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Updated: Aug 19, 2026
Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells
Published on: October 20, 2023
Evaluation of time-dependent inactivation of CYP3A in cryopreserved human hepatocytes
Ping Zhao1, Kent L Kunze, Caroline A Lee
1Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, La Jolla Laboratories, 10724 Science Center Dr., San Diego, CA 92121, USA. ping.zhao2@pfizer.com
Abstract:
Irreversible CYP3A inhibition by drugs constitutes one of the major causes of inhibition-based drug interactions. We evaluated time-dependent inactivation of CYP3A in cryopreserved human hepatocytes for six structurally diverse compounds known to exhibit this property. Inactivation kinetic parameters were also determined using human liver microsomes. Except for diclofenac, which did not cause CYP3A inactivation either in microsomes or in hepatocytes at concentrations up to 100 microM, time-dependent inactivation was observed in hepatocytes for amprenavir, diltiazem, erythromycin, raloxifene, and troleandomycin. The observed inactivation potency in hepatocytes (observed IC50) was compared with the potency predicted using microsomal parameters (predicted IC50). Despite satisfactory prediction for troleandomycin (1.35 and 2.14 microM for the predicted and observed IC50, respectively), over-prediction of inactivation was observed for raloxifene, amprenavir, and erythromycin (observed IC50 values 6.2-, 55-, and 7.8-fold higher, respectively, than the predicted IC50). By contrast, the observed IC50 for diltiazem in hepatocytes was approximately 4-fold lower than the IC50 predicted from microsomal data (under-prediction). After correcting for factors including nonspecific binding and inactivator consumption, prediction was significantly improved for raloxifene (the observed IC50 then became 2-fold higher than the predicted IC50) and for amprenavir to a lesser extent. A specific P-glycoprotein inhibitor, 4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-N-[2-(3.4-dimethoxyphenyl)ethyl]-6,7-dimethoxyquinazolin-2-amine (CP-100356), modulated the observed CYP3A inactivation potency by erythromycin and troleandomycin. In summary, these studies reveal three important factors that must be considered when microsomal inactivation parameters are used to predict inhibition-based drug interactions in intact cell systems.
Insights
Drug interactions involving irreversible CYP3A inhibition are common. This study evaluated time-dependent inactivation in hepatocytes, finding predictions from microsomes varied, highlighting factors for accurate drug interaction assessment.
Area of Science:
- Pharmacology
- Drug Metabolism
- Biochemistry
Background:
- Irreversible inhibition of Cytochrome P450 3A (CYP3A) is a significant cause of drug-drug interactions.
- Understanding drug inactivation kinetics is crucial for predicting these interactions.
Purpose of the Study:
- To evaluate time-dependent inactivation of CYP3A in human hepatocytes for six known inactivators.
- To compare in vivo inactivation potency with predictions derived from in vitro microsomal data.
- To identify factors influencing the accuracy of these predictions.
Main Methods:
- Incubation of cryopreserved human hepatocytes and liver microsomes with six diverse drug compounds.
- Determination of inactivation kinetic parameters (IC50 values).
- Comparison of observed hepatocyte IC50 with predicted IC50 from microsomal data, with adjustments for binding and consumption.
Main Results:
- Time-dependent CYP3A inactivation was observed in hepatocytes for amprenavir, diltiazem, erythromycin, raloxifene, and troleandomycin.
- Predictions based on microsomal data showed variable accuracy, with over-prediction for raloxifene, amprenavir, and erythromycin, and under-prediction for diltiazem.
- Adjustments for nonspecific binding and inactivator consumption improved prediction accuracy for raloxifene and amprenavir.
- A P-glycoprotein inhibitor modulated inactivation potency for erythromycin and troleandomycin.
Conclusions:
- Microsomal inactivation parameters alone are insufficient for accurately predicting CYP3A inhibition in intact cells.
- Nonspecific binding, inactivator consumption, and transporter effects (like P-glycoprotein) are critical factors influencing drug interaction predictions.
- Accurate prediction of drug-drug interactions requires considering these additional factors in intact cell systems.
