CN116440131A - 培非替尼或其药学上可接受的盐的新用途 - Google Patents
培非替尼或其药学上可接受的盐的新用途 Download PDFInfo
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- CN116440131A CN116440131A CN202310286561.1A CN202310286561A CN116440131A CN 116440131 A CN116440131 A CN 116440131A CN 202310286561 A CN202310286561 A CN 202310286561A CN 116440131 A CN116440131 A CN 116440131A
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Abstract
本发明涉及培非替尼或其药学上可接受的盐在制备用于治疗滑膜炎的药物中的用途。本发明利用培非替尼或其药学上可接受的盐能够有效治疗滑膜炎。另外,本发明还涉及含有培非替尼或其药学上可接受的盐的药物制剂在制备用于治疗滑膜炎的药物中的用途。
Description
技术领域
本发明涉及药物领域,具体涉及培非替尼或其药学上可接受的盐在制备用于治疗滑膜炎的药物中的用途。
背景技术
色素沉着绒毛结节性滑膜炎(pigmented villonodullar synovitis,PVNS),也称弥漫型腱鞘巨细胞瘤(diffuse type-giant cell tumor,TGCT),是一种良性的关节内软组织肿瘤,病理特征是滑膜绒毛膜病变增生,伴有铁黄色素沉着和炎症细胞浸润,具有局部侵袭性。临床表现为关节长期肿胀疼痛,活动受限,穿刺会显示血性关节液。发病率为4/百万,多见于膝关节。
目前,PVNS的治疗方式以手术切除为主,但术后容易复发,复发率高达46%,且缺乏有效治疗药物。目前唯一获批治疗药物培西达替尼(Pexidartinib)的三期临床试验数据显示,有效率仅为39%。
因此,临床迫切需要治疗效果好的PVNS治疗药。
发明内容
本发明的目的是克服现有技术的缺点,提供疗效好的滑膜炎治疗药,即培非替尼或其药学上可接受的盐,其可用于制备治疗滑膜炎的药物。
为了实现以上目的,本发明提供如下技术方案。
培非替尼或其药学上可接受的盐在制备用于治疗滑膜炎的药物中的用途。
本发明利用培非替尼或其药学上可接受的盐能够有效治疗滑膜炎。所述培非替尼的结构式如下:
在本发明的一些实施例中,所述滑膜炎可选自色素沉着绒毛结节性滑膜炎(PVNS)。
PVNS滑膜主要由成纤维细胞样滑膜细胞(fibroblast-like synoviocytes,FLS)、多核巨细胞、淋巴细胞和巨噬细胞组成。其中,FLS是构成滑膜层结构的主要细胞类型。正常滑膜仅有1~2层FLS,而在PVNS滑膜中,FLS异常活化增生。同时,PVNS患者滑液中的炎症因子,包括白介素(interleukin,IL)-1β和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)明显上调。PVNS的转录组学研究显示,PVNS滑膜有显著的炎症反应,包括免疫细胞浸润和细胞因子分泌增加,还具有肿瘤表型。大量炎症细胞,包括T细胞、自然杀伤(naturalkiller,NK)细胞、NKT细胞和B细胞被从血液募集到滑膜,破骨细胞生成和巨噬细胞激活在PVNS滑膜明显增加。活化的巨噬细胞释放大量炎症因子,与活化的FLS形成级联放大反应,与滑膜炎症、高复发率、侵袭和软骨破坏密切相关。因此,抑制FLS和巨噬细胞异常活化,在PVNS治疗中具有重要意义。
Janus激酶(Janus kinase,JAK)-信号转导因子和转录激活因子(signaltransducers and activators of transcription,STAT)通路是重要的细胞因子信号转导通路之一,在细胞增殖、分化、凋亡和炎症反应等许多生物学过程中发挥重要调节作用。该通路主要由三个成分组成:酪氨酸激酶偶联受体、非受体酪氨酸激酶JAK和转录因子STAT。受体与配体结合后发生二聚化,激活与其偶联的JAK,活化的JAK使下游STAT磷酸化,两个磷酸化的STAT分子形成二聚体进入细胞核,与相应靶基因启动子结合,调控目的基因的转录和表达。与正常滑膜组织相比,PVNS滑膜组织中p-JAK、p-STAT1和p-STAT3表达显著增加,JAK-STAT通路显著活化,进一步促进细胞增殖,引起促炎细胞因子大量表达,从而促进滑膜炎症并最终导致关节破坏。因此,抑制JAK-STAT通路可以同时阻断多种炎症因子的信号转导,在PVNS治疗中具有重要意义。
发明人发现,培非替尼或其药学上可接受的盐能够抑制PVNS FLS和巨噬细胞的JAK-STAT通路活化,进而抑制FLS和巨噬细胞异常活化。包括抑制FLS增殖,诱导FLS死亡、凋亡,抑制FLS迁移,诱导巨噬细胞死亡,抑制FLS和巨噬细胞分泌炎症因子,从而改善患者滑膜炎症和关节破坏。因此,利用培非替尼或其药学上可接受的盐能够有效治疗PVNS。
