(T1330-02-10) Genetic Polymorphism Based Inhibition on CYP450 by Radix Puerariae lobatae in Healthy Subjects

Purpose: Since previous in vitro and limited clinical findings from us and others suggested that Radix Puerariae lobatae (Gegen) might have impacts on cytochrome (CYP) 450 isoenzymes, we have conducted a preliminary clinical study to evaluate the effect of orally administered Gegen on CYPs enzymes in healthy volunteers using cocktail approach and found that two-week treatment of Gegen could significantly inhibit CYP2C19 in human, especially in poor metabolizers. To further evaluate the genetic polymorphism impact on such inhibition of CYP by Gegen, further clinical investigations were conducted to 1) evaluate whether there existed any dose dependency of such inhibition on CYP by Gegen; 2) identify the genetic mutation on CYP2C19 poor metabolizers.

Methods: About fifty two healthy subjects (26 males and 26 females) were randomized into two groups (14 males & 14 females for low dose Gegen group; 12 males & 12 females for high Gegen dose group ) for investigations on the effect of Gegen at low (3.3 g/time,t.i.d) or high doses (5 g/time, t.i.d) on the cocktail probe drugs for six CYPs (caffeine 100 mg, efavirenz 50 mg, losartan 12.5 mg, omeprazole 10 mg, metoprolol 12.5 mg, and midazolam 2 mg as the probe drugs for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, respectively). All the healthy volunteers received a single oral dose of the six-probe cocktail as baseline (Session I) followed by a 14-day oral treatment with Gegen at the dose of 3.3g/5g per time, t.i.d. On Day 14, 2h after the Gegen dosing, each healthy volunteer received another dose of the six-probe cocktail (Session II). Blood samples were collected in each session before dosing (0 h), 0.25, 0.50, 0.75, 1, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h after dosing of the six-probe cocktail to obtain plasma samples for analyses of the probe and their metabolite concentrations by our previously developed LC-MS/MS method [1]. Pharmacokinetic parameters were then calculated using a non-compartmental model. To further verify the pharmacogenetic impact, identification for the variant of CYP2C19 in those subjects with a reduced-function CYP2C19 genotype using PCR followed by Sanger sequencing and comparisons with reference genes from NCBI were conducted. In the meantime, to evaluate the impact of Gegen on the anticoagulant effects, plasma thromboxane B2 (TXB2)/thrombomodulin (CD141) levels were quantified by commercial enzyme-linked immunosorbent assay kits (ab133022/ab46508, Abcam Plc) and prothrombin time (PT) was determined according to the supplier’s manual. All measurements were conducted in triplicate. Correlation analyses on enzyme activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (represented by corresponding AUC_{metabolite (0-72h)}/AUC_{probe drugs (0-72h)}), TXB2, CD141 and PT from different treatment groups were conducted using R studio.

Results: As indicated in Table 1, among the six CYP450 isoenzymes, CYP2C19 demonstrated the strongest dose-dependent inhibition by Gegen. CYP2C19 could be only significantly inhibited by high dose Gegen in humans especially in non-poor metabolizers of CYP2C19 (AUC_{metabolites (0-72h)}/AUC_{probe drugs(0-72h})≥1 in Session I) and females (p < 0.05) with no impact by low dose Gegen. In the further pharmacogenetic study, single nucleotide mutation in Exon 1, Exon 5 and Exon 7 and mutation I331V were observed for most of the poor metabolizers. On the other hand, CYP1A2 was induced by both low and high doses of Gegen with no dose dependency observed. Furthermore, only the CYP1A2 enzyme was found to be significantly induced by low dose Gegen in humans, especially in non-poor metabolizers of CYP1A2 and in males (p < 0.05), and significant induction of CYP1A2 by high dose Gegen was observed in poor metabolizers of CYP1A2 (AUC_{metabolites (0-72h)}/AUC_{probe drugs(0-72h}) <1 in Session I) (p < 0.05). Additionally, CYP2D6 could be only significantly inhibited by low dose Gegen in non-poor metabolizers of CYP2D6 (p < 0.05) with no impact by high dose Gegen. As to CYP2B6, CYP2C9 and CYP3A4, minimal impact were observed after Gegen treatments. As shown in Fig. 1, plasma TXB2 levels were found to be significantly decreased via co-administration with Gegen at both doses with no dose dependency observed, while for CD141 levels determination, only the high dose group was found to have a significant decrease. Further correlation analyses indicated a positive correlation between CYP2C19 activity and CD141 levels, while negative correlations of TXB2 levels with CYP1A2 activity, and with CD141 levels. Moreover, correlations of PT values were found to be negative with CYP1A2, and positive with CYP2B6, although no significant impact on the PT values was found by both low and high doses of Gegen (Fig. 2).

Conclusion: Among the six major CYPs, orally administered Gegen could only inhibit CYP2C19 in a genotype and dose-dependent manner. Considering its extensive clinical applications, further studies that could provide genetic screening guidances on the safe use of Gegen in individuals are warranted.

References: [1] J. Zhang, Y. Zhang, Q. B. Song, J. H. Li, Y. S. Lai, S. K. Wo, Z. Zuo. Clinical evaluations of potential CYP450 inhibition by Radix Puerariae lobatae (Gegen) in healthy subjects. American Association of Pharmaceutical Scientists 2023 PharmSci 360, Orlando, FL, United States.

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


Table 1. Impact of Gegen at low and high doses on the CYP450 enzyme- mediated probe drug metabolisms


Fig. 1. Comparison of plasma concentrations of thromboxane B2 (TXB2, Upper) and Thrombomodulin (CD141, Middle) and Prothrombin Time (PT, Bottom) in healthy subjects at the pre-dose of Session I (Open Column) and 72h post-dose in Session II (Slash column). *p < 0.05, ** p < 0.01, **** p < 0.0001.



Fig. 2. Correlation analyses of enzyme activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP2B6 (represented by corresponding AUCmetabolite (0-72h)/AUCprobe drugs (0-72h)), Prothrombin Time, Levles of Thromboxane B2 and Thrombomodulin from different treatment groups. D: probe drugs, D.H: probe drugs + Gegen, Pre: pre-dose, Post: post-dose, red and blue circle represented negative and positive correlation respectively with significant level p<0.05. Insignificant correlations were left blank.