Modulation of Neuronally Induced Contractions (EFS) of Human Airways

Drug Discovery Assay – reference number: B131

Overview

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Assay Description

This assay assesses whether test articles can modify EFS-induced contractions in human isolated airway strips with atropine/formoterol as reference compounds.

Figure 1: Cumulative concentration response curve to formoterol and vehicle in human isolated airways stimulated with Electrical Field Stimulation (EFS). Results are expressed as a percentage inhibition of the baseline EFS response.

Figure 2: Cumulative concentration response curve to atropine in EFS stimulated isolated human bronchi (n=4) demonstrating abolishment of the EFS responses. The Log EC₅₀ = −7.00 ± 0.35 M (mean ± S.E.M). Maximum relaxation was 110.8 ± 21.26 % at 1 × 10^-5 M.

Testing Information

Introduction

The specific results that will be provided are the effects of increasing concentrations of test articles on EFS-induced contractile responses of human isolated airways.

Test Article Requirements

Test article(s) to be provided by the Sponsor in storable aliquots at required test concentrations with information on diluent vehicle used. Stock solutions are prepared in distilled water unless otherwise requested. Bath volumes are 25 mL; sponsor to provide sufficient test article to run the entire study.

Suggested Testing

In duplicate at 6 concentrations.

Study Outline

Rationale and Experimental Design

This assay assesses whether test articles can modify neuronally-mediated contractions in isolated human airways, with formoterol or atropine as reference compounds.

Exclusion Criteria

Data presented here are from healthy lungs.  Exclusion criteria can be put as required for example asthma, COPD, fibrosis etc. Furthermore, tissues which do not respond to the standard pharmacology checks will be excluded.

Standardisation and Qualification

All individual airway strips are initially processed through standardisation and qualification procedures to ensure functionality, prior to starting the study protocol.

Airway strips are processed through a standardisation procedure to reduce signal variability prior to pharmacological intervention. This ensures that tissue is maintained under appropriate physiological tension throughout the experiments.

Following standardisation, the airway strips are stimulated with EFS to ensure robust contractile responses.

Airway strips that pass the standardisation and qualification pass/fail criteria will then progress to the study protocol.

Contractility Assay

Modulation of EFS-induced contractions

To assess the ability of each test article to modulate EFS-induced contractility, 6 point cumulative concentration response curves (CCRCs) will be performed for each test article. A positive control compound (and vehicle) CCRC will also be run to allow direct comparison with test articles.

An example of the conditions assessed for 3 test articles are detailed below (each condition can be run in duplicate):

• Representative test article vehicle CCRC
• Test article 1 CCRC
• Test article 2 CCRC
• Test article 3 CCRC
• Positive control CCRC

Analysis

Responses shall be expressed either in milligrams or grams tension, or as a % of baseline or maximum response. Statistical analysis will be performed (where appropriate) using GraphPad Prism, with the results being shown in graphical form in the final report.

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