Case objective

Tuberculosis remains a global health threat, necessitating the development of effective treatments. BTZ-043, a promising benzothiazinone antibiotic, has shown potential in combatting Mycobacterium tuberculosis by targeting decaprenylphosphoryl-β-ᴅ-ribose-2ʹ-epimerase (DprE1). Encouraged by phase 2a success, BTZ-043´s phase 2b/c trials were underway and the planning for phase 3 trials was driving the need for deeper comprehension of the metabolism of BTZ-043 through a human absorption, metabolism and excretion (hAME) study. Due to BTZ-043´s complicated metabolism and the range of its metastable and unstable biotransformation products, a European research institute requested comprehensive support for this study.

Our approach

A hAME study was conducted successfully involving four healthy male volunteers to whom a single oral dose of 500 mg BTZ-043 blended with 3.74 MBq of [¹⁴C]-BTZ-043 was administered. Comprehensive assessments, including mass balance, plasma radioactivity and metabolite profiling across plasma, urine and faecal samples, were undertaken. Special precautions addressed the hydride Meisenheimer complex-related metabolite instability, crucial for accurate pharmacokinetic evaluation. Therefore, classical sample extraction procedures for radio profiling could not be applied for these metabolites.

Solutions were found in separate LC-MS bioanalytical methods for all main metabolites in plasma and in urine by spiking to an excess of blank plasma and applying the respective plasma bioanalytical methods. By AUC clustering for the main metabolites (cluster 1: parent drug, M1 and M2; cluster 2: M3, M4 and M10), we could prove that the total cluster AUCs remained constant across quantitative methods, and were statistically not different from the total cluster AUCs measured by liquid chromatography mass spectrometry (LC-MS). The results confirmed that the bioanalytical data by LC-MS were best to be used in the overall metabolite exposure evaluations rather than AUCs generated by radiometry data.

Our findings

BTZ-043 exhibited substantial absorption, minimally 66 %, based on urinary excretion data. BTZ-043 was extensively metabolised, primarily by reduction, hydrolysis and oxidation. Structurally characterised metabolites pointed at the pathways of nitro-reduction (M1, M10), hydride Meisenheimer formation (M2, M3), hydrolysis and ketoreduction (M3, M4, M10), hydroxylation and oxidation, glucuronidation, sulfation as well as combinations thereof.

Excretion of [¹⁴C] drug-related material in sum (mass balance; top graph) and as individual metabolites (bottom graph) in urine and feces.

Metabolite profiles of [¹⁴C]-BTZ-043 in human plasma, urine and feces.

Comparison of plasma (left table) and urine (right table) AUCs of Cluster-1 and Cluster-2 components quantified by [¹⁴C]-metabolite profiling and by validated UPLC-MS/MS methods using tailored stabilisation of analytes.

Conclusion and impact

The hAME study provided critical insights into pharmacokinetics and metabolism of BTZ-043, shedding light on its elimination pathways and metabolite profiles. The integration of radiometry and LC-MS data, as well as the application of an AUC metabolite cluster approach facilitated a comprehensive understanding of the metabolite exposure assessment.

Facing similar challenges?

Source

Dr. Piet Swart, Dr. Dieter Gallemann, Dr. Matthias Bader and Dr. Heike Wiese contributed to:

Piet Swart et al., Comprehensive evaluation of BTZ-043: from hAME study to metabolite exposures: handling the challenge of unstable hydride Meisenheimer complex metabolites, 50th Open Meeting 2024, DMDG, 3 September 2024, Heslington Campus East & Campus West, University of York, UK

About BTZ-043

BTZ-043 was discovered at the Leibniz Institute for Natural Product Research and Infection Biology (Leibniz-HKI) in Jena, Germany, and developed in collaboration with the Institute of Infectious Diseases and Tropical Medicine at the LMU University Hospital in Munich, Germany. Hapila GmbH, based in Gera, Germany, provided BTZ-043 and all analytical standards for the Leibniz Institute and LMU Klinikum.