(T1430-02-08) Ontogeny of Drug Metabolizing Enzymes and Its Impact on Valproic Acid Hepatotoxicity: Insights from In Vitro and PBPK Modeling Studies

Purpose: Valproic acid (VPA) is a widely utilized anticonvulsant in both pediatric and adult populations. Despite its broad therapeutic application, VPA has been associated with severe hepatotoxicity, predominantly in pediatric patients. The precise mechanisms underlying valproate-induced hepatotoxicity remain elusive. However, substantial evidence points to the involvement of the parent compound and its unsaturated metabolite, 4-ene-valproic acid (4-ene-VPA), which is primarily produced through phase I metabolism by cytochrome P450 (CYP) enzymes. Furthermore, in pediatric patients, particularly neonates and infants, there is a predisposition towards CYP-mediated oxidation of VPA. This is attributed to the immature phase II metabolic activity of UDP-glucuronosyltransferase (UGT) enzymes, leading to insufficient elimination of VPA metabolites. This study integrates in vitro data from HepG2 cells transfected with metabolic enzymes and physiologically based pharmacokinetic (PBPK) models of VPA and 4-ene-VPA. This approach simulates potential differences in VPA metabolic pathways between adults and pediatric patients, enabling the exploration of the role of ontogeny in valproate hepatotoxicity.

Methods: Physiologically based pharmacokinetic (PBPK) models for VPA and 4-ene-VPA in both adults and pediatric patients were constructed and executed using the Simcyp PBPK Simulator. Sensitivity analysis was performed to assess the impact of CYP enzymes and UGT2B7 on the area under the curve (AUC) of VPA and 4-ene-VPA, utilizing a sensitivity index. Human hepatocellular carcinoma HepG2 cells transfected with VPA metabolism-associated enzymes—cytochrome P450 (CYP) 2C9 and 2E1, and UDP-glucuronosyltransferase (UGT) 2B7—were employed as experimental models to simulate the differential metabolic pathways between pediatric and adult patients.

Results: The sensitivity analysis revealed that CYP2C9 is the predominant enzyme involved in the metabolism of both VPA and 4-ene-VPA in pediatric patients. The limited activity of UGT2B7 in this age group results in a greater dependence on oxidative pathways mediated by CYP2C9 and CYP2E1, potentially contributing to a heightened risk of hepatotoxicity. In adults, UGT2B7 assumes a more prominent role, promoting glucuronidation and facilitating safer drug elimination. The balanced enzymatic contributions of CYP2C9 and UGT2B7 in adults may indicate a reduced risk of hepatotoxicity compared to pediatric patients. Valproic acid (VPA) increased mitochondrial superoxide production and induced cell death in HepG2 cells transfected with either CYP2C9 or CYP2E1, representing the primary VPA metabolic pathways in pediatric patients. In contrast, HepG2 cells co-transfected with CYP2C9 and UGT2B7, or CYP2E1 and UGT2B7, which represent the major metabolic pathways in adults, exhibited minimal cell injury compared to cells transfected with CYP2C9 or CYP2E1 alone. Furthermore, VPA-associated lipid accumulation was observed exclusively in HepG2 cells transfected with CYP2C9. Notably, no lipid accumulation occurred in HepG2 cells co-transfected with CYP2E1 and UGT2B7.

Conclusion: The in vitro data demonstrated that VPA-induced cell injuries in HepG2 cells transfected with either CYP2C9 or CYP2E1 were more severe compared to those in HepG2 cells co-transfected with CYP2C9 and UGT2B7 or CYP2E1 and UGT2B7. This finding suggests that young children are more susceptible to valproate hepatotoxicity. The PBPK models developed for VPA and 4-ene-VPA further underscored the significance of ontogeny in the differing metabolic pathways between pediatric and adult patients. However, additional research is necessary to identify and elucidate other factors that may influence the absorption, distribution, metabolism, and excretion of VPA in pediatric patients.

References: 1. Huang, Y. T., Huang, Y. M., Kung, F. L., Lin, C. J., Jao, T., & Ho, Y. F. (2023). Physiologically based mechanistic insight into differential risk of valproate hepatotoxicity between children and adults: A focus on ontogeny impact. CPT: pharmacometrics & systems pharmacology, 12(12), 1960–1971. https://doi.org/10.1002/psp4.13045
2. Ghodke-Puranik, Y., Thorn, C. F., Lamba, J. K., Leeder, J. S., Song, W., Birnbaum, A. K., Altman, R. B., & Klein, T. E. (2013). Valproic acid pathway: pharmacokinetics and pharmacodynamics. Pharmacogenetics and genomics, 23(4), 236–241. https://doi.org/10.1097/FPC.0b013e32835ea0b2

Acknowledgements: The authors are indebted to Dr. Ian Gardner at the Simcyp for his valuable comments. This study would not have been possible without Dr. David Yu-Shen Lai and Mrs. Amy Huei-Mei Lin's constant encouragement and support. Dr. Shiew-Mei Huang at the US Food and Drug Administration (FDA) is especially acknowledged for supporting the corresponding author as an ORISE fellow in 2017 to explore PBPK research. Certara UK Limited (Simcyp Division) granted access to the Simcyp Simulators through a sponsored academic license (subject to conditions).

Hypothesis about mechanism of VPA hepatotoxicity in pediatric.

The GSA to compare the influence of Clint,CYP2A6, Clint,CYP2B6, Clint,CYP2C9, Clint,CYP2E1, and Clint,UGT2B7 on the simulated AUC of VPA and 4-ene-VPA.

Effect of different drug-metabolizing enzymes expression on valproate hepatotoxicity. (A) cell viability (B) mitochondrial superoxide levels (C) lipid accumulation. Data were presented as mean ± SD from 3 independent experiments. *P < 0.05 compared with untreated (**P < 0.01 and ***P < 0.001). #P < 0.05 compared with HepG2 cells transfected with phase Ⅰ enzymes (##P < 0.01 and ###P < 0.001).