CN116874545B - 偶联药物及其制备方法与在制备治疗类风湿关节炎滑膜药物中的应用 - Google Patents
偶联药物及其制备方法与在制备治疗类风湿关节炎滑膜药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,涉及偶联药物及其制备方法与在制备治疗类风湿关节炎滑膜药物中的应用。偶联药物化学结构式如下所示:。本发明提供的偶联药物不仅能够治疗RA的炎症,而且能够治疗RA的滑膜侵袭以及骨破坏。
Description
技术领域
本发明属于生物医药技术领域,涉及偶联药物及其制备方法与在制备治疗类风湿关节炎滑膜药物中的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
类风湿关节炎(rheumatoid arthritis, RA)是一类以慢性、进行性、侵袭性关节炎为主要表现的全身性自身免疫病,若不经过正规治疗,病情会逐渐发展,最终会导致关节畸形、功能丧失、具有很高的致残率。据发明人研究了解,目前治疗RA缺乏特效药,即使改善病情的抗风湿药物(氨甲喋呤)疗效确切,但容易引发心血管和呼吸系统疾病;同时,目前的治疗药物主要是抑制RA患者体内的炎症反应,对于致残性的RA骨破坏则缺乏较好的治疗药物。
发明内容
为了解决现有技术的不足,本发明的目的是提供偶联药物及其制备方法与在制备治疗类风湿关节炎滑膜药物中的应用,本发明提供的偶联药物不仅能够治疗RA的炎症,而且能够治疗RA的滑膜侵袭以及骨破坏。
为了实现上述目的,本发明的技术方案为:
一方面,一种偶联药物,其化学结构式如下所示:
。
成纤维样滑膜细胞(Fibroblast-like synoviocytes, FLS)的凋亡异常及其介导的炎症反应在促进 RA病变进展中发挥重要作用。RA FLS 分泌大量 IL-1、IL-6 等炎性因子,介导滑膜炎症;增殖能力旺盛并抵御凋亡,引起滑膜组织异常增生;分泌血管内皮生长因子等促进血管翳的形成;产生多种不同的基质金属蛋白酶(Matrixmetalloproteinases, MMPs),促进其迁移和侵袭能力,造成软骨和骨破坏, 进而导致关节畸形甚至关节功能丧失。成纤维细胞活化蛋白(fibroblast activation protein,FAP)是一种Ⅱ型跨膜丝氨酸蛋白水解酶,在多种内源性多肽及多肽类药物的代谢中起着至关重要的作用。研究表明,FAP既可以作为肿瘤的潜在靶点,又可以作为类风湿性关节炎等疾病早期诊断的生物学标志物。
鉴于FAP是成纤维细胞特异性标记物,成纤维细胞活化蛋白抑制剂(fibroblastactivation protein inhibitor, FAPI)可以特异性被FAP阳性的细胞摄取,在识别RAFLSs中具有较好的特异性和敏感性,本发明将治疗药物偶联到FAPI上用于治疗RA,可显著提高药物进入病变关节的浓度和疗效、减轻对其他器官的毒副作用。
阿霉素(Doxorubicin,Dox或DOX),具有较强的抗肿瘤作用,能抑制肿瘤细胞DNA和RNA的合成,有强烈的细胞毒性作用,临床用于缓解多种肿瘤,包括急性白血病、恶性淋巴瘤、乳腺癌、肺癌等,而关于其在类风湿关节炎中的功能尚不明确。本发明经过研究意外地发现,以FAPI作为载体,通过偶联阿霉素(FAPI-Dox)特异性抑制RA FLSs活性,从而达到延缓疾病进展、治疗疾病的功效。
另一方面,一种上述偶联药物的制备方法,包括按照如下反应路线获得所述偶联药物的步骤;
。
具体地,将3,3'-二硫代二丙酸进行分子内酸酐化反应获得化合物12,将化合物12与DOX进行酰胺化反应获得化合物13,将化合物13与化合物11进行酰胺化反应,即得。
在一些实施方式中,化合物11按照如下反应路线合成;
。
