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In Vitro ADME

The assessment of the Absorption, Distribution, Metabolism, and Excretion (ADME) properties of compounds is a critical component in supporting the development of compounds within Discovery and Development.

Absorption

Permeability Assays

The permeability of drugs is an important component in the oral absorption of drugs. The permeability of a drug across a membrane is dependent on the passive permeability as well as the susceptibility of the drug to efflux or uptake by drug transporter proteins. At Sygnature Discovery, two assays to study permeability, PAMPA and Caco-2, are available.

The Caco-2 cell system is a well-characterized intestinal in vitro model which makes it possible to evaluate the ability of chemicals to cross the intestinal barrier, as well as to study their transport mechanisms.

  • Permeability values estimated with Caco-2  correlate well with human in vivo absorption data for many drugs and chemicals. As a result, we use this model to predict oral absorption in humans.
  • Sygnature Discovery’s bi-directional Caco-2 permeability assay can predict in vivo absorption of drugs across the gut wall by measuring the rate of transport of a compound across the Caco-2 cell line and can also provide an indicator as to whether a compound undergoes active efflux.

Intestinal/Lung Tissue Metabolic Stability

In addition to the liver, which is the primary site of metabolism, metabolism can also occur in tissues including GI tract, lung, skin, nasal mucosa to impact absorption.

  • First-pass metabolism that includes both intestinal and hepatic metabolism following oral dosing of a compound can impact on oral bioavailability. If inhalation is the proposed route, lung metabolism can play a significant role.  Sygnature’s intestinal metabolic stability or lung metabolic stability assay uses subcellular fractions such as microsomes from human and all pre-clinical-species to assess intestinal metabolism.

Distribution

Protein binding

Several in vitro binding assays are available which include Plasma Protein Binding (PPB), Brain Tissue Binding and Blood Plasma Partitioning (BPP). The flexibility to adapt protocols based on specific customer requirements is possible.

Plasma Protein Binding

Drugs may bind to a wide variety of plasma proteins, including α-glycoprotein and albumin, and the degree of binding can impact on the pharmacokinetics and pharmacodynamics parameters of a drug, given that only the free drug in plasma is available for passive diffusion to extravascular or tissue sites where it is available to elicit a pharmacological effect on the target.

The leading approach for assessing plasma protein binding is an assay utilising the Rapid Equilibrium Dialysis (RED) device as the impact of non-specific binding is minimised when compared to other methods such as ultrafiltration and HT-dialysis, which are relatively slow to reach equilibrium. Sygnature Discovery’s Plasma Protein Binding assay uses the RED device to measure the percentage binding of a test compound to plasma proteins in human and preclinical species.

Brain Tissue Binding

The composition of brain and plasma are very different, with plasma having twice as much protein than brain and brain having 20-fold more lipids than plasma. For this reason, free fraction in plasma is not a suitable surrogate for determining brain unbound concentrations. And it is the brain unbound concentrations that dictates receptor occupancy and hence target engagement for example.

Sygnature Discovery’s Brain Tissue Binding assay uses the RED device to measure the percentage binding of a test compound to brain tissue from rat. Brain tissue binding is species independent, and as such, brain tissue binding of rat can be used to obtain binding of other species and strains in drug discovery.

Blood Plasma Partitioning

Knowledge of Blood Plasma Partitioning (BPP) of compounds enables a rational choice of appropriate biological fluid, either whole blood, plasma, or serum, for bioanalysis of in vivo PK samples or for use in the correction in the scaling of in vitro clearance data to in vivo.

Sygnature Discovery’s Blood Plasma Partitioning assay offers a specific and robust assay to measure these parameters in a variety of species using fresh blood from human and preclinical species.

Drug Transporter Assays

The movement of many drugs and endogenous molecules across the cell membrane can be impacted by uptake or efflux protein transporters. These drug transporters exist in many tissues including, but not limited to, the brain, intestine, liver and kidney.

To assess the impact of P-glycoprotein (P-gp; MDR1) on the brain penetration of compounds, the Madine Darby Canine Kidney cell line expressing human P-gp (MDCKII-MDR1) assay is available at Sygnature Discovery.

Other validated transporter assays available at Sygnature Discovery that will help in assessing the interactions of your compounds with drug transporters include NTCP, OCT1, OCT2, OAT1, OAT3 MATE1, OATP1B1 and OATP1B3. Other transporters are available on request.

Metabolism

Poor metabolic properties are a major barrier to pre-clinical and clinical development. Short-lived compounds may require excessively regular dosing to maintain a concentration in the bloodstream or the target tissue that is sufficient to elicit a therapeutic effect. A slowly metabolised drug may persist in the body for long periods, causing accumulation and potential toxicity. It is, therefore, crucial to accurately assess compound metabolism during the discovery optimisation process.

In vitro metabolic screening provides a cost-effective and efficient strategy to evaluate compound metabolism during hit-to-lead and lead-optimisation stages of discovery. These tests help to narrow down the number of clinically relevant compounds selected for more extensive (and costly) in vivo PK profiling.

Our fully validated and robust in vitro methodologies provide an understanding of compound metabolic liabilities and when coupled with more detailed Metabolite Identification studies can facilitate lead structure optimisation.

Metabolic Stability

Metabolism occurs primarily through the cytochrome P450 (CYP) family of enzymes located in the hepatic endoplasmic reticulum but can also occur through non-CYP enzymes, including esterases, Phase II glucuronosyl- and sulfo-transferases.

For compounds which have a particularly low clearance, accurate extrapolation of the in vitro intrinsic clearance to the in vivo situation can be challenging using the standard methods. To this end, we have validated a low clearance hepatocyte stability method to specifically address this issue using plated hepatocytes over longer incubation times.

CYP450 Inhibition

Potential drug-drug interactions between metabolizing enzymes and investigational drugs are usually assessed during the optimisation process.

The contribution of a specific metabolizing enzyme to an investigational drug’s clearance is considered significant if the enzyme is responsible for > 25% of the drug’s elimination. It is recommended that phenotyping studies and CYP induction tests are conducted during candidate selection to investigate detrimental interactions between metabolizing enzymes and lead candidates.

  • Reaction Phenotyping
  • Cytochrome P450 Induction

CYP independent metabolism

Such as a flavin containing mono-oxygenases (FMO), monoamine-oxidases (MAO), carboxylesterases (CE), aldehyde oxidase (AO), UDP glucuronosyltransferases (UGT), sulfotransferases (SULT) or N-acetyltransferases (NAT). Adequate approaches using liver microsomes, S9, cytosol, or hepatocytes with enzyme-selective inhibitors are used. Alternatively, this can be done by using recombinant enzymes.

 


Although in vivo PK information is necessary to best assess the metabolic profile of drug candidates, the potential for interactions between metabolizing enzymes and investigational drugs using in vitro metabolic studies is helpful to inform the need for and design of in vivo PK studies.

In combination with protein binding studies including microsomal protein binding to understand the free concentration available in the microsomal incubations, in vitro intrinsic clearance (CLint) from microsomes and/or hepatocytes can be used for in vitro in vivo extrapolation (IVIVE) and subsequent prediction of in vivo PK.

With access to a range of in-silico models, our scientists make use of in vitro data to rank and select compounds for progression and further validation saving time, reducing project costs and expediting the optimisation process.

Excretion

With access to some of the most sensitive UPLC-MS instruments in the market, such as the Waters Xevo-G2-XS QToF and the Sciex 5500 Qtrap, analysis of bile, faeces or urine samples as well as tissue can be performed to quantify compound and metabolites levels.

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