Prediction of relative in vivo metabolite exposure from in vitro data using two model drugs: dextromethorphan and omeprazole

Abstract

Metabolites can have pharmacological or toxicological effects, inhibit metabolic enzymes, and be used as probes of drug-drug interactions or specific cytochrome P450 (P450) phenotypes. Thus, better understanding and prediction methods are needed to characterize metabolite exposures in vivo. This study aimed to test whether in vitro data could be used to predict and rationalize in vivo metabolite exposures using two model drugs and P450 probes: dextromethorphan and omeprazole with their primary metabolites dextrorphan, 5-hydroxyomeprazole (5OH-omeprazole), and omeprazole sulfone. Relative metabolite exposures were predicted using metabolite formation and elimination clearances. For dextrorphan, the formation clearances of dextrorphan glucuronide and 3-hydroxymorphinan from dextrorphan in human liver microsomes were used to predict metabolite (dextrorphan) clearance. For 5OH-omeprazole and omeprazole sulfone, the depletion rates of the metabolites in human hepatocytes were used to predict metabolite clearance. Dextrorphan/dextromethorphan in vivo metabolite/parent area under the plasma concentration versus time curve ratio (AUC(m)/AUC(p)) was overpredicted by 2.1-fold, whereas 5OH-omeprazole/omeprazole and omeprazole sulfone/omeprazole were predicted within 0.75- and 1.1-fold, respectively. The effect of inhibition or induction of the metabolite's formation and elimination on the AUC(m)/AUC(p) ratio was simulated. The simulations showed that unless metabolite clearance pathways are characterized, interpretation of the metabolic ratios is exceedingly difficult. This study shows that relative in vivo metabolite exposure can be predicted from in vitro data and characterization of secondary metabolism of probe metabolites is critical for interpretation of phenotypic data.

Fig. 1.

Metabolic scheme of dextromethorphan. The enzyme responsible for the formation of each metabolite and the metabolite/parent pair used as an in vivo activity measure for the specified P450 isoform is indicated. UGT indicates proposed UDP-glucuronosyltransferase metabolism.

Fig. 2.

Metabolic scheme of omeprazole. The enzyme responsible for the formation of each metabolite and the metabolite/parent pair used as an in vivo activity measure for the specified P450 isoform are indicated.

Fig. 3.

Characterization of in vitro formation and metabolism of dextrorphan. Formation of dextrorphan from dextromethorphan (A), 3-hydroxymorphinan from dextrorphan (B), and dextrorphan-O-glucuronide from dextrorphan (C) in pooled HLM. Dextrorphan-O-glucuronide is shown both with (■) and without (●) the presence of 2% BSA. D, formation of dextrorphan-O-glucuronide after incubation of dextrorphan with 12 rUGT isoforms. Error bars represent the S.D. of three measurements (A–C) and the range of two measurements (D).

Fig. 4.

Characterization of in vitro formation and depletion of 5OH-omeprazole and omeprazole sulfone in pooled HLM. Formation (A) and depletion (B) of 5OH-omeprazole. Formation (C) and depletion (D) of omeprazole sulfone. B, the percentage remaining of 5OH-omeprazole over time is provided with (■) and without (●) NADPH. Error bars represent the S.D. of three measurements.

Fig. 5.

Characterization of in vitro formation and depletion of 5OH-omeprazole and omeprazole sulfone in hepatocytes. Formation of 5OH-omeprazole (A) and omeprazole sulfone (C) from omeprazole. The percentage remaining of 5OH-omeprazole (B) and omeprazole sulfone (D). Error bars represent the S.D. of four measurements.

Fig. 6.

Simulation of the effects of inhibition and induction of probe metabolite clearance in vivo on AUC_m/AUC_p activity measures. The AUC_m/AUC_p for a generic metabolite/parent pair in which E1 is solely responsible for forming the probe metabolite and the probe metabolite is eliminated by enzymes E1 and E2 as described under Materials and Methods is simulated under the conditions of E1 induction, E2 induction, E1 inhibition, and E2 inhibition. Left, the condition in which E1 is the only enzyme that clears the metabolite (f_m,E1 = 1 and f_m,E2 = 0 for metabolite elimination) is shown as the dark blue line, the condition in which both E1 and E2 have equal responsibility for clearing the metabolite (f_m,E1 = 0.5 and f_m,E2 = 0.5) is shown as the green line, and the condition in which E2 is the only enzyme that clears the metabolite (f_m,E1 = 0 and f_m,E2 = 1) is shown as the purple line. Right, the effect of f_m,E1 (the fraction of the metabolite clearance by the same enzyme which forms it) on the magnitude of change in AUC_m/AUC_p after a 2-fold (light blue line), 5-fold (black line), and 10-fold (red line) increase (for induction) or decrease (for inhibition) in the specified enzyme activity.