CN1391471A - 用于治疗肿瘤的去甲二氢愈创木酸衍生物 - Google Patents
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Abstract
本发明公开了去甲二氢愈创木酸衍生物及其用于治疗肿瘤的方法。
Description
本说明书描述和要求保护的本发明部分是国家健康研究院的资助下完成的。美国政府对本发明享有一定权利。
发明背景
1.发明领域
本发明涉及去甲二氢愈创木酸(nordihydroguaiaretic acid)衍生物,特别是含有天然存在的氨基酸取代基的衍生物,用于治疗肿瘤和病毒感染的用途。
2.背景信息
癌发生是受各种遗传和外遗传因素影响的多阶段事件,并且以暴发自不同组织的不受控制的细胞生长为特征。全球性抗癌研究的目的在于开发高度有效地减弱肿瘤生长,对宿主没有毒性,并且对于大多数患者可获得的临床治疗方法。聚焦于对正在分裂的细胞独特的靶物的抑制作用的药物应该是有效的化学治疗药物而没有明显的副作用的危险。
在进行细胞循环时,细胞穿过很多关卡。为了通过这些关卡的每一个,必须符合特定的标准。在G2/M转换中,最必要的调节物是依赖细胞周期蛋白的激酶CDC2。该激酶紧密结合调节蛋白细胞周期蛋白B,并且该复合物,也称之为促成熟因子(MPF),负责刺激导致细胞进入早期分裂前期的种种事件(1)。意料之中的是,MPF的任一成分的失去或失活将阻断从G2出来的细胞进程。
MPF的表达和活性在不同水平调节。细胞周期B蛋白水平通过细胞周期的G1期和S期缓慢升高,在G2期至M期过渡过程达到最高,并且在有丝分裂期间锐减(2)。另一方面,在细胞周期中总是存在CDC2蛋白,尽管在G2期的最后阶段其水平稍微升高(3)。蛋白质的活性依赖于与适当的细胞周期蛋白的结合,以及依赖于磷酸酶CDC25C对其抑制位点的去磷酸化作用(4,5)。已经证明该去磷酸化作用衰竭引起响应于放射或化学作用对DNA损伤的G2停滞。最近的证据还提示DNA损伤之后任何残留活性CDC2可以传输到细胞核之外(6)。
已经证明植物木脂体去甲二氢愈创木酸(NDGA)的大量天然存在的衍生物通过抑制病毒转录而阻断病毒复制。这项早期工作证明最初从Larrea Tridentata分离并且接着化学合成的NDGA衍生物通过灭活其Sp1-依赖性启动子能够抑制HIV(7,8),HSV(9)和HPV转录物的产生。出人意料地,这些衍生物中的一个,四-O-甲基NDGA,表现出也诱导哺乳动物细胞系中细胞周期停滞。下文给出的证据证明M4N能够诱导哺乳动物细胞的G2停滞而没有检测到毒性,并且支持了该停滞是由于依赖于细胞周期蛋白的激酶CDC2的抑制作用的观点。
人乳头瘤病毒(HPV)感染在很多类型的鳞状上皮细胞中引起非调控性细胞生长,导致从良性乳头瘤(疣)到子宫颈(cervical)的癌,阴茎癌和口腔癌的病痛折磨。这些癌症与HPV的紧密相关性以及感染的广泛发生指出了开发抗HPV治疗的重要性。
大部分,如果不是全部的话,病毒,包括那些复制活性突变体,是依赖宿主的。它们需要某些支持病毒生长的细胞因子的参与。宿主细胞因子,与病毒蛋白质不一样,不处于突变压力下并且一般情况下结构不可变。因此,阻断这些细胞因子在病毒生活周期的不同阶段的利用的化合物可能是作为突变不敏感抗病毒药物的好的侯选者。已经综述了几项利用细胞因子作为HIV-1抑制作用的可供选择的靶物的研究(11)。
