CN113493415B - 一种青藤碱衍生物代谢产物及其制备方法、药物组合物和用途 - Google Patents
一种青藤碱衍生物代谢产物及其制备方法、药物组合物和用途 Download PDFInfo
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Abstract
本发明属于医药技术领域,具体公开了一种如式(Ⅰ)的青藤碱衍生物代谢产物sino‑wcj‑43‑M1和制备方法,及其在制备预防或治疗再生障碍性贫血药物中的应用。药代动力学研究发现,sino‑wcj‑43‑M1是sino‑wcj‑43在体内的主要存在形式。利用酯酶对sino‑wcj‑43进行水解,经分离纯化可得到新化合物sino‑wcj‑43‑M1,或以盐酸青藤碱为原料,采用化学合成方法合成sino‑wcj‑43‑M1。该化合物具有与sino‑wcj‑43类似的明显的治疗再生障碍性贫血的作用,可以单体或药用组合物形式在再生障碍性贫血的临床治疗中应用。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种青藤碱衍生物代谢产物—新化合物sino-wcj-43-M1或其药学上可接受的盐及其制备方法,及含有该衍生物代谢产物或其药学上可接受的盐的药物组合物及其在制备预防或治疗再生障碍性贫血中的应用。
背景技术
再生障碍性贫血,简称再障(aplastic anemia,AA),是一种较为严重的血液系统疾病,是由物理、化学、生物或其它原因所致的骨髓造血功能衰竭性疾病,临床主要表现为全血细胞减少所致的贫血、出血和感染,严重时可危及生命。造血干细胞移植是目前唯一能够治愈再障的治疗方案,但由于移植配型难,治疗风险大,大部分患者只能采取药物进行免疫抑制治疗。目前认为,免疫功能紊乱在再障的发生发展过程中发挥关键性作用。临床常用的免疫抑制剂包括ATG/ALG、环孢素A(cyclosporin A,CsA)、环磷酰胺等。然而,免疫抑制剂对免疫系统的整体抑制会给患者带来严重的不良反应,且价格昂贵、复发率高,治疗效果不尽人意。因此寻找更为有效的治疗途径和药物是再障治疗的迫切需求。
青藤碱衍生物sino-wcj-43是我所自主研发的新型小分子抗再生障碍性贫血化合物,关于sino-wcj-43的合成及抗炎、治疗再生障碍性贫血的药理作用已于2017年申请国家发明专利,申请号为CN201710472411.4。sino-wcj-43具有合成工艺简单稳定、生物利用度高、毒性小、疗效确切的特点,具有实际开发价值。
发明内容
本发明解决的技术问题是提供一种具有新的化学结构特征的青藤碱衍生物代谢产物sino-wcj-43-M1及其药学上可接受的盐,其制备方法、药物组合物和用途。
为解决本发明的技术问题,本发明提供如下技术方案:
本发明技术方案的第一方面是提供了一种具有新的化学结构特征的青藤碱衍生物代谢产物sino-wcj-43-M1及其药学上可接受的盐,其结构如式(Ⅰ)所示:
本发明技术方案的第二方面提供了所述式(Ⅰ)化合物的生物合成制备方法:利用酯酶水解sino-wcj-43 1位羟甲基酯键,获得青藤碱衍生物代谢产物,
本发明青藤碱衍生物代谢产物sino-wcj-43-M1,结构新颖,分子式为C29H31NO6,命名为1-hydroxymethylene-4-cinnamyloxy-7,8-didehydro-3,7-dimethoxy-17-methyl-morphinan-6-one,1-羟基亚甲基-4-桂皮酰氧基-7,8-二去氢-3,7-二甲氧基-17-甲基-吗啡喃-6-酮。生物学合成步骤如下:
1)采用酯酶催化sino-wcj-43水解为sino-wcj-43-M1。反应体系为PBS缓冲液(pH=7.4)、猪肝酯酶25U/mL、sino-wcj-43 0.5mg/mL,37℃水浴反应2h。
2)反应液中加入等体积乙腈沉淀蛋白,乙酸乙酯萃取,制备液相分离纯化,浓缩干燥,得白色固体sino-wcj-43-M1。
本发明技术方案的第三方面提供了所述式(Ⅰ)化合物的化学合成制备方法:以盐酸青藤碱sino为原料,与多聚甲醛加热回流反应生成式1所示1-羟基亚甲基青藤碱;1-羟基亚甲基青藤碱与叔丁基二甲基氯硅烷,咪唑和4-二甲氨基吡啶反应得到分离得中间体2;
