Scaled up supply of O- and N- sulfate conjugates
Phase 2 conjugated metabolites such as sulfates can be screened for and scaled-up using a variety of methods including microbial biotransformation, mammalian biotransformation and chemical synthesis. The chemical sulfation screens work well for both aliphatic and aromatic alcohols. Metabolites can be supplied at microgram to multi mg scale, and at gram scale via the microbial and chemical synthesis routes.
Sulfated metabolites using biotransformation
Sulfated metabolites of several drugs have been produced by microbes in Hypha’s panel, such as O-sulfated metabolites of apomorphine and genistein, Scale-up of the apomorphine biotransformation generated 3.2 mg of the sulfate conjugate for definitive MetID. A glucose conjugate was produced in parallel, demonstrating broader conjugative capabilities of microbial biotransformation.
To demonstrate N-sulfation, the dopamine receptor antagonist metoclopramide was screened against Hypha’s microbial and S9 panels. Metoclopramide has significant individual variation in metabolism, featuring multiple metabolites generated via both oxidative and conjugative pathways. Argikar et al. reported ten principal metabolites comprising five oxidation products, four glucuronides and one sulfate conjugate. Sulfate conjugation occurs at the primary amine in vivo with marked variability in levels due to polymorphism of human sulfotransferases. The sulfated metabolite of metoclopramide was produced by two fungal species but not detected in in vitro liver S9 incubations, despite being a known circulating metabolite. Glucuronides were produced by both microbial and S9 systems, with several microbes also generating oxidative metabolites and other sugar conjugates.
A project for a large pharma client involved supply of two sulfated metabolites in mg amounts. Following a dual system screening approach, more than 50mg of an O-sulfate was produced from porcine S9 fractions and more than 10mg of an N-sulfate via microbial biotransformation. Employing the best yielding path for creating each sulfated metabolite meant that requirements were successfully met for supply of both metabolites at high purity.
Lorcaserin is a selective 5-HT2C receptor agonist acting on the hypothalamus to reduce appetite and treat obesity, but which has now been withdrawn from the market. Lorcaserin N-sulfamate (M1) is the major circulating metabolite in plasma with lorcaserin N-carbamoyl glucuronide (M5) being the major excretory metabolite in urine. Screening of lorcaserin against Hypha’s panels of microbes and liver S9 preps gave scalable routes to accessing both metabolites.
Access to multiple metabolites needed to support clinical development is not always straightforward, and can sometimes mean that more than one technique needs to be applied to fulfil requirements. In one such project, a US pharma client required > 200 mg of three metabolites of a drug; an N-glucuronide (M1), an indirect O-glucuronide (M2b) and a hydroxylated metabolite (M8b). As part of this project, multiple components of Hypha’s one-stop metabolite shop were employed, including chemical synthesis, microbial biotransformation as well as purification and structure elucidation by NMR.
In Chapter 4 of the book on “Identification and quantification of drugs, metabolites, drug metabolizing enzymes and transporters”, Hypha authors summarise the different methods employed for producing metabolites of drugs, illustrated with representative examples from the literature and work undertaken at Hypha. The chapter also includes a discussion and examples of the use of NMR spectroscopy for structure elucidation of metabolites.