One-Stop Metabolite Shop

Microbial Biotransformation

Metabolite Synthesis using Microbial Biotransformation

For difficult-to-synthesise metabolites, we employ our talented microorganism panels to create metabolites of drugs and agrochemicals. Microbial biotransformation will produce most mammalian metabolites as it is well-established that microbial organisms mimic mammalian metabolism of small molecules due to evolutionarily-related enzyme systems. The relevance of the technique to agrochemical metabolism is also established, due largely to the representation of soil microbes in our strain panels. The microbial route provides a cost-efficient method for scaling mg to gram scale amounts, as the microbes make their own co-factors.

Microbial biotransformation is highly effective for making many metabolites

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

Case Studies

Production of a human disproportionate hydroxylated metabolite

Ingenol disoxate is a chemically-stable topical dermal clinical drug candidate that was being developed by one of Hypha’s clients for treating actinic keratosis. Profiling of ingenol disoxate against multiple species of hepatocytes, revealed M27 as a predominant metabolite, particularly in human hepatocytes. Although accurate mass spectroscopy indicated the metabolite was mono-hydroxylated in the ingenol moiety, the precise location of the hydroxyl group could not be identified. Consequently, chemical synthesis was not feasible, nor were bioanalytical quantification and further biological testing possible. Hypha subsequently solved these issues for the client using microbial biotransformation.

Hypha’s microbial screen highlighted fourteen strains that produced oxidised metabolites of ingenol disoxate. After precise chromatographic matching by the client, the best-yielding strain was scaled up to produce a target amount of 10-50 mg of the human metabolite M27.

Ingenol disoxate poster image

Following scale-up, 86 mg of M27 was supplied to the client at >99% purity, who identified the metabolite as 16-hydroxyingenol disoxate. In addition to this material, 5 mg of a dihydroxylated derivative was supplied for evaluation. A subsequent request for more M27 for further studies needed a minimum of 200 mg, with Hypha supplying 415 mg of M27 to the client at 99% purity.
Having plentiful supply of this material meant that scientists were able to confirm the structure of the major metabolite via NMR spectroscopy and undertake PK profiling assays, as well as respond immediately to a later request by the FDA for drug-drug interaction studies to be undertaken with the metabolite.

Reference
Carlsen et al., 2016. Biosynthesis, structural identification and quantification of low pg/ml levels of a major human metabolite of a dermal drug candidate. European Bioanalysis Forum, Barcelona, Nov. 2016.

Production of metabolites of epacadostat arising from multiple pathways

Reference
Boer et al., 2016. Roles of UGT, P450 and Gut Microbiota in the Metabolism of Epacadostat in Humans. Drug Metab. Dispos. 44(10),1668-1674.

Formation and scale-up of human metabolites formed through mixed metabolic pathways is possible using Hypha’s microbial biotransformation system.

In vivo human metabolism of 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 cleaved secondary metabolite M12.

Hypha’s microbial panels provided a route to achieve formation of all three human metabolites, with several strains shown able to effectively biotransform EPA. Different strains and dosing regimens were found to be optimal for production of each metabolite. Although M12 was produced by the microbes, it was more easily synthesised.

Scale-up of the most productive biotransforming strains for M9 and M11 enabled the supply of 112mg of the glucuronide and 69mg of the gut metabolite at 95% purity to the client, which permitted the qualification of the metabolites for their drug-drug interaction potential.

Key Features of Hypha’s One-stop Metabolite shop

Routes to access phase I and phase 2 metabolites, including mixed pathway and multi-step metabolites, as well as low-turnover/accumulating metabolites

Purification and structure elucidation using NMR spectroscopy

Provision of Certificates of Analysis

Provides a tool for MetID

Applicable to broad structural types and complex molecules, including both natural products and synthetic compounds

Provides a solution for synthetically challenging metabolites when synthesis is not possible or too time consuming

Reproducible and scalable process to gram scale for pharmacology/toxicology testing

Formulation know-how for poorly-soluble compounds

Metabolites are produced on a simple fee for-service basis, i.e. there are no downstream terms or royalties

Resources

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

It is well known that metabolites of drug compounds may have different efficacy and properties to that of the parent compound. Investigation and production of these metabolites is critical for exploration and understanding of SAR, and to ensure thorough patent coverage. Metabolites can also be sourced to create antibodies used in development of assays for therapeutic drug monitoring.

The FDA’s 2016 MIST guidance states that phase 2 conjugates are generally pharmacologically inactive, however where a potentially toxic conjugate, such as an acyl glucuronide is formed, additional safety assessments may be needed. Idiosyncratic drug toxicity of carboxylic acid-containing drugs can be caused by the formation of reactive acyl glucuronides,1 which have the ability to directly acylate proteins and undergo intramolecular rearrangement producing reactive aldehydes leading to protein glycation.

Our other one-stop shop solutions for accessing and characterising metabolites

Hypha’s One-Stop Metabolite Shop enables synthesis, purification and characterization of all the main types of mammalian phase 1 and 2 metabolites.

Chemical Synthesis

Late-stage chemical glucuronidation

Recombinant enzymes

PolyCYPs, hrCYPs, AOX, FMOs

Mammalian biotransformation

Panels of liver S9s / microsomes

Purification & structure elucidation

COAs, acquisition / interpretation of NMR data

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

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