WO2019238107A1 - 红景天苷及其衍生物在制备眼科纤维化细胞外基质蛋白异常疾病的抑制剂药物中的应用 - Google Patents

红景天苷及其衍生物在制备眼科纤维化细胞外基质蛋白异常疾病的抑制剂药物中的应用 Download PDF

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WO2019238107A1
WO2019238107A1 PCT/CN2019/091203 CN2019091203W WO2019238107A1 WO 2019238107 A1 WO2019238107 A1 WO 2019238107A1 CN 2019091203 W CN2019091203 W CN 2019091203W WO 2019238107 A1 WO2019238107 A1 WO 2019238107A1
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salidroside
medicament
derivative
concentration
extracellular matrix
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PCT/CN2019/091203
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French (fr)
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郭涛
范先群
范宇晨
郭丽
彭玉豪
魏嘉洪
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上海交通大学医学院附属第九人民医院
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Priority to EP19819593.5A priority Critical patent/EP3808352A4/en
Priority to US17/252,265 priority patent/US20210260091A1/en
Publication of WO2019238107A1 publication Critical patent/WO2019238107A1/zh

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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the invention belongs to the field of use of salidroside and its derivatives, and particularly relates to the application of salidroside and its derivatives in the preparation of an inhibitory drug for abnormal diseases of ophthalmic fibrosis extracellular matrix protein.
  • fibrosis of trabecular meshwork caused by abnormal accumulation of fibronectin (FN) laminin (LA) and other factors is closely related to trabecular meshwork thickening, narrowing or occlusion of the mesh.
  • cytokines secreted by trabecular meshwork such as Transforming Growth Factor- ⁇ 2 ((TGF- ⁇ 2), etc., also affect the outflow of aqueous humor to varying degrees.
  • ocular hypotensive drugs can be divided into two major categories according to their mechanism of action: inhibition of aqueous humor production and promotion of aqueous humor outflow through the uveoscleral pathway. Glaucoma surgery also only focuses on creating pathways to increase the rate of aqueous humor outflow. These The characteristics of treatment methods are that although they can reduce intraocular pressure, they are not directly targeted at the pathological mechanism of glaucoma trabecular meshwork aqueous outflow obstruction, which can be said to treat the symptoms but not the root cause, so patients must be treated for life.
  • anti-metabolic drugs such as mitomycin C and 5-fluorouracil are used during surgery to prevent scarring of the filter tract, but these drugs may still cause a series of complications, such as postoperative filtration bleb leakage, corneal edema, conjunctiva, Scleral necrosis, endophthalmitis, etc. Therefore, drugs such as MMC and 5-FU still have deficiencies in preventing scar formation in the filter tract after glaucoma surgery.
  • TGF- ⁇ transforming growth factor ⁇
  • basic fibroblast growth factor The former regulates epithelial-mesenchymal transdifferentiation of lens epithelial cells, causing abnormalities of extracellular matrix proteins and capsular membranes.
  • Fibrosis the latter regulates the regeneration of the lens structure and cell proliferation, which becomes the main reason that affects the visual function of patients after cataract surgery.
  • the choice of intraocular lenses with different designs before surgery can reduce the incidence of posterior cataracts.
  • Proliferative vitreoretinopathy is a common proliferative lesion in ophthalmology. Trauma, diabetes, retinal detachment and retinal detachment can all cause proliferative lesions. Its main pathological features are proliferation of retinal pigment epithelial cells and glial cells, abnormalities of extracellular matrix proteins, and formation of subretinal and anterior retinal fibrocyte membranes. After the retina is damaged, retinal pigment epithelial cells (retinal pigment epithelial (RPE), glial cells, macrophages, etc.) migrate under the action of cytokines and gather in the vitreous cavity and under the retina.
  • RPE retinal pigment epithelial
  • Salidroside is an important active ingredient extracted from Rhodiola, CAS: 10338-51-9, English name: Salidroside 2- (4-HYDROXYPHENYL) ETHYL-BETA-D-GLUCOPYRANOSIDE, in antioxidant, Anti-inflammatory, anti-ischemia-reperfusion injury, relaxation of vascular smooth muscle and anti-fibrosis of liver, lung, and kidney can all play a better role.