化合物2与肉桂酸,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶反应得到中间体3;化合物3与氟化氢和吡啶反应得到目标化合物sino-wcj-43-M1。
具体化学合成步骤如下:
1)称取盐酸青藤碱10.0g,溶于200ml水中,搅拌下加入20.0g多聚甲醛(质量2-3倍于盐酸青藤碱),加热回流反应4h-12h,TLC监测反应过程。反应结束后,用CHCl3萃取(100mL×3或者萃取次数可增加),合并有机相,无水Na2SO4干燥,过滤,蒸除溶剂,得白色固体,得到1-羟基亚甲基青藤碱。
2)称取1-羟基亚甲基青藤碱250mg(1eq),溶于10ml四氢呋喃中,搅拌下加入叔丁基二甲基氯硅烷840mg(8eq),咪唑380mg(8eq),4-二甲氨基吡啶170mg(0.2eq),室温下搅拌,TLC监测反应。约4小时后,薄层色谱显示原料化合物1基本消失,向反应液中加入10ml水停止反应,将液体转移至分液漏斗中,加入(20ml×3次)乙酸乙酯萃取,合并有机层,用无水硫酸钠干燥,浓缩,柱层析(二氯甲烷/甲醇=50/1)分离得到白色发泡状固体化合物2。
3)称取化合物2约150mg(1eq)溶于10ml二氯甲烷中,搅拌下加入肉桂酸178mg(3.8eq),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐200mg(3.3eq),4-二甲氨基吡啶116mg(0.3eq),室温下搅拌,TLC监测反应。约1.5小时后,薄层色谱显示原料化合物2基本消失,向反应液中加入10ml水停止反应,将液体转移至分液漏斗中,加入(20ml×3次)二氯甲烷萃取,合并有机层后用饱和氯化铵溶液3×30ml洗涤,氯化铵水层合并后用20ml二氯甲烷萃取一次。合并有机层,用无水硫酸钠干燥,浓缩,柱层析(乙酸乙酯/石油醚=100/1)分离得到淡黄色油状液体化合物3。
4)称取化合物3约30mg(1eq)溶于2ml乙腈中,将氟化氢0.25ml(60eq)和吡啶1.0ml(60eq)混匀冷却至室温后,加入到反应液中,室温下搅拌,TLC监测反应。约2小时后,薄层色谱显示原料化合物3基本消失,向反应液中加入乙酸乙酯5ml稀释反应液,将反应液转移至分液漏斗中,加入(5ml×3次)饱和碳酸氢钠溶液洗涤,合并水相,水相用乙酸乙酯(15ml×3次)萃取。合并有机层,用无水硫酸钠干燥,浓缩,柱层析(二氯甲烷/甲醇=25/1)分离得到淡黄色发泡状固体,然后将淡黄色发泡状固体加入至5ml乙酸乙酯中搅拌混匀,得白色悬浊液,搅拌20分钟,滤出固体,用乙酸乙酯和石油醚洗后,干燥得白色粉末状固体化合物sino-wcj-43-M1(路线图中的结构式M1即为本发明中的结构式(I))。
本发明技术方案的第四方面是提供了一种药物组合物,其含有治疗有效剂量的式(Ⅰ)的青藤碱衍生物代谢产物sino-wcj-43-M1及其药学上可接受的盐及药用载体。
本发明还涉及一种含有药物有效剂量所述的化合物和药效学上可接受的载体的药物组合物。用于此目的时,如果需要,可与一种或多种固体或液体药物赋形剂和/或辅剂结合,制成可作为人药使用的适当施用形式或剂量形式。
根据本发明,本发明化合物可以以异构体的形式存在,而且通常所述的“本发明化合物”包括该化合物的异构体。
本发明的药物组合物可以单位剂量形式给药,给药途径可为肠道或非肠道,如口服、肌肉、皮下、鼻腔、口腔粘膜、皮肤、腹膜或直肠等。
本发明的药物组合物的给药途径可为注射给药。注射包括静脉注射、肌肉注射、皮下注射、皮内注射和穴位注射等。给药剂型可以是液体剂型、固体剂型。如液体剂型可以是真溶液类、胶体类、微粒剂型、乳剂剂型、混悬剂型。其他剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、栓剂、冻干粉针剂等。
