Synthesis of Drug and Environmental Fate (eFate) Metabolites
Hypha are go-to experts in provision of phase I and phase II metabolites to pharma and agrochemical companies. We have worked with over 110 companies and organisations worldwide to provide metabolites for discovery and development stage projects.
Our “One-stop Metabolite Shop” concept comprises a comprehensive suite of technologies to enable synthesis of even the most difficult-to-synthesise metabolite. Human and other mammalian phase I and phase II metabolism of drugs and agrochemicals can be synthesised using a variety of methods to maximise the chances of success, including chemical synthesis, mammalian liver fractions (S9s / microsomes), microbial biotransformation, recombinant enzymes such as PolyCYPs, as well as the purification of metabolites from biological matrices. Hypha scientists are also experts in structural elucidation via NMR spectroscopy.
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 phase I mechanisms and phase II conjugates. We are typically asked to produce the following metabolite types:
- Oxidised metabolites
- CYP-mediated metabolites
- Non-CYP metabolites e.g. via AOX, FMOs
- Acyl glucuronides
- N-carbamoyl glucuronides
- Sulfated metabolites
- GSH conjugates
Our latest developments include the development of our PolyCYPs®+ metabolite screening kits and a late-stage chemical glucuronidation screen, suitable for synthesis of all types of glucuronide metabolites. The chemical screen features tailored deprotection strategies to improve the synthesis of acyl glucuronides and some unstable N-glucuronides, and is complementary to our existing successful microbial and mammalian biotransformation routes for generating phase II conjugates.
The extended PolyCYPs®+ kits comprise 18 diverse CYP isoforms cloned from some of Hypha’s bacteria, as well as complimentary inclusion of two other phase 1 enzymes – human aldehyde oxidase (AOX1) and the main hepatic flavin monooxygenase – FMO3.
All human FMO isoforms 1 to 5 have been cloned and successfully expressed in E.coli. Please contact us for more information on how to access these.
Case Study – Provision of multiple human phase I drug metabolites using multiple methods
This project undertaken for Eli Lilly illustrates the benefit of using different techniques to fulfil the synthesis of all metabolites required for a project.
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.
Difficult-to-synthesise drug metabolites
For metabolites that are difficult to synthesise chemically, we can employ our scalable microorganism panels and a suite of liver fractions (S9/microsomes) obtained from different species. We are experts in production of metabolites by microbial biotransformation and have worked with over 110 clients to provide support for drugs and agrochemicals in discovery and development.
Through a partnership between Hypha and Selcia, [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. Download a one page summary here.
A recent project for a client involved preparing a 14C radiolabelled CYP metabolite of a drug for transporter and metabolism study assays. Chemical synthesis of the metabolite was challenging since it required multiple steps as well as stereospecific reactions, which was time and cost consuming. Thus microbial biotransformation was successfully employed to produce the radiolabelled material.
Metabolites of agrochemicals
In addition to drug metabolites, Hypha’s biotransformation platform is also effective for the production of animal and plant metabolites of agrochemicals, including glycosides. 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.