  • the present invention aims to study whether salidroside and its derivatives can inhibit related cell fibrosis in the occurrence and development of ophthalmic diseases, and fundamentally treat and prevent ophthalmic diseases of patients.
  • the technical problem to be solved by the present invention is to provide pharmacological use of salidroside and its derivative as a disease inhibitor for treating and preventing abnormalities of extracellular matrix proteins such as ophthalmic fibrosis, especially salidroside and its derivative.
  • POAG primary open-angle glaucoma
  • the purpose of the present invention is the application of salidroside and its derivatives in the preparation of an inhibitory agent for abnormal diseases of ophthalmic fibrosis extracellular matrix protein.
  • the ophthalmic fibrosis abnormality of extracellular matrix protein is one of glaucoma, conjunctival scarring, pseudoptosis, eye skin scar, senile macular degeneration, diabetic retinopathy, and posterior cataract Or several.
  • abnormal diseases of ophthalmic fibrosis extracellular matrix protein are surgical treatment failure caused by scarring of the filter tract after trabecular meshwork resection and scarring of the filter tract after trabecular meshwork resection.
  • another object of the present invention is a disease inhibitor drug for treating and preventing abnormalities of extracellular matrix protein in ophthalmic fibrosis, comprising an effective amount of salidroside as an active ingredient in a tablet, capsule, pill, oral solution, Injections, eye drops or ointments.
  • the medicament contains a pharmaceutically acceptable excipient.
  • the injections include intravenous injections, intramuscular injections, subcutaneous injections, periocular injections, post-eyeball injections, subconjunctival injections, scleral injections, and intraocular injections.
  • the drug is an ointment, wherein the concentration of salidroside and its derivative is
  • the drug is an ointment, wherein the concentration of salidroside and its derivative is
  • the drug is an eye drop, wherein the concentration of salidroside and its derivative is
  • the drug is an eye drop, wherein the concentration of salidroside and its derivative is
  • the drug is an intraocular injection, wherein the concentration of salidroside and its derivative is
  • the drug is a subconjunctival injection, wherein the concentration of salidroside and its derivative is
  • the drug is a subscleral injection, wherein the concentration of salidroside and its derivative is
  • the drug is an injection in a vitreous cavity, wherein the concentration of salidroside and its derivative is
  • the drug is an injection in the anterior chamber, wherein the concentration of salidroside and its derivative is
  • the present application (1) explores the regulation mechanism of salidroside on the secretion of extracellular matrix protein of human normal trabecular meshwork induced by dexamethasone.
  • Salidroside has been proven to reduce tissue fibrosis in liver, lung, kidney and other organs in previous experimental studies. Recent research has shown that salidroside also has a certain role in anti-fibrosis. Salidroside can inhibit fibrosis caused by TGF ⁇ / smad signaling pathway in organs such as liver, lung and kidney. Our previous research found that dexamethasone can induce the expression of transforming growth factor ⁇ (TGF ⁇ ), fibronectin (FN), type IV collagen (COL-IV), and laminin (LN) in cells to increase red, causing cells Fibrosis (please give the source of the relevant proof or experimental data). After salidroside intervention, the expression levels of TGF ⁇ , FN, COL-IV and LN decreased.
  • TGF ⁇ transforming growth factor ⁇
  • FN fibronectin
  • COL-IV type IV collagen
  • LN laminin
  • the invention provides the application of a disease inhibitor medicine for treating and preventing abnormalities of extracellular matrix proteins such as ophthalmic fibrosis.
  • the drug is used to inhibit the tissue fibrosis of trabecular meshwork and other tissues through salidroside.
  • Therapeutic effects in chemically related diseases such as glaucoma, conjunctival scarring, pseudo-blepharoptosis, eye skin scars, age-related macular degeneration, diabetic retinopathy, and other abnormalities of extracellular matrix protein, filter channels after trabeculectomy Failure of surgical treatment due to scarring and prevention of scarring of the filter tract after trabeculectomy. This may provide new treatment ideas for ophthalmic-related fibrotic diseases.