具体地,6-羟基喹啉-4-羧酸与1-溴-4-氯丙烷进行取代反应获得化合物9,化合物9与N-叔丁氧基羰基哌嗪进行取代反应获得化合物10,化合物10与化合物8进行酰胺化反应获得化合物11。
在一些实施方式中,化合物8按照如下反应路线合成;
。
具体地,1-(叔丁基)2-甲基(2S,4R)-4-羟基吡咯烷-1,2-二羧酸酯进行还原反应获得化合物1,化合物1进行氟化反应获得化合物2,化合物2进行水解反应获得化合物3,化合物3与氨进行酰胺化反应获得化合物4,化合物4进行Boc脱保护反应获得化合物5,化合物5与N-(叔丁氧基羰基)甘氨酸(Boc-甘氨酸)进行酰胺化反应获得化合物6,化合物6脱水获得化合物7,化合物7进行Boc脱保护反应获得化合物8。
第三方面,一种药物组合物,包括上述偶联药物、所述偶联药物的药用盐或所述偶联药物的溶剂化物。
本发明所述药用盐可以为盐酸盐、硫酸盐、乙酸盐、枸橼酸盐、甲苯磺酸盐等。
第四方面,一种药物制剂,包括活性成分和药用辅料,所述活性成分为上述偶联药物或上述药物组合物。
本发明所述的药用辅料可以为药用载体或赋形剂。所述药用载体可以为氧化铝、硬脂酸铝、卵磷脂、血清蛋白、磷酸盐缓冲溶液、聚乙二醇等。药用载体在药物制剂中的重量百分数可以为1~98%,通常为75~85%。本发明所述赋形剂可以为粘合剂、润滑剂、填充剂、崩解剂、增溶剂等。本发明所述的药物制剂的剂型可以为片剂、颗粒剂、溶液剂、注射剂等。
第五方面,一种上述偶联药物、药物组合物或药物制剂在制备治疗类风湿关节炎滑膜药物中的应用。
具体地,所述治疗类风湿关节炎滑膜药物为治疗类风湿关节炎滑膜侵袭和/或治疗类风湿关节炎骨破坏的药物。
本发明的有益效果为:
通过动物实验表明,本发明提供的偶联药物FAPI-Dox,不仅能够明显缓解骨松质层疏松、减少滑膜侵袭,而且能够改善软骨退化,减轻骨侵蚀的程度。
通过细胞增殖实验表明,本发明提供的偶联药物FAPI-Dox能够抑制RA FLSs细胞的增殖;通过实时荧光定量实验表明,本发明提供的偶联药物FAPI-Dox能够降低炎性因子TNF-α、IL-1β、MMP1、CCL2、MMP3的表达水平;通过细胞迁移和侵袭实验表明,本发明提供的偶联药物FAPI-Dox能够降低RA FLSs的迁移和侵袭能力;通过诱导血管内皮细胞成环表明,本发明提供的偶联药物FAPI-Dox能够抑制细胞血管翳的形成。
综上,本发明提供的偶联药物FAPI-Dox对类风湿关节炎的治疗作用。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明实施例中制备偶联药物FAPI-Dox的1H NMR谱图;
图2为本发明实施例中制备偶联药物FAPI-Dox的13C NMR谱图;
图3为本发明实施例中制备偶联药物FAPI-Dox的质谱图;
图4为本发明实施例中FAPI-Dox缓解胶原诱导性关节炎小鼠症状示意图,A为FAPI-Dox治疗缓解CIA小鼠爪子厚度情况,B为FAPI-Dox治疗改善CIA小鼠关节炎指数情况,C为FAPI-Dox治疗改善CIA小鼠爪子关节炎症状情况;
图5为本发明实施例中FAPI-Dox缓解胶原诱导性关节炎小鼠骨破坏程度的Micro-CT分析示意图,A为Micro-CT分析FAPI-Dox治疗后CIA小鼠骨破坏程度,B为Micro-CT分析FAPI-Dox治疗后CIA小鼠的相对骨体积,C为Micro-CT分析FAPI-Dox治疗后CIA小鼠的骨小梁骨密度,D为Micro-CT分析FAPI-Dox治疗后CIA小鼠的骨小梁分离度;
图6为本发明实施例中FAPI-Dox缓解胶原诱导性关节炎小鼠骨破坏的组织学染色示意图,A为HE染色分析FAPI-Dox治疗后CIA小鼠软骨组织结构图,B为番红固绿染色分析FAPI-Dox治疗后CIA小鼠软骨结构,C为甲苯胺蓝染色分析FAPI-Dox治疗后CIA小鼠软骨结构;
