CN110403925A - 化学小分子4-氨基联苯在防治骨关节炎的应用 - Google Patents
化学小分子4-氨基联苯在防治骨关节炎的应用 Download PDFInfo
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Abstract
本发明公开了4‑氨基联苯(4‑ABP)作为小分子化合物在治疗骨关节炎的应用。实验表明,关节局部应用4‑ABP可有效修复小鼠DMM所致软骨损伤,可提高Collagen II、CD44、CD105蛋白的表达水平,降低Collagen Ⅹ的表达量,增加软骨厚度及软骨基质的表达。因此本发明证明了4‑ABP可有效减少骨关节炎时软骨损伤,延缓软骨退变。本发明提供的4‑氨基联苯(4‑ABP)小分子化合物在治疗骨关节炎的应用可为医学研究及组织工程学研究等提供全新的促进损伤软骨再生的方案,该发明也包括含4‑ABP的片剂、注射剂、粉针剂等对骨关节炎等软骨退变与损伤的治疗作用。
Description
技术领域
本发明属于细胞医药领域,具体而言,本发明涉及4-氨基联苯(4-ABP)减轻骨关节炎损伤及修复损伤的应用。
背景技术
骨关节炎(osteoarthritis,OA)主要分成两大类:由局部创伤引起的创伤性OA,或因个体老化所致的老年性OA,其中后者在老年及肥胖人群中的发病率极高。OA主要病理特征在于关节软骨的退行性病变、滑膜炎症、软骨下骨损伤、骨重塑、骨赘形成、细胞外基质降解以及其他关节结构的改变,最终导致关节疼痛、僵硬甚至是运动功能障碍等,严重影响生活质量。
年龄的增长、遗传因素和机械应激均是引起OA的危险因素。随年龄增长,OA的发病率逐渐升高;且女性的发病率明显高于男性,可能由于女性体内激素的原因。据流行病学研究显示,在40岁之前,OA发病率较低,最常见的是继发性骨关节炎,通常是由于创伤;在40~60岁间,OA发病率增加,且呈线性增长。据统计,60岁以上的人群中,有9.6%的男性和18%的女性可能患有骨关节炎;而75岁以上人群中,骨关节炎的发病率为80%。同时,遗传、外伤、肥胖、职业等因素也是引起OA的主要原因。
骨关节炎的发病部位主要在关节软骨、软骨下骨及滑膜(图1)。关节软骨的成分包含软骨细胞和细胞外基质(extracellular matrix, ECM)。目前普遍认为健康的软骨细胞外基质主要有II型胶原蛋白(Collagen II,COL2)和蛋白聚糖(Aggrecan,ACAN)组成,它们为组织提供了拉力支撑,并在负载下提供了软骨的抗压和减震能力,有助于维持软骨细胞外环境和软骨结构的稳态。在OA的发病过程中,软骨的组成结构发生改变,如:软骨细胞发生死亡、软骨细胞受到炎性细胞因子的刺激从而导致细胞外基质的降解,细胞外基质的合成与分解失去平衡,基本结构被破坏,主要表现在ACAN含量降低,胶原蛋白的类型从II型胶原变为I型胶原。另外,有研究显示,炎性细胞因子,如白细胞介素-1β(Interleukin-1β,IL-1β)、肿瘤坏死因子-α(Tumor Necrosis Factor-α,TNF-α)和白细胞介素-6(interleukin6,IL-6),可刺激软骨细胞分泌一种蛋白水解酶基质金属蛋白酶(matrixmetallaproteinases,MMPs),从而引发软骨基质的改变,导致软骨炎症或病变。因此,许多研究通过抑制这些炎性细胞因子的表达,并增加软骨基质的表达作为治疗OA的方式。目前对于OA的发病机理仍多有不清,处于探索阶段,有待进一步的详细研究。
