Phenotypic and metabolic investigation of a CSF-1R kinase receptor inhibitor (BLZ945) and its pharmacologically active metabolite

Abstract

1. 4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide (BLZ945) is a small molecule inhibitor of CSF-1R kinase activity within osteoclasts designed to prevent skeletal related events in metastatic disease. Key metabolites were enzymatically and structurally characterized to understand the metabolic fate of BLZ945 and pharmacological implications. The relative intrinsic clearances for metabolites were derived from in vitro studies using human hepatocytes, microsomes and phenotyped with recombinant P450 enzymes.

2. Formation of a pharmacologically active metabolite (M9) was observed in human hepatocytes. The M9 metabolite is a structural isomer (diastereomer) of BLZ945 and is about 4-fold less potent. This isomer was enzymatically formed via P450 oxidation of the BLZ945 hydroxyl group, followed by aldo–keto reduction to the alcohol (M9).

3. Two reaction phenotyping approaches based on fractional clearances were applied to BLZ945 using hepatocytes and liver microsomes. The fraction metabolized (fm) or contribution ratio was determined for each metabolic reaction type (oxidation, glucuronidation or isomerization) as well as for each metabolite. The results quantitatively illustrate contribution ratios of the involved enzymes and pathways, e.g. the isomerization to metabolite M9 accounted for 24% intrinsic clearance in human hepatocytes. In summary, contribution ratios for the Phase I and Phase II pathways can be determined in hepatocytes.

• Contribution ratio
• enzyme phenotyping
• in vitro clearance
• in vitro metabolism
• metabolic isomerization

Acknowledgements

We gratefully acknowledge the Novartis Isotope Lab Synthesis and Analytics groups (Ines Rodriguez, Albrecht Glaenzel, Yves Metz, Raphael Ruetsch and Patrick Bross), for the synthesis and release of BLZ945, Frederick Lozach & Christine Douglas von Daeniken for their supportive work on metabolism, Song Lin for supporting the discovery phase metabolism identification work while at Novartis, and Matthias Kittelmann for the biochemical preparation of the glucuronide metabolite standard M6.

Declaration of interest

Krauser, Jin, Walles, Pfaar, Sutton, Wiesmann, Wolf, Camenisch and Swart participated in research study design and data interpretation. Graf, Pflimlin-Fritschy and Wolf conducted experiments and data interpretation. Krauser, Jin, Walles, Pfaar, Wiesmann and Sutton contributed in the preparation of this article. The authors report no conflict of interests.