WO2020173484A1 - 一种眼内手术用前房灌注液及其用途 - Google Patents

一种眼内手术用前房灌注液及其用途 Download PDF

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WO2020173484A1
WO2020173484A1 PCT/CN2020/076984 CN2020076984W WO2020173484A1 WO 2020173484 A1 WO2020173484 A1 WO 2020173484A1 CN 2020076984 W CN2020076984 W CN 2020076984W WO 2020173484 A1 WO2020173484 A1 WO 2020173484A1
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intraocular
anterior chamber
corneal endothelial
perfusion
glutamine
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李程
金梦怡
洪滢
刘祖国
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Xiamen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F9/00—Methods or devices for treatment of the eyes; Devices for putting in contact-lenses; Devices to correct squinting; Apparatus to guide the blind; Protective devices for the eyes, carried on the body or in the hand
    • A61F9/007—Methods or devices for eye surgery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2217/00—General characteristics of surgical instruments
    • A61B2217/002—Auxiliary appliance
    • A61B2217/007—Auxiliary appliance with irrigation system

Definitions

  • the invention belongs to the field of medicine, and specifically relates to an anterior chamber perfusion solution for intraocular surgery and its use.
  • Intraocular surgeries such as extracapsular cataract extraction, intraocular lens implantation, and vitrectomy require a long period of fluid perfusion in order to balance the intraocular pressure.
  • the conventional intraocular perfusion solution is an ophthalmic balanced salt solution, which lacks nutrients and antioxidant effects.
  • long-term contact with corneal endothelial cells can easily cause cell inactivation or even large areas of apoptosis and shedding.
  • the vision recovery of patients after transplantation is closely related to the activity of corneal endothelial cells.
  • Glutamine (Glutamine, Gin) is the most abundant free amino acid in the body, accounting for about 60% of the total amount of free amino acids in the human body. It is also a nitrogen source for many biosynthetic pathways. Gin is used in parenteral nutrition to regulate the tight junctions between intestinal cells. protein and essential to the integrity of the connection, can effectively protect the intestinal barrier function 2. However, Gin and stored in the process of heat sterilization unstable nature, and low solubility in aqueous solutions, which limits its wide application 3. L-alanyl-L-glutamine (L-alanyl-L-glutamine, Ala-Gin), also known as glutathione, is an amino acid chemical agent that can release glutamine in the body. It is currently mainly used after surgery There is no report on the application of parenteral and enteral nutrition supplementation, tissue and organ ischemia-reperfusion injury in intraocular surgery.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide an anterior chamber perfusion solution for intraocular surgery and its use. Under the action of balancing intraocular pressure, it also contains a certain amount of L-alanyl- L-glutamine solves the shortcomings of unstable nature and low solubility of glutamine; the anterior chamber perfusion solution for intraocular surgery not only protects the corneal endothelial cells from mechanical damage, ischemia and hypoxia, but also It provides nutrition and is used for intraoperative perfusion. When the perfusate acts on corneal endothelial cells, it can preventively reverse corneal endothelial cell apoptosis and exfoliation, protect the corneal endothelial barrier function, and achieve better surgical results.
  • One of the technical solutions adopted by the present invention to solve its technical problems is:
  • An anterior chamber perfusion solution for intraocular surgery is an ophthalmic balanced salt solution containing 0.217 ⁇ 1.090 mg/mL L-alanyl-L-glutamine.
  • the ophthalmic balanced salt solution contains 5.95 ⁇ 6.00mg/mL sodium chloride, 0.29 ⁇ 0.30mg/mL potassium chloride, 0.19 ⁇ 0.11 ⁇ /11 calcium chloride, 3.05 ⁇ 3.15mg/mL mL of sodium lactate.
  • Ala-Gin is stable in nature, easily soluble in water, can withstand high temperature disinfection, and can quickly decompose and release Gin in the body to play its role. It has high bioavailability and wide application range, which makes up for the shortcomings of Gin, especially for sudden trauma.
  • the patient has an important role and has been used as a guideline for parenteral nutrition.
  • the anterior chamber perfusion solution for intraocular surgery of the present invention also contains a certain amount of Ala-Gin under the action of balancing intraocular pressure.
  • Ala-Gin is stable after heat sterilization and has high solubility in water.