另外,PVNS患者的1号和2号染色体均存在1p13染色体易位,而集落刺激因子1(colony-stimulating factor 1,CSF-1)是染色体1p13断点的基因。CSF-1易位导致CSF-1在PVNS部分具有恶性克隆倾向的细胞过表达。PVNS滑膜的CSF-1通过旁分泌和自分泌的方式作用于表达集落刺激因子1受体(colony-stimulating factor 1receptor,CSF-1R)的巨噬细胞,招募巨噬细胞在滑膜聚集,这一配体-受体的作用机制为靶向抑制药物筛选提供了潜在的靶点。
发明人发现,培非替尼或其药学上可接受的盐对巨噬细胞的CSF-1R的表达有显著的抑制作用,能够抑制CSF-1R活化,减弱其活性,阻止CSF-1与受体结合,抑制其信号转导,从而抑制巨噬细胞在滑膜的募集及滑膜增生。
在本发明的一些实施例中,培非替尼或其药学上可接受的盐可以经口施用或注射施用。
在本发明的一些实施例中,以按游离碱计,所述培非替尼或其药学上可接受的盐的施用剂量可为10~500mg/天,例如可为10~50mg/天、50~150mg/天、150~250mg/天、250~350mg/天、350~450mg/天或450~500mg/天。活性化合物可在宽剂量范围内有效,并且一般以药学上有效量施用。然而,应了解,化合物事实上施用的量通常将由医师根据有关环境来决定,包括待治疗的疾患、所选施用途径、实际施用的化合物、个别患者的年龄、体重和反应、患者症状的严重度等等。
本发明还提供含有培非替尼或其药学上可接受的盐的药物制剂在制备用于治疗滑膜炎的药物中的用途。
在本发明的一些实施例中,所述滑膜炎可选自色素沉着绒毛结节性滑膜炎。
在本发明的一些实施例中,所述药物制剂的剂型可包括注射剂、片剂、冲剂、颗粒剂、丸剂、胶囊、悬浮剂、乳剂中的任一种。上述各种剂型可以根据药物制剂领域的常规工艺制备而成。
在本发明的一些实施例中,所述药物制剂还可包括药学上可接受的载体。所述药学上可接受的载体是指药物制剂领域常规的药物载体,例如可选自填充剂、粘合剂、崩解剂、润滑剂、助悬剂、润湿剂、色素、矫味剂、溶剂、表面活性剂中的一种或几种。
本发明所述填充剂包括但不限于淀粉、微晶纤维素、蔗糖、糊精、乳糖、糖粉、葡萄糖等;所述润滑剂包括但不限于硬脂酸镁、硬脂酸、氯化钠、油酸钠、月桂醇硫酸钠、泊洛沙姆等;所述粘合剂包括但不限于水、乙醇、淀粉浆、糖浆、羟丙基甲基纤维素、羧甲基纤维素钠、海藻酸钠、聚乙烯吡咯烷酮等;所述崩解剂包括但不限于干淀粉、泡腾混合物(即碳酸氢钠和枸橼酸的混合物)、酒石酸、低取代羟丙基纤维素等;所述助悬剂包括但不限于多糖如金合欢胶、琼脂、藻酸、纤维素醚和羧甲基甲壳酯等;所述溶剂包括但不限于水、平衡的盐溶液等。
另外,培非替尼或其药学上可接受的盐还可以与其他活性药物联合使用,以更好地治疗PVNS治疗。因此,在本发明的一些实施例中,所述药物制剂还可包括其他药物,所述其他药物可选自CSF-1R抑制剂、TNF阻断剂、酪氨酸激酶抑制剂、非甾体类抗炎药、止疼药和消肿药中的一种或多种。例如,所述CSF-1R抑制剂可包括培西达替尼;所述TNF阻断剂可包括英夫利昔单抗;所述酪氨酸激酶抑制剂可包括伊马替尼、尼罗替尼;所述非甾体类抗炎药可包括依托考昔、西乐葆、凯芬;所述止疼药可包括舒敏片;所述消肿药可包括迈之灵。
术语解释
如本文所用,术语“药学上可接受的盐”是指所公开化合物的衍生物,其中母体化合物通过将所存在的酸或碱部分转变成其盐形式而改性。药学上可接受的盐的实例包括(但不限于)例如胺的碱性残余物的无机酸盐或有机酸盐;例如羧酸的酸性残余物的碱盐或有机盐等等。本申请的药学上可接受的盐包括例如由无毒无机酸或有机酸形成的母体化合物的常规无毒盐。本申请的药学上可接受的盐可以通过常规化学方法由含有碱性或酸性部分的母体化合物合成。一般来说,此类盐可以通过使这些化合物的游离酸或游离碱形式与化学计算量的适当碱或酸在水中或有机溶剂中或两者的混合物中反应来制备;一般来说,非水性介质,如乙醚、乙酸乙酯、醇(例如甲醇、乙醇、异丙醇或丁醇)或乙腈(ACN)是优选的。
如本文所用,术语“治疗”是指以下中一种或多种:(1)抑制疾病;例如抑制正经历或展示疾病、疾患或病症的病变或症状的个体中的疾病、疾患或病症(即阻止病变和/或症状进一步发展);(2)改善疾病;例如,改善正经历或展示疾病、疾患或病症的病变或症状的个体中的疾病、疾患或病症(即逆转病变和/或症状),例如降低疾病的严重度;或(3)预防易患所述疾病、疾患或病症但尚未经历或显示疾病的病变或症状的个体中的疾病、疾患或病症。在一些实施方案中,治疗是指抑制或改善疾病。在一些实施方案中,治疗是指预防疾病。
相比现有技术,本发明的有益效果:
本发明提供了培非替尼或其药学上可接受的盐在制备用于治疗滑膜炎的药物中的新用途,培非替尼或其药学上可接受的盐能够抑制FLS增殖并诱导FLS死亡、凋亡,抑制FLS迁移,诱导巨噬细胞死亡,并且还能够抑制FLS和巨噬细胞分泌炎症因子,因此利用培非替尼或其药学上可接受的盐能够有效治疗滑膜炎,从而扩展培非替尼的适应症,为后续培非替尼开展相关临床实验,继而发展为滑膜炎治疗药物提供依据。
