Modelling atopic dermatitis in healthy human skin for the characterization of topical compounds.

Suitable human models for the development and characterization of topical compounds for inflammatory skin diseases such as atopic dermatitis are not readily available to date. We describe here the development of a translational model involving healthy human skin mimicking major aspects of AD and its application for the characterization of topical Janus kinase inhibitors. Full thickness human abdominal skin obtained from plastic surgery stimulated in vitro with IL4 and IL13 shows molecular features of AD. This is evidenced by STAT6 phosphorylation assessed by immunohistochemistry and analysis of skin lysates. Broad transcriptome changes assessed by AmpliSeq followed by gene set variation analysis showed a consistent upregulation of gene signatures characterizing AD in this model. Topical application of experimental formulations of compounds targeting the JAK pathway to full thickness skin normalizes the molecular features of AD induced by IL4 and IL13 stimulation. The inhibitory effects of topical JAK inhibitors on molecular features of AD are supported by pharmacokinetic analysis. The model described here is suited for the characterization of topical compounds for AD and has the potential to be extended to other inflammatory skin diseases and pathophysiological pathways.

IL-26 Potentiates Type 2 Skin Inflammation in the Presence of IL-1ß.

Atopic dermatitis (AD) is a debilitating inflammatory skin disorder. Biologics targeting the IL-4/IL-13 axis are effective in AD, but there is still a large proportion of patients who do not respond to IL-4R blockade. Further exploration of potentially pathogenic T-cell-derived cytokines in AD may lead to new effective treatments. This study aimed to investigate the downstream effects of IL-26 on skin in the context of type 2 skin inflammation. We found that IL-26 alone exhibited limited inflammatory activity in the skin. However, in the presence of IL-1ß, IL-26 potentiated the secretion of TSLP, CXCL1, and CCL20 from human epidermis through Jak/signal transducer and activator of transcription signaling. Moreover, in an in vivo AD-like skin inflammation model, IL-26 exacerbated skin pathology and locally increased type 2 cytokines, most notably of IL13 in skin T helper cells. Neutralization of IL-1ß abrogated IL-26-mediated effects, indicating that the presence of IL-1ß is required for full IL-26 downstream action in vivo. These findings suggest that the presence of IL-1ß enables IL-26 to be a key amplifier of inflammation in the skin. As such, IL-26 may contribute to the development and pathogenesis of inflammatory skin disorders such as AD.

Design of a Supersoft Topical JAK Inhibitor, Which Is Effective in Human Skin but Rapidly Deactivated in Blood.

We describe the discovery and characterization of the supersoft topical JAK inhibitor 3(R), which is potent in biochemical and cellular assays as well as in human skin models. In blood, the neutral ester 3(R) is rapidly hydrolyzed (t1/2 ∼ 6 min) to the corresponding charged carboxylic acid 4 exhibiting >30-fold reduced permeability. Consequently, acid 4 does not reach the intracellular JAK kinases and is inactive in cellular assays and in blood. Thus, hydrolysis by blood esterases leads to the rapid deactivation of topically active ester 3(R) at a rate beyond the maximal hepatic clearance.

Adriforant is a functional antagonist of histamine receptor 4 and attenuates itch and skin inflammation in mice.

BACKGROUND: Histamine has been postulated to play a role in atopic dermatitis via histamine receptor 4, mediating pruritic and inflammatory effects. The H4R antagonist adriforant (PF-3893787 or ZPL389) indicated clinical efficacy in a Ph2a study in atopic dermatitis. Preclinical investigations of adriforant had been scarce as experiments in transfectants with H4R from several species suggested partial agonism, not seen in human cells. OBJECTIVE: During the Ph2b trial in AD, we performed experiments to understand the pharmacology of adriforant in primary murine cells and in vivo models. We assessed its effects on ERK phosphorylation and transcriptional changes in bone marrow-derived mast cells, histamine-dependent Ca2+ flux in neurons and histamine-induced itch response. In addition, its impact on MC903-induced skin inflammation was evaluated. RESULTS: We show that, contrary to transfectants, adriforant is a competitive antagonist of the murine histamine receptor 4, antagonizes histamine-induced ERK phosphorylation, normalizes histamine-induced transcriptional changes in mast cells and reduces histamine-dependent Ca2+ flux in neurons. Administration to mice reduces acute histamine-induced itch response. In addition, adriforant ameliorates inflammation in the mouse MC903 model. CONCLUSIONS: Our results suggest that functional inhibition of histamine receptor 4 by adriforant reduces itch and inflammation in vivo. The effects observed in mice, however, did not translate to clinical efficacy in patients as the Ph2b clinical trial with adriforant did not meet pre-specified efficacy endpoints. Given the complex pathogenesis of AD, antagonism of histamine receptor 4 alone appears insufficient to reduce disease severity in AD patients, despite the effects seen in mouse models.

Discovery and Characterization of the Topical Soft JAK Inhibitor CEE321 for Atopic Dermatitis.