图7为本发明实施例中FAPI-Dox对类风湿关节炎滑膜成纤维细胞增殖能力的影响,A为不同浓度FAPI-Dox作用于RA FLSs后CCK8检测细胞增殖情况,B为EdU处理后经免疫荧光检测FAPI-Dox作用于RA FLSs的细胞增殖情况,C为免疫荧光染色的统计图;
图8为本发明实施例中FAPI-Dox对类风湿关节炎滑膜成纤维细胞分泌炎性因子能力的影响,A为qRT-PCR检测FAPI-Dox作用后RA FLSs细胞分泌炎性因子TNF-α水平,B为qRT-PCR检测FAPI-Dox作用后RA FLSs细胞分泌炎性因子IL-1β水平,C为qRT-PCR检测FAPI-Dox作用后RA FLSs细胞分泌炎性因子MMP1水平,D为qRT-PCR检测FAPI-Dox作用后RA FLSs细胞分泌炎性因子CCL2水平,E为qRT-PCR检测FAPI-Dox作用后RA FLSs细胞分泌炎性因子MMP3水平;
图9为本发明实施例中FAPI-Dox对类风湿关节炎滑膜成纤维细胞生物学功能影响的示意图, A为Transwell法检测FAPI-Dox对RA FLSs细胞侵袭能力的影响,B为侵袭实验统计图,C为Transwell法检测FAPI-Dox对RA FLSs细胞迁移能力的影响,D为迁移实验统计图,E为FAPI-Dox对CRL-1730细胞血管形成的影响,F为血管形成实验统计图。
具体实施方式
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例详细说明本发明的技术方案。
实施例
偶联药物FAPI-Dox的合成:
一、化合物8的合成:
反应路线如下:
具体步骤为:
步骤1:0°C条件下,将 1,3,5-三氯-1,3,5-三嗪-2,4,6-三酮(3.78 g, 16.27mmol)添加到25 ml含有1-(叔丁基)2-甲基(2S,4R)-4-羟基吡咯烷-1,2-二羧酸酯(3.8 g,15.49 mmol)的DCM溶液中,然后添加催化 TEMPO(0.024 g,0.155mmol)。5 分钟后,使混合物达到室温,再搅拌 30 分钟,并在硅藻土上过滤。用 20 ml 饱和碳酸钾溶液、硫代硫酸钠、盐水清洗有机层,用无水硫酸钠干燥,过滤并蒸发。粗化合物1为未经进一步净化而使用。
步骤2:将步骤1获得1-(叔丁基)2-甲基(S)4-氧代吡咯烷-1,2-二酸酯溶液(化合物1,0.23 g, 0.946mmol) 溶于DCM(3 ml)中,在配备有氮气和搅拌棒的 25 ml 烧瓶中,在室温下用2 ml含有二乙氨基三氟化硫(DAST,0.197 ml,1.607 mmol)DCM溶液处理。添加乙醇(0.011 ml,0.189 mmol),并在室温下将混合物搅拌18小时。将溶液倒入饱和碳酸氢钠中,停止二氧化碳释放后,将其提取到DCM(3×15 ml)中,干燥(Na2SO4),过滤,并在真空中蒸发。在 DCM 的硅胶上进行色谱分析,得到一种淡黄色的油状化合物2。
步骤3:将从步骤2获得的化合物2(1.51 g,5.69 mmol)溶解在 6 ml 1M 氢氧化钾溶液中。将溶液搅拌过夜。用乙醚洗涤混合物,酸化,用乙酸乙酯萃取,用盐水洗涤,用硫酸钠干燥,过滤并蒸发,得到略带褐色的晶体化合物3。未经进一步纯化即可使用。
步骤4:在 50 mL 圆底烧瓶中,在 15°C 下将步骤3中获得的化合物3(1.6 g,6.37mmol)溶解在10 mL二氯甲烷中。然后添加1-羟基吡咯烷-2,5-二酮(HONSu)(0.806 g,7.01mmol)。在剧烈搅拌下向形成的悬浮液中添加N,N-二环己基碳二亚胺(DCC)(1.445 g,7.01mmol),继续搅拌形成悬浮物。让混合物达到室温并搅拌 30 分钟,然后在甲醇(2.002 ml,14.01mmol)中添加 7N 氨,并再搅拌 20 分钟。在蒸发挥发性成分之前,向烧瓶中添加1勺硅藻土。向残渣中添加冷乙酸乙酯,并在硅藻土上过滤。滤液用饱和碳酸氢钠洗涤。形成的微黄色晶体化合物4,未经进一步纯化即可使用。