另外,相比现有的治疗PVNS的药物培西达替尼,培非替尼诱导PVNS FLS死亡和凋亡以及抑制炎症因子表达的效果更好,并且培非替尼诱导THP-1分化的巨噬细胞死亡和抑制炎症因子表达的效果也更好,因而对于PVNS的疗效更优。
附图说明
图1为正常滑膜组织与PVNS滑膜组织中JAK-STAT通路活化情况的比较。实验重复3次,图1为其中一次代表性结果。
图2为培非替尼对PVNS FLS增殖的作用。培非替尼预处理PVNS FLS 1h后给予TNF-α(A)或IL-6/sIL-6R(B)刺激,通过CCK-8试剂检测细胞活力计算培非替尼抑制PVNS FLS增殖的IC50。n=6。
图3为培非替尼对PVNS FLS死亡和凋亡的作用。A:通过CCK-8试剂检测细胞活力计算培非替尼引起PVNS FLS死亡的IC50。B:通过LDH活性试剂盒检测培非替尼引起PVNS FLS死亡的作用。C:通过caspase-3/7活性试剂盒检测并计算培非替尼引起PVNS FLS凋亡的IC50。D:通过CCK-8试剂检测细胞活力评价培西达替尼引起PVNS FLS死亡的作用。n=6。
图4为培非替尼对PVNS FLS表达IL-6(A)、MCP-1(单核细胞趋化蛋白-1)(B)和MMP-3(基质金属蛋白酶-3)(C)的作用。n=4。
图5为培非替尼对PVNS FLS迁移的作用。****P<0.0001,每组3孔,每孔采集4个视野。
图6为培非替尼对PVNS FLS的JAK-STAT通路活化的作用。用IL-6/sIL-6R以不同时长刺激PVNS FLS(A),用不同浓度培非替尼处理PVNS FLS(B),检测JAK-STAT通路相关蛋白表达情况。
图7为培非替尼对THP-1(人单核细胞白血病细胞)分化的巨噬细胞死亡的作用。通过CCK-8试剂检测细胞活力值计算培非替尼(A)和培西达替尼(B)引起THP-1分化的巨噬细胞死亡的IC50。n=6。
图8为培非替尼对THP-1分化的巨噬细胞表达IL-1β(A)、IL-6(B)和TNF-α(C)的作用。n=4。
图9为培非替尼对THP-1分化的巨噬细胞的JAK-STAT通路活化的作用。用LPS(脂多糖)以不同时长刺激THP-1分化的巨噬细胞(A),用不同浓度培非替尼处理THP-1分化的巨噬细胞(B),检测JAK-STAT通路相关蛋白表达情况。
图10为培非替尼对THP-1分化的巨噬细胞的CSF-1R活化的作用。用CSF-1以不同时长刺激THP-1分化的巨噬细胞(A),用不同浓度培西达替尼(B)和培非替尼(C)处理THP-1分化的巨噬细胞,检测p-CSF-1R和CSF-1R表达情况。
图11为PVNS原代滑膜巨噬细胞的鉴定。A:巨噬细胞特征性蛋白CD68,B:DAPI,C:merge。
图12为培非替尼对PVNS原代滑膜巨噬细胞表达IL-1β(A)、IL-6(B)和TNF-α(C)的作用。n=4。
具体实施方式
下面参照具体的实施例对本发明做进一步说明。应当理解,此处所描述的具体实施例仅用于解释本发明,并不用于限定本发明的范围。本领域的技术人员将容易识别多个非关键参数,这些参数可以变化或修改,结果基本上相同。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道购买得到的常规产品。下面实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为市售产品。
实施例1:正常滑膜组织与PVNS滑膜组织中JAK-STAT通路活化情况的比较
通过手术获得正常滑膜组织和PVNS滑膜组织(其中正常滑膜组织取自前交叉韧带损伤/半月板损伤患者的膝关节),剔除脂肪、血管和肌腱。对滑膜组织称重,按重量体积比1:7加入RIPA裂解液(含蛋白酶抑制剂和磷酸酶抑制剂),用磁珠匀浆仪匀浆15s,冰上静置30min,以12000g在4℃离心10min,弃去沉淀吸取上清于新的预冷离心管中,采用BCA蛋白定量试剂盒测定蛋白浓度。蛋白样品按30μg上样。根据测得的蛋白浓度,用RIPA裂解液将各样品稀释至统一浓度,加入总体积四分之一体积的4*上样缓冲液(loading buffer),充分混匀,置于金属浴中以98℃加热5分钟使其变性,即为制备好的组织蛋白样液。制胶(10%分离胶,5%浓缩胶),将蛋白样品加入相应的泳道中电泳分离蛋白,然后通过电泳转移(300mA,3h)到PVDF膜上。转膜结束后,将PVNS膜以5%脱脂奶粉室温摇动封闭1h。封闭完成后,用TBST缓冲液洗膜三次,每次5min,根据标记物(marker)的位置裁切含有目的蛋白的条带,放入抗体孵育盒,加入稀释好的一抗,4℃摇动孵育过夜。次日回收一抗,用TBST缓冲液洗膜三次,每次5min,加入稀释好的HRP标记的二抗,室温摇动孵育1h。孵育结束后,用TBST缓冲液洗膜三次,每次5min。将ECL发光液均匀滴加在膜上,应用化学发光显影仪进行成像,检测膜上结合蛋白的表达水平。
