Synthesis of CYP metabolites
Production of oxidised metabolites of ruxolitinib by microbial biotransformation
Ruxolitinib is metabolised in humans largely by CYP3A4, and to a lesser extent by CYP2C9, to a number of oxidised metabolites. While it was possible for low milligram amounts of metabolites to be made using chemical synthesis, larger amounts were required for absolute stereochemical assignment by X-ray crystallography and for further biological testing.
Many microbes were able to produce some of the desired metabolites; however, one strain in particular was able to produce all the cyclopentyl 2-hydroxy- and 3-hydroxy metabolites plus the respective keto derivatives, and so was scaled up to enable the purification of up to 120 mg of each metabolite.
Several of these metabolites were found to possess pharmacological activity, including two metabolites resulting from hydroxylation at the 2- and 3-position on the cyclopentyl moiety, and the third a ketone resulting from further oxidation at the 3-position.
Hydroxylated CYP metabolites of ruxolitinib produced by Hypha’s microbial biotransformation system
CYP metabolites by PolyCYPs
A number of recombinant enzymes can also be employed to produce CYP metabolites. These include PolyCYPs enzymes which have been mined from some of Hypha’s talented actinomycete bacteria and expressed in E.coli, providing a diverse set of CYPs effective for producing human and other mammalian CYP-mediated metabolites. They are soluble enzymes which are generally easy to express and scale-up. We also have a panel of human recombinant CYPs available.
PolyCYPs+ metabolite kits contain 20 enzymes effective for producing a wide range of phase 1 metabolites. In addition to 18 PolyCYPs enzymes, the kit also contains human aldehyde oxidase (AOX1) and the main human hepatic flavin-containing monooxygenase (FMO3), with the other human FMO isoforms also available at Hypha. Reactions are scalable either by resupply of lyophilized enzymes for mg scale production in-house, or larger scale production up to gram scale at Hypha, with optional purification and structure elucidation.
PolyCYPs+ kits contain 20 enzymes effective for producing a wide range of phase 1 metabolites. As well as 18 PolyCYPs enzymes, the kit also contains human aldehyde oxidase (AOX1) and the main human hepatic flavin-containing monooxygenase (FMO3), with other human FMO isoforms also available from Hypha. PolyCYPs® enzymes have been mined from talented actinomycete bacteria in Hypha’s biotransformation panel, providing a wide diversity of CYPs that oxidize a variety of drug compounds. Both human and other mammalian CYP-mediated metabolites can be produced.
Metabolite identification is an integral part of both preclinical and clinical drug discovery and development. Synthesis of drug metabolites is often required to support definitive identification, preclinical safety studies and clinical trials.
Ingenol disoxate is a chemically-stable drug developed by LEO Pharma, effective at treating actinic keratosis topically and currently in Phase 3 clinical trials. Profiling of ingenol disoxate against multiple species of hepatocytes revealed M27 as a predominant metabolite, particularly in human hepatocytes. Although accurate mass spectrometry indicated the metabolite was monohydroxylated in the ingenol moiety, the precise location of the hydroxyl group could not be identified. Consequently, chemical synthesis was not feasible, nor biological quantification and further biological testing possible.
Find out about synthesis of other metabolite types
Hypha Discovery has been a valuable metabolite ID partner. Hypha have provided biotransformation, metabolite purification and structure elucidation answers to some of our most challenging metabolism and metabolite ID problems. We really appreciate the breadth of expertise available at Hypha Discovery and will definitely reach out for future work.
Director of DMPK
US Pharma Company
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Hypha Discovery is a UK-based CRO supporting pharmaceutical and agrochemical companies worldwide through the production of metabolites and new derivatives of drugs and agrochemicals in discovery and development.