In vitro human Gut/Liver models | ADME confidence before FIH

Imagine a world where developing new medicines is faster, cheaper, and less reliant on the use of animals for testing. Novel multi-organ in vitro human Gut/Liver models, tiny replicas of their human equivalents are an essential part of this journey.

By unlocking a deeper understanding of how our bodies Absorb, Distribute and Metabolize drugs (three of the four ADME pillars) and their potential bioavailability, these advanced models provide a unique path forward to mitigate risk before clinical trials. Pre-clinical miscalculations of these critical pharmacokinetic factors can cause a cascade of problems that impact trial outcomes, drug development timelines, and costs.

The most cited reasons for drugs failing in the clinic are lack of efficacy and safety concerns, both of which are influenced by dose. Could success rates improve if preclinical estimations were more accurate?

New approach methodologies (NAMs), such as Organ-on-a-chip (OOC), are being readily adopted in drug discovery and development pipelines to address inherent ADME workflow challenges and to improve predictivity. In this blog, we will first explore why traditional approaches constrain our ability to deliver accurate human ADME estimations, followed by the benefits of human in vitro Gut/Liver models, and their potential to move the needle towards safer and more effective clinical trials.

Why is estimating human drug ADME a challenge?

Currently, a combination of simple in vitro assays that either model the gut (Caco-2 cell line) or the liver (liver microsomes and suspension hepatocytes), and in vivo animal models are used. However, significant limitations exist with both approaches.

Caco-2 cells have been the workhorse for assessing in vitro intestinal permeability for a long time, but they cannot account for liver metabolism. The cell line also has absent or low levels of enzyme and transporter expression. Conversely, liver microsomes and suspension hepatocytes are used for in vitro drug metabolism screening studies, but they do not consider intestinal absorption. Collectively these limit the accuracy of in vitro-derived estimations.

diagram seperate | in vitro human gut/liver models

Furthermore, human bioavailability estimations from in vivo animal models weakly correlate with human-derived data, as demonstrated in a seminal study investigating 184 compounds, (R²=0.34)¹. Human-relevant approaches that combine oral absorption and hepatic metabolism are required to estimate drug bioavailability more accurately before first-in-human (FIH) trials.

Why does the problem of estimating drug ADME and bioavailability in humans remain?

One of the most important innovations in the field has been the adoption of computational or in silico tools to screen for new compounds and predict their ADME behavior using physiological-based pharmacokinetic (PBPK) modeling. These models are becoming increasingly important prior to trials in humans. However, their ability to accurately predict outcomes in the clinic is often constrained by the quality of the input data from prior ADME studies.

OOC technologies, also known as microphysiological systems (MPS), are designed to recapitulate the structural and functional biomarkers of cells and tissues in a more physiologically relevant manner through the culture of primary human cells on perfused scaffolds. But their potential in the field of ADME has been held back because of several challenges.

Firstly, maintaining differentiated intestinal enterocytes from human donors is challenging. By their very nature, intestinal epithelial cells have a short life span with a renewal cycle of 4-5 days. The intestinal lining is highly dynamic; enterocytes move towards the intestinal villus tip, become fully differentiated, and only then gain absorptive function. Because of the high turnover rate, isolated enterocytes de-differentiate quickly, making it challenging to conduct absorption studies in vitro. Additional challenges also come from the inherent microbial population in the intestinal lining, which can lead to contamination by bacteria isolated alongside human enterocyte cells².

Secondly, generating enough metabolic capacity or “horsepower” to reproduce phase 1 and 2 drug metabolism by in vitro human liver models requires continual culture perfusion (to mimic the bloodstream) and scale. Half a million primary cells perfused by large volumes of media (ranging between 1-2 mL) are required to achieve this goal which we have made possible via our FDA-recognized and industry-proven PhysioMimix^® Liver-on-a-chip model^3,4.

