WO2023160499A1 - Peptide, ophthalmic pharmaceutical composition containing same and use thereof - Google Patents

Abstract

The present disclosure relates to a peptide, an ophthalmic pharmaceutical composition containing same, the use of the peptide or the ophthalmic pharmaceutical composition in the manufacture of a drug for treating, alleviating or ameliorating ocular surface diseases and/or conditions associated with corneal neovascularization, the use of the peptide or the ophthalmic pharmaceutical composition in the manufacture of a drug for treating, alleviating or ameliorating ocular surface diseases and/or conditions associated with corneal epithelial injury, and the use of the peptide or the ophthalmic pharmaceutical composition in the manufacture of a drug for treating, alleviating or ameliorating ocular surface diseases and/or conditions associated with dry eyes.

Description

一种肽、含有所述肽的眼用药物组合物及其用途Peptide, ophthalmic pharmaceutical composition containing said peptide and use thereof 技术领域technical field

本公开涉及生物技术和药学领域，具体涉及一种肽；包含所述肽的眼用药物组合物；所述肽或眼用药物组合物在制造用于治疗、缓解或改善与角膜新生血管相关的眼表疾病和/或病症的药物中的用途；所述肽或眼用药物组合物在制造用于治疗、缓解或改善与角膜上皮损伤相关的眼表疾病和/或病症的药物中的用途；以及所述肽或眼用药物组合物在制造用于治疗、缓解或改善与干眼相关的眼表疾病和/或病症的药物中的用途。The present disclosure relates to the fields of biotechnology and pharmacy, and specifically relates to a peptide; an ophthalmic pharmaceutical composition comprising the peptide; Use in medicine for ocular surface diseases and/or disorders; use of the peptide or ophthalmic pharmaceutical composition in the manufacture of medicines for treating, alleviating or improving ocular surface diseases and/or disorders associated with corneal epithelial damage; And the use of the peptide or ophthalmic pharmaceutical composition in the manufacture of medicines for treating, alleviating or improving ocular surface diseases and/or conditions associated with dry eye.

背景技术Background technique

早在1984年Nelson就提出了眼表疾病的概念。从解剖学上说，眼表包括上下睑缘间的整个黏膜上皮衬里。从组织学上讲，这一上皮层覆盖了两个主要的区域，即角膜和结膜。眼表的主要功能是保证睁眼状态下的清晰视觉。它包括角膜、结膜上皮和泪膜。这三者关系紧密，相互影响。任何一者的改变都将导致眼表的不稳定。因此，从狭义的角度，眼表疾病仅指角、结膜上皮的疾病。而从相对广义的角度说，眼表疾病应包括角结膜浅层疾病和泪膜功能异常导致的疾病。As early as 1984, Nelson proposed the concept of ocular surface disease. Anatomically, the ocular surface includes the entire mucosal epithelial lining between the upper and lower eyelid margins. Histologically, this epithelial layer covers two main areas, the cornea and the conjunctiva. The main function of the eye surface is to ensure clear vision when the eyes are open. It includes the cornea, conjunctival epithelium and tear film. These three are closely related and influence each other. Changes in either will lead to instability of the ocular surface. Therefore, from a narrow perspective, ocular surface diseases only refer to diseases of the horn and conjunctival epithelium. From a relatively broad perspective, ocular surface diseases should include superficial corneal and conjunctival diseases and diseases caused by abnormal tear film function.

角膜是眼睛的窗户，它的透明及无血管状态是保持最佳视觉所必需的生理基础。化学伤、机械伤、感染等因素会导致角膜发生病理性改变，毛细血管从角膜缘入侵进而形成角膜新生血管(CNV)，降低角膜透明度并影响视力。全世界每年约有140万角膜疾病患者发生新生血管，其中约12％的患者丧失视力。由于其发病人数庞大，而且引起失明，CNV一直是全世界角膜病领域研究的热点，也是急需解决的重大疾病之一。目前，角膜新生血管的治疗包括局部滴用糖皮质激素和非甾体类抗炎药物，对新生血管进行电凝、激光光凝和光动力疗法等手术治疗，绷带镜、湿室镜、泪点栓塞和眼睑闭合等器械治疗，但这些治疗方法临床效果不甚理想。因此，亟需对角膜新生血管有良好治疗效果且毒副作用较小或无毒副作用的治疗手段，尤其是局部应用药物。The cornea is the window of the eye, and its transparent, avascular state is the physiological basis necessary for optimal vision. Chemical injury, mechanical injury, infection and other factors can lead to pathological changes in the cornea. Capillaries invade from the limbus to form corneal neovascularization (CNV), reducing corneal transparency and affecting vision. About 1.4 million patients with corneal diseases develop new blood vessels every year in the world, and about 12% of them lose their vision. Because of its large number of patients and its blindness, CNV has always been a research hotspot in the field of corneal diseases all over the world, and it is also one of the major diseases that need to be solved urgently. At present, the treatment of corneal neovascularization includes local dripping of glucocorticoids and non-steroidal anti-inflammatory drugs, electrocoagulation, laser photocoagulation, and photodynamic therapy for neovascularization, bandage endoscopy, wet room endoscopy, and punctal embolization. And eyelid closure and other device treatment, but the clinical effect of these treatment methods is not satisfactory. Therefore, there is an urgent need for a therapeutic method with good therapeutic effect on corneal neovascularization and less or no toxic side effects, especially local application of drugs.

角膜上皮缺损是一种常见的眼科疾病，其是由各种因素导致的角膜上皮屏障功能与完整性被破坏，引起角膜上皮细胞层部分或全层缺失的病理状态。角膜上皮缺损可表现为角膜上皮弥漫性点状脱落或糜烂，角膜上皮反复脱落和缺损，伴有不同程度的眼表炎症反应。严重者可导致角膜基质病变，影响视功能。现有的治疗包括润滑液、眼膏、绷 带镜、羊膜、自体血清滴眼液以及角膜移植。这些治疗存在各种问题，并且角膜上皮不能完全愈合。理想的治疗是局部应用药物促进上皮增长或迁移从而加速角膜愈合。Corneal epithelial defect is a common ophthalmic disease, which is a pathological state in which the function and integrity of the corneal epithelial barrier are destroyed due to various factors, resulting in partial or full-thickness loss of the corneal epithelial cell layer. Corneal epithelial defects can manifest as diffuse punctate detachment or erosion of the corneal epithelium, repeated detachment and defect of the corneal epithelium, accompanied by varying degrees of ocular surface inflammation. Severe cases can lead to corneal stromal lesions and affect visual function. Available treatments include lubricating fluids, eye ointments, bandages Glasses, amniotic membrane, autologous serum eye drops, and corneal transplant. There are various problems with these treatments, and the corneal epithelium does not fully heal. The ideal treatment is a topical drug that promotes epithelial growth or migration to accelerate corneal healing.

干眼是眼科门诊最常见的疾病，其主要特征是泪膜不稳定、眼表发炎、眼部不适和/或由于泪液或眼表异常或对眼睛表面的伤害。干眼的症状包括干燥、异物感、发红、瘙痒、烧灼感、眼痛、视力模糊、畏光和/或流泪。干眼会影响阅读、使用电脑、看电视、驾驶等日常活动，并造成长期眼部不适，严重影响生活质量。严重的干眼可引起角膜炎、角膜新生血管、角膜溃疡和视力障碍，从而导致失明。改变生活方式可治疗干眼(例如，减少对电子屏幕的接触、生活环境的改变等)。然而，改变生活方式而不影响其他领域通常是不切实际或不可能的。药物治疗可包括润滑组合物(例如人工泪液等)治疗、抗炎(例如糖皮质激素、非甾体抗炎药(NSAID)、免疫抑制剂如环孢菌素A等)治疗和抗菌(例如局部治疗和全身抗生素)治疗。然而，由于干眼的病理生理机制复杂，对机制缺乏明确认识，这些治疗方法存在疗效差、应答者比例有限、起效慢、副作用显著、可行性低等诸多局限性。Dry eye is the most common condition seen in ophthalmology clinics and is characterized by instability of the tear film, ocular surface inflammation, ocular discomfort and/or damage to the ocular surface due to tear fluid or ocular surface abnormalities. Symptoms of dry eye include dryness, foreign body sensation, redness, itching, burning, eye pain, blurred vision, photophobia, and/or tearing. Dry eye can affect daily activities such as reading, using computers, watching TV, driving, etc., and cause long-term eye discomfort, seriously affecting the quality of life. Severe dry eye can cause keratitis, corneal neovascularization, corneal ulcers, and visual impairment, leading to blindness. Dry eye can be treated with lifestyle changes (eg, reduced exposure to electronic screens, changes in living environment, etc.). However, it is often impractical or impossible to make lifestyle changes without affecting other areas. Drug therapy may include treatment with lubricating compositions (e.g., artificial tears, etc.), anti-inflammatory (e.g., glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs), immunosuppressants such as cyclosporine A, etc.) therapy, and antibacterial (e.g., topical treatment and systemic antibiotics). However, due to the complex pathophysiological mechanism of dry eye and the lack of clear understanding of the mechanism, these treatments have many limitations such as poor efficacy, limited proportion of responders, slow onset, significant side effects, and low feasibility.

综上，对于角膜新生血管、角膜上皮缺损、干眼迫切需要开发一种治疗效果好、可长期使用、无明显局部或全身毒副作用的眼科治疗药物。In summary, for corneal neovascularization, corneal epithelial defect, and dry eye, there is an urgent need to develop an ophthalmic drug with good therapeutic effect, long-term use, and no obvious local or systemic side effects.

发明内容Contents of the invention

为了解决上述技术问题，发明人进行了深入的研究，终于发现了解决的技术方案，具体地，本公开提供了一种下式所代表的肽，In order to solve the above technical problems, the inventors conducted in-depth research and finally found a technical solution. Specifically, the present disclosure provides a peptide represented by the following formula,

Xaa-Xaa-Xaa-Xaa-Gln，Xaa-Xaa-Xaa-Xaa-Gln,

其中，in,

第1-3位的Xaa彼此独立地不存在或代表Ala、Gln、Gly、Glu、Tyr、Leu、Ser、Met或Pro；且Xaa at positions 1-3 are independently absent or represent Ala, Gln, Gly, Glu, Tyr, Leu, Ser, Met or Pro; and

第4位的Xaa代表Ala、Gln、Gly、Ser、Thr、Pro或Glu。Xaa at the 4th position represents Ala, Gln, Gly, Ser, Thr, Pro or Glu.

本公开还提供了一种眼用药物组合物，其包含本公开的肽，以及眼科可接受的赋形剂。 The present disclosure also provides an ophthalmic pharmaceutical composition comprising the peptide of the present disclosure, and an ophthalmologically acceptable excipient.

本公开还提供了本公开的肽或本公开的眼用药物组合物在制造用于治疗、缓解或改善与角膜新生血管相关的眼表疾病和/或病症的药物中的用途。The present disclosure also provides the use of the peptide of the present disclosure or the ophthalmic pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating, alleviating or improving ocular surface diseases and/or disorders associated with corneal neovascularization.

本公开还提供了本公开的肽或本公开的眼用药物组合物在制造用于治疗、缓解或改善与角膜上皮损伤相关的眼表疾病和/或病症的药物中的用途。The present disclosure also provides the use of the peptide of the present disclosure or the ophthalmic pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating, alleviating or improving ocular surface diseases and/or disorders associated with corneal epithelial damage.

本公开还提供了本公开的肽或本公开的眼用药物组合物在制造用于治疗、缓解或改善与干眼相关的眼表疾病和/或病症的药物中的用途。The present disclosure also provides the use of the peptide of the present disclosure or the ophthalmic pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating, alleviating or improving ocular surface diseases and/or conditions associated with dry eye.

附图说明Description of drawings

以下提供附图的简要说明，其用于说明本文所公开的示例性实施方式，而不是为了限制这些实施方式。A brief description of the accompanying drawings is provided below to illustrate exemplary embodiments disclosed herein and not to limit these embodiments.

图1示出了本公开的滴眼液对小鼠角膜新生血管的影响。Figure 1 shows the effect of eye drops of the present disclosure on corneal neovascularization in mice.

图2和图3示出了本公开的滴眼液对小鼠角膜上皮损伤的影响。Figures 2 and 3 show the effect of eye drops of the present disclosure on corneal epithelial damage in mice.

图4示出了本公开的滴眼液对干眼小鼠模型泪液分泌量的影响。Fig. 4 shows the effect of the eye drops of the present disclosure on the amount of tear secretion in a mouse model of dry eye.

