Synthesis of Drug and Environmental Fate (eFate) Metabolites

We are able to synthesise mammalian phase I and phase II metabolism using a variety of methods to maximise the chances of success, including chemical synthesis, mammalian liver fractions (S9s / microsomes), microbial biotransformation and recombinant enzymes (PolyCYPs). Clients regularly ask Hypha to produce metabolites for unambiguous identification, use as quantitation standards or larger amounts for pharmacology and toxicology studies to satisfy regulatory guidelines, including the FDA’s MIST guidelines.

Metabolites arising from a variety of pathways are accessible, including both CYP and non-CYP derived mechanisms. We can also produce phase II conjugates such as N-glucuronides, O-glucuronides, acyl glucuronides and sulfated metabolites.

References 1) Smith & Rosazza, Arch. Biochem Biophys. (1974) 161: 551-558. 2) Azerad, R., Adv Biochem Eng Biotechnol (1999) 63, 169.

Microbial biotransformation

For difficult-to-synthesise metabolites, we employ our talented microorganism panels to create drug or environmental fate metabolites. Microbial biotransformation will produce most mammalian metabolites as it is well-established that microbial organisms mimic mammalian metabolism of small molecules1, 2 due to evolutionarily-related enzyme systems. We have been able to demonstrate that our biotransformation panel are able to undertake these common reactions, amongst many others. The microbial route provides a cost efficient method for scaling larger mg to gram scale amounts, as the microbes make their own co-factors.

Metabolite Synthesis using Liver Fractions

We are also 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 bescaled 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.

1) Smith & Rosazza, Arch. Biochem Biophys. (1974) 161: 551-558.
2) Azerad, R., Adv Biochem Eng Biotechnol (1999) 63, 169.

Metabolite match rate >90%
Scale-up reproducibility rate 97%

Key features:

  • Phase I metabolites produced from both CYP and non-CYP mediated pathways, including aldehyde oxidase mediated metabolites
  • Production of Phase II conjugates, including N-,O- & acyl glucuronide and sulfated metabolites
  • Multiple metabolites can be captured in a single screen
  • Reproducible and scalable process up to gram quantities
  • Provides a tool for MetID
  • Target molecules can be produced at mg/g quantities for pharmacology/toxicology and to satisfy MIST guidelines
  • Applicable to broad structural types and complex molecules, including natural products and synthetic compounds
  • Formulation know-how for poorly-soluble compounds
  • Provides a solution for synthetically challenging metabolites when synthesis is not possible or too costly / time-consuming
  • Metabolites are produced on a simple fee-for-service basis, i.e. there are no downstream terms or royalties

Case Study – Provision of multiple human metabolites via biotransformation

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.

Download a copy of the poster here.

Accessing metabolites of agrochemical products

Hypha’s biotransformation platform is also effective for the production of environmental fate derivatives of agrochemicals. Our processes have been specially adapted to accept and metabolise pesticides which typically have poor aqueous solubility.

Download a newsletter on accessing oxidised metabolites of agrochemical products via microbial biotransformation using mammalian metabolites of napropamide and imidacloprid as case studies.

Contact Us to discuss how we can help your project succeed

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