CN101111241B - 抗肿瘤制备物和抗肿瘤制备物的用途 - Google Patents

抗肿瘤制备物和抗肿瘤制备物的用途 Download PDF

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CN101111241B
CN101111241B CN2006800019675A CN200680001967A CN101111241B CN 101111241 B CN101111241 B CN 101111241B CN 2006800019675 A CN2006800019675 A CN 2006800019675A CN 200680001967 A CN200680001967 A CN 200680001967A CN 101111241 B CN101111241 B CN 101111241B
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马蒂纳·坎德费尔-塞尔森
沃伊切赫·热斯基
斯特凡·彼任诺夫斯基
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Abstract

本发明解决了抗肿瘤制备物的问题。该解决方案的关键在于所述制备物含有α-酮戊二酸(AKG)或/和谷胺酰胺或/和谷氨酸或/和鸟氨酸的α-酮戊二酸或/和谷胺酰胺与其它氨基酸的二肽或/和谷胺酰胺与其它氨基酸的三肽或/和谷氨酸与其它氨基酸的二肽和三肽或/和α-酮戊二酸或/和谷胺酰胺或/和谷氨酸的单价和二价金属盐及其它或/和鸟氨酸的α-酮戊二酸。

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抗肿瘤制备物和抗肿瘤制备物的用途
技术领域
本发明的目的是抗肿瘤制备物和抗肿瘤制备物的用途。
背景技术
尽管在过去的几年中化疗、放疗和免疫疗法有显著进展，但有效的抗肿瘤疗法的问题仍然是目前医学的严峻挑战。流行病学研究显示在高度发达国家中三分之一的人口患有不同的肿瘤性疾病(neoplasticdiseases)。在此群体中每四个病例中有一个是致死性的。治疗中实际应用的细胞抑制剂(cytostatics)具有发生副作用的风险，限制了其有效性并降低了患者的生活质量。
因此开发特异性作用于肿瘤细胞同时对正常细胞具有保护作用的新药是尤其迫切的挑战。
此抗肿瘤制备物的区别之处在于，其含有α-酮戊二酸(alpha-ketoglutarate，AKG)或/和谷胺酰胺或/和谷氨酸或/和鸟氨酸的α-酮戊二酸或/和谷胺酰胺与其它氨基酸的二肽或/和谷胺酰胺与其它氨基酸的三肽或/和谷氨酸与其它氨基酸的二肽和三肽或/和α-酮戊二酸或/和谷胺酰胺或/和谷氨酸的单价和二价金属盐及其它或/和α-酮戊二酸的鸟氨酸。
抗肿瘤制备物的用途在于其用于预防肿瘤性疾病，其中该制备物含有α-酮戊二酸(AKG)或/和谷胺酰胺或/和谷氨酸或/和鸟氨酸的α-酮戊二酸或/和谷胺酰胺与其它氨基酸的二肽或/和谷胺酰胺与其它氨基酸的三肽或/和谷氨酸与其它氨基酸的二肽和三肽或/和α-酮戊二酸或/和谷胺酰胺或/和谷氨酸的单价和二价金属盐及其它或/和α-酮戊二酸的鸟氨酸。
抗肿瘤制备物的其它用途在于其用于抑制转移，其中该制备物含有α-酮戊二酸(AKG)或/和谷胺酰胺或/和谷氨酸或/和鸟氨酸的α-酮戊二酸或/和谷胺酰胺与其它氨基酸的二肽或/和谷胺酰胺与其它氨基酸的三肽或/和谷氨酸与其它氨基酸的二肽和三肽或/和α-酮戊二酸或/和谷胺酰胺或/和谷氨酸的单价和二价金属盐及其它或/和鸟氨酸的α-酮戊二酸。
所述制备物及其用于抑制肿瘤细胞的转移，反映了AKG在转移抑制中的潜在作用。添加到膳食中的所述制备物起到支持用于肿瘤治疗的现有方法的肿瘤辅助治疗(neo-adiuvant)的作用。它可通过与抗肿瘤药物的协同作用以及同时对正常细胞的保护作用来改善患者生活质量。
