Case Studies and Client Projects

Many metabolites of drugs are wholly or partly responsible for both on-target or off-target in vivo activity. Metabolites of some drugs may initially be considered pharmacologically inactive, yet further investigation yields surprising alternative effects via different mechanisms. Off-target effects might synergize with the on-target effects or have other beneficial outcomes which could enhance or broaden the indication of the drug.¹

Formation of N-oxide metabolites is one of the major pathways for metabolism of tertiary nitrogen-containing drugs. Some N-oxide metabolites have similar or greater pharmacological activity to the parent drug and thus require exposure assessment. They can also be unstable and can revert to the parent drug.¹ Conversion of an N-oxide metabolite back to the parent in vivo is a well- known phenomenon which may result in an altered tissue distribution of the metabolite and parent drug, such as that proposed for tamoxifen,² or cause adverse reactions as reported for soratenib.³ In the lab, it is possible to reduce the potential for conversion of N-oxide metabolites to the parent through careful sample handling, as described for the clinically-significant metabolite loxapine N-oxide.⁴

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.

Lorcaserin is a selective 5-HT_2C receptor agonist acting on the hypothalamus to reduce appetite and treat obesity, but which has now been withdrawn from the market.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.

There has been a notable increase in metabolism of new drug candidates through non-CYP phase I pathways such as those mediated via aldehyde oxidase (AO).¹ Further, mixed AO/P450 substrates may be subject to metabolic shunting, an important consideration during toxicology and DDI assessment of these drugs.² Access to metabolites may thus be important to consider for drugs with mixed metabolism.

If clearance mechanisms of the test drug results in sufficient quantities of the major metabolites in biological material such as faeces or urine, purification and subsequent identification of metabolites from such matrices is possible. One such project undertaken at Hypha resulted in tens of milligrams of the R- and S-O-glucuronides of carisbamate, a neuromodulator developed by SK Life Science, which were purified to >95% purity from 150 ml of urine using a three-step purification method.