结果如图1所示,与正常组相比,PVNS滑膜组织中p-JAK、p-STAT1和p-STAT3表达显著增加,表明JAK-STAT通路显著活化。
实施例2:培非替尼对PVNS FLS增殖的作用
通过手术获得PVNS滑膜组织,剔除脂肪、血管和肌腱,剪碎后加入装有Ⅰ型胶原酶和DNase I(脱氧核糖核酸酶I)(1mg/mL)的DMEM培养基的离心管中。将离心管放入震荡恒温摇床中,以37℃,250r/min消化40min,400目筛网过滤,收集滤液,离心(300g,5min)去除滤液收集细胞,重悬于红细胞裂解液中去除红细胞,离心(300g,5min)去除破碎的红细胞。其余细胞重悬于含10% FBS(胎牛血清)的DMEM(高糖)培养基中,37℃、5% CO2培养24h后换液,以弃去未贴壁的细胞,剩余贴壁细胞即为PVNS FLS。继续培养,每2~3天换液一次,待细胞长满后用0.25%胰酶消化传代,使用第3~7代细胞用于实验。
(A)将PVNS FLS以5×103个/孔接种于96孔板,用含10%FBS的DMEM(高糖)培养基孵育24h使细胞贴壁。弃去原培养基,加入含不同浓度培非替尼(0、0.003、0.01、0.03、0.1、0.3、1、3和10μmol/L)的无血清DMEM培养基预孵育1h,然后给予TNF-α(10ng/mL)刺激72h。孵育结束前3h每孔加入10μL CCK-8试剂,水平轻轻摇晃96孔板使液体混匀。继续孵育3h后,采用酶标仪读取每孔在450nm的吸光度,用待测孔减去仅加培养基的对照孔在450nm的吸光度,计算待测孔细胞活力值,进而计算培非替尼抑制PVNS FLS增殖的IC50。
(B)将PVNS FLS以5×103个/孔接种于96孔板,用含10%FBS的DMEM(高糖)培养基孵育24h使细胞贴壁。弃去原培养基,加入含不同浓度培非替尼(0、0.003、0.01、0.03、0.1、0.3、1和3μmol/L)的无血清DMEM培养基预孵育1h,然后给予IL-6(100ng/mL)和sIL-6R(人可溶性白介素6受体,100ng/mL)刺激72h。孵育结束前3h每孔加入10μL CCK-8试剂,水平轻轻摇晃96孔板使液体混匀。继续孵育3h后,采用酶标仪读取每孔在450nm的吸光度,用待测孔减去仅加培养基的对照孔在450nm的吸光度,计算待测孔细胞活力值,进而计算培非替尼抑制PVNS FLS增殖的IC50。
给予TNF-α刺激、IL-6和sIL-6R刺激均可引起PVNS FLS增殖。如图2所示,培非替尼预处理PVNS FLS 1h后给予TNF-α(10ng/mL)刺激72h,抑制PVNS FLS增殖的IC50为2.078μmol/L(图2A),培非替尼预处理PVNS FLS 1h后给予IL-6(100ng/mL)和sIL-6R(100ng/mL)刺激72h,抑制PVNS FLS增殖的IC50为0.2451μmol/L(图2B)。
实施例3:培非替尼对PVNS FLS死亡和凋亡的作用
(A)将PVNS FLS以5×103个/孔接种于96孔板,用含10% FBS的DMEM(高糖)培养基孵育24h使细胞贴壁。弃去原培养基,加入含不同浓度培非替尼(0、0.03、0.1、0.3、1、3、10、30、100和300μmol/L)的无血清DMEM培养基孵育24h。孵育结束前3h每孔加入10μL CCK-8试剂,水平轻轻摇晃96孔板使液体混匀。继续孵育3h后,采用酶标仪读取每孔在450nm的吸光度,用待测孔减去仅加培养基的对照孔在450nm的吸光度,计算待测孔细胞活力值,进而计算培非替尼引起PVNS FLS死亡的IC50。
(B)将PVNS FLS以2×105个/孔接种于24孔板,用含10% FBS的DMEM(高糖)培养基孵育24h使细胞贴壁。弃去原培养基,加入含不同浓度培非替尼(0、0.03、0.1、0.3、1、3、10、30、100和300μmol/L)的无血清DMEM培养基孵育24h。提前预热酶标仪至37℃,吸取待测孔培养基40μL加到96孔酶标板中,每孔加入200μL新鲜配制的LDH检测试剂(上海景源医疗器械有限公司)(乳酸脱氢酶试剂2和乳酸脱氢酶试剂1按1:5混合),水平轻轻摇晃96孔板使液体混匀,放入酶标仪中检测340nm波长紫外吸收光,每隔1分钟读取一次检测值,共检测7个时间点,计算其斜率,通过与对照孔比较可得各孔LDH相对变化值,进而评价培非替尼对于PVNS FLS死亡的作用。
(C)将PVNS FLS以2×105个/孔接种于24孔板,用含10% FBS的DMEM(高糖)培养基孵育24h使细胞贴壁。弃去原培养基,加入含不同浓度培非替尼(0、0.03、0.1、0.3、1、3、10、30、100和300μmol/L)的无血清DMEM培养基孵育24h。每孔加入200μL的caspase-3/7检测试剂,室温避光摇晃孵育30min。每孔吸取100μL溶液转移至白色不透明96孔板,使用酶标仪检测化学发光值,通过与对照孔比较可得各孔caspase-3/7活性相对变化值,进而计算培非替尼引起PVNS FLS凋亡的IC50。
