High IC₅₀ Problem-solution Of Low Solubility Inhibitors In Cytochrome P450 Inhibition Assays

Objective: To establish CYP inhibition model in vitro using human liver microsomes, research the high IC50 problem of low solubility inhibitors and improve the determination accuracy of low solubility inhibitor in cytochrome P450 inhibition, reduce the risk ans cost of low solubilty in drug discovery process, avoid the influence of drug safety caused by drug-drug interaction.Methods: After pre-incubation with human liver microsomes and inhibitors(CYP 1A2: Furafylline, CYP2C9: Sulfaphenazole, 2C19:(+)-N-3-Benzylnirvanol, 2D6: Quinidine, 3A4: Ketoconazole), co-incubated with specific probe substrates(CYP 1A2: Phenacetin, CYP2C9: Diclofenac, 2C19: S-Mephenytoin, 2D6: Dextromethorphan, 3A4: Testosterone). Five metabolites(CYP 1A2: Acetaminophen, CYP2C9: 4’-Hydroxydiclofenac, 2C19, 4’-Hydroxymephenytoin, 2D6: Dextrorphan, 3A4: 6β-Hydroxytestosterone) of specific probe substrates metabolized by CYP450 enzymes were measured by LC-MS/MS. Calculated the half maximal inhibitory concentration(IC50) of inhibitors on different subtypes of CYP enzymes, to evaluate the inhibiting ability of different inhibitors. We established CYP inhibition experimental model with one point and multiplate point inhibitor concentration, and compared. After experimental model established, select the inhibitors which with different solubility, using single factor analysis, to investigate the influence to IC50 value by changing the inhibitor solvent, liver microsomes concentration and inhibitor concentration. After single factor analysis study, and then changing the inhibitor solvent, liver microsomes concentration and inhibitor concentration at the same time to investigate the influence to IC50 value.Results:(1) We established successfully CYP inhibition experimental model using human liver microsomes. The IC50 value were calculated in one point and multiplate point CYP inhibition model, the linearly dependent coefficient is up to 0.94, indicating the IC50 value of one point and multiplate point inhibitor concentration have the same accurate and reliable.(2) Selected the inhibitors with different solubility, in single factor analysis assay, when we changed H2 O to DMSO, the inhibitos which have high solubility such as sulfaphenazole, erythromycin and fluconazole, the IC50 value of CYP2C9 and CYP3A4 didn’t have significant changes. But the inhibitos which have poor solubility such as sorafenib, felodipine, terfenadine and amiodarone, the IC50 value of CYP2C9 and CYP3A4 have significant significant changes. It shows that the IC50 value of low solubility inhibitor was significantly decreased by changing solvent from H2 O to DMSO.In another single factor analysis assay, when we reduced the microsome concentrations from 0.5mg/ml to 0.05mg/ml, the IC50 value of sulfaphenazole and fluconazole on CYP2C9, erythromycin on CYP3A4 didn’t have significant changes; the IC50 value of fluconazole on CYP3A4 have significant changes(P<0.05). This indicated that when the inhibitors have high solubility, different inhibitors have different effects on IC50 values. But the inhibitos which have poor solubility such as sorafenib, felodipine, terfenadine and amiodarone, the IC50 value of CYP2C9 and CYP3A4 have significant changes(P<0.05). It testified that the IC50 value of low solubility inhibitor was significantly decreased by reducing microsome concentration from 0.5mg/ml to 0.05mg/ml.In the third single factor analysis assay, we reduced the inhibitor concentrations from 10μM to 3μM, all of IC50 value of high solubility inhibitors didn’t have significant changes, expect the IC50 of erythromycin on CYP3A4 had significant change but didn’t have biological difference. The inhibitos which have poor solubility such as sorafenib and amiodarone, the IC50 value of CYP2C9 have significant changes(P<0.05). The value of sorafenib and amiodarone on CYP3A4 didn’t have significant changes, but there is a decreasing trend. It shows that reduced the inhibitor concentrations from 10μM to 3μM can decrease the IC50 value.(3) Selected the inhibitors which have different solubility, comprehensive study on the inhibitor solvents, microsome and inhibitor concentrations, compared with optimization, the IC50 value of all inhibitos had high significant change(P<0.01). The IC50 of high solubility inhibitors have 2~3 times changes, poor solubility inhibitors have more than 10 times changes show significant biological differences. It testified that there is a significant influence of IC50 value of poor solubility inhibitors when we changed the inhibitors solvents, microsome and inhibitor concentrations at the same time.Conclusion: We have established successfully CYP inhibition experimental model by using human liver microsomes. The IC50 value were calculated in one point and multiplate point CYP inhibition model, the linearly dependent coefficient is up to 0.94, one point CYP inhibition model can greatly save the cost and improve the efficiency of measurement. All of inhibitor solvents, microsome and inhibitor concentrations had significant effect on IC50 value of inhibitors in human microsome CYP inhibition. Increased the proportion of organic solvent in inhibitor solvent, reduced the human liver microsome concentration, and reduced the inhibitor concentration can reduce the IC50 value to the poor solubility inhibitor. If changed these three factors at the same time will get smaller and more accurate IC50 value, and improve the accuracy and success rate of NCEs screening.