申请人早期报道过从灌丛(Larrea tridentata)分离的3′-O-甲基化NDGA(即Mal.4)能特异性阻断人细胞培养物中基础HIV转录,Tat-调节的反式激活,和HIV复制(12,13,14)。Mal.4通过干扰转录因子Sp1与HIV病毒原模板的启动子的结合而施展其作用。Mal.4的靶物的图谱位置是核苷酸-87至-40,HIV长末端重复序列(LTR)的Sp1结合位点。没有修饰的NDGA在体外不抑制HIV转录并且对Sp1结合没有影响(12)。
但是植物木脂体的分离和纯化是劳动密集型的并且耗费大。预期植物木脂体在控制人Sp1-调节的病毒和肿瘤生长中可能的临床应用,低成本大规模地以没有甲基化的NDGA作为母体底物化学合成九种不同的甲基化NDGA活性(15)。药物浓度低于30μM时,发现四-O-甲基NDG在通过抑制Sp1调节的病毒原转录和反式激活的复制HIV的控制中最有效(15)。因此,该项研究扩展到对单纯疱疹病毒(HSV-1和HSV-2)生长的控制(16)。单纯疱疹立即早期(IE)ICP4基因对于HSV复制是必需的(17)。其启动子区具有8个Sp1共有区结合位点(18),其中的五个对于ICP4基因表达是必需的。因此,这使得ICP4基因是对于该项试验的好的侯选物。申请人发现两种3-O-甲基NDGA(Mal.4)和四-O-甲基NDGA(M4N)在非洲绿猴肾细胞株系细胞中通过阻断Sp1蛋白质结合ICP4启动子而是HSV ICP4基因表达的有效转录抑制剂,这一点通过电泳迁移率变动分析来证明(16)。
当在感染的非洲绿猴肾细胞株系细胞中测定M4N和Mal.4的抗-HSV活性并且与9-(2-羟乙氧甲基)鸟嘌呤(无环鸟苷,ACV)的相比时,申请人发现M4N的IC50对于HSV-1的第10代和HSV-2的第4代在11.7μM至4μM之间变化而没有更高药物浓度的要求的明显升高的趋势。但是,对于ACV,IC50从对于病毒第一代的7μM提高到对于HSV-1的第10代的444μM并且对于HSV-2的第4代升高到>88μM,表明其在非洲绿猴肾细胞株系细胞中快速产生的抗ACV抗药性。结果,当选择系数,S.I.(TC50/IC50)对于M4N保持相对稳定时,对于ACV的S.I.在非洲绿猴肾细胞株系细胞中病毒传代之后降低60倍(16)。因此,M4N是一种突变不敏感药物。其能有效地抑制ACV抗性HSV(16)。
由于Sp1是重要的细胞转录因子这一事实(9),应该寻求该类化合物对Sp1-调节的细胞基因的表达的可能的抑制作用。一旦Mal.4稳定结合其结合位点,其不能替代Sp1(2)。因此,看起来可能是NDGA衍生物对增殖细胞中Sp1-调节基因的作用应该比对静态细胞中Sp1-调节的管家基因的表达的作用大。在前一情况下,DNA合成期间,该药物能与Sp1蛋白质竞争基因启动子中的Sp1位点,而在后一种情况下,该药物可能对Sp1蛋白质已经稳定结合它们的启动子的管家基因的转录的染色质有极小作用。事实已证明是这种情况。正如下面将证明的,通过用9600个表达基因进行的基因阵列研究,申请人发现大多数Sp1调节基因的产物保持在相似水平,不受培养物中宫颈癌细胞C3的药物治疗的影响(FIG.5)。即使这样,如果必须全身使用该药物,则相对低的M4N的选择性系数自然限制了其应用到最低有效浓度。另一方面,人乳头状瘤病毒最初通过HPV E6/E7基因的Sp1调控的表达诱导实体宫颈瘤和口腔肿瘤(10)。申请人推论:如果药物能够原位送递,并且只保留在肿瘤区,则可以利用高浓度的药物来有效破坏肿瘤,而对患者几乎没有损伤。
发明概述
