Hydroxylated Human Metabolites of Tivantinib Produced by an Enzyme in Hypha’s PolyCYPs Cytochrome P450 kit

Presented at: ISSX meeting, 2017, Cologne, Germany

A cell-free kit of cytochrome P450 enzymes and ferredoxin/ferredoxin reductase redox partners, termed PolyCYPs^®, is being developed for generating scalable quantities of oxidised metabolites. P450 cytochromes in the kit have been derived from some of Hypha’s most talented biotransforming bacteria and are capable of generating human and other mammalian metabolites of drug compounds.

The hydroxylating abilities of one P450 enzyme from the kit, PolyCYP 6.1, which has been cloned from an actinomycete species into E.coli together with redox partners, is illustrated using tivantinib. Tivantinib is an experimental anti-cancer drug which is extensively metabolised in humans, with two major circulating metabolites M4 and M5. M4 and M5 are epimers formed by hydroxylation at the benzyl position of the tricyclic ring of tivantinib and comprise 19% and 12% of the total area under the concentration-time curve (AUC) respectively, implicating these metabolites under the FDA MIST guideline.

Recombinant enzyme PolyCYP 6.1 was able to produce all four of the hydroxylated human metabolites originally observed in the wild-type microbial biotransformation, from which the cytochrome was derived. As well as utility for providing sufficient material for MetID, reactions can be scaled to produce milligram to gram quantities of metabolites and novel derivatives for further evaluation.