图5示出了本公开的滴眼液对干眼小鼠模型抗炎作用的影响。Figure 5 shows the effect of the eye drops of the present disclosure on the anti-inflammatory effect of a mouse model of dry eye.

图6示出了本公开的滴眼液对干眼小鼠模型角膜上皮屏障功能的影响。Fig. 6 shows the effect of the eye drops of the present disclosure on the corneal epithelial barrier function of a mouse model of dry eye.

图7示出了本公开的滴眼液对干眼小鼠模型结膜杯状细胞的影响。Figure 7 shows the effect of eye drops of the present disclosure on conjunctival goblet cells in a dry eye mouse model.

具体实施方式Detailed ways

除非另有定义，否则本文使用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常理解的相同的含义。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

在本文中使用时，表述“A和/或B”包括3种情况：(1)A；(2)B；以及(3)A和B。As used herein, the expression "A and/or B" includes 3 cases: (1) A; (2) B; and (3) A and B.

在本文中使用时，除非另有说明或与上下文相矛盾，否则提及肽、二肽、三肽、四肽、五肽时均涵盖这些肽本身及其药学上可接受的盐。As used herein, unless otherwise stated or contradicted by context, references to peptides, dipeptides, tripeptides, tetrapeptides, pentapeptides encompass these peptides themselves and their pharmaceutically acceptable salts.

在本文中使用时，表述“药学上可接受的盐”包括与药学上可接受的酸形成的酸加成盐，以及与药学上可接受的碱形成的盐。所述药学上可接受的酸包括无机酸，例如盐 酸、氢溴酸、硫酸、磷酸、硝酸等，和有机酸，例如乙酸、柠檬酸、枸橼酸、富马酸、苹果酸等。所述与药学上可接受的碱形成的盐包括但不限于铵盐、钠盐、钾盐、镁盐、钙盐等。As used herein, the expression "pharmaceutically acceptable salt" includes acid addition salts formed with pharmaceutically acceptable acids, as well as salts formed with pharmaceutically acceptable bases. Such pharmaceutically acceptable acids include inorganic acids such as salts acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc., and organic acids such as acetic acid, citric acid, citric acid, fumaric acid, malic acid, etc. The salts formed with pharmaceutically acceptable bases include, but are not limited to, ammonium salts, sodium salts, potassium salts, magnesium salts, calcium salts, and the like.

本发明的发明人出乎意料地发现，包含二肽Gln-Gln、三肽Tyr-Gln-Gln、四肽Leu-Gln-Ser-Gln或五肽Gln-Met-Gln-Pro-Gln的滴眼液均能够有效抑制角膜新生血管和抑制角膜上皮损伤，并且对于干眼具有显著的改善或治疗作用，从而指示二肽Gln-Gln、三肽Tyr-Gln-Gln、四肽Leu-Gln-Ser-Gln、五肽Gln-Met-Gln-Pro-Gln用于治疗、缓解或改善与角膜新生血管相关的眼表疾病和/或病症，与角膜上皮损伤相关的眼表疾病和/或病症，以及与干眼相关的眼表疾病和/或病症的用途。The inventors of the present invention unexpectedly found that eye drops comprising dipeptide Gln-Gln, tripeptide Tyr-Gln-Gln, tetrapeptide Leu-Gln-Ser-Gln or pentapeptide Gln-Met-Gln-Pro-Gln All solutions can effectively inhibit corneal neovascularization and corneal epithelial damage, and have a significant improvement or therapeutic effect on dry eye, thus indicating dipeptide Gln-Gln, tripeptide Tyr-Gln-Gln, tetrapeptide Leu-Gln-Ser- Gln, pentapeptide Gln-Met-Gln-Pro-Gln are used to treat, alleviate or improve ocular surface diseases and/or diseases related to corneal neovascularization, ocular surface diseases and/or diseases related to corneal epithelial damage, and Use for diseases and/or disorders of the ocular surface associated with dry eye.

在本文中使用时，表述“眼表疾病和/或病症”指的是角结膜浅层疾病和泪膜功能异常导致的疾病。As used herein, the expression "ocular surface diseases and/or disorders" refers to diseases of the superficial keratoconjunctiva and diseases resulting from abnormal function of the tear film.

根据本公开的一种实施方式，可以提供一种下式所代表的肽，According to one embodiment of the present disclosure, a peptide represented by the following formula can be provided,

Xaa-Xaa-Xaa-Xaa-Gln，Xaa-Xaa-Xaa-Xaa-Gln,

其中，in,

第1-3位的Xaa彼此独立地不存在或代表Ala、Gln、Gly、Glu、Tyr、Leu、Ser、Met或Pro；且Xaa at positions 1-3 are independently absent or represent Ala, Gln, Gly, Glu, Tyr, Leu, Ser, Met or Pro; and

第4位的Xaa代表Ala、Gln、Gly、Ser、Thr、Pro或Glu。Xaa at the 4th position represents Ala, Gln, Gly, Ser, Thr, Pro or Glu.

在本文中使用时，“第1-3位的Xaa彼此独立地……”表示第1位的Xaa、第2位的Xaa、第3位的Xaa各自的选择不会彼此影响。也就是说，第1位的Xaa、第2位的Xaa、第3位的Xaa可以相同，也可以不同；第1位的Xaa代表哪种氨基酸与第2位的Xaa、第3位的Xaa分别代表哪种氨基酸没有影响，反之亦然。As used herein, "Xaa at positions 1-3 independently of each other..." means that the respective selections of Xaa at position 1, Xaa at position 2, and Xaa at position 3 do not affect each other. That is to say, the Xaa at the first position, the Xaa at the second position, and the Xaa at the third position can be the same or different; It does not matter which amino acid is represented and vice versa.

根据本公开的一种实施方式，可以提供一种肽，其选自由二肽Gln-Gln和Gly-Gln、三肽Tyr-Gln-Gln和Gln-Ala-Gln、四肽Leu-Gln-Ser-Gln(SEQ ID NO.1)和Ala-Gln-Ala-Gln(SEQ ID NO.3)、以及五肽Gln-Met-Gln-Pro-Gln(SEQ ID NO.2)和Gln-Ala-Gln-Ala-Gln(SEQ ID NO.4)组成的组中的一种。 According to one embodiment of the present disclosure, a peptide can be provided, which is selected from dipeptides Gln-Gln and Gly-Gln, tripeptides Tyr-Gln-Gln and Gln-Ala-Gln, tetrapeptides Leu-Gln-Ser- Gln (SEQ ID NO.1) and Ala-Gln-Ala-Gln (SEQ ID NO.3), and the pentapeptide Gln-Met-Gln-Pro-Gln (SEQ ID NO.2) and Gln-Ala-Gln- One of the group consisting of Ala-Gln (SEQ ID NO.4).

根据本公开的一种实施方式，可以提供一种肽，其选自由二肽Gln-Gln、三肽Tyr-Gln-Gln、四肽Leu-Gln-Ser-Gln和五肽Gln-Met-Gln-Pro-Gln组成的组中的一种。According to one embodiment of the present disclosure, a peptide selected from dipeptide Gln-Gln, tripeptide Tyr-Gln-Gln, tetrapeptide Leu-Gln-Ser-Gln and pentapeptide Gln-Met-Gln- One of the group consisting of Pro-Gln.

根据本公开的一种实施方式，可以提供一种眼用药物组合物，其包含一种或更多种本公开的肽，以及眼科可接受的赋形剂。According to one embodiment of the present disclosure, an ophthalmic pharmaceutical composition may be provided, which comprises one or more peptides of the present disclosure, and an ophthalmologically acceptable excipient.

在一些实施方式中，眼用药物组合物中本公开的肽的浓度为0.1％w/v至8％w/v。在一些实施方式中，眼用药物组合物中本公开的肽的浓度为0.5％w/v至2％w/v。In some embodiments, the concentration of the disclosed peptides in the ophthalmic pharmaceutical composition is 0.1% w/v to 8% w/v. In some embodiments, the concentration of the disclosed peptides in the ophthalmic pharmaceutical composition is 0.5% w/v to 2% w/v.

在一些实施方式中，眼用药物组合物为等渗或低渗的形式。In some embodiments, ophthalmic pharmaceutical compositions are in isotonic or hypotonic form.

在本文中使用时，“等渗”是指眼用药物组合物的渗透压与眼表上皮细胞的渗透压相同或大致相同。在本文中使用时，“低渗”是指眼用药物组合物的渗透压低于眼表上皮细胞的渗透压。As used herein, "isotonic" means that the osmotic pressure of the ophthalmic pharmaceutical composition is the same or about the same as that of the ocular surface epithelial cells. As used herein, "hypotonic" means that the osmotic pressure of the ophthalmic pharmaceutical composition is lower than that of the ocular surface epithelial cells.

在一些实施方式中，眼用药物组合物为滴眼液、混悬液、凝胶剂、眼膏、乳剂、眼罩、眼贴、眼膜、眼霜、喷雾剂、注射剂或植入剂的形式。In some embodiments, the ophthalmic pharmaceutical composition is in the form of eye drops, suspensions, gels, eye ointments, emulsions, eye patches, eye patches, eye masks, eye creams, sprays, injections, or implants.

在一些实施方式中，所述眼科可接受的赋形剂包括渗透压调节剂、抑菌剂、增粘剂、pH调节剂、稳定剂、表面活性剂、保湿剂等中的一种或多种。In some embodiments, the ophthalmologically acceptable excipients include one or more of osmotic pressure regulators, bacteriostats, thickeners, pH regulators, stabilizers, surfactants, humectants, etc. .

在一些实施方式中，渗透压调节剂选自月桂氮卓酮、氯化钠、氯化钾、硼酸、硼砂、硫酸钠、硫酸钾、硝酸钠、硝酸钾、醋酸钠、甘露醇、甘油、丙二醇、山梨醇、2-(4-正辛基苯乙基)-2-氨基丙二醇盐酸盐、葡萄糖、蔗糖、果糖和乳糖中的一种或多种。在一些实施方式中，渗透压调节剂为月桂氮卓酮、硼酸、甘油、丙二醇和/或氯化钠。在一些实施方式中，渗透压调节剂选自氯化钠、氯化钾、硼酸、硼砂、硫酸钠、硫酸钾、硝酸钠、硝酸钾、醋酸钠、甘露醇、甘油、丙二醇、2-(4-正辛基苯乙基)-2-氨基丙二醇盐酸盐和葡萄糖中的一种或多种。在一些实施方式中，渗透压调节剂为氯化钠。In some embodiments, the osmotic pressure regulator is selected from the group consisting of laurocaprazine, sodium chloride, potassium chloride, boric acid, borax, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, sodium acetate, mannitol, glycerin, propylene glycol One or more of sorbitol, 2-(4-n-octylphenethyl)-2-aminopropanediol hydrochloride, glucose, sucrose, fructose and lactose. In some embodiments, the osmolarity regulator is laurocaprazine, boric acid, glycerin, propylene glycol, and/or sodium chloride. In some embodiments, the osmotic pressure regulator is selected from sodium chloride, potassium chloride, boric acid, borax, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, sodium acetate, mannitol, glycerol, propylene glycol, 2-(4 - one or more of n-octylphenethyl)-2-aminopropanediol hydrochloride and glucose. In some embodiments, the osmotic pressure regulator is sodium chloride.

在一些实施方式中，抑菌剂选自苯扎氯铵、苯扎溴铵、季铵盐-73、醋酸氯己定、葡萄糖氯己定、三氯叔丁醇、苯氧乙醇、羟苯甲酯、羟苯乙酯和羟苯丙酯中的一种或多种。在一些实施方式中，抑菌剂为苯扎氯铵、季铵盐-73、苯氧乙醇和/或羟苯乙酯。在一些实施方式中，抑菌剂选自苯扎氯铵、苯扎溴铵、醋酸氯己定、葡萄糖氯己定、三氯叔丁醇、苯氧乙醇、羟苯甲酯、羟苯乙酯和羟苯丙酯中的一种或多种。在一些实施方式中，抑菌剂为苯扎氯铵。 In some embodiments, the bacteriostatic agent is selected from benzalkonium chloride, benzalkonium bromide, quaternium-73, chlorhexidine acetate, chlorhexidine dextrose, chlorobutanol, phenoxyethanol, paraben One or more of esters, ethylparaben and propylparaben. In some embodiments, the bacteriostatic agent is benzalkonium chloride, quaternium-73, phenoxyethanol, and/or ethylparaben. In some embodiments, the bacteriostatic agent is selected from benzalkonium chloride, benzalkonium bromide, chlorhexidine acetate, chlorhexidine dextrose, chlorobutanol, phenoxyethanol, methylparaben, ethylparaben and one or more of propylparaben. In some embodiments, the bacteriostatic agent is benzalkonium chloride.