步骤5:将 9.54 ml 三氟乙酸添加到10 ml含有化合物4(1.25 g,5 mmol)的二氯甲烷溶液中。溶液在蒸发前搅拌1小时。残渣用乙醚洗涤,得到白色晶体化合物5。
步骤6:将 HATU(12.47 g,32.8 mmol)溶解在 20 ml DMF 中,并添加至30 ml含有N-(叔丁氧基羰基)甘氨酸(5.75 g,32.8 mmol)和 DIPEA(5.43 ml, 32.65mmol)的DCM溶液中。 10 分钟后,将化合物5(5.1 g,27.3mmol)溶液添加至40 ml含有DIPEA(9.1 ml,54.4mmol)的DCM中。 3 小时后,混浊的混合物消失过滤掉。滤液冷却后再次过滤。结合的残留物用洗涤液洗涤 DCM 和水,由乙酸乙酯再结晶,获得化合物6。
步骤7:在 50 ml 圆底瓶中,将化合物6(0.720 g,2.343 mmol)加入至-15°C 的干燥 THF 中溶解。然后添加吡啶,然后逐滴添加5 ml 2,2,2-三氟乙酸酐(TFAA)(0.094 ml,0.664 mmol)的DCM溶液,滴加完毕后,升温至室温,将反应混合物搅拌 90 分钟。用 1 M 盐酸水溶液洗涤反应混合物。然后用饱和碳酸氢钠和盐水冲洗有机层三次,用硫酸钠干燥并蒸发。使用柱色谱法(己烷-乙酸乙酯 2-3)纯化粗混合物,得到淡黄色油状化合物7。
步骤8:将对甲苯磺酸-水合物(3.59 g,18.87 mmol)添加到 35ml含有化合物7(3.9 g,13.48 mmol)的乙腈冷却(0℃)溶液中。将混合物缓慢加热至室温并搅拌 24 小时。将残留物蒸发至干燥,并用冷乙醚和冷乙酸乙酯洗涤,然后再次干燥,以产生白色晶体化合物8。
二、化合物11的合成:
反应路线如下:
具体步骤为:
步骤1:将 20.2µL(30.1 mg; 176µmol) 1-溴-4-氯丙烷添加到含有 9.52 mg(50.3µmol) 6-羟基喹啉-4-羧酸和 159 mg(489µmol)碳酸铯的悬浮液中,并在 250µL DMF中加热至 60°C 过夜。将反应混合物冷却至室温,用 250µL 水和 500µL 乙腈稀释,然后添加 100µL 6 M NaOH。酯完全水解完成后,通过 HPLC(5-40%)直接纯化反应混合物。冻干后获得 10.1 mg(35.8µmol;71%)的化合物9。
步骤2:将 10.3 mg(36.7µmol)化合物9、 39.2 mg(210µmol) N-叔丁氧基羰基哌嗪和 37.4 mg(226µmol)碘化钾溶解在 250µL DMF 中。反应在 60°C 下摇动一夜。在通过HPLC 纯化产物之前,用 750µL 水稀释所得悬浮液。冷冻干燥后,获得 17.7 mg(32.5µmol;89%)的化合物10。
步骤3:将 100µL含有13.3 mg(35.0µmol)HBTU的DMF溶液添加到 100µL含有12.5mg(30.0µmol)化合物10、 7.87 mg(58.3µmol)HOBt 和 13.7µL(10.1 mg;75µmol) DIPEA的DMF溶液中。15 分钟后,加入 17.3 mg(37.5µmol)化合物8和 13.7µL(10.1 mg;75µmol)二苯甲酸二乙酯(100µL DMF)。用 850µL 水淬灭反应,并通过 HPLC 纯化。冷冻干燥得到13.2 mg(22.4µmol;75%) 目标化合物11。
三、偶联药物FAPI-Dox(化合物14)的合成:
反应路线如下:
具体步骤为:
