CN116687904A - 截短侧耳素抗生素在制备抗肿瘤及逆转分子靶向治疗耐药的药物中的应用 - Google Patents
截短侧耳素抗生素在制备抗肿瘤及逆转分子靶向治疗耐药的药物中的应用 Download PDFInfo
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Abstract
本发明属于医药领域，涉及截短侧耳素抗生素在制备抗肿瘤及逆转分子靶向治疗耐药的药物中的应用；在HepG2肝癌细胞中，Lefamulinacetate能够促进sorafenib引起的肝癌细胞促凋亡、抑增殖作用，增加sorafenib的细胞杀伤效果；在裸鼠皮下移植瘤模型中，Lefamulinacetate能够增加sorafenib引起的抑制体内肿瘤生长的疗效，降低瘤体重量以及肿瘤组织中细胞死亡面积百分比、KI67阳性细胞数，但对裸鼠体重、血浆ALT、AST、BUN、CRE含量均无影响、对裸鼠心、肝、脾、肺、肾各脏器也无损伤，说明Lefamulinacetate具有良好的逆转肝细胞癌sorafenib耐药的作用；Lefamulinacetate具有效果良好，安全性较高的优点，且充分发挥老药新用的优势，有望发展成为治疗分子靶向治疗耐药肝细胞癌的有效药物。
Description
技术领域
本发明属于医药领域，涉及截短侧耳素抗生素在制备抗肿瘤及逆转分子靶向治疗耐药的药物中的应用。
背景技术
肝细胞肝癌(Hepatocellular carcinoma，HCC)是最常见的原发性肝脏肿瘤，其发病率约占原发性肝癌的70％至90％，死亡率居第2位，全球发病率不断上升，严重威胁人民的生命和健康。目前，以肝切除术、肝移植和局部消融为代表的外科手术治疗是治疗早期HCC患者的首选方法和最有效措施，但由于早期诊断困难、病情进展迅速、复发转移率高及缺乏有效的治疗方法等原因，大部分HCC患者一经诊断即为中晚期，中晚期肝癌患者和大多数肝功能较差的肝癌患者不宜采用手术切除治疗，只能通过肝动脉化疗栓塞(transcatheter arterial chemoembolization，TACE)、放化疗等手段来延长生存时间。
索拉菲尼是一种多靶点酪氨酸激酶抑制剂，是第一个被FDA批准用于治疗晚期HCC的分子靶向药物，既可通过阻断由RAF/MEK/ERK介导的细胞信号传导通路而直接抑制肿瘤细胞的增殖，还可以通过抑制VEGFR和血小板衍生生长因子(PDGF)受体而阻断肿瘤新生血管的形成，间接地抑制肿瘤细胞的生长，体内外研究中均展现出良好的抗HCC效果，促进晚期肝癌患者生存。然而在临床治疗中原发性及获得性耐药现象的产生不仅限制了HCC患者从索拉菲尼治疗中长久获益，还使疾病恶化，病程加速。索拉菲尼在HCC中的原发性和获得性耐药涉及多种机制，包括自噬、上皮-间充质转化、癌症干细胞、肿瘤微环境和表观遗传调控，并可能涉及许多信号通路，如Wnt/β-catenin、TGFβ、Ras/MEK/ERK、PI3K/Akt、TNFα/NF-κB和JAK/STAT通路，但sorafenib耐药机制在体内及HCC患者中研究较少，因此探究HCC索拉菲尼耐药问题，并寻求可能的逆转策略具有非常重要的临床意义。
新药研发周期长、投入大、风险高，“老药新用”在一定程度上可弥补这些局限性，针对具体疾病的药物发现，以化学小分子药物为例，从体外细胞模型上获得具有活性的苗头化合物，到最终走向临床应用，需要挺过多层面的活性与药效评价、靶向性与作用机制探索、毒性与安全性评价、代谢性能评价等“九死一生”的艰难历程。而已经挺过重重关卡考验的老药至少可以迅速满足药物安全性评价和代谢方面的基本条件，因此有望加速新药发现的进程。以老药为骨架开展优化，可以极大缩短苗头化合物发现、先导化合物优化乃至候选药物的临床前研究周期，同时基于明确的药代动力学性质及安全性等参数，可以有效降低临床研究失败的风险。目前关于“老药新用”的研究已经取得了许多成果，例如阿司匹林的抗癌、预防老年痴呆、降血糖等多种新用途；但目前对于逆转sorafenib耐药肝细胞癌的药物的研究大多局限于天然产物，关于逆转肝细胞癌耐药的临床用药研究较少，且作用机制尚不清楚。