本发明的组合物可以制成普通制剂、也可以是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将单位给药剂型制成片剂,可以广泛使用本领域公知的各种载体。关于载体的例子是,例如稀释剂与吸收剂,如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化纳、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等;湿润剂与粘合剂,如水、甘泊、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素纳、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等;崩解剂,例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢纳与枸橼酸、碳酸钙、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等;崩解抑制剂,例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等;吸收促进剂,例如季铵盐、十二烷基硫酸纳等;润滑剂,例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片,例如糖包衣片、薄膜包衣片、肠溶包衣片,或双层片和多层片。
为了将给药单元制成丸剂,可以广泛使用本领域公知的各种载体。关于载体的例子是,例如稀释剂与吸收剂,如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等;粘合剂,如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等;崩解剂,如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。
为了将给药单元制成栓剂,可以广泛使用本领域公知的各种载体。关于载体的例子是,例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酶、明胶、半合成甘油酶等。
为了将给药单元制成胶囊,将有效成分与上述的各种载体混合,并将由此得到的混合物置于硬的明胶胶囊或软胶囊中。也可将有效成分制成微囊剂,混悬于水性介质中形成混悬剂,亦可装入硬胶囊中或制成注射剂应用。
例如,将本发明的组合物制成注射用制剂,如溶液剂、混悬剂溶液剂、乳剂、冻干粉针剂,这种制剂可以是含水或非水的,可含一种和/或多种药效学上可接受的载体、稀释剂、粘合剂、润滑剂、防腐剂、表面活性剂或分散剂。如稀释剂可选自水、乙醇、聚乙二醇、1,3一丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酶等。另外,为了制备等渗注射液,可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油,此外,还可以添加常规的助溶剂、缓冲剂、pH调节剂等。这些辅料是本领域常用的。
此外如需要,也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。
本发明药用组合物的给药剂量取决于许多因素,例如所要预防或治疗疾病的性质和严重程度,患者或动物的性别、年龄、体重、性格及个体反应,给药途径、给药次数等,因此本发明的治疗剂量可以有大范围的变化。一般来讲,本发明化合物使用剂量是本领域技术人员公知的。可以根据本发明药用组合物中最后的制剂中所含有的实际有效药物数量,加以适当的调整,以达到其治疗有效量的要求,完成本发明的治疗再生障碍性贫血的目的。
通常对体重约75公斤患者,所给本发明化合物的日剂量为0.001mg/kg体重~200mg/kg体重,优选1mg/kg体重~100mg/kg体重。上述剂量可以单一剂量形式或分成几个,例如二、三或四个剂量形式给药,这受限于给药医生的临床经验以及给药方案。本发明的化合物或组合物可单独服用,或与其它治疗药物或对症药物合并使用。
本发明技术方案的第五方面是提供了第一方面所述式(Ⅰ)的青藤碱衍生物代谢产物sino-wcj-43-M1及其药学上可接受的盐和第四方面所述药物组合物在制备预防或治疗再生障碍性贫血药物中的应用。所述再生障碍性贫血包括急性、慢性再生障碍性贫血。
有益技术效果:
本发明通过药代动力学研究发现,青藤碱衍生物sino-wcj-43经口服给药后在体内经酯酶迅速水解,主要以代谢产物sino-wcj-43-M1的形式存在,该代谢产物结构新颖,化学结构如式I所示。研究显示,该代谢产物在体外能有效抑制CD8+T淋巴细胞的激活以及造血负调控因子IFN-γ、TNF-α的释放,皮下给药具有抑制小鼠耳部急性炎症的作用,口服给药可显著改善再障模型小鼠的动物生存状态与贫血症状、恢复骨髓造血功能,具有与sino-wcj-43类似的抗再生障碍性贫血的作用与较小的毒性,具有深入研究与开发的价值。有关该代谢产物的结构、制备方法及其药理活性研究迄今未见报道。具体而言,其有益技术效果如下:
