ADME Studies: Determining Promising Drug Compounds

Published on: 

Experts discuss factors that can affect the outcome of an ADME study, signs that a drug compound shows promise, red flags, and key parameters to determine safety and efficacy.

Absorption, distribution, metabolism, and excretion (ADME) studies are a key part of drug discovery and preclinical phases, as this evaluation of pharmacological properties determines the efficacy and safety of a given drug compound. Some say this process defines the disposition of a drug. Pharmaceutical Technology dives into ADME studies in this exclusive interview with Dr. Yassen Abbas, lead scientist, CN Bio, an organ-on-a-chip (OOC) company that designs and manufactures microphysiological systems.

ADME studies: overview

PharmTech: How would you describe the process of ADME studies? What does that typically look like?

Abbas (CN Bio): ADME studies are performed during drug discovery and preclinical phases to optimize drug properties, support candidate selection, and subsequently inform the design of the clinical phase. Experiments are first performed in vitro and investigate parameters such as [the] extent of protein binding, [the] likelihood of inhibition of major drug metabolizing enzymes, and understanding of the metabolic stability of a drug. Then they are followed by animal (i.e., in-vivo) studies, which provide more insight into a drug’s behavior in a whole organism, complete with the immune and circulatory systems. Combined, these preclinical studies enable decision-makers to make a go-no-go choice to move the drug into the late preclinical stage and clinical trials or abandon the project.

Unfortunately, the clinical translatability of data derived using standard preclinical in-vitro and in-vivo approaches remain constrained by certain limitations. Where preclinical ADME predictions are inaccurate, this can result in poor efficacy or unforeseen toxicity in the clinic. Due to their simplicity, most in-vitro ADME assays are limited by poor physiological relevance. Animal models circumvent this but are in turn limited by a host of interspecies differences—for example, in the expression profile of cytochrome p450 enzymes, which affect their predictability. The use of complementary in-silico modeling to overcome these limitations remains constrained by the quality of the model’s input data.

Over the past decade, OOC technologies have been developed to bridge the gap between traditional in-vitro assays and in-vivo tissue functionality in humans. Here, primary human cells—cultured in physiologically-relevant combinations and perfused by fluidic flow to mimic the bloodstream—form three-dimensional microtissues that function and respond to drugs in a similar way to humans. For liver-on-a-chip models, also known as liver microphysiological systems, the inclusion of fluidic flow is critical to their success, helping to maintain their metabolic competency and viability for much longer (at least two to three weeks) compared to static in-vitro 2D cultures. Amazingly, different organ models can now be connected to simulate key ADME processes, such as drug absorption by the intestine, then hepatic metabolism by the liver. Samples collected from these models can be sent for analytical chemistry analysis using liquid chromatography/mass spectrometry to determine the drug concentration over time. The data can then be fed into in-silico models to determine pharmacokinetic (PK) parameters, such as hepatic clearance, intestinal clearance, and intestinal permeability (1). Here, the quality of the PK parameters generated by the in-silico models is dependent on the ability of the biological models used to predict behavior in humans.

Factors impacting ADME studies

PharmTech: What can affect the outcome of an ADME study?


Abbas (CN Bio): The lack of human-relevant preclinical models is a major cause of drug failure—in-vitro models being too restrictive and lacking the complexity of a living organism whilst animals utilize non-human pathways, notably in their immunity, metabolism, and microbiome, all of which can impact their response to drugs. As such, the output quality of an early drug development ADME study generally depends on the quality of the biological model used to extrapolate key PK parameters.

The success of a study can also depend on the chemical properties of the drugs being studied. Those that have a high partition coefficient, LogP (>3), a measure of lipophilicity, exhibit poor solubility in aqueous solutions, and high-binding affinities to proteins. These drugs typically have poor intestinal permeability and low oral bioavailability. Where there is a concern, experiments are performed in vitro without cells but in media to quantify the free fraction of a drug. Routinely performing ADME experiments in serum-free media (to limit drug binding) is key to successfully assessing a drug’s true properties. It is also important to consider the composition of the material used for cell culture as some regularly used 2D, 3D, and OOC cell-culture materials, such as polydimethylsiloxane, have high non-specific binding properties and, therefore, can impact data accuracy.