Lefamulin acetate是一种截短侧耳素抗生素，为FDA批准的用于治疗社区获得性肺炎community-acquiredbacterial pneumonia(CABP)的药物。其作用机制为Lefamulinacetate通过与50S bacterial ribosome的肽基转移酶中心结合来抑制蛋白质合成，从而阻止了转移RNA与肽转移的结合，实现抗菌作用。
本发明基于已构建的肝癌sorafenib耐药细胞株HepG2 SR，对FDA批准的已上市临床药物库的药物进行sorafenib增敏活性筛选研究，发现截短侧耳素类抗生素药物Valnemulin HCl、Retapamulin、Lefamulin acetate均具有不同程度的增敏作用，表明该类母核药物具有潜在的逆转肝细胞癌sorafenib耐药的活性。鉴于前两者分别具有兽药、外用药的局限性，本实验室以Lefamulin acetate为研究对象，对其逆转肝细胞癌sorafenib耐药作用进行了深入地研究，并在考察药物安全性的基础上，从体内动物模型中进一步验证了药物Lefamulin acetate对HepG2皮下异位移植瘤模型中增加体内sorafenib疗效的作用。所述药物Lefamulin acetate目前在临床治疗过程中不良反应较小，其逆转肝细胞癌分子靶向治疗耐药的作用，迄今尚未见有国内外的相关文献报道。
发明内容
本发明通过肿瘤细胞模型和小鼠肿瘤模型，发现截短侧耳素抗生素Lefamulinacetate通过直接结合ILF3蛋白在逆转肝细胞癌分子靶向治疗耐药中的新用途。
截短侧耳素抗生素在制备逆转分子靶向治疗肝癌产生耐药的药物中的应用。
所述肝癌为对sorafenib治疗不敏感或耐药的肝癌。
截短侧耳素抗生素为Valnemulin HCl、Retapamulin或Lefamulin acetate。
一种药物组合物在治疗逆转sorafenib治疗肝癌产生耐药的药物中应用，其特征在于所述的药物组合物包括Lefamulin acetate或其药学上可接受的盐或酯与临床肝细胞癌分子靶向治疗药物sorafenib。
本发明用于与sorafenib联合治疗肝细胞癌的药物时，已上市临床用药Lefamulinacetate口服或非口服给药均属安全有效的。口服用药为片剂；非口服用药为注射液针剂。
本发明所述截短侧耳素抗生素Lefamulin acetate给药剂量，可根据给药方式、患病程度、患者年龄、有无既往病史等因素综合考虑进行调整。
有益效果。
本发明涉及了Lefamulin acetate的新用途，具体来说，本发明以肝癌sorafenib耐药细胞株HepG2 SR作为药物筛选细胞模型，将FDA批准的已上市临床药物库中的药物与sorafenib进行联合用药，利用CCK8测定细胞存活率首次发现截短侧耳素抗生素Valnemulin HCl、Retapamulin、Lefamulin acetate对sorafenib均具有不同程度的增敏作用。鉴于给药方式及患者用药的便捷性、实用性综合考量，选择Lefamulin acetate作为进一步研究对象。本发明通过使用HepG2细胞进行体外活性研究，在细胞中，分为对照组、给药sorafenib组、给药Lefamulin acetate组、sorafenib与Lefamulin acetate联合用药组，给药48小时后的HepG2细胞，Annexin-V/PI双染流式细胞术检测细胞凋亡率；CCK8、EDU、克隆形成检测细胞增殖情况。
为进一步评价药物在体内增加sorafenib抗肝癌效果，本发明通过使用HepG2细胞构建裸鼠皮下移植瘤模型，证实Lefamulin acetate增加sorafenib治疗肝细胞癌效果的药理活性。在动物模型中，采用4～6周龄Balb/c裸鼠，后侧翼接种HepG2细胞，待肿瘤形成可触及时开始给药，分为溶剂组、给药sorafenib组、给药Lefamulin acetate组、sorafenib与Lefamulin acetate联合用药组，给药3周后取裸鼠血液进行生化指标分析；取裸鼠心、肝、脾、肺、肾各器官进行HE染色；取裸鼠肿瘤组织进行HE染色、KI67免疫组织化学染色；检测裸鼠血液中ALT、AST、BUN、CRE含量。