在一些实施方式中，增粘剂选自玻璃酸钠、三乙醇胺、卡姆波、羧甲基纤维素钠、甲基纤维素、聚乙二醇、聚乙烯醇和聚维酮中的一种或多种。在一些实施方式中，增粘剂是玻璃酸钠、卡姆波940和/或三乙醇胺。在一些实施方式中，增粘剂选自玻璃酸钠、羧甲基纤维素钠、甲基纤维素、聚乙二醇、聚乙烯醇和聚维酮中的一种或多种。在一些实施方式中，增粘剂是玻璃酸钠。In some embodiments, the thickening agent is selected from one or more of sodium hyaluronate, triethanolamine, carbomer, sodium carboxymethylcellulose, methylcellulose, polyethylene glycol, polyvinyl alcohol and povidone Various. In some embodiments, the tackifier is sodium hyaluronate, Kamper 940, and/or triethanolamine. In some embodiments, the thickener is selected from one or more of sodium hyaluronate, sodium carboxymethylcellulose, methylcellulose, polyethylene glycol, polyvinyl alcohol and povidone. In some embodiments, the tackifier is sodium hyaluronate.

在一些实施方式中，pH调节剂选自磷酸二氢钠、磷酸氢二钠、磷酸二氢钾、磷酸氢二钾、硼酸、硼砂、醋酸、醋酸钠、柠檬酸、柠檬酸钠、酒石酸、酒石酸钠、碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾、氢氧化钠、氢氧化钾、盐酸和磷酸中的一种或多种。在一些实施方式中，pH调节剂选自碳酸钠、碳酸氢钠、氢氧化钠中的一种或多种。在一些实施方式中，pH调节剂为氢氧化钠。In some embodiments, the pH regulator is selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, tartaric acid One or more of sodium, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid and phosphoric acid. In some embodiments, the pH regulator is selected from one or more of sodium carbonate, sodium bicarbonate, and sodium hydroxide. In some embodiments, the pH adjusting agent is sodium hydroxide.

在一些实施方式中，稳定剂选自依地酸二钠，依地酸二钠钙，依地酸二钾，依地酸二胺，2,3-二巯基-1-丙磺酸，二巯基琥珀酸，二巯基丙醇，甲磺酸去铁胺，α-硫辛酸，次氮基三乙酸酯，青霉胺，乙二醇二甲基丙烯酸酯，油酸钠，无水亚硫酸钠，抗坏血酸钠，去铁胺，苹果酸，柠檬酸，琥珀酸，苹果酸、柠檬酸和琥珀酸的钠盐、钙盐和镁盐中的一种或多种。在一些实施方式中，稳定剂为依地酸二钠。In some embodiments, the stabilizer is selected from edetate disodium, edetate disodium calcium, edetate dipotassium, edetate diamine, 2,3-dimercapto-1-propanesulfonic acid, dimercapto Succinic Acid, Dimercaptopropanol, Desferoxamine Methanesulfonate, Alpha Lipoic Acid, Nitrilotriacetate, Penicillamine, Ethylene Glycol Dimethacrylate, Sodium Oleate, Anhydrous Sodium Sulfite, Ascorbic Acid Sodium, deferoxamine, malic acid, citric acid, succinic acid, one or more of the sodium, calcium, and magnesium salts of malic acid, citric acid, and succinic acid. In some embodiments, the stabilizer is edetate disodium.

在一些实施方式中，表面活性剂选自泊洛沙姆、十二烷基硫酸钠、吐温-20、吐温-40、吐温-60、吐温-65、吐温-80、吐温-85、卵磷脂、聚氧乙烯蓖麻油、聚氧乙烯氢化蓖麻油、聚乙烯吡咯烷酮、聚乙二醇类和聚乙二醇15-羟基硬脂酸酯中的一种或多种。在一些实施方式中，表面活性剂选自吐温-80。In some embodiments, the surfactant is selected from poloxamer, sodium lauryl sulfate, Tween-20, Tween-40, Tween-60, Tween-65, Tween-80, Tween -85, one or more of lecithin, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyvinylpyrrolidone, polyethylene glycols, and polyethylene glycol 15-hydroxystearate. In some embodiments, the surfactant is selected from Tween-80.

在一些实施方式中，保湿剂选自甘油、丁二醇、聚乙二醇、丙二醇、乙二醇、木糖醇、聚丙二醇、山梨糖醇、吡咯烷酮羧酸钠、乳酸钠、尿素、羟乙基尿素、甲基葡糖聚醚、甲壳质衍生物、海藻糖、银耳多糖、透明质酸钠、泛醇、神经酰胺、天然植物提取物等中的一种或多种。In some embodiments, the humectant is selected from glycerin, butylene glycol, polyethylene glycol, propylene glycol, ethylene glycol, xylitol, polypropylene glycol, sorbitol, sodium pyrrolidone carboxylate, sodium lactate, urea, hydroxyethyl One or more of urea, methyl glucosan, chitin derivatives, trehalose, tremella polysaccharide, sodium hyaluronate, panthenol, ceramide, natural plant extracts, etc.

根据本公开的一种实施方式，可以提供本公开的肽或本公开的眼用药物组合物在制造用于治疗、缓解或改善与角膜新生血管相关的眼表疾病和/或病症的药物中的用途。According to one embodiment of the present disclosure, the use of the peptide of the present disclosure or the ophthalmic pharmaceutical composition of the present disclosure in the manufacture of drugs for treating, alleviating or improving ocular surface diseases and/or disorders related to corneal neovascularization can be provided. use.

在一些实施方式中，“与角膜新生血管相关的眼表疾病和/或病症”包括但不限于沙眼、角膜炎、角膜变性、角膜营养不良、角膜先天性疾病、角膜溃疡、佩戴隐形眼镜所 致角膜病变、眼化学性烧伤、角膜缘干细胞缺乏、过敏、眼外伤、自身免疫性疾病、角膜移植排斥反应等。In some embodiments, "ocular surface diseases and/or conditions associated with corneal neovascularization" include, but are not limited to, trachoma, keratitis, corneal degeneration, corneal dystrophy, congenital corneal disease, corneal ulcer, contact lens wear Keratopathy, ocular chemical burns, limbal stem cell deficiency, allergies, eye trauma, autoimmune diseases, corneal transplant rejection, etc.

根据本公开的一种实施方式，可以提供本公开的肽或本公开的眼用药物组合物在制造用于治疗、缓解或改善与角膜上皮损伤相关的眼表疾病和/或病症的药物中的用途。According to one embodiment of the present disclosure, the use of the peptide of the present disclosure or the ophthalmic pharmaceutical composition of the present disclosure in the manufacture of drugs for treating, alleviating or improving ocular surface diseases and/or disorders related to corneal epithelial damage can be provided. use.

在一些实施方式中，“与角膜上皮损伤相关的眼表疾病和/或病症”包括但不限于角膜上皮和基底膜营养不良、角膜基质营养不良、格子状角膜营养不良、角膜内皮营养不良、眼外伤、复发性角膜上皮糜烂、眼化学伤、眼部热灼伤、辅助性眼损伤、电击伤性眼病、应激性眼损伤、各种类型的角膜炎、各种类型的干眼、反复发作的单纯疱疹病毒性角膜炎、糖尿病角膜病变及面神经或三叉神经手术后角膜神经功能异常、严重的结膜炎和反复的过敏性眼表炎性、自身免疫性疾病所致角膜病变、Steven-Johnson综合征、综合征、瘢痕性类天疱疮、眼睑内翻、倒睫、睑缘炎、睑板腺功能障碍、眼睑闭合不全、角膜带状变性、Salzmann结节状角膜变性、角膜内皮失代偿、药物所致角膜上皮损伤、长期佩戴角膜接触镜致角膜损伤、全身代谢性疾病所致角膜病变、角膜软化症。In some embodiments, "ocular surface diseases and/or disorders associated with corneal epithelial damage" include, but are not limited to, corneal epithelial and basement membrane dystrophy, corneal stromal dystrophy, lattice corneal dystrophy, corneal endothelial dystrophy, ocular Trauma, recurrent corneal epithelial erosion, ocular chemical injury, ocular thermal burn, auxiliary eye injury, electrical injury ophthalmopathy, stress eye injury, various types of keratitis, various types of dry eye, recurrent Herpes simplex keratitis, diabetic keratopathy and corneal nerve dysfunction after facial nerve or trigeminal nerve surgery, severe conjunctivitis and recurrent allergic ocular surface inflammation, keratopathy caused by autoimmune diseases, Steven-Johnson syndrome , Syndrome, cicatricial pemphigoid, entropion, trichiasis, blepharitis, meibomian gland dysfunction, eyelid insufficiency, corneal band degeneration, Salzmann nodular corneal degeneration, corneal endothelial decompensation, drugs Corneal epithelial damage, corneal damage caused by long-term wearing of contact lenses, corneal lesions and corneal softening caused by systemic metabolic diseases.

根据本公开的一种实施方式，可以提供本公开的肽或本公开的眼用药物组合物在制造用于治疗、缓解或改善与干眼相关的眼表疾病和/或病症的药物中的用途。According to one embodiment of the present disclosure, use of the peptide of the present disclosure or the ophthalmic pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating, alleviating or improving ocular surface diseases and/or conditions associated with dry eye can be provided .

在一些实施方式中，“与干眼相关的眼表疾病和/或病症”包括但不限于综合征、Steven-Johnson综合征、移植物抗宿主病、各种***和胶原血管病、严重的肝功能异常、甲状腺功能异常、糖尿病及痛风、维生素A缺乏、雄激素缺乏、感染性结膜炎、过敏性结膜炎、角膜上皮基底膜下神经纤维丛密度异常，泪腺、睑板腺、眼表上皮细胞(杯状细胞)及角膜神经功能异常，螨虫、眼睑皮肤及结膜松弛症、泪阜部增生、眼睑痉挛、眼型痤疮、环境因素所致干眼(空气污染、光污染、射线、高海拔、低湿度及强风力)、视频终端性干眼、包括各种手术导致泪腺、副泪腺、睑板腺、眼表上皮细胞、角膜上皮基底膜下神经纤维丛损伤及缺失性睑缘炎、睑缘结构异常、各种手术引起泪液动力学异常、泪道管径扩大、泪小点位置异常、睑缘缺损、激光角膜屈光手术、白内障摘除手术等导致干眼、药物相关性干眼、焦虑、抑郁等情绪导致的干眼、睑板腺功能障碍、睑缘炎、化学性眼外伤、热烧伤及角膜缘功能障碍、瞬目异常(如瞬目频率降低、不完全瞬目等)、泪液排出异常、结膜松弛及眼睑异常、神经麻痹性或暴露性眼睑闭合不全、干燥综合征所致干眼、干燥性角结膜炎、水液缺乏型干眼(ADDE)、脂质异常 型干眼、黏蛋白异常型干眼、泪液动力学异常型干眼、混合型干眼、蒸发型干眼(EDE)、过敏性结膜炎相关干眼、病毒性结膜炎后干眼、白内障术后干眼、手术相关干眼以及隐形眼镜佩戴相关干眼。In some embodiments, "ocular surface diseases and/or conditions associated with dry eye" include, but are not limited to Syndrome, Steven-Johnson syndrome, graft-versus-host disease, various connective tissue and collagen vascular diseases, severe liver dysfunction, thyroid dysfunction, diabetes and gout, vitamin A deficiency, androgen deficiency, infectious conjunctivitis , allergic conjunctivitis, abnormal density of nerve fiber bundles under the corneal epithelial basement membrane, abnormal function of lacrimal glands, meibomian glands, ocular surface epithelial cells (goblet cells) and corneal nerves, mites, eyelid skin and conjunctivochalasis, lacrimal caruncle Hyperplasia, blepharospasm, ocular acne, dry eye caused by environmental factors (air pollution, light pollution, rays, high altitude, low humidity and strong wind), video terminal dry eye, including lacrimal glands, accessory lacrimal glands, eyelids caused by various operations Lamellar glands, ocular surface epithelial cells, corneal epithelial subbasal membrane nerve fiber plexus injury and absent blepharitis, eyelid margin structural abnormalities, tear fluid dynamics abnormalities caused by various operations, lacrimal duct diameter expansion, punctum location abnormalities, Dry eye caused by eyelid margin defect, laser corneal refractive surgery, cataract extraction surgery, etc., drug-related dry eye, dry eye caused by anxiety, depression and other emotions, meibomian gland dysfunction, blepharitis, chemical eye trauma, heat Burns and limbal dysfunction, abnormal blinking (such as reduced blinking frequency, incomplete blinking, etc.), abnormal tear drainage, conjunctival relaxation and eyelid abnormalities, nerve paralysis or exposed eyelid insufficiency, dryness caused by Sjogren's syndrome Eyes, keratoconjunctivitis sicca, aqueous depleted dry eye (ADDE), lipid abnormalities dry eye, dry eye with abnormal mucin, dry eye with abnormal tear dynamics, mixed dry eye, evaporative dry eye (EDE), dry eye associated with allergic conjunctivitis, dry eye after viral conjunctivitis, cataract surgery Post-dry eye, surgery-related dry eye, and contact lens wear-related dry eye.