  • Ala-Gin can prevent myocardial, intestinal, liver or cerebral ischemia-reperfusion injury 4 _ 7 , in addition ala-Gin in parenteral nutrition can increase the expression of Claudin and to maintain mucosal thickness and villous height 8,9 to retain intestinal barrier function.
  • Ala-Gin in different physiological environments May also inhibit the release of inflammatory factors, reduce inflammation, promote cell proliferation and to prevent apoptosis and oxidative injury 10 - 13 o intraocular surgery the anterior chamber of the present invention is applied to a perfusion perfusate anterior chamber, both It has a protective effect on the mechanical damage of corneal endothelial cells and ischemia and hypoxia during the operation.
  • Figure 1 is a photograph of corneal endothelial cell apoptosis detected by TUNEL method in each in vitro experimental group and control group.
  • A in vitro normal group
  • B 1h control group
  • C 2h control group
  • D 1h experimental group
  • E 2h experiment group.
  • Figure 2 is a photograph of the F-actin detection of the corneal endothelial cytoskeleton in each in vitro experimental group and control group by immunofluorescence staining.
  • A in vitro normal group
  • B 1h control group
  • C 2h control group
  • D 1h experimental group
  • E 2h experimental group.
  • Figure 3 shows the morphology of corneal endothelial cells after in vivo confocal detection of anterior chamber perfusion.
  • A normal group
  • B 1h control group
  • C 1h experimental group.
  • the anterior chamber perfusion solution for intraocular surgery in this example contains the following components per 1 mL:
  • mice were killed by cervical dislocation and the eyeballs were removed. After washing with normal saline, the mouse cornea was cut along the limbus to remove the iris and ciliary body. The operation of separating the cornea is performed under an operating microscope to prevent mechanical damage to the cornea.
  • the cornea was quickly moved into the container containing the perfusion solution obtained in Example 1, at this time, it was counted as 0 o'clock, the cornea was taken out after 1 h and 2 h, fixed with 4% paraformaldehyde at 4 ° C overnight, and cell apoptosis was detected by TUNEL method , F-actin staining by immunofluorescence staining method to view the corneal endothelial cytoskeleton.
  • TUNEL method F-actin staining by immunofluorescence staining method to view the corneal endothelial cytoskeleton.
  • the cornea Separate the cornea according to the above steps, and quickly move the cornea into the conventional perfusion solution (an ophthalmic balanced salt solution, that is, without adding L-alanyl-L-glutamine, the remaining components and contents are the same as those of the intraocular surgery in Example 1 Atrial perfusion solution is the same). At this time, it is counted as 0 o'clock. After 1h and 2h, the cornea is taken out and fixed with 4% paraformaldehyde at 4 ° C overnight. Apoptosis is detected by TUNEL method, and F-actin staining is used for immunofluorescence staining. Corneal endothelial cytoskeleton.
  • the conventional perfusion solution an ophthalmic balanced salt solution, that is, without adding L-alanyl-L-glutamine, the remaining components and contents are the same as those of the intraocular surgery in Example 1 Atrial perfusion solution is the same. At this time, it is counted as 0 o'clock. After 1h and 2
  • the cornea was separated according to the above steps, without any treatment, fixed with 4% paraformaldehyde at 4 ° C overnight, cell apoptosis was detected by TUNEL method, and the corneal endothelial cytoskeleton was viewed by immunofluorescence staining with F-actin.
  • mice were anesthetized with 1% pentobarbital 5mL/kg.
  • the compound tropikamide eye drops were dispersed, and the eyelid was opened with a lid opener.
  • a lid opener At the 4 o'clock position of the corneal limbus, use a 32G needle at a 15° angle
  • the anterior chamber was punctured 2mm from the limbus, and a 34G metal perfusion needle was inserted and fixed to avoid contact with the corneal endothelium and the surface of the iris.
  • Example 1 The perfusion solution obtained in Example 1 was quickly connected to the perfusion needle, and the anterior chamber was perfused at a stable flow rate, 10 drops per minute. At this time, the perfusion was stopped after 1 hour, and the corneal endothelial cell photo was taken immediately by in vivo confocal.
  • corneal endothelial cell photos were taken with in vivo confocal.
