An enzyme experiment can produce different results when the test compound has time to undergo metabolism before the probe substrate is added. A 2018 study of the synthetic cannabinoid EAM-2201 illustrates why forensic toxicologists should examine incubation conditions when evaluating a proposed drug interaction.
The investigators measured probe-drug metabolism in pooled human liver microsomes. Direct inhibition was negligible for the tested CYP activities at EAM-2201 concentrations up to 50 micromolar. After a 30-minute preincubation with microsomes and NADPH, inhibition became evident for CYP2C8, CYP2C9, CYP2C19, and CYP3A4.
NADPH supplies a cofactor needed for CYP-mediated metabolism. Further experiments varying preincubation time and EAM-2201 concentration supported time-dependent inhibition of these four enzymes. The paper also reported competitive inhibition of UGT1A3 in separate glucuronidation experiments.
The authors cited EAM-2201 plasma concentrations of 0.26–4.1 nanomolar from recreational-use and postmortem reports. Those values and the experimental micromolar concentrations require careful contextual comparison. Relevant unbound concentrations at an enzyme site, tissue distribution, exposure duration, and metabolites affect translation from a microsomal experiment to a person.
An expert reviewing a claimed interaction can identify which enzyme was tested, whether metabolic preincubation occurred, and how the proposed human exposure compares with the experiment. These results provide a mechanistic reason to investigate interactions. Estimating an interaction’s magnitude in a particular case requires additional exposure and clinical evidence.
Read the scientific paper in full. DOI 10.3390/molecules23040920.