| ObjectiveEpilepsy is one of the most common chronic serious neurological diseases,affecting approximately 65 million people worldwide.In China,about 9 million people suffer from epilepsy,and over 600,000 to 700,000 new patients are diagnosed every year.Carbamazepine as a broad-spectrum antiepileptic drug is one of the most prescribed drugs for the treatment of grand mal seizure disorder and refractory epilepsy.However,the treatment window of carbamazepine is narrow,and the effective blood concentration range is 4-12 mg/L,which is easy to induce drug-drug adverse reactions when taken with other drugs.Carbamazepine is mainly metabolized by cytochrome P450 enzyme(CYP)3A4 to produce carbamazepine 10,11-epoxide,which plays an antiepileptic effect.Previous studies have shown that traditional Chinese medicine has been widely used in epilepsy treatment.Therefore,it is necessary to evaluate the effect of active ingredients in Chinese medicine on CYP3A4 mediated carbamazepine metabolism.However,many clinical data of Chinese herbal medicine-drug interactions come from the non-carbamazepine CYP3A4 probe response.Therefore,this study established a CYP3A4-mediated carbamazepine in vitro assay to accurately assess the risk of clinical Chinese herbal medicine-carbamazepine interactions and guide clinical rational drug to use.Methods1.This study is based on the optimization of liquid chromatography technology to establish a CYP3A4-mediated carbamazepine and its main metabolite carbamazepine 10,11-epoxide detection method in vitro.2.In this study,the risk assessment of Chinese herbal medicine-carbamazepine interactions was performed from 65 natural products of Chinese medicine,which based on the specific CYP3A4 probe substrate reaction based on carbamazepine established in vitro.3.The monomer components with inhibitory action were selected from traditional Chinese medicine.The interaction prediction between traditional Chinese medicine monomer components and carbamazepine was performed through inhibition kinetic experiments and proved with molecular simulation docking technique.Results1.This study established a CYP3A4-mediated detection method for carbamazepine and major metabolites in vitro.The concentration of the product was linear in the range of 0.4-100 μM.Methodological verification showed that the method has strong specificity,good precision,and high recovery.2.While the establishment of CYP3A4-mediated carbamazepine evaluation model in vitro,the screening of carbamazepine interactions from active ingredients of Chinese herbal medicines was carried out from 65 kinds of natural products of traditional Chinese medicine.It was found that carvacrol strongly inhibited CYP3A4 mediated carbamazepine 10,11-epoxidation with residual activity of 22.7±1.4%.The half inhibitory concentration(IC50)of the inhibition was further measured.Carvacrol produces a concentration-dependent inhibition of its catalyzed reaction,with an IC50 value of 15.3±1.4 μM.3.The types of reversible inhibition and kinetic parameters of carvacrol were determined.It was found that the kinetic reaction of carbamazepine 10,11-epoxide mediated by CYP3A4 conformed to the characteristics of noncompetitive inhibition as revealed by increased Vmax and almost unchanged Km with the increase of carvacrol concentration.By nonlinear fitting,the reversible inhibition constant Ki was 14.1 ± 1.0 μ M..Conclusions1.This study established a CYP3A4-mediated carbamazepine epoxidation probe reaction detection method,which is simple,convenient,accurate,and easy to operate.2.Using the CYP3A4 probe substrate reaction based on carbamazepine specificity established in this study to screen out that carvacrol in aromatic plants and carbamazepine may cause serious drug-drug interactions.3.Inhibition of carvacrol further studies showed that carvacrol had a non-competitive inhibitory effect on CYP3A4-mediated carbamazepine. |
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