Metabolite Synthesis

Gut Metabolites

Gut Metabolites

Microbiota in the human gut are increasingly being considered as a virtual organ which plays a critical role in drug metabolism. The majority are obligate anaerobes from the genera Bacteriodes, Clostridium, Lactobacillus, Escherichia and Bifidobacteria, together with an assortment of other microorganisms [1]. The role of this “organ” in the biotransformation of some drugs to form gut metabolites is complicated by variability in the individual composition of microbial species in the gastrointestinal tract. This potentially can cause differences in the drug response.

Direct microbiome-derived metabolism can lead to deactivation, reactivation through enterohepatic recycling, or biotransformation of a drug to a toxic metabolite. Metabolic reactions mediated by the microbiota that are known to significantly impact the biologic activity of drugs and xenobiotics involve reduction, hydrolysis, dihydroxylation, acetylation, deacetylation, proteolysis, deconjugation, and deglycosylation processes [2].

References

[1] Wilson ID and Nicholson JK,  2017. Gut microbiome interactions with drug metabolism, efficacy, and toxicity. Transl Res. 179, 204-222. doi: 10.1016/j.trsl.2016.08.002.

[2] Sousa et al., 2008.  The gastrointestinal microbiota as a site for the biotransformation of drugs. Int J Pharm 363 (1-2), 1-25. doi: 10.1016/j.ijpharm.2008.07.009.

Screening and scale-up service for MetID

Biotransformation of drugs using pooled human faecal material

Hypha’s human faecal pool can be used to screen for gut metabolites of drugs, as exemplified by the reduction of sulfasalazine and nizatidine. In these case studies, drugs were incubated in human faecal material derived from several mixed sex donors under oxygen depleted conditions. The reduced metabolites were easily detected by LC-MS.

Human faecal incubations can be scaled up to provide material for purification of metabolites prior to structure elucidation by cryoprobe NMR.

We have undertaken a number of projects for clients where identification of a suspected gut-derived metabolite of a drug in clinical trials was needed, sometimes revealing surprising structures. These have included reduced and unusual conjugated metabolites.

As well as gut microbiota-mediated metabolites, sometimes a conjugate can be formed in the complex matrix of faecal material, without direct involvement of enzymes produced by bacteria in the gut.

Reduced metabolites of sulfasalazine and nizatidine in the gut.

Case study - production of a reduced metabolite of epacadostat

Metabolism of epacadostat

A reduced metabolite of epacadostat, M11, was surprisingly found to be produced by one of Hypha’s microbes under aerobic conditions. The metabolite was identical to that produced in a human faecal incubation. Microbial biotransformation of epacadostat also yielded a major O-glucuronide M9, and a metabolite M12 derived from CYP mediated metabolism of M11 [3].

The O-glucuronide M9 is subject to enterohepatic recycling, involving hydrolysis of the glucuronide by gut bacteria enzymes. This is followed by the absorption of the drug back into systemic circulation, thereby leading to a prolonged elimination half-life and altered pharmacokinetics and pharmacodynamics. 

Reference

[3] Boer at al., 2016. Metabolism of Epacadostat in Humans.

Key Features

Human origin faecal material

Pooled mixed sex source

Screen and scale-up: 100s µg to mg amounts for MetID

Expertise in purification from complex mixtures

Structure elucidation by cryoprobe NMR

Resources

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

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.

Metabolism of drugs often results in the formation of major circulating metabolites derived from mixed clearance pathways, and can include both primary and secondary metabolites. Human metabolism of Incyte’s investigational new drug epacadostat (EPA) forms 3 major circulating plasma metabolites (Boer et al., 2016). Glucuronidation of EPA to form M9 is the dominant metabolic pathway, in conjunction with formation of an amidine M11 and an N-dealkylated metabolite, M12. Boer and co-workers showed that reductive metabolism by gut microbiota results in M11, which is then absorbed and further modified by CYP enzymes to form the secondary metabolite M12.

Test tubes with dripper
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We have been very much impressed with the experience at Hypha Discovery. Hypha has provided us not only the services with high quality but also superior expertise in the field. From the study design, flow, production estimation to the timeline of the final product delivery, every single step was well planned. We were able to receive the high quality product in time. The communication was always quick and effective. There is no doubt that we will work with Hypha in the future when needed.

Yan Zhang, Senior Director DMPK

Nuvation Bio, USA

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