Synthesis of O-, N- and acyl-glucuronides
Purification of glucuronides
We are experts in purifying glucuronides from biological matrices and complex mixtures. This is possible even where small amounts of metabolite are present, such as in one client project where we isolated 0.4 mg of a glucuronide from 24L of urine. The pure material was subsequently used to determine the structure by 2D NMR spectroscopy.
Why is glucuronidation an important route of drug metabolism?
Glucuronidation is the predominant phase 2 metabolic route for a variety of drugs and agrochemicals catalysed by UDP-glucuronosyltransferases (UGTs) into hydrophilic conjugates that are eliminated from the cell by efflux transporters. UGTs are a family of membrane-bound proteins located in the endoplasmic reticulum in the cells of various tissues, with UGT 1A and UGT 2B subfamilies responsible for most drug metabolism.
Although many UGTs are expressed in human liver, some are also expressed extrahepatically and some are only expressed in certain organs (Ge et al., 2016). UGTs catalyse the transfer of glucuronic acid to a variety of O-, N-, C– and S– containing parent substrates and have become increasingly of interest in the elimination of drugs.
Although generally pharmacologically inactive, some acyl glucuronides of carboxylic acid containing drugs can form acyl migration isomers, which result in transacylation and glycation reactions with proteins causing idiosyncratic adverse drug reactions.
Several classes of glucuronides have been shown to interact with CYPs, in particular CYP2C8 due to its distinctive active site (Ma et al., 2017).
Some glucuronides are subject to enterohepatic recycling, such as the O-glucuronide of epacadostat made by one of Hypha’s microbial strains (Boer et al., 2016), where hydrolysis of the glucuronide by gut bacteria can occur. 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.
Glucuronide synthesis client case study
Clearance of a GPR40 partial agonist backup candidate to MK-8666 by glucuronidation had the potential for conjugation to occur either at the alcohol functionality (ether glucuronide) or at the carboxylic acid (acyl glucuronide). Synthesis of both glucuronides was needed for stability testing.
Although the chemical synthesis of the acyl glucuronide was facile, the tertiary alcohol was refractory to generation of the O-glucuronide, presumably due to steric hindrance. Microbial biotransformation was successful in generating both glucuronides (Salter et al., 2018).
Synthesis of the O-glucuronide through a small 50 ml scale microbial biotransformation yielded 0.46 mg of pure material, the structure of which was confirmed by NMR spectroscopy. The O-glucuronide synthesised was shown to have identical chromatographic retention time and molecular ion to a reference peak produced by incubation of the parent drug with recombinant human UGT 1A4.
Why access glucuronides?
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.
We’ve observed an increase in requests for synthesis of N-glucuronides over the last couple of years. We speculate that this may be due to the increasing use of N-heterocyclic chemistry in the design of new small molecule drugs, and pan company strategies to reduce CYP metabolism. The situation is further complicated by the high interspecies variability in formation of some N-glucuronides, especially aliphatic tertiary amines and aromatic N-heterocycles. UGT1A4 and UGT2B10 are key enzymes responsible for N-glucuronidation reactions in humans, rates of which can be much higher than in other animals. To compound this, synthesis of N-glucuronides is not always straightforward, and can be further muddied by metabolite stability issues, complicating interpretation of data.
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.
Find out about synthesis of other metabolite types
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.
Joshua Day Ph.D, Director of Chemistry
Crestone, Inc., Colorado, USA
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Hypha Discovery is a UK-based CRO supporting pharmaceutical and agrochemical companies worldwide through the production of metabolites and new derivatives of drugs and agrochemicals in discovery and development.