Drug bioavailability

There are many reasons, however, the repertoire and expression of enzymes that drive human drug metabolism differ compared to those of commonly used animal species. This is also true for transporters that are responsible for the uptake or efflux of drugs through the small intestine. In addition, there are differences in the physiology of certain animal species compared to the human. For example, rats do not have a gall bladder – an important component of the hepatobiliary system.

In their 2014 publication, Musther et al, demonstrate how poorly animals predict this key parameter. For 184 drugs investigated, the R² value of animal model estimations versus actual clinically derived human bioavailability were shown to be 0.34.

We expect animal models to continue to be an important part of ADME studies for the foreseeable future, however, the complementary use of the PhysioMimix Bioavailability assay will add confidence to the data generated with animals or query their findings.

In their 2014 publication, Musther et al, demonstrate how poorly animals predict this key parameter For 184 drugs investigated, the R² value of animal model estimations versus actual clinically derived human bioavailability was shown to be just 0.34.

A deeper and earlier knowledge of human bioavailability enables issues to be addressed before the start of costly preclinical studies. In vivo models are only an approximation of what’s likely to happen in the human as they lack human relevance; and traditional in vitro assays are limited by physiological relevance. Advanced human models, however, address these limitations, enabling you to bridge the gap to confidently select safer and more effective drug candidates.

For predictive modeling, two main parameters are required: hepatic clearance rate and compound permeability through the intestinal barrier. We are working on a mathematical model that will take data generated from the Multi-chip Dual-organ consumable plate and generate fittings for the parameters. Flow rates can easily be adjusted by the user via the PhysioMimix Multi-organ System’s controller unit, which allows us to understand the effects of different mixing rates.

To learn more, read our Multi-Organ Application Note: Drug metabolism in a gut-liver microphysiological system. Note this study utilizes our Caco-2 Gut/ Liver-on-a-chip model

We have characterized the activity of the CYP3A4 enzyme, one of the main drivers of drug metabolism in humans, for quality control purposes during culture to ensure that there is sufficient and maintained activity throughout the experiment. This has also been independently validated by the CDER group at the FDA in the following study by Rubiano et al, 2021.

We can take data generated from the PhysioMimix® Bioavailability assay (run by a PhysioMimix Multi-Organ System, utilizing Multi-chip Dual-organ consumable plates), and conduct parameter fitting to estimate the hepatic clearance rate and permeability of drugs across the intestinal barrier. Here, parameter fitting is the process of computing a model’s parameter values from measured values. By utilizing mathematical modelling, the data generated from one bioavailability experiment can be maximized e.g., intestinal and hepatic clearance rate. We recommend reading this publication by Roche (Milani et al., 2022) who developed a mathematical model on the compound mycophenolate mofetil after profiling bioavailability using the Caco-2 Gut/Liver-on-a-chip.

Yes, using the Gut/Liver-on-a-chip, or microphysiological system (MPS) as it is also known, we have demonstrated that bioavailability can be profiled for low clearance compounds, which are defined as drugs that have an intrinsic clearance rate of <5ml/min/kg.

To learn more, read our Multi-Organ Application Note: Drug metabolism in a gut-liver microphysiological system. Note this study utilizes our Caco-2 Gut/ Liver-on-a-chip model

We have not applied allometric scaling to data generated using either the Liver-on-a-chip or the Gut/Liver-on-a-chip.

Using the primary Human RepliGut/Liver-on-a-chip model, we have tested Temocapril and Midazolam, who’s human ADME properties were not adequately predicted by existing in vitro, or in vivo models. Temocapril is a pro-drug designed to be resistant to intestinal hydrolysis. It is metabolized to Temocaprilat by carboxylesterase 1 (CES1). The isoenzyme pattern in human is well-characterized with the liver and intestine expressing CES1 and CES2 respectively.

In Caco-2 cells, there is a miss-match as CES1 is predominantly expressed, resulting in an overestimation of drug clearance. In contrast, the more human representative ratios of both CES1 & CES2 expression by the primary human RepliGut model make it more relevant for pro-drug studies. Midazolam predominately undergoes intestinal clearance by the CYP3A4 enzyme. When compared to the equivalent Caco-2/Liver-on-a-chip model, the primary RepliGut/Liver-on-a-chip model delivered oral bioavailability estimations that more closely represented human clinical observations

For further reading please view our latest Application Note

It is well known that the repertoire and expression of enzymes that drive metabolism differ with genetic diversity. One of the benefits of MPS over in vivo animal studies is that you can run experiments using primary cells from multiple donors to account for/investigate the impact of genetic diversity.

Yes, we can take samples in the gut apical compartment as well as in the systematic circulation of the plate to determine the fraction absorbed of a drug added and the amount available to the hepatocytes. Slight adjustments to the design of the experiment are required to calculate Fa and FaFg.

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Combination dosing Gut/Liver-on-a-chip models (also known as Gut/Liver microphysiological systems) has not been tried, but where LC-MS analysis is able to differentiate between compounds, the bioavailability of drug combinations could be estimated using a modified version of this approach. The benefit of MPS is that many more dosing regimens and concentrations can be tested to finely tune estimations compared to animal studies.

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Serum-free media is used during the human bioavailability in vitro experiment to limit drug binding with proteins. Additionally, it is worth noting that we avoid the use of Polydimethylsiloxane (PDMS) in our Multi-Chip Dual-organ plates consumable plates, which are made using Cyclic Olefin Copolymer (COC) instead.

By using serum-free media and PDMS-free consumable plates. Although PDMS is widely used, it has limitations. Unfortunately, PDMS has very high non-specific binding properties, which poses a challenge for human bioavailability in vitro testing. Customers report that up to 70% of drugs can bind to the plastic, which impacts data accuracy. It is possible to saturate PDMS plasticware ahead of experiments, however this isn’t ideal as adds more complexity with respect to assay set up and poses a contamination risk. When developing our PhysioMimix OOC Systems and Multi-chip consumable plates, we decided to move away from PDMS and use Cyclic Olefin Copolymer (COC) instead. For cell culture applications, COC, is currently one of the most inert materials in the market. and is therefore more suitable for estimating bioavailability in vitro, although we advise performing exploratory studies to check for non-specific binding should there be concerns.

We also recommend running a cell-free condition, where the compound is added but no cells are seeded in the Gut/Liver-on-a-chip. This allows users to monitor and consider any drug loss. Whilst we expect drug binding to the COC plastic to be low, we understand that it is important to show this experimentally, enabling increased confidence when testing human bioavailability in vitro.

Please read the following references to learn more: van Midwoud et al, 2012, Tsamandouras et al., 2017, McAleer et al, 2019.

Our Lung/Liver model is very exciting, and it’s something that we can apply to (more or less!) anything people are interested in. In the first instance we designed this dual organ system for COVID-19 research as funded by our Innovate UK grant. In particular, we looked at the crosstalk between the lung and liver when we infected the lung with COVID-19 pseudoparticles. Through this we’ve been able to elucidate over time how the liver responds to inflammatory signals given by the lung during the challenge. In the future this could also be applied to drug studies, whether that be ADME, toxicity, bioavailability or pharmacology – there’s a whole host of different applications that could be applied to the multi-organ system.