Another contributing factor to poor data accuracy is in-vitro cell culture media evaporation. High levels of evaporation can make the interpretation of PK data challenging, especially for low clearance compounds. To prevent evaporation, some cell culture systems have incorporated an additional well containing sterile water along the consumable plate’s edge to limit cell culture media loss.

Red flags

PharmTech: What are early signs/red flags that an ADME study is going poorly?

Abbas (CN Bio): A critical point in drug discovery where red flags are identified is the transition from in-vitro [to] in-vivo studies. PK data generated in vitro that cannot be reproduced in animal models will require further work to establish the cause.

Signs of toxicity are major red flags to look out for particularly if these are observed at, or close to, the dose required to produce the desired therapeutic effect. Subsequent work to increase bioavailability/reduce dose by optimizing the drug formulation may be required. If toxicity is identified in animals, additional studies will be required to determine if this is likely to be a species-specific response and the potential effect in humans. It may, however, prove difficult to progress to clinical trials where toxicity has been observed in animal models due to concerns over risk.

Human single- and multi-OOC models are uniquely placed to investigate these red flags in more detail. For example, it is possible to observe the toxicity of drug candidates to intestinal or liver cells using OOC ADME assays. To ensure that toxic effects are indeed caused by the administration of a drug, quality control experiments before and after drugs are added can be performed. For the liver, a drop in the cytochrome P450 3A4 (CYP3A4) activity, albumin, and a rise in lactate dehydrogenase—a marker of cytotoxicity—is indicative. In our intestinal model, we can detect the loss of cellular barrier integrity by measuring electrical resistance across cells. Where a drug is flagged as potentially toxic in our ADME assays, we investigate further by assessing its potential to cause drug-induced liver injury. Here, we report dose-dependent toxicity across multiple concentrations using a wide range of output parameters, including clinically relevant biomarkers (e.g., alanine transaminase and aspartate aminotransferase) to provide a mechanistic fingerprint.

Signs of promise

PharmTech: What are signs that an ADME study is successful and a drug shows promise?

Abbas (CN Bio): The goal of an ADME study is to understand how much of the drug is available to reach the target organ and to accurately predict ADME behavior in humans before clinical trials. A measure of success is the safe administration of a drug candidate to human volunteers based on a pre-determined dose extrapolated from ADME studies and in-silico modeling. The challenge for the pharmaceutical sector is how to improve the predictability of preclinical ADME studies to reduce the attrition rate of drugs that fail to make it to market. Here, the success or failure of an ADME study depends largely on the chemistry of the compound and the human relevance of the models and assays used.

Determining safety and efficacy

PharmTech: What parameters do you follow for ADME studies to determine the safety and efficacy of a drug? Is there a range for what is considered acceptable?

Abbas (CN Bio): Many parameters reported by ADME studies are used to determine the safety and efficacy of a drug. One important parameter to identify is the metabolic property of the drug. For example, drugs that don’t inhibit CYP3A4—and are not a major substrate of the CYP3A4 metabolizing enzyme—are considered a success because the risk of drug–drug interactions are reduced.

The key parameter, however, is bioavailability … Until recently, it has not been possible to measure bioavailability in vitro due to the limitations of standard approaches. To derive this value, a drug must be exposed to complex physiological processes, such as drug absorption through a biological barrier and first-pass metabolism. It is now, however, possible to do exactly this using an organ-on-a-chip approach. Our gut/liver multi-organ model, for example, recreates the transit of a drug that is dosed orally and intravenously to report bioavailability. These powerful new in-vitro models can either be used ahead of in-vivo animal studies to identify red flags early in the process, such as drugs with low bioavailability; and because of their high human relevance, they can be used to investigate cross-species concerns where toxicity has been identified in in-vivo animal studies.


1. N. Milani, et al., Lab on a Chip, DOI:10.1039/D2LC00276K (July 14, 2022).

About the author

Meg Rivers is a former senior editor for Pharmaceutical Technology, Pharmaceutical Technology Europe, and BioPharm International.