CN1498105A - 含有类维生素a受体配体和选择的细胞毒性剂的用于治疗癌症的协同联合体系 - Google Patents
含有类维生素a受体配体和选择的细胞毒性剂的用于治疗癌症的协同联合体系 Download PDFInfo
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Abstract
本发明提供含有选择的细胞毒性剂和RAα/β选择性激动剂或RAR泛拮抗剂的化疗联合体系,其用于治疗癌症,并用于降低选择的细胞毒性剂的有效细胞毒性剂量。
Description
相关申请
按照美国专利法35§119(e),本发明要求享有2001年3月22日提交的第60/277,754号美国临时申请的优先权。
发明领域
本发明涉及治疗癌症的化疗策略,其中视黄酸受体(RAR)配体(特别是α/β选择性激动剂或RAR泛拮抗剂(pan antagonist))与微管蛋白聚合剂(如紫杉烷类)联合给药。如此中所证明,RARα/β选择性激动剂和RAR泛拮抗剂是有效的抗癌剂,并且与也激活RARγ的RAR泛激动剂以及与天然类维生素A(例如激活RARγ和RXR的全反式视黄酸)相比,它们显示出较低的毒性。而且,RARα/β选择性激动剂和RAR泛拮抗剂与微管蛋白聚合剂(特别是紫杉烷类)协同作用,可显著地降低诱导许多肿瘤细胞系的细胞毒性所需要的紫杉烷类的有效量。据信:与RARα/β选择性激动剂的这种协同作用部分地与对Bcl-2表达/磷酸化作用以及JNK和AP1的活性相关。因此,对于能增强Bc1-2磷酸化作用的其它试剂,也可预期到与RARα/β选择性激动剂类似的协同作用。
发明背景
类维生素A是维生素A的生物活性衍生物,在胚胎发生期间以及在成年生活期间,维生素A介导广泛的生理功能(Vitamin Ain Healthand Disease,Blomhoff编辑,Institute for Nutrition Research,University of Oslo,Oslo Norway,1994;Kastner等人Cell 199583:859-869;Giguere等人。Ann.N.Y.Acad.Sci.1996 785:12-22)。天然类维生素A主要由全反式视黄酸(ATRA)和9-顺视黄酸构成,它们通过维生素A的不可逆氧化作用从维生素A加工得来(Vitamin Ain Health and Disease,Blomhoff编辑,Institute forNutrition Research,University of Oslo,Oslo Norway,1994;Napli,J.L.Faseb J.199610:993-1001).另外,已经设计出保持了天然配体许多生物特性的多个合成配体。
在细胞水平上,类维生素A配体能够调节许多正常和恶性细胞的增生、分化和凋亡(Vitamin A in Health and Disease,Blomhoff编辑,Institute for Nutrition Research,University of Oslo,Oslo Norway,1994;Lotan,R.Sem.in Cancer Biol.1991 2:197-208;Love,J.M.和Gudas,L.J.Curr.Opinion in Cell Biol.1994 6:825-831;Lotan,R.Faseb J.1996 10:1031-1039;和,Fitzgerald等人Cancer Res.1997 57:2642-2650)。类维生素A类的生物活性由两类核受体——视黄酸受体(RARs)和类维生素AX受体(RXRs)介导。RAR和RXR两类受体各有三种同型(isotype),分别命名为α、β和γ,它们由不同的基因编码(Chambon,P.Sem.Cell Biol.19945:115-125;Chambon,P.Faseb J.1996 10:940-954)。RAR和RXR是核激素受体(NHR)总科的成员,并充当配体依赖性的转录因子,它们能够通过作为RAR/RXR杂二聚体复合物结合到特定类维生素A反应元件(RARE)DNA序列调控靶基因表达,其中所述特定的类维生素A反应元件(RARE)DNA序列由PuG(G/A)(T/A)CA(n)1-5PuG(G/T)TCA共有序列的直接重复所组成(Gronemeyer,H.,和Moras,D.Nature 1995 375:190-191)。RAR/RXR杂二聚体的活性可以是阻遏的,也可以是刺激的,这分别取决于其分离的(非配位的)或完全的(结合配体的)构型。