*4.4.1 Anti IL-1*

The IL-1 cytokine family in general and IL-1α and IL-1β, specifically have pro-inflammatory effects, which are neutralized by using the IL-1R antagonist. [59]. Several IL-1 mutations (NLRP3 genes) are collectively defined as auto-inflammatory syndromes, which cause the increased secretion of IL-1β. This is associated with a heterogeneous syndrome (NLRP3-AID (consisting of familial cold autoinflammatory syndrome, Muckle–Wells syndrome, and chronic infantile neurological, cutaneous and articular syndrome. The urticarial-like rash is one of most common hallmarks of these syndromes [60, 61]. IL-1 inhibitors, such as canakinumab (monoclonal antibody against of IL-1β), anakinra (recombinant IL-1R antagonist), and rilonacept (IL-1α/β blocker) are very effective in reducing inflammation and the clinical spectrum of these syndromes [62]. It is worth mentioning, that the emerging knowledge regarding the use of IL-1-blocking agents in the treatment of Schnitzler's syndrome, is characterized by the presence of urticarial rash and systemic inflammation [63, 64]. In on-going clinical trials, the effectiveness and safety of RPH-104 (a novel molecule against IL-1β), rilonacept, and canakinumab has been confirmed in Schnitzler syndrome (NCT04213274), acquired cold-contact urticaria (NCT02171416), and CSU (NCT01635127). The results of these trials have not yet been published. In few sporadic reports, anti-IL-1drugs were shown to

be beneficial in CSU patients, who remained resistant to all classical therapies for CSU [65]. A new somatic mutation in NLRP3 was recently reported in two elderly patients with long-standing, refractory CSU associated with fever and increased CRP. Both of these patients improved dramatically following the usage of anakinra. As a result, it is assumed assumed that in patients with refractory urticaria and markers of systemic inflammation (a possible underlying NLRP3-related disorder), anti-IL-1 treatment requires further evaluation [66].

#### *4.4.2 Anti-IL-4/13*

In the process of Th2 differentiation several cytokines are produced. The most important cytokines in this process are interleukin-4 and IL-13 [59]. Dupilumab, a new monoclonal antibody directed against the alpha subunit of IL-4 and IL-13 receptors, was recently approved for the treatment of asthma, nasal polyposis, and atopic dermatitis [67]. Increased levels of IL-4 were recently demonstrated in patients with CSU, thereby suggesting a pathogenic role of both Th1/Th2 responses and raising the option of treating CSU with Dupilumab [68]. A recent case report involving six patients with concomitant atopic dermatitis and CSU who were refractory to high dose of omalizumab (600 mg\4 weeks) documented their successful treated with Dupilumab. In this report, it was postulated that the beneficial therapeutic effect of Dupilumab could be the result of its blocking Th2 inflammatory pathways by inhibiting IL-4 and IL-13, respective [69]. Currently, there are three ongoing, phase II/III clinical studies investigating the efficacy and safety of Dupilumab in CSU (NCT03749135, NCT04180488 (EFC16461-CUPID)) and cholinergic urticaria (NCT03749148) unresponsive to a high dosage of antihistamines and omalizumab.

#### *4.4.3 Anti IL-5*

Eosinophils, are considered to have a pivotal role in the pathogenesis of CSU. Many reports have demonstrated elevated numbers of eosinophils in urticarial lesions when compared with normal skin. Their contribution to CSU pathogenesis is probably achieved through interactions with mast cells, the secretion of histamine and other inflammatory mediators and the activation of the coagulation cascade [70]. The important role of interleukin-5 (IL-5) in eosinophil development and maturation, as well as in increased chemotaxis towards skin urticarial lesions has been well documented [59]. Several monoclonal antibodies were recently approved for the treatment of eosinophil related airway diseases (e.g. asthma, Churg-Strauss syndrome, nasal polyposis etc.) by targeting IL-5 (reslizumab, mepolizumab) or its receptor, IL-5R (benralizumab). These drugs were recently used in three CSU patients who were refractory to classical therapies; two patients responded well and showed a significant improvement with Reslizumab and mepolizumab [71, 72], while the other patient who suffered from symptomatic dermographism (SD) benefited from their treatment with benralizumab [73]. In a recent single-blind, repeated measures study, 12 CSU patients were treated with benralizumab (30 mg subcutaneously) every 4 weeks for 12 weeks following a single dose of a placebo. Among the nine patients who completed the study, five had complete response. Their UAS7 and CU-Q2oL scores improved significantly with benralizumab when compared with the placebo [74]. Gene-expression analysis in patients with CSU following benralizumab treatment demonstrated the normalization of SIGLEC-8 expression and IL-4/5 induced inflammation [75]. Although the results imply that eosinophils play a role in CSU, the exact mechanism of action has not yet been understood. Two clinical trials investigating the efficacy of benralizumab

(NCT03183024) and mepolizumab (NCT03494881) in CSU are still in progress, and their results are not yet available. Regarding benralizumab, a phase IIb study (ARROYO Trial- D3259C00001) is set to start soon.