  • the beneficial effect of the present invention is that the present invention provides an application of a disease inhibitor medicine for treating and preventing abnormalities of extracellular matrix proteins such as ophthalmic fibrosis, and inhibits the expression of extracellular matrix proteins in the eye to play an anti-fibrotic effect. Inhibition of diseases related to extracellular matrix proteins such as glaucoma and related ocular fibrosis can effectively delay and inhibit fibrosis.
  • the present invention utilizes the extracellular matrix protein inhibitor salidroside and its derivatives to prevent fibrosis. Can obviously play a role in inhibiting fibrosis.
  • Figure 1 shows the change of FN expression after salidroside addition.
  • Figure 2 shows the change of COL-IV expression after salidroside addition.
  • Figure 3 shows the change of LN expression after salidroside addition.
  • Fig. 4 is a graph showing changes in intraocular pressure after salidroside intervention in high intraocular pressure mice.
  • FIG. 5 is a graph showing changes in intraocular pressure after salidroside treatment in high intraocular pressure mice.
  • 1Cell plating Human normal trabecular meshwork cells (NTM) are cultured with F12 containing 10% fetal bovine serum (FBS), and human normal trabecular meshwork cells in a good growth state will be fused to 75% -80% At that time, the cells were digested with trypsin, and seeded into a 6-well plate and 2 ml of culture solution was added.
  • NTM Human normal trabecular meshwork cells
  • FBS fetal bovine serum
  • 3Extracellular matrix protein gene and protein secretion detection of trabecular meshwork design primer sequences of laminin (LN), type IV collagen (Col-IV) and fibronectin (FN) genes, and detect LN, COL by RT-PCR -IV, FN messenger RNA expression; using Anti-LN, Anti-Col-IV, and Anti-FN antibodies, ICC was used to detect trabecular meshwork extracellular matrix protein expression levels.
  • LN laminin
  • Col-IV type IV collagen
  • FN fibronectin
  • Figures 1 to 3 illustrate the effects of salidroside on the extracellular matrix of human trabecular meshwork induced by dexamethasone using real-time PCR.
  • dexamethasone can increase the normal extracellular matrix of human trabecular meshwork, and the addition of salidroside can reduce the extracellular matrix of trabecular meshwork.
  • "*" In the figure means P ⁇ 0.05.
  • mice 8-12 weeks old and weigh about 10-15 grams, all of which meet the first-class animal standards. Mice and feed were provided by the Experimental Animal Center of the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. The room temperature is 15-20 ° C, the relative humidity is 55-75%, the light is maintained for 12 hours, and the animals can drink and drink freely. Inclusion criteria: 1 No eye disease. 2 The pupils of both eyes directly reflect light and indirectly reflect light;
  • mice 5. Measure the weight of each mouse. Anesthetize the mice by intraperitoneal injection of 4% chloral hydrate. The anesthetic dose is 10 ⁇ l / g.
  • TGF ⁇ 2 over-expression adenovirus into the vitreous cavity of the model mice at an injection dose of 3 ⁇ l at a concentration of 3 ⁇ 107 pfu / ⁇ l, and inject the same dose and concentration of empty adenovirus into the vitreous cavity of the contralateral eye;
  • the salidroside intervention group started the administration immediately after the model was established.
  • the salidroside treatment group started the administration immediately after the intraocular pressure in the model group increased and became stable.
  • the salidroside was injected intraperitoneally at 40 mg / kg. 1 time daily.
  • the left eye drops were used as eye drops Su Sutan, 3 times a day, 2 drops each time, and the intraocular pressure was measured 4 hours after each administration for two weeks; the three groups of the present invention selected the left eye drops With the eye drops of the present invention, three drops a day, two drops each time, and the intraocular pressure was measured once every 4 hours after each administration for two weeks; the right eyes of the positive drug group and the present invention group were used as control eyes daily. The third time, 2 drops each time, also measured once every 4 hours after administration, lasting for two weeks; the control group's eyes twice a day, 2 drops each time, also measured once every 4 hours after administration For two weeks.