进一步探究Lefamulin acetate增敏sorafenib的机制，采用蛋白质谱分析发现Lefamulin acetate结合的蛋白，并用Docking、DARTS、CETSA证实Lefamulin acetate与ILF3的结合，Western-Blot检测Lefamulin acetate对ILF3蛋白的影响。
细胞与动物模型的结果表明：
1.联合给药Lefamulin acetate与sorafenib的细胞中凋亡率与单独给药sorafenib相比明显增加，说明Lefamulin acetate能增加sorafenib促进肝癌细胞凋亡的作用。
2.联合给药Lefamulin acetate与sorafenib的细胞中增殖速度较慢，EDU阳性细胞数、克隆形成数与单独给药sorafenib相比明显降低，说明Lefamulin acetate能增加sorafenib抑制肝癌细胞增殖的作用。
3.动物模型中各组小鼠体重无明显下降，心、肝、脾、肺、肾各脏器均无发生明显损伤，血浆ALT、AST、BUN、CRE水平均无显著差异，且无中毒迹象及死亡发生，表明药物在肿瘤模型的安全性较高，这不仅为化合物体内药效学评价提供了依据，也该类药物在临床上的新用途开发奠定了基础。
4.体内药效学表明，联合给药Lefamulin acetate与sorafenib的裸鼠肿瘤生长较慢，重量小；肿瘤组织病理切片染色结果表明Lefamulin acetate与Sorafenib协同增加肝癌细胞死亡面积百分比，KI67阳性细胞数减少，表明体内肝癌细胞增殖被抑制，说明Lefamulin acetate可通过增加sorafenib敏感性从而治疗HCC。
5.机制探究显示，Lefamulin acetate直接与ILF3蛋白结合，但对ILF3表达量无影响，药物能够降低ILF3下游靶基因表达，提示Lefamulin acetate可能通过与ILF3靶蛋白直接结合阻断其与下游耐药靶基因启动子区的结合进而抑制其转录活性，发挥增敏作用。
由此，本发明提供截短侧耳素抗生素Lefamulin acetate无论是安全性，还是有效性在逆转肝细胞癌分子靶向治疗耐药中具有良好的市场前景和临床价值
附图说明
图1、HepG2和HepG2SR(sorafenib耐药)细胞中给予不同浓度sorafenib后的IC50结果。
图2、HepG2SR细胞中在Lefamulin acetate存在或不存在条件下给予不同浓度sorafenib后IC50结果，A为Valnemulin HCl、B为Retapamulin、C为Lefamulin acetate(10μM)、D为Lefamulin(10和20μM)、E为Retapamulin(10和20μM)。
图3、HepG2细胞中分别给予DMSO、sorafenib、Lefamulin acetate、sorafenib+Lefamulin acetate 48h后细胞凋亡率。
图4、HepG2细胞中分别给予DMSO、sorafenib、Lefamulin acetate、sorafenib+Lefamulin acetate 24、48、72h后细胞吸光度值。
图5、HepG2细胞中分别给予DMSO、sorafenib、Lefamulin acetate、sorafenib+Lefamulin acetate 24h后细胞EDU染色结果。
图6、HepG2细胞中分别给予DMSO、sorafenib、Lefamulin acetate、sorafenib+Lefamulin acetate 24h后，继续培养2～3周细胞克隆形成结果。
图7、皮下移植瘤裸鼠分别给予Vehicle、sorafenib、Lefamulin acetate、sorafenib+Lefamulin acetate 3周后体重变化曲线。
图8、皮下移植瘤裸鼠分别给予Vehicle、sorafenib、Lefamulin acetate、sorafenib+Lefamulin acetate 3周后心、肝、脾、肺、肾组织HE染色图片。