The JAK kinases JAK1, JAK2, JAK3, and TYK2 play key roles in cytokine signaling. Activation of the JAK/STAT pathways is linked to many diseases involving the immune system, including atopic dermatitis. As systemic JAK inhibitor pharmacology is associated with side effects, topical administration to the skin has been considered to locally restrict the site of action. Several orally bioavailable JAK inhibitors repurposed for topical use have been recently approved or are in clinical development. Here, we disclose our clinical candidate CEE321, which is a potent pan JAK inhibitor in enzyme and cellular assays. In contrast to repurposed oral drugs, CEE321 does not display high potency in blood and has a high clearance in vivo. Therefore, we consider CEE321 to be a "soft drug". When applied topically to human skin that was stimulated with the cytokines IL4 and IL13 ex vivo, CEE321 potently inhibited biomarkers relevant to atopic dermatitis.

Hepatic stellate cells suppress NK cell-sustained breast cancer dormancy.

The persistence of undetectable disseminated tumour cells (DTCs) after primary tumour resection poses a major challenge to effective cancer treatment1-3. These enduring dormant DTCs are seeds of future metastases, and the mechanisms that switch them from dormancy to outgrowth require definition. Because cancer dormancy provides a unique therapeutic window for preventing metastatic disease, a comprehensive understanding of the distribution, composition and dynamics of reservoirs of dormant DTCs is imperative. Here we show that different tissue-specific microenvironments restrain or allow the progression of breast cancer in the liver-a frequent site of metastasis4 that is often associated with a poor prognosis5. Using mouse models, we show that there is a selective increase in natural killer (NK) cells in the dormant milieu. Adjuvant interleukin-15-based immunotherapy ensures an abundant pool of NK cells that sustains dormancy through interferon-γ signalling, thereby preventing hepatic metastases and prolonging survival. Exit from dormancy follows a marked contraction of the NK cell compartment and the concurrent accumulation of activated hepatic stellate cells (aHSCs). Our proteomics studies on liver co-cultures implicate the aHSC-secreted chemokine CXCL12 in the induction of NK cell quiescence through its cognate receptor CXCR4. CXCL12 expression and aHSC abundance are closely correlated in patients with liver metastases. Our data identify the interplay between NK cells and aHSCs as a master switch of cancer dormancy, and suggest that therapies aimed at normalizing the NK cell pool might succeed in preventing metastatic outgrowth.

Intravenous thrombolysis for ischemic stroke in the Veneto region: the gap between eligibility and reality.

Intravenous thrombolysis (IVT) is the treatment of choice for most patients with acute ischemic stroke. According to the recently updated guidelines, IVT should be administered in absence of absolute exclusion criteria. We aimed to assess the proportion of ischemic strokes potentially eligible and actually treated with IVT, and to explore the reasons for not administering IVT. We prospectively collected and analyzed data from 1184 consecutive ischemic stroke patients admitted to the 22 Stroke Units (SUs) of the Veneto region from September 18th to December 10th 2017. Patients were treated with IVT according to the current Italian guidelines. For untreated patients, the reasons for not administering IVT were reported by each center in a predefined model including absolute and/or relative exclusion criteria and other possible reasons. Out of 841 (71%) patients who presented within 4.5 h of stroke onset, 704 (59%) had no other absolute exclusion criteria and were therefore potentially eligible for IVT according to the current guidelines. However, only 323 (27%) patients were eventually treated with IVT. Among 861 (73%) untreated patients, 480 had at least one absolute exclusion criterion, 283 only relative exclusion criteria, 56 only other reasons, and 42 a combination of relative exclusion criteria and other reasons. Our study showed that only 46% (323/704) of the potentially eligible patients were actually treated with IVT in the SUs of the Veneto region. All healthcare professionals involved in the acute stroke pathway should make an effort to bridge this gap between eligibility and reality.

Introduction of direct oral anticoagulant within 7 days of stroke onset: a nomogram to predict the probability of 3-month modified Rankin Scale score > 2.

In clinical practice, direct oral anticoagulants (DOACs) are often started earlier (≤ 7 days) than in randomized clinical trials after stroke. We aimed to develop a nomogram model incorporating time of DOAC introduction ≤ 7 days of stroke onset in combination with different degrees of stroke radiological/neurological severity at the time of treatment to predict the probability of unfavorable outcome. We conducted a multicenter prospective study including 344 patients who started DOAC 1-7 days after atrial fibrillation-related stroke onset. Computed tomography scan 24-36 h after stroke onset was performed in all patients before starting DOAC. Unfavorable outcome was defined as modified Rankin Scale (mRS) score > 2 at 3 months. Based on multivariate logistic model, the nomogram was generated. We assessed the discriminative performance by using the area under the receiver operating characteristic curve (AUC-ROC) and calibration of risk prediction model by using the Hosmer-Lemeshow test. Onset-to-treatment time for DOAC (OR: 1.21, p = 0.030), NIH Stroke Scale (NIHSS) score at the time of treatment (OR: 1.00 for NIHSS = 0-5; OR: 2.67, p = 0.016 for NIHSS = 6-9; OR: 26.70, p < 0.001 for NIHSS = 10-14; OR: 57.48, p < 0.001 for NIHSS ≥ 15), size infarct (OR: 1.00 for small infarct; OR: 2.26, p = 0.023 for medium infarct; OR: 3.40, p = 0.005 for large infarct), and age ≥ 80 years (OR: 1.96, p = 0.028) remained independent predictors of unfavorable outcome to compose the nomogram. The AUC-ROC of nomogram was 0.858. Calibration was good (p = 2.889 for the Hosmer-Lemeshow test). The combination of onset-to-treatment time of DOAC with stroke radiological/neurological severity at the time of treatment and old age may predict the probability of unfavorable outcome.