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Abstract

本发明提供了了红景天苷及其衍生物在制备治疗及预防眼科纤维化等细胞外基质蛋白异常的疾病抑制剂药物的应用。本发明通过抑制眼内细胞外基质蛋白的表达起到抗纤维化作用而治疗及预防青光眼等相关眼部纤维化等疾病。本发明还提供了制备一种治疗及预防眼科纤维化等细胞外基质蛋白异常的疾病抑制剂药物，包含有效量的红景天苷及其衍生物为活性成分。

Description

红景天苷及其衍生物在制备眼科纤维化细胞外基质蛋白异常疾病的抑制剂药物中的应用 技术领域
本发明属于红景天苷及其衍生物的用途领域，具体涉及红景天苷及其衍生物在制备眼科纤维化细胞外基质蛋白异常疾病的抑制剂药物中的应用。
背景技术
眼科常见的各种增生性病变中，尽管发病机制、治疗方式和预后各不相同，但其共同之处是许多细胞和细胞因子的激活和刺激，引起细胞外基质蛋白异常，导致细胞的迁移和增殖，对临床的治疗和预后造成了极大的困难。
原发性开角型青光眼、青光眼术后滤过泡瘢痕化、增生性玻璃体视网膜病变、增生性糖尿病视网膜病变、后发性白内障等疾病是眼科常见的增生性病变。尽管它们的发病机制、治疗方式和预后各不相同，但其共同之处是许多细胞和细胞因子一起参与病变的发生和发展，病变区炎性因子的激活和刺激，引起细胞外基质蛋白异常，导致细胞的迁移和增殖，给眼科医生的临床工作和手术预后带来了极大的挑战。
1、在原发性开角型青光眼中，房水排出途径的病理改变特别是小梁网的改变会引起房水流出受阻和眼压升高，这些改变与小梁网细胞的细胞外基质蛋白比如纤维连接蛋白(FN)层粘连蛋白(LA)等异常累积引起的小梁网纤维化和小梁网增厚，网眼变窄或闭塞有密切关系。此外小梁网分泌的一些细胞因子比如Transforming Growth Factor-β2((TGF-β2)等也不同程度的影响着房水的流出。目前青光眼的治疗只局限于通过降低眼压来缓解症状。临床上常用的降眼压药物可以按它们的作用机制分为两大类：抑制房水生成和促进经葡萄膜巩膜途径的房水外流。青光眼手术也只注重创建通路以增加房水外流的速率。这些治疗方法的特点是它们虽然可以降低眼压，但都没有直接针对青光眼小梁网房水外流阻塞的病理机制进行治疗，可以说是治标不治本，因此患者必须终身用药。此外，由于小梁网的病理改变持续加重，这些治疗手段通常在治疗几年后疗效便会逐渐降低，从而不能有效控制眼压，致使患者必须联合使用多种药物并加以手术治疗。
2、青光眼最常用的手术方式为小梁切除术，但目前仍然存在一些问题影响手术的成功率，最常见的是术后滤过区Tenon's囊纤维增殖，细胞外基质蛋白异常，造成滤过道的瘢痕化，阻碍房水经由滤过道的正常循环，无法降低眼压。研究发现，引起青光眼术后滤过道瘢痕化的原因主要是滤过区Tenon's囊成纤维细胞发生增殖、收缩和迁移，细胞外基质合成增加所致，此外许多细胞因子参与并诱导成纤维细胞的增殖。所以术中会使用抗代谢药物如丝裂霉素C、5－氟尿嘧啶等防止滤过道瘢痕化，但这些药物仍然可能引起一系列并发症，如术后滤过泡渗漏、角膜水肿、结膜、巩膜坏死、眼内炎等。因此，MMC和5－FU等药物在防止青光眼术后滤过道瘢痕化的形成仍存在缺陷。
