(W1030-10-65) Clinical Evaluations of Potential CYP450 Inhibition by Radix Puerariae lobatae (Gegen) in Healthy Subjects

Purpose: Based on our preclinical findings, it is hypothesized that puerarin containing Radix Puerariae lobatae (Gegen) could inhibit the CYP 450s and offset the activities of anticoagulation and antiplatelet drugs via alteration of the relevant biomarkers. Our current study was designed aiming to evaluate the potential inhibition of CYPs enzymes by coadministered Gegen in healthy subjects.

Methods: An in vivo cocktail approach using caffeine (100 mg), losartan (12.5 mg), omeprazole (10 mg), metoprolol (12.5 mg), midazolam (2 mg), and efavirenz (50 mg) as the probe drugs for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP2B6, respectively, was adopted. A specific LC-MS/MS assay was developed and validated for the determination of plasma concentrations of these probe drugs and their corresponding metabolites following oral administrations in healthy volunteers. In the randomized, parallel study under fasting conditions, all healthy subjects (11 male and 13 female) underwent two study sessions. Session I served as the baseline session, in which a single oral dose of the cocktail probe drugs was administered. After about two weeks washout period, subjects underwent Session II, which involved a multi-dose of Gegen granules (equivalent to 5 g crude herb/person/time, t.i.d. ) for 14 days followed by a single oral dose of the cocktail probe drugs as those in Session I at 2h post Gegen administration on day 14. Blood was collected in each session before dosing (0 h) and 0.25, 0.50, 0.75, 1, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h after dosing of cocktail probe drugs to obtain plasma samples for analyses of the probe and their metabolites concentrations by LC-MS/MS method developed above. The non-compartmental model was used to analyze the plasma concentration versus time profiles to calculate the corresponding pharmacokinetic parameters.

Results: The developed LC-MS/MS method for the simultaneous determination of the six probe drugs and their metabolites was selective for all tested analytes with no interference effect observed. Additionally, assays were linear over a wide range (2.0-1000 ng/ml for all analytes except for caffeine and its metabolite paraxanthine (31.2-1000 ng/ml) and lower limits of quantification for all analytes varied between 2.0-31.2 ng/mL in plasma precision and the inter-/intra day assay accuracies all less than 15% (Table 1). All probe drugs and their metabolites were found stable in plasma during three freeze-thaw cycles. The clinical study results in Table 2 demonstrated that the CYP2C19 enzyme could be significantly inhibited by Gegen with a 56.9% inhibition on omeprazole metabolism in humans and 59.3% of inhibition in those who are non-poor metabolizers of CYP2C19 (AUC_{metabolites (0-72h)}/AUC_{probe drugs(0-72h}) ≥1 in Session I) (p < 0.05). Additionally, the use of Gegen could slightly inhibit CYP2D6 (15.6% inhibition on metoprolol metabolism) and induce CYP1A2, CYP3A4 and CYP2B6 (20.0%, 15.2% and 11.1% increase on caffeine, midazolam and efavirenz metabolism, respectively). Moreover, in poor metabolizers of CYP1A2 (AUC_{metabolites (0-72h)}/AUC_{probe drugs(0-72h}) < 1 in Session I), CYP1A2 enzyme could be significantly induced by Gegen with a 187.0% increase on caffeine metabolism (p < 0.05). The effect of Gegen on CYP2C9 could be minimal due to limited changes ( < 1%) in losartan metabolism after Session II.

Conclusion: A cocktail approach was adopted to monitor the impact of Gegen at its clinical dose on the activity of six major CYP450 enzymes. We for the first time revealed that two weeks treatment of Gegen could significantly inhibit CYP2C19 in humans, especially in extensive metabolizers. In addition, slight inhibition on CYP2D6 and inductions on CYP1A2, CYP3A4 and CYP2B6 were observed after Gegen treatment. Furthermore, inductions of CYP1A2 by Gegen were found to be significant in poor metabolizers. These findings implied that special attention should be paid to the use of Gegen when co-administrated with drugs that were substrates of CYP2C19, CYP2D6, CYP1A2, CYP3A4 and CYP2B6.

Acknowledgements: Health and Medical Research Fund (Reference No.: 17180741) from Health Bureau of the Government of Hong Kong SAR.

Table 1. Precision and accuracy for determination of the studied analytes in human plasma by LC/MS/MS

Table 2 Effects of Gegen on the CYP 450 enzymes mediated probe drug metabolisms