目前针对骨关节炎的治疗包括非药物治疗、药物治疗以及手术。对于早期OA患者,通常建议使用非药物手段进行治疗,如:物理治疗以及适当的锻炼,肥胖患者应进行减肥,还可使用拐杖以减轻关节的负荷。对于终末期OA,目前只有通过手术的方法进行治疗,如:人工关节置换术,通常需要花费高昂的治疗费用且可能出现并发症。而对于处于中期OA的患者通常采取药物治疗,包括非甾体类抗炎药(nonsteroidal anti-inflammatory drugs,NSAIDS)、对乙酰氨基酚、曲马多和阿片类药物、硫酸软骨素和氨基葡萄糖等,可起到缓解症状的作用,但仅有有限的证据表明这些药物可延缓疾病的进展。一些防止OA的新型药物或制剂,如阿达木单抗(抗TNF-a单抗)、托珠单抗(IL-6受体单抗)和MOR-103(抗粒细胞-巨噬细胞集落刺激因子单抗)、甲氨蝶呤和维生素D, 目前多仍处于实验研究或临床试验的阶段。关节腔注射给药是治疗骨关节炎的重要方式,可提高药物在局部组织的有效浓度及有效性,同时也提高药物的安全性,包括糖皮质激素、透明质酸钠,及干细胞制剂等,都已在临床前试验或临床工作中得以实施。
迄今为止,上述多数治疗骨关节炎的潜在药物都具有一定的局限性,一般仅能达到减缓关节疼痛的效果,而未能达到根本减轻退变,或促进损伤组织的再生修复作用。因此,现阶段迫切需要能够有效改善疾病的骨关炎药物(disease-modifyingosteoarthritis drugs,DMOADs),其既能延缓关节结构进展又可缓解疾病症状。
化学小分子Kartogenin (KGN) 被证明可软骨保护,已在美国获得专利保护。多项研究证实,KGN单独注射或与生物支架结合使用,均可促进组织工程软骨再生。Kang ML等人用壳聚糖与KGN偶联***(CHI-KGN)体外诱导hBMMSCS成软骨分化,并将CHI-KGN注射到OA大鼠关节内,有效减少了关节软骨的退变。此外,Mohan G等人研究了KGN对OA大鼠软骨和软骨下骨的预防作用,在OA造模一周后每周接受关节腔内注射125µM KGN治疗,结果表明KGN可预防OA大鼠软骨及软骨下骨的退变。迄今KGN治疗骨关节炎还处于实验阶段,对于其作用机制研究非常有限。
实验证实,受试动物口服和静脉注射KGN后,应用高压液相色谱-质谱(HPLC-MS)技术,在在其血液中能检测到KGN及其水解产物4-ABP,;而受试动物软骨组织内未予检测到KGN,但能检测到KGN水解产物4-ABP(图2)。进一步测试表明,化学合成的小分子4-ABP本身具有较KGN相似或更强的介导软骨再生修复的作用(附图-3),即在相同的摩尔浓度下,局部使用(即关节腔注射)4-ABP后,已有损伤退变的软骨比同样使用KGN的软骨组织更容易修复再生。
虽然有研究报道4-氨基联苯(4-ABP)是烟草等环境污染物中的可能引起膀胱癌的成分之一,但接触后癌症的发生可能需数年或数十年;有研究证据还表明4-ABP是一种DNA反应性致癌物,即其致癌毒性依赖于肝脏相关酶的活化,而且单次口服剂量的4-ABP达15mg / kg的水平在5年内都未发现诱发***。因此,本说明书提出在相对封闭的关节腔内局部使用而不是***性使用,可避免4-ABP暴露于肝脏,从而可以保障4-ABP的最大有效性和安全性。
本发明涉及的4-氨基联苯(4-ABP)在关节炎的治疗研究中未见有相关报道。
发明内容
骨与软骨的修复受到广泛的关注,本发明解决的问题是研究证实4-氨基联苯(4-ABP)既能延缓关节炎所致的组织结构破环又可修复损伤的组织结构。本发明证实了软骨损伤过程后,使用4-ABP诱导软骨形成的能力比KGN更为强大。因此,4-氨基联苯(4-ABP)的这一特性使其成为临床开发新型软骨修复药物的潜在有效成分。
本发明的目的在于发现并证实小分子化合物4-氨基联苯(4-ABP)具有较高的促损伤软骨形成的能力。该小分子化合药物的发现弥补了现有治疗骨关节炎药物存在的缺陷——仅能减缓病症而未能达到治疗软骨损伤的效果,为临床软骨损伤修复治疗提供了新途径。
骨关节炎最直接的伤害是导致关节软骨的损伤,为了实现上述发明目的,本发明采用如下技术方案:
利用DMM软骨缺损模型模拟骨关节炎导致的软骨损伤,检测关节腔注射KGN和4-ABP对损伤软骨的修复作用。
1. 随机选取48只10周龄的雄性C57BL/6小鼠,取其一腿进行DMM造模。
2. 选取12只同等条件的小鼠对其一腿进行假手术处理作为对照组(Vehicle)。