步骤1:将1g 3,3'-二硫代二丙酸溶解于10ml 醋酐中,然后在室温下搅拌 6h,浓缩以去除醋酐,得到化合物12。
步骤2:将化合物12(2eq)和 Dox(1eq)溶解于 20mlDMF,然后添加 DIPEA(3eq),在室温下搅拌 1h,浓缩,粗品用硅胶柱纯化(DCM:MeOH=15:1),得到化合物13。
步骤3:将化合物11(1eq)溶解在400µL乙腈中,并添加适量4-甲苯磺酸-水合物,在45°C下摇动2小时,然后在减压下去除挥发物。再加入化合物13(1eq)和HATU(1.2eq),最后加入DIPEA(3eq),室温反应1小时,减压下去除挥发物后通过HPLC纯化。冷冻干燥后获得目标产物,如图1~3所示。
细胞学实验
1)利用ELISA检测细胞培养上清中细胞因子的分泌水平;
(1-1)稀释标准品:按照0、1、10、50、100、200、500、800、1000 ng/mL的浓度梯度对标准品进行稀释,用于绘制标准曲线。
(1-2)加标准品和待测样品:将收集得到的C57BL/6小鼠血清放入低温离心机中高速离心,取上清,将血清和配制好的不同浓度的蛋白标准品按照100µl/孔的量加入各待测孔,37℃温箱孵育90分钟,此过程要用封口膜将ELSIA板封好,防止样品挥发,影响实验结果。
(1-3)洗板并拍干后,加生物素化的抗体:将试剂盒中提供的生物素化抗体按照100µl/孔的量加入除了空白孔之外的待测孔中,用封口膜封闭好,37℃温箱孵育1小时。
(1-4)洗板并拍干后,加入酶反应化合物。
(1-5)洗板并拍干后,先后加入显色剂和终止液各100µ1/孔;轻轻摇晃混匀,将检测板放入酶标仪中,450nM波长检测各孔数值。
(1-6)根据标准品的OD值绘制标准曲线,并计算样品中FAPI分泌水平。
2)利用免疫组织化学检测滑膜组织中FAPI的表达,利用HE染色检测滑膜组织的损伤程度;
(2-1)取新鲜组织经固定、脱水、浸蜡和包埋等,制成石蜡切片。石蜡切片置于67℃烘箱中,烘片2小时,脱蜡至水,用pH7.4的PBS冲洗三次,每次3分钟(3×3’)。
(2-2)取一定量pH=6.0柠檬酸盐缓冲液,加入微波盒中,微波加热至沸腾,将脱蜡水化后的组织切片置于耐高温塑料切片架上,放入已沸腾的缓冲液中,中档微波处理10分钟,取出微波盒流水自然泠却,从缓冲液中取出玻片,先用蒸馏水冲洗两次,之后用PBS冲洗2×3’。
(2-3)每张切片加1滴3%H2O2,室温下孵育10分钟,以阻断内源性过氧化物酶的活性。PBS冲洗3×3’。
(2-4)除去PBS液,每张切片加1滴相应的第一抗体(相应稀释倍数),室温下孵育2小时。
(2-5)PBS冲洗3×5’。除去PBS液,每张切片加1滴聚合物增强剂,室温下孵育20分钟。PBS冲洗3×3’。
(2-6)除去PBS液,每张切片加1滴酶标二抗,室温下孵育30分钟。PBS冲洗3×5’。
(2-7)除去PBS液,每张切片加1滴新鲜配制的DAB液(二氨基联苯胺),显微镜下观察5分钟。
(2-8)苏木素复染,0.1%HCl分化,自来水冲洗,蓝化,切片经梯度酒精脱水干燥,二甲苯透明,中性树胶封固,晾干后观察。
3)利用Transwell实验检测细胞迁移和细胞侵袭;
(3-1)血清饥饿处理:将处于对数生长期的RASFs换为含0.5%血清的培养基,37℃,5%CO2孵箱中培养12-24 h。
(3-2)制备细胞悬液:终止消化后离心弃去培养液,用0.5%血清培养基制备细胞悬液。计数细胞,调整细胞密度至1-10 ×105/ml。
(3-3)接种细胞:
a.取含有RASFs的细胞悬液200µl加入Transwell小室,注意枪头垂直,将细胞悬液缓慢滴入小室。
b.24孔板下室一般加入600µl完全培养基(保证下层培养液和小室间无气泡产生)。
c.培养细胞:将细胞培养板置于含37℃,5%CO2的细胞培养箱中培养24h。(此时应注意种板后将细胞培养板平稳放入孵箱中,使小室保持水平)。
