Hypha Discovery: metabolite synthesis and purification specialists
WHAT'S NEW
Synthesis of deuterated metabolites
We've completed a number of projects in which deuterated metabolites were required. A recent example was the synthesis of a sequential hydroxylated-sulfated metabolite where microbial biotransformation was used to generate the correct isomer of an aryl hydroxylated intermediate, followed by a late-stage chemical sulfation of the purified material. This late-stage method enabled hundreds of milligrams of the deuterated metabolite to be produced from the deuterated parent drug compound.
Accessing CYP metabolites
In our case study this quarter we look at the methods used to access CYP3A4-mediated hydroxylated metabolites of the agrochemical imidacloprid, in which PolyCYPs was successful in generating the major 5-hydroxy metabolite. Read more in the link below.
Purification challenges
Some projects we undertake involve purification of metabolites from biological matrices. This can prove a challenge where metabolites are present at only low levels. One such client project involved the purification of 0.4 mg of a non-UV-active metabolite from 24L of urine, followed by 2D-NMR structural elucidation. A great job by the chemists at Hypha!
Meeting the challenge of synthesizing glucuronides
We were delighted to receive this feedback from another happy client who needed both N- and O-glucuronides of their drug compound:
“Hypha Discovery did a fantastic job synthesizing N- and O-glucuronides of our clinical stage drug substance. The project updates were detailed, our questions were answered in a timely manner, and the overall timeline was maintained. Hypha was highly recommended to us and I would not hesitate to recommend them to a colleague.”
Director of Chemistry, US pharma company
SCIENTIFIC PAPER FEATURE
Dose-dependent metabolic switching
Looking at the structure of tropifexor, a farnesoid X receptor (FXR) agonist for the treatment of nonalcoholic steatohepatitis (NASH), immediately raises thoughts about formation of an acyl glucuronide metabolite.
In fact, tropifexor is predominantly eliminated by metabolism and indeed the acyl glucuronide (M22) was observed during in vitro studies, although not in the human radiolabelled ADME study described in the paper by scientists at Novartis (see reference below).
Investigations with M22 showed that this acyl glucuronide is stable in human plasma with < 5% hydrolyzed back to the parent by 5 hours during an incubation, and in addition a low rate of intramolecular acyl migration under the incubation conditions was evident from mass spec studies. However, one concern with biliary excreted glucuronides is that they can revert to the parent drug through the action of β-glucuronidase secreted by bacteria in the gut.
An ex vivo experiment with M22 showed it was almost entirely converted back to tropifexor after 20 hours of incubation under anaerobic conditions, so providing a plausible explanation as to why this metabolite was not detected in human in vivo experiments. The authors suggest that M22 could have been formed in the liver, excreted in the bile and passed back into the intestinal tract, where it was deconjugated by β-glucuronidase expressed in microflora, and observed as intact tropifexor in faeces.
Interestingly it turns out that the primary metabolic clearance pathway is dependent on the dose of the drug, with a switch from glucuronidation at higher concentrations to oxidative metabolism dominating at the more clinically relevant lower doses, highlighting the importance of the interplay between different biotransformation mechanisms in the body.
The authors point out that in vivo and in vitro experiments should be carefully designed for optimum understanding of disposition processes during drug development.
Reference
Wang-Lakshman et al., 2021. Evaluation of the Absorption, Metabolism, and Excretion of a Single Oral 1 mg Dose of Tropifexor in Healthy Male Subjects and the Concentration Dependency of Tropifexor Metabolism. Drug Metabolism and Disposition May 5, 2021, DMD-AR-2020-000349; DOI: https://doi.org/10.1124/dmd.120.000349
About Hypha
Hypha are experts in the scalable synthesis, purification and structure elucidation of metabolites of drugs and agrochemicals. We work with pharma and agrochemical companies worldwide to fulfil our clients' needs to obtain metabolites for definitive MetID and for further biological testing. Applying a comprehensive portfolio of methods, including microbial and liver S9 / microsomal biotransformation methods, chemical synthesis, and recombinant enzymes, Hypha has the ability to produce gram amounts of synthetically challenging metabolites and oxidised derivatives of drugs. Combined with strong capabilities in purification chemistry and structural elucidation using state-of-the-art NMR spectroscopy, we are able to deliver an end-to-end service to clients.