3. DMM手术1个月后,将DMM造模的小鼠随机分为3组,每组12只,即:Model组(DMM造模)、KGN组(DMM造模后关节腔内注射10µM KGN)、4-ABP组(DMM造模后关节腔内注射10 µM4-ABP)。
4. 给药治疗:每周一次关节腔注射给药,连续给药4周。
5. 取样检测:将小鼠处死,取膝关节处进行组织学评价。软骨组织切片番红固绿染色;软骨组织切片进行Collagen X免疫组化染色;Collagen II、CD44与CD105进行组化免疫荧光染色。
4-氨基联苯(4-ABP)作为药物在治疗骨关节炎的应用。
作为本发明的进一步改进:所述药物服用方法是关节腔注射或与生物材料结合后关节局部的注射或移植。
作为本发明的进一步改进:所述药物的制药方式为片剂、注射剂,粉针剂。
作为本发明的进一步改进:所述的药物包含药学上有效剂量的4-氨基联苯和药学上可接受的辅料。
本发明公开了4-氨基联苯(4-ABP)作为小分子化合物药物在治疗骨关节炎导致的软骨缺损的应用。与现有技术相比,本发明的有益效果如下:连续给药4周治疗DMM软骨损伤小鼠,4-ABP组较KGN组蕃红染色阳性着色范围变大,表明可促进明显的软骨有修复再生。进一步的,软骨损伤给予4-ABP治疗,较之KGN组,4-ABP可令软骨组织内 Collagen II、CD44、CD105表达自发上调,而作为软骨损伤标记物的Collagen X表达量下调。4-ABP促软骨再生能力的提高,能够治愈软骨缺损,对于骨关节炎导致的软骨损伤治疗研究有十分重要的意义。本结果说明了4-氨基联苯(4-ABP)在软骨损伤修复方面有着促进作用。本发明为医药学及组织工程学领域骨关节炎治疗相关研究提供了新的方向。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,以下将对实施例或现有技术描述中所需要使用的附图进行简单的介绍。
图1 关节软骨损伤与退变基本病理特征。
图2 口服KGN后受试动物体内检测KGN或其代谢产物4-ABP的HPLC-MS分析。
图3为本实施例中关节腔内注射KGN、4-ABP对DMM关节软骨损伤的修复作用比较对比图。
图3中分组情况,Vehicle组:正常对照组,未进行DMM造模,展示正常软骨组织;Model组:DMM模型组,阴性对照组,展示损伤的软骨组织;KGN组:DMM后关节腔注射KGN药物,作为阳性对照组;4-ABP组:DMM后关节腔注射4-ABP药物。
图中各项指标染色检测情况,膝关节石蜡切片番红固绿染色:红色部分展示的为软骨组织,绿色部分展示的为骨组织;Collagen Ⅱ免疫荧光染色:绿色荧光为Collagen Ⅱ的表达,展示了软骨细胞数量;CD44/105荧光染色:红色荧光为CD44表达,绿色荧光为CD105表达,两者共表达区为软骨组织内干细胞的再生。Collagen Ⅹ免疫组化染色:棕黄色染色为Collagen X的表达,Collagen X为损伤软骨中表达,正常软骨中Collagen X表达量极低。
具体实施方式
现结合图3说明与实施例对本发明进一步说明,以下实施例仅为本发明的优选实施例,以便于更好地理解本发明,因而不应视为限定本发明的范围。对于本领域的技术人员来说,本发明可以有各种更改和变化,凡在本发明的精神和原则之内,所作的任何修改、等同替换或改进等,均应包含在本发明的保护范围之内。下列实施例中未注明具体条件者按照常规条件或制造商建议的条件进行,所用试剂或仪器未注明生产厂商者,均为可通过市售购买获得的常规产品。
实施例1
本实施例中的内侧半月板失稳模型(DMM)可模拟关节炎导致的软骨缺损。
1)取36只10周龄雄性C57BL/6J小鼠麻醉,备皮消毒皮肤,沿膝关节内侧切开皮肤,剥离肌肉组织,将关节腔韧带推向外侧,暴露股骨和胫骨,分离关节腔中脂肪组织,暴露内侧半月板韧带,然后用精细剪刀将其剪断,复位韧带及肌肉组织,缝合肌肉及皮肤,封闭关节腔。
2)术后给予青霉素钠(22000U/100g)肌肉注射,预防感染。
3)假手术组12只C57BL/6J小鼠仅打开关节腔后进行缝合,不进行韧带剪断。