(3-4)染色观察:
a.用棉签擦去基质胶和上室内的细胞。
b. 24孔板每孔加入1mL 4%多聚甲醛,将小室放入其中固定5-10min,用棉签擦除上室内的多聚甲醛和上室内的细胞。
c.避光操作:24孔板每孔加入600µL 0.1%结晶紫染液,将小室放入其中,染色20min,蒸馏水冲洗掉多余染液。
(3-5)镜下观察并拍照。
a.封片:用刀片将小室底部的膜延边缘切下。
b.在盖玻片上滴一滴中性树胶,用镊子将膜平铺于中性树胶上,在膜上再滴一小滴中性树胶。
c.盖玻片轻轻盖住膜,注意将气泡赶净。
(3-6)结果处理:在相同倍数下数五个视野的细胞数,求其平均值,再进行比较。
细胞迁移实验:具体步骤同上,区别是在小室上层铺入100微升基质胶。
动物实验
选取10-14周龄DBA/1小鼠为研究对象,采用胶原对小鼠进行两次胶原注射,免疫诱导关节炎症状,称为CIA小鼠。在评价FAPI抗体的预防性作用时,将其在初次免疫的当天注射;评价其治疗效果时,是当小鼠关节炎指数达到6时注射。
结果:
1、FAPI-Dox缓解胶原诱导性关节炎小鼠的骨破坏程度。CIA关节炎小鼠构建成功后,使用FAPI、Dox、FAPI-Dox对其进行治疗,治疗期间,测量记录鼠爪厚度、对小鼠的关节肿胀程度进行评分以及对鼠爪进行拍照。结果如图4A-C所示,FAPI-Dox的治疗相较于Dox和FAPI的治疗组更能缓解CIA关节小鼠的爪子厚度以及关节的肿胀程度,对CIA关节炎小鼠具有更好的治疗效果。采用Micro-CT对Ctrl组,DMSO组,Dox、FAPI、FAPI-Dox治疗组小鼠分别进行了骨密度相关参数的测定,3D扫描的结果表明(图5A),FAPI-Dox可以更好的缓解CIA关节炎小鼠的骨破坏程度。通过分析骨密度的相关参数(图5B-D),DMSO处理的CIA组的相对骨体积、骨小梁骨密度显著降低,骨小梁分离度升高。而FAPI、Dox、FAPI-Dox治疗后这种趋势被逆转,相对骨体积、骨小梁骨密度显著升高,骨小梁分离度降低,尤其在FAPI-Dox治疗组这一趋势更为明显。类风湿关节炎的关节中慢性炎症环境会导致滑膜增生,侵入软骨-骨交界处的关节周骨,导致骨侵蚀和软骨退化。对骨组织切片进行苏木精-伊红(HE)染色、番红固绿染色、甲苯胺蓝染色,研究骨关节组织形态的变化。苏木素的嗜酸性可使细胞核染成蓝色,嗜碱性的伊红可使胞浆染成红色从而显示骨组织基本的形态结构。HE的染色结果表明(图6A),Ctrl组关节软骨表面比较光滑,潮线清晰较完整,骨细胞分布均匀,骨松质层密度适中,无滑膜侵袭;CIA组软骨表面粗糙,完整性遭到破坏,潮线扭曲不完整,软骨细胞增生,骨松质层疏松,滑膜增生侵袭严重;FAPI、Dox、FAPI-Dox治疗组与DMSO组相比,软骨表面变得光滑,潮线清晰完整,骨松质层疏松的情况得到缓解,滑膜侵袭减少,FAPI-Dox的治疗作用更明显。番红与多糖中阴离子基结合呈红色。番红的着色间接反映了基质中蛋白多糖的含量和分布。当软骨受损时,软骨糖蛋白被释放出来,导致基质成分分布不均,所以番红的着色轻微或根本不着色。嗜酸性染料的固绿与软骨下骨胶原纤维结合,呈绿色。番红固绿染色结果显示(图6B),与DMSO组相比,FAPI、Dox、FAPI-Dox治疗组红色深,阳性范围大,固绿的范围减少,FAPI-Dox的趋势更明显,表明FAPI-Dox改善了软骨退化,减轻了骨侵蚀的程度。甲苯胺蓝与软骨基质中的糖胺聚糖结合呈紫蓝色,软骨下骨基质呈浅蓝色。甲苯胺蓝染色结果显示(图6C),与CIA组相比,FAPI-Dox治疗组紫蓝色深,阳性范围最大。体内实验结果表明FAPI-Dox治疗类风湿关节炎的潜力。