  • TUNEL method to detect cell apoptosis Place the cornea obtained in the above steps in a 1.5mL centrifuge tube with the endothelial cells facing up. After washing with 1XPBS, fix it with cold acetone for 3min; wash with 1XPBS and then permeate the membrane with 1% TD-Buffer at room temperature for 10min, repeat 3 times; after washing with 1XPBS, Incubate with Equilibration Buffer 4 ° C for 1h, then incubate with the rTDT solution prepared in the kit at 4 ° C in the dark for 4 hours; incubate with 2XSSC solution 4 ° C in the dark for 40min to stop the reaction, wash with 1XPBS and mount the slides with DAPI.
  • the nucleus of apoptotic corneal endothelial cells was stained green under the microscope, and the nucleus of normal endothelial cells was blue.
  • the nucleus of normal endothelial cells was blue.
  • the normal corneal endothelial cells are arranged tightly under microscope, and the cytoskeleton has a regular double-track structure.
  • the corneal endothelial cytoskeleton has a regular double-track structure.
  • the corneal endothelial cytoskeleton rearranged in the control group at 2h, a large number of endothelial cells fell off, the integrity and barrier function of the corneal endothelium were destroyed, and the corneal endothelial cytoskeleton of the experimental group did not change significantly at 2h.
  • results of in vivo confocal examination As shown in Figure 3, the corneal endothelial cells of the normal group were hexagonal, with equal size, and the cell connections were tight; in the experimental group, after the anterior chamber perfusion, the corneal endothelial cells had increased pleomorphism and the cell boundaries were clear. There was no exfoliation of endothelial cells. The connection is complete; in the control group, after anterior chamber perfusion, the corneal endothelial cells lose their normal hexagonal shape, the intercellular spaces are enlarged, the boundaries are unclear, and the nucleus is highly reflective.
  • the intraocular perfusion solution of the present invention not only maintains the original role of perfusion and balances intraocular pressure, but has no toxic and side effects on intraocular tissues.
  • the addition of a certain concentration of Ala-Gin can also protect against ischemia and hypoxia during surgery. It can also provide nutrients, prevent corneal endothelial cell apoptosis and peeling, maintain the morphology of corneal endothelial cells, protect the integrity and barrier function of corneal endothelium, and obtain better surgical results.
  • the invention discloses an anterior chamber perfusion solution for intraocular surgery and its use. Under the action of balancing intraocular pressure, it also contains a quantitative amount of L-alanyl-L-glutamine. Extracapsular cataract extraction, intraocular lens implantation, vitrectomy and other intraocular operations require a longer period of fluid perfusion.
  • the commonly used ophthalmic perfusate is a balanced salt solution, which lacks nutrients and anti-oxidation effects, and has a negative effect on corneal endothelial cells. The impact cannot be ignored.
  • the anterior chamber perfusion solution for intraocular surgery provided by the present invention is used for intraoperative perfusion, which not only has a protective effect on the mechanical damage and ischemia and hypoxia of intraoperative corneal endothelial cells, but also provides nutrients and can preventively reverse the corneal endothelium Exfoliation of cell apoptosis, protects the barrier function of the corneal endothelium, achieves better surgical results, and has good industrial applicability.

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Abstract

本发明公开了一种眼内手术用前房灌注液及其用途，在平衡眼内压的作用下，还含有一定量的L-丙氨酰-L-谷氨酰胺。白内障囊外摘除术、人工晶状体植入、玻璃体切除术等眼内手术均需较长时间的液体灌注，而常用眼科灌注液为平衡盐溶液，缺乏营养物质及抗氧化作用，对角膜内皮细胞的影响不容忽视。本发明提供的眼内手术用前房灌注液应用于术中灌注，既对术中角膜内皮细胞的机械损伤和缺血缺氧有保护作用，又能提供营养物质，可以预防性的逆转角膜内皮细胞凋亡剥脱，保护角膜内皮屏障功能，达到更好的手术效果。

Description

—种眼内手术用前房灌注液及其用途 技术领域
本发明属于医药领域，具体涉及一种眼内手术用前房灌注液及其用途。
背景技术
随着显微镜技术的发展， 内眼手术日趋成熟，数量逐年上升，挽救了很多失明 患者。 白内障囊外摘除术、 人工晶状体植入、 玻璃体切除术等眼内手术为了平衡眼 内压均需较长时间的液体灌注。 常规眼内灌注液为眼科平衡盐溶液，该溶液缺乏营 养物质及抗氧化作用 ，在较长手术过程中 ，与角膜内皮细胞长时间接触，容易使得 细胞失去活性甚至发生大片凋亡脱落，而角膜移植术后患者的视力恢复与角膜内皮 细胞的活性有很大关系。 谷氨酰胺 ( Glutamine , Gin )是体内含量最丰富的游离氨 基酸，约占人体游离氨基酸总量的 60% ,还是许多生物合成途径的氮源 Gin在肠 外营养中对于调节肠道细胞间紧密连接蛋白和连接完整性至关重要，能有效保护肠 道屏障功能 2。 然而， Gin在储存和加热灭菌过程中性质不稳定，并且在水溶液中溶 解度低，这限制了它的广泛应用 3。 L-丙氨酰 -L-谷氨酰胺 ( L-alanyl-L-glutamine , Ala-Gin ) ,又名丙谷二肽，属于氨基酸类的化学药剂 ，在体内可以释放谷氨酰胺， 目前主要应用于手术后的肠外和肠内营养补充、 组织器官缺血再灌注损伤的保护 ， 还没有在眼内手术中作为前房灌注液应用的报道。
