Case Studies

Case Studies and Client Projects

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Formation and scale-up of human metabolites formed through mixed metabolic pathways is possible using Hypha’s microbial biocatalysis system. In vivo human metabolism of Incyte’s IND epacadostat (EPA) forms 3 major circulating metabolites, from both primary and secondary pathways. Glucuronidation of EPA forms M9, the dominant metabolic pathway, in conjunction with formation of an amidine M11 and an N-dealkylated metabolite, M12. Boer et al. showed reductive metabolism by gut microbiota results in M11, which is absorbed and further modified by CYP enzymes to form the secondary metabolite M12.

Hypha’s microbial biocatalysis process is effective at generating metabolites at up to gram scale. Through Hypha and Selcia’s partnership, [13C], [14C], [2H], [3H] and [15N]-labelled metabolites can be accessed to support regulatory, development or research projects in the pharma and crop protection industries. Hypha establishes optimized processes using unlabelled or stable labelled parent substrates, which can then be transferred to Selcia’s state-of-the-art radiochemistry labs for the production of radiolabelled metabolites.

In this case study at least 2 mg of a monohydroxylated metabolite (M4), originally observed in rat liver microsomes, was required by a US pharma company. Screening of the parent compound against 22 PolyCYPs enzymes and 23 microbes revealed the production of two main monohydroxylated products, one of which was M4. M4 was best produced by PolyCYPs 152 and 359, as well as by bacterial species 45. The other monohydroxylated metabolite was produced by a different PolyCYPs isoform, (PolyCYP 350) and bacterial species 1.

Phase 2 conjugated metabolites such as sulfates can be screened for and scaled-up using a variety of methods including microbial biotransformation, mammalian biotransformation and chemical synthesis. The chemical sulfation screens work well for both aliphatic and aromatic alcohols. Metabolites can be supplied at microgram to multi mg scale, and at gram scale via the microbial and chemical synthesis routes.

Once a target metabolite or oxidised derivative has been synthesised by one or more PolyCYPs enzymes in the screening kit, a scale-up reaction with the best performing isoform is performed in order to access mg amounts of material for MetID and biological testing. The quickest and most cost-effective route for generating low mg amount of product is through the use of scale-up vials. Higher amounts of product can be generated using either a recombinant E.coli cell paste or through fermentation of a recombinant streptomyces clone expressing the isoform responsible for the biotransformation.

Hypha’s PolyCYPs kits are in routine use by pharma and agchem companies for producing human and other mammalian metabolites. One application involves use of PolyCYPs for creating radiolabelled metabolites for direct comparison with the radio profiles from mass balance and distribution study samples, necessary for regulatory filing. PolyCYPs provides a clean route for scalable access to more of the CYP-derived metabolites observed in these matrices, for definitive MetID and any tox studies deemed necessary. This is especially useful where low concentrations or unstable metabolites in the mass balance sample make structural identification difficult.

Many metabolites of drugs are wholly or partly responsible for both on-target or off-target in vivo activity. Metabolites of some drugs may initially be considered pharmacologically inactive, yet further investigation yields surprising alternative effects via different mechanisms. Off-target effects might synergize with the on-target effects or have other beneficial outcomes which could enhance or broaden the indication of the drug.1

Formation of N-oxide metabolites is one of the major pathways for metabolism of tertiary nitrogen-containing drugs. Some N-oxide metabolites have similar or greater pharmacological activity to the parent drug and thus require exposure assessment. They can also be unstable and can revert to the parent drug.1 Conversion of an N-oxide metabolite back to the parent in vivo is a well- known phenomenon which may result in an altered tissue distribution of the metabolite and parent drug, such as that proposed for tamoxifen,2 or cause adverse reactions as reported for soratenib.3 In the lab, it is possible to reduce the potential for conversion of N-oxide metabolites to the parent through careful sample handling, as described for the clinically-significant metabolite loxapine N-oxide.4

We’ve observed an increase in requests for synthesis of N-glucuronides over the last couple of years. We speculate that this may be due to the increasing use of N-heterocyclic chemistry in the design of new small molecule drugs, and pan company strategies to reduce CYP metabolism. The situation is further complicated by the high interspecies variability in formation of some N-glucuronides, especially aliphatic tertiary amines and aromatic N-heterocycles. UGT1A4 and UGT2B10 are key enzymes responsible for N-glucuronidation reactions in humans, rates of which can be much higher than in other animals. To compound this, synthesis of N-glucuronides is not always straightforward, and can be further muddied by metabolite stability issues, complicating interpretation of data.

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