PROOF OF CONCEPT FOR ANTIMICROBIAL PROPERTIES OF LACRITIN IN VIVO

描述（由申请人提供）：在过去的六十年中，抗生素已成为治疗和预防细菌感染的关键组成部分。从那时起，新抗生素的不断开发对于限制毒性、促进更广谱的治疗、增加使用的便利性和减少清除感染的时间是必要的。发现新抗生素制剂的另一个驱动力是微生物耐药性的发展和抗生素覆盖范围的扩大。这一点最容易从对第二代和第三代氟喹诺酮类药物的耐药性的出现中看出，需要后续开发和应用第四代氟喹诺酮类药物。为了保持领先的耐药性，制药行业修改现有的抗生素，并开发新类型和类别的抗生素制剂。蛋白质抗菌剂和激动剂分别代表了眼表感染和病理生理学的新治疗方法。由泪腺和唾液腺以及眼表上皮细胞产生的新型人类泪液蛋白Lacritin具有包裹这两个区域的多种功能。它在低微摩尔浓度下对革兰氏阴性菌和革兰氏阳性菌均具有杀菌作用。此外，在低纳摩尔浓度下，它促进MUC 16的产生、细胞增殖和对炎性细胞因子的保护。催泪素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，集中在泪腺分泌颗粒中，有13个已知的直向同源物。除泪腺功能单位和唾液腺（可能还有甲状腺）外，没有其他器官正常表达催乳素。在泪腺中表达最强。因此，泪蛋白可被视为泪腺功能单位的几乎唯一的生长因子。泪腺功能单位是一个综合系统，包括泪腺、眼表（角膜、结膜和睑板腺）和眼睑，以及连接它们的感觉和运动神经（2007 International Dry Eye Workshop，Lemp，2007）。我们的方法：基于这些在体外阐明的抗菌特性，我们假设，催泪素具有显着的潜力，作为一种抗菌剂，局部应用催泪素将增加天然的抗微生物活性的眼泪。作为眼特异性分泌前糖蛋白，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。我们已经制备了几种在易于制造、稳定性或功效方面可能更合适的催泪蛋白构建体，并且已经选择N-65催泪蛋白用于这些概念验证研究。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催乳素可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 lacritin可作为局部滴眼液中的抗菌防腐剂。N-65 lacritin在我们的概念验证标准中的任一项或两项中均被视为成功。这些实验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视力丧失的严重眼部感染。虽然目前的抗生素是有效的，但如果耐药性发展并使今天的抗生素无效，则有必要开发新的药物以确保安全有效的治疗。我们的研究代表了一种新的眼部感染治疗方法，该方法将评估人类泪液中一种新型蛋白质的抗生素特性，该蛋白质在导致严重眼部感染的细菌中显示出非常有希望的体外结果。

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.