3、白内障患者通过超声乳化吸除联合人工晶状体植入术获得了较为显著的疗效，多数患者的视力较术前得到不同程度的提高。但是术中保留的部分晶状体前囊膜及完整的后囊膜，使残留的晶状体上皮细胞出现增殖、迁移和分化而产生晶状体前囊膜纤维化，转化为成纤维细胞，导致后发性白内障形成，是白内障术后常见的并发症之一。在此过程中，转化生长因子β(TG F－β)与碱性成纤维生长因子之间的平衡破坏，前者调控晶状体上皮细胞的上皮－间叶转分化，使细胞外基质蛋白异常，囊膜纤维化；后者调控晶状体结构的再生和细胞增殖，成为白内障术后影响患者视功能的主要原因。通过术前选择不同设计的人工晶状体能够减少后发性白内障的发生，通过Nd:YAG激光可以治疗后发性白内障，但仍有可能引起炎症、眼压升高、人工晶状体损伤、黄斑水肿及视网膜脱离等并发症。仍需寻找更有效地方法和药物预防和治疗后发性白内障的发生发展。
4、增生性玻璃体视网膜病变(proliferative vitreoretinopathy，PVR)是眼科常见的增殖性病变，外伤、糖尿病、视网膜脱离及视网膜脱离术后等都可引起增生性病变。其主要病理特征为视网膜色素上皮细胞和神经胶质细胞增殖，细胞外基质蛋白异常，并伴有视网膜下和视网膜前膜纤维细胞膜形成。视网膜受到损伤后，视网膜色素上皮细胞(retinal pigment epithelial，RPE)、神经胶质细胞、巨噬细胞等在细胞因子的作用下发生迁移，聚集在玻璃体腔和视网膜下，在此过程中，细胞发生显型转变，形成具有收缩功能的纤维增殖膜，最终导致视网膜脱离。目前，手术切除增殖膜可明显提高手术成功率，但仍然有许多患者的术后视力不理想。尤其是严重的PVR无论是手术方式还是术后预后情况都 不理想，常需要多次手术。近年来，许多学者研究PVR的基因治疗，但是由于技术尚不完善，载体的靶向性和组织特异性差、转导成功率低以及安全性问题，还未能完全应用于临床。由此可见，眼科疾病术后增殖性病变为眼科医生的临床治疗工作带来了重大挑战，因此，许多学者尝试寻找更安全有效的药物能够预防和治疗增殖性病变。
红景天苷是从红景天中提取出的一种重要活性成分，CAS:10338-51-9，英文名称：Salidroside 2-(4-HYDROXYPHENYL)ETHYL-BETA-D-GLUCOPYRANOSIDE，在抗氧化，抗炎，抗缺血再灌注损伤，舒张血管平滑肌以及在抗肝肺肾纤维化等方面均能起到比较好的作用。
本发明旨在研究红景天苷及其衍生物能够在眼科疾病的发生发展中抑制相关细胞纤维化，从根本上治疗及预防患者的眼科疾病。
发明内容
本发明所要解决的技术问题是提供发红景天苷及其衍生物作为一种治疗及预防眼科纤维化等细胞外基质蛋白异常的疾病抑制剂的药理用途，尤其在于红景天苷及其衍生物在制备治疗及预防小梁网纤维化抑制药物中的应用，及其在制备降低原发性开角青光眼(POAG)中眼压的相关应用，同时也探索红景天苷及其衍生物在眼科其他纤维化相关的等细胞外基质蛋白异常的疾病中的应用。
本发明的目的，红景天苷及其衍生物在制备眼科纤维化细胞外基质蛋白异常疾病的抑制剂药物中的应用。
进一步的，所述的眼科纤维化细胞外基质蛋白异常疾病为青光眼、结膜瘢痕化、假性上睑下垂、眼部皮肤疤痕、老年性黄斑变性、糖尿病视网膜病变、后发性白内障中的一种或几种。
进一步的，所述的眼科纤维化细胞外基质蛋白异常疾病为小梁网切除术后滤过道疤痕化导致的手术治疗失败及小梁网切除术后滤过道疤痕化。
另外，本发明的另一目的，一种治疗及预防眼科纤维化细胞外基质蛋白异常的疾病抑制剂药物，包含有效量的红景天苷为活性成分的片剂、胶囊、丸剂、口服液、针剂、眼药水或眼药膏。
进一步的，所述药物中包含有药学上可接受的辅料。
