Relaxation in Human Lower Esophageal Sphincter

The specific results that will be provided are the effects of increasing concentrations of test articles on the LES.

Turnaround Time

For a standard experiment with n=3, tissue procurement is estimated to be 8-10 weeks. These timelines are a guide based on standard tissue criteria; any client-imposed alterations to the criteria may alter these estimates.

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. Sponsor to provide sufficient test article to run the entire study.

Suggested Testing

We suggest test conditions are assessed in triplet as a minimum.

Study Outline

Rationale and Experimental Design

The experiment assesses whether test articles cause tonal changes in the lower esophageal sphincter compared with a reference compound.

Exclusion Criteria

No specific exclusion criteria are in place other than to reject macroscopically diseased/necrotic tissue.

Standardization and Qualification

Tissues which do not respond to stimulation by the reference compounds will be excluded.

Lower Esophageal Sphincter Relaxation Assays

Option 1 − IC50 Determination

To assess the ability of each test article to elicit smooth muscle relaxation, 6-point cumulative concentration response curves will be performed for each test article. These concentration response curves (CCRC’s) will be performed following pre-contraction with an appropriate agent, such as Carbachol. A positive control compound and representative test article 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 will be run in triplicate muscle strips):

Representative test article vehicle CCRC

• Test article 1 CCRC
• Test article 2 CCRC
• Test article 3 CCRC
• Positive control CCRC

Option 2 − pA2 Determination

To assess the ability of a test article to antagonize an agonist mediated relaxation response; 6-point cumulative concentration response curves (CCRC’s) will firstly be performed for the reference agonist, following pre-contraction with an appropriate agent, such as Carbachol. Following a wash out and recovery period, muscle strips will be incubated with the test item (3 different concentrations of test article will be assessed), positive control or test article vehicle before the same 6-point cumulative concentration response curve will be repeated for the reference agonist.

An example of the conditions assessed for 3 test articles are detailed below (each condition will be run in triplicate muscle strips):

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

Supplementary Option

To assess the involvement of a specific receptor subtype in any observed responses, the concentration of test article eliciting the largest response can subsequently (following a wash out and recovery period) be tested in the presence of a specific antagonist. This supplementary option will incur an extra charge.

Analysis

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

Relaxation of LES in response to vehicle and control

Back to the Drug Discovery Assay Catalog »