Metabolite Synthesis using Liver Fractions
As part of our suite of technologies for producing drug metabolites and agrochemicals, we are able to produce human metabolites using scalable liver S9 and microsome incubations. We ensure a broad mammalian metabolic coverage comprising 8-10 different species in the initial screen. These are typically evaluated alongside our microbial panels allowing the best yielding and most cost effective process to be scaled up to deliver high purity metabolites. Using a panel of hepatic S9 and microsomal fractions to complement our successful microbial biotransformation platform boosts the success rate from 86% to over 90% coverage of target metabolites.
Case Study – Provision of multiple human phase I metabolites via biotransformation
This project undertaken for Eli Lilly illustrates the benefit of using different techniques to fulfil the synthesis of all metabolites required for a project.
There has been a notable increase in metabolism of new drug candidates through non-CYP phase I pathways such as those mediated via aldehyde oxidase (AO).1 Further, mixed AO/P450 substrates may be subject to metabolic shunting an important consideration during toxicology and DDI assessment of these drugs.2 Access to metabolites may thus be important to consider for drugs with mixed metabolism.
Zhou et al. presented a poster at the 2018 ISSX meeting in Montreal on “Elimination of [14C]-LY3023414 by Aldehyde Oxidase and CYP Enzymes in Humans Following Oral Administration.” Both AO and CYP enzymes were responsible for the metabolic clearance of LY3023414 with the non-CYP enzymes mediating approximately half of the clearance of the drug. The predominant metabolic clearance pathways were aromatic hydroxylation of the quinoline moiety (M2), N-demethylation (M5) and quinoline oxidation with N-demethylation (M12).
No metabolism was observed when tested vs 5 human recombinant CYPs, however screening of LY3023414 against a subset of Hypha’s biotransforming strains generated a number of metabolites. The best microbial strain was scaled-up to 6L to access target metabolites M2 and M4. Subsequent incubation of the synthesised intermediate M5 vs Cyno S9 enabled production of a further target metabolite M12. Metabolites were purified to > 95% purity by Hypha and the structures confirmed by LC-MS and NMR. The AO mediated hydroxylated metabolite (M2, 20.1mg) and an N-oxide (M4, 66.3mg) were made via microbial biosynthesis and a CYP/AO mediated metabolite (M12, 18.4mg) was generated through liver S9 incubations.
1Rashidi & Soltani, 2017. Expert Opin. Drug Discovery 12 (3), 305-316.
2Crouch et al., 2016. Drug Metab. Dispos. 44, 1296-1303.
Contact Us to discuss how we can help your project succeed.