(T1530-02-10) Validating Gut-On-A-Chip-Model: Comparison of Caco-2 Transwell and Gut-On-A-Chip Models for Oral Drug Delivery

Purpose: Oral drug delivery is the preferred method for patients, but successful formulation requires adequate solubility and permeability. Despite the notable progress made in predictive solubility and dissolution tools, the validation of permeability assays lags behind. Specifically, the existing tools for assessing permeability have not undergone extensive validation comparable to the gold-standard Caco-2 Transwell assay. In fact, the Caco-2 Transwell assay is the only in vitro method approved by the Food and Drug Administration (FDA) guidelines. The FDA Modernization Act 2.0 and the National Center for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs) have reaffirmed the need for better and more predictive in vitro models. To this end, complex in vitro models such as microfluidic Chip systems have been investigated. To appropriately use Chip model in drug development, there is a need for validation using model compounds to ensure accuracy and reproducibility. With the goal of using complex formulation for oral drug development, we have performed validation of Gut-On-Chip Model using small molecules, peptides, and permeation enhancers (PEs).

Methods: We first evaluated Caco-2 intestinal permeability assay in Transwells and a commercially available microfluidic Chip using 19 representative Biopharmaceutics Classification System (BCS) Class I-IV compounds. For each selected compound, we performed a comprehensive viability test, quantified its apparent permeability (P_app), and established an in vitro in vivo correlation (IVIVC) to the human fraction absorbed (f_a) in both culture conditions. Furthermore, we accessed baseline permeability of model peptides (insulin and octreotide) in the presence of two established intestinal permeation markers (sodium caprate and sucrose monolaurate) in variable conditions including fasted and fed state simulated intestinal fluids (FaSSIF and FeSSIF).

Results: In the presence of different small molecule drugs, permeability differences were observed across the models between high and low permeability drugs (E.g. antipyrine (Transwell P<sub>app: 38.5 ± 6.1 × 10^-8 cm/s vs Chip P<sub>app: 32.9 ± 11.3 × 10^-8 cm/s) vs nadolol (Transwell P<sub>app: 0.6 ± 0.1 × 10^-7 cm/s vs Chip P<sub>app: 3 ± 1.2 × 10^-7 cm/s)). However, the in vitro in vivo correlation (IVIVC; Papp vs. fa) of the Transwell model (r² = 0.59–0.83) was similar to the Chip model (r² = 0.41–0.79), highlighting similar levels of predictivity. Comparing to literature, our Chip P<sub>app data aligned more closely to native tissues assessed in Ussing chambers. We further investigated the applicability of the Chip model for assessing the permeability of peptides, specifically Insulin and Octreotide, in the presence of permeation enhancers (PEs). We introduced FaSSIF and FeSSIF into the Chip model and compared the results with the traditional Transwell assay. The baseline permeability for FITC-Dextran (3 kDa, FD3) was also determined in Transwell (5.3 ± 0.8 x 10^-8 cm/s) vs Chip (3.2 ± 1.8 x 10^-7 cm/s). Interestingly, we observed that the Chip model resulted in a 3-fold increase in basal FD3 for Insulin and a 20-fold increase for Octreotide compared to a 4-fold increase for Insulin and a substantial 4000-fold increase for Octreotide in the Transwell assay.

Conclusion: We conducted a comprehensive analysis of 19 reference compounds, following the FDA Biopharmaceutics Classification System, using an established Chip system as a preclinical tool for evaluating drug permeability. Both the Transwell and Chip models demonstrated differences in permeability, as observed with antipyrine and nadolol, showing a nearly 500-fold difference in Transwells compared to a 40-fold difference in Chips. Yet, when considering in vitro-in vivo correlation, both models exhibited similar predictive characteristics. Both the Transwell and Chip models have valuable applications in the pharmaceutical industry. Transwells can be utilized for high-throughput discovery efforts, while Chips offer a complementary model bridging the gap between in vitro and in vivo studies. This study represents the first comprehensive validation of a commercial Gut-on-a-Chip model as a predictive tool for assessing oral absorption, aiming to minimize reliance on animal models.  In addition, our study aimed to compare the effectiveness of 2D static Transwell models using Caco-2 cells and 3D microfluidic Gut-on-a-Chip models in assessing intestinal permeability enhancers (PEs)  We established the baseline characteristics for Gut-on-a-Chip experiments, including basal P<sub>app (3.2 ± 1.8 x 10^-7 cm/s), flow rate (125 μl/h), and effective concentrations of two model PEs. Comparative analysis of the predictive capabilities of these models, along with ex vivo and in vivo models, suggested that Caco-2 Gut-on-a-Chip is more closely aligned with these models than typical in vitro models. This model serves as a powerful addition to the in vitro toolkit in drug discovery and development, offering the potential to reduce reliance on animal models, consistent with the principles of the 3R Principle. This validation study represents the first of its kind for a Gut-on-a-Chip model and highlights its utility in the pharmaceutical industry as a supplementary system to address knowledge gaps and refine the heavy dependence on animal models.

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Acknowledgements: Declaration of competing interest: All authors are employees or former employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA and may own stock or hold stock options in Merck & Co., Inc., Rahway, NJ, US.
We thank Yufang He and Marc Durante of Merck 3D Technologies for providing support and laboratory space to conduct this research. The authors acknowledge support from the MRL Postdoctoral Research Program.