上文针对本公开的肽和眼用药物组合物所述的各种实施方式和优选项可以相互组合(只要它们彼此之间不是内在矛盾的)，并且适用于本公开的用途，由此组合而形成的各种实施方式都视为本公开的一部分。The various embodiments and preferences described above for the peptides and ophthalmic pharmaceutical compositions of the present disclosure may be combined with each other (as long as they are not inherently contradictory to each other) and are applicable to the use of the present disclosure, thus combining The various embodiments formed are considered a part of this disclosure.

下面将结合实施例以例证的方式更清楚、明确地阐述本公开的技术方案。应该理解的是，这些实施例仅用于例证的目的，绝不旨在限制本公开的保护范围。本公开的保护范围仅通过权利要求来限定。The technical solution of the present disclosure will be described more clearly and definitely in an exemplary manner below in conjunction with the embodiments. It should be understood that these examples are for illustration purposes only, and are by no means intended to limit the protection scope of the present disclosure. The scope of protection of the present disclosure is limited only by the claims.

实施例Example

除非另有说明，否则以下实施例中所采用的试剂和仪器均为可以通过市购获得的常规产品。除非另有说明，否则按照常规条件或制造商建议的条件进行实验。实施例中使用的二肽(Gly-Gln、Gln-Gln)、三肽(Tyr-Gln-Gln、Gln-Ala-Gln)四肽(Leu-Gln-Ser-Gln、Ala-Gln-Ala-Gln)和五肽(Gln-Met-Gln-Pro-Gln、Gln-Ala-Gln-Ala-Gln)均表示肽本身，购买自中国科学院上海药物研究所苏州药物创新研究院。Unless otherwise stated, the reagents and instruments used in the following examples are commercially available conventional products. Experiments were performed under conventional conditions or conditions recommended by the manufacturer unless otherwise stated. Dipeptide (Gly-Gln, Gln-Gln), tripeptide (Tyr-Gln-Gln, Gln-Ala-Gln) tetrapeptide (Leu-Gln-Ser-Gln, Ala-Gln-Ala-Gln) used in the embodiment ) and pentapeptides (Gln-Met-Gln-Pro-Gln, Gln-Ala-Gln-Ala-Gln) both represent the peptide itself and were purchased from Suzhou Institute of Pharmaceutical Innovation, Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

实施例1：本公开的滴眼液的制备Embodiment 1: Preparation of eye drops of the present disclosure

按照以下配方和制备方法制备本公开的滴眼液：The eye drops of the present disclosure are prepared according to the following formula and preparation method:

表1.二肽滴眼液的配方
Table 1. Formula of dipeptide eye drops

二肽滴眼液的制备方法：按表1中所述比例称取氯化钠、依地酸二钠、苯扎氯铵，加入注射用水700mL搅拌溶解。溶解后，加入比例的玻璃酸钠，继续搅拌至玻璃酸钠溶解，然后加入二肽搅拌溶解。用0.1M氢氧化钠调节pH值至7.0，补加注射用水至1000mL，用0.22μm微孔膜过滤，在无菌条件下灌入5ml瓶中的无菌滴眼液中。The preparation method of dipeptide eye drops: weigh sodium chloride, edetate disodium, benzalkonium chloride according to the ratio stated in Table 1, add 700mL of water for injection and stir to dissolve. After dissolving, add the appropriate proportion of sodium hyaluronate, continue to stir until the sodium hyaluronate dissolves, then add the dipeptide and stir to dissolve. Adjust the pH value to 7.0 with 0.1M sodium hydroxide, add water for injection to 1000mL, filter through a 0.22μm microporous membrane, and pour it into the sterile eye drops in a 5ml bottle under aseptic conditions.

表2.三肽滴眼液的配方
Table 2. The formulation of tripeptide eye drops

三肽滴眼液的制备方法：按表2中所述比例称取氯化钠、依地酸二钠、苯扎氯铵，加入注射用水700mL搅拌溶解。溶解后，加入比例的玻璃酸钠，继续搅拌至玻璃酸钠溶解，然后加入三肽搅拌溶解。用0.1M氢氧化钠调节pH值至7.0，补加注射用水至1000mL，用0.22μm微孔膜过滤，在无菌条件下灌入5ml瓶中的无菌滴眼液中。The preparation method of tripeptide eye drops: weigh sodium chloride, edetate disodium, benzalkonium chloride according to the ratio stated in Table 2, add 700mL of water for injection and stir to dissolve. After dissolving, add the appropriate proportion of sodium hyaluronate, continue to stir until the sodium hyaluronate is dissolved, then add the tripeptide and stir to dissolve. Adjust the pH value to 7.0 with 0.1M sodium hydroxide, add water for injection to 1000mL, filter through a 0.22μm microporous membrane, and pour it into the sterile eye drops in a 5ml bottle under aseptic conditions.

表3四肽滴眼液的配方

The formula of table 3 tetrapeptide eye drops

四肽滴眼液的配方：按表3中所述比例称取氯化钠、依地酸二钠、苯扎氯铵，加入注射用水700mL搅拌溶解。溶解后，加入玻璃酸钠，继续搅拌至玻璃酸钠溶解，然后加入四肽搅拌溶解。用0.1M氢氧化钠调节pH值至7.0，补加注射用水至1000mL，用0.22μm微孔膜过滤，在无菌条件下灌入5ml瓶中的无菌滴眼液中。The formula of tetrapeptide eye drops: weigh sodium chloride, edetate disodium, benzalkonium chloride according to the ratio stated in Table 3, add 700mL of water for injection and stir to dissolve. After dissolving, add sodium hyaluronate, continue to stir until sodium hyaluronate dissolves, then add tetrapeptide and stir to dissolve. Adjust the pH value to 7.0 with 0.1M sodium hydroxide, add water for injection to 1000mL, filter through a 0.22μm microporous membrane, and pour it into the sterile eye drops in a 5ml bottle under aseptic conditions.

表4五肽滴眼液的配方
The formula of table 4 pentapeptide eye drops

按上述比例称取氯化钠、依地酸二钠、苯扎氯铵，加入注射用水700mL搅拌溶解。溶解后，加入玻璃酸钠，继续搅拌至玻璃酸钠溶解，然后加入五肽搅拌溶解。用0.1M氢氧化钠调节pH值至7.0，补加注射用水至1000mL，用0.22μm微孔膜过滤，在无菌条件下灌入5ml瓶中的无菌滴眼液中。Weigh sodium chloride, edetate disodium, and benzalkonium chloride according to the above ratio, add 700 mL of water for injection and stir to dissolve. After dissolving, add sodium hyaluronate, continue to stir until sodium hyaluronate is dissolved, then add pentapeptide and stir to dissolve. Adjust the pH value to 7.0 with 0.1M sodium hydroxide, add water for injection to 1000mL, filter through a 0.22μm microporous membrane, and pour it into the sterile eye drops in a 5ml bottle under aseptic conditions.

实施例2：刺激性试验Embodiment 2: irritation test

(1)皮肤刺激性试验(1) Skin irritation test

将30只健康新西兰兔随机分为5组，6只/组，雌雄各半，分别用于进行单次给药和多次给药皮肤刺激性实验，每个实验又分为2组，即完整皮肤组和破损皮肤组，均采用同体左右自身对照法进行比较。新西兰兔背部用8.0％硫化钠水溶液脱毛，每只家兔背部做2个脱毛部位(范围约为3.0cm×3.0cm)，剔除脱毛区域出现红斑、水肿或破损情况的新西兰兔。脱毛24h后，建立破损皮肤模型：先用温水洗净、碘伏消毒脱毛区域后， 用无菌针头在其上轻划“#”号，以皮肤出现轻微密集渗血为度造成皮肤破损；建立完整皮肤模型：选择皮肤区完好，无损伤、无异常处。30 healthy New Zealand rabbits were randomly divided into 5 groups, 6/group, half male and half female, respectively used for single administration and multiple administration skin irritation experiments, and each experiment was further divided into 2 groups, namely complete The skin group and the damaged skin group were compared by the same body left and right self-control method. The back of New Zealand rabbits was depilated with 8.0% sodium sulfide aqueous solution, and two depilated parts (range about 3.0cm×3.0cm) were made on the back of each rabbit, and the New Zealand rabbits with erythema, edema or damage in the depilated area were excluded. After 24 hours of hair removal, establish a damaged skin model: first wash it with warm water and disinfect the hair removal area with iodophor, Use a sterile needle to scratch the sign "#" lightly on it, and cause skin damage to the degree of slight intensive bleeding on the skin; establish a complete skin model: select a skin area that is intact, without damage, and without abnormalities.

将8％Gln-Gln滴眼液、8％Tyr-Gln-Gln滴眼液、8％Leu-Gln-Ser-Gln滴眼液和8％Gln-Met-Gln-Pro-Gln滴眼液和溶液赋形剂均匀涂抹到不同的新西兰兔脱毛区域(破损皮肤和完整皮肤组)，表面覆以薄纱布，用无刺激性的透气无纺布胶带包扎固定。单次给药皮肤刺激性实验(给药1次)和多次给药皮肤刺激性实验(连续给药14d，每天给药1次)。具体步骤：受试物接触皮肤24h，每天分别于在给药前和除药后0.5、1h肉眼观察，记录不同试药部位皮肤反应(红斑、水肿)情况。末次除药后72h后，安乐死所有动物，取给药部位皮肤组织，做组织病理学检查。参考皮肤刺激反应评分标准统计评分，根据计算分值评价皮肤刺激强度。8% Gln-Gln eye drops, 8% Tyr-Gln-Gln eye drops, 8% Leu-Gln-Ser-Gln eye drops and 8% Gln-Met-Gln-Pro-Gln eye drops and solutions The excipients were evenly applied to different depilatory areas of New Zealand rabbits (broken skin and intact skin group), the surface was covered with gauze, and fixed with a non-irritating breathable non-woven tape. Single-dose skin irritation test (administration once) and multiple-dose skin irritation test (continuous administration for 14 days, administration once a day). Specific steps: the test substance was in contact with the skin for 24 hours, observed with the naked eye before administration and 0.5 and 1 hour after drug removal, and recorded the skin reactions (erythema, edema) at different test drug sites. 72 hours after the last drug removal, all the animals were euthanized, and the skin tissue of the administration site was taken for histopathological examination. Refer to the statistical score of the skin irritation reaction scoring standard, and evaluate the skin irritation intensity according to the calculated score.

表5皮肤刺激、过敏反应评分标准
Table 5 Scoring criteria for skin irritation and allergic reactions

表6皮肤刺激强度评价标准
Table 6 Evaluation Criteria for Skin Irritation Intensity

在本试验条件下，对普通级新西兰兔完整皮肤、破损皮肤的单次涂敷给药，给药部位肉眼观察的平均刺激分数均为0，为无刺激。具体结果见表7。Under the conditions of this test, the average irritation score of the administration site is 0, which means no irritation to the intact skin and damaged skin of ordinary grade New Zealand rabbits. The specific results are shown in Table 7.

对普通级新西兰兔完整皮肤、破损皮肤的重复涂敷给药，每天1次，连续给药14天。给药部位肉眼观察的平均刺激分数均为0，为无刺激；组织病理学检查的平均刺激分数为0，为无刺激。具体结果见表8。Repeated coating and administration to the intact skin and damaged skin of ordinary grade New Zealand rabbits, once a day, for 14 consecutive days. The average irritation score of the administration site is 0, which means no irritation; the average irritation score of histopathological examination is 0, which means no irritation. The specific results are shown in Table 8.

