In Vitro Safety Pharmacology Profiling
Safety & Toxicology

In Vitro Safety Pharmacology Profiling

Reaction Biology’s In Vitro Evaluation of Safety and Toxicity (InVEST) assays allows for off-target screening of new drug candidates for early safety pharmacology assessment. Inhibition of unwanted targets is often associated with adverse drug reactions in animal models and clinical studies. In vitro safety pharmacology profiling involves the screening of compounds against a broad range of targets that may cause adverse drug reactions in humans including receptors, transporters, enzymes, and ion channels.

Predicting potential safety liabilities early in the drug discovery process is integral for lead compound selection. In vitro safety screening enables selectivity-focused structure-activity relationship studies to mitigate off-target effects while retaining or increasing the compound’s potency at the primary target.

In addition to over 100 individual targets available for testing, Reaction Biology offers several pre-selected safety panels including InVEST44 (covering 44 well-established targets for early and broad assessment of safety liabilities), and CYP and PDE panels (for profiling against the most important CYP and PDE isoforms to deliver early guidance on a compound’s toxicity)

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Validated Targets by Family

Our validated targets include receptors, transporters, enzymes, and ion channels.

Early safety profiling includes more than 100 targets across 10 target families for broad coverage of potential adverse drug effects. All of our selected targets are clinically relevant. Their inhibition was shown to cause potentially serious health problems.

In-vitro-safety-screening

The InVEST44™ Panel

The InVEST44 panel covers a well-published set of 44 targets spanning core biological systems and secondary organs, enabling identification of common off-target interactions for a comprehensive early assessment of a drug candidate’s safety.

Key Features

  • Ten-day turnaround time from a US-based facility
  • Early identification of off-target interactions
  • Targets 44 critical molecular sites, including (24) GPCRs, (7) ion channels, (8) enzymes, (3) transporters, and (2) nuclear receptors
  • Comprehensive coverage of core and secondary organs and systems
  • Minimizes risk of adverse effects in drug development
  • Monthly panel runs supported by a world-class client service and scheduling team
  • Multiple concentrations and assay formats, plus individual targets on-demand

 Click here to download the InVEST44 target list.

The InVEST CYP Panel

The InVEST CYP panel provides profiling against the most important CYP isoforms to deliver early guidance on a compound’s toxicity. Our CYP450 inhibition assays are performed with the Vivid™ CYP450 assay technology to detect the activity of CYP enzymes in a simple “mix-and-read” fluorescent assay which is suitable for high-throughput analysis.

Key Features

  • Ten-day turnaround time from a US-based facility
  • Targets 14 CYP isoforms
  • Early identification of off-target interactions
  • Minimizes risk of adverse effects in drug development
  • Ad-hoc panel administered by a world-class client service and scheduling team
  • Multiple concentrations and assay formats, plus individual targets on-demand

Safety Pharmacology Targets

We consistently establish more targets for our InVEST Panel. Please reach out to inquire if the target of your interest is currently in development.

Optional: Functional Ion Channel Panel

In addition to compound profiling with the InVEST panel, clients have the option to investigate the cardiac liability of new drug candidates against our ion channel panel. Performed with whole-cell patch clamp technique, the results reveal whether a compound inhibits cardiac ion channel function in live cells. Advanced cardiac safety assays including testing on the CiPA panel to comply with regulatory guidelines as well as testing on cardiac tissue or isolated hearts can be performed. Please read more on our cardiac safety assessment website.

Additional information about InVEST Screening

  • Assay setup
  • Assay formats
  • Screening schedule
  • Example data
Assay setup

Setups: Standard panel testing consists of single concentration testing in duplicates. Alternate testing formats may be available upon request.

Controls:
Binding assays: Total binding (DMSO vehicle) and non-specific binding (containing saturating concentration of reference compound) and a full concentration-response curve with an appropriate reference compound are conducted with each target.
Enzymatic activity assays: No inhibitor control (DMSO vehicle) and a full concentration-response curve with an appropriate reference compound are conducted with each target.

Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to the commencement of the studies.

Report: The raw data, % inhibition, and control compound IC50 values will be reported in Excel format.  Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.

Screening facility: This assay is performed at our screening facility in Malvern, PA, US.

