(M1330-02-07) Understanding the Influence of Drug Metabolizing Enzyme Activity on Small Molecule Stability Within a Commonly Used Preclinical Tumor Model

Purpose: While unbound plasma concentrations are often used as a surrogate for unbound tumor concentrations early in a drug discovery effort, ADME properties such as tumor clearance and permeability can affect the relationship between unbound plasma and unbound tumor concentration. Tumor cells have been found to express a host of Phase 1 and Phase 2 drug metabolizing enzymes, including members of the cytochrome P450 and UDP-glucuronosyltransferase superfamilies, which are known to metabolize a wide range of therapeutic enzymes. As such, the expression of P450 enzymes in tumor tissue may play a role in decreasing drug concentrations within the tumor, ultimately decreasing the effectiveness of chemotherapeutic drugs. Unfortunately, only a limited number of studies have evaluated the expression and activity of DMEs in cultured tumor cell lines.

Methods: In this study we utilized the CT26 tumor cell line, a syngeneic murine colorectal carcinoma derived from BALB/c mice, which is commonly used in oncology drug discovery. Specifically, we measured the in vitro intrinsic clearance of selected P450 substrates and chemotherapeutic drugs and utilized minimal physiologically-based pharmacokinetics (PBPK) modeling to simulate combinations of tumor permeability and clearance that could lower intracellular tumor concentrations. We further elucidated the qualitative expression of DMEs in the CT26 cell line using traditional bottom-up proteomic analysis.

Results: Our results showed that four of the seven P450 substrates exhibited metabolite formation in CT26 cells, confirming Cyp activity in the tumor cells. HRMS based proteomics analysis showed increased expression of Cyp20a1 and Cyp2c55 among other proteins in the CT26 tissue, relative to their levels in control tissue. Subsequently, PBPK models were used to evaluate the combined effects of tumor clearance and permeability on the resulting tumor concentrations of a given drug.

Conclusion: Ultimately, the work demonstrates the importance of understanding the role of tumor metabolism on unbound drug concentrations which are able to then interact within their pharmacological targets.  While the overall metabolic rates observed in CT26 cells were modest in this study, further research is needed to determine if this observation is true in other commonly used tumor cell lines in drug discovery research.

References: Yu LJ, Matias J, Scudiero DA, Hite KM, Monks A, Sausville EA, Waxman DJ. 2001. P450 enzyme expression patterns in the NCI Human Tumor Cell Line Panel. Drug Metabolism and Disposition, 29:304-312.

Enzyme Kinetics Plot

Proteomics Analysis

PBPK Modeling