所述制备物的作用及其用途的实例以实施发明的方式给出：图1-表明用AKG刺激A549细胞增殖的曲线，图2-AKG对C6细胞的增殖和图3-AKG刺激对HT-29细胞的增殖，而图4表示在环磷酰胺存在下AKG对人肿瘤细胞A549的增殖，而图5-表示在异环磷酰胺存在下AKG对人肿瘤细胞A549的增殖，图6-在噻替派存在下AKG对人肿瘤细胞A549的增殖，图7-由AKG对C6细胞迁移的抑制。
肿瘤细胞的培养：
A549-人肺癌肿瘤细胞，从the Institute of the Immunology and theExperimental Therapy of Polish Academy of Science in Wroclaw得到的连续细胞系
HT-29-人大肠癌肿瘤细胞，从the Institute of the Immunology andthe Experimental Therapy of Polish Academy of Science in Wroclaw得到的连续细胞系
C6-大鼠脑癌(神经胶质瘤)肿瘤细胞，从the Department ofNeonatology，Humboldt University，Berlin，Germany得到的连续细胞系培养基
A549细胞系培养在培养基DMEM:F-12HAM(2:1)中，HT-29和C6细胞培养在培养基DMEM中。向培养基中加入10％胎牛血清(FBS)、青霉素100i.u./ml和链霉素100μg/ml。培养基DMEM:F-12HAM、DMEM由Sigma Company生产(Sigma，St.Louis，MO，U.S.A.)。胎牛血清(FBS)由Life Technologies Company(Life Technologies，Karlsruhe，Germany)生产。其它试剂由Sigma Company生产。
细胞培养物的制备
在37℃解冻保存在组织库液氮中的细胞，然后将细胞倒入含有适当培养基的塑料瓶中。将它们在温度37℃、5％CO2流的培养箱中培养。细胞繁殖后将液体倒出，用PBS(不含钙离子和镁离子)洗细胞并用0.25％胰蛋白酶溶液+EDTA处理，以得到试验所需的细胞悬液。
在细胞培养中评价AKG的抗增殖活性
将早前制备的密度为1×104个细胞/ml(A549)、4×104个细胞/ml(HT-29)和0.5×104个细胞/ml(C6)的细胞在培养基中的悬液以体积为100μl/孔倒入平底96-孔微板(NUNC company，Roskilde，Denmark)中。细胞粘附后(24小时)小心地将液体倒掉，然后添加不同浓度AKG和受检细胞抑制剂(环磷酰胺、异环磷酰胺、噻替派)的含10％FBS的液体(100μl/孔)。板中的培养物在37℃、95％空气和5％CO2的气氛中培养96小时。采用MTT法评价受检物质的抗增殖活性。
MTT法(根据试剂盒“细胞增殖试剂盒III”，Boehringer Manheim)
该方法用于在细胞毒性和抗增殖物质的研究中测定细胞的增殖和活力。在代谢活性细胞中四氮唑黄色盐MTT在线粒体脱水酶作用下被还原成甲臜蓝。不溶于水的甲臜结晶在细胞中累积，并且为了其溶解，需要采用有机去污剂，破坏膜并同时溶解染料。为此目的，采用pH7.4的缓冲液SDS-HCl。在96-孔板酶标仪中在波长570nm处定量评价所释放染料的浓度。颜色强度与活细胞的量成正比。
将浓度5mg/ml的MTT的PBS溶液添加到塑料板的每个孔中，剂量为15μl/孔。在温度37℃下培养该板3小时。然后添加剂量为100μl/孔的缓冲液SDS-HCl并将板于37℃放置过夜。次日用E-max读出仪(MolecularDevices Corporation，Menlo Park，CA，U.S.A.)评价结果。
用“划痕试验”法(wound assay)评价细胞迁移度
该方法用于评价体外影响细胞活动性的物质的活性。其用于研究创伤愈合、血管生成和肿瘤转移。