RAR/RXR杂二聚体的分离构型是一种允许结构,允许蛋白质的相互作用界面引发大的共阻遏物多蛋白质复合物的形成,包括募集NCOR和组蛋白去乙酰基转移酶,这两者一起共同将染色质维持于凝聚的无转录活性的结构以抑制目标基因的表达(Xu等人Curr.Opinion in Genetic and Development 1999 9:140-147;Hu,X.和Lazar,M.A.Trends in Endocrin.和Metab.200011:6-10;Robyr等人.Mol.Endocrin.2000 14:329-347)。相反,激动剂配体与RARs的结合涉及该配体结合区域的构形转变(LBD;Renaud等人Nature 1995 378:681-689),从而导致共阻遏物复合物不稳定,并导致共活化剂(包括p160蛋白质家族、CBP/p300和多蛋白复合物TRAP/DRIP/ARC)的同时募集。若干这样的因子具有组蛋白乙酰基转移酶活性,能使随后全-RAR/RXR杂二聚体与基础转录复合物的连接以引发目标基因表达所需要的染色质发生解凝(Xu等人Curr.Opinion in Genetic and Development 1999 9:140-147;Robyr等人。Mol.Endocrin.2000 14:329-347)。
在许多致癌作用的动物模型中,ATRA和合成类维生素A受体配体已被证明能够促进肿瘤消退,并在受急性前髓细胞性白血病折磨的患者中显示出了功效(Chomienne等人Faseb J 1996 10:1025-1030;Fenaux等人,Leukemia 2000 14:1371-1377)。13-顺视黄酸与紫杉醇(paclitaxel)或顺铂和/或α-干扰素的联合已经进入治疗头和颈部鳞状细胞癌、非小细胞肺癌和前列腺癌的I/II期试验阶段(Gravis等人Anticancer Drugs 1999 10:369-374;DiPaola等人.J.Clin.Oncol.1999 17:2213-2218)。RAR激动剂,ALRT1550,AP1的有效抑制剂,也已经进入包括对患有晚期癌症(包括非小细胞肺癌、甲状腺癌、肾癌、子宫平滑肌肉瘤、前列腺癌和腺样体囊癌患者)的多种群体的I/II期研究阶段(Soignet等人Proc.Annu.Meet.Am.Soc.Clin.Oncol.1998 17:A826)。
与紫杉醇和ATRA联合的胞嘧啶阿拉伯糖苷也被建议作为急性成髓细胞白血病的联合化学治疗。最近,已证明ATRA能使乳腺癌细胞系对紫杉醇和阿霉素的细胞杀死作用敏感(Wang等人Cancer Res.200060:2040-2048)。
ATRA、13-顺视黄酸和ALRT1550都是RAR泛激动剂,这意味着它们瞄准并激活α、β和γRAR同型。而且,ATRA原位转变成属于泛RAR和RXR激动剂的9-顺-RA。
这三个RAR同型中,RARβ-2同型与类维生素A的肿瘤抑制作用相关联。RARβ-2产生于RARβ基因启动子P2的可选择启动子的应用(Chambon,P.Sem.In Cell Biol.1994 5:115-125),其包含强RARE,表明RARβ表达被视黄酸上调节,并将RARβ2作为支承视黄酸的生长抑制作用的潜在的靶基因。与调节细胞生长的作用相一致,F9畸胎瘤细胞中的RARβ基因失活导致视黄酸依赖型生长抑制的丧失(Faria等.J.Biol.Chem.1999 274:26783-26788),然而,就细胞凋亡的生长抑制和诱导来说,乳腺癌细胞中RARβ的反转录病毒介导的表达与视黄酸反应性的强烈升高有关(Seewaldt等人,CellGrowth and Differentiation 19956:1077-1088)。这些观察结果明显表明RARβ属于类维生素A作用的重要介质,并表明其调节作用可以有效地减少肿瘤细胞的生长。
然而,在包括乳腺癌和肺肿瘤在内的癌症发展的早期阶段,RARβ2表达的丧失已有报道(Widschwendter等人,Cancer Res.1997 57:4158-4161)。相信这种丧失是起因于由灭活RARβ2启动子的甲基化和/或去乙酰基化作用引起的染色质的阻抑状态,而非起因于基因的改变(Arapshian等,Oncogene 2000 19:4066-4070)。单纯的RARβ激动剂不对细胞增生产生影响。
相反,RARα和γ以及RXRα的表达在恶性组织中基本上没有改变。然而,认为是类维生素A主要毒性、并限制其用做治疗药剂的类维生素A的粘膜皮肤毒性作用与RARγ的活性有关。
发明概述
