Metabolite Synthesis

Synthesis of CYP metabolites

CYP Metabolites

Hypha can produce human and other mammalian CYP metabolites using a variety of methods in our one-stop metabolite shop, including microbial biotransformation, mammalian biotransformation and using recombinant enzymes such as PolyCYPs and human recombinant CYPs.

Cytochrome P450 (CYPs) are monoxygenases that are involved in oxidative metabolism of a wide range of drugs and agrochemicals. They catalyse a variety of reactions including hydroxylation, epoxidation, N– and O-dealkylation and Bayer-Villiger oxidation. Various CYP isoforms are found in the liver and other tissues of the body, with the more prolific human CYP isoforms involved in drug metabolism comprising CYP3A4, CYP2D6, CYP2C9, CYP1A1/2, CYP2B6, CYP2C19, CYP2C8, CYP2A6, CYP2E1 and CYP2J2 (Zanger and Schwab, 2013).

CYP Metabolites by whole cell microbial biotransformation

Wild-type microbes have broad oxidation capabilities due to the high number of CYP enzymes that can be expressed (25− 50 for bacterial species, 100+ for fungi). Hydroxylated and other oxidised derivatives can be produced, including aliphatic and aromatic hydroxylated derivatives.

Case Study

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.

Ruxo metabolties diagram
Hydroxylated CYP metabolites of ruxolitinib produced by Hypha’s microbial biotransformation system
PolyCYPs screening kit

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.

Resources

Explore our library of resources comprising brochures, case studies, posters and publications about the work we do.

Access a brochure on Hypha’s PolyCYPs+ kits. The 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.

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.

Test tubes with dripper
251A3171-DE01-48EB-AA27-BD82BC5AC10DCreated with sketchtool.

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

Ready to begin? Our scientists are available to talk through your requirements

Contact Us

Don't forget to subscribe to our newsletter and blog!

Newsletter Signup
Which do you wish to signup for?