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In Vitro ADME

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

Absorption

Permeability Assays

Drug permeability 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, a number of assays to study permeability are available:

Distribution

Protein Binding

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

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 pharmacokinetic and pharmacodynamic parameters of a drug. 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. In this system the impact of non-specific binding is minimised compared to other methods such as ultrafiltration and HT-dialysis, which are relatively slow to reach equilibrium. Sygnature’s Plasma Protein Binding assay uses RED to measure the percentage binding of a test compound to plasma proteins in human and preclinical species.

Brain Tissue Binding

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

Sygnature’s Brain Tissue Binding assay uses RED to measure the percentage binding of a test compound to brain tissue. 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 to correct scaling of in vitro clearance data to in vivo.

Sygnature’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.

Metabolism

Metabolic Stability

Metabolism of small molecules 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 and Phase II glucuronosyl- and sulfo-transferases for instance.

Intestinal/Lung Tissue Metabolic Stability

In addition to the liver, metabolism can also occur in other tissues, including the GI tract, lung, skin and nasal mucosa, affecting 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 preclinical species to assess intestinal metabolism.

Drug Interaction Assays

CYP450 Inhibition

Potential drug-drug interactions between metabolising 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, or earlier where a particular liability is suspected, to investigate detrimental interactions.

CYP Induction

Cytochrome P450 (CYP) induction by a drug can accelerate the metabolism of a co-administered victim drug significantly, causing serious drug-drug interactions.

To date induction of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 have been described. Whilst CYP1A2 induction involves the Aryl hydrocarbon receptor (AhR) and induction of CYP2B6 is mediated by the constitutive androstane receptor (CAR), the CYP2C isoforms and CYP3A4 are co-induced via the pregnane X receptor (PXR). Therefore, the current FDA guidelines suggest to investigate induction of CYP1A2, CYP2B6 and CYP3A4.

Traditionally, CYP induction has been measured via the assessment of enzyme activity in human hepatocytes. In order to address inter-individual variability, hepatocyte preparations from at least three donors had to be used.

HepaRG is an immortalised human hepatic cell line highly rated by scientists for its hepatocyte-like phenotype, e.g. its expression of drug-metabolising enzymes drug transporters and transcription factors.

Therefore, HepaRG provide an excellent, highly reproducible and cost-efficient system to study CYP induction during drug discovery or during the early phases of drug development.

Determination of mRNA levels by quantitative revers-transcription polymerase chain reaction (qRT-PCR) has proven superior over enzyme activity, because the latter could lead to false negative results when CYP induction was masked by simultaneous inhibition.

Excretion

With access to some of the most sensitive UPLC-MS instruments in the industry, such as 3 Sciex 5500 Qtrap and 3 Q Exactive Focus, analysis of bile, faeces or urine samples as well as tissue can be performed to quantify compound and metabolite levels.

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