(D)将PVNS FLS以5×103个/孔接种于96孔板,用含10% FBS的DMEM(高糖)培养基孵育24h使细胞贴壁。弃去原培养基,加入含不同浓度培西达替尼(0、0.1、0.3、1、3、10、30、100和300μmol/L)的无血清DMEM培养基孵育24h。孵育结束前3h每孔加入10μL CCK-8试剂,水平轻轻摇晃96孔板使液体混匀。继续孵育3h后,采用酶标仪读取每孔在450nm的吸光度,用待测孔减去仅加培养基的对照孔在450nm的吸光度,计算待测孔细胞活力值,进而评价培西达替尼对于PVNS FLS死亡的作用。
如图3所示,培非替尼处理24h引起PVNS FLS死亡的IC50为23.58μmol/L(图3A),对细胞上清液LDH活性的检测印证了培非替尼引起PVNS FLS死亡的结果(图3B)。培非替尼处理24h引起PVNS FLS凋亡的IC50为28.9μmol/L(图3C)。PVNS治疗药培西达替尼处理24h则没有显著引起PVNS FLS死亡。
实施例4:培非替尼对PVNS FLS表达IL-6、MCP-1和MMP-3的作用
将PVNS FLS以5×105个/孔接种于6孔板,用含10% FBS的DMEM(高糖)培养基孵育24h使细胞贴壁。弃去原培养基,加入含不同浓度培非替尼(0、0.03、0.1、0.3、1、3和10μmol/L)或不同浓度培西达替尼(1、3和10μmol/L)的无血清DMEM培养基预孵育1h,然后给予TNF-α(10ng/mL)刺激24h。细胞处理结束后,弃去培养基,用PBS缓冲液洗涤细胞1次,每孔加入1mLTRIzol试剂,反复吹吸使细胞充分裂解。将液体转移至无RNAse的1.5mL离心管中,每孔加入0.2mL氯仿,剧烈震荡30s使之充分混匀,室温静置5min,以12000g在4℃离心10min。将上层水相吸出至新的无RNAse的1.5mL离心管中,加入等体积异丙醇,充分颠倒混匀后冰上静置10min,以12000g在4℃离心10min。离心后可在管底观察到白色沉淀,小心弃去上清,加入1mL 75%乙醇,轻柔颠倒混匀10s以洗涤沉淀,以12000g在4℃离心10min。重复洗涤一次后小心吸干乙醇,开盖干燥约10min。加入30μL无RNAse水溶解沉淀,使用Nanodrop检测RNA浓度及纯度。在无RNase的八连排中加入逆转录体系,每孔为:RNA 14μL,随机引物(Randomprimer)2μL,R Mix 20μL,E Mix 2μL和gDNA remover 2μL。将体系混匀,采用PCR仪进行逆转录,程序为:25℃10min,42℃30min,85℃5s。逆转录终止后,将产物平衡至室温,在无RNase的八连排中加入qPCR体系,每孔为:5uL 2*SYBR Green master mix,0.5μL引物(上、下游引物各0.25μL),3.5μL无RNAse水和1μLcDNA。离心使液体汇集在孔底,上机进行qPCR,程序为:95℃5min;95℃10s,60℃20s,72℃20s,35个循环。对结果使用2-ΔΔCT进行相对定量分析,得到IL-6、MCP-1和MMP-3的mRNA表达水平,进而计算培非替尼抑制其表达的IC50。
给予TNF-α刺激可促进PVNS FLS表达IL-6、MCP-1和MMP-3。不同浓度培非替尼预处理1h后给予TNF-α(10ng/mL)刺激24h,如图4所示,培非替尼抑制IL-6表达的IC50为0.804μmol/L(图4A),抑制MCP-1表达的IC50为3.75μmol/L(图4B),抑制MMP-3表达的IC50为0.06μmol/L(图4C),效果均优于培西达替尼(图4A~C)。
实施例5:培非替尼对PVNS FLS迁移的作用
通过划痕实验检测PVNS FLS的迁移能力。用记号笔细端过6孔板的孔中央划线,将PVNS FLS以6×105个/孔接种于6孔板,用含10% FBS的DMEM(高糖)培养基培养至细胞融合率达95%。弃去原培养基,用无血清DMEM培养基饥饿过夜。用200μL枪头垂直于先前记号笔所划细线,在孔中划两道线制造划痕。每孔用PBS缓冲液洗三遍去除细胞碎片,于划痕和细线交界处拍摄4张照片(0h)。加入含培非替尼(0、1和3μmol/L)的无血清DMEM培养基预孵育1h,然后除对照组外,每组给予TNF-α(10ng/mL)刺激48h,每孔于划痕和细线交界处拍摄4张照片(48h)。使用ImageJ软件进行结果分析,计算每孔迁移面积并统计。
如图5所示,TNF-α刺激促进PVNS FLS迁移,培非替尼(1和3μmol/L)均显著抑制PVNS FLS迁移。
实施例6:培非替尼对PVNS FLS的JAK-STAT通路活化的作用