图9、皮下移植瘤裸鼠分别给予Vehicle、sorafenib、Lefamulin acetate、sorafenib+Lefamulin acetate 3周后血浆ALT(A)、AST(B)、CRE(C)、BUN(D)含量。
图10、皮下移植瘤裸鼠分别给予Vehicle、sorafenib、Lefamulin acetate、sorafenib+Lefamulin acetate 3周后肿瘤生长曲线(A)、瘤体积变化(B)、瘤体重量(C)。
图11、皮下移植瘤裸鼠分别给予Vehicle、sorafenib、Lefamulin acetate、sorafenib+Lefamulin acetate 3周后肿瘤组织切片HE染色图片(A)、KI67免疫组织化学染色图片(B)。
图12、Lefamulin acetate与ILF3蛋白的直接结合指征图片，包括Docking(A)、DARTS(B)、CETSA(C)。
图13、Lefamulin acetate对ILF3蛋白表达量的影响图片(A)，Lefamulin acetate对ILF3下游靶基因的mRNA水平影响(B为IL-2，C为IL13，D为Sunvivin。
具体实施方式
下面结合附图和实施例对本发明进行详细描述，但所述内容是对本发明的解释而不是限定；下述实施例中的实验方法，除非另有指明，所涉及原料和试剂均为普通市售品，皆可通过市场购买获得。
实施例1逆转肝细胞癌耐药的药物筛选
1.实验方法
本发明通过药物浓度梯度递增法和持续给药法构建耐受索拉菲尼的肝癌耐药细胞株，具体过程为先以IC50浓度sorafenib刺激细胞，每天换含药物的培养基继续培养，约2～4周细胞生长正常后以0.5μM梯度增加sorafenib浓度，约3～6个月后耐药株构建成功，并通过CCK8法表征耐药株的成功构建。耐药株构建成功后，37℃，5％CO2条件下培养，待细胞汇合度约90％后，以每孔约1×104个细胞铺于96孔板中，待细胞贴壁后吸出培养基，加入含药的培养基，分组为FDA批准的上市临床药物库与sorafenib联合使用组以及单独sorafenib组，给药48h后加入CCK8，37℃避光孵育2～4h后通过酶标仪在450nm处测定吸光度值监测细胞活力，通过将联合用药组与单独sorafenib组对比发现增敏sorafenib的药物。
2.实验结果
本发明首先成功构建了肝癌耐药细胞株(图1)，耐药指数RI为2.78；并在耐药株中发现截短侧耳素抗生素药物Valnemulin HCl、Retapamulin、Lefamulin acetate能够显著降低肝癌细胞存活率，综合考虑临床用药的便捷及实用性，选择Lefamulin acetate进行进一步研究，通过进一步测定IC50值发现联合用药后sorafenib IC50值显著降低，且药物联合指数(CI)小于1，表明Lefamulin acetate可增加sorafenib杀死肝癌细胞的敏感性(图2)，同时进一步研究发现Lefamulin acetate对另外两个肝癌治疗分子靶向治疗药物Levatinib、Regorafenib也具有增敏作用(图2)。
实施例2Lefamulin acetate对肝癌细胞的体外增敏活性研究
1.实验方法
1.1Annexin V-PI细胞凋亡率测定
使用AnnexinV-PI细胞凋亡试剂盒对细胞凋亡率进行测定。将HepG2细胞以每孔约3×105个细胞接种至6孔板，待细胞过夜贴壁后Lefamulin acetate、sorafenib处理细胞48h，收集细胞后使用FITC偶联的AnnexinV和PI室温避光孵育15min，采用流式细胞仪进行细胞凋亡率分析。
1.2细胞增殖曲线测定
HepG2细胞以每孔约5×103个细胞接种至96孔板，待细胞过夜贴壁后加入药物处理，分别于24h、48h、72h采用CCK8测定吸光度值。
1.3EDU渗入实验
