Development of topical rapamycin as a novel treatment for skin disorders

描述（由申请人提供）：自从在皮肤疾病单纯大疱性表皮松解症（EBS）中发现角蛋白5 （K5）和K14基因的潜在突变以来的十五年里，已经有大量的额外研究确定了许多上皮易碎性疾病的遗传基础，54个人类角蛋白基因中的20个现在与遗传疾病有关，包括皮肤和指甲疾病先天性厚甲癣（PC）。尽管有这些重大发现，但在开发这些遗传性皮肤病的治疗方法方面进展甚微。我们最近发现，诱导性角蛋白（K6a、K6b、K16和K17）的任何一种突变都可能导致PC，它们的5'非翻译区含有调控基元，这可能使它们在大环内酯类雷帕霉素（西鲁莫司）治疗后容易下调。事实上，经western blot分析，用雷帕霉素处理人HaCaT角质形成细胞可导致K6a表达降低90%。一项针对三名PC患者的小型雷帕霉素标签外试验显示，当达到治疗性雷帕霉素血槽水平时，PC症状显著减轻，足底疼痛的神经血管结构显著缓解。不幸的是，这些患者受到全身给药雷帕霉素引起的众所周知的副作用的影响，两名患者因此过早退出研究。本提案的目的是确定角化细胞中雷帕霉素治疗后角蛋白基因表达下调的途径，并开发雷帕霉素前药偶联物（包括皮肤穿透雷帕霉素衍生物），可以局部高浓度局部应用，从而避免全身治疗带来的不良副作用。斯坦福大学（和Cellgate）的Paul Wender博士及其同事成功地将环孢素（一种不穿透皮肤的药物）转化为环孢素转运蛋白偶联物，利用低精氨酸或脂质转运蛋白与释放游离药物的连接体，容易穿透人体皮肤的多层，这方面的丰富经验将被纳入雷帕霉素偶联物的设计中。局部递送雷帕霉素衍生物以下调诱导角蛋白表达的能力可能具有更广泛的应用，因为雷帕霉素已被证明对牛皮癣患者有益。尽管对大量遗传性皮肤病的潜在基因和突变有了令人兴奋的发现，但很少有新的临床治疗方法出现。本提案的目的是利用最近的发现，雷帕霉素选择性地抑制角蛋白的表达参与皮肤疾病先天性肿甲。在第一阶段，我们验证了参与角蛋白mRNA翻译的雷帕霉素敏感通路，并制备了含有皮肤穿透转运蛋白元件的雷帕霉素前药。在第二阶段，我们利用动物模型优化配方，局部递送雷帕霉素和雷帕霉素偶联物，为临床试验做准备。理论上，开发的平台技术应该适用于其他皮肤疾病，包括由雷帕霉素敏感角蛋白过度或不适当表达引起的牛皮癣。

DESCRIPTION (provided by applicant): In the decade and a half since the discovery of the underlying mutations in the keratin 5 (K5) and K14 genes in the skin disorder epidermolysis bullosa simplex (EBS), there has been a plethora of additional research identifying the genetic bases of many epithelial fragility disorders with 20 of the 54 human keratin genes now linked to genetic diseases, including the skin and nail disorder pachyonychia congenita (PC). Despite these significant discoveries, little progress has been made in developing therapeutics for these genodermatoses. We have recently discovered that the inducible keratins (K6a, K6b, K16, and K17), mutation in any of which can result in PC, contain regulatory motifs in their 5' untranslated regions that may make them susceptible to downregulation following treatment with the macrolide rapamycin (sirulimus). Indeed, treatment of human HaCaT keratinocytes with rapamycin led to a 90% decrease in K6a expression as determined by western blot analysis. A small rapamycin off-label trial in three PC patients showed marked reduction in PC symptoms when therapeutic rapamycin blood trough levels were achieved, with striking resolution observed for painful neurovascular structures on the soles of the feet. These patients, unfortunately, were affected by the well-known side effects arising from systemic administration of rapamycin and two patients withdrew prematurely from the study for these reasons. The purpose of this proposal is to define the pathway involved in downregulation of keratin gene expression following rapamycin treatment in keratinocytes and to develop rapamycin prodrug conjugates (including skin penetrating rapamycin derivatives) that can be applied topically at high local concentrations, thereby avoiding the undesirable side effects resulting from systemic treatment. The wealth of experience achieved by Dr. Paul Wender and colleagues at Stanford University (and Cellgate) in successfully converting cyclosporine, an agent that does not penetrate skin, into cyclosporine transporter conjugates that readily penetrate multiple layers of human skin using oligoarginine or lipid transporters with linkers that release free drug will be incorporated into the rapamycin-conjugate design. The ability to topically deliver rapamycin derivatives to downregulate expression of the inducible keratins may have much broader application, as rapamycin has been shown to be of benefit to patients suffering from psoriasis. PUBLIC HEALTH RELEVANCE Despite the exciting discoveries of the underlying genes and mutations responsible for a large number of genodermatoses, few if any novel clinical treatments have emerged. The purpose of this proposal is to exploit the recent discovery that rapamycin selectively inhibits expression of keratins involved in the skin disorder pachyonychia congenita. In Phase 1, we validate the rapamycin-sensitive pathway involved in keratin mRNA translation and prepare rapamycin prodrugs with skin-penetrating transporter elements and in Phase 2, we utilize animal models to optimize formulations to topically delivery rapamycin and rapamycin conjugates in preparation for a clinical trial. In theory, the platform technology developed should be applicable to other skin disorders including psoriasis caused by over- or inappropriate expression of rapamycin-sensitive keratins.