Vasodilatation in human chorionic plate arteries (PDE5)

Drug Discovery Assay – reference number: B009

Overview

Assay Description

The experiment assesses whether test articles cause vasodilatation in human placenta vessels with sildenafil as a reference compound.

Chorionic plate arteries supply blood and nutrients to the growing foetus making them essential for healthy development. Any disruption to blood flow can cause adverse effects to the foetus.

Figure 1: Cumulative concentration response curve to sildenafil and its vehicle in healthy isolated human chorionic plate arteries pre-constricted with 3 × 10⁻⁸M U46619, demonstrating a concentration dependent relaxation response. The Maximum relaxation response was 87.17 ± 11.59% at 1 × 10⁻⁵M for sildenafil, compared to a maximum relaxation response of 24.99 ± 4.06% at 1 × 10⁻⁶M for the vehicle.

Testing Information

Introduction

The specific results that will be provided are the effects of increasing concentrations of test articles on the contractile state of human isolated chorionic plate arteries.

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 5mL; sponsor to provide sufficient test article to run the entire study.

Suggested Testing

In duplicate at 6 concentrations.

Study Outline

Rationale and Experimental Design

The experiment assesses whether test articles cause vasodilatation in human chorionic plate arteries with sildenafil as a reference compound.

Exclusion Criteria

No specific exclusion criteria are in place other than to reject macroscopically diseased/necrotic tissue. Furthermore, tissues which do not respond to the standard pharmacology checks will be excluded.

Standardisation and Qualification

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

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

Following standardisation, vessels are “woken up” by 2 successive high potassium salt solution challenges with washes and recovery periods following each challenge. This process standardises the basal resting tone and ensures robust contractile responses.

Vessels are then challenged with an appropriate vasoconstrictive agent before endothelial function is assessed by the addition of the appropriate endothelial dependent vasorelaxant agent. Test tissues are then washed, and the responses allowed to return to baseline.

Vessels which pass the standardisation and qualification pass/fail criteria will then progress to the study protocol.

Agonist Relaxation Assays

To assess the ability of each test article to elicit vasodilatation, 6 point cumulative concentration response curves (CCRC’s) will be performed for each test article. These CCRC’s will be performed following pre-constriction with an appropriate vasoconstrictive agent. 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 duplicate vessels):

• Representative test article vehicle CCRC

• Test article 1 CCRC

• Test article 2 CCRC

• Test article 3 CCRC

• Positive control CCRC

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.

Antagonist Relaxation Assays

Option 1 − IC₅₀ Determination

To assess the ability of each item to antagonise an agonist mediated vasorelaxation response, 6 point cumulative concentration response curves (CCRC’s) will be performed for each test article. These CCRC’s will be performed following pre-constriction and relaxation with an appropriate vasorelaxant reference agonist. 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 duplicate vessels):

• 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 antagonise an agonist mediated vasorelaxation response; 6 point cumulative concentration response curves (CCRC’s) will firstly be performed for the reference agonist, following pre-constriction with an appropriate vasoconstrictive agent.

Following a wash out and recovery period, vessels will be incubated with the test article (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 duplicate vessels):

• 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

Analysis

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