Evaluation of pharmacological responses to subcutaneous injection of adalimumab in InflammaSkin®, a full-thickness human ex vivo skin model reproducing key features of psoriatic lesions

USING INFLAMMASKIN EX VIVO SKIN MODEL

Preclinical skin models closely mimicking inflammatory features in diseased skin such as psoriasis are needed to support the development of anti-inflammatory drugs in dermatology. Approaches based on the incorporation of immune cells in reconstructed skin models are now developed. However, these skin models lack tissue complexity and diversity of immune cells and do not support the delivery of subcutaneously-delivered therapeutic compounds. Genoskin has developed and characterized InflammaSkin®, a T-cell driven skin inflammation model for psoriasis based on the activation and differentiation into Th17/Th1 phenotype of skin resident T cells. This model is composed of epidermis, dermis and adipose tissue, enabling subcutaneous delivery of biologics. In this study, we aimed at assessing pharmacological response of the model to subcutaneous administration of adalimumab.

Production of InflammaSkin® model from healthy ex vivo skin biopsies

 HypoSkin® and InflammaSkin® models were produced from healthy ex vivo skin samples of one donor (Female, 67 years old) and cultured in standard cell culture conditions for 7 days.
From day 4 to day 6, 23 μL Betamethasone was topically applied at the surface of skin models.
At day 3, 35 μL Adalimumab at either 5 nM or 50 nM were subcutaneously injected in the hypodermis of the models.
The study was performed on 3 models for each condition. 

A serie of 4 illustrations showing the production of InflammaSkin® model from resident T cell in situ activation to the therapeutic treatment

From a healthy skin biopsy to an injectable inflammation assay

Improvement of skin structure and downregulation of psoriasis-associated markers in a pilot study following adalimumab subcutaneous injection

 Hematoxylin and Eosin (H&E) staining was performed on 5 μm thickness paraffin-embedded skin cross-sections. Scale bar 100 μm. 

A series of 5 H7E staining photos showing how InflammaSkin® assay responds to the injection of adalimumab

 Quantitative analysis of skin structure integrity was performed through attribution of a score depending on defects detected in the models on H&E stained sections:
0: Uncultured skin without any changes
1: Early signs of keratinocytes separation (spongiosis)
2: Advanced separation and volume increase of keratinocytes (hypertrophy) / Early signs of dermis disorganization
3: Nuclei condensation (pyknosis) / Advanced dermis disorganization
4: Cytoplasms vacuolation
5: Stratum basale detachment from dermis / Release of stratum spinosum 

Two graphics showing how InflammaSkin® assay reacts to adalimumab injection

 Anti-S100A7 / Keratin 16 (K16) double immunofluorescence staining was performed on 5 μM thickness paraffin-embedded skin cross-sections. For each marker, normalized positive area expressed was quantitatively analyzed. For each condition, single replicates values, mean and standard deviation are presented. 

Anti-100A7/K16 double immunofluorescence staining showing how InflammaSkin® reacts to adalimumab

Dose-depending downregulation of IL-17A, IL-22 and INF-γ but not IL-27 release

Levels of cytokines released into the culture media were measured with the Mesoscale (Rockville, USA) human V-Plex Proinflammatory and Th17 Panels. For each analyzed cytokine, concentrations in each replicate, as well as mean values and standard deviation, are presented. 

A series of 4 graphs showing the level of IL-17A, IL-22, IL-27 and INF-γ in InflammaSkin® with and without injection of adalimumab

Conclusion

InflammaSkin® is a new fully human ex vivo skin
model of psoriasis with unique key characteristics:
– Disease-relevant, complex biological matrix based on the NativeSkin® model
– Includes adipose tissue with controlled thickness to support reproducible subcutaneous injection
– Recapitulates cellular activities and inflammatory features as found in psoriatic lesions
– Successfully responds to subcutaneous injection of biologics

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