Relaxation in human ureter (Adrenoceptor – Isoprenaline)

Drug Discovery Assay – reference number: B071

Overview

Assay Description

This assay assesses whether test articles cause relaxation in isolated human ureter, with isoprenaline as a reference compound.

The ureter connects the kidneys to the urinary bladder. The ureter has a smooth muscle layer which propels urine to the bladder, via peristalsis, a phenomenon that can be observed in vitro.

Testing Information

Introduction

The specific results that will be provided are the effects of increasing concentrations of test articles on the magnitude of the pre-constrictor response in isolated human ureter muscle.

Turnaround Time

6 weeks from sample receipt for n = 3 donors.

Timelines are a guide based on REPROCELL 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 25mL; 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 cause relaxation in isolated human ureter, with isoprenaline 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 muscle strips are initially processed through standardisation and qualification procedures to ensure functionality, prior to starting the study protocol.

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

Following standardisation, the muscle strips are challenged with an appropriate agent to ensure robust contractile responses. Test tissues are then washed, and the responses allowed to return to baseline.

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

Smooth Muscle Contractility Assay

Agonist Relaxation Assays

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. 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 muscle strips):

• 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 test article to antagonise an agonist mediated relaxation response, 6 point cumulative concentration response curves will be performed for each test item. These concentration response curves (CCRC’s) will be performed following pre- contraction with an appropriate agent and then relaxation with the appropriate 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 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 antagonise 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. Following a wash out and recovery period, muscle strips 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 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

Analysis

Responses shall be expressed either in milligrams or grams tension, or as a % relaxation of the pre-constriction 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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