(A)将PVNS FLS以5×105个/孔接种于6孔板,用含10% FBS的DMEM(高糖)培养基孵育24h使细胞贴壁。弃去原培养基,加入含IL-6(100ng/mL)和sIL-6R(100ng/mL)的无血清DMEM培养基,刺激不同时长(5min、10min、15min、20min、30min、1h、4h、8h和12h)。细胞处理结束后,弃去培养基,用PBS缓冲液洗涤细胞1次,每孔加入RIPA裂解液(含蛋白酶抑制剂和磷酸酶抑制剂),反复吹吸使细胞充分裂解,收集细胞裂解液于离心管中,以12000g在4℃离心10min,弃去沉淀吸取上清于新的预冷离心管中,采用BCA蛋白定量试剂盒测定蛋白浓度,检测目的蛋白的表达水平(具体方法见实施例1)。
(B)将PVNS FLS以5×105个/孔接种于6孔板,用含10% FBS的DMEM(高糖)培养基孵育24h使细胞贴壁。弃去原培养基,加入含培非替尼(0、0.3、1、3和10μmol/L)的无血清DMEM培养基预孵育1h,然后以IL-6(100ng/mL)和sIL-6R(100ng/mL)刺激15min。细胞处理结束后,弃去培养基,用PBS缓冲液洗涤细胞1次,每孔加入RIPA裂解液(含蛋白酶抑制剂和磷酸酶抑制剂),反复吹吸使细胞充分裂解,收集细胞裂解液于离心管中,以12000g在4℃离心10min,弃去沉淀吸取上清于新的预冷离心管中,采用BCA蛋白定量试剂盒测定蛋白浓度,检测目的蛋白的表达水平(具体方法见实施例1)。
如图6所示,IL-6(100ng/mL)和sIL-6R(100ng/mL)刺激PVNS FLS 10~30min可引起p-JAK1、p-STAT1和p-STAT3表达显著增加,即引起JAK-STAT通路活化(图6A)。接下来用不同浓度培非替尼预处理PVNS FLS 1h,再用IL-6(100ng/mL)和sIL-6R(100ng/mL)刺激15min,检测JAK-STAT通路活化情况。结果显示,培非替尼剂量依赖性抑制PVNS FLS的p-JAK1、p-STAT1和p-STAT3表达,即抑制JAK-STAT通路活化(图6B)。
实施例7:培非替尼对THP-1分化的巨噬细胞死亡的作用
(A)将THP-1以104个/孔接种于96孔板,用含PMA(20ng/mL)和10% FBS的RPMI-1640培养基孵育48h使其分化为巨噬细胞。弃去原培养基,加入含培非替尼(0、0.03、0.1、0.3、1、3、10、30、100和300μmol/L)的无血清RPMI-1640培养基孵育24h。孵育结束前3h每孔加入10μL CCK-8试剂,水平轻轻摇晃96孔板使液体混匀。继续孵育3h后,采用酶标仪读取每孔在450nm的吸光度,用待测孔减去仅加培养基的对照孔在450nm的吸光度,计算待测孔细胞活力值,进而计算培非替尼引起THP-1分化的巨噬细胞死亡的IC50。
(B)将THP-1以104个/孔接种于96孔板,用含PMA(20ng/mL)和10% FBS的RPMI-1640培养基孵育48h使其分化为巨噬细胞。弃去原培养基,加入含培西达替尼(0、0.03、0.1、0.3、1、3、10、30、100和300μmol/L)的无血清RPMI-1640培养基孵育24h。孵育结束前3h每孔加入10μL CCK-8试剂,水平轻轻摇晃96孔板使液体混匀。继续孵育3h后,采用酶标仪读取每孔在450nm的吸光度,用待测孔减去仅加培养基的对照孔在450nm的吸光度,计算待测孔细胞活力值,进而计算培西达替尼引起THP-1分化的巨噬细胞死亡的IC50。
如图7所示,培非替尼处理24h引起THP-1分化的巨噬细胞死亡的IC50为16.55μmol/L(图7A),PVNS治疗药培西达替尼处理24h引起THP-1分化的巨噬细胞死亡的IC50为30.07μmol/L(图7B)。与培西达替尼相比,培非替尼引起THP-1分化的巨噬细胞死亡效果更显著。
实施例8:培非替尼对THP-1分化的巨噬细胞表达IL-1β、IL-6和TNF-α的作用
将THP-1以6×105个/孔接种于12孔板,用含PMA(20ng/mL)和10% FBS的RPMI-1640培养基孵育48h使其分化为巨噬细胞。弃去原培养基,加入含不同浓度培非替尼(0、0.003、0.01、0.03、0.1、0.3、1、3、10和30μmol/L)或不同浓度培西达替尼(1、3和10μmol/L)的无血清RPMI-1640培养基预孵育1h,然后给予LPS(100ng/mL)刺激4h。
细胞处理结束后,收细胞RNA并逆转录成cDNA,通过qPCR检测并计算得到IL-1β、IL-6和TNF-α的mRNA表达水平,进而计算培非替尼抑制其表达的IC50(详细方法见实施例4)。
给予LPS刺激可引起THP-1分化的巨噬细胞表达IL-1β、IL-6和TNF-α。不同浓度培非替尼预处理1h后给予LPS(100ng/mL)刺激4h,如图8所示,培非替尼抑制IL-1β表达的IC50为3.876μmol/L(图8A),抑制IL-6表达的IC50为0.1807μmol/L(图8B),抑制TNF-α表达的IC50为4.62μmol/L(图8C),抑制效果均优于培西达替尼(图8A~C)。
实施例9:培非替尼对THP-1分化的巨噬细胞的JAK-STAT通路活化的作用