表7单次给药刺激性试验肉眼观察动物皮肤刺激反应积分均值
Table 7 Single-administration irritation test The mean value of the animal skin irritation reaction points observed with the naked eye

注：时间表示给药当天除药后30min和60min，24h、48h、72h表示除药后24h、48h、72h。 Note: Time means 30min and 60min after drug removal on the day of administration, 24h, 48h, 72h means 24h, 48h, 72h after drug removal.

表8重复给药刺激性试验动物皮肤刺激反应积分均值
Table 8 Repeated administration irritation test animal skin irritation reaction integral mean value

注：D1-D14表示给药第1-14天，末24h-72h表示末次除药后24h-72hNote: D1-D14 means the 1st-14th day of administration, and the last 24h-72h means 24h-72h after the last drug removal

(2)皮肤过敏性试验(2) Skin allergy test

将48只健康豚鼠随机分为6组，即溶液赋形剂组、8％Gln-Gln滴眼液组、8％Tyr-Gln-Gln滴眼液组、8％Leu-Gln-Ser-Gln滴眼液组和8％Gln-Met-Gln-Pro-Gln滴眼液组、阳性对照组，每组8只，雌雄各半。豚鼠背部用10％硫化钠乙醇溶液脱毛，脱毛范围约3.0cm×3.0cm。脱毛24h后筛查，选皮肤无损伤者进行实验。给药方式同皮肤刺激性试验。阳性对照组在每只豚鼠脱毛区涂以1％的2,4-二硝基氯苯0.2ml，持续6h后，温水清洗。第7天、第14天用同样方法再致敏，共计致敏3次。末次给受试物致敏后14d，再次将受试物(阳性对照组为0.1％2,4-二硝基氯苯)涂抹在对应组别的豚鼠脱毛区，持续6h后，用温水清洗受试部位，立即观察皮肤过敏反应情况，分别在清洗受试物24、48、72h后记录一次，按皮肤过敏反应评分标准(表5)对过敏情况进行评分。依据致敏发生 率评价受试物致敏强度，其中平均分值＝(红斑形成总分+水肿形成总分)/合计的动物数，致敏率％＝(出现红斑、水肿或全身过敏反应的动物例数/受试动物总数)×100％。48 healthy guinea pigs were randomly divided into 6 groups, namely solution vehicle group, 8% Gln-Gln eye drop group, 8% Tyr-Gln-Gln eye drop group, 8% Leu-Gln-Ser-Gln drop group Eye drop group, 8% Gln-Met-Gln-Pro-Gln eye drop group, positive control group, 8 rats in each group, half male and half male. The back of the guinea pig was depilated with 10% sodium sulfide ethanol solution, and the depilated area was about 3.0cm×3.0cm. Screen after 24 hours of hair removal, and select those with no skin damage for the experiment. The administration method is the same as the skin irritation test. In the positive control group, 0.2ml of 1% 2,4-dinitrochlorobenzene was applied to the depilated area of each guinea pig for 6 hours, and then washed with warm water. On the 7th day and the 14th day, the same method was used to sensitize again, a total of 3 times of sensitization. 14 days after the last sensitization to the test substance, apply the test substance (0.1% 2,4-dinitrochlorobenzene for the positive control group) on the hair removal area of the corresponding group of guinea pigs again, and after 6 hours, wash the subject with warm water. Immediately observe the skin allergic reaction at the test site, record once after cleaning the test object for 24, 48, and 72 hours respectively, and score the allergic reaction according to the skin allergic reaction scoring standard (Table 5). Occurs according to sensitization Rate evaluates the sensitization intensity of the test substance, wherein the average score=(the total score of erythema formation+the total score of edema formation)/the total number of animals, the sensitization rate%=(the number of animals with erythema, edema or systemic allergic reaction/ Total number of animals tested)×100%.

表9致敏强度
Table 9 Sensitization intensity

试验期间所有动物一般观察未见明显异常。阳性对照组8例(8/8)动物每次致敏除药后1、24小时给药部位均可见轻度至重度的红斑及水肿症状，致敏第4天～末次致敏后第7天，动物给药部位可见白色至暗红色大小不一的结痂。致敏发生率为100％，分级为V级，致敏强度为极强致敏。During the test period, all animals were generally observed without obvious abnormalities. In the positive control group, 8 cases (8/8) of animals showed mild to severe erythema and edema symptoms at the administration site 1 and 24 hours after each sensitization and drug removal, from the 4th day of sensitization to the 7th day after the last sensitization , White to dark red scabs of various sizes can be seen at the administration site of the animal. The incidence rate of sensitization was 100%, the classification was V grade, and the sensitization intensity was extremely strong sensitization.

溶液赋形剂组、8％Gln-Gln滴眼液组、8％Tyr-Gln-Gln滴眼液组、8％Leu-Gln-Ser-Gln滴眼液组和8％Gln-Met-Gln-Pro-Gln滴眼液组，各组：激发给药除药后1、24、48小时，8例(8/8)动物给药部位皮肤均未见过敏反应症状，平均分为0，致敏发生率为0％，分级为I级，致敏强度为弱致敏。具体结果见表10。Solution vehicle group, 8% Gln-Gln eye drop group, 8% Tyr-Gln-Gln eye drop group, 8% Leu-Gln-Ser-Gln eye drop group and 8% Gln-Met-Gln- Pro-Gln eye drops group, each group: 1, 24, and 48 hours after stimulating administration and removing the drug, 8 cases (8/8) of animals had no allergic reaction symptoms on the skin of the administration site, and the average score was 0, sensitization The incidence rate was 0%, the classification was grade I, and the sensitization intensity was weak sensitization. The specific results are shown in Table 10.

表10试验激发除药后皮肤过敏反应评分统计表

Table 10 test stimulates the statistical table of skin allergic reaction score after drug removal

实施例3：本公开的滴眼液抑制小鼠角膜新生血管模型的作用研究Example 3: Research on the effect of the eye drops of the present disclosure on inhibiting corneal neovascularization in mice

1、实验目的1. Purpose of the experiment

考察本公开的滴眼液对小鼠角膜新生血管模型的治疗作用。The therapeutic effect of the eye drops of the present disclosure on a mouse corneal neovascularization model was investigated.

2、实验材料2. Experimental materials

2.1试验品2.1 Test product

上述实施例1中制备的各含0.1％和8％的Gln-Gln滴眼液、Tyr-Gln-Gln滴眼液、Leu-Gln-Ser-Gln滴眼液和Gln-Met-Gln-Pro-Gln滴眼液。试验过程中直接滴眼给药。The Gln-Gln eye drops, Tyr-Gln-Gln eye drops, Leu-Gln-Ser-Gln eye drops and Gln-Met-Gln-Pro- Gln eye drops. Eye drops were administered directly during the test.

2.2对照品2.2 Reference substance

双氯芬酸钠滴眼液(Diclofenac Sodium Eye Drops，简称Diclofenac或双氯)，沈阳兴齐眼药股份有限公司。Diclofenac Sodium Eye Drops (Diclofenac Sodium Eye Drops, referred to as Diclofenac or diclofenac), Shenyang Xingqi Eye Medicine Co., Ltd.

2.3试剂2.3 Reagents

NaOH，1mol/L，NaOH 4g溶解于100mL双蒸水中，高温高压消毒后备用。NaOH购自天津市大茂化学试剂厂；NaCl购自台山市新宁制药有限公司；盐酸丙美卡因滴眼液购自s.a.ALCON-COUVREUR n.v.；舒泰50购自法国维克公司；速眠新II购自敦化市圣达动物药品有限公司；荧光素钠眼科检测试纸购自天津晶明新技术开发有限公司。NaOH, 1mol/L, 4g of NaOH was dissolved in 100mL of double distilled water, sterilized by high temperature and high pressure for later use. NaOH was purchased from Tianjin Damao Chemical Reagent Factory; NaCl was purchased from Taishan Xinning Pharmaceutical Co., Ltd; proparacaine hydrochloride eye drops were purchased from s.a. New II was purchased from Dunhua Shengda Animal Pharmaceutical Co., Ltd.; fluorescein sodium ophthalmic test paper was purchased from Tianjin Jingming New Technology Development Co., Ltd.

2.4仪器2.4 Instruments

ZCS-3电子天平(分度值：0.1g)购自瑞安市浩展衡器有限公司；LDZX-50KBS立式压力蒸汽灭菌锅购自上海申安医疗器械厂。ZCS-3 electronic balance (division value: 0.1g) was purchased from Ruian Haozhan Weighing Instrument Co., Ltd.; LDZX-50KBS vertical pressure steam sterilizer was purchased from Shanghai Shen'an Medical Instrument Factory.

2.5实验动物2.5 Experimental animals

SPF级KM小鼠，雄性，由广东省医学实验动物中心提供，实验动物生产许可证号为SCXK(粤)2018-0002。动物实验环境：广东药科大学(大学城)实验动物中心，实验动物使用许可证号：SYXK(粤)2017-0125。SPF-grade KM mice, male, were provided by the Guangdong Medical Experimental Animal Center, and the experimental animal production license number is SCXK (Guangdong) 2018-0002. Animal experiment environment: Experimental Animal Center of Guangdong Pharmaceutical University (University Town), experimental animal use license number: SYXK (Guangdong) 2017-0125.

3、实验方法3. Experimental method

3.1给药方法3.1 Administration method

滴眼给药，1滴(约5μL)/眼/次，3次/天，间隔4h/次，连续7日。Eye drop administration, 1 drop (about 5 μL)/eye/time, 3 times/day, with an interval of 4h/time, for 7 consecutive days.

3.2测定方法 3.2 Determination method

取检疫合格的、经裂隙灯显微镜检查证实无眼前节病变的SPF级雄性小鼠(18～22g)。试验前2日，以0.3％氧氟沙星眼药水滴眼，4次/天，1滴/眼/次，连续2日，以维持眼部清洁无菌状态。以体重按区组分组法分为正常对照组、模型对照组、双氯芬酸钠组、各含0.1％及8％的Gln-Gln组、Tyr-Gln-Gln组、Leu-Gln-Ser-Gln组、Gln-Met-Gln-Pro-Gln组，8只/组。SPF-grade male mice (18-22 g) that had passed quarantine inspection and had no anterior segment lesions confirmed by slit-lamp microscopy were taken. 2 days before the test, 0.3% ofloxacin eye drops were instilled into the eyes, 4 times/day, 1 drop/eye/time, for 2 consecutive days to maintain the clean and sterile state of the eyes. Divided into normal control group, model control group, diclofenac sodium group, each containing 0.1% and 8% Gln-Gln group, Tyr-Gln-Gln group, Leu-Gln-Ser-Gln group, Gln-Met-Gln-Pro-Gln group, 8 animals/group.

将舒泰50(5mL)、速眠新II(5mL)与40mL生理盐水混合后，按1mL/kg肌肉注射麻醉，用盐酸丙美卡因滴眼液行角膜表面麻醉。将直径为2mm的单层圆形滤纸浸于1mol/L NaOH溶液中，去除多余液体后迅速贴于小鼠角膜中央30s，随后立即用生理盐水冲洗结膜囊1min以清除多余的NaOH，再用滤纸小心擦干眼睛，用眼科镊以平行于角膜缘的角度旋转刮掉角膜中央区表面的全部角膜上皮细胞，并滴荧光素钠生理盐水溶液进行显色检验。正常对照组用生理盐水代NaOH，其他同模型组动物。筛选建模成功的小鼠入组；建模后的当日即按照试验设计给予相应的药物，滴眼给药，1滴(约5μL)/眼/次，3次/天，间隔4h/次，连续7日。After mixing Sutai 50 (5mL), Sumianxin II (5mL) and 40mL normal saline, intramuscular injection of 1mL/kg was used for anesthesia, and proparacaine hydrochloride eye drops were used for corneal surface anesthesia. Soak a single-layer round filter paper with a diameter of 2 mm in 1 mol/L NaOH solution, remove excess liquid, and quickly stick it to the center of the mouse cornea for 30 seconds, then immediately rinse the conjunctival sac with normal saline for 1 min to remove excess NaOH, and then use the filter paper Carefully dry the eyes, use ophthalmic tweezers to rotate and scrape off all the corneal epithelial cells on the surface of the central cornea at an angle parallel to the limbus, and drop fluorescein sodium saline solution for color development test. The normal control group was replaced by NaOH with saline, and the other animals were in the same model group. The mice with successful modeling were screened and enrolled; on the day after modeling, the corresponding drugs were administered according to the experimental design, eye drops, 1 drop (about 5 μL)/eye/time, 3 times/day, with an interval of 4h/time, 7 consecutive days.

