Abstract/Summary

Background: The development of in vitro and ex vivo models to mimic human illness is important, not only for scientific understanding and investigating therapeutic approaches, but also to mitigate animal testing, bridge the inter-species translational gap, and reduce costly development and clinical trial time and expense. The life span of a new molecular entity from inception to commercial introduction can span over 15 years, with high failure rates at key milestones including safety, stability, thermodynamics, and clinical efficacy. Dermatological development accounts for $50 million dollars in clinical trial expense alone. This expense is heavily dependent upon the later stage clinical trial aspect depending on number of patients needed to establish treatment effects and number of pivotal trials required to support marketing approval, each variable contributing to higher accrued costs of running the trial (Moore et al., 2020). While in vitro models can facilitate high-throughput and cost-efficient evaluation of novel therapeutics, more complex ex vivo systems can better predict both desirable and adverse in vivo effects. The lack of translation from simpler in vitro models to complex in vivo disease as well as the discrepancies between animal physiology and human due to immune system, skin pathology, and drug interaction lend to the need for more complex ex vivo models to better characterise the human disease state. Aim: The objective of the work described in this thesis was to develop a human ex vivo skin culture (HESC) model to explore the pathophysiology of inflammatory dermatoses and for preclinical testing of potential therapeutic treatments. Results: Here a human ex vivo skin culture model is described in which pathological tissue integrity, barrier function, and metabolic stability over time has been characterised and shown success up to 9 days in culture. Following exogenous stimulation, tissue integrity and ability to induce inflammatory gene expression including interferons, interleukins, chemokines, and antimicrobial pepetides was retained, and stimulant concentrations and duration were optimised to correlate with published data from clinical biopsies of inflammatory dermatitis and psoriasis patients. The validity and utility of the model was demonstrated when challenged with 5 drugs; clobetasol, calcitriol, pimecrolimus, crisaborole, and tofacitinib; where inflammatory biomarkers were regulated in a manner consistent with the drugs’ reported in vivo mechanisms of action. The steroid clobetasol propionate inhibited multiple signaling pathways including IFNg, GM-CSF, IL13, IL31, CCL26, MMP12, IL17a and IL8 corresponding to published clinical trial data of clobetasol effect on AD lesions with treatment (Guttman-Yassky et al., 2017). The vitamin D3 analogue calcitriol inhibited Th2-cell specific cytokines IL13 and IL31 and significantly increased CCL26 and S100A12 gene expression, illustrating its Th2-dominant activity. Crisaborole, the PDE4 inhibitor, significantly inhibited all Th2 associated biomarkers IL13, IL31, MMP12, and GM-CSF and increased CCL26 gene expression, analogous to the gene expression profiles reported by Bissonnette, et al clinical application. Calcineurin inhibition by pimecrolimus following Th1-mediated stimulation reduced IFNg, CXCL10, S100A12 and GM-CSF. This reduction in inflammatory activity correlates to the clinical reduction of AD flares in children and adults and a mean Eczema Area Severity Index (EASI) reduction between 38-71 % in adults and 47-82 % in children in multiple clinical studies (Breuer et al., 2005). Tofacitinib significantly inhibited IL31, CCL26 and MMP12 gene expression with Th2 stimulation, confirming that JAK inhibitors show promise in Th2-driven AD patients (He and Guttman-Yassky, 2019). Tissue responses to established therapies of pimecrolimus (Elidel) and clobetasol propionate (Dermovate) were evaluated using the human ex vivo skin culture, assessing pharmacodynamic changes in gene expression alongside the pharmacokinetics of drug penetration with both products showing time dependent efficacies. Similarities included reduction of IFNg 95% in vivo compare to 97% in the HESC model by clobetasol and reduction of IL13 68% in vivo compare to 70% in the HESC model by pimecrolimus. Conclusion: Through characterisation of the HESC model including tissue integrity, viability, cellular activity, inflammatory stimulation, demographic variability and topical therapeutic action, a comprehensive understanding of the capabilities of the model as a preclinical ex vivo drug development tool have been established. The translation of the HESC model to in vivo clinical data justifies the use of human ex vivo skin culture in an inflammatory state in early development i.e., New Chemical Entity and formulation screening and optimisation and the characterisation and comparison of such drugs and formulations with those already marketed has the potential of de-risking costly and time-consuming clinical trials. In addition, upon further validation such inflammatory HESC models may provide an ex vivo approach to demonstrate therapeutic bioequivalence for commonly prescribed drugs allowing for faster and cost-efficient regulatory approval.