2、FAPI-Dox对类风湿关节炎滑膜成纤维细胞生物学功能的影响。本课题以RAFLSs为研究对象,评价FAPI-Dox抗类风湿性关节炎的作用。为了获得FAPI-Dox的最佳浓度,首先使用CCK-8试剂盒进行细胞计数。如图7A所示,当FAPI-Dox的浓度在200nM -200μM时,可浓度依赖性的抑制细胞的增殖。因此,选择100μM的FAPI-Dox用于后续的细胞分析。在RAFLSs中,给予IL-1β(10ng/ml) 和 TNF-α(10ng/ml)刺激,可活化细胞增殖、分泌炎性因子以及迁移侵袭等细胞表型。RA的主要特征是滑膜细胞的异常增生,本实施例采用EdU实验进一步探究了FAPI-Dox对细胞增殖的影响。如图7B结果所示蓝色荧光代表DAPI,红色荧光代表新增殖的RA FLSs细胞,图7C为统计图,与对照组相比,IL-1β和 TNF-α刺激可显著促进RAFLSs增殖,而FAPI-Dox可以抑制这种增殖。实时荧光定量实验(RT-q PCR)检测RA FLSs中炎性因子的表达,如图8,与对照比相比, IL-1β和 TNF-α刺激组的炎性因子TNF-α、IL-1β、MMP1、CCL2、MMP3的表达水平显著升高;然而,在刺激条件下,这种增加在FAPI-Dox治疗后大多被逆转,证明FAPI-Dox可以抑制RA FLSs的炎性因子分泌。由于RA的病理特征是滑膜细胞的异常增生并侵袭至周围的软骨及骨组织,本实施例通过Transwell法检测FAPI-Dox对RAFLSs迁移和侵袭能力的影响。如图9A-D所示,在IL-1β和TNF-α的刺激下,RA FLSs的迁移和侵袭能力都显著升高,而加入FAPI-Dox后,RA FLSs的迁移(图9A-B)和侵袭能力(图9C-D)有所降低。RA的另一个主要特征是炎性细胞浸润,诱导血管内皮细胞成环,形成血管翳。本实施例研究了FAPI-Dox对细胞成环的影响。如图9E-F结果显示,经IL-1β、TNF-α和DMSO处理的RA FLSs培养上清孵育后的CRL-1730细胞的血管形成数目显著增加,而经IL-1β、TNF-α和FAPI-Dox处理的RA FLSs培养上清孵育的CRL-1730细胞的血管形成数目有所降低。表明FAPI-Dox能抑制细胞血管翳的形成。细胞实验结果进一步显示出FAPI-Dox对类风湿关节炎的治疗作用。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,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、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种偶联药物,其特征是,其化学结构式如下所示:
。
2.一种权利要求1所述的偶联药物的制备方法,其特征是,包括按照如下反应路线获得所述偶联药物的步骤;
3.如权利要求2所述的偶联药物的制备方法,其特征是,化合物11按照如下反应路线合成;
。
4.一种药物组合物,其特征是,包括权利要求1所述的偶联药物或所述偶联药物的药用盐。
5.如权利要求4所述的药物组合物,其特征是,所述药用盐为盐酸盐、硫酸盐、乙酸盐、枸橼酸盐或甲苯磺酸盐。
6.一种药物制剂,包括活性成分和药用辅料,其特征是,所述活性成分为权利要求1所述的偶联药物或权利要求4或5所述的药物组合物。
7.如权利要求6所述的药物制剂,其特征是,所述的药用辅料为药用载体或赋形剂。
8.一种权利要求1所述的偶联药物、权利要求4或5所述的药物组合物或权利要求6或7所述的药物制剂在制备治疗类风湿关节炎滑膜药物中的应用。
9.如权利要求8所述的偶联药物、药物组合物或药物制剂在制备治疗类风湿关节炎滑膜药物中的应用,其特征是,所述治疗类风湿关节炎滑膜药物为治疗类风湿关节炎滑膜侵袭和/或治疗类风湿关节炎骨破坏的药物。
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