(1)本发明为青藤碱衍生物代谢产物sino-wcj-43-M1,结构新颖;(2)本发明制备方法新颖,原料易得,制备工艺简单,易于规范化生产;(3)本发明能有效防治再生障碍性贫血,sino-wcj-43-M1在体外能有效抑制CD8+T淋巴细胞激活以及造血负调控因子IFN-γ、TNF-α的释放;皮下单次给药可抑制巴豆油诱导的小鼠急性耳炎,显示对免疫系统的调节作用;口服给药可显著提高再障模型小鼠的生存率、体重、进食量和胸腺脏器指数,改善动物生存状态,升高外周血红细胞、白细胞以及血小板的含量,缓解贫血症状;增加骨髓有核细胞计数,恢复骨髓造血功能,具有与sino-wcj-43类似的抗再生障碍性贫血的作用,有进一步开发成防治再生障碍性贫血药物的潜力;(4)本发明具有相对较小的毒性。
附图说明
图1化合物1的1H NMR图谱
图2化合物2的1H NMR图谱
图3化合物3的1H NMR图谱
图4化合物M1的1H NMR图谱
图5化合物M1的质谱
图6大鼠口服给药(100mg/kg)后血浆中药物含量随时间变化曲线(n=3)
具体实施方式
以下实施例对本发明作进一步的说明,但本发明并不限于这些实施例。
一、sino-wcj-43-M1的制备方法
实施例1:sino-wcj-43-M1的生物合成
步骤一:采用酯酶催化sino-wcj-43水解为sino-wcj-43-M1。反应体系为PBS缓冲液(pH=7.4)、猪肝酯酶25U/mL、sino-wcj-43 0.5mg/mL,37℃水浴反应2h,经HPLC-UV检测,水解反应转化率可达90%以上。
步骤二:反应液中加入等体积乙腈沉淀蛋白,5500rpm离心10min去除蛋白沉淀,上清液减压蒸去有机溶剂,乙酸乙酯萃取5次,减压蒸干,少量流动相溶解,利用制备液相分离纯化sino-wcj-43-M1。制备柱为Reprosil-Pur Basic C18(250×10mm,5μm,Beim Brueckle14-D-72119Ammerbuch,Germany),流动相为27%水(0.02%二乙胺)-73%乙腈等度洗脱,流速5mL/min,UV检测波长283nm,sino-wcj-43-M1出峰时间为5.7min,收集流份,减压蒸干,得白色固体。经UV、MS、NMR等方法进行测定分析,确定结构如式Ⅰ。
实施例2:sino-wcj-43-M1的化学合成
分子式:C29H31NO6
命名:1-hydroxymethylene-4-cinnamyloxy-7,8-didehydro-3,7-dimethoxy-17-methyl-morphinan-6-one,1-羟基亚甲基-4-桂皮酰氧基-7,8-二去氢-3,7-二甲氧基-17-甲基-吗啡喃-6-酮。
以盐酸青藤碱sino为原料,与多聚甲醛加热回流反应生成式1所示1-羟基亚甲基青藤碱;1-羟基亚甲基青藤碱与叔丁基二甲基氯硅烷,咪唑和4-二甲氨基吡啶反应得到分离得中间体2;化合物2与肉桂酸,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-二甲氨基吡啶反应得到中间体3;化合物3与氟化氢和吡啶反应得到目标化合物sino-wcj-43-M1。
步骤一,称取盐酸青藤碱10.0g,溶于200ml水中,搅拌下加入20.0g多聚甲醛(质量2-3倍于盐酸青藤碱),加热回流反应4h-12h,TLC监测反应过程。反应结束后,用CHCl3萃取(100mL×3或者萃取次数可增加),合并有机相,无水Na2SO4干燥,过滤,蒸除溶剂,得白色固体,得到1-羟基亚甲基青藤碱(1)。化合物1的1H NMR(400MHz,DMSO-d6)δ8.28(s,1H),6.82(s,1H),5.68(d,J=2.00Hz,1H),4.91(s,1H),4.38(ABq,J=13.50Hz,2H),4.18(d,J=15.60Hz,1H),3.72(s,3H),3.34(s,3H),3.14(s,1H),2.77-2.86(m,2H),2.41-2.45(m,1H),2.35-2.37(m,2H),2.27(s,3H),1.82-1.87(m,1H),1.71-1.77(m,2H)。(见图1)