Compound requirements: In brief, for a standard project with a final test concentration of 10 µM we require 100 µl of a 10 mM stock solution in DMSO or 5 mg powder. Less material per test is needed for large-scale screening. Please refer to our FAQs for information regarding compound preparation and shipping.

Please see our FAQ page to access our assay condition form (US facility).
Assay formats

Radioligand Binding Assay

A. Commercially sourced membrane preparations and prepared rat brain membranes expressing the target receptors, respectively, are placed in multi-well plates. B. 3H-labelled ligands incubate with the membrane preparations in the presence and absence of test compounds for 90 minutes. C. The solution is pulled through a filter trapping the membranes and bound ligands. Unbound ligands are not retained on the filter. The radioactivity in the filter is measured via scintillation counting as a readout of ligand-target receptor binding.

 

Fluorescence Polarization

The fluorescence polarization (FP) assays are binding assays where a ligand conjugated to a fluorophore binds to the target receptor.
A. The FP assay is performed with either soluble protein or (in the case of hERG) membrane preparations. B. The client compound and the fluorescent ligand incubate with the target. C. Ligand binding is quantified by measuring the amount of polarized light emitted by the fluorophores in the well.

Principle: Light waves, passed through certain crystalline materials (polarizers), have their electrical vectors oriented in a single plane and are thus polarized. The fluorophores linked to the ligand absorb the light and emit fluorescence. Soluble, small fluorescent ligands have a high rotational movement (Brownian movement) that is shorter than their fluorescence decay time resulting in their emitted fluorescence light being depolarized. Fluorescent ligands bound to the larger receptor have a rotational movement that is slower than their fluorescence decay time thus, their emitted light remains polarized.

Cell-based transcriptional regulation

Receptor-specific reporter cells express the luciferase reporter gene functionally linked to a bona fide receptor-responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in receptor activity. This assay format is suitable to investigate agonistic and antagonistic responses of the target receptor.

Enzymatic activity

Assays for testing the activity of an enzymatic target measure modifications to the target substrate. More information can be found for cytochrome P450, phosphodiesterases, or protease targets.

Screening schedule

Testing of the InVEST44 Panel is performed on a monthly basis.

Please see the upcoming screening dates here.

Example data

Fluorescence Polarization Assay with a Nuclear Receptor

Progesterone Receptor Fluorescence Polarization: Results of three independent experiments of progesterone binding are shown. 95% CV shown as dashed lines. Sigmoid fit (solid line) parameters: IC50=36.9 nM, Hill slope=−2.13. 95% confidence intervals: EC50: 30.4 to 44.8 nM, Hillslope: ‑3.0 to ‑1.2.

Fluorescence Polarization Assay with an Ion Channel

hERG Receptor Fluorescence Polarization: Results of six independent experiments of E-4031 binding to hERG containing membranes are shown.Average of 6 independent experiments. 95% CV shown as dashed lines. Competing drug: E-4031. Sigmoid fit (solid line) parameters: IC50=20.9 nM, Hill slope=−1.46. 95% confidence intervals: IC50: 14.7 to 29.7 nM, Hillslope: ‑-2.08 to ‑0.84.

Radioligand Assay with a GPCR

Histamine H1 Receptor Radioligand Binding Assay: Results of one experiment performed in duplicates of binding of reference compound pyrilamine to Histamine H1 receptor are shown.

Sigmoid fit parameters: EC50=1.25 nM, Hill slope=−0.99.

 

Enzymatic Activity Assay with a PDE

Three reference compounds IBMX, methoxyquinazoline, and Rolipram were tested against the activity of cAMP-specific cyclic phosphodiesterase 4A (PDE4A). Concentration-response curves are shown with semi-log concentrations in singlicates with the following parameters:

IBMX: IC50= 1.4e-05, hillslope= -0.72
Methoxyquinazoline: IC50= 7.82e-07, hillslope= -0.86
Rolipram: IC50= 1.1e-06, hillslope= -0.86

Enzymatic Activity Assay with a Protease

Reference inhibitor gabexate mesylate (GM) was tested against thrombin for IC50 value determination with semi-log concentrations in singlicate. The fluorescent substrate Pefafluor TH containing AMC was used as a substrate. Parameters: IC50= 5.9e-07, hillslope= -0.94

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