Targeting Galectin-3 with novel drugs for treatment of eczema

描述（由申请人提供）：湿疹影响广泛的人群年龄组，在过去三十年中，工业化国家的患病率增加了两到三倍，目前儿童的发病率估计为15- 20%。研究表明，湿疹是一种复杂的疾病，涉及多种免疫和炎症途径，包括T细胞，树突状细胞，肥大细胞和嗜酸性粒细胞，以及角质形成细胞。湿疹的治疗仍然是一个挑战，特别是在患有严重疾病的患者中。目前的治疗包括局部应用糖皮质激素和钙调磷酸酶抑制剂（免疫调节剂），以及全身性治疗糖皮质激素，免疫抑制剂和钙调磷酸酶抑制剂。由于长期使用糖皮质激素和免疫抑制剂的重大健康问题，局部免疫调节剂获得广泛接受。然而，FDA提出的安全性问题可能会限制这些免疫调节剂的使用。动物凝集素家族，称为半乳糖凝集素，是具有保守的碳水化合物识别结构域的β-半乳糖苷结合动物凝集素。它们在进化上是高度保守的（在线虫和哺乳动物中发现），一些显示出广泛的组织分布，而另一些则更有选择性地由组织表达。半乳糖凝集素没有信号序列，也没有跨膜结构域，存在于细胞质和细胞核中，但也存在于细胞外空间中。半乳糖凝集素通过蛋白质-蛋白质相互作用与其他蛋白质结合而在细胞内起作用。半乳糖凝集素-3的典型表达包括上皮细胞和免疫及炎性细胞。半乳糖凝集素-3是一种独特的嵌合结构，它由一个富含脯氨酸的重复序列和一个C端碳水化合物识别结构域组成。半乳糖凝集素-3通过其N-末端结构域与多价寡聚体结合形成寡聚体。通过对转基因小鼠和人体组织的广泛研究，我们已经确定半乳糖凝集素-3是参与皮肤炎症的关键分子。我们已经开始表征针对半乳糖凝集素-3治疗湿疹的药物。这些分子是一种新建立的，但不断增长的一类药物，称为适体（RNA寡核苷酸），并可能提供比传统疗法更好的安全性和特异性。公共卫生相关性：特应性皮炎是一种常见的慢性炎症性皮肤病。在美国，1980年以后出生的人口中有15-20%受到这种疾病的影响。特应性皮炎的治疗仍然是一个挑战，显然需要新的治疗方法。目前的模型表明，特应性皮炎是由于在急性期Th 2介导的炎症反应，T细胞和树突状细胞发挥重要作用。半乳糖凝集素-3是动物凝集素家族的一员，由其对2-半乳糖苷和共有氨基酸序列的亲和力定义。它由许多细胞类型表达，包括T细胞和树突状细胞。与其他半乳糖凝集素一样，半乳糖凝集素-3没有经典的信号序列，存在于细胞内，但可以分泌。已经通过使用外源添加的半乳糖凝集素-3证明了许多细胞外功能，其可以通过交联细胞表面聚糖来诱导跨膜信号转导。此外，大量证据表明内源性半乳糖凝集素-3通过细胞内作用（在不存在蛋白质分泌的情况下）参与各种细胞功能的调节。我们通过研究半乳糖凝集素-3-缺陷（gal 3-/-）小鼠获得了关于内源性半乳糖凝集素-3的功能的重要信息，并证明了半乳糖凝集素-3促进过敏性炎症。特别地，我们有数据证明半乳糖凝集素-3在人类特应性皮炎的小鼠模型中的关键作用：Gal 3-/-小鼠表现出Th 1-极化应答和显著减少的疾病。因此，我们认为半乳糖凝集素-3是治疗特应性皮炎以及其他过敏性疾病的靶标。在这里，我们建议开发半乳糖凝集素-3特异性RNA适体作为治疗特应性皮炎的新药。我们选择了这种技术，因为RNA适体与低分子量化合物相比，更可能对半乳糖凝集素-3具有更高的亲和力，并且对半乳糖凝集素-3的特异性更高。此外，RNA适体可用于靶向细胞外或细胞内半乳糖凝集素-3，并可局部应用。具体目标1：开发半乳糖凝集素-3特异性RNA适体作为有效抑制剂具体目标2：证明半乳糖凝集素-3适体在体外抑制半乳糖凝集素-3功能中的效力1.选择可在体外抑制半乳糖凝集素-3功能的半乳糖凝集素-3 RNA适体2.具体目的3：通过使用人特应性皮炎的小鼠模型1确定半乳糖凝集素-3适体用于治疗过敏性皮肤炎症的功效。确定靶向细胞外半乳糖凝集素-3是否有效抑制过敏性皮炎2。在小鼠模型中评价半乳糖凝集素-3 RNA适体的体内作用。我们希望拟议的研究将明确验证半乳糖凝集素-3作为治疗特应性皮炎的靶点，并导致开发这种疾病的新疗法。

DESCRIPTION (provided by applicant): Eczema affects a wide population age group, and prevalence of the disease has increased two- to three-fold during the past three decades in industrialized countries, where the current incidence in children is estimated to be 15-20%. Studies suggest that eczema is a complex disease, involving multiple immunologic and inflammatory pathways that include T cells, dendritic cells, mast cells and eosinophils, as well as keratinocytes. Treatment of eczema continues to be a challenge, especially in patients with severe disease. Current therapies include topical application of glucocorticoids and calcineurin inhibitors (immunomodulators), and systemic treatment with glucocorticoids, immunosuppressants and calcineurin inhibitors. Due to significant health concerns of long-term use of glucocorticoids and immunosuppressants, topical immunomodulators gained wide acceptance. However, safety issues raised by the FDA may limit use of these immunomodulators. A family of animal lectins, called galectins, is beta-galactoside-binding animal lectins with conserved carbohydrate-recognition domains. They are evolutionarily highly conserved (found in nematodes and mammals), and some show wide tissue distribution, while others are more selectively expressed by tissue. Galectins have no signal sequence and no transmembrane domain and are found in the cytosol and nucleus, but also in extracellular spaces. Galectins function intracellularly by binding to other proteins through protein-protein interactions. Typical expression of galectin- 3 includes epithelia and immune and inflammatory cells. Galectin-3 is unique among others in possessing chimeric structure consisting of an N-terminal region that contains proline-rich repeats and a C-terminal carbohydrate-recognition