Optimized LC-MS/MS quantification of tuberculosis drug candidate macozinone (PBTZ169), its dearomatized Meisenheimer Complex and other metabolites, in human plasma and urine.

Tuberculosis, and especially multidrug-resistant tuberculosis (MDR-TB), is a major global health threat which emphasizes the need to develop new agents to improve and shorten treatment of this difficult-to-manage infectious disease. Among the new agents, macozinone (PBTZ169) is one of the most promising candidates, showing extraordinary potency in vitro and in murine models against drug-susceptible and drug-resistant Mycobacterium tuberculosis. A previous analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed by our group to support phase I clinical trials of PBTZ169. These plasma sample analyses revealed the presence of several additional metabolites among which the most prominent was H2PBTZ, a reduced species obtained by dearomatization of macozinone, one of the first examples of Meisenheimer Complex (MC) metabolites identified in mammals. Identification of these new metabolites required the optimization of our original method for enhancing the selectivity between isobaric metabolites as well as for ensuring optimal stability for H2PBTZ analyses. Sample preparation methods were also developed for plasma and urine, followed by extensive quantitative validation in accordance with international bioanalytical method recommendations, which include selectivity, linearity, qualitative and quantitative matrix effect, trueness, precision and the establishment of accuracy profiles using ß-expectation tolerance intervals for known and newer analytes. The newly optimized methods have been applied in a subsequent Phase Ib clinical trial conducted in our University Hospital with healthy subjects. H2PBTZ was found to be the most abundant species circulating in plasma, underscoring the importance of measuring accurately and precisely this unprecedented metabolite. Low concentrations were found in urine for all monitored analytes, suggesting extensive metabolism before renal excretion.

Development and validation of a multiplex UHPLC-MS/MS method for the determination of the investigational antibiotic against multi-resistant tuberculosis macozinone (PBTZ169) and five active metabolites in human plasma.

The emergence of Mycobacterium tuberculosis strains resistant to current first-line antibiotic regimens constitutes a major global health threat. New treatments against multidrug-resistant tuberculosis (MDR-TB) are thus eagerly needed in particular in countries with a high MDR-TB prevalence. In this context, macozinone (PBTZ169), a promising drug candidate with an unique mode of action and highly potent in vitro tuberculocidal properties against MDR Mycobacterium strains, has now reached the clinical phase and has been notably tested in healthy male volunteers in Switzerland. To that endeavor, a multiplex UHPLC-MS/MS method has been developed for the sensitive and accurate human plasma levels determination of PBTZ169 along with five metabolites retaining in vitro anti-TB activity. Plasma protein precipitation with methanol was carried out as a simplified sample clean-up procedure followed by direct injection of the undiluted supernatant for the bioanalysis of the six analytes within 5 min, using 1.8 µm reversed-phase chromatography coupled to triple quadrupole mass spectrometry employing electrospray ionization in the positive mode. Stable isotopically-labelled PBTZ169 was used as internal standard (ISTD), while metabolites could be reliably quantified using two unlabeled chemical analogues selected as ISTD from a large in-house analogous compounds library. The overall methodology was fully validated according to current recommendations (FDA, EMEA) for bioanalytical methods, which include selectivity, carryover, qualitative and quantitative matrix effect, extraction recovery, process efficiency, trueness, precision, accuracy profiles, method and instrument detection limits, integrity to dilution, anticoagulant comparison and short- and long-term stabilities. Stability studies on the reduced metabolite H2-PBTZ169 have shown no significant impact on the actual PBTZ169 concentrations determined with the proposed assay. This simplified, rapid, sensitive and robust methodology has been applied to the bioanalysis of human plasma samples collected within the frame of a phase I clinical study in healthy volunteers receiving PBTZ169.