(M1130-02-08) Conserving Biorelevant Enzymatic Activity during In Vitro Analysis of Biotransformable Drugs

Purpose: The end goal in nearly all in-vitro research is to try to establish a relationship between that seen in the simulated environment, with that seen in vivo. Ensuring these relationships exist, enriches the data being produced by the in vitro platforms and increases the likelihood of candidate success later in the development process. Often, in vitro experiments focus on a specific parameter or characteristic to compare drug candidates such as dissolution or solubility, however, these types of tests often miss the functional aspect of testing, not accounting for the interplay these parameters have in vivo ^[1]. In addition to this, the biorelevant setting needed to simulate the stress conditions encountered in vivo are often not represented. The purpose of this work was to incorporate active enzymes into a simulated artificial extracellular matrix (ECM) used in our in vitro subcutaneous injection site simulator (SCISSOR) machine ^[2],[3] (Fig 1), to study the behaviour and performance of pharmaceutical agents that undergo enzyme-mediated inactive-to-active transitions in vivo, in a functional assay providing a dynamic test for more in depth drug performance evaluation.

Methods: Esterase from bovine pancreas (Sigma, USA) was incorporated into an artificial extracellular matrix (ECM) provided by Pion Inc. The enzymatic activity was assessed by tracking the conversion of methylparaben (MP) to 4-hydroxybenzoic acid (4H) using an Acquity Arc HPLC system (Waters®) following the injection of an MP solution into various esterase-containing media. Once enzymatic activity and stability were confirmed, SCISSOR assays were conducted to validate the utility of this approach for pre-clinical drug development applications

Results: Esterase-mediated conversion of methylparaben (MP) to 4-hydroxybenzoic acid (4H) was monitored in biorelevant bicarbonate buffer, a glycosaminoglycan-based extracellular matrix (ECM), and within the SCISSOR assay. Enzymatic activities of 0.5–1 pmol/min were sustained for at least 24 hours in all tested environments. Notably, in the absence of the hydrogel-like ECM, esterase activity was three orders of magnitude higher, emphasizing the distinct functional constraints imposed by the SCISSOR assay environment compared to traditional solubilization techniques (fig 2). The glycosaminoglycan-based ECM within SCISSOR provided stable integration of active enzymes while preserving the mechanical and optical properties essential for consistent bolus formation and reliable post-injection analysis. Control samples in buffer further highlighted the impact of the hydrogel-like ECM on esterase performance, demonstrating significant differences in activity between these environments.

Conclusion: In this study, we demonstrated that esterase—an enzyme capable of acting on at least 54% of FDA-approved small molecule prodrugs ^[4] —retains its enzymatic activity throughout the duration of a 1-day SCISSOR assay. Unlike traditional methods focused on solubility or dissolution, these findings provide initial validation of the platform for in situ analysis of prodrugs in a functional in-vitro environment. Further investigation of formulated prodrugs within this model will shed light on how SCISSOR's integrated analytics perform as injectates undergo their inactive-to-active transitions, offering deeper insights into in vitro-in vivo relationships.

References: [1] 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.
[2] 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.
[3] 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.
[4] 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.


Figure 1. Schematic of SCISSOR chamber set-up: A) Donor/Cartridge containing the artificial extracellular matrix (and Esterase) simulating the subcutaneous space; B) Acceptor/Chamber containing bicarbonate buffer simulating uptake to the blood and lymphatics; C) Hypodermic needle for sample injection


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.