每只小鼠均佩戴自制项圈。每日裂隙灯显微镜下观察各组小鼠眼表情况。Each mouse wore a homemade collar. The ocular surface conditions of the mice in each group were observed daily under a slit lamp microscope.

3.3观察指标：3.3 Observation indicators:

1)角膜新生血管面积(CNV)测量1) Corneal neovascularization area (CNV) measurement

分别于建模后第3、7、14天(D3、D7、D14)：用游标卡尺测量自角、巩膜缘长出的新生血管长度，每次测量以连续弯度小，与角膜切线垂直的最长新生血管为准，每个角膜在不同象限测量值，取其平均值得出不同组、不同时期角膜新生血管的长度。参照公式S＝C/12×3.1416×[r²-(r-L)²]计算CNV，其中C代表新生血管累及角膜的圆周钟点数，L代表生血管从角膜缘侵入角膜的长度，r代表角膜半径(小鼠角膜半径约1.5mm)。On the 3rd, 7th, and 14th day after modeling (D3, D7, D14): Measure the length of new blood vessels growing from the corneal limbus and scleral limbus with a vernier caliper. For each measurement, the longest curvature is small and perpendicular to the corneal tangent The new blood vessels shall prevail, each cornea is measured in different quadrants, and the average value is taken to obtain the length of corneal new blood vessels in different groups and different periods. Calculate CNV by referring to the formula S=C/12×3.1416×[r ² -(rL) ² ], where C represents the number of hours in the circumference of the cornea with neovascularization, L represents the length of angiogenesis from the corneal limbus into the cornea, and r represents the radius of the cornea (The radius of mouse cornea is about 1.5mm).

2)角膜炎症反应评分2) Corneal inflammatory response score

分别于建模后第3、7、14天：角膜缘血管环充血程度：(1)无充血，0分；(2)角膜缘血管环轻度充血但宽度小于1mm，1分；(3)角膜缘血管环中度充血且宽度1～2mm，2分；(4)角膜缘血管环重度充血且宽度超过2mm，3分。中央角膜水肿程度：(1)无水肿，0分；(2)有但虹膜纹理清晰，1分；(3)有且虹膜纹理不清晰；有且瞳孔不可见，3分。周围角膜水肿程度：(1)无水肿，0分；(2)有但虹膜纹理清晰，1分；(3)有且虹膜纹理不清晰；有且虹膜不可见，3分。炎症指数为角膜缘血管环充血程度、中央角膜水肿程度和外周角膜水肿程度三项得分总和除以9的数值。On the 3rd, 7th, and 14th days after modeling respectively: degree of hyperemia of the limbal vascular ring: (1) no hyperemia, 0 points; (2) mild hyperemia of the limbal vascular ring but the width is less than 1 mm, 1 point; (3) The limbal vascular ring is moderately congested with a width of 1-2 mm, 2 points; (4) the limbal vascular ring is severely congested with a width of more than 2 mm, 3 points. The degree of central corneal edema: (1) no edema, 0 points; (2) yes but clear iris texture, 1 point; (3) yes and unclear iris texture; yes and no pupil visible, 3 points. The degree of peripheral corneal edema: (1) no edema, 0 points; (2) yes but clear iris texture, 1 point; (3) yes and unclear iris texture; yes and no iris visible, 3 points. Inflammation index was calculated by dividing the sum of the three scores of limbal vascular ring congestion, central corneal edema and peripheral corneal edema by 9.

3.4统计学方法 3.4 Statistical methods

计量资料以均数±标准差表示，多组间均数的比较先采用单因素方差分析(One-Way ANOVA)；方差齐时，组间均数两两比较用SNK法；方差不齐时，组间均数两两比较用Dunnett’s T3法；由SPSS15.0完成，α＝0.05。Measurement data are expressed as mean ± standard deviation Said that the comparison of the means among multiple groups was firstly performed by One-Way ANOVA; when the variances were homogeneous, the pairwise comparison of the means between groups was performed using the SNK method; Dunnett's T3 method; completed by SPSS15.0, α=0.05.

4、实验结果4. Experimental results

碱烧伤后，打破了小鼠角膜原有的“免疫赦免”状态，导致毛细血管由角膜缘向角膜内生长，形成CNV。本试验中模型对照组，D7、D14小鼠角膜CNV和角膜炎症指数均比D3升高且统计学差异显著(P<0.01)。治疗后，与模型对照组比，0.1％和8％的Gln-Gln组、Tyr-Gln-Gln组、Leu-Gln-Ser-Gln组、Gln-Met-Gln-Pro-Gln组的CNV和角膜炎症指数均不同程度降低且统计学差异显著(P<0.01)。因此，实施例3证明了，本公开的眼用药物组合物迅速、有效地抑制了角膜新生血管，效果显著。具体结果见表11、表12和图1。After alkali burn, the original "immune pardon" state of the mouse cornea was broken, causing capillaries to grow from the limbus to the cornea, forming CNV. In the model control group in this experiment, the corneal CNV and corneal inflammation index of D7 and D14 mice were higher than those of D3, and the difference was statistically significant (P<0.01). After treatment, compared with the model control group, the CNV and corneal Inflammation indexes were all decreased in varying degrees and the difference was statistically significant (P<0.01). Therefore, Example 3 proves that the ophthalmic pharmaceutical composition of the present disclosure inhibits corneal neovascularization rapidly and effectively, and the effect is remarkable. The specific results are shown in Table 11, Table 12 and Figure 1.

表11本公开的滴眼液对碱烧伤小鼠角膜CNV(mm²)的影响(n＝8)
Table 11 Effect of eye drops of the present disclosure on corneal CNV (mm ² ) of alkali-burned mice ( n=8)

注：与模型对照组相比，**P<0.01；模型对照组的组内相比，与D3相比，##P<0.01。Note: Compared with the model control group, **P<0.01; compared with the model control group within the group, compared with D3, ##P<0.01.

表12本公开的滴眼液对碱烧伤小鼠角膜炎症指数的影响(n＝8)
Table 12 The impact of the eye drops of the present disclosure on the corneal inflammation index of alkali-burned mice ( n=8)

注：组间相比，与模型对照组相比，**P<0.01；模型对照组的组内相比，与D3相比，##P<0.01。 Note: Compared between groups, compared with the model control group, **P<0.01; compared within the model control group, compared with D3, ##P<0.01.

实施例4：本公开的滴眼液抑制小鼠角膜上皮损伤模型的作用研究Example 4: Research on the effect of the eye drops of the present disclosure on inhibiting the mouse corneal epithelial injury model

1、实验目的:考察本公开的滴眼液对小鼠角膜上皮损伤的治疗作用。1. Experimental purpose: investigate the therapeutic effect of eye drops of the present disclosure on mouse corneal epithelial injury.

2、实验材料2. Experimental materials

2.1试验品2.1 Test product

上述实施例1中制备的各含0.1％和8％的Gln-Gln滴眼液、Tyr-Gln-Gln滴眼液、Leu-Gln-Ser-Gln滴眼液和Gln-Met-Gln-Pro-Gln滴眼液。试验过程中直接滴眼给药。The Gln-Gln eye drops, Tyr-Gln-Gln eye drops, Leu-Gln-Ser-Gln eye drops and Gln-Met-Gln-Pro- Gln eye drops. Eye drops were administered directly during the test.

2.2试剂2.2 Reagents

盐酸丙美卡因滴眼液购自s.a.ALCON-COUVREUR n.v.；NaCl购自台山市新宁制药有限公司；舒泰50购自法国维克公司；速眠新II购自敦化市圣达动物药品有限公司；荧光素钠眼科检测试纸购自天津晶明新技术开发有限公司。Proparacaine hydrochloride eye drops were purchased from s.a.ALCON-COUVREUR n.v.; NaCl was purchased from Taishan Xinning Pharmaceutical Co., Ltd.; Shutai 50 was purchased from France Vic; Sumianxin II was purchased from Dunhua Shengda Animal Pharmaceutical Co., Ltd. Company; fluorescein sodium ophthalmic test paper was purchased from Tianjin Jingming New Technology Development Co., Ltd.

2.3仪器2.3 Instruments

ZCS-3电子天平(分度值：0.1g)购自瑞安市浩展衡器有限公司；LDZX-50KBS立式压力蒸汽灭菌锅购自上海申安医疗器械厂。ZCS-3 electronic balance (division value: 0.1g) was purchased from Ruian Haozhan Weighing Instrument Co., Ltd.; LDZX-50KBS vertical pressure steam sterilizer was purchased from Shanghai Shen'an Medical Instrument Factory.

2.4实验动物2.4 Experimental animals

SPF级KM小鼠，雄性，由广东省医学实验动物中心提供，实验动物生产许可证号为SCXK(粤)2018-0002。动物实验环境：广东药科大学(大学城)实验动物中心，实验动物使用许可证号：SYXK(粤)2017-0125。SPF-grade KM mice, male, were provided by the Guangdong Medical Experimental Animal Center, and the experimental animal production license number is SCXK (Guangdong) 2018-0002. Animal experiment environment: Experimental Animal Center of Guangdong Pharmaceutical University (University Town), experimental animal use license number: SYXK (Guangdong) 2017-0125.

3、实验方法3. Experimental method

3.1给药方法3.1 Administration method

滴眼给药，1滴(约5μL)/眼/次，3次/天，间隔4h/次，连续3日。Eye drop administration, 1 drop (about 5 μL)/eye/time, 3 times/day, with an interval of 4h/time, for 3 consecutive days.

3.2测定方法3.2 Determination method

取检疫合格的、经裂隙灯显微镜检查证实无眼前节病变的SPF级雄性小鼠(18～22g)。以体重按区组分组法分模型对照组、各含0.1％及8％的Gln-Gln组、Tyr-Gln-Gln组、Leu-Gln-Ser-Gln组、Gln-Met-Gln-Pro-Gln组，8只/组。SPF-grade male mice (18-22 g) that had passed quarantine inspection and had no anterior segment lesions confirmed by slit-lamp microscopy were taken. Divide the model control group, each containing 0.1% and 8% Gln-Gln group, Tyr-Gln-Gln group, Leu-Gln-Ser-Gln group, Gln-Met-Gln-Pro-Gln Group, 8/group.

将舒泰50(5mL)、速眠新II(5mL)与40mL生理盐水混合后，按1mL/kg肌肉注射麻醉。用盐酸丙美卡因滴眼液行角膜表面麻醉，使用已消毒的手术刀刮挫法除去一侧的部分角膜上皮，直径约1.5mm范围，以造成小鼠角膜上皮损伤，但同时保持角膜缘区上皮完整。术后使用生理盐水冲洗结膜囊。使用100g/L荧光素钠溶液着染角膜上皮，拍摄。建模、拍照后即按照试验设计给予相应的药物，5μL/眼/次，1次/4小时，3次/天。拍摄的图像使用Image Pro Plus 6.0软件计算角膜上皮损伤率。 After mixing Shutai 50 (5mL), Sumianxin II (5mL) and 40mL normal saline, inject 1mL/kg intramuscularly for anesthesia. Corneal surface anesthesia was performed with proparacaine hydrochloride eye drops, and part of the corneal epithelium on one side was removed by scraping with a sterilized scalpel. The epithelium is intact. The conjunctival sac was flushed with saline after surgery. The corneal epithelium was stained with 100g/L fluorescein sodium solution and photographed. After modeling and photographing, the corresponding drugs were administered according to the experimental design, 5 μL/eye/time, once/4 hours, 3 times/day. The captured images were calculated using Image Pro Plus 6.0 software to calculate the corneal epithelial damage rate.

3.4统计学方法3.4 Statistical methods

计量资料以均数±标准差表示，多组间均数的比较先采用单因素方差分析(One-Way ANOVA)；方差齐时，组间均数两两比较用SNK法；方差不齐时，组间均数两两比较用Dunnett’s T3法；由SPSS15.0完成，α＝0.05。Measurement data are expressed as mean ± standard deviation Said that the comparison of the means among multiple groups was firstly performed by One-Way ANOVA; when the variances were homogeneous, the pairwise comparison of the means between groups was performed using the SNK method; Dunnett's T3 method; completed by SPSS15.0, α=0.05.

