Metabolic clearance of select opioids and opioid antagonists using hepatic spheroids and recombinant cytochrome P450 enzymes.

The opioid crisis is a pressing public health issue, exacerbated by the emergence of more potent synthetic opioids, particularly fentanyl and its analogs. While competitive antagonists exist, their efficacy against synthetic opioids is largely unknown. Furthermore, due to the short durations of action of current antagonists, renarcotization remains a concern. In this study, metabolic activity was characterized for fentanyl-class opioids and common opioid antagonists using multiple in vitro systems, namely, cytochrome P450 (CYP) enzymes and hepatic spheroids, after which an in vitro-in vivo correlation was applied to convert in vitro metabolic activity to predictive in vivo intrinsic clearance. For all substrates, intrinsic hepatic metabolism was higher than the composite of CYP activities, due to fundamental differences between whole cells and single enzymatic reactions. Of the CYP isozymes investigated, 3A4 yielded the highest absolute and relative metabolism across all substrates, with largely negligible contributions from 2D6 and 2C19. Comparative analysis highlighted elevated lipophilicity and diminished CYP3A4 activity as potential considerations for the development of more efficacious opioid antagonists. Finally, antagonists with a high degree of molecular similarity exhibited comparable clearance, providing a basis for structure-metabolism relationships. Together, these results provide multiple screening criteria for early stage drug discovery involving opioid countermeasures.

Assessment of naloxone as a therapeutic for inhaled carfentanil in the ferret.

Carfentanil is a powerful synthetic opioid that is approximately 100 times more potent than fentanyl and 10,000 times more potent than morphine. Carfentanil was originally intended to be used as a sedative for big game animals in a veterinary setting, but it is becoming increasingly recognized as a public health concern. We set out to investigate the effectiveness of naloxone against a potentially lethal dose of inhaled carfentanil in male ferrets. Ferrets were implanted with telemetry devices to study cardiac parameters and exposed to aerosolized carfentanil in a whole-body plethysmography chamber to record respiratory parameters. We observed profound respiratory depression in exposed animals, which led to apneic periods constituting 24-31 % of the exposure period. Concomitant with these apneic periods, we also observed cardiac abnormalities in the form of premature junctional contractions (PJCs). At our acute exposure dose, lethal in 3 % of our animals, naïve ferrets were unresponsive and incapacitated for a total of 126.1 ± 24.6 min. When administered intramuscularly at human equivalent doses (HEDs) of either 5 mg or 10 mg, naloxone significantly reduced the time that ferrets were incapacitated following exposure, although we observed no significant difference in the reduction of time that the animals were incapacitated between the treatment groups. Naloxone was able to quickly resolve the respiratory depression, significantly reducing the frequency of apneic periods in carfentanil-exposed ferrets. Our results suggest that naloxone, when administered via intramuscular injection following incapacitation, is a viable treatment against the effects of a potentially lethal dose of inhaled carfentanil.