进一步的，所述的针剂包括经静脉注射针剂、肌肉注射针剂、皮下注射针 剂、眼周注射针剂、眼球后注射针剂、結膜下注射针剂、巩膜注射针剂、眼内注射针剂。
进一步的，所述药物为眼药膏，其中红景天苷及其衍生物的浓度为
Figure PCTCN2019091203-appb-000001
进一步的，所述药物为眼药膏，其中红景天苷及其衍生物的浓度为
Figure PCTCN2019091203-appb-000002
进一步的，所述药物为眼药水，其中红景天苷及其衍生物的浓度为
Figure PCTCN2019091203-appb-000003
进一步的，所述药物为眼药水，其中红景天苷及其衍生物的浓度为
Figure PCTCN2019091203-appb-000004
进一步的，所述药物为眼内注射针剂，其中红景天苷及其衍生物的浓度为
Figure PCTCN2019091203-appb-000005
进一步的，所述药物为結膜下注射针剂，其中红景天苷及其衍生物的浓度为
Figure PCTCN2019091203-appb-000006
进一步的，所述药物为巩膜下注射针剂，其中红景天苷及其衍生物的浓度为
Figure PCTCN2019091203-appb-000007
进一步的，所述药物为玻璃体腔内注射针剂，其中红景天苷及其衍生物的浓度为
Figure PCTCN2019091203-appb-000008
进一步的，所述药物为前房内注射针剂，其中红景天苷及其衍生物的浓度为
Figure PCTCN2019091203-appb-000009
本申请(1)探讨红景天苷对地塞米松诱导的人正常小梁网细胞外基质蛋白分泌的调控机制。
(2)在小鼠腺病毒载体TGFβ2过表达法构建的POAG模型中，验证红景天苷对青光眼相关因子，纤维化的小梁网以及高眼压的调控作用。
在过往实验研究中红景天苷被证实在肝、肺、肾等脏器中有减轻组织纤维化的作用，在最新研究表明，红景天苷在抗纤维化方面也具有一定的作用。红景天苷在肝、肺、肾等器官可以抑制TGFβ/smad信号通路引起的纤维化。我们前期研究发现地塞米松可诱导细胞中转化生长因子β(TGFβ)、纤维连接蛋白(FN)、IV型胶原(COL-IV)、层粘连蛋白(LN)等表达量升高红，引起细胞纤维化(请给出相关证明文献出处或者实验数据)。红景天苷干预后，TGFβ、 FN、COL-IV、LN表达含量降低。
本发明提供了一种治疗及预防眼科纤维化等细胞外基质蛋白异常的疾病抑制剂药物的应用，通过红景天苷抑制小梁网等组织纤维化，探索红景天苷在其他眼组织纤维化相关疾病中的治疗作用例如青光眼、结膜瘢痕化，假性上睑下垂，眼部皮肤疤痕、老年性黄斑变性，糖尿病视网膜病变等细胞外基质蛋白异常的疾病，小梁网切除术后滤过道疤痕化导致的手术治疗失败及小梁网切除术后滤过道疤痕化的预防。这可能为眼科相关的纤维化疾病提供新的治疗思路。
本发明的有益效果是：本发明提供了一种治疗及预防眼科纤维化等细胞外基质蛋白异常的疾病抑制剂药物的应用，通过抑制眼内细胞外基质蛋白的表达起到抗纤维化作用而抑制青光眼等相关眼部纤维化等细胞外基质蛋白异常的疾病，可以有效地延缓、抑制纤维化，本发明利用细胞外基质蛋白抑制剂红景天苷及其衍生物起到抗纤维化作用，可明显起到抑制纤维化的作用。
附图说明
图1是红景天苷加入后FN表达改变。
图2是红景天苷加入后COL-IV表达改变。
图3是红景天苷加入后LN表达改变。
