ADMEt Evaluation of Anti-tuberculosis Compounds and New Methodologies Development

All content on this webpage originated from other webpages, Please check the medicilon's website for details. Medicilon offers fully integrated pharmaceutical services for the global scientific community. We focus on providing an exceptional client-centered experience and advancing the drug discovery process.

Email: Marketing@medicilon.com.cn Website: www.medicilon.com

Drug metabolism assays were used to study existing anti-tuberculosis (TB) drugs and to extend the half-life of experimental anti-TB compounds in internal drug discovery projects. A new cell model using HepaRG cells was developed and validated to study the induction potential of an experimental compound on liver enzymes. A multiplexed cytotoxicity assay was developed and validated with HepG2 cells to monitor three toxicity markers from the same culture. Finally, research was initiated to develop a novel TB active metabolite assay (TAMA) for monitoring the anti-TB activity of drug metabolites without the need for structural identification and synthesis.

Initial work involved the establishment of a panel of ADMEt assays. This panel assessed microsome stability, protein binding, plasma stability, gastric acid stability, cytochrome (CYP) P450 inhibition and induction, multiplexed cytotoxicity using HepG2 cells and metabolite toxicity using HepaRG cells. All of the assays were developed and validated in 96-well format in order to increase the throughput.

This panel of ADMEt assays was then applied to retrospective drug metabolism studies on existing anti-TB drugs. A bio-analytical method to detect TB drugs was developed and validated, which could simultaneously quantitate 15 existing anti-TB drugs from human serum plasma samples in one run. The method was given a name of ‘detection of multiple TB drugs’ (DMTD). The development and validation of DMTD followed FDA guidelines and appeared to be the first bio-analytical method that could detect more than 10 drugs in one run for anti-tuberculosis drugs. Compared with a previously reported method, which could simultaneously quantitate four anti-TB drugs in one run, DMTD gave an equal, or up to 10-fold lower, limit of quantitation (LOQ). Therefore, DMTD offered higher throughput and greater sensitivity than the previously published method.

DMTD was then applied in the assessment of the in vitro stability for all 15 established and experimental anti-TB drugs in both mouse and human microsomes. Five drugs were relatively unstable in both mouse and human microsomes; four of those drugs had previously been reported to be metabolically unstable and to have active metabolites. An extensive literature search failed to reveal any reports of metabolic stability for LL3858, which is in Phase I clinical trial for the treatment of TB. The experimental data, however, showed that LL3858 was metabolically unstable, and its metabolites were identified. This lays the groundwork for improving the drug-like properties of this series including the determination of the anti-TB activity of the (synthesized) metabolites.

The second series was based on a tetrahydroindazole template with a low micromolar minimum inhibitory concentration (MIC). Similar compounds have been previously reported to have promising anti-TB activity but to be relatively unstable in liver microsomes. However, the metabolites of this type of compound have yet to be identified. Through metabolite identification, the tert-butyl group of tetrahydroindazole was identified as the major metabolism site for hydroxylation. The metabolites were then synthesized but showed little anti-TB activity. Guided by microsomal stability data, a series of compounds were designed to block the metabolism site. A new lead compound, with a trifluoromethoxy group replacing the tert-butyl group, showed the best drug-like profile among all analogs synthesized with a 5-fold increase in microsomal stability, and similar anti-TB activity to the original screening hit. When administered orally to mice, plasma concentrations exceeded the MIC for more than 24 hours. Further research confirmed the compound to be active in inhibiting the growth of intracellular M. tuberculosis and to be effective against strains resistant to individual established TB drugs development.