1 C alder, P. C. Glutamine and the immune system. Clin Nutr 13, 2-8 (1994).
2 Li, N. & Neu, J. Glutamine deprivation alters intestinal tight junctions via a PI3-K/Akt mediated pathway in Caco-2 cells. J Nutr 139, 710-714, doi: 10.3945/jn.108.101485 (2009).
3 Stehle P, P. P., Furst P lsotachophoretic analysis of a synthetic dipeptide L-alanyl-glutamine evidence for stability during heat sterilization Journal of Chromatography 294, 507-512 (1984).
4 Liu, S., Yang, Y., Song, Y. Q., Geng, J. & Chen, Q. L. Protective effects of N(2)LalanylLglutamine mediated by the JAK2/STAT3 signaling pathway on myocardial ischemia reperfusion. Mol Med Rep 17, 5102-5108, doi:10.3892/mmr.2018.8543 (2018).
5 Tazuke, Y., Wasa, M., Shimizu, Y., Wang, H. S. & Okada, A. Alanyl-glutamine-supplemented parenteral nutrition prevents intestinal ischemia-reperfusion injury in rats. JPEN J Parenter Enteral Nutr 27, 110-115, doi:10.1177/0148607103027002110 (2003).
6 Araujo Junior, R. J. et al. Preconditioning with L-alanyl-glutamine reduces hepatic ischemia-reperfusion injury in rats. Acta Cir Bras 26 Suppl 1, 8-13 (2011).
7 Vasconcelos, P. R., Guimaraes, A. B., Campelo, M. W., Vasconcelos, P. R. & Guimaraes, S. B.
Preconditioning with L-alanyl-glutamine upon cerebral edema and hypocampus red neurons counting in rats subjected to brain ischemia/reperfusion injury. Acta Cir Bras 30, 199-203, doi:10.1590/S0102-865020150030000006 (2015).
8 Liu, Y. W., Bai, M. X., Ma, Y. X. & Jiang, Z. M. Effects of alanyl-glutamine on intestinal adaptation and bacterial translocation in rats after 60% intestinal resection. Clin Nutr 16, 75-78 (1997).
9 Zhang, X. et al. Alanyl-glutamine ameliorates lipopolysaccharide-induced inflammation and barrier function injury in bovine jejunum epithelial cells. Biochem Cell Biol, doi:10.1139/bcb-2018-0320 (2019).
10 Cameiro, B. A. et al. Caspase and bid involvement in Clostridium difficile toxin A-induced apoptosis and modulation of toxin A effects by glutamine and alanyl-glutamine in vivo and in vitro. Infect Immun 74, 81-87, doi: 10.1128/IAI.74.1.81-87.2006 (2006).
11 Hou, Y. C., Chu, C. C., Ko, T. L., Yeh, C. L. & Yeh, S. L. Effects of alanyl-glutamine dipeptide on the expression of colon-inflammatory mediators during the recovery phase of colitis induced by dextran sulfate sodium. Eur J Nutr 52, 1089-1098, doi: 10.1007/s00394-012-0416-3 (2013).
12 Haynes, T. E. et al. L-Glutamine or L-alanyl-L-glutamine prevents oxidant- or endotoxin-induced death of neonatal enterocytes. Amino Acids 37, 131-142, doi:10.1007/s00726-009-0243-x (2009).
