Accounting for metabolites is a key activity in drug development programs. Sometimes biotransformations result in metabolites that might not be spotted initially. Reasons for these “missing” metabolites are described in this paper, along with real case studies and advice on how to best to “expect the unexpected”.
Reasons for missing metabolites comprise:
- Metabolites invisible in LC-MS
- Metabolites with reduced ionization in mass spectrometry
- Biotransformation mediated loss of radiolabel
- Bioactivation leading to reactive metabolite protein or DNA adducts
- Non-selective covalent binding
- Unexpected major metabolites in circulation resulting from low in vivo clearance, slow and sequential metabolism or extrahepatic metabolism.
- Other scenarios such as metabolite dimerization and the challenge of oxidation-reduction equilibrium of thiol-containing drugs and metabolites are also highlighted.
Given analytical detection systems are key, the benefit gained from the extra sensitivity of AMS (accelerator mass spectrometry) is discussed for capturing slow-forming or long lived metabolites in radiolabelled ADME studies.Other tactics taken to “uncover” missing metabolites include derivatisation e.g. of cyanide when released. Trapping agents are described for reactive metabolites that could ordinarily be overlooked due to quenching with endogenous small molecules and protein nucleophiles.
For low clearance or slowly formed metabolites, long-term hepatocyte cultures are advised, and for any human specific metabolite that might be missed, the recommendation is to look at plasma samples from the phase I MAD (multiple ascending dose) study in detail. Equally, any gut microbiome derived biotransformations need to be considered as resulting metabolites can also sometimes be absorbed into circulation and appear as a major metabolite.
Dual radiolabelling is also raised as a solution to capture metabolites that might be lost to the “dark side” on hydrolysis, in instances where a drug is labelled in only one part of the molecule.
Finally, structural complexity of metabolites arising from “non-standard” biotransformations is often best solved using NMR spectroscopy.
The Importance of Tracking “Missing” Metabolites: How and Why? Shuai Wang, T. Eric Ballard, Lisa J. Christopher, Robert S. Foti, Chungang Gu, S. Cyrus Khojasteh, Joyce Liu, Shuguang Ma, Bin Ma, R. Scott Obach, Simone Schadt, Zhoupeng Zhang, and Donglu Zhang. Journal of Medicinal Chemistry Article ASAP. DOI: 10.1021/acs.jmedchem.3c01293,