PROOF OF CONCEPT FOR ANTIMICROBIAL PROPERTIES OF LACRITIN IN VIVO

LACRITIN 体内抗菌特性的概念验证

• 批准号: 7800684
• 负责人: JOHN D SHEPPARD
• 金额: $ 12.88万
• 依托单位: EYERX RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7800684
• 关键词: Agonist; Animal Model; Antibiotics; Antimicrobial Resistance; Area; Bacteria; Bacterial Infections; Blindness; Cell Proliferation; Clinical Trials; Cornea; Defensins; Development; Dose; Drops; Drug Industry; Educational workshop; Encapsulated; Ensure; Epithelial Cells; Eye; Eye Infections; Eyedrops; Fluoroquinolones; Functional disorder; Generations; Glycoproteins; Goals; Gram-Positive Bacteria; Growth Factor; Human; Hyperemia; In Vitro; Infection; Inflammatory; International; Iris; Keratitis; Lacrimal gland structure; Light; Mediator of activation protein; Modeling; Motor; Natural Immunity; Nerve; New Agents; Organ; Oryctolagus cuniculus; Phase; Production; Property; Prophylactic treatment; Proteins; Pseudomonas aeruginosa; Reaction; Reflex action; Research; Resistance; Resistance development; Salivary Glands; Secretory Vesicles; Sensory; Staphylococcus aureus; Surface; System; Thyroid Gland; Time; Tissues; Topical application; Toxic effect; Variant; Vascularization; Vertebrates; anterior chamber; antimicrobial; antimicrobial drug; antimicrobial peptide; aqueous; bactericide; base; cathelicidin; conjunctiva; cytokine; driving force; effective therapy; extracellular; eye dryness; in vivo; irritation; lacrimal; meibomian gland; microbial; novel; novel therapeutic intervention; ocular surface; prevent; public health relevance; research study; synthetic peptide; tear proteins

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.

描述（由申请人提供）：在过去的六十年里，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，有必要不断开发新的抗生素来限制毒性、推广更广谱的治疗、增加使用便利性并减少清除感染的时间。发现新抗生素药物的另一个驱动力是微生物耐药性的发展和抗生素覆盖率差距的扩大。这可以从第二代和第三代氟喹诺酮类抗生素耐药性的出现中最容易看出，这需要第四代氟喹诺酮类药物的后续开发和应用。为了保持领先于耐药性，制药行业改进现有抗生素并开发新类型和类别的抗生素药物。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。泪素是一种新型人类泪液蛋白，由泪腺、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。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，它还能促进 MUC16 的产生、细胞增殖和针对炎症细胞因子的保护。泪泌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 糖蛋白，集中在泪腺分泌颗粒中，具有 13 个已知的直系同源物。除了泪腺功能单位和唾液腺（可能还有甲状腺）之外，没有其他器官通常表达泪泌素。泪腺中的表达最强。因此，泪素可以被视为泪腺功能单位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、眼表面（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 年国际干眼研讨会，Lemp，2007 年）。我们的方法：根据体外阐明的这些抗菌特性，我们假设泪蛋白具有作为抗菌剂的巨大潜力，并且局部应用泪蛋白将增强泪液中的天然抗菌活性。作为一种眼部特异性的分泌前糖蛋白，乳泌素具有独特的作用机制。然而，天然糖蛋白，乳泌素，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适合的lacritin构建体，并选择了N-65lacritin用于这些概念验证研究。该第一阶段提案的长期目标具体目标包括： 1. 证明 N-65 泪泌素可以减少兔角膜炎模型中已形成的铜绿假单胞菌和/或金黄色葡萄球菌感染。 2. 证明 N-65 乳泌素可以在外用滴眼液中用作抗菌防腐剂。 N-65 催乳素可能被认为在我们的概念验证标准中的一个或两个方面是成功的。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。尽管目前的抗生素是有效的，但有必要开发新的药物，以确保在耐药性出现并使当今的抗生素失效时可以进行安全有效的治疗。我们的研究代表了一种治疗眼部感染的新方法，该方法将评估人类泪液中一种新型蛋白质的抗生素特性，该蛋白质在体外对引起严重眼部感染的细菌显示出非常有希望的结果。