本发明的目的是提供用于治疗癌症的协同化疗联合体系。在本发明的一个实施方案中,该协同联合体系包含RARα/β选择性激动剂和增强Bc1-2的磷酸化作用的第二药物,例如微管蛋白聚合剂,优选紫杉烷(taxane)。在本发明的另一个实施方案中,该协同联合体系包含RAR泛拮抗剂和微管蛋白聚合剂,其中微管蛋白聚合剂优选紫杉烷。
本发明的另一个目的是,通过将选择的细胞毒性剂与RARα/β选择性激动剂或RAR泛拮抗剂联合给药,降低为肿瘤细胞中诱导细胞毒性所需要的该选择的细胞毒性剂的有效量。
本发明还有一个目的是提供一种用这些协同化疗联合体系治疗患者的癌症的方法。在一个实施方案中,将RARα/β选择性激动剂与增强Bc1-2磷酸化作用的第二药物(例如微管蛋白聚合剂,优选紫杉烷)联合给药于患者。在另一实施方案中,将RAR泛拮抗剂与微管蛋白聚合剂(优选紫杉烷)联合给药于患者。
附图的简要描述
图1为可用于本发明的RARα/β选择性激动剂的一种举例性说明的合成方案。
图2为可用于本发明的RAR泛拮抗剂的一种举例性说明的合成方案。
发明的详细描述
本发明涉及RARα/β选择性激动剂和RAR泛拮抗剂以及它们在协同提高肿瘤细胞对选择的细胞毒性剂的敏感性中的应用。
就本发明的目的来说,术语“RARα/β选择性激动剂”意指这样的化合物,它能激活视黄酸受体的α和β同型,但很少或不显示视黄酸受体γ同型的活性,或确切地说抑制视黄酸受体的γ同型活性。可用于本发明的RARα/β选择性激动剂的例子通常如式I所示:
式I
其中R1是氢或甲基,R是顺-CH=CH-C(CH3)3或顺-CH=CH-C(CH3)2CH2CH3。更具体的RARα/β选择性激动剂的例子描述于式Ia和式Ib:
式Ia
式Ib
式Ia及它的合成详述于WO 00/17147中,其教导的全部内容在此引入本文。其供治疗皮肤疾病和作为抗肿瘤剂之用。本领域技术人员在阅读本发明公开的内容后会理解:除了在本文中所具体举例说明的以外,其它的RARα/β选择性激动剂也能够用于本发明。例如,对RARα和RARβ显示选择性性的其它化合物例子被描述于WO 97/48672。按照例如描述于WO 97/48672和Gehin等人(Chemistry and Biology1999 6:519-529)的方法,可按常规方式测定化合物激活视黄酸受体的α和β同型但很少或不显示γ同型活性的能力。
术语“RAR泛拮抗剂”意指抑制视黄酸受体的α、β和γ同型的化合物。举例性的RAR泛拮抗剂描述于式II:
式II
这种化合物可由本领域的技术人员按照所熟知的方法予以常规合成。合成这种化合物的方法的例子描述于实施例2中。另外,本领域的技术人员在阅读本发明的公开内容将会理解,显示RAR泛拮抗活性的其它化合物也可予以使用。另外的RAR泛拮抗剂的实例被公开于WO97/48672中。具有这种活性的其它化合物也可由本领域的技术人员按照如描述于WO 97/48672中的方法以常规方式进行识别。
除了他们的游离酸形式以外,式I、Ia、Ib和II化合物的可药用盐也可用于本发明的方法和组合物。这样的可药用盐包括但不限于与下列物质形成的盐:碱金属如钠、钾和锂;碱土金属如钙和镁;有机碱如二环己基胺、三丁胺和吡啶等;以及氨基酸如精氨酸和赖氨酸等。
“RAR泛激动剂”意指能激活视黄酸受体的α、β和γ同型的化合物。RAR泛激动剂的例子包括但不限于ATRA、13顺视黄酸和ALRT1550。尽管已有报道RAR泛激动剂抑制肿瘤的发展,然而这些治疗却被有害的毒性所限制,这些毒性包括但不限于:粘膜皮肤毒性、头疼、致畸、肌肉骨骼毒性、dylipidemias、皮肤刺激和肝(细胞)毒性。被看作为泛激动剂的主要的毒性、并限制其用做治疗剂的泛激动剂的粘膜皮肤毒性与RARγ的活性有关。而且,该天然类维生素AATRA被原位转变成属于泛RAR和RXR激动剂的9顺-RA。在施用ATRA后,RXR的激活也已经与非所希望的副作用联系起来。
“选择的细胞毒性剂”意指紫杉烷或以与紫杉烷类相似的机理起作用的其它药物。因此,这个术语意指包括其它微管蛋白聚合剂在内。而且,对于含有RARα/β选择性激动剂的协同组合物来说,这个术语意指包括能增强Bc1-2磷酸化作用的其它药物在内。在一个优选的实施方案中,该选择的细胞毒性剂是紫杉醇或多西他噻(docetaxel)。
如此中所证明,RARα/β选择性激动剂和RAR泛拮抗剂与选择的细胞毒性剂协同作用,降低诱导肿瘤细胞毒性所需要的选择的细胞毒性剂的有效量。在本发明的一个实施方案中,该协同联合体系包含RARα/β选择性激动剂和增强Bcl-2磷酸化作用的第二药剂,例如微管蛋白聚合剂,优选紫杉烷。在本发明的另一实施方案中,该协同联合体系包含RAR泛拮抗剂和微管蛋白聚合剂,其中优选紫杉烷。