4、实验结果4. Experimental results

由表13可知，角膜上皮损伤建模后，随时间延长，其损伤程度呈现先加重再逐渐减轻的趋势，即角膜上皮损伤有自愈能力。0.1％和8％的给药组(Gln-Gln组、Tyr-Gln-Gln组、Leu-Gln-Ser-Gln组、Gln-Met-Gln-Pro-Gln组)均促进了角膜上皮损伤的修复，且给药后当日(D3：首次给药后8h)即能起效。其中与模型对照组比，治疗后D3、D7时各给药组均显著减少了小鼠角膜上皮损伤率(P<0.05)，治疗后D4、D6时各给药组均显著减少了小鼠角膜上皮皮损伤率(P<0.01)。因此，实施例4证明了，本公开的眼用药物组合物迅速、有效地促进了角膜上皮损伤的修复，效果显著。具体见表13和图2、图3。It can be seen from Table 13 that after the corneal epithelial injury was modeled, the degree of the injury showed a trend of aggravating first and then gradually reducing as time went on, that is, the corneal epithelial injury had self-healing ability. 0.1% and 8% administration groups (Gln-Gln group, Tyr-Gln-Gln group, Leu-Gln-Ser-Gln group, Gln-Met-Gln-Pro-Gln group) all promoted the repair of corneal epithelial damage , and the day after administration (D3: 8h after the first administration) can be effective. Among them, compared with the model control group, each administration group significantly reduced the corneal epithelial injury rate of mice on D3 and D7 after treatment (P<0.05), and each administration group significantly reduced the mouse corneal epithelial injury rate on D4 and D6 after treatment. Epithelial injury rate (P<0.01). Therefore, Example 4 proves that the ophthalmic pharmaceutical composition of the present disclosure can rapidly and effectively promote the repair of corneal epithelial damage, and the effect is remarkable. See Table 13 and Figure 2 and Figure 3 for details.

表13本公开的滴眼液对小鼠角膜上皮损伤之损伤率的影响(n＝8，)
Table 13 Effect of the eye drops of the present disclosure on the damage rate of corneal epithelial damage in mice (n=8, )

注：①每日给药3次，间隔4h/次；于给药前拍照，拍照时间为治疗前、D3、D4、D6、D7、D9，即每日的第一次和第三次给药前分别拍照，拍照2次/日；如D3表示第3次给药前进行拍照。②损伤率＝100×(治疗前的损伤面积-治疗后的损伤面积)÷治疗前的损伤面积。③与模型对照组相比，*P<0.05，**P<0.01。Note: ①Administer 3 times a day, with an interval of 4h/time; take pictures before administration, and the time of taking photos is before treatment, D3, D4, D6, D7, D9, that is, the first and third administration of the day Take pictures before each dose, and take pictures twice a day; for example, D3 means to take pictures before the third administration. ②Injury rate=100×(injury area before treatment-injury area after treatment)÷injury area before treatment. ③Compared with the model control group, *P<0.05, **P<0.01.

从治愈率上分析，0.1％和8％的给药组(Gln-Gln组、Tyr-Gln-Gln组、Leu-Gln-Ser-Gln组、Gln-Met-Gln-Pro-Gln组)均提高了高角膜上皮损伤的治愈率。与模型对照组比，Gln-Gln组(D3)、0.1％和8％的给药组(Gln-Gln组、Tyr-Gln-Gln组、Leu-Gln-Ser-Gln组、Gln-Met-Gln-Pro-Gln组)(D4、D6、D7)均显著提高了角膜上皮损伤的治愈率(P<0.01或P<0.05)。具体结果见表14。 From the cure rate analysis, 0.1% and 8% administration groups (Gln-Gln group, Tyr-Gln-Gln group, Leu-Gln-Ser-Gln group, Gln-Met-Gln-Pro-Gln group) all improve High healing rate of corneal epithelial injury. Compared with the model control group, Gln-Gln group (D3), 0.1% and 8% administration groups (Gln-Gln group, Tyr-Gln-Gln group, Leu-Gln-Ser-Gln group, Gln-Met-Gln group -Pro-Gln group) (D4, D6, D7) all significantly improved the healing rate of corneal epithelial injury (P<0.01 or P<0.05). The specific results are shown in Table 14.

表14本公开的滴眼液对小鼠角膜上皮损伤治愈率的影响(n＝8。)
Table 14 Effect of the eye drops of the present disclosure on the healing rate of corneal epithelial injury in mice (n=8. )

注：①治愈率＝100×(治疗前损伤率-治疗后率)÷治疗前损伤率。②与模型对照组相比，*P<0.05。Note: ①Cure rate=100×(injury rate before treatment-after treatment rate)÷injury rate before treatment. ②Compared with model control group, *P<0.05.

实施例5：本公开的滴眼液抑制小鼠干眼模型的作用研究Example 5: Research on the effect of the eye drops of the present disclosure on inhibiting dry eye model in mice

1、实验目的:本公开的滴眼液对小鼠干眼模型的防治作用。1. Experimental purpose: the prevention and treatment effect of eye drops of the present disclosure on mouse dry eye model.

2、实验材料2. Experimental materials

2.1试验品2.1 Test product

上述实施例1中制备的各含0.1％和8％的Gln-Gln滴眼液、Tyr-Gln-Gln滴眼液、Leu-Gln-Ser-Gln滴眼液和Gln-Met-Gln-Pro-Gln滴眼液。试验过程中直接滴眼给药。The Gln-Gln eye drops, Tyr-Gln-Gln eye drops, Leu-Gln-Ser-Gln eye drops and Gln-Met-Gln-Pro- Gln eye drops. Eye drops were administered directly during the test.

2.2试剂2.2 Reagents

生理盐水购自厦门闽研生物科技有限公司；荧光素钠眼科检测试纸购自天津晶明新技术开发有限公司；氢溴酸东莨菪碱注射液购自大连美仑生物技术有限公司；Zone-Quick酚红棉线购自Yokota，Japan；无水乙醇购自中国医药集团有限公司；Mouse IFN-γ、IL-17、IL-13、TNF-α、IL-1βPlatinum ELISA试剂盒购自eBioscience，USA；Anti-MMP-3 antibody购自Santa Cruz，货号Sc6839；Anti-MMP-9 antibody购自Abcam，货号ab38898；Goat anti-rat antibody购自BD Pharmingen，货号559286。Normal saline was purchased from Xiamen Minyan Biotechnology Co., Ltd.; sodium fluorescein ophthalmic test paper was purchased from Tianjin Jingming New Technology Development Co., Ltd.; scopolamine hydrobromide injection was purchased from Dalian Meilun Biotechnology Co., Ltd.; Zone-Quick phenol red Cotton thread was purchased from Yokota, Japan; absolute ethanol was purchased from China Pharmaceutical Group Co., Ltd.; Mouse IFN-γ, IL-17, IL-13, TNF-α, IL-1β Platinum ELISA kits were purchased from eBioscience, USA; Anti-MMP -3 antibody was purchased from Santa Cruz, product number Sc6839; Anti-MMP-9 antibody was purchased from Abcam, product number ab38898; Goat anti-rat antibody was purchased from BD Pharmingen, product number 559286.

2.3仪器2.3 Instruments

LDZX-50KBS立式压力蒸汽灭菌锅购自上海申安医疗器械厂；PCR仪购自Biometra Thermocycler，Germany；DY-6138EB除湿机购自宁波德业科技股份有限公司；Leica组织石蜡包埋机、手动轮转式石蜡切片机、冰冻切片机购自德国徕卡，Germany；LC96荧光定量PCR仪购自Applied Biosystems，USA；荧光显微镜购自Nikon，Japan。LDZX-50KBS vertical pressure steam sterilizer was purchased from Shanghai Shen’an Medical Equipment Factory; PCR instrument was purchased from Biometra Thermocycler, Germany; DY-6138EB dehumidifier was purchased from Ningbo Deye Technology Co., Ltd.; Leica tissue paraffin embedding machine, Manual rotary paraffin slicer and cryostat were purchased from Leica, Germany; LC96 fluorescent quantitative PCR instrument was purchased from Applied Biosystems, USA; fluorescence microscope was purchased from Nikon, Japan.

2.4实验动物 2.4 Experimental animals

SPF级C57/BL小鼠，雌性，由厦门大学实验动物中心提供，实验动物生产许可证号为SCXK(闽)2018-0003。动物实验环境：厦门大学实验动物中心屏障环境动物房，实验动物使用许可证号：SYXK(闽)2018-0009。SPF-grade C57/BL mice, female, were provided by the Experimental Animal Center of Xiamen University, and the experimental animal production license number is SCXK (Min) 2018-0003. Animal experiment environment: barrier environment animal room of Xiamen University Experimental Animal Center, experimental animal use license number: SYXK (Fujian) 2018-0009.

3、实验方法3. Experimental method

3.1分组方法3.1 Grouping method

取检疫合格的、经裂隙灯显微镜检查无病变的SPF级C57/BL雌性小鼠(10-12周龄)。以体重按区组分组法分为正常对照组(NS)、模型对照组(DS5)、各含0.1％和8％的Gln-Gln组、Tyr-Gln-Gln组、Leu-Gln-Ser-Gln组、Gln-Met-Gln-Pro-Gln组，8只/组。SPF-grade C57/BL female mice (10-12 weeks old) that had passed the quarantine inspection and had no lesions by slit-lamp microscopy were taken. Divided into normal control group (NS), model control group (DS5), Gln-Gln group containing 0.1% and 8%, Tyr-Gln-Gln group, Leu-Gln-Ser-Gln group respectively group, Gln-Met-Gln-Pro-Gln group, 8 rats/group.

3.2给药方法3.2 Administration method

滴眼给药，1滴(约5μL)/眼/次，3次/天，间隔4h/次，连续5日。Eye drop administration, 1 drop (about 5 μL)/eye/time, 3 times/day, with an interval of 4h/time, for 5 consecutive days.

3.3测定方法3.3 Determination method

取检疫合格的SPF级10-12周龄、C57/BL雌性小鼠。除正常对照组(8只)于正常饲养环境下饲养、皮下注射同剂量的生理盐水外，其余小鼠均饲养于人工干燥环境(相对湿度＜40％，温度21-23℃)中，并皮下注射东莨菪碱氢溴酸盐(0.5mg/0.2ml，200μl/次，4次/天，3小时/次，连续5天)，诱导小鼠干眼模型。造模的同时并按试验设计给予相应的药物，1滴(约5μL)/眼/次，3次/天，间隔4h/次，连续5日。10-12 weeks old, C57/BL female mice of SPF grade qualified for quarantine were taken. Except for the normal control group (8 mice) which were reared in a normal feeding environment and injected subcutaneously with the same dose of normal saline, the rest of the mice were raised in an artificial dry environment (relative humidity <40%, temperature 21-23°C), and injected subcutaneously. Inject scopolamine hydrobromide (0.5 mg/0.2 ml, 200 μl/time, 4 times/day, 3 hours/time, for 5 consecutive days) to induce dry eye model in mice. At the same time as the model was established, the corresponding drugs were administered according to the experimental design, 1 drop (about 5 μL)/eye/time, 3 times/day, with an interval of 4 hours/time, for 5 consecutive days.

试验结束前，用酚红棉线法测小鼠泪液分泌量；试验结束后，脱颈椎处死小鼠，取眼及附属组织，PAS染色法检测结膜中杯状细胞数量，ELISA法测小鼠结膜中IL-1β、TNF-α、IFN-γ、IL-17A、IL-13的表达，QRT-PCR法测小鼠角膜中MMP-3和MMP-9。Before the end of the experiment, the tear secretion of the mice was measured by the phenol red cotton thread method; after the end of the experiment, the mice were sacrificed by dislocation of the cervical vertebrae, and the eyes and accessory tissues were collected. IL-1β, TNF-α, IFN-γ, IL-17A, IL-13 expression, QRT-PCR method to detect MMP-3 and MMP-9 in mouse cornea.

3.4统计学方法3.4 Statistical methods

计量资料以均数±标准差表示，多组间均数的比较先采用单因素方差分析(One-Way ANOVA)；方差齐时，组间均数两两比较用SNK法；方差不齐时，组间均数两两比较用Dunnett’s T3法；由SPSS15.0完成，α＝0.05。Measurement data are expressed as mean ± standard deviation Said that the comparison of the means among multiple groups was firstly performed by One-Way ANOVA; when the variances were homogeneous, the pairwise comparison of the means between groups was performed using the SNK method; Dunnett's T3 method; completed by SPSS15.0, α=0.05.

