CYP450 inhibition
Application note
We offer a comprehensive range of standardized in vitro ADMET assays that follow the latest global and regional regulatory guidance to support Hit to Lead and Lead Optimization.
Key points:
- Test item characterization and evaluation of physicochemical properties, permeability, protein binding, metabolism, and drug-drug interactions
- Rapid data turnaround to support design make test cycle
- Assays developed to minimize compound usage and reduce cost
- State-of-the-art equipment, automated data management and interpretation by highly experienced scientists
- High compound throughput via robotic liquid handling systems
- Tailored service to client requirements with guidance on best techniques and project management
In vitro ADMET assays
For more bespoke assays, contact our ADMET and DMPK services team – CLS-ADMET.services@malvernpanalytical.com
Below is a list of the in vitro ADME screening assays we provide:
Physicochemical screening
Solubility
Low solubility is a limiting factor for absorption and bioavailability. By identifying poorly soluble drug compounds early in the development process, developers can evaluate the resources and additional time needed to progress a therapy.
By using the shake flask method, we determine kinetic and thermodynamic solubility, helping you to choose the most promising drug compounds to take through development in the most effective way.
LogD
LogD assays simulate the ability of chemicals to passively diffuse across biological membranes. Our assay uses the shake flask methodology and provides quantitative LogD7.4 values against a five-point standard curve.
Chromatographic hydrophobicity index (CHI)
Using reverse-phase liquid chromatography (RP-LC) this assay uses physiochemical methods to determine hydrophobicity delivering an accurate CHILogD7.4 value.
Chemical stability
This early-stage assay can determine the likely stability of drug compound by assessing its stability in buffer solutions. Hydrolysis, oxidation, light-catalyzed degradation and many other factors can be assessed with buffer solutions of varying temperature and pH. This can flag potential shelf-life issues as well as stability at the different pH’s which your drug product may encounter in vivo. Weak candidates can be removed from drug development pipelines before evaluation in vivo.
You may also like to take a look at our OECD 117 OECD 123 and OECD 107 methods which comprise part of our extensive array of regulatory physicochemical tests.
Protein binding
Plasma Protein binding
When drugs bind to proteins within blood, plasma and tissues it reduces the drug free fraction that can permeate cell membranes and generate pharmacological effects.
Equilibrium dialysis is effective at determining protein binding affinities and the influence on dosing, efficacy, clearance rate and drug-drug interactions.
Blood plasma partitioning ratio
In vivo analysis of drug concentration and pharmacokinetic properties are often determined in plasma, but these figures can be misleading if compounds have a high affinity for blood components, such as hemoglobin or cytosol binding proteins. By measuring concentration in the whole blood, we provide a blood plasma partitioning ratio, demonstrating red blood cell interactions and providing the complete picture of pharmacokinetic parameters in plasma.
Other metrics are available upon request.
In vitro metabolism
Plasma stability
Rapid metabolism lowers drug exposure at the therapeutic target. Furthermore unstable compounds can cause issues in in vitro plasma protein binding and in vivo pharmacokinetic studies as they continue to degrade after blood samples are taken. Plasma stability assays identify these compounds early in development as part of prodrug screening programs, providing an accurate measure of test compound degradation over time.
Microsome and S9 stability
Most drugs are biotransformed within the body by metabolic enzymes; the rate of this metabolism determines how much of the drug is exposed to its target. Microsome assays help to determine the stability of drug compounds by assessing the effects of phase I enzymes. In addition, S9 assays can determine the impact of phase I and II enzymes in the cytosolic liver fraction and provide a cost-effective alternative to hepatocyte assays.
Hepatocyte stability
Metabolic stability is directly correlated to both therapeutic toxicity and efficacy. By testing drug compounds in isolated, whole hepatocytes, the entire complement of drug-metabolizing enzymes can be assessed with phase I and II coordinated activity. Comparison of datasets across species contributes to predictions of drug exposure in humans.
CYP 450 inhibition
This assay measures the inhibitory potency (IC50) of drug compounds against the five main drug-metabolizing enzymes: CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4.
We also provide an extended assay for the seven FDA-recommended drug-metabolizing enzymes which includes CYP2B6 and CYP2C8.
Time-dependent inhibition (TDI)
By measuring the amount of irreversible and quasi-irreversible CYP metabolism, the loss of enzyme function can be used to assess clinically relevant drug-drug interactions. TDI percentages are quantitatively calculated for each test compound against CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4.
CYP induction
Metabolic drug-drug interactions following co-administration of xenobiotics are known to result in either increased toxicity or reduced efficacy. Understanding a drug's liability to induce Cytochrome P450 enzymes is required to guide drug development.
We offer qPCR (mRNA) analysis and enzyme activity profiles in primary hepatocytes from both human and animal species.
Permeability and transporters
Caco-2 permeability
Using Caco-2, an immortal human colon carcinoma cell line, drug permeability can be assessed in an environment similar to the epithelial cells present in the small intestine. Apparent permeability (Papp) figures are calculated, measuring efflux and the activation of P-glycoprotein (P-gp), BCRP or MRP2 cell membrane transporters.
MDCK permeability
Using a monolayer of canine kidney cell lines overexpressing human transporters, permeability and active transport can be assessed for drug compounds. By effectively mimicking the barrier between the intestinal lumen and blood supply from the hepatic portal vein, Papp is determined and compounds that are P-gp/BCRP substrates are identified.
Toxicity
ADMET screening for cytotoxicity
Using the HepG2 epithelial cell line, this assay measures cell viability in the presence of the test compound. Using fluorometric/colorimetric measures, we determine the cellular metabolic activity and mitochondrial viability of cells to give a viable cell count.
hERG inhibition
The large inner cavity of the human ether-a-go-go-related gene (hERG) potassium channel makes it particularly sensitive to drug binding. The inhibition of this channel, present in cardiac tissue, can lead to a loss of function and Long QT Syndrome (LQTS). With cardiovascular safety issues being the main reason for drug development candidate failure, this is an important assay early in the drug development process. Our assay provides concentration-response curves so that developers can be confident that inhibition lies within permissible levels.