(M1330-05-32) Simulating Prodrug Biotransformation In Vitro: Conserving Enzymatic Activity within the SCISSOR N3 Platform

Purpose: Preclinical in vitro studies aim to reflect in vivo outcomes that will be seen in humans, but often overlook key functional dynamics and bioactivity present at the site of application. Traditional testing of dissolution, solubility, and predicted bioavailability does not include functioning biomolecules, significantly deviating from the environment found in vivo. [1] This difference is a main contributor to the deviation of animal vs. human responses for biologics. [1,2] The purpose of this work was to incorporate active enzyme into an in vitro subcutaneous injection site simulator (SCISSOR™), which has been shown to produce a more accurate in vitro-in vivo correlation as compared to minipigs and monkeys for subcutaneously injected monoclonal antibodies. [2-4] Validating the ability to conserve the activity of esterase, an enzyme that acts on >50% of FDA approved prodrugs, [5] can allow for the characterization of behavior and performance of biotransformable drugs (e.g. prodrugs). Herein, we describe how we integrated esterase into the SCISSOR, ensured stable activity throughout an assay, and employed the newly developed protocol to assay a group of blinded prodrugs being developed for subcutaneous delivery. If the validation set is expanded upon, the SCISSOR will continue to not only bridge the gap between in vitro results and in vivo bioavailability, but also significantly minimize the amount of animal testing needed for accurate in vivo estimation.[6]

Methods: Esterase from bovine pancreas was obtained from Sigma, USA and homogenized within an artificial extracellular matrix (ECM) obtained from Pion Inc. The enzymatic activity was calculated by monitoring the conversion of methylparaben (MP) to 4-hydroxybenzoic acid (4H) via Acquity Arc HPLC (Waters®) after injection of an MP solution into multiple esterase-containing media. After confirmation of the enzymatic activity and stability, representative SCISSOR assays were carried out to demonstrate the applicability of this methodology within the pre-clinical drug development pipeline.

Results: Monitoring the esterase-mediated conversion of MP into 4H in biorelevant bicarbonate buffer, a glycosaminoglycan-based ECM, and within a SCISSOR assay all resulted in enzymatic activities of 0.5-1 pmol/min for a minimum of 24 hours. Without the steric hinderance caused by the hydrogel-like environment, the esterase achieved activity 3 orders of magnitude greater, highlighting the differences between the SCISSOR assay and traditional solubilization techniques. The SCISSOR’s glycosaminoglycan-based ECM allowed the stable integration of active enzymes, as well as maintained the mechanical and optical properties that allow for reproducible bolus formation and seamless post-injection analysis. Control samples in buffer showed activity three orders of magnitude higher than artificial ECM samples with MP, highlighting the impact of the hydrogel-like environment on esterase function. Whilst this study only focused on a small set of samples, it was found that for a set of the rank-order release rates of the prodrugs resulted in a positive correlation with the molecular weight of the -R group being cleaved by the enzyme

Conclusion: This study demonstrates that esterase (acting on over 54% of FDA-approved prodrugs) can maintain its activity throughout a SCISSOR assay, highlighting the ability to use enzymes in a dynamic in vitro setting to better establish the in vitro-in vivo relationship for testing prodrugs. Current and future efforts are looking to model the release of commercial prodrug formulations using SCISSOR.

References: (1) U.S. Pharmacopeia. DISSOLUTION. United States Pharmacopeia 2011.
(2) Fralish, Z.; Chen, A.; Khan, S.; Zhou, P.; Reker, D. The Landscape of Small-Molecule Prodrugs. Nat Rev Drug Discov 2024, 23 (5), 365–380. https://doi.org/10.1038/s41573-024-00914-7.
(3) Bown, H. K.; Bonn, C.; Yohe, S.; Yadav, D. B.; Patapoff, T. W.; Daugherty, A.; Mrsny, R. J. In Vitro Model for Predicting Bioavailability of Subcutaneously Injected Monoclonal Antibodies. Journal of Controlled Release 2018, 273, 13–20. https://doi.org/10.1016/j.jconrel.2018.01.015.
(4) Gomes, C.; Gridley, K.; Anastasiou, I.; Sinkó, B.; Mrsny, R. J. Hydrogel Formats to Model Potential Drug Interactions Occurring at the Subcutaneous Injection Site. European Journal of Pharmaceutics and Biopharmaceutics 2024, 199, 114308. https://doi.org/10.1016/j.ejpb.2024.114308.
(5) Sánchez-Félix, M.; Burke, M.; Chen, H. H.; Patterson, C.; Mittal, S. Predicting Bioavailability of Monoclonal Antibodies after Subcutaneous Administration: Open Innovation Challenge. Adv Drug Deliv Rev 2020, 167, 66–77. https://doi.org/10.1016/j.addr.2020.05.009.
(6) Food and Drug Administration. Roadmap to Reducing Animal Testing in Preclinical Safety Studies. FDA Roadmap. 2025 https://www.fda.gov/media/186092/

Table 1. Summary of 0.9 mL samples that were transferred to the HPLC with a 0.1 mL addition of 1 μmol/mL MP

Figure 2. A) Calibration curve of MP and 4H with their linear regression fits. B) Initial sample chromatogram from Control sample in HPLC. C) Final sample chromatogram from Control sample in HPLC. D) Bar graph indicating activity from each sample based on time-resolved concentration data of MP conversion to 4H.