CN113058035B - 替莫唑胺及其活性产物mtic的声动力新应用 - Google Patents

替莫唑胺及其活性产物mtic的声动力新应用 Download PDF

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CN113058035B
CN113058035B CN202110302764.6A CN202110302764A CN113058035B CN 113058035 B CN113058035 B CN 113058035B CN 202110302764 A CN202110302764 A CN 202110302764A CN 113058035 B CN113058035 B CN 113058035B
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闫华
王银松
徐立霞
王钒臣
周岩
宋思蓉
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Abstract

本发明属于医药技术领域,具体涉及一种替莫唑胺及其活性产物MTIC的声动力新应用,替莫唑胺及其活性产物MTIC的声动力新应用具体为利用超声协同替莫唑胺用于治疗肿瘤,能够通过引起单线态氧1O2的产生进而增强替莫唑胺抗肿瘤疗效;激发的超声频率为50kHz‑1MHz;超声强度为0.5W/cm2‑2W/cm2;激发的超声时间为30‑60s。TMZ(替莫唑胺)具有较好的声敏性,超声协同TMZ能够通过引起单线态氧的产生进而增强TMZ对胶质瘤和黑色素瘤细胞的毒性作用。本发明利用超声激发TMZ活性产物MTIC声动力开发抗肿瘤作用的新疗法,将具有不可估量的临床应用价值。