13 Nosworthy, M. G., Dodge, M. E., Bertolo, R. F. & Brunton, J. A. Enterally delivered dipeptides improve small intestinal inflammatory status in a piglet model of intestinal resection. Clin Nutr 35, 852-858, doi: 10.1016/j.clnu.2015.05.013 (2016).
发明内容
本发明的目的在于克服现有技术的不足之处，提供了一种眼内手术用前房灌注液 及其用途，在平衡眼内压的作用下，还含有一定量的 L-丙氨酰 -L-谷氨酰胺，解决了 谷氨酰胺性质不稳定、 溶解度低的缺点；该眼内手术用前房灌注液既对术中角膜内皮 细胞的机械损伤和缺血缺氧有保护作用 ，又能提供营养，应用于术中灌注，以该灌注 液作用于角膜内皮细胞时，可以预防性的逆转角膜内皮细胞凋亡剥脱，保护角膜内皮 屏障功能，达到更好的手术效果。 本发明解决其技术问题所采用的技术方案之一是：
—种眼内手术用前房灌注液，为含有 0.217〜 1.090 mg/mL的 L-丙氨酰 -L-谷氨酰 胺的眼科平衡盐溶液。
—实施例中 ：为含有 1.085〜 1.087 mg/mL的 L-丙氨酰 -L-谷氨酰胺的眼科平衡盐 溶液。
—实施例中 ：所述眼科平衡盐溶液含有 5.95〜 6.00mg/mL 的氯化钠 ， 0.29〜 0.30mg/mL的氯化钾， 0.19〜0.2011^/11 的氯化钙， 3.05〜 3.15mg/mL的乳酸钠。
本发明解决其技术问题所采用的技术方案之二是：
_种上述的眼内手术用前房灌注液制剂的用途。
L-丙氨酰 -L-谷氨酰胺分子结构如下：
Figure imgf000005_0001
Ala-Gin性质稳定， 易溶于水， 能耐受高温消毒，在体内可迅速分解释放 Gin发挥 其作用， 生物利用度高、 适用范围广， 弥补了 Gin的不足特别是对突发性创伤的病人 具有重要作用， 已作为肠外营养指南用药。 本发明的眼内手术用前房灌注液，在平衡 眼内压的作用下，还含有一定量的 Ala-Gin。 Ala- Gin作为 Gin的合成二肽，在加热灭 菌后性质稳定，在水中溶解度高，研究表明 ， Ala-Gin 可以预防心肌、 肠、 肝或脑缺 血再灌注引起的损伤 4_7，此外 Ala-Gin在肠外营养中可以增加紧密连接蛋白的表达， 并维持肠粘膜厚度和绒毛高度以保留肠屏障功能 8,9。 此外， Ala-Gin在不同生理环境 下还可以抑制炎性因子的释放，减轻炎症反应，促进细胞增殖，并防止细胞凋亡和氧 化损伤 10-13 o 本发明的眼内手术用前房灌注液应用于术中前房灌注，既对术中角膜内 皮细胞的机械损伤和缺血缺氧有保护作用 ，又能提供营养物质，并在手术应激的情况 下保护细胞，提高细胞抗氧化、 抗凋亡能力 ，预防性的逆转角膜内皮细胞凋亡剥脱， 保护角膜内皮屏障功能，达到更好的手术效果。
附图说明
下面结合附图和实施例对本发明作进_步说明。
图 1为 TUNEL法检测各体外实验组和对照组角膜内皮细胞凋亡照片 ，图中 A : 体外正常组； B : lh对照组； C : 2h对照组； D : lh实验组； E : 2h实验组。
图 2为免疫荧光染色法检测各体外实验组和对照组角膜内皮细胞骨架 F-actin照 片 ，图中 A :体外正常组； B : lh对照组； C : 2h对照组； D : lh实验组； E : 2h实 验组。
图 3为活体共聚焦检测前房灌注后角膜内皮细胞的形态，图中 A :正常组； B : lh对照组； C : lh实验组。 具体实施方式
下面通过实施例具体说明本发明的内容：