取对数生长期的HepG2细胞，0.25％的胰酶消化离心后用完全培养基稀释，细胞计数仪计数，调整细胞密度约为1.0×105cells/mL，然后每孔100μL接种96孔板中。在37℃、5％CO2培养箱中培养24h后，将EdU用无血清培养基稀释成20μM的溶液加入培养板中孵育6h。弃培养基，PBS小心清洗3次细胞，4％的多聚甲醛于通风橱固定15min后弃固定液，洗涤，通透液(0.3％Triton X-100的PBS)室温孵育10min，去除通透液，每孔用0.1ml洗涤液洗涤细胞1-2次，每次3-5分钟，然后每孔加入50μL Click反应液，轻轻摇晃培养板以确保反应混合物可以均匀覆盖样品。室温避光孵育30min，吸除Click反应液，用洗涤液洗涤3次，Hochest33342染色液室温孵育10min，洗涤液洗涤3次，随后用高内涵进行拍照检测。
1.4克隆形成实验
取对数生长期的HepG2细胞，0.25％胰蛋白酶消化，弃去消化液，用完全培养基轻轻吹打混匀，计数，调整细胞密度至2×103cells/mL，然后每孔2mL分别接种于6孔培养板中，培养箱中孵育，2～3天更新一次培养液，并于倒置显微镜下观察克隆形成过程。约两周后，待培养板中出现肉眼可见的克隆体时结束培养，弃去培养液，PBS洗涤2次后，4％多聚甲醛固定15min，弃固定液，并使用PBS清洗2次，于每孔加入1mL的结晶紫染色液染色20min左右，最后PBS洗去多余的染色液，将孔板置于空气中干燥，拍照。
2.实验结果
结果显示，Lefamulin acetate与sorafenib联合用药组细胞凋亡率显著增加(图3)，增殖速度减慢(图4)，EDU阳性细胞数减少(图5)，克隆形成数增加(图6)，表明Lefamulinacetate能够显著增加sorafenib对肝癌细胞的促凋亡、抑增殖作用，具有一定的增敏活性。
实施例3Lefamulin acetate体内增加sorafenib抗HCC疗效
1.实验方法
1.1裸鼠皮下移植瘤模型构建方法
细胞培养箱中培养足量的HepG2细胞。将处于对数生长期的上述细胞用0.25％的胰酶消化后，1000rpm离心5min后弃上清，计数，PBS清洗细胞沉淀2次，接着用无血清培养基和Matrigel基质胶混合物(1：1)重悬细胞成细胞密度为2×107cells/mL的细胞混悬液，充分混匀置于冰上。裸鼠荷瘤模型采取皮下接种方法。取无胸腺雄性BALB/c裸鼠右后侧腹股沟部作为成瘤部位穿刺点，70％酒精消毒穿刺部位，提起裸鼠皮肤，1mL无菌微量注射器吸取0.2mL细胞混悬液迅速注入腹股沟部位。
1.2观察成瘤及肿瘤生长情况
细胞接种后待成瘤部位长成约5mm3的瘤体视为成瘤，即造模成功。每2～3天用数显卡尺测量皮下移植瘤的最长径(L)和与之垂直的最短径(W)，计算平均瘤体积(TumorVolume,TV)：TV＝0.5×L×W2。待瘤体积约100mm3时开始随机分组给药，每组6只，分为溶剂组(灌胃蓖麻油：95％乙醇：水＝1:1:6，腹腔注射生理盐水)、灌胃sorafenib组(30mg/kg/day)、腹腔注射Lefamulin acetate低剂量组(25mg/kg/day)、腹腔注射Lefamulin acetate高剂量组(50mg/kg/day)、灌胃sorafenib组(30mg/kg/day)+腹腔注射Lefamulin acetate低剂量组(25mg/kg/day)、灌胃sorafenib组(30mg/kg/day)+腹腔注射Lefamulin acetate高剂量组(50mg/kg/day)。根据瘤体积绘制移植瘤生长曲线。给药3周后结束实验，取材前12h禁食不禁水，异氟烷吸入麻醉裸鼠，眼球取血，颈椎脱臼处死，摘取肿瘤组织称重、拍照后一部分置于4％多聚甲醛溶液中浸泡固定，石蜡包埋用于病理切片H&E染色及免疫组织化学染色，另一部分液氮速冻后放入-80℃冰箱中，用于后续实验检测。
1.3血浆样本采集
小鼠麻醉后摘眼球取血于1.5ml EP管中(加入肝素钠抗凝剂)，静置30min后4℃环境中4000rpm离心10min，轻轻转移上层透明的血浆，-80℃保存用于后续生化指标检测。
2.实验结果
2.1Lefamulin acetate对裸鼠无毒
