PROOF OF CONCEPT FOR ANTIMICROBIAL PROPERTIES OF LACRITIN IN VIVO

描述(申请人提供)：在过去的60年里，抗生素在细菌感染的治疗和预防中都是一个关键组成部分。从那时起，为了限制毒性，推广更广泛的光谱治疗，增加使用的便利性，并缩短感染清除的时间，不断开发新的抗生素是必要的。发现新抗生素的另一个驱动力是微生物耐药性的发展和抗生素覆盖面差距的扩大。这一点最容易从第二代和第三代氟喹诺酮类药物耐药性的出现中看出，这需要随后开发和应用第四代氟喹诺酮类药物。为了保持耐药性的领先地位，制药业修改了现有的抗生素，并开发了新类型和新类别的抗生素制剂。蛋白质抗菌剂和激动剂分别代表了治疗眼表感染和病理生理学的新途径。Lacritin是一种新的人类泪液蛋白，由泪腺和唾液腺以及眼表上皮细胞产生，具有包裹这两个区域的多种功能。在低微摩尔浓度下，对革兰氏阴性菌和革兰氏阳性菌均有杀菌作用。此外，在低纳摩尔浓度下，它促进MUC16的产生、细胞增殖和对炎性细胞因子的保护。泪蛋白的发现源于对泪液分泌功能的细胞外介质的筛选。它是一种12.3 kDa的糖蛋白，集中在泪液分泌颗粒中，有13个已知的同源物。除了泪腺功能单位和唾液腺(可能还有甲状腺)以外，没有其他器官正常表达催乳素。泪腺中表达最强。因此，Lacritin几乎可以被视为泪腺功能单位的唯一生长因子。泪腺功能单位是一个完整的系统，包括泪腺、眼表(角膜、结膜和眼眉腺)和眼盖，以及连接它们的感觉神经和运动神经“(2007年国际干眼研讨会，LEMP，2007年)。我们的方法：基于体外阐明的这些抗菌特性，我们假设lacritin具有作为抗菌剂的巨大潜力，局部应用lacritin将增强泪液中的天然抗菌活性。作为一种眼部特异的视网膜前糖蛋白，lacritin具有独特的作用机制。然而，天然糖蛋白lacritin可能不是最合适的形式。我们已经准备了几种在制造简单性、稳定性或有效性方面可能更适合的催乳素结构，并选择了N-65催乳素用于这些概念研究的证明。这一第一阶段提案的长期目标具体目标包括：1.在兔角膜炎模型中证明N-65 lacritin可以减少已建立的铜绿假单胞菌和/或金黄色葡萄球菌感染。2.证明N-65半乳糖可以作为外用滴眼液的抗菌防腐剂。在我们的两个概念验证标准中，N-65 lacritin可以被认为是成功的。我们从这些实验中获得的结果将指导我们确定商业开发的范围和第二阶段提案的具体目标。 公共卫生相关性：抗生素滴剂通常用于预防和治疗可能导致失明的严重眼部感染。虽然目前的抗生素是有效的，但开发新的药剂是必要的，以确保在耐药性发展并使当今的抗生素无效时有安全有效的治疗方法。我们的研究代表了一种治疗眼睛感染的新方法，它将评估人类眼泪中一种新蛋白质的抗生素特性，该蛋白质在导致严重眼睛感染的细菌的体外实验中显示出非常有希望的结果。

DESCRIPTION (provided by applicant): Over the past sixty years, antibiotics have comprised a key component in both the treatment and prophylaxis of bacterial infections. Since that time the continuous development of new antibiotics has been necessary to limit toxicity, promote broader spectrum therapy, increase convenience of use, and decrease the time to clear the infection. Another driving force in the discovery of new antibiotic agents is the development of microbial resistance and widening gaps in antibiotic coverage. This can most readily be seen in the emergence of antimicrobial resistance to second and third generation fluoroquinolones requiring the subsequent development and application of fourth generation fluoroquinolones. In an effort to stay ahead of resistance, the pharmaceutical industry modifies existing antibiotics and develops new types and classes of antibiotic agents. Protein antimicrobials and agonists respectively represent a new therapeutic approach to ocular surface infection and pathophysiology. Lacritin, a novel human tear protein produced by lacrimal and salivary glands and by epithelial cells of the ocular surface, has multiple functions that encapsulate both areas. It is bacteriocidal for both gram negative and gram positive bacteria at low micromolar concentrations. Also, at low nanomolar concentrations it promotes MUC16 production, cell proliferation and protection against inflammatory cytokines. The discovery of lacritin emerged from a screen for extracellular mediators of lacrimal secretory function. It is a 12.3 kDa glycoprotein, concentrated in lacrimal secretory granules, and has 13 known orthologues. No other organs, beyond the lacrimal functional unit and salivary gland (and possibly the thyroid gland) normally express lacritin. Expression is strongest in lacrimal gland. Lacritin can thus be viewed as an almost exclusive growth factor of the lacrimal functional unit. The lacrimal functional unit is an integrated system comprising the lacrimal glands, ocular surface (cornea, conjunctiva and meibomian glands) and lids, and the sensory and motor nerves that connects them' (2007 International Dry Eye Workshop, Lemp, 2007). Our approach: Based upon these antimicrobial properties elucidated in vitro, we hypothesize that lacritin possesses significant potential as an antimicrobial agent and that topical application of lacritin will augment the native antimicrobial activity in tears. As an ocular-specific presecretory glycoprotein, lacritin has a unique mechanism of action. However, the native glycoprotein, lacritin, may not be the most suitable form. We have prepared several lacritin constructs which may be more suitable with regard to ease of manufacture, stability or efficacy and have selected N-65 lacritin for these proofs of concept studies. The long-term goals Specific Aims for this Phase I proposal include: 1. A demonstration that N-65 lacritin can reduce established Pseudomonas aeruginosa and/or Staphylococcus aureus infections in a rabbit keratitis model. 2. A demonstration that N-65 lacritin can serve as an antimicrobial preservative in topical eye drops. N-65 lacritin may be deemed successful in either or both of our criteria for proof of concept. Our results from these experiments will guide us in defining the scope of commercial development and specific aims for a Phase II proposal. PUBLIC HEALTH RELEVANCE: Antibiotic drops are commonly used to prevent and treat serious eye infections that may result in loss of vision. Although current antibiotics are effective, development of new agents is necessary to ensure that safe and effective treatments are available if resistance develops and renders today's antibiotics ineffective. Our research represents a new therapeutic approach to ocular infection that will evaluate the antibiotic properties of a novel protein in human tears which shows very promising in vitro results with bacteria that cause serious eye infections.