步骤二,称取1-羟基亚甲基青藤碱250mg(1eq),溶于10ml四氢呋喃中,搅拌下加入叔丁基二甲基氯硅烷840mg(8eq),咪唑380mg(8eq),4-二甲氨基吡啶170mg(0.2eq),室温下搅拌,TLC监测反应。约4小时后,薄层色谱显示原料化合物1基本消失,向反应液中加入10ml水停止反应,将液体转移至分液漏斗中,加入(20ml×3次)乙酸乙酯萃取,合并有机层,用无水硫酸钠干燥,浓缩,柱层析(二氯甲烷/甲醇=50/1)分离得到白色发泡状固体化合物2。化合物2的1H NMR(500MHz,CDCl3)δ6.82(s,1H),5.44(s,1H),4.56(ABq,J=13.50Hz 2H),4.37(d,J=15.50Hz,1H),3.80(s,3H),3.45(s,3H),3.25(s,1H),3.01(s,1H),2.91(d,J=18.00Hz,1H),2.53(dd,J=12.00,2.50Hz,1H),2.49(d,J=5.50Hz,1H),2.44-2.46(m,1H),2.41(s,3H),2.01-2.06(m,1H),1.94(d,J=12.50Hz,1H),1.85-1.89(m,1H),0.93(s,9H),0.08(s,6H)。(见图2)
步骤三,称取化合物2约150mg(1eq)溶于10ml二氯甲烷中,搅拌下加入肉桂酸178mg(3.8eq),1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐200mg(3.3eq),4-二甲氨基吡啶116mg(0.3eq),室温下搅拌,TLC监测反应。约1.5小时后,薄层色谱显示原料化合物2基本消失,向反应液中加入10ml水停止反应,将液体转移至分液漏斗中,加入(20ml×3次)二氯甲烷萃取,合并有机层后用饱和氯化铵溶液3×30ml洗涤,氯化铵水层合并后用20ml二氯甲烷萃取一次。合并有机层,用无水硫酸钠干燥,浓缩,柱层析(乙酸乙酯/石油醚=100/1)分离得到淡黄色油状液体化合物3。化合物3的1H NMR(400MHz,CDCl3)δ8.02(d,J=16.00Hz,1H),7.64-7.67(m,2H),7.41-7.43(m,3H),7.05(s,1H),6.69(d,J=16.00Hz,1H),5.43(d,J=2.00Hz,1H),4.63(ABq,J=13.50Hz,2H),3.95(d,J=15.20Hz,1H),3.75(s,3H),3.55-3.57(m,1H),3.48(s,3H),3.30(s,1H),2.97(d,J=18.40Hz,1H),2.69-2.82(m,2H),2.56(s,3H),,2.29-2.35(m,1H),2.07(dd,J=13.20,4.40Hz,1H),2.02(s,1H),1.66-1.74(m,1H),0.95(s,9H),0.12(s,6H)。(见图3)
步骤四,称取化合物3约30mg(1eq)溶于2ml乙腈中,将氟化氢0.25ml(60eq)和吡啶1.0ml(60eq)混匀冷却至室温后,加入到反应液中,室温下搅拌,TLC监测反应。约2小时后,薄层色谱显示原料化合物3基本消失,向反应液中加入乙酸乙酯5ml稀释反应液,将反应液转移至分液漏斗中,加入(5ml×3次)饱和碳酸氢钠溶液洗涤,合并水相,水相用乙酸乙酯(15ml×3次)萃取。合并有机层,用无水硫酸钠干燥,浓缩,柱层析(二氯甲烷/甲醇=25/1)分离得到淡黄色发泡状固体,然后将淡黄色发泡状固体加入至5ml乙酸乙酯中搅拌混匀,得白色悬浊液,搅拌20分钟,滤出固体,用乙酸乙酯和石油醚洗后,干燥得白色粉末状固体化合物sino-wcj-43-M1。化合物M1的1H NMR(500MHz,CDCl3)δ8.02(d,J=16.00Hz,1H),7.65(s,2H),7.43(s,3H),6.98(s,1H),6.69(d,J=16.00Hz,1H),5.47(s,1H),4.67(ABq,J=13.50Hz,2H),4.49(s,1H),3.95(d,J=16.00Hz,1H),3.75(s,3H),3.48(s,3H),3.39(s,1H),3.07-3.15(m,2H),2.67-2.75(m,2H),2.53(d,J=16.00Hz,1H),2.44(s,3H),2.22(s,1H),1.95-1.99(m,1H),1.66(s,1H)。(见图4);ESI-MS(m/z):490.22(M+H)+。(见图5)