据信,这些协同联合体系能够有效地治疗癌症,并且能够有效地降低与该联合体系中的选择的细胞毒性剂有关的毒副作用。另外,RARα/β选择性激动剂或RAR泛拮抗剂在本发明联合体系中的使用消除或降低了在给药RAR泛激动剂和ATRA后与RARγ和RXR的激活有关的毒副作用。因此,本发明也涉及通过将RARα/β选择性激动剂或RAR泛拮抗剂与选择的细胞毒性剂联合给药于患者以治疗癌症的方法。在这些方法中,优选将RARα/β选择性激动剂或RAR泛拮抗剂与微管蛋白聚合剂联合给药,最优选与紫杉烷联合给药。或者,对于RARα/β选择性激动剂,选择的细胞毒性剂也可以包含增强Bc1-2磷酸化作用的药物。
“癌”意指包括肿瘤、瘤形成、和任何其它的恶性组织和细胞。
本发明也涉及含有选择的细胞毒性剂和RARα/β选择性激动剂或RAR泛拮抗剂的药用组合物。在优选的实施方案中,该选择的细胞毒性剂包括微管蛋白聚合剂,最优选紫杉烷。另一方面,对于含有RARα/β选择性激动剂的组合物来说,该选择的细胞毒性剂也可以含有增强Bc1-2磷酸化作用的药剂。
本发明组合物可以在单个的可药用制剂中含有这两种组分。或者,这些组分可独立配制,并与另一种组分联合给药。本领域技术人员熟知的各种可药用剂型都可以用于本发明。选择用于本发明的适宜制剂可由本领域技术人员根据给药的方式和组合物成分的溶解度特性决定。
就本发明的目的而言,所谓“联合”是指,将RARα/β选择性激动剂或RAR泛拮抗剂在选择的细胞毒性剂给药以前、同时、或以后给药于患者。在优选的实施方案中,将RARα/β选择性激动剂或RAR泛拮抗剂在选择的细胞毒性剂给药以前或与其同时给药于患者。本文中所用的术语“同时”或“与其同时”是指将RARα/β选择性激动剂或RAR泛拮抗剂和选择的细胞毒性剂彼此在24小时、优选12小时、更优选6小时、和最优选3小时或更短的时间以内给药。
当与现有的化疗药剂联合给药时,RARα/β选择性激动剂和RAR泛拮抗剂以相加作用或协同作用影响细胞生长的能力首先在三种不同的人肿瘤细胞系中予以评估。在用紫杉醇和下面药剂之一共同治疗的细胞中进行细胞生长的抑制比较:RAR泛激动剂(见式III)、式Ia RARα/β选择性激动剂、RARα激动剂(见式IV)、RARβ激动剂(见式V)、RARγ激动剂(见式VI)、式II RAR泛拮抗剂和RXR泛激动剂(见式VII)。式III至VII的结构描述如下:
式III
式IV
式V
式VI
式VII
如表1所示,式Ia的RARα/β选择性激动剂显著地降低为诱导细胞毒性所需要的紫杉醇的有效量。在有式Ia的情况下,紫杉醇的IC50在MCF7、OVCAR3和SQCC-Y1细胞中分别降低了8、15和32倍。这些数值与将紫杉醇和式III的RAR泛激动剂联合所获得的数值非常相似,这样就支持了RARα/β同型在类维生素A的肿瘤抑制活性中担主要角色。式II的RAR泛拮抗剂也降低诱导细胞毒性所需要的紫杉醇的有效量。相反,单纯的RARα激动剂(式IV)只能把紫杉醇的IC50(表1)降低2至4倍,这,与式Ia相比,和它对RARα具有低亲和性是一致的。单纯的RARβ激动剂(式V)、RAR-γ-选择性激动剂(式VI)和单纯的RXR激动剂(式VII)影响紫杉醇的细胞毒性都不超过2至3倍,从而表明:RARα/β配体与紫杉醇的协同效应主要由RARα(表1)引起。然而,在OVCAR3细胞中γ-选择性激动剂表现出一个例外,其把紫杉醇的IC50降低了8倍。
表1:类维生素A/紫杉醇协同抑制细胞生长所需要的选择性
生长抑制([3H]-胸苷结合) | |||
紫杉醇 | |||
选择性 | IC50 | 功效 | |
MCF7 | 无 | 0.070 | 100 |
RAR泛激动剂(式III) | 0.008 | 100 | |
RARα/β激动剂(式Ia) | 0.009 | 98 | |
RARα激动剂(式IV) | 0.017 | 100 | |
RARβ激动剂(式V) | 0.064 | 100 | |
RARγ激动剂(式VI) | 0.044 | 100 | |
RAR泛拮抗剂(式II) | 0.007 | 100 | |
RXR泛激动剂(式VII) | 0.068 | 100 | |
SQCC-Y1 | 无 | 0.160 | 100 |
RAR泛激动剂(式III) | 0.008 | 100 | |
RARα/β激动剂(式Ia) | 0.005 | 100 | |
RARα激动剂(式IV) | 0.05 | 100 | |
RARβ激动剂(式V) | 0.113 | 100 | |