实施例 1眼内手术用前房灌注液的制备：
本实施例的眼内手术用前房灌注液每 lmL含有如下成分：
氯化钠 5.95〜 6.00mg
氯化钾 0.29〜 0.30mg
氯化钙 0.19〜 0.20mg 乳酸钠 3.05〜 3.15mg
L-丙氨酰 -L-谷氨酰胺 0.217〜 1.086mg。
实施例 2灌注液处理角膜实验及前房灌注实验
一、 实验对象
健康清洁级 6~8周龄 ICR小鼠 30只，体重 20〜 25g，雌雄不限。
二、 实验试剂
L-丙氨酷 -L-谷氨酷胺、 Invitrogenä Alexa Fluorä 594 Phalloidin、 Triton X- 100、 牛血清白蛋白（BSA X 磷酸盐缓冲液（PBS）、 戊巴比妥、 丙酮、 复方托吡卡胺滴眼 液、 DAPI染色液。
三、 实验仪器
手术显微镜、 正置多光子激光共聚焦系统 Olympus FV1000MPE-B、、 海德堡共焦 激光角膜显微镜。
四、 实验方法
1. 分离角膜
颈椎脱臼法处死小鼠，摘除眼球。 生理盐水冲洗后，将小鼠角膜沿角膜缘剪下， 除去虹膜、 睫状体等。 分离角膜的操作均在手术显微镜下进行，防止对角膜的机械损 伤。
2. 处理角膜
2.1体外实验组
将角膜迅速移入装有实施例 1所得灌注液的容器中 ，此时计为 0时，于 1 h和 2h 后取出角膜， 4%多聚甲醛 4°C固定过夜，通过 TUNEL法检测细胞凋亡，免疫荧光染 色法 F-actin染色查看角膜内皮细胞骨架。 2.2体外对照组
按以上步骤分离角膜，将角膜迅速移入常规灌注液（为眼科平衡盐溶液，即不添 加 L-丙氨酰 -L-谷氨酰胺，其余组分及含量与实施例 1的眼内手术用前房灌注液相同） 中 ，此时计为 0时，于 l h和 2h后取出角膜，4%多聚甲醛 4°C固定过夜，通过 TUNEL 法检测细胞凋亡，免疫荧光染色法 F-actin染色查看角膜内皮细胞骨架。
2.3体外正常组
按以上步骤分离角膜，不经过任何处理，4%多聚甲醛 4°C固定过夜，通过 TUNEL 法检测细胞凋亡，免疫荧光染色法 F-actin染色查看角膜内皮细胞骨架。
3. 前房灌注
通过 1%戊巴比妥 5mL/kg将小鼠麻醉，在室温下，复方托吡卡胺滴眼液散曈，开 睑器开睑，在角膜缘 4点方位，用 32G针头以 15°角距角膜缘 2mm处穿刺入前房，置 入 34G的金属灌注针头并固定，避免接触角膜内皮层及虹膜表面。
3.1体内实验组
迅速将实施例 1所得灌注液与灌注针头相连，以稳定流速进行前房灌注，每分钟 10滴，此时计为 0时，于 lh后停止灌注，立即采用活体共聚焦拍摄角膜内皮细胞照 片。
3.2体内对照组
迅速将常规灌注液与灌注针头相连，以相同流速进行前房灌注，此时计为 0时， 于 lh后停止灌注，立即采用活体共聚焦拍摄角膜内皮细胞照片。
3.3体内正常组
不经过任何灌注处理，采用活体共聚焦拍摄角膜内皮细胞照片。
4. TUNEL法检测细胞凋亡 将上述步骤得到的角膜放置在 1.5mL离心管内 内皮细胞面朝上 ，1XPBS冲洗后 ， 用冷丙酮固定 3min ;1XPBS冲洗后 用 1%TD-Buffer室温透膜 lOmin，重复 3次；1XPBS 冲洗后 ，与 Equilibration Buffer 4 °C卿育 lh，再与试剂盒配制的 rTDT溶液 4°C避光卿 育 4h ;与 2XSSC溶液 4°C避光孵育 40min终止反应 ， 1XPBS冲洗后 DAPI封片。
5. 全角膜平铺片的免疫荧光染色
将上述步骤得到的角膜放置在装有 4%多聚甲醛的 1.5mL离心管后 ， 内皮细胞面 朝上 ， 4°C过夜， 1XPBS冲洗后 ，用冷丙酮固定 3min ; 1XPBS冲洗后用 1%TD-Buffer 透膜 3次，每次 lOmin ;在常温下 ，将角膜放入 2% BSA溶液中封闭处理 1小时。 对 于鬼笔环肽染色，将封闭后的角膜与 F-actin(l:150)染色液室温孵育 90min : 1XPBS冲 洗后 DAPI封片 ， 随即使用激光共聚焦显微镜扫描记录。