二、大鼠口服sino-wcj-43及sino-wcj-43-M1后血浆药代动力学研究
实验例1:
实验方法:雄性SD大鼠每组5只,口服给药50mg/kg,实验前禁食12h,自由饮水。于给药后10、30min、1、2、4、6、8、12、24h目内眦静脉丛连续取血,肝素抗凝,离心分离血浆,乙腈沉淀蛋白,采用HPLC测定血浆中sino-wcj-43和sino-wcj-43-M1的含量。所测实验数据应用WinNonlin 6.3软件计算药代动力学参数。
实验结果:大鼠灌胃给予sino-wcj-43后,在100-5000ng/ml定量范围内血浆中未检测到原型药,但可检测到代谢产物sino-wcj-43-M1,在给药4h达峰,平均血药峰浓度为2626ng/ml,AUC0-t为10281h*ng/ml(图1)。该实验结果表明,药物经口服给药后,代谢产物sino-wcj-43-M1是药物在体内的主要存在形式。
大鼠灌胃给予sino-wcj-43-M1后,经HPLC测定,血浆中可检测到sino-wcj-43-M1,未检测到其它代谢产物。表明代谢产物经口服给药后,sino-wcj-43-M1仍是药物在体内的主要存在形式。
三、初步毒理学实验
实验例2:sino-wcj-43-M1急性毒性实验
实验方法:雄性昆明种小鼠,(20±2)g,随机分为正常组、青藤碱各剂量给药组(0.51、0.64、0.80、1.00、1.25、1.56、1.95、2.44、3.05g/kg)、sino-wcj-43-M1各剂量给药组(5、10g/kg),sino-wcj-43各剂量给药组(5、10g/kg),每组10只。给药前禁食12h,自由饮水,灌胃给药1次,停药后观察2周,计算小鼠半数致死量LD50。
实验结果:sino-wcj-43和sino-wcj-43-M1各剂量组灌胃给药后小鼠精神状态良好,均未出现中毒反应,给药后14天内无动物死亡,最大给药量为10g/kg,未能测得LD50,青藤碱LD50为1.07g/kg。实验结果表明,与青藤碱相比,青藤碱衍生物sino-wcj-43与代谢产物sino-wcj-43-M1的急性毒性均较低。
四、药理学实验:
实验例3:sino-wcj-43-M1体外对CD8+T淋巴细胞激活的抑制作用
实验方法:取balb/c小鼠淋巴结和脾脏制备单细胞悬液,磁珠分选CD8+T细胞,接种于96孔板中,2×105个/孔,培养体系为RPMI-1640、10%FBS、50μM 2-巯基乙醇、1μg/mlCD3/CD28抗体(T细胞激活剂),加入不同浓度化合物,于37℃、5%CO2培养箱中培养72h。实验分为正常组、模型组、阳性药组(环孢素CsA 50nM)、低中高浓度给药组。采用CCK-8法测定细胞活力,收集上清液,采用ELISA试剂盒测定药物对IFN-γ、TNF-α释放的影响。
实验结果:免疫功能失调导致CD8+T淋巴细胞功能亢进是AA的重要病理环节,激活的CD8+T细胞可与骨髓造血干细胞直接作用而将其杀伤,还可通过大量释放IFN-γ、TNF-α抑制造血干细胞功能,从而导致骨髓造血抑制。本实验结果见表1,与正常组相比,模型组CD8+T细胞活力升高,IFN-γ、TNF-α释放量增加,10、30μM sino-wcj-43-M1可显著抑制CD8+T细胞活力的升高,3、10、30μM sino-wcj-43-M1可显著抑制IFN-γ、TNF-α释放的增加,表明sino-wcj-43-M1在体外对CD8+T淋巴细胞激活、造血负调控因子的释放具有抑制作用。
表1 sino-wcj-43-M1体外对CD8+T淋巴细胞激活的抑制作用(Mean±SD,n=6-9)
备注:###p<0.001vs正常对照组;**p<0.01,***p<0.001vs模型对照组
实验例4:sino-wcj-43-M1对巴豆油诱导小鼠耳炎的影响
