Preclinical pharmacokinetics of KBF611, a new antituberculosis agent in mice and rabbits, and comparison with thiacetazone.

Thiacetazone (TAZ), one of the oldest known antituberculosis drugs, causes severe skin reactions in patients co-infected with tuberculosis and human immunodeficiency virus (HIV). KBF611 is a new fluorinated thiacetazone analogue that has shown strong antituberculosis effects. In order to provide valuable information for subsequent preclinical development, pharmacokinetics of KBF611 and its analogue (TAZ) were studied and compared in two animal species (mice and rabbits) following intravenous and oral administration, and pharmacokinetic parameters were characterized. According to the calculated parameters, KBF611 showed a more favourable pharmacokinetics profile than TAZ in terms of half-life (0.89 h compared with 0.57 in mice, p < 0.05, and 2.71 compared with 0.98 in rabbits, p < 0.001) and volume of distribution (1.45 l kg(-1) compared with 0.86 l kg(-1) in mice, p < 0.05, and 1.01 l kg(-1) compared with 0.41 l kg(-1) in rabbits, p < 0.001) for tuberculosis therapy. In rabbits, the oral bioavailability of KBF611 was markedly lower than mice (39% compared with 82%), which may be attributed to a higher presystemic metabolism in rabbit liver. The results of in vivo studies on the metabolism of KBF611, supported by liquid chromatography-mass spectrometry (LC-MS) analysis, showed that the incorporation of a fluorine atom to the TAZ structure made the molecule susceptible to N-deacetylation, a pathway not seen in TAZ metabolism. In summary, KBF611 could be considered a suitable candidate for further preclinical and clinical evaluation.

Human flavin-containing monooxygenase 2.1 catalyzes oxygenation of the antitubercular drugs thiacetazone and ethionamide.

The second-line antitubercular drugs thiacetazone (TAZ) and ethionamide (ETA) are bioactivated by the mycobacterial enzyme EtaA. We report here that human flavin-containing monooxygenase 2.1 (FMO2.1), which is expressed predominantly in the lung, catalyzes oxygenation of TAZ. The metabolites generated, the sulfenic acid, sulfinic acid, and carbodiimide derivatives, are the same as those produced by EtaA and human FMO1 and FMO3. Two of the metabolites, the sulfenic acid and carbodiimide, are known to be harmful to mammalian cells. FMO2.1 also catalyzes oxygenation of ETA, producing the S-oxide. We have developed a novel spectrophotometric assay for TAZ oxygenation. The assay was used to determine kinetic parameters for TAZ oxygenation catalyzed by human FMO1, FMO2.1, and FMO3 and by EtaA. Although the K(M) values for the four enzyme-catalyzed reactions are similar, k(cat) and, consequently, k(cat)/K(M) (the specificity constant) for FMO2.1-catalyzed TAZ oxygenation are much higher than those of FMO1, FMO3, or EtaA. This indicates that FMO2.1 is more effective in catalyzing TAZ oxygenation than are the other three enzymes and thus is likely to contribute substantially to the metabolism of TAZ, decreasing the availability of the prodrug to mycobacteria and producing toxic metabolites. Because of a genetic polymorphism, Europeans and Asians lack FMO2.1. However, in sub-Saharan Africa, a region in which tuberculosis is a major health problem, a substantial proportion of individuals express FMO2.1. Thus, our results may explain some of the observed interindividual differences in response to TAZ and ETA and have implications for the treatment of tuberculosis in sub-Saharan Africa.

Isocratic high-performance liquid chromatographic determination of thiacetazone by direct injection of plasma into an internal surface reversed-phase column.

