivable leprosy bacillus can be performed by radiorespirometric analysis of viable Mycobacterium leprae freshly harvested from nude mice (5)(6)(7)(9)(10)(11)(12). In vitro systems have many advantages over the traditional mouse footpad system (22), especially with regard to time, expense, quantity of drug required, and independence of mouse pharmacokinetics.…”