Metabolite synthesis via 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.
Hypha are the go-to experts in microbial biotransformation
- 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
1) Smith & Rosazza, Arch. Biochem Biophys. (1974) 161: 551-558.
2) Azerad, R., Adv Biochem Eng Biotechnol (1999) 63, 169.
Our recent publications featuring the application of biotransformation in drug discovery
Shanu-Wilson, J., Evans, L., Wrigley, S., Steele, J., Atherton, J., Boer, J., 2020. Biotransformation: Impact and Application of Metabolism in Drug Discovery. ACS Medicinal Chemistry Letters, 11: 2087-2107.
Evans, L., Phipps, R., Shanu-Wilson, J., Steele, J., Wrigley, S., 2020. Chapter 4 Metabolite generation and characterization by NMR. In: Identification and quantification of drugs, metabolites, drug metabolizing enzymes and transporters. Second edition. Eds Shuguang Ma and Swapan Chowdhury. Elsevier Science. ISBN: 9780128200186
Salter, R., Beshore, D.C., Colletti, S.L., Evans, L., Gong, Y., Helmy, R., Liu, Y., Maciolek, C.M., Martin, G., Pajkovic, N., Phipps, R., Small, J., Steele, J., de Vries, R., Williams, H., Martin, I.J., 2018. Microbial biotransformation – an important tool for the study of drug metabolism. Xenobiotica, 49:8, 877-886.
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.
In recent years, FDA guidance has advised initiating human metabolite profiling earlier in drug development, emphasizing the importance of metabolite identification and quantification to evaluate a drug metabolite’s safety and pharmacological activity. Praliciguat (IW-1973) is a soluble guanylate cyclase (sGC) stimulator in Phase 2 clinical trials for diabetic nephropathy and heart failure with preserved ejection fraction (HFpEF). During studies on metabolism of praliciguat in preclinical species and in human hepatocytes, a prominent direct O-glucuronide metabolite was detected. Initial attempts by Cyclerion to isolate the metabolite in rat bile, via administration of praliciguat to bile duct-cannulated rats, lacked scalability and resulted in low yields. Access to larger quantities of this glucuronide metabolite for structure elucidation and pharmacological activity was achieved through microbial biotransformation of the parent compound where nearly 100% of praliciguat was biotransformed to a single O-glucuronide to provide a total of 184 mg of purified praliciguat-glucuronide. The purified glucuronide metabolite produced by the actinomycete strain enabled the structural elucidation of the position of the glucuronide conjugate and was used for determining the identification and circulating concentrations of this metabolite in clinical samples.
Accessing metabolites of agrochemicals
Hypha’s biotransformation platform is also effective for the production of environmental fate derivatives and metabolites of agrochemicals. Our processes have been specially adapted to accept and metabolise pesticides which typically have poor aqueous solubility.
In one project, two glucuronidated metabolites of the herbicide napropamide, which would have been too complex to prepare by traditional synthetic methods, were produced by microbial biotransformation at Hypha. Tens of milligrams of the metabolites were purified by preparative HPLC to >95% purity and their structures confirmed by NMR spectroscopy.
Download a summary on accessing oxidised metabolites of agrochemical products via microbial biotransformation using mammalian metabolites of napropamide and imidacloprid as case studies.
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