Drug Discovery Assay Services Assay: Vasoconstriction in atherosclerotic human subcutaneous resistance arteries (Angiotensin receptor)

Code: B037
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Assay type:Blood Vessels
Tissue:Human Resistance Artery (Atherosclerotic)
Target:Angiotensin receptor
Control Compound:PDA-angiotensin 1
Study Type:Wire Myography
Functional Endpoint:Vasoconstriction

Assay Description

The experiment assesses whether test articles cause vasoconstriction in human subcutaneous resistance arteries with PDA-angiotensin 1 as a reference compound.

Resistance arteries are involved in regulating organ-specific blood flow throughout the body and offer the greatest resistance to blood flowing into tissues. Resistance arteries therefore play an important role in the regulation of arterial blood pressure. Atherosclerosis is the formation of fatty lesions or fibrous plaques in the smooth muscle of arteries. This causes a narrowing of the vessels, an increase in blood pressure and an increased risk of myocardial infarction and stroke.

Example assay graph

Image Legend

Figure 1: Cumulative concentration response curve to PDA-Ang 1 in isolated human subcutaneous resistance arteries, demonstrating a concentration dependent contractile response.

The Log EC50 = −8.2 ± 0.5 M (6.7 nM) (mean ± S.E.M), with a maximum contractile response of

266.4 ± 45.84 % at 3 × 10−7 M.

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 subcutaneous arteries.

Turnaround Time

10-12 weeks from sample receipt for n = 3 donors.

Timelines are a guide based on Biopta's standard tissue criteria; any client imposed deviations to the standard tissue criteria can alter the project timelines.

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 5 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

The experiment assesses whether test articles cause vasoconstriction in human subcutaneous resistance arteries with PDA-angiotensin 1 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 Constriction Assays

To assess their ability to elicit vasoconstriction, six point cumulative concentration response curves (CCRC's) will be performed for each test article. These CCRC's will be performed from resting baseline tension. 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
  • Positive control CCRC
  • Test article 1 CCRC
  • Test article 2 CCRC
  • Test article 3 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 Constriction Assays

Option 1 − IC50 determination

To assess the ability of each item to antagonise an agonist mediated vasoconstriction response, six 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 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 vasoconstriction response; six point cumulative concentration response curves (CCRC's) will firstly be performed for the vasoconstrictive reference agonist.

Following a wash out and recovery period, vessels will be incubated with the test article (3 different concentrations of test item 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 (eg. KPSS). Statistical analysis (where appropriate) will be performed using GraphPad Prism, with the results being shown in graphical form in the final report.

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