图4为红景天苷对高眼压小鼠干预后眼压变化图。
图5为红景天苷对高眼压小鼠治疗后眼压变化图。
具体实施方式
下面进一步说明本发明的实施例。
实施例1
阐明红景天苷对青光眼相关因子及小梁网细胞外基质分泌的调控机制
①细胞铺板：人正常小梁网细胞(Normal Trabecular Meshwork Cells，NTM)用含10％胎牛血清(FBS)的F12培养，待生长状态良好的人正常小梁网细胞融合至75％-80％时，将细胞用胰酶消化，铺种到6孔板内加2ml培养液。
②实验分组：将人正常小梁网细胞正常培养作为空白对照组，将人正常小梁网细胞加入地塞米松作为实验对照组，将人正常小梁网细胞加入地塞米松和不同 浓度红景天苷作为实验组(实验对照组和实验组培养液中地塞米松浓度均为10-7mol/l，红景天苷浓度分别为1，3，10，30，100umol/l)，培养小梁网细胞至第0，3，6，12，24，48h。
③小梁网细胞外基质蛋白基因和蛋白分泌检测：设计层粘连蛋白(LN)、IV型胶原蛋白(Col-IV)和纤维连接蛋白(FN)基因的引物序列，RT-PCR检测LN、COL-IV、FN的信使RNA表达；利用Anti-LN、Anti-Col-IV、Anti-FN抗体，ICC检测小梁网细胞外基质蛋白表达水平。
图1至图3说明：用Real time PCR法检测红景天苷对经地塞米松诱导的人小梁网细胞外基质的影响。结果发现，地塞米松可以使人正常小梁网细胞外基质增多，而加入红景天苷后，可以使小梁网细胞外基质减少。图中“*”表示P<0.05。
实施例2
1.、将红景天苷粉末溶解于生理盐水中，配置得40mg/kg红景天苷溶液；
2、选取雄性Balb/cJ小鼠8～12周龄，体重约10～15克，均符合一级动物标准。小鼠及饲料均由上海交通大学医学院附属第九人民医院实验动物中心提供。饲养室温15～20℃，相对湿度55～75％，12h光照维持，自由摄食饮水。纳入标准：①无眼部疾病。②双眼瞳孔直接对光反射和间接对光反射正常；
4、测量每只小鼠在清醒状态时左右两只眼的眼压，并进行记录；
5、对每只小鼠进行体重测量，采用4％水合氯醛腹腔注射方式麻醉小鼠，麻醉剂量为10μl/g；
6、对造模小鼠玻璃体腔进行TGFβ2过表达腺病毒注射，注射剂量为3μl，浓度为3×107pfu/μl，对侧眼玻璃体腔注射相同剂量及浓度的空载腺病毒；
7、造模后随机分为模型组(n＝6)、红景天苷干预组(n＝10)、红景天苷治疗组(n＝10)以及正常对照组(n＝6)，其中红景天苷干预组组于造模后立即开始给药，红景天苷治疗组待模型组眼压升高且处于稳定状态后立即开始给药，按40mg/kg红景天苷腹腔注射，每日1次。
8、于造模当天测量小鼠眼压一次，造模后第二天测量小鼠眼压一次，之后每隔 一段时间测量一次小鼠眼压，避免因眼压测量过于频繁导致小鼠眼角膜造成损伤，此外，腹腔注射红景天苷应于每日测量小鼠眼压之后，避免由于给药对眼压造成影响，另外每次测量眼压应于同一时间段进行，避免昼夜变化对小鼠眼压造成影响。
红景天苷对高眼压模型小鼠眼压干预结果，红景天苷干预后第11天，干预组的眼压低于模型组且具有统计学意义(图4)。
红景天苷治疗后高眼压模型小鼠眼压的结果，红景天苷治疗后第18天，治疗组的眼压低于模型组且具有统计学意义(图5)。
实施例3
1.将红景天苷粉末溶解于生理盐水中，分配配置得
Figure PCTCN2019091203-appb-000010
红景天苷滴眼液.