Description

替莫唑胺及其活性产物MTIC的声动力新应用
技术领域
本发明属于医药技术领域,具体涉及一种替莫唑胺及其活性产物MTIC的声动力新应用。
背景技术
目前替莫唑胺(TMZ)是被广泛应用于抗脑恶性胶质瘤和黑色素瘤治疗的一线常用药物,属于第2代口服烷化剂。口服后吸收迅速,平均半衰期为1.8小时,生物利用度接近100%,可透过血脑屏障,其脑脊液/血浆药物浓度比接近30%~40%。TMZ本身没有直接的抗肿瘤活性,其通过在体循环生理pH状态下开环转化为中间活性产物MTIC(3-甲基-(三嗪-1-)咪唑-4-甲酰胺),MTIC进一步分解为AIC和重氮甲烷,从而产生抗肿瘤作用。然而,逐渐获得的TMZ耐药性在很大程度上限制了TMZ对肿瘤患者的益处。贸然提高药物浓度来实现药效会对正常组织细胞造成伤害。因此,开发新的安全的提高TMZ抗肿瘤作用的新疗法,迫在眉睫。
声动力疗法(Sonodynamic Therapy,简称SDT)是一种无创而精准的新的肿瘤治疗方法,是在无创条件下用超声激活声敏剂,使发生超声化学反应产生单线态氧,从肿瘤细胞夺取电子破坏肿瘤细胞。声敏剂也成为SDT的关键角色,除了声敏性,还应该具有安全性好、容易操作、能被人体快速吸收且不会产生永久性伤害等特点。对于适用于脑恶性胶质瘤治疗的声敏剂,还应该具有易于穿透血脑屏障等特点。目前尚没有应用于临床脑恶性胶质瘤治疗的有效声敏剂。
发明内容
本发明的目的在于克服现有技术中的缺点,提供一种替莫唑胺及其活性产物MTIC的声动力新应用。
为实现上述目的,本发明采用的技术方案为:
一种替莫唑胺及其活性产物MTIC的声动力新应用,具体包括:利用超声协同莫唑胺用于治疗肿瘤,能够通过引起单线态氧1O2的产生进而增强替莫唑胺抗肿瘤疗效;激发的超声频率为50kHz-1MHz;超声强度为0.5W/cm2-2W/cm2;激发的超声时间为30-60s。
作为优选,激发的超声强度1W/cm2,超声时间60s。
本发明还包括制备作为治疗肿瘤的药物的应用。
所述的肿瘤为脑胶质瘤和黑色素瘤。
与现有技术相比,本发明的有益效果是:
TMZ(替莫唑胺)具有较好的声敏性,超声协同TMZ能够通过引起单线态氧的产生进而增强TMZ对胶质瘤和黑色素瘤细胞的毒性作用。本发明利用超声激发TMZ活性产物MTIC声动力开发抗肿瘤作用的新疗法,将具有不可估量的临床应用价值。
附图说明
图1是不同超声条件对肿瘤细胞存活率的影响;
图2是超声结合TMZ对肿瘤细胞存活率的影响;
图3是超声结合TMZ体内肿瘤生长曲线图;
图4是超声结合TMZ对肿瘤细胞内单线态氧产生的影响图;
图5是TMZ、MTIC、AIC体外单线态氧生成检测图。
具体实施方式
为了使本技术领域的技术人员更好地理解本发明的技术方案,下面结合附图和最佳实施例对本发明作进一步的详细说明。
实施例1:分析不同超声条件对肿瘤细胞存活率的影响。
以胶质瘤细胞U87为模型,取对数生长期的U87胶质瘤细胞,经胰酶消化后,按1×105的密度接种到12孔板里,细胞贴壁后,给予超声(强度为0、0.5、1、1.5、2W/cm2;超声时间为30s和60s)激发。24小时后,每孔加入CCK-8溶液,孵育1小时后吸取上清转移至96孔板后酶标仪测定OD450。
如图1所示,随着超声强度的增加,细胞的存活率明显降低,尤其到达1.5W/cm2以上时,超声30s的存活率为60%,而超声60s的存活率仅为40%;而当超声强度保持一致时,超声60s的存活率均低于超声30s的存活率。由此,从安全性出发,我们后续实验选取超声强度1W/cm2,超声时间60s。
实施例2:分析超声结合TMZ对U87肿瘤细胞存活率的影响。
以胶质瘤细胞U87为模型,取对数生长期的U87胶质瘤细胞,经胰酶消化后,按1×105的密度接种到12孔板里,细胞贴壁后,加入不同浓度TMZ(Solarbio,IT1330)(0、50、100、200、400、800μM),1小时后给予超声(强度为1W/cm2,时间为60s)激发。24小时后,每孔加入CCK-8溶液,孵育1小时后吸取上清转移至96孔板后酶标仪测定OD450。
如图2所示,随着TMZ浓度的增加,细胞的活力明显降低,TMZ浓度400μM时细胞存活率为下降到60%;而联合超声干预后,明显增强了TMZ的毒性作用,TMZ浓度400μM时细胞存活率仅为37%。该部分实验说明,超声增强了肿瘤细胞对TMZ的敏感性。
实施例3:超声结合TMZ体内肿瘤生长曲线图:
将U87(TMZ耐药细胞株)按照107个/100μL植入1只Bal-c裸鼠(4周Bal-c裸鼠)右侧腹股沟处,待最大直径约为10mm时,于无菌条件下取出,修剪去除脂肪和坏死组织,立即置入无血清培养液中,将瘤体剪碎至1-2mm3大小,植入第2批次24只裸鼠。将24只随机分成4组,空白对照组、单纯超声组、替莫唑胺组、超声+替莫唑胺组,每组6只。植瘤第7天腹腔注射TMZ(5mg/kg),在注射药物1h后对小鼠进行超声治疗(1.0MHz,2W/cm2,2min);TMZ是连续注射5天,超声是2天干预1次。分别在0days,2days,4days,6days,用游标卡尺测量肿瘤结节的最长径(a)和最短径(b),根据公式V=1/6π(ab2)计算肿瘤体积,绘制肿瘤生长曲线。
如图3所示,因选择的肿瘤是TMZ耐药U87细胞株,所以,单纯TMZ疗效并不理想,同时单纯超声的抑瘤效果也不明显。但是,超声+替莫唑胺联合组效果显著,皮下肿瘤生长明显受到抑制。该部分结果说明,超声结合替莫唑胺起到了协同的抑制肿瘤生长的效果。
实施例4:分析超声结合TMZ对肿瘤细胞内单线态氧产生的影响:
以U87胶质瘤细胞为模型,利用单线态氧试剂盒(贝博,BB-47055)检测空白对照组、单纯超声组、TMZ组和TMZ联合超声组细胞中单线态氧浓度。具体步骤如下:
取对数生长期的U87胶质瘤细胞,经胰酶消化后,按2×104的密度接种到已铺入细胞爬片的24孔板里,细胞贴壁后,加入含200μM TMZ的完全培养基,1小时后给予超声(强度为1W/cm2,时间为60s)激发。6小时后,弃去含药培养基,加入含单线态氧检测试剂的无血清培养基,孵育1小时后,加入PBS冲洗,待多聚甲醛固定后,取出爬片,在用含DAPI的防荧光淬灭剂封片后,放置于共聚焦显微镜下观察并拍照。
如图4所示,空白对照组、TMZ组和抑制剂组,细胞内单线态氧的表达量很低;而TMZ联合超声组细胞中单线态氧的表达量最高,强于单纯超声组。结果说明,超声激发了TMZ的声动力。
实施例5:分析TMZ、MTIC、AIC体外单线态氧生成情况
具体步骤:取浓度为4mg/ml的TMZ溶于PBS(0.01M,pH 5.5)中,震荡混匀后在室温下避光静置1小时,使用酸性PBS来确保TMZ不会水解成MTIC;取浓度为4mg/ml的TMZ溶于PBS(0.01M,pH 7.4)中,震荡混匀后在室温下避光静置1小时,以完全转化为MTIC(MW 182.18);取浓度为2.6mg/ml的AIC(MW126.12)溶于PBS(0.01M,pH 7.4)中,震荡混匀后在室温下避光静置1小时。分别向以上试剂中加入单线态氧检测试剂SOSG,震荡混匀后置于超声探头上。于超声的第0、2、4、6、8、10分钟取出部分液体,于分光光度仪中检测该溶液在激发波长Ex=504nm,发射波长Em=525nm下的紫外吸收,获得吸光度值;
如图5所示,随着超声时间的延长,MTIC产生单线态氧的浓度明显增加,超声10分钟MTIC单线态氧浓度增加了20倍,远远超过TMZ和AIC产生的单线态氧浓度。结果表明,TMZ声动力指的是在体循环生理pH状态下TMZ的活性产物MTIC的声动力,而不是TMZ本身。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。

Claims (5)

1.一种替莫唑胺及其活性产物MTIC作为制备声敏剂的应用。
2.根据权利要求1所述的替莫唑胺及其活性产物MTIC作为制备声敏剂的应用,其特征在于,利用超声协同替莫唑胺用于治疗肿瘤,能够通过引起单线态氧1O2的产生进而增强替莫唑胺抗肿瘤疗效;激发的超声频率为50kHz-1MHz;超声强度为0.5W/cm2-2W/cm2;激发的超声时间为30-60s。
3.根据权利要求2所述的替莫唑胺及其活性产物MTIC作为制备声敏剂的应用,其特征在于,激发的超声强度1W/cm2,超声时间60s。
4.根据权利要求1所述的替莫唑胺及其活性产物MTIC作为制备声敏剂的应用,其特征在于,制备用于肿瘤声动力治疗。
5.根据权利要求4所述的替莫唑胺及其活性产物MTIC作为制备声敏剂的应用,其特征在于,所述的肿瘤为脑胶质瘤和/或黑色素瘤。
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Citations (1)

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WO2000033823A2 (en) * 1998-12-07 2000-06-15 Schering Corporation Methods of using temozolomide in the treatment of cancers

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