RARγ激动剂(式VI) | 0.088 | 100 | |
RAR泛拮抗剂(式II) | 0.037 | 100 | |
RXR泛激动剂(式VII) | 0.111 | 100 | |
OVCAR3 | 无 | 0.6 | 99 |
RAR泛激动剂(式III) | 0.080 | 99 | |
RARα/β激动剂(式Ia) | 0.040 | 100 | |
RARα激动剂(式IV) | 0.251 | 100 | |
RARβ激动剂(式V) | 0.2 | 100 | |
RARγ激动剂(式VI) | 0.079 | 100 | |
RAR泛拮抗剂(式II) | 0.100 | 100 | |
RXR泛激动剂(式VII) | 0.22 | 100 |
在此表中,功效被定义为与DMSO相比较的抑制百分率(%)。将全部IC50以纳摩尔值来表示。该结果是2到5个试验的平均数。与IC50有关的标准偏差为10-20%。
为了比较MCF7在有选择性RARα/β激动剂、RAR泛拮抗剂或ATRA存在的情况下形成集落的能力,还进行了另外的实验。在有式Ia选择性RARα/β激动剂存在的情况下生长的集落清楚地显示了类维生素A的分化作用。由载体DMSO处理的细胞形成了非常紧密的集落,该细胞显示细胞质受到极大约束,并且大部分集落看起来轮廓非常清晰。相反,用式Ia的RARα/β激动剂处理MCF7细胞却导致集落和细胞两者形态的显著变化。集落显得分散,但细胞体积增大而显示出细胞质,并趋向于构成上皮状结构。用式II的RAR泛拮抗剂处理MCF7细胞也显著损坏集落的形成。该集落仍然小,有两个明显的细胞群,其中一个凋亡了,另一个处于分化过程中,显示原生质伸展。而且,用RAR泛拮抗剂预先处理这些细胞三天会显著提高该细胞对紫杉醇的敏感性。ATRA也损坏集落的形成。然而,与RARα/β选择性激动剂相反,由ATRA处理的集落显示出高水平的细胞凋亡,据信这反映了ATRA原位转变为9顺-RA后随之而来的RXR活性。
对紫杉醇和RARα/β选择性激动剂或RAR泛拮抗剂两者对这些药剂共有的信号转导途径的作用进行分析。在这些试验中,首先检测紫杉醇/类维生素A的共同处理对抗凋亡蛋白Bc1-2的表达水平和磷酸化状况的作用,Bc1-2是一种类维生素A和紫杉醇信号传输均涉及的蛋白质。用紫杉醇处理的MCF7细胞在Bc1-2的磷酸化方面表现出剂量依赖性的增强。向细胞中添加选择性α/β激动剂导致Bc1-2蛋白质水平的下调,而单独的RAR泛拮抗剂却对Bc1-2水平或磷酸化没有影响。紫杉醇与选择性RAR激动剂联合导致Bc1-2水平的下调和磷酸化状态的增加。相反,紫杉醇和RAR泛拮抗剂联合仅涉及Bc1-2磷酸化的增加而不调整它的水平。这些结果表明,紫杉醇与RARα/β选择性激动剂的协同效应可部分归因于Bcl2的下调水平和磷酸化的相加效应,这种相加效应导致促细胞凋亡的Bax活性的释放、线粒体膜的渗透作用和细胞凋亡的诱导。因此,相信以与紫杉醇相似的方式增强Bc1-2磷酸化的其它药剂,也将显示与RARα/β选择性激动剂类似的协同活性。
相反,RAR泛拮抗剂并不影响Bc1-2蛋白的水平或紫杉醇诱导的Bc1-2的磷酸化,从而表明微管蛋白聚合剂如紫杉醇和类维生素A拮抗剂之间的协同作用可以采用不同的路径。
对紫杉醇与RARα/β激动剂或RAR泛拮抗剂对微管蛋白聚合的联合效应也进行了分析。从不可溶的聚合的微管蛋白库中分离出可溶性非聚合微管蛋白库,并通过Western印迹法用抗-α微管蛋白抗体对之进行分析。观察到由紫杉醇诱导的与剂量有关的微管蛋白的聚合作用。然而,选择性RAR激动剂或拮抗剂并无任何作用。而且,紫杉醇与任何类维生素A的联合并未增强紫杉醇独自的作用。因此,就细胞骨架诱导细胞毒性的作用而言,类维生素A未显示出与紫杉醇对其产生协同作用。
一些近来的报道已经描述了Taxol诱导对Jun N-末端激酶(JNK)活性的刺激作用,导致c-Jun磷酸化作用的增强和AP1转录复合物活性的相关增加(Lee等人。J.Biol.Chem.1998 273:28253-28260;Wang等人。J.Biol.Chem.1998 273:4928-2936)。类维生素A已表明能够调整和抑制AP1转录活性(Chen等人。EMBO J.1995 14:1187-1197)。在该文章的上下文中,分析了紫杉醇与类维生素A对MCF7细胞中的JNK激酶活性和AP1转录活性的联合效果。通过免疫沉淀的JNKc-Jun的磷酸化作用表明:用紫杉醇处理的MCF7细胞导致JNK的活性平均增加2.5倍,而单独的类维生素A对JNK活性却没有影响。