将悬在添加10％血清(FBS)的培养基中的C6细胞(1×106)倒入直径4cm的培养板中(NUNC，Roskilde，Denmark)。次日在相同细胞层中，用自动吸液器的末端制造缺陷(划痕)并用PBS溶液漂洗板两次以除去未粘附细胞。然后将溶解在培养基中的AKG(10和20mM)添加到制备好的培养物中。在37℃和95％空气及5％CO2的潮湿气氛中将板子培养24小时。随后采用May-Grünwald-Giemza法将培养物显色。然后采用Olympus BX51显微镜(Olympus Optical CO.，LTD，Tokyo，Japan)，并使用软件
Figure S06801967520070711D000041
(Soft Imaging System GmbH，Münster，Germany)进行显微分析。细胞迁移度在细胞计数法中作为在细胞层中早前所作划痕周围群集的细胞数来评价。评价8张照片上至少50个所选视野。结果：评价AKG的抗增殖活性
在不同种类的肿瘤细胞中评价培养物中AKG的抗增殖活性：肺癌细胞(A549)、大肠癌细胞(HT-29)和神经胶质瘤细胞(C6)。以0.5、1、2.5、5、10和20mM浓度的AKG处理细胞96小时。
受检物质相对于所有肿瘤细胞类型具有抗增殖活性(图-图1、图2、图3)。与对照组相比，在A549细胞系中AKG在浓度2.5mM时观察到统计学显著的(4.5％)细胞生长抑制。该作用与AKG的剂量相关并且当AKG剂量为5mM、10mM和20mM时抑制作用分别为7.8％、12.4％、17.5％(图1中的图)。AKG剂量为2.5mM、5mM、10mM和20mM时对神经胶质瘤细胞(C6)的生长抑制分别为12.6％、7.9％、16％和19.8％。AKG浓度在1至10mM之间时，其对大肠癌细胞(HT-29)的生长抑制不具有线性特征。剂量1mM对细胞生长的抑制为11.8％。剂量5mM(11.8％)和10mM(11.5％)得到相似的结果。仅20mM剂量引起了这些细胞生长的显著抑制(25％)(图-图3)。
评价AKG和抗肿瘤药物之间的相互作用
在肺癌细胞(A549)培养中对AKG与用于癌症化疗的常用细胞抑制剂之间的相互作用进行研究。为此目的，用下述细胞抑制剂处理细胞：环磷酰胺(1.5mM)、异环磷酰胺(1.5mM)和噻替派(5μM)，单独以及与AKG(5、10和20mM)组合使用。观察到AKG对所用化疗剂的细胞抑制活性的加和作用(结果见图4、图5和图6)。浓度1.5mM的环磷酰胺对A549细胞的生长抑制为21.4％。AKG的加入使其细胞抑制活性分别增加6.4％、9.8％和14.4％(图4)。异环磷酰胺(1.5mM)对细胞生长的抑制为7.3％。添加AKG(5、10和20mM)后其作用分别增加5％、5.3％和8.8％(图5)。AKG还增强了噻替派(5μM-25.9％)的细胞抑制活性，分别为4.2％(5mM)、8％(10mM)和11.2％(20mM)(结果如图6所示)。
AKG对肿瘤细胞迁移的影响
在“划痕试验”模型中研究肿瘤细胞的运动性。照片1中呈现C6细胞层中的划痕(A)、无AKG培养24小时后划痕处细胞群体(B)和在20mM AKG存在下对细胞迁移的显著抑制(C)。在图7中显示在均匀细胞层中迁移至一个已有缺陷视野的平均细胞数。在10mM和20mMAKG存在下显示出对细胞迁移的统计学显著的抑制。
统计学分析
采用t-student检验进行统计学分析。＊p<0.05，＊＊p<0.01，＊＊＊p<0.001。

Claims (2)

1.α-酮戊二酸(AKG)、α-酮戊二酸的单价或二价金属盐和/或α-酮戊二酸的鸟氨酸作为抗肿瘤制备物中仅有的活性成分在制备用于治疗或预防肿瘤性疾病的药物制剂中的用途。
2.α-酮戊二酸(AKG)、α-酮戊二酸的单价或二价金属盐和/或α-酮戊二酸的鸟氨酸作为抗肿瘤制备物中仅有的活性成分在制备用于抑制癌症转移的药物制剂中的用途。
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