实验方法:取18-20g的雄性昆明种小鼠,随机分组,各组动物别于左耳两面涂巴豆油0.02ml;30分钟后,各组动物分别皮下注射给予100mg/kg、200mg/kg体重受试化合物,模型对照组给予等体积溶媒;给药4h后,脱颈处死小鼠,沿耳廓基线剪下双耳,直径6mm打孔器分别取下左右耳相同位置的耳片,分析天平称重,计算耳肿胀度(耳肿胀度=左耳片重量-右耳片重量)和耳肿胀抑制率[耳肿胀抑制率(%)=(模型组平均耳肿胀度-给药组耳肿胀度)/模型组平均耳肿胀组×100%]。
实验结果:实验结果如表2所示,100mg/kg、200mg/kg sino-wcj-43-M1可减轻巴豆油致小鼠急性耳炎的耳肿胀程度,与模型组相比具有显著性差异,显示对免疫系统的调节作用,与等剂量原型药作用效果一致。
表2 sino-wcj-43-M1对巴豆油诱导小鼠耳炎的影响(Mean±SD,n=8)
备注:*p<0.05,**p<0.01vs模型组
实验例5:sino-wcj-43-M1对免疫介导的再生障碍性贫血的作用
实验方法:雄性BALB/c小鼠,(20±2)g,随机分为正常组、模型组、阳性药组(环孢素10mg/kg)、sino-wcj-43组(10mg/kg)、sino-wcj-43-M1组(7mg/kg、10mg/kg)。除空白组外,各组动物采用钴-60γ射线(5.8Gy)全身照射+DBA/2小鼠胸腺-淋巴结免疫细胞2:1静脉注射的方法制备再生障碍性贫血模型;造模当日开始灌胃给药,每天1次,连续14天。动物每周称进食量和体重,造模第14天,小鼠目内眦静脉从采血,EDTA-K2抗凝,五分类血细胞分析仪检测血液常规,取小鼠胸腺和脾脏,称其湿重,计算脏器指数(脏器指数=脏器重量/当日体重);取小鼠右侧股骨骨髓,裂解红细胞后采用流式细胞仪进行骨髓有核细胞计数。
实验结果:
(1)sino-wcj-43-M1对再生障碍性贫血小鼠体重、进食量与死亡率的影响
表3结果显示,与正常组比较,再障模型小鼠造模第7天、14天体重显著降低,sino-wcj-43及sino-wcj-43-M1对体重下降有显著改善作用,且sino-wcj-43-M1的作用呈剂量依赖关系,阳性药对小鼠体重下降虽有缓解趋势,但未见显著性差异。进食量结果如表4所示,与正常组比较,再障模型小鼠造模第7天、14天进食量显著降低,在造模第14天,sino-wcj-43-M1的10mg/kg剂量组可显著增加再障小鼠进食量,提示sino-wcj-43-M1可改善再障引起的小鼠消化功能损伤,改善动物生存质量。等剂量阳性药及sino-wcj-43对小鼠进食量虽有增加趋势,但未见显著性差异。死亡率结果如表5所示,sino-wcj-43-M1和sino-wcj-43均可降低模型动物的死亡率,与阳性药相比具有优势。
表3 sino-wcj-43-M1对再生障碍性贫血小鼠体重生长的影响(Mean±SD,n=9-10)
备注:#p<0.05,##p<0.01vs正常对照组;*p<0.05,**p<0.01vs模型对照组
表4 sino-wcj-43-M1对再生障碍性贫血小鼠进食量的影响(Mean±SD,n=9-14)
备注:#p<0.05,##p<0.01vs正常对照组;*p<0.05,**p<0.01vs模型对照组
表5 sino-wcj-43-M1对再生障碍性贫血小鼠死亡率的影响(n=9-14)
(2)sino-wcj-43-M1对再生障碍性贫血小鼠胸腺、脾脏脏器指数的影响
胸腺、脾脏脏器指数可反映动物的免疫功能状态。实验结果如表6所示,与正常组比较,再障模型小鼠脾脏与胸腺指数均显著下降,阳性药、sino-wcj-43和sino-wcj-43-M1均可显著增加模型小鼠的胸腺指数,说明各药物均具有改善再障小鼠免疫功能低下的作用,且sino-wcj-43-M1的作用呈剂量依赖关系。
表6 sino-wcj-43-M1对再生障碍性贫血小鼠免疫脏器指数的影响(Mean±SD,n=9-10)
备注:#p<0.05,##p<0.01vs正常对照组;*p<0.05,**p<0.01vs模型对照组
(3)sino-wcj-43-M1对再生障碍性贫血小鼠外周血液常规检查结果的影响
外周血全血细胞减少是再障贫血的主要特征,反映了骨髓造血功能的衰竭情况。外周血各项指标尤其是红细胞、血红蛋白、白细胞和血小板计数的升高是再障得到有效治疗的直接证据和药理学检测的主要药效学指标。小鼠外周血的各项指标结果见表7A、7B及7C。
①sino-wcj-43-M1对再生障碍性贫血小鼠外周血红细胞计数和血红蛋白含量的影响