开始给药后每天记录裸鼠体重，身体状况(如呼吸是否急促、皮肤是否出现出血点等)以及是否有死亡。给药结束后收集各组裸鼠的血浆及心、肝、脾、肺、肾各脏器，检测血浆生化指标，观察脏器病理染色，结果显示给药后各组小鼠体重无明显下降(图7)，血浆生化指标显示各组裸鼠肝功能(ALT、AST)和肾功能(CRE、BUN)均正常(图8)，HE染色结果显示各脏器无中毒迹象及死亡发生(图9)，表明药物安全性较高。
2.2Lefamulin acetate能显著增加体内sorafenib的抗肝癌效果
肿瘤生长曲线及瘤重结果显示Lefamulin acetate与sorafenib联合给药组裸鼠肿瘤生长更慢(图10)，瘤体重量更轻(图11)，结果表明Lefamulin acetate与sorafenib联合给药能协同抑制体内肝癌生长，肿瘤组织HE染色及KI67免疫组织化学染色结果显示Lefamulin acetate与sorafenib联用可协同增加肝癌细胞死亡面积百分比，抑制体内肝癌细胞增殖(KI67阳性细胞数减少)，说明Lefamulin acetate能够增加sorafenib的疗效。
实施例4Lefamulin acetate的靶蛋白研究
1.实验方法
1.1分子对接模拟(Docking)
使用基于ILF3蛋白质结构(PDB：2L33)的对接算法及Lefamulin acetate的结构式采用薛定谔模型进行分子对接模拟。
1.2药物亲和反应的靶点稳定性(DARTS)
利用裂解缓冲液收集细胞并分离总蛋白。细胞裂解液在18000×g下4℃离心10min。上清液用10×TNC缓冲液(500mM Tris-HCl,pH 8.0,500mM NaCl,100mM CaCl 2)1:10稀释，并用不同浓度的Lefamulin acetate或DMSO处理作为对照。室温孵育2h后，在37℃下加入pronase再孵育5min。通过加入蛋白酶抑制剂和SDS-PAGE加载缓冲液，然后进行免疫印迹，停止反应。
1.3细胞热转移实验(CETSA)
HepG2细胞在10厘米培养皿中培养，直到70-80％汇合。然后用Lefamulin acetate或DMSO处理细胞，孵育6h。收集细胞，PBS洗涤，在添加蛋白酶抑制剂的PBS中重悬至5×106个细胞/ml。取每个细胞悬液100μl分装到PCR管中，用热循环器在40-68℃下加热3分钟，立即在液氮中冻融裂解。细胞裂解液在4℃,15000×g离心15分钟澄清。免疫印迹法分析上清液。
1.4ILF3蛋白表达测定
HepG2细胞接种至细胞培养皿中，待细胞过夜贴壁后加入药物处理，48h后收集细胞，依据蛋白提取试剂盒操作说明Western Blot技术检测细胞中ILF3的表达。
2.实验结果
分子对接模拟、DARTS、CETSA结果显示Lefamulin acetate与ILF3蛋白直接结合，分数为-7，(图12)，Western Blot结果显示Lefamulin acetate对ILF3的蛋白表达量无影响(图13)，表明Lefamulin acetate通过与ILF3直接结合增敏sorafenib，具体机制为药物与ILF3结合后阻断ILF3与其下游耐药靶基因启动子区结合进而抑制其转录活性，发挥增敏作用。
Claims (5)
1.截短侧耳素抗生素在制备抗肿瘤及逆转分子靶向药物耐受的药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述肿瘤包括肺癌、肝癌、乳腺癌、结直肠癌、胃癌、胰腺癌，白血病和肾癌。
3.根据权利要求1所述的应用，其特征为所述肿瘤对sorafenib、levatinib、regorafenib治疗不敏感或耐药的肿瘤。
4.根据权利要求1或2所述的应用，其特征在于截短侧耳素抗生素为ValnemulinHCl、Retapamulin或Lefamulinacetate。
5.一种药物组合物在治疗肿瘤以及逆转分子靶向药物耐药的药物中应用，其特征在于所述的药物组合物包括截短侧耳素抗生素或其药学上可接受的盐或酯与临床分子靶向治疗药物。
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