domain. Galectin-3 forms oligomers through its N-terminal domain upon binding to multivalent saccharides. Through extensive studies of transgenic mice and human tissues, we have identified galectin-3 as a key molecule involved in skin inflammation. We have begun to characterize drugs targeting galectin-3 for treatment of eczema. These molecules are a newly-established, but growing class of drugs called aptamers (RNA oligonucleotides), and may offer improved safety and specificity over traditional therapies. PUBLIC HEALTH RELEVANCE: Atopic dermatitis is a common chronic inflammatory skin disease. In the US, 15-20% of the population born after 1980 is affected by this disease. Treatment of atopic dermatitis continues to be a challenge and new therapies are clearly needed. The current model suggests that atopic dermatitis is attributable to a Th2-mediated inflammatory response in the acute phase, and both T cells and dendritic cells play important roles. Galectin-3 is a member of a family of animal lectins defined by their affinity for 2-galactosides and consensus amino acid sequences. It is expressed by a number of cell types, including T cells and dendritic cells. Like other galectins, galectin-3 does not have a classical signal sequence and is present inside the cells, but it can be secreted. A number of extracellular functions have been demonstrated by using exogenously added galectin-3, which can induce transmembrane signal transduction by crosslinking cell surface glycans. In addition, a great deal of evidence implicates endogenous galectin-3 in the regulation of various cellular functions through intracellular actions (in the absence of protein secretion). We have obtained important information on the functions of endogenous galectin-3 by studying galectin-3-deficient (gal3-/-) mice, and demonstrated that galectin-3 promotes allergic inflammation. In particular, we have data demonstrating a critical role for galectin-3 in a mouse model of human atopic dermatitis: Gal3-/- mice exhibit Th1-polarized responses and significantly reduced disease Thus, we believe that galectin-3 is a target for treatment of atopic dermatitis, as well as other allergic diseases. Here we propose developing galectin-3 specific RNA aptamers as novel drugs for treatment of atopic dermatitis. We have chosen this technology, because RNA aptamers are more likely to have higher affinity for galectin-3 and more specific for galectin-3 over other galectins than low molecular weight compounds. Moreover, RNA aptamers can be used to target either extracellular or intracellular galectin-3 and may be applied topically. Specific Aim 1: Develop galectin-3-specific RNA aptamers as potent inhibitors Specific Aim 2: Demonstrate the potency of galectin-3 aptamers in suppressing galectin-3 functions in vitro 1. Selection of galectin-3 RNA aptamers that can inhibit galectin-3 functions in vitro 2. Determine the potency of galectin-3 RNA aptamers in inhibition of human Th2 responses in vitro Specific Aim 3: Determine the efficacy of galectin-3 aptamers for treatment of allergic skin inflammation by using a mouse model of human atopic dermatitis 1. Determine whether targeting extracellular galectin-3 is effective in suppressing allergic dermatitis 2. Evaluate the in vivo effects of galectin-3 RNA aptamers in a mouse model. We hope the proposed studies will definitively validate galectin-3 as a target for treating atopic dermatitis and lead to the development of a novel therapy for this disease.