ADMEt Evaluation of Anti-tuberculosis Compounds and New Methodologies Development

Tuberculosis (TB) is among the top three life-threatening infectious diseases in developing countries. Therefore, there is a great need to discover and develop novel anti-TB drugs. The current dissertation research focused on overcoming challenges: drug metabolism, induction of liver enzymes, and toxicity. 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. Of particular interest, 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. Compared with a previously reported method, 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. The established drug metabolism assays were used to study existing anti-TB drugs and to extend the half-life of experimental anti-TB compounds in internal drug discovery projects. This work lead to the first time characterization of “soft spots” of compound LL5858 from phase I clinical trial. Furthermore, two successful cases of ‘hit-to-lead’ optimization demonstrated the effectiveness of using drug metabolism studies to guide analoging efforts aimed at improving the drug-like properties of anti-TB compounds with stability enhancement by ten folds and five folds for isoxazole and tetrahydroindazole series. A new cell model using HepaRG cells was developed and validated to study the induction potential of an experimental compound on liver enzymes. Compared with the cost of primary hepatocytes-based induction assay, this induction assay reduced the cost 50-fold. The study further reduced the leading cell culture time from 4 weeks (published induction protocol) to 2 weeks before the treatment with inducers. 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. Once fully developed, this novel assay will have many applications. It will serve to increase compound libraries’ chemical diversity and to consider revisiting previously screened libraries.