Assessment of Cytochrome P450 Metabolic Clearance Using Hepatocyte Suspension

Cai, Jingjing; Shalan, Hadil

doi:10.1007/978-1-0716-1542-3_15

“…The quality of the in vitro input data is not explicitly taken into account in this guidance document yet. Nonetheless, effective protocols for performing in vitro kinetic studies to derive values for CLint, Papp, and Fup are available in the scientific literature (For example Watanabe et al, 2018;Cai and Shalan, 2021;Hubatsch et al, 2007;Black et al, 2021) . We highly recommend that these high-quality protocols would be formalized to describe the applicability domain/use in a regulatory context.…”

Section: Discussionmentioning

confidence: 99%

Impact of in vitro experimental variation in kinetic parameters on physiologically based kinetic (PBK) model simulations_suppl1

Punt

¹

2022

ALTEX

0

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In vitro toxicokinetic data are critical in meeting an increased regulatory need to improve chemical safety evaluations towards a better understanding of internal human chemical exposure and toxicity. In vitro intrinsic hepatic clearance (CLint), the fraction unbound in plasma (Fup), and the intestinal apparent permeability (Papp) are important parameters as input in a physiologically based kinetic (PBK) model to make first estimates of internal exposure after oral dosing. In the present study we explored the experimental variation in the values for these parameters as reported in the literature. Furthermore, the impact that this experimental variation has on PBK model predictions of maximum plasma concentration (Cmax) and the area under the concentration time curve (AUC0-24h) was determined. As a result of the experimental variation in CLint, Papp, and Fup, the predicted variation in Cmax for individual compounds ranged between 1.4-to 28-fold and the predicted variation in AUC0-24h ranged between 1.4-and 23-fold. These results indicate that there are still some important steps to take to achieve robust data that can be used in regulatory applications. To gain regulatory acceptance of in vitro kinetic data and PBK models based on in vitro input data, the boundaries in experimental conditions as well as the applicability domain and the use of different in vitro kinetic models need to be described in guidance documents.

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“…The same hepatocyte metabolic stability assay conditions were applied to manually run experiments as previously described with minor modifications (Cai and Shalan, 2021). Briefly, test compounds were diluted to 2 µM in DMEM buffer, and 50 µL was added in triplicate to each 96-well plate-one plate per time point.…”

Section: Manual Hepatocyte Stability Assaymentioning

confidence: 99%

“…Clearance is an essential pharmacokinetic parameter necessary to understand how quickly a drug gets metabolized and eliminated from the body. The liver is a major metabolic organ that accounts for approximately 60% of marketed drug clearance via the major cytochrome P450 enzyme CYP3A4 (Eijk et al, 2019;Cai and Shalan, 2021). Therefore, hepatic metabolic stability assays evaluating hepatic clearance are critical in the early stage of drug discovery as they provide an efficient route for metabolic liability screening and allow for the extrapolation of in vivo clearance data from in vitro results (Cai and Shalan, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…The liver is a major metabolic organ that accounts for approximately 60% of marketed drug clearance via the major cytochrome P450 enzyme CYP3A4 (Eijk et al, 2019;Cai and Shalan, 2021). Therefore, hepatic metabolic stability assays evaluating hepatic clearance are critical in the early stage of drug discovery as they provide an efficient route for metabolic liability screening and allow for the extrapolation of in vivo clearance data from in vitro results (Cai and Shalan, 2021). Metabolic stability assays are normally performed by incubating the compound in liver subcellular fractions and monitoring the parent compound depletion over time.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

An Integrated Hepatocyte Stability Assay for Simultaneous Metabolic Stability Assessment and Metabolite Profiling

Leung,

Liu,

Cunico

et al. 2024

Drug Metab Dispos

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The determination of metabolic stability is critical for drug discovery programs, allowing for the optimization of chemical entities and compound prioritization. As such, it is common to perform high-volume in vitro metabolic stability experiments early in the lead optimization process to understand metabolic liabilities. Additional metabolite identification experiments are subsequently performed for a more comprehensive understanding of the metabolic clearance routes to aid medicinal chemists in the structural design of compounds. Collectively, these experiments require extensive sample preparation and a substantial amount of time and resources.To overcome the challenges, a high-throughput integrated assay for simultaneous hepatocyte metabolic stability assessment and metabolite profiling was developed. This assay platform consists of four parts: 1) an automated liquid-handling system for sample preparation and incubation; 2) a liquid chromatography and high-resolution mass spectrometry-based system to simultaneously monitor the parent compound depletion and metabolite formation; 3) an automated data analysis and report system for hepatic clearance assessment; and 4) a streamlined auto-batch processing for software-based metabolite profiling. The assay platform was evaluated using eight control compounds with various metabolic rates and biotransformation routes in hepatocytes across three species. Multiple sample preparation and data analysis steps were evaluated and validated for accuracy, repeatability, and metabolite coverage. The combined utility of an automated liquid-handling instrument, a high-resolution mass spectrometer, and multiple streamlined data processing software improves the process of these highly demanding screening assays, and allows for simultaneous determination of metabolic stability and metabolite profiles for more efficient lead optimization during early drug discovery.

show abstract

Impact of in vitro experimental variation in kinetic parameters on physiologically based kinetic (PBK) model simulations

Punt

¹

2022

ALTEX

View full text Add to dashboard Cite

In vitro toxicokinetic data are critical in meeting an increased regulatory need to improve chemical safety evaluations towards a better understanding of internal human chemical exposure and toxicity. In vitro intrinsic hepatic clearance (CLint), the fraction unbound in plasma (Fup), and the intestinal apparent permeability (Papp) are important parameters as input in a physiologically based kinetic (PBK) model to make first estimates of internal exposure after oral dosing. In the present study we explored the experimental variation in the values for these parameters as reported in the literature. Furthermore, the impact that this experimental variation has on PBK model predictions of maximum plasma concentration (Cmax) and the area under the concentration time curve (AUC0-24h) was determined. As a result of the experimental variation in CLint, Papp, and Fup, the predicted variation in Cmax for individual compounds ranged between 1.4-to 28-fold and the predicted variation in AUC0-24h ranged between 1.4-and 23-fold. These results indicate that there are still some important steps to take to achieve robust data that can be used in regulatory applications. To gain regulatory acceptance of in vitro kinetic data and PBK models based on in vitro input data, the boundaries in experimental conditions as well as the applicability domain and the use of different in vitro kinetic models need to be described in guidance documents.

show abstract

Assessment of Cytochrome P450 Metabolic Clearance Using Hepatocyte Suspension

Cited by 3 publications

References 18 publications

Impact of in vitro experimental variation in kinetic parameters on physiologically based kinetic (PBK) model simulations_suppl1

Impact of in vitro experimental variation in kinetic parameters on physiologically based kinetic (PBK) model simulations_suppl1

An Integrated Hepatocyte Stability Assay for Simultaneous Metabolic Stability Assessment and Metabolite Profiling

Impact of in vitro experimental variation in kinetic parameters on physiologically based kinetic (PBK) model simulations

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