相反,紫杉醇与α/β选择性激动剂的联合只是略微增强由紫杉醇诱发的JNK活性,使酶的活性产生最大4倍的增加。通过使用稳定MCF7细胞系稳定表达AP1报道基因(AP1)5x-tk-Luc,在转录复合物活性的水平上,进一步分析了对JNK的这些作用。在MCF7细胞中,由佛波醇酯(PMA)诱导的这种受体结构的有效活性,以与剂量有关的方式受到选择性RARα/β激动剂(式Ia)的抑制,达到了应用50nM类维生素A最大效应的30-45%。相反,当作为单一药剂加入时,紫杉醇对PMA所诱导的AP1转录活性进一步增强了20%。相应于紫杉醇和PMA的联合,紫杉醇与类维生素A的联合阻止紫杉醇对PMA所诱导的胶原酶启动子活性的作用,导致抑制30至50%的AP1转录因子活性。这样,紫杉醇与RARα/β选择性激动剂的联合提高了紫杉醇对Jun-N-末端激酶活性的作用,然而,由于类维生素A诱导的AP1的负交互调节,而与随后发生的AP1转录激活相分离。这些观察结果和对Bc1-2的作用一起构成了这些药剂的有效协同细胞毒性作用。紫杉醇与式II的RAR泛拮抗剂的联合并未改变由紫杉醇所诱导的JNK活性。然而,这种联合的确提高了紫杉醇/PMA或AP1活性的作用。
提供如下非限制性实施例来进一步举例说明本发明。
实施例
实施例1:材料
用于这些试验的细胞系包括T47D、HT-3、UMSCC-25、MCF-7、OyCAR3、HCT-1 16和N-87,其来自ATCC并保存于ATCC推荐的介质和浆液浓度中。按照Garrigues等人规定的方法获得H3396细胞系(Am.J.Pathol.1993 142:607-622),并保存于补充有10% FBS(Gibco-BRL)的RPMI中。从Anderson Cancer Center(Houston,Texas)处获得MDA-PCA.2B和SQCC-YI。改造成抗药的A2780S和它的p27转基因变种A2780p27、HCT-116TYPK(Taxol-抗性)和HCTVM46、SAN-1、M109及其Taxol-抗性变种M109TX和PAT-7(Taxol抗性)细胞系也予以使用。所有的细胞毒性剂和佛波醇12-肉豆蔻酸酯13-乙酸酯(PNIA)都购自Sigma-Aldrich。用于这些试验的小鼠抗-aBc1-2、抗-a-微管蛋白和抗-人JNK抗体分别购自BiosourceInternational、Sigma-Aldrich和BD-Pharmingen。从UpstateBiotechnology处获得c-jun(1-169)-GST融合蛋白质。
实施例2:合成RAR配体
用于这些试验的RARα/β选择性激动剂和RAR的合成途径和结构概述于图1中。式Ia、式Ib和式III的二氢萘基化合物的合成涉及所要求的烯醇triflates 1a或1b与各种乙炔的Sonogashira偶合反应。在3,3-二甲基-丁炔、二异丙胺、碘化铜(I)和氯化双(三苯膦)钯(II)的存在下,合成化合物2a和2b。同样,用3,3-二甲基-戊-1-炔(Van Boom等人Rec.Trav.Chim.1965 84:31)来制备化合物2c。应用10%的硫酸钡上的钯氢化2a和2c中的叁键,然后在甲基酯部分进行皂化以产生需要的式Ia和Ib的顺式-乙烯衍生物。同样,通过乙炔2b的皂化作用合成式III的RAR泛激动剂。
按照图2所示的合成方法制备制备式II的RAR泛拮抗剂,4-(5,6-二氢-5,5-二甲基-6-氧代-8-(3-甲基-苯基)-蒽-2-基)-苯甲酸。其描述如下。4-(5,6-二氢-5,5-二甲基-6-氧代-8-(3-甲基-苯基-)-蒽-2-基)-苯甲酸甲酯
用二氧化硒(0.186g,1.672mmol)处理搅拌的4-(5,6-二氢-5,5-二甲基-8-(3-甲基-苯基)-蒽-2-基)苯甲酸甲酯(0.181g,0.418mmol)在二噁烷(6mL)中的溶液,并将该混合物回流5小时。然后把该反应混合物用乙酸乙酯稀释,用盐水洗涤,用硫酸镁干燥,过滤并浓缩。将剩余物用硅胶色谱法纯化,获得标题物质(0.155g,83%)。
1H NMR 400MHZ C6D6δppm:1.69(6H,s,2×-CH3),2,14(3H,s,-CH3)6.38(1H,s,H-7′),7.03-7.19(3H,m,芳香H),7.33(2H,J=8.4Hz,H-3和H-5),7.48-7.49(2H,m,芳香H),7.59(1H,d,J=9.1Hz,芳香H),7.78(1H,s,芳香H),7.891H,s,芳香H),8.24(2H,d,J=8.4Hz,H-2和H-6)。
4-(5,6-二氢-5,5-二甲基-5-氧代-8-(3-甲基-苯基)-蒽-2-基)-苯甲酸