(A)将THP-1以6×105个/孔接种于12孔板,用含PMA(20ng/mL)和10% FBS的RPMI-1640培养基孵育48h使其分化为巨噬细胞。弃去原培养基,加入含LPS(100ng/mL)的无血清RPMI-1640培养基,刺激不同时长(5min、10min、15min、20min、30min、1h、4h、8h和12h)。细胞处理结束后,弃去培养基,用PBS缓冲液洗涤细胞1次,每孔加入RIPA裂解液(含蛋白酶抑制剂和磷酸酶抑制剂),反复吹吸使细胞充分裂解,收集细胞裂解液于离心管中,以12000g在4℃离心10min,弃去沉淀吸取上清于新的预冷离心管中,采用BCA蛋白定量试剂盒测定蛋白浓度,检测目的蛋白的表达水平(具体方法见实施例1)。
(B)将THP-1以6×105个/孔接种于12孔板,用含PMA(20ng/mL)和10% FBS的RPMI-1640培养基孵育48h使其分化为巨噬细胞。弃去原培养基,加入含培非替尼(0、0.01、0.03、0.1和0.3μmol/L)的无血清RPMI-1640培养基预孵育1h,然后以LPS(100ng/mL)刺激4h。细胞处理结束后,弃去培养基,用PBS缓冲液洗涤细胞1次,每孔加入RIPA裂解液(含蛋白酶抑制剂和磷酸酶抑制剂),反复吹吸使细胞充分裂解,收集细胞裂解液于离心管中,以12000g在4℃离心10min,弃去沉淀吸取上清于新的预冷离心管中,采用BCA蛋白定量试剂盒测定蛋白浓度,检测目的蛋白的表达水平(具体方法见实施例1)。
如图9所示,LPS(100ng/mL)刺激THP-1分化的巨噬细胞4h可引起p-JAK1、p-STAT1和p-STAT3表达显著增加,即引起JAK-STAT通路活化(图9A)。接下来用不同浓度培非替尼预处理THP-1分化的巨噬细胞1h,再用LPS(100ng/mL)刺激4h,检测JAK-STAT通路活化情况。结果显示,培非替尼剂量依赖性抑制THP-1分化的巨噬细胞的JAK-STAT通路活化(图9B)。
实施例10:培非替尼对THP-1分化的巨噬细胞的CSF-1R活化的作用
(A)将THP-1以6×105个/孔接种于12孔板,用含PMA(20ng/mL)和10% FBS的RPMI-1640培养基孵育48h使其分化为巨噬细胞,然后以不含PMA的RPMI-1640培养基孵育48h。弃去原培养基,加入含CSF-1(100ng/mL)的无血清RPMI-1640培养基刺激不同时长(5、10和15min)。细胞处理结束后,弃去培养基,用PBS洗涤细胞1次,每孔加入RIPA裂解液(含蛋白酶抑制剂和磷酸酶抑制剂),反复吹吸使细胞充分裂解,收集细胞裂解液于离心管中,以12000g在4℃离心10min,弃去沉淀吸取上清于新的预冷离心管中,采用BCA蛋白定量试剂盒测定蛋白浓度,检测目的蛋白的表达水平(具体方法见实施例1)。
(B)将THP-1以6×105个/孔接种于12孔板,用含PMA(20ng/mL)和10% FBS的RPMI-1640培养基孵育48h使其分化为巨噬细胞,然后以不含PMA的RPMI-1640培养基孵育48h。弃去原培养基,加入含培西达替尼(0、0.3、1、3和10μmol/L)的无血清RPMI-1640培养基预孵育1h,然后以CSF-1(100ng/mL)刺激5min。细胞处理结束后,弃去培养基,用PBS缓冲液洗涤细胞1次,每孔加入RIPA裂解液(含蛋白酶抑制剂和磷酸酶抑制剂),反复吹吸使细胞充分裂解,收集细胞裂解液于离心管中,以12000g在4℃离心10min,弃去沉淀吸取上清于新的预冷离心管中,采用BCA蛋白定量试剂盒测定蛋白浓度,检测目的蛋白的表达水平(具体方法见实施例1)。
(C)将THP-1以6×105个/孔接种于12孔板,用含PMA(20ng/mL)和10% FBS的RPMI-1640培养基孵育48h使其分化为巨噬细胞,然后以不含PMA的RPMI-1640培养基孵育48h。弃去原培养基,加入含培非替尼(0、0.3、1、3和10μmol/L)的无血清RPMI-1640培养基预孵育1h,然后以CSF-1(100ng/mL)刺激5min。细胞处理结束后,弃去培养基,用PBS缓冲液洗涤细胞1次,每孔加入RIPA裂解液(含蛋白酶抑制剂和磷酸酶抑制剂),反复吹吸使细胞充分裂解,收集细胞裂解液于离心管中,以12000g在4℃离心10min,弃去沉淀吸取上清于新的预冷离心管中,采用BCA蛋白定量试剂盒测定蛋白浓度,检测目的蛋白的表达水平(具体方法见实施例1)。
如图10所示,CSF-1(100ng/mL)刺激THP-1分化的巨噬细胞5min可引起p-CSF-1R表达显著增加,即引起CSF-1R活化(图10A)。培西达替尼是CSF-1R特异性抑制剂,预处理THP-1分化的巨噬细胞1h后以CSF-1(100ng/mL)刺激5min,对p-CSF-1R的表达有显著抑制作用(图10B)。培非替尼预处理THP-1分化的巨噬细胞1h后以CSF-1(100ng/mL)刺激5min,在10μmol/L时对p-CSF-1R的表达有显著抑制作用(图10C)。
实施例11:PVNS原代滑膜巨噬细胞的鉴定
