Hypha Discovery: Metabolite synthesis, purification and identification experts
WHAT'S NEW - Q2 2022
GSH Conjugates
Do you need to access GSH conjugates for identification by NMR spectroscopy? These phase 2 metabolites can be produced by PolyCYPs enzymes and by human recombinant CYPs when reactions are conducted in the presence of glutathione.
Case study - clozapine reactivity
The antipsychotic clozapine is extensively metabolised by multiple CYPs, including formation of GSH conjugates via formation of a reactive nitrenium ion. This bioactivation is considered to be responsible for several adverse drug reactions and is mainly driven by CYP3A4 in human liver.
Clozapine was incubated in a PolyCYPs screening kit and recombinant human CYPs in the presence of glutathione, and the extracts analysed by LC-MS. Two clozapine GSH conjugates were formed by 5 PolyCYPs enzymes. The metabolites co-chromatographed with those produced via CYP3A4 and CYP1A2.
New diversification kit
A kit containing 8 promiscuous PolyCYPs isoforms is now available. This kit is particularly useful for rapid screening of lead compounds for oxidised derivatives. Scale-up vials of each isoform can also be kept in stock in client freezers to enable immediate scale-up for structure elucidation and biological testing.
Purification from human plasma and urine
We love purification challenges, both small and large scale.
In a recent metabolite project, we isolated < 0.1 mg of a key drug metabolite at high purity (>95% by LC-UV and NMR) from a small amount of human plasma. The structure was resolved using cryoprobe NMR spectroscopy and shown to be a result of oxidation and reduction biotransformations at two different positions. The pure plasma-derived metabolite was then used to confirm homology with the chemically synthesised metabolite.
Scaling up
A different project involved purification of milligram amounts of an AO metabolite from litres of human urine. It was subsequently found that the metabolite could be produced by human recombinant aldehyde oxidase (AO) and by two microbes. One of these microbes could convert the parent drug to the AO metabolite at a 67% conversion. Following a successful dose escalation experiment, over 800 mgs of the metabolite was purified from a 2L microbial biotransformation.
As exemplified in this project, microbes are often a scalable and cost effective route to obtain large quantities of metabolites for biological testing and to act as analytical standards. Microbes mimic mammalian systems to produce both phase 1 and 2 metabolites.
Latest Metabolite Tales Blogs
Metabolism of t-butyl groups in drugs
Julia Shanu-Wilson looks at metabolism of drugs in which CYPs catalyse oxidation of t-butyl groups, together with some of the strategies used to reduce clearance.
Orphan CYPs in personalised medicine
Sam Coe highlights the prospect of exploiting orphan cytochrome P450s for development of targeted anti-cancer agents through a prodrug strategy.
Underappreciated hydroxyl in drug discovery
Consultant Rob Young explores the potential impact of introducing hydroxyl groups in drugs and discusses the physical consequences of its introduction.
About Hypha
Hypha are experts in the scalable synthesis, purification and structure elucidation of metabolites and impurities 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.