用氢氧化钠水溶液(5N,0.5mL)处理搅拌的溶解于乙醇(2mL)和四氢呋喃(2mL)中的4-(5,6-二氢-5,5-二甲基-6-氧代-8-(3-甲基-苯基)蒽-2-基)-苯甲酸甲酯(0.015g,0.034mmol)溶液,并在室温把该混合物搅拌一夜。然后把溶剂蒸发掉,将剩余物用乙酸乙酯(4mL)稀释并用1N盐酸酸化。把有机层用盐水洗涤,用无水硫酸镁干燥,并过滤和浓缩。
1H NMR 400MHZ DMSO-d6δppm:1.59(6H,s,2×-CH3),2.43(3H,s,-CH3),6.12(1H,s,H-7′),7.35-7.39,7.46-7.50(3H和1H,3组m峰,芳香H),7.91(1H,s,芳香H),7.96-8.04(5H,m,芳香H),8.09(1H,d,J=8.6Hz,芳香H),8.25和8.34(1H和1H,2s,芳香H)。
实施例3:促有丝分裂的分析
细胞增生用[3H]胸苷结合予以量化。把细胞放置于含有10%胎牛血清(FBS)的培养基的96孔培养皿中,让其粘附过夜。第二天移去细胞培养基,并将指定化合物加到补充以10%已被活性碳吸收过的FBS(HyClone)的新鲜培养基中。暴露三天后更换这些含有化合物的培养基。处理6天后,向每一个孔中加入4μCi/ml的[3H]胸苷(NEN lifeScience Products)2-4小时。然后,使细胞受胰蛋白酶化作用并将其收集在GF/B玻璃纤维过滤器上。将结合到DNA的[3H]胸苷在闪烁Topcount计数器(Packard)中进行测量。结果可以表示为与DMSO载体比较的抑制百分率。IC50定义为,诱导50%抑制的化合物浓度,而该功效相当于与DMSO进行比较该化合物获得的最大抑制。该结果是2到5个独立试验的平均数。
实施例4:贴壁依赖性的集落形成测试
为了测定RAR激动剂对贴壁依赖性集落的大小、数目和形态的作用,把MCF7细胞放置于6孔培养皿(500个细胞/孔)中,其中含有补充有10%FBS的DMEM,培养5天。移去培养基,然后把化合物加到补充有5%的已被活性碳吸收过的FBS(HyClone)的培养基中。三天后更换含有化合物的培养基,并使培养物再生长三天。暴露于化合物6天后,将集落用结晶紫染色。对集落予以清点以确定集落的面积和数目,并在光学显微镜下予以拍照以评估对集落的大小和形态的影响。所获结果代表两个独立试验。按照由Sambrook等人所描述的方法(MOLECULAR CLONING:A LABORATORY MANUAL,2nd Ed.;ColdSpring Harbor Laboratory Press,Cold Spring Harbor,N.Y.(1989)),制备免疫印迹的MCF-7细胞溶解产物用于Western印迹。通过把200μl含有蛋白酶抑制物混合剂(Roche)的溶解缓冲剂(Tris-HCl,PH6.8,20mM,EDTA,1mM,NP-40 0.5%)加入到6孔培养皿的每一个孔中,制备细胞溶解产物。通过在4EC以1000转/分钟离心5分钟将溶胞产物澄清,把上清液用Laemmli样本缓冲剂予以调整,然后用10或15%的SDS-聚丙烯酰胺胶体电泳(PAGE)处理,然后转移到硝化纤维素膜(Biorad)上。用标准方法处理膜,然后用一次抗体在室温培育1小时或在4EC培育过夜。用与HRP结合的山羊抗-IgG二次抗体和化学发光检测系统(ECL,Amersham Pharmacia Biotech)使结合抗体显像。
实施例5:微管蛋白聚合作用分析
按照Giannakakou等人所描述的方法(J.Biol.Chem.1997272:17118-17125)测定类维生素A和紫杉醇曝露对游离和聚合微管蛋白含量的影响。把MCF7细胞放置于6孔培养皿中,其中含有补充10%FBS的DMEM,使其粘附过夜。第二天移去细胞培养基,把指定化合物加到补充有5%已被活性碳吸收了的FBS(HyClone)的新鲜培养基中。在有类维生素A存在的情况下使细胞生长3天,然后加入紫杉醇培养1小时。萃取可溶性的和聚合的微管蛋白,并通过如上面所描述的Western印迹法用抗-α微管蛋白抗体(n=2)对各自水平予以评估。
实施例6:AP1受体分析
