Challenges with Large or Poorly Permeabilized Drugs
Primary hepatocytes are the gold standard for in vitro metabolism studies, but their cell membranes can restrict the uptake of large or poorly permeable drugs, making it difficult to assess metabolism for these compounds. Traditional hepatocyte models rely on passive diffusion or active transport for intracellular drug entry, meaning that drugs with poor membrane permeability may not properly engage key metabolic enzymes like cytochrome P450s (CYPs) or UDP-glucuronosyltransferases (UGTs).
For example, PROTACs, due to their size and high polar surface area, have limited passive diffusion across cell membranes. Poor permeability can lead to an underestimation of metabolism by intracellular CYP enzymes, affecting clearance predictions and DDI risk assessments3. As a result, the drug’s actual metabolic potential may be underestimated, leading to inaccurate assumptions about half-life and dosing. This can contribute to suboptimal therapeutic levels or increase the likelihood of unexpected adverse events in clinical trials2-5. Similarly, ADCs (~150 kDa) rely on receptor-mediated endocytosis for cellular uptake. Because transporter expression can vary across donors, metabolism studies using primary hepatocytes may introduce unwanted variability that complicates clearance predictions.
Since traditional hepatocytes may not fully capture the metabolism of these drug types, permeabilized hepatocytes provide a novel solution by removing membrane barriers and transporter-mediated entry. This allows for direct enzyme interactions, allowing researchers to effectively evaluate permeability-limited metabolism and DDI of large or poorly permeabilized drugs6.
Advantages of Permeabilized Hepatocytes
The permeabilized cell membrane of Gentest® MetMax® hepatocytes allows drug compounds to bypass membrane barriers and directly access intracellular metabolic enzymes without transporter limitations. This overcomes challenges for studying the metabolism of large or poorly permeable compounds that may struggle to enter intact hepatocytes. Since the cell membrane barrier is removed, the observed metabolic rate in permeabilized hepatocytes more accurately reflects the drug’s intrinsic metabolic capacity2.
Additionally, this approach is particularly valuable for studying drugs that undergo metabolic activation, as it allows researchers to detect toxic metabolites that could contribute to drug-induced liver injury (DILI)5. Identifying potential safety risks early in drug development reduces the likelihood of late-stage failures.
Conclusion
As drug discovery continues to evolve with more complex modalities, new in vitro tools are needed to assess their metabolism. A clear understanding of metabolism is critical for predicting clearance, optimizing dosing, and ensuring patient safety. Permeabilized hepatocytes are a powerful addition to the drug metabolism toolbox, providing direct enzyme access for large or poorly permeable drugs that may otherwise be limited in traditional models.
By integrating permeabilized hepatocytes into drug metabolism workflows, researchers can gain deeper insights into the metabolic fate of complex therapeutics and generate more accurate data. As the industry continues to develop next-generation therapies, innovative in vitro models like MetMax® hepatocytes will play a critical role in bridging the gap between preclinical metabolism studies and successful clinical translation.
References
- Koziolek M, Augustijns P, Berger C, et al. Challenges in Permeability Assessment for Oral Drug Product Development. Pharmaceutics. 2023;15(10):2397. Published 2023 Sep 28. doi:10.3390/pharmaceutics15102397
- Zhang S, Orozco CC, Tang LWT, et al. Characterization and Applications of Permeabilized Hepatocytes in Drug Discovery. AAPS J. 2024;26(3):38. Published 2024 Mar 28. doi:10.1208/s12248-024-00907-9
- Liping Ma L. Tackling the DMPK challenges of developing PROTAC drugs. Drug Discovery & Development. April 15, 2022. Accessed February 21, 2025. https://www.drugdiscoverytrends.com/tackling-the-dmpk-challenges-of-developing-protac-drugs/
- Wegler C, Matsson P, Krogstad V, et al. Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes. Mol Pharm. 2021 ;18(4) :1792-1805. doi: 10.1021/acs.molpharmaceut.1c00053
- Wei H, Li AP. Permeabilized Cryopreserved Human Hepatocytes as an Exogenous Metabolic System in a Novel Metabolism-Dependent Cytotoxicity Assay for the Evaluation of Metabolic Activation and Detoxification of Drugs Associated with Drug-Induced Liver Injuries: Results with Acetaminophen, Amiodarone, Cyclophosphamide, Ketoconazole, Nefazodone, and Troglitazone. Drug Metab Dispos. 2022;50(2):140-149. doi:10.1124/dmd.121.000645
- Sawant-Basak A, Obach Emerging Models of Drug Metabolism, Transporters, and Toxicity. Drug Metab Dispos. 2018;46(11):1556-1561. doi:10.1124/dmd.118.084293