This report describes the determination of thiacetazone in human and rat plasma by direct-injection high-performance liquid chromatography (HPLC). Plasma filtrate (50 microliters) was injected directly into the internal surface reversed-phase (ISRP) mixed-functional phenyl column (Capcell Pak, 50 x 4.6 mm, 5 microns) and eluted with an aqueous mobile phase containing 7.5% acetonitrile at a flow-rate of 1 ml/min. With UV detection at 322 nm, thiacetazone eluted at 11.0 min whereas endogenous interferences eluted before 5 min. The lower detection limit for a 50-microliter sample at a signal-to-noise ratio of 5 was 63 ng/ml, which was several hundred fold lower than its cytotoxic concentrations determined from in vitro cell line studies. At a concentration range of 0.17 to 2.7 micrograms/ml, the recovery of thiacetazone was 98.0 +/- 4.4% (mean +/- S.D.). The intra- and inter-day coefficients of variation were 3.0 +/- 1.4% and 4.2 +/- 2.1%, respectively. This method was successfully applied to study the pharmacokinetics of thiacetazone in rats. The direct injection method is simple, requires small sample volume and does not require sample extraction, internal standard, or gradient elution.

Pharmacokinetic evaluation of thiacetazone.

STUDY OBJECTIVE: To investigate the steady-state pharmacokinetics of thiacetazone (TB-1), which is active in vitro against Mycobacterium avium complex (MAC). DESIGN: Open-label phase I study. SETTING: Specialized referral hospital. PATIENTS: Twelve healthy men and women. INTERVENTIONS: Oral TB-1 150 mg/day was administered for 7 days, followed by blood and urine collection over 48 hours. MEASUREMENTS AND MAIN RESULTS: The serum concentration versus time curves of TB-1 showed sustained concentrations, with maximum values of 1.59 +/- 0.47 micrograms/ml, time to maximum 3.30 +/- 1.18 hours, and serum half-life of 15-16 hours. Less than 25% of TB-1 was recovered unchanged in the urine over 48 hours. Rashes occurred in two subjects at the end of the 7-day dosing period and resolved without progression or sequelae. CONCLUSIONS: Based on these data, we initiated a phase II study of TB-I in patients with pulmonary MAC infection who do not have the acquired immunodeficiency syndrome.

Characteristics of a cytosolic arylacylamidase metabolizing thiacetazone.

The N-deacetylation of thiacetazone, an antitubercular drug possessing hepatotoxic side effects, by an exclusively cytosolic arylacylamidase has been identified in the liver and kidney of rat by monitoring the appearance of its metabolite p-aminobenzaldehydethiosemicarbazone spectrophotometrically. Studies toward its characterization in liver cytosol revealed that the hydrolase possesses a broad pH optimum ranging from 6.0 to 9.0. The Km and Vmax values for the N-deacetylation of thiacetazone are 5.7 x 10(-4) M and 0.123 nmol of p-aminobenzaldehydethiosemicarbazone formed/min/mg cytosolic protein, respectively. The ability to metabolize thiacetazone was the same in the livers of cat, mouse and human, but lagged significantly in that of rat. Among the biodegradable esters examined as potential rivals of thiacetazone, only aspirin competitively inhibited thiacetazone hydrolysis (Ki = 2.1 x 10(-4) M). Discrimination of cytosolic thiacetazone N-deacetylase from nonspecific p-nitrophenylacetate esterase on the basis of their differential reactivity toward various inhibitors and activators disclosed that low concentrations of p-chloromercuribenzoate, AgNO3 and CuSO4 selectively undermine the activity of thiacetazone N-deacetylase, whereas SKF 525-A, ZnSO4 and FeCl3 are effective inhibitors of p-nitrophenylacetate esterase. However, divalent ions (Ca++ and Mg++) and EDTA failed to alter the activity of the enzyme. Besides, thiacetazone metabolism was significantly retarded upon exposure to malathion. Notably, Nal/Kl stimulated the N-deacetylase activity as a function of iodide concentration. The hydrolysis of thiacetazone in the liver and kidney remained uninduced by phenobarbital, 3-methylcholanthrene or benzo(a)pyrene (80 mg/kg, p.o., 8 days).(ABSTRACT TRUNCATED AT 250 WORDS)