与正常组比较,再障模型小鼠红细胞计数、血红蛋白含量、红细胞压积均显著下降,表现出明显的贫血症状,阳性药、sino-wcj-43和sino-wcj-43-M1对以上三项指标均具有显著改善作用,且sino-wcj-43-M1的作用呈剂量依赖关系。
表7A sino-wcj-43-M1对再生障碍性贫血小鼠外周血红细胞计数和血红蛋白含量的影响(Mean±SD,n=9-10)
备注:红细胞(RBC),血红蛋白(HGB),血红细胞压积(HCT);#p<0.05,##p<0.01vs正常对照组;*p<0.05,**p<0.01vs模型对照组。
②sino-wcj-43-M1对再生障碍性贫血小鼠外周血中白细胞数和淋巴细胞数的影响
与正常组比较,再障模型小鼠白细胞总数、淋巴细胞、单核细胞、中性粒细胞、嗜酸性、嗜碱性粒细胞等各分类细胞计数均显著下降,sino-wcj-43-M1在10mg/kg剂量下可显著升高白细胞总数和淋巴细胞计数,对单核细胞、中性粒细胞、嗜酸性粒细胞计数也有一定升高趋势与sino-wcj-43的药效作用基本一致。
表7B sino-wcj-43-M1对再生障碍性贫血小鼠外周血中白细胞分类计数的影响(Mean±SD,n=9-10)
备注:白细胞计数(WBC),淋巴细胞计数(LYM#),单核细胞计数(Mon#),中性粒细胞计数(NEUT#),嗜酸性粒细胞计数(Eos#),嗜碱性粒细胞计数(Bas#);#p<0.05,##p<0.01vs正常对照组;*p<0.05,**p<0.01vs模型对照组。
③sino-wcj-43-M1对再生障碍性贫血小鼠外周血血小板计数的影响
与正常组比较,再障模型小鼠血小板计数、血小板压积均显著下降,阳性药、sino-wcj-43和sino-wcj-43-M1对血小板计数、血小板压积均有显著升高作用。
表7C sino-wcj-43-M1对再生障碍性贫血小鼠外周血中血小板的影响(Mean±SD,n=9-10)
备注:血小板计数(PLT),血小板压积(PCT);#p<0.05,##p<0.01vs正常对照组;*p<0.05,**p<0.01vs模型对照组。
(5)sino-wcj-43-M1对再生障碍性贫血小鼠骨髓有核细胞计数的影响
实验结果如表8所示,与正常对照组比较,再障模型小鼠骨髓有核细胞计数显著降低,表明造血组织功能减退,阳性药、sino-wcj-43和sino-wcj-43-M1可显著升高模型小鼠的骨髓有核细胞计数,表明药物对再生障碍性贫血小鼠骨髓造血功能的减退具有改善作用。
表8 sino-wcj-43-M1对再生障碍性贫血小鼠骨髓有核细胞计数的影响(Mean±SD,n=8)
备注:#p<0.05,##p<0.01vs正常对照组;*p<0.05,**p<0.01vs模型对照组
综上所述,sino-wcj-43-M1可降低再生障碍性贫血模型小鼠的死亡率,增加再障小鼠进食量与体重,改善动物生存状态;增加胸腺指数,恢复免疫器官功能;提高外周血红细胞计数、血红蛋白含量、白细胞计数和血小板计数,改善贫血症状;升高骨髓有核细胞计数,改善骨髓造血功能。以上结果表明,青藤碱衍生物代谢产物sino-wcj-43-M1具有与原型药sino-wcj-43类似的抗再生障碍性贫血药效作用,且在改善动物生存状态方面与阳性药相比更具优势。
Claims (7)
1.一种青藤碱衍生物代谢产物及其药学上可接受的盐,其特征在于,所述的结构如式(Ⅰ)所示,
2.制备权利要求1所述的青藤碱衍生物代谢产物的生物合成方法,其特征在于,包括以下步骤:利用酯酶水解青藤碱衍生物sino-wcj-43 1位羟甲基酯键,获得青藤碱衍生物代谢产物;
3.一种药物组合物,其特征在于,所述的药物组合物由有效剂量的权利要求1所述的青藤碱衍生物代谢产物或其药学上可接受的盐和药学上可接受的载体或辅料组成。
4.根据权利要求3的药物组合物,其特征在于,所述的药物组合物选自片剂、胶囊、丸剂、注射剂。
5.根据权利要求3的药物组合物,其特征在于,所述药物组合物选自缓释制剂、控释制剂、及各种微粒给药系统。
6.权利要求1所述的青藤碱衍生物代谢产物或其药学上可接受的盐在制备治疗和/或预防再生障碍性贫血药物中的应用。
7.根据权利要求6的应用,其特征在于,所述的再生障碍性贫血选自:急性再生障碍性贫血、慢性再生障碍性贫血。
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