2、选取体重2.0～3.0kg健康成年新西兰白兔50只，雌雄兼用，雌雄各半，用1％地塞米松滴眼液对治疗组的白兔造模，采用滴双眼的方法，每日5次，诱导时间为3周。第2周开始监测眼压，以每日固定时间的眼压(IOP)>22mmHg并持续1周纳入诱发成功模型。50只兔双眼全部造模成功，平均眼压(26.07±2.03)mmHg(100只眼)，随机分为5组，每组10只，即正常对照组、阳性药组(采用苏为坦，0.004％曲伏前列腺素滴眼液)、本发明药物的三组(20uM、25uM、30uM红景天苷药物)。
阳性药组选择左眼滴用滴眼液苏为坦，每日第三次，每次2滴，每次给药后4h测眼压一次，持续两周；本发明的三组选择左眼滴用本发明滴眼液，每日第三次，每次2滴，每次给药后4h测眼压一次，持续两周；阳性药组和本发明组的右眼均作为对照眼，每日第三次，每次2滴，也是每次给药后4h测眼压一次，持续两周；对照组双眼每日第三次，每次2滴，也是每次给药后4h测眼压一次，持续两周。
对比实验日起一周后，每组每只眼每次测眼压的平均值见表1，从表1数据中看出，与作为模型对照组的右眼相比，阳性药组与本发明组的测得的左眼眼压均具显著性差异(P<0.01)。本发明组效果优于阳性药组。
表1眼压平均值
  左眼 右眼 IOP降低率
生理盐水对照组 21.39±1.09 21.75±1.38 ——
阳性对照组 17.59±1.06 22.10±1.24 20.41％
20uM眼药水 15.57±1.52 22.56±1.72 30.98％
25uM眼药水 15.07±1.19 23.03±1.02 34.56％
30uM眼药水 14.37±1.54 22.88±1.09 37.19％
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都涵盖在本发明范围内。

Claims (15)

  1. 红景天苷及其衍生物在制备眼科纤维化细胞外基质蛋白异常疾病的抑制剂药物中的应用。
  2. 根据权利要求书1所述的应用，其特征在于，所述的眼科纤维化细胞外基质蛋白异常疾病为青光眼、结膜瘢痕化、假性上睑下垂、眼部皮肤疤痕、老年性黄斑变性、糖尿病视网膜病变、后发性白内障中的一种或几种。
  3. 根据权利要求书1所述的应用，其特征在于，所述的眼科纤维化细胞外基质蛋白异常疾病为小梁网切除术后滤过道疤痕化导致的手术治疗失败及小梁网切除术后滤过道疤痕化。
  4. 一种治疗及预防眼科纤维化细胞外基质蛋白异常的疾病抑制剂药物，包含有效量的红景天苷为活性成分的片剂、胶囊、丸剂、口服液、针剂、眼药水或眼药膏。
  5. 根据权利要求4所述的药物，其特征在于，所述药物中包含有药学上可接受的辅料。
  6. 根据权利要求4所述的药物，其特征在于，所述的针剂包括经静脉注射针剂、肌肉注射针剂、皮下注射针剂、眼周注射针剂、眼球后注射针剂、結膜下注射针剂、巩膜注射针剂、眼内注射针剂。
  7. 根据权利要求5所述的药物，其特征在于，所述药物为眼药膏，其中红景天苷及其衍生物的浓度为
    Figure PCTCN2019091203-appb-100001
  8. 根据权利要求7所述的药物，其特征在于，所述药物为眼药膏，其中红景天苷及其衍生物的浓度为
    Figure PCTCN2019091203-appb-100002
  9. 根据权利要求4所述的药物，其特征在于，所述药物为眼药水，其中红景天苷及其衍生物的浓度为
    Figure PCTCN2019091203-appb-100003
  10. 根据权利要求9所述的药物，其特征在于，所述药物为眼药水，其中红景天苷及其衍生物的浓度为
    Figure PCTCN2019091203-appb-100004
  11. 根据权利要求4所述的药物，其特征在于，所述药物为眼内注射针剂，其中红景天苷及其衍生物的浓度为
    Figure PCTCN2019091203-appb-100005
  12. 根据权利要求4所述的药物，其特征在于，所述药物为結膜下注射针剂，其中红景天苷及其衍生物的浓度为
    Figure PCTCN2019091203-appb-100006
  13. 根据权利要求4所述的药物，其特征在于，所述药物为巩膜下注射针剂，其中红景天苷及其衍生物的浓度为
    Figure PCTCN2019091203-appb-100007
  14. 根据权利要求11所述的药物，其特征在于，所述药物为玻璃体腔内注射针剂，其中红景天苷及其衍生物的浓度为
    Figure PCTCN2019091203-appb-100008
  15. 根据权利要求11所述的药物，其特征在于，所述药物为前房内注射针剂，其中红景天苷及其衍生物的浓度为
    Figure PCTCN2019091203-appb-100009
PCT/CN2019/091203 2018-06-15 2019-06-14 红景天苷及其衍生物在制备眼科纤维化细胞外基质蛋白异常疾病的抑制剂药物中的应用 WO2019238107A1 (zh)

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