通过手术获得PVNS滑膜组织,消化得到单细胞并裂解去除红细胞,通过CD14阳性筛选试剂盒获得CD14+细胞。在12孔板的每孔中心滴加20μL培养基,放入无菌小玻片,使小玻片与孔底完全贴合。以4×105个/孔接种CD14+细胞,用含CSF-1(20ng/mL)和10% FBS的RPMI-1640培养基培养48h将其诱导成巨噬细胞后用于实验。
弃去培养基,PBS缓冲液轻柔洗3次,每孔加入0.5mL预冷的4%多聚甲醛,室温固定20min。PBS缓冲液轻柔洗3次,每孔加入0.5mL含0.2% Triton X-100的PBS缓冲液,冰上静置10min。PBS缓冲液轻柔洗3次,每孔加入0.5mL含1% BSA的PBS缓冲液,室温封闭1h。用封闭液将anti-CD68抗体以1:200稀释,按40μL/玻片滴加在封口膜上。从12孔板中取出小玻片,细胞面朝下放在封口膜的抗体液体上,使各玻片与抗体充分均匀接触,4℃孵育过夜。次日取下玻片,细胞面朝上置于12孔板中。PBS缓冲液轻柔洗3次,滴加1:1000稀释的荧光素二抗,37℃孵育30min。PBS缓冲液轻柔洗3次,取洁净载玻片,滴加20μL含DAPI染液的抗荧光淬灭封片剂,取出小玻片并封片,在荧光显微镜下观察并拍照。
CD68是巨噬细胞的特征性蛋白。如图11所示,培养的原代滑膜巨噬细胞CD68染色阳性,符合巨噬细胞的特征。
实施例12:培非替尼对PVNS原代滑膜巨噬细胞表达IL-1β、IL-6和TNF-α的作用
将分选自PVNS滑膜的CD14+细胞以6×105个/孔接种于12孔板,用含CSF-1(20ng/mL)和10% FBS的RPMI-1640培养基孵育48h使其分化为巨噬细胞。弃去原培养基,加入含不同浓度培非替尼(0、0.03、0.1、0.3、1、3、10和30μmol/L)的无血清RPMI-1640培养基预孵育1h,然后给予LPS(100ng/mL)刺激4h。
细胞处理结束后,收细胞RNA并逆转录成cDNA,通过qPCR检测并计算得到IL-1β、IL-6和TNF-α的mRNA表达水平,进而计算培非替尼抑制其表达的IC50(详细方法见实施例4)。
给予LPS刺激可引起PVNS原代滑膜巨噬细胞表达IL-1β、IL-6和TNF-α。不同浓度培非替尼预处理1h后给予LPS(100ng/mL)刺激4h,如图12所示,培非替尼抑制IL-1β表达的IC50为7.813μmol/L(图12A),抑制IL-6表达的IC50为0.9339μmol/L(图12B),抑制TNF-α表达的IC50为0.068μmol/L(图12C)。
以上实施例的实验结果表明,培非替尼对于PVNS FLS具有以下作用:抑制增殖、引起死亡、抑制迁移、抑制炎症因子表达和抑制JAK-STAT通路活化;培非替尼对于THP-1分化的巨噬细胞具有以下作用:引起死亡、抑制炎症因子表达、抑制JAK-STAT通路活化和抑制CSF-1R活化;培非替尼对于PVNS滑膜原代巨噬细胞具有以下作用:抑制炎症因子表达。
另外,本发明应用药物培非替尼处理与PVNS滑膜病变密切相关的细胞,得到的体外细胞实验结果还表明,与现有药物培西达替尼相比,药物培非替尼对于PVNS FLS,引起死亡和抑制炎症因子表达的效果更优;并且对于THP-1分化的巨噬细胞,引起死亡和抑制炎症因子表达的效果同样更好,这都表明培非替尼对于PVNS的疗效更优。这为后续培非替尼开展相关临床实验,继而发展为PVNS治疗药物提供依据。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (10)
1.培非替尼或其药学上可接受的盐在制备用于治疗滑膜炎的药物中的用途。
2.根据权利要求1所述的用途,其特征在于,所述滑膜炎选自色素沉着绒毛结节性滑膜炎。
3.根据权利要求1或2所述的用途,其特征在于,所述培非替尼或其药学上可接受的盐抑制FLS增殖并诱导FLS死亡、凋亡;
所述培非替尼或其药学上可接受的盐诱导巨噬细胞死亡。
4.根据权利要求1或2所述的用途,其特征在于,所述培非替尼或其药学上可接受的盐经口施用或注射施用。
5.根据权利要求4所述的用途,其特征在于,以按游离碱计,所述培非替尼或其药学上可接受的盐的施用剂量为10~500mg/天。
6.含有培非替尼或其药学上可接受的盐的药物制剂在制备用于治疗滑膜炎的药物中的用途。
7.根据权利要求6所述的用途,其特征在于,所述滑膜炎选自色素沉着绒毛结节性滑膜炎。
8.根据权利要求6或7所述的用途,其特征在于,所述药物制剂的剂型包括注射剂、片剂、冲剂、颗粒剂、丸剂、胶囊、悬浮剂、乳剂中的任一种。
9.根据权利要求6或7所述的用途,其特征在于,所述药物制剂还包括药学上可接受的载体。
10.根据权利要求6或7所述的用途,其特征在于,所述药物制剂还包括其他药物,所述其他药物选自CSF-1R抑制剂、TNF阻断剂、酪氨酸激酶抑制剂、非甾体类抗炎药、止疼药和消肿药中的一种或多种。
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