Skin culture induced psoriasis model

Drug Discovery Assay – reference number: B113

This experiment assesses whether test articles cause a reduction in inflammatory cytokine release in psoriasis compared with a control compound, for example an ROR-ɣ inhibitor (systemic) or betamethasone (topical). It uses healthy skin punch biopsies which are induced to display a psoriasis phenotype using a cocktail that induces the Th17/IL-17 pathway. We then explore test article effect on the expression on inflammatory cytokines via ELISA and/or rtPCR.

Assay Type: Skin
Tissue: Human Skin Biopsies (Healthy)
Target:

TLR4/TLR2 Pathway/ Glucocorticoid receptors/ PDE4

Control Compound: Fluticasone/ Roflumilast
Study Type: Ex vivo cultures
Functional Endpoint: Cytokine release/ Gene Expression

Testing Information

Introduction

The specific results that will be provided are on the anti-inflammatory effect of test articles across an induced psoriasis model using human fresh skin.

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 deionized 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.

Diagram showing pathways activated by TH17 stimulation in human fresh tissue explants


Study Outline: Rationale and Experimental Design

Option 1: Systemic administration of test article

Full-thickness skin biopsies are achieved via punch-biopsy of residual surgical tissue. Biopsies are taken at a size of 3mm2 and transferred onto a mesh transwell in a 12 well plate. The dermis of the skin is submerged in specially fortified media, leaving the epidermis exposed to air. Biopsies are incubated in optimum conditions for a maximum of 24 hrs, then cultured in the presence of the inflammatory cocktail and the test article for a further 24 hrs. Tissue media is collected for phenotype analysis by ELISA and/or the biopsy set is collected for rtPCR.

An example of the conditions assessed for 3 test articles are detailed below (it is recommended a minimum of triplicates should be used for each condition):

  • Non-stimulated control
  • Stimulated control
  • Test article 1
  • Test article 2
  • Test article 3
  • Positive control

Option 2: Topical application of test article

Full-thickness skin biopsies are achieved via punch-biopsy of residual surgical tissue. Biopsies are taken at a size of 8mm2 and transferred onto a mesh transwell in a 12 well plate. The dermis of the skin is submerged in specially fortified media, leaving the epidermis exposed to air. Biopsies are incubated in optimum conditions for a maximum of 24 hrs, then cultured in the presence of the inflammatory cocktail (added to the media) and the test article (added to the exposed skin surface) for a further 24 hrs. Tissue media is collected for phenotype analysis by ELISA and/or the biopsy set is collected for rtPCR.

An example of the conditions assessed for 3 test articles are detailed below (it is recommended a minimum of triplicates should be used for each condition):

  • Non-stimulated control
  • Stimulated control
  • Test article 1
  • Test article 2
  • Test article 3
  • Positive control

Exclusion Criteria

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

Standardization and Qualification

Tissue that does not respond to stimulation with our TH17 inflammatory cocktail LPS would be deemed non-functional.

Analysis

Cytokine analysis: Supernatant samples can be analyzed for specific analytes of your choice by a multiplex ELISA platform. Each analyte will be quantified by interpolation against a standard curve generated on the same 96 well analysis plate.

Other forms of end point analysis are available such as gene expression or immunohistochemistry.

The set up for a typical full-thickbness human skin tissue explant experiment


Example data from Skin culture induced psoriasis model

Induction of IL-17A gene expression and secretion (systemic)

Induction of IL-17A Gene Expression and Secreation after 24hrsData is presented as mean ± SEM in bar format with the individual donor means presented in the scatter. Analysis was conducted by two-way ANOVA. Three subjects were evaluated in quadruplicate measurements. Asterisks indicate significant (P < 0.05, for one, P < 0.01 for two, P < 0.001 for three and P < 0.0001 for four) differences versus the control group. Multiple comparisons across all test cocktails were evaluated with the Tukey test. A, Actin gene expression was used as the endogenous control and expression levels are therefore adjusted by those of actin using the ΔCT method. ; B, Dotted line represents the LLOQ of the ELISA.

Inhibition of TH17 cell differentiation by RORγ inhibitor (systemic)

17 cell differentiation and it’s inhibition by a RORγ inhibitorData is presented as mean ± SEM. Analysis was conducted by two-tailed unpaired t-test. Three subjects were evaluated in quadruplicate measurements. Asterisks indicate significant (P < 0.05, for one, P < 0.01 for two, P < 0.001 for three and P < 0.0001 for four) differences versus the DMSO control group.

Results from topical application of SOC formulationsData from topical application of SOC formulations

Data is presented as mean ± SEM in bar format with the individual donor means presented in the scatter. Analysis was conducted by two-way ANOVA . Three subjects were evaluated in quadruplicate measurements. Multiple comparisons were evaluated with the Dunnett’s test. Asterisks indicate significant (P < 0.05, for one, P < 0.01 for two, P < 0.001 for three and P < 0.0001 for four) differences versus the untreated control group.


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