4、实验结果4. Experimental results

4.1增加泪液分泌作用4.1 Increase tear secretion

经干燥环境联合东莨菪碱皮下注射5天诱导的模型对照组小鼠的泪液分泌量较正常对照组明显下降(P<0.01)。与模型对照组相比，经0.1％和8％的Gln-Gln、Tyr-Gln-Gln、Leu-Gln-Ser-Gln、Gln-Met-Gln-Pro-Gln局部滴眼处理后小鼠的泪液分泌量均显著增加The lacrimal secretion of mice in the model control group induced by dry environment combined with subcutaneous injection of scopolamine for 5 days was significantly lower than that in the normal control group (P<0.01). Compared with the model control group, the tear fluid of mice treated with 0.1% and 8% Gln-Gln, Tyr-Gln-Gln, Leu-Gln-Ser-Gln, Gln-Met-Gln-Pro-Gln topical eye drops significantly increased secretion

(P<0.01)，具体见表15和图4。(P<0.01), see Table 15 and Figure 4 for details.

表15本公开的滴眼液对干眼小鼠模型泪液分泌量的影响(n＝8，)
Table 15 The impact of the eye drops of the present disclosure on the amount of tear secretion in a mouse model of dry eye (n=8, )

注：与模型对照组相比，**P<0.01。Note: Compared with the model control group, **P<0.01.

4.2抗炎作用4.2 Anti-inflammatory effect

IL-1β、TNF-α、IFN-γ和IL-17A四种炎症因子在小鼠干眼模型对照组小鼠结膜中的表达量明显高于正常对照组(P<0.05或P<0.01)，提示小鼠干眼模型出现了炎症反应。0.1％和8％的治疗组(Gln-Gln组、Tyr-Gln-Gln组、Leu-Gln-Ser-Gln组、Gln-Met-Gln-Pro-Gln组)小鼠结膜中IL-1β、TNF-α、IFN-γ和IL-17A四种炎症因子的表达量均有不同程度的降低(P<0.05或P<0.01)，证明本公开的滴眼液抑制了干眼中的炎症，减少了炎性因子对眼组织的损伤。The expressions of IL-1β, TNF-α, IFN-γ and IL-17A in the conjunctiva of mice in the mouse dry eye model control group were significantly higher than those in the normal control group (P<0.05 or P<0.01), It suggested that the mouse dry eye model had an inflammatory response. IL-1β, TNF -α, IFN-γ and IL-17A four kinds of inflammatory factor expression levels all have different degrees of reduction (P<0.05 or P<0.01), prove that the eye drops of the present disclosure have suppressed the inflammation in the dry eye, reduced inflammation Sexual factor damage to eye tissue.

IL-13在模型对照组小鼠结膜中的表达量明显低于正常对照组(P<0.01)，0.1％和8％的Gln-Gln组、Tyr-Gln-Gln组、Leu-Gln-Ser-Gln组、Gln-Met-Gln-Pro-Gln组小鼠结膜中的表达量明显高于模型对照组(P<0.01)。具体结果见表16和图5。 The expression of IL-13 in the conjunctiva of the model control group was significantly lower than that of the normal control group (P<0.01), and the expression of IL-13 in the 0.1% and 8% Gln-Gln group, Tyr-Gln-Gln group, Leu-Gln-Ser- The expression levels in the conjunctiva of mice in Gln group and Gln-Met-Gln-Pro-Gln group were significantly higher than those in the model control group (P<0.01). The specific results are shown in Table 16 and Figure 5.

表16本公开的滴眼液对干眼小鼠模型抗炎作用的分析(n＝8，)
Table 16 Analysis of the anti-inflammatory effect of the eye drops of the present disclosure on the dry eye mouse model (n=8, )

注：与模型对照组相比，*P<0.05，**P<0.01。Note: Compared with the model control group, *P<0.05, **P<0.01.

4.3减轻干眼模型小鼠的角膜上皮屏障功能破坏4.3 Alleviate the damage of corneal epithelial barrier function in dry eye model mice

QRT-PCR的结果显示：与正常对照组相比，干眼的模型对照组小鼠角膜上皮MMP-3和MMP-9的mRNA的表达水平明显升高(P<0.01)。经滴眼给药治疗后，0.1％和8％的给药组小鼠角膜上皮MMP-3和MMP-9的mRNA表达水平均不同程度的降低(P<0.05或P<0.01)。具体见表17和图6。The results of QRT-PCR showed that compared with the normal control group, the expression levels of MMP-3 and MMP-9 mRNAs in the corneal epithelium of mice in the dry eye model control group were significantly increased (P<0.01). After eye drop administration, the mRNA expression levels of MMP-3 and MMP-9 in the corneal epithelium of mice in the 0.1% and 8% administration groups decreased in different degrees (P<0.05 or P<0.01). See Table 17 and Figure 6 for details.

表17本公开的滴眼液对干眼小鼠模型角膜上皮屏障功能修复作用的影响(n＝8,)

Table 17 The effect of the eye drops of the present disclosure on the repair of the corneal epithelial barrier function in a mouse model of dry eye (n=8, )

注：与模型对照组相比，*P<0.05，**P<0.01。Note: Compared with the model control group, *P<0.05, **P<0.01.

4.4增加结膜杯状细胞数量4.4 Increase the number of conjunctival goblet cells

黏蛋白是由结膜杯状细胞所分泌的、是泪膜的重要组成成分之一。本实验以PAS染色以检测结膜杯状细胞的数量，结果显示：与正常对照组相比，模型对照组小鼠的结膜杯状细胞数量显著减少(P<0.01)。经点眼治疗后，0.1％和8％的治疗组的结膜杯状细胞数量较模型对照组明显增加(P<0.05)。具体见表18和图7。Mucin is secreted by conjunctival goblet cells and is one of the important components of the tear film. In this experiment, PAS staining was used to detect the number of conjunctival goblet cells. The results showed that compared with the normal control group, the number of conjunctival goblet cells in the model control group was significantly reduced (P<0.01). After eyedrop treatment, the number of conjunctival goblet cells in the 0.1% and 8% treatment groups was significantly higher than that in the model control group (P<0.05). See Table 18 and Figure 7 for details.

表18本公开的滴眼液对干眼小鼠模型结膜杯状细胞的影响(n＝8，)
Table 18 Effects of the eye drops of the present disclosure on conjunctival goblet cells in a mouse model of dry eye (n=8, )

注：与模型对照组相比，*P<0.05，**P<0.01。Note: Compared with the model control group, *P<0.05, **P<0.01.

综上，实施例5证明了，本公开的眼用药物组合物迅速、有效地增加了泪液分泌、降低了眼部炎症因子的表达、改善了角膜上皮屏障功能并且增加了结膜杯状细胞的数量，从而能够用于治疗、改善或缓解干眼。In summary, Example 5 proves that the ophthalmic pharmaceutical composition of the present disclosure rapidly and effectively increases tear secretion, reduces the expression of ocular inflammatory factors, improves the corneal epithelial barrier function and increases the number of conjunctival goblet cells , so as to be used for treating, improving or alleviating dry eye.

尽管具体的实施方式已被描述，然而对于申请人或其它本领技术人员而言，可能存在或目前无法预见上述实施方式的替代、修改、变化、改进和实质等效物。因此，提交的所附的权利要求以及可能被修改的权利要求旨在涵盖所有这样的替代、修改、变化、改进和实质等效物。 While specific embodiments have been described, there may be or are currently unforeseeable alternatives, modifications, variations, improvements and substantial equivalents of the above described embodiments to the applicant or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to embrace all such alternatives, modifications, variations, improvements and substantial equivalents.

Claims (10)

1. 一种下式所代表的肽，A peptide represented by the formula,

   Xaa-Xaa-Xaa-Xaa-Gln，Xaa-Xaa-Xaa-Xaa-Gln,

   其中，in,

   第1-3位的Xaa彼此独立地不存在或代表Ala、Gln、Gly、Glu、Tyr、Leu、Ser、Met或Pro；且Xaa at positions 1-3 are independently absent or represent Ala, Gln, Gly, Glu, Tyr, Leu, Ser, Met or Pro; and

   第4位的Xaa代表Ala、Gln、Gly、Ser、Thr、Pro或Glu。Xaa at the 4th position represents Ala, Gln, Gly, Ser, Thr, Pro or Glu.
2. 如权利要求1所述的肽，所述肽选自由二肽Gln-Gln和Gly-Gln、三肽Tyr-Gln-Gln和Gln-Ala-Gln、四肽Leu-Gln-Ser-Gln(SEQ ID NO.1)和Ala-Gln-Ala-Gln(SEQ ID NO.3)、以及五肽Gln-Met-Gln-Pro-Gln(SEQ ID NO.2)和Gln-Ala-Gln-Ala-Gln(SEQ ID NO.4)组成的组中的一种。The peptide of claim 1, which is selected from dipeptides Gln-Gln and Gly-Gln, tripeptides Tyr-Gln-Gln and Gln-Ala-Gln, tetrapeptides Leu-Gln-Ser-Gln (SEQ ID NO.1) and Ala-Gln-Ala-Gln (SEQ ID NO.3), and pentapeptide Gln-Met-Gln-Pro-Gln (SEQ ID NO.2) and Gln-Ala-Gln-Ala-Gln ( A kind of in the group that SEQ ID NO.4) forms.
3. 一种眼用药物组合物，其包含一种或更多种如权利要求1或2所述的肽，以及眼科可接受的赋形剂。An ophthalmic pharmaceutical composition comprising one or more peptides according to claim 1 or 2, and an ophthalmologically acceptable excipient.
4. 如权利要求3所述的眼用药物组合物，其中如权利要求1或2所述的肽的浓度为0.1％w/v至8％w/v。The ophthalmic pharmaceutical composition according to claim 3, wherein the concentration of the peptide according to claim 1 or 2 is 0.1% w/v to 8% w/v.
5. 如权利要求3所述的眼用药物组合物，其中如权利要求1或2所述的肽的浓度为0.5％w/v至2％w/v。The ophthalmic pharmaceutical composition according to claim 3, wherein the concentration of the peptide according to claim 1 or 2 is 0.5% w/v to 2% w/v.
6. 如权利要求3-5中任一项所述的眼用药物组合物，其中所述眼用药物组合物为等渗或低渗的形式。The ophthalmic pharmaceutical composition according to any one of claims 3-5, wherein said ophthalmic pharmaceutical composition is in isotonic or hypotonic form.
7. 如权利要求3-6中任一项所述的眼用药物组合物，其中所述眼用药物组合物为滴眼液、混悬液、凝胶剂、眼膏、乳剂、眼罩、眼贴、眼膜、眼霜、喷雾剂、注射剂或植入剂的形式。The ophthalmic pharmaceutical composition according to any one of claims 3-6, wherein the ophthalmic pharmaceutical composition is eye drops, suspensions, gels, eye ointments, emulsions, eye masks, eye patches, In the form of an eye mask, eye cream, spray, injection, or implant.
8. 如权利要求1或2所述的肽或如权利要求3-7中任一项所述的眼用药物组合物在制造用于治疗、缓解或改善与角膜新生血管相关的眼表疾病和/或病症的药物中的用途。The peptide as claimed in claim 1 or 2 or the ophthalmic pharmaceutical composition as described in any one of claims 3-7 are used in the manufacture of treating, alleviating or improving ocular surface diseases associated with corneal neovascularization and/or Use in medicines for disorders.
9. 如权利要求1或2所述的肽或如权利要求3-7中任一项所述的眼用药物组合物在制造用于治疗、缓解或改善与角膜上皮损伤相关的眼表疾病和/或病症的药物中的用途。The peptide as claimed in claim 1 or 2 or the ophthalmic pharmaceutical composition as described in any one of claims 3-7 are used in the manufacture of treating, alleviating or improving ocular surface diseases associated with corneal epithelial damage and/or Use in medicines for disorders.
10. 如权利要求1或2所述的肽或如权利要求3-7中任一项所述的眼用药物组合物在制造用于治疗、缓解或改善与干眼相关的眼表疾病和/或病症的药物中的用途。 The peptide as claimed in claim 1 or 2 or the ophthalmic pharmaceutical composition as described in any one of claims 3-7 are used in the manufacture of treating, alleviating or improving ocular surface diseases and/or diseases associated with dry eye use in medicines.