五、 实验结果
1. TUNEL法检测细胞凋亡
如图 1 ，镜下观察凋亡角膜内皮细胞的细胞核被染成绿色 ， 正常内皮细胞核呈蓝 色。 lh时 ， 实验组和对照组的仅有少量凋亡细胞， 随着实验时间的延长，在 2h时 ， 对照组角膜内皮内可见大量的凋亡细胞核， 实验组仅有少量荧光。
2. 免疫荧光染色检测细胞骨架
如图 2A，镜下观察正常角膜内皮细胞排列规则紧密 ，细胞骨架呈规则双轨样结 构。 lh时 ，对照组存在散在的角膜内皮细胞丢失，实验组角膜内皮完整，细胞骨架排 列规则。 随着实验延长，对照组在 2h时角膜内皮细胞骨架重排， 大量内皮细胞脱落， 角膜内皮的完整性以及屏障功能被破坏， 实验组在 2h 时角膜内皮细胞骨架未见明显 改变。
3. 活体共聚焦检查结果 如图 3 ， 正常组角膜内皮细胞呈六边形 ， 大小均等 ，细胞间连接紧密 ；实验组在 前房灌注后 ，角膜内皮细胞多形性增加 ，细胞边界清晰，未见内皮细胞剥脱，细胞间 连接完整；对照组在前房灌注后 ，角膜内皮细胞丧失正常六边形 ，细胞间间隙扩大 ， 边界不清 ，细胞核高反光。
本发明中的眼内灌注液既保持了原有灌注、 平衡眼内压的作用 ，对眼内组织无毒 副作用 ，加入一定浓度的 Ala-Gin , 又能对手术过程中缺血缺氧起保护作用 ，还能提 供营养物质 ， 可以预防性的减少角膜内皮细胞凋亡剥脱，能够很好的维持角膜内皮细 胞形态 ，保护角膜内皮的完整性和屏障功能，从而得到更好的手术效果， 是一种良好 的新型眼内手术用前房灌注液。
以上所述 ，仅为本发明较佳实施例而已 ，故不能依此限定本发明实施的范围 ， 即依本发明专利范围及说明书内容所作的等效变化与修饰， 皆应仍属本发明涵盖的 范围内。 工业实用性
本发明公开了 _种眼内手术用前房灌注液及其用途 ，在平衡眼内压的作用下 ， 还含有 _定量的 L-丙氨酰 -L-谷氨酰胺。 白内障囊外摘除术、 人工晶状体植入、 玻璃 体切除术等眼内手术均需较长时间的液体灌注 ， 而常用眼科灌注液为平衡盐溶液， 缺乏营养物质及抗氧化作用 ，对角膜内皮细胞的影响不容忽视。 本发明提供的眼内 手术用前房灌注液应用于术中灌注 ，既对术中角膜内皮细胞的机械损伤和缺血缺氧 有保护作用 ， 又能提供营养物质 ， 可以预防性的逆转角膜内皮细胞凋亡剥脱 ，保护 角膜内皮屏障功能，达到更好的手术效果，具有良好的工业实用性。

Claims

权 利 要 求
1 . —种眼内手术用前房灌注液，其特征在于：为含有0.217〜1.090 11^/11 的1^ 丙氨酰 -L-谷氨酰胺的眼科平衡盐溶液。
2 .根据权利要求 1所述的眼内手术用前房灌注液，其特征在于：为含有 1.085〜 1.087mg/mL的 L-丙氨酰 -L-谷氨酰胺的眼科平衡盐溶液。
3 .根据权利要求 1或 2所述的眼内手术用前房灌注液，其特征在于：所述眼科 平衡盐溶液含有 5.95〜 6.00mg/mL 的氯化钠 ， 0.29〜 0.30mg/mL 的氯化钾 ， 0.19〜 0.20mg/mL的氯化钙， 3.05〜 3.15mg/mL的乳酸钠。
4 . —种权利要求 1至 3中任一项所述的眼内手术用前房灌注液制剂的用途。
PCT/CN2020/076984 2019-02-28 2020-02-27 一种眼内手术用前房灌注液及其用途 Ceased WO2020173484A1 (zh)

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