Treatment of Chronic Itch in Atopic Dermatitis With Opioid Antagonist Naltrexone

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Specific aims: A1 Evaluate the underlying mechanism of itch severity in terms of circadian rhythm by collecting epidermis at different circadian stages using suction blisters. This will be done in AD patients with chronic itch. The cells will be isolated from part of the suction blisters and used to...

Specific aims: A1 Evaluate the underlying mechanism of itch severity in terms of circadian rhythm by collecting epidermis at different circadian stages using suction blisters. This will be done in AD patients with chronic itch. The cells will be isolated from part of the suction blisters and used to isolate and sequence RNA to evaluate and compare the differential protein and RNA expression in suction blister taken from symptomatic & non-symptomatic areas in patients with AD at different time points. The investigators will then correlate the itch severity symptoms to clock gene and opioid receptor levels and RNA expression differential pattern. A1 will be evaluated by study arm A and the results from this study will serve to decide on time point taking the suction blisters for the therapeutic/mechanistic studies (arm B and C). A2 Investigate the mechanistic role and therapeutic efficacy of opioid receptors in chronic itch. For this purpose, a cross-over, placebo-controlled design will be used to treat patients with opioid receptor antagonist (Naltrexone) by expanding on our existing trial. The investigators will collect, evaluate, quantify and compare the differential protein and RNA expression in epidermis taken by suction blisters from symptomatic and non-symptomatic areas in patients with AD on Placebo or Naltrexone treatment. Transcriptome analysis will be focused, but not restricted to, analysis of neuroinflammatory and neuronal markers (including cytokines, GPCR and opioid receptors). Confirmation will occur using in-situ hybridization of biopsies to correlate specific cell type and location and qPCR expression of in vitro cultures. The binding studies using fluorescent labeled opioid ligands and internalization pattern will be carried out. A3 Test the underlying mechanisms of the opioid system with nerve-keratinocyte interaction and possible effects on itch transduction from skin to nerves by using an in vitro neuron-keratinocyte co-culture model. Moreover, the investigators plan to validate itch-causing networks and pathways found in A2 using this in vitro model and will be able to evaluate calcium flux threshold changes after exposure to external stimuli (e.g. light, or mechanic stimuli) onto keratinocytes and neuronal somata under different opioid exposure conditions. The FC derived sensory neurons will be co-cultured with different patient KC. The investigators will test the reaction pattern of KC and transmission of the signal to connected peripheral sensory neurons by Fluo-4 AM Calcium imaging and electrophysiology. The reaction will be responding to the odorant Sandalore, as the investigators have previously published that Sandalore induces these desired calcium fluxes. Apply the in vitro epithelial/nerve model to validate possible itch pathways identified by epigenetic analysis in the clinical trials by confirming the expression pattern with qPCR and functional assays on cells with CRISPR knockout of the RNA targets, opioid trafficking, calcium imaging and electrophysiology. Finally, further develop co-cultures between human skin and iPS-derived human peripheral neurons to perform functional studies.

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