在用(AP1)5x-tk-Luc报道基因(IGBMC,France)稳定转染的MCF7细胞中评估AP1转录活性。(AP1)5x相当于在重复5次串联排列的胶原酶基因API反应元件,tk是最小的胸苷激酶启动子,Luc为萤虫素酶基因的编码序列。把MCF7细胞以400,000细胞/孔的密度放置于12孔培养皿中,其中补充有10% FBS的DMEM,使其粘附过夜。第二天移去培养基,将细胞用PBS洗涤并补充含有0.5%已被活性碳吸收过的FBS(HyClone)的DMEM培养基。24小时后,在有或没有ATRA、式Ia或式III的RAR泛激动剂(浓度范围为0.1,1和50nM)的情况下,并在它们与或不与紫杉醇(1μM)联合的情况下,用PMA(10nM)处理细胞6小时。通过使用Packard的Luc-lite系统,按照制造商的教导,测定荧光素酶的活性。结果表示为与PMA(10nM)相比较的百分比活性,其中PMA被确定为100%活性。所提供的数据是三个独立试验的平均值±sem(平均标准误差)。
实施例7:Jun N-末端激酶测试
按照Coso等人描述的方法(Cell 1995 81:1137-1146),对其稍加调整,测定JNK的活性。把MCF-7细胞放置于6孔培养皿中,其中含有补充有10% FBS的DMEM,使其粘附过夜。第二天,把细胞转换到补充有0.5%已被活性碳吸收过的FBS(HyClone)的DMEM中,加入100nM所指示的类维生素A或0.1% DMSO培养细胞24小时,然后在有1μM紫杉醇存在的情况下再培育2到3个小时。按所述从所述的每个孔中制备细胞溶解产物,并通过离心作用予以澄清。先用溶胞缓冲剂洗涤,然后在4℃用1μg的抗-hJNK(Pharmingen)和25μl的蛋白质G琼脂糖珠(Gibco-BRL)培育过夜,从每一溶胞产物中免疫沉淀出JNK。通过在4℃以10,000转/每分钟的离心2分钟免疫复合物,并用含有1%NP40和2mM Na3VO4的PBS洗涤3次,用100mM Tris-HCl pH7.5,0.5mM LiCl洗涤一次,然后用激酶反应缓冲剂(KRB:12.5mM MOPSpH7.5,12.5 mMβ-磷酸甘油脂,7.5mM MgCl2,0.5mM EGTA,0.5mMNaF,0.5mM Na3VO4)洗涤一次,得到丸状物。将该丸状物重悬浮于含有5μCi[γ-33P]ATP(NEN Life Science Products)、10μM冷ATP、3.3mM DTT和2μg的GST-c-Jun(1-169)融合蛋白的30μl KRB中,在30℃进行激酶测试30分钟。加入10μl Laemmli样品缓冲液终止激酶反应。用10% SDS-PAGE和磷酰化GST-c-Jun解离,通过放射自显影成像,用光密度测定法定量。结果以3次独立实验的平均值±sem表示。
Claims (20)
1.治疗患者的癌症的方法,其包括对需要的患者施用:
a)选择的细胞毒性剂;和
b)RARα/β选择性激动剂或RAR泛拮抗剂。
2.权利要求1的方法,其中选择的细胞毒性剂是微管蛋白聚合剂。
3.权利要求1的方法,其中选择的细胞毒性剂是紫杉烷。
4.权利要求1的方法,其中选择的细胞毒性剂是紫杉醇。
5.权利要求1的方法,其中将RARα/β选择性激动剂或RAR泛拮抗剂在把选择的细胞毒性剂给药于患者之前或者在其同时施用于患者。
6.权利要求1的方法,其包括施用:
a)增强Bc1-2磷酸化作用的选择的细胞毒性剂;和
b)RARα/β选择性激动剂。
7.权利要求1的方法,其中RARα/β选择性激动剂选自:式Ia和式Ib以及它们的可药用盐。
8.权利要求1的方法,其中RAR泛拮抗剂选自:式II和其可药用盐。
9.降低在肿瘤细胞中诱导细胞毒性所需要的选择的细胞毒性剂的有效量的方法,包括对细胞给予:
a)选择的细胞毒性剂;和
b)RARα/β选择性激动剂或RAR泛拮抗剂。
10.权利要求9的方法,其中选择的细胞毒性剂是微管蛋白聚合剂。
11.权利要求9的方法,其中选择的细胞毒性剂是紫杉烷。
12.权利要求9的方法,其中选择的细胞毒性剂是紫杉醇。
13.权利要求9的方法,其中RARα/β选择性激动剂选自:式Ia和式Ib以及它们的可药用盐。
14.权利要求9的方法,其中RAR泛拮抗剂选自:式II以及它们的可药用盐。
15.权利要求9的方法,其包括给予:
a)增强Bc1-2磷酸化作用的选择的细胞毒性剂;和
b)RARα/β选择性激动剂。
16.药物组合物,含有:
a)选择的细胞毒性剂;和
b)RARα/β选择性激动剂或RAR泛拮抗剂。
17.权利要求16的药物组合物,其中选择的细胞毒性剂是微管蛋白聚合剂。
18.权利要求16的药物组合物,其中选择的细胞毒性剂是紫杉烷。
19.权利要求16的药物组合物,含有:
a)增强Bc1-2磷酸化作用的选择的细胞毒性剂;和
b)RARα/β选择性激动剂。
20.式II的化合物或它的可药用盐。
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