Finally, the kinetics of drug absorption and metabolism by the gut can impact the fraction of a drug that is absorbed and available to the liver. Therefore, efforts to improve the in vitro to in vivo translation (IVIVE) of drug efficacy, safety and pharmacokinetic data have led to the emergence of more complex models where multiple organs, such as gut and liver, are fluidically linked to simulate drug absorption and first-pass metabolism.

gut liver 4 | in vitro human gut/liver models

dual plate tl6 well v3 | in vitro human gut/liver models

One of the major challenges when co-culturing two or more tissues together is establishing the conditions that maintain the functionality of both tissue types. We initially linked the established Caco-2 gut model to our industry-proven Liver-on-a-chip model⁵ using a chemically defined media that maintains hepatic metabolic functionality and intestinal barrier integrity.

cnb416 fig3 v1 e1719527856701 | in vitro human gut/liver models

Enabling human oral bioavailability to be profiled in vitro, rather than inferred from isolated gut and liver assays, the model provides a step forward. However, it remains limited by absent or low levels of enzyme and transporter expression. For some drug types, this limitation of the Caco-2 cell line results in failure to accurately profile a drug’s ADME behavior and bioavailability in humans.

What’s the solution? An in vitro human Gut/Liver model

To address the limitations of the Caco-2 cells, we have successfully developed a PhysioMimix primary in vitro human Gut/Liver-on-a-chip model in collaboration with Altis Biosystems. Altis Biosystems’ RepliGut^®-Planar Jejunum model recreates the intestinal barrier using primary cells isolated from the human jejunum and expanded on a biomimetic scaffold. Primary gut tissue is then interconnected with our Liver-on-a-chip model using dual-organ supporting media, which is added to the basolateral side of the RepliGut’s Transwell^® and to the liver compartment of our PhysioMimix Multi-chip Dual-organ plate. Multi-chip Dual-organ plates enable six RepliGut/Liver-on-a-chip models to be cultured per plate. Up to three plates can be run simultaneously by one PhysioMimix Multi-organ System (18 replicates/run).

Using well-studied drug compounds, we have demonstrated the improved predictive capacity of this primary in vitro human Gut/Liver model for profiling the ADME behavior of oral drugs compared to an equivalent Caco-2 Gut/Liver model. For the full study results, please read our Application Note.

diagram combine | in vitro human gut/liver models

This in vitro human Gut/Liver model now enables intestinal absorption and hepatic clearance to be studied within a system that more precisely recapitulates the human process – versus traditional approaches operating in isolation. The added benefit is that the approach enables bioavailability estimations to be made too.

By combining data generated using this fully human in vitro Gut/Liver model with in silico computational modelling tools, the in vivo pharmacokinetic parameters of orally administered drugs can now be more accurately estimated⁵.

in vivo predictions | in vitro human gut/liver models

Here’s our recommendation for using the human in vitro Gut/Liver model

We recommend utilizing this advanced human in vitro Gut/Liver model to change the risk calculus and gain confidence in your lead candidates. Using more human and physiologically relevant in vitro test models in lead optimization, or before clinical trials, ensures that only the candidate compounds with optimal drug absorption and bioavailability profiles are progressed into in vivo animal or FIH studies. Additionally, the approach can be used to refine the preclinical design, save costs, and align with the 3Rs (to support reductions in the number of animals required).

Acknowledgement of where OOC is today

OOC/MPS models do have their limitations as they operate in small systems rather than a whole functioning organism, however, they significantly move the needle towards recapitulating the way that a human interacts with drugs before in vivo studies.

OOC/MPS models should be used in a complementary manner to overcome the physiological-relevance limitations of traditional in vitro approaches and the human-relevance limitations of animal studies. By doing so, they enable a “bigger picture” view for more informed decision-making about the optimal therapeutic window (maximal efficacy/minimized toxicity), and to reduce the risk of identifying poor oral bioavailability during first-in-human studies for increased confidence in clinical success.

So, don’t delay! The longer you wait the greater the risk of cascading problems down the line from miscalculated pre-clinical estimations. The PhysioMimix primary human in vitro Gut/Liver model can be accessed via our ADME Contract Research Services, with an “in-a-box” kit for use in your own laboratory coming soon.