实施例2
关节腔注射给药治疗软骨缺损。
1)DMM手术1个月后,将DMM造模的小鼠随机分为3组,每组12只,即:Model组(DMM造模)、KGN组(DMM造模后关节腔内注射100µM KGN)、4-ABP组(DMM造模后关节腔内注射100µM4-ABP)。
2)给药治疗:每周一次关节腔注射给药,连续进行4周给药治疗。
实施例3
关节软骨组织再生修复的检测。
(1)骨组织切片番红固绿染色。
1)50~60℃烤片2小时。
2)脱蜡复水:分别用二甲苯Ⅰ、Ⅱ各脱蜡10min,依次100%乙醇、100%乙醇、95%乙醇、85%乙醇、70%乙醇、50%乙醇各复水5min,流水冲洗5min。
3)固绿染色:0.05%固绿染色1~5min;1%冰醋酸快速洗涤10s~1min;去离子水洗干净遗留的酸溶液,显微镜下观察固绿染色情况,若着色浅则重新染色。
4)番红染色:0.5%番红O染色5~10min;95%乙醇脱色30s,显微镜下观察着色情况,染色理想则继续,否则水洗后重新染色。
5)脱水封片:100%乙醇、100%乙醇、二甲苯、二甲苯各浸泡30s,显微镜观察染色结果,若未达到效果重新染色;若染色理想,则用中性树脂胶封片,烘干。
(2)骨组织切片Collagen X免疫组化染色。
1)50~60℃烤片2小时。
2)石蜡切片脱蜡复水:分别用二甲苯Ⅰ、Ⅱ各脱蜡10min,依次100%乙醇、100%乙醇、95%乙醇、85%乙醇、70%乙醇、50%乙醇各复水5min,流水冲洗5min。
3)冰冻切片水化:PBS润洗5~10min,去除包埋剂。
4)用组化笔标记样本。
5)抗原修复:滴加骨组织抗原修复液,37℃,孵育1.5h-3h,PBS洗两次。
6)阻断内源性过氧化物酶:3% 过氧化氢溶液孵育10min,PBS洗两次。
7)封闭:5% BSA(PBS溶解),60min。
8)滴加Collagen X一抗 (Abcam,1:500),放入湿盒中,4℃过夜,PBS洗3次。
9)滴加辣根过氧化物酶标记的二抗(Abcam,1:2000),室温孵育1h,PBS洗3次。
10)滴加DAB显色液,反应10~60s,着色后停止反应,充分水洗。
11)脱水封片:70%乙醇、85%乙醇、95%乙醇、100%乙醇梯度脱水,二甲苯透明,中性树脂胶封片,显微镜观察。
(3)骨组织切片Collagen II、CD44、CD105免疫荧光染色。
1)50~60℃烤片2小时。
2)石蜡切片脱蜡复水:分别用二甲苯Ⅰ、Ⅱ各脱蜡10min,依次100%乙醇、100%乙醇、95%乙醇、85%乙醇、70%乙醇、50%乙醇各复水5min,流水冲洗5min。
3)冰冻切片水化:PBS润洗5~10min,去除包埋剂。
4)用组化笔标记样本。
5)抗原修复:滴加骨组织抗原修复液,37℃,孵育1.5h-3h,PBS洗两次。
6)阻断内源性过氧化物酶:3% 过氧化氢溶液孵育10min,PBS洗两次。
7)封闭:5% BSA(PBS溶解),60min。
8)滴加Collagen II、CD44、CD105一抗 (Abcam,1:500),放入湿盒中,4℃过夜,PBS洗3次。
9)滴加荧光二抗(Abcam,1:500),室温孵育1h,PBS洗3次。
10)使用70%甘油封片照相。
Claims (5)
1.4-氨基联苯(4-ABP)在调控软骨再生、防治骨关节炎中的应用。
2.根据权利要求1所述的应用,其特征在于:所述药物以天然化合物4-氨基联苯(4-ABP)、人工合成4-氨基联苯(4-ABP)为活性成分。
3.如权利要求2所述的具有4-氨基联苯(4-ABP)活性化合物的制备药物,应用于软骨再生和防治骨关节炎的治疗。
4.根据权利要求1所述的应用,其特征在于:所述的药物为片剂、注射液或粉针剂。
5.根据权利要求1所述的应用,其特征在于:所述的药物包含药学上有效剂量的4-氨基联苯(4-ABP)和药学上可接受的辅料。
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