因此,本发明的一个目的是提供用于治疗动物,特别是哺乳动物,最特别是人的癌症和非癌性肿瘤的化合物和组合物。根据本发明的该方面,提供抑制肿瘤生长的新的去甲二氢愈创木酸衍生物。
去甲二氢愈创木酸衍生物是指下面结构的化合物
其中R1,R2,R3和R4独立地代表-OH,-OCH3,-O(C=O)CH3,或一种氨基酸残基,但不同时都代表-OH。氨基酸取代基尤其包括丙氨酸,精氨酸,天冬酰胺,天冬氨酸,半胱氨酸,谷氨酸,谷氨酰胺,甘氨酸,组氨酸,异亮氨酸,亮氨酸,赖氨酸,甲硫氨酸,苯丙氨酸,脯氨酸,丝氨酸,苏氨酸,色氨酸,酪氨酸,缬氨酸,5-羟基赖氨酸,4-羟基脯氨酸,甲状腺素,3-甲基组氨酸,ε-N-甲基赖氨酸,ε-N,N,N-三甲基赖氨酸,氨基己二酸,γ-羧基谷氨酸,磷酸丝氨酸,磷酸苏氨酸,磷酸酪氨酸,N-甲基精氨酸和N-乙酰基赖氨酸。
本发明应用的特别优选的化合物是M4N和G4N,其如图1。
本发明的另一个目的是提供一种利用这些新的衍生物,和通过本领域已知的但是迄今为止还没有用于治疗肿瘤的相似衍生物治疗癌症和非癌症肿瘤的方法。该方法应该对抗含有依赖细胞周期蛋白的激酶CDC2的快速增殖的细胞类型特别有效。因此,本发明的另一个目的是提供抑制真核细胞周期,特别是动物细胞,更特别是哺乳动物细胞,最特别人细胞中CDC2的方法。
要治疗的肿瘤包括对上述根据本发明方法使用的化合物敏感的任何肿瘤。特别是,这包括对依赖于细胞周期蛋白的激酶CDC2周期的抑制作用敏感的快速分裂着的癌性和良性肿瘤。
术语“癌性肿瘤”意指包括任何可能转移或者可能还没有转移的恶性肿瘤。术语“非癌症肿瘤”意指包括任何良性肿瘤。按照本领域技术人员常规理解使用这些术语。
癌症生物学(Cancer Biology)(Raymond W.Ruddon,Cancer Biology,第三版,牛津大学出版社,1995,这里引作参考)的表1-1中可以找到通过本发明的组合物和方法可以治疗的良性和恶性肿瘤的各种例子。要治疗的肿瘤包括已知是病毒起源的那些和已知是非病毒起源的那些。预期本发明的组合物和方法在治疗实体肿瘤中特别有用。
本发明的另一个目的是提供一种抑制依赖细胞周期蛋白的激酶CDC2周期的方法。该方法将在抑制细胞增殖,特别是快速分裂着的细胞类型中的细胞增殖中是有用的。
在优选的实施方案中,这里描述的化合物和组合物用于治疗HPV-诱导的肿瘤。HPV-诱导的肿瘤特别包括,但不限于,HPV感染相关的宫颈癌,口腔癌,阴茎癌和头颈癌。该方法包括对癌性或非癌性HPV-诱导的肿瘤局部施用去甲二氢愈创木酸衍生物,特别是四-O-甲基去甲二氢愈创木酸(M4N)和四甘氨酰基去甲二氢愈创木酸衍生物(G4N)(tetraglycinalnordihydroguaiaretic acid)。
本发明的另一个目的是提供一种通过施用含有氨基酸取代基的式I的化合物抑制病毒复制和生长的方法。优选在本发明中使用的是其中氨基酸取代基R1,R2,R3和R4是相同的的化合物。
本发明涉及对肿瘤局部注射施用M4N,G4N和其它衍生物,其一般同药物可接受稀释剂,赋形剂和载体一起。在优选的实施方案中,M4N以DMSO溶液的形式注射给肿瘤,而以PBS溶液的形式施用G4N。G4N的使用将补充M4N的使用,特别是对较大的肿瘤(>2cm3),由于其水溶性,其允许它扩散到肿瘤的较大区域。根据本发明,可以类似使用其它水溶性和水不溶性去甲二氢愈创木酸衍生物。这些也可以在用于全身送递的脂质基的制剂中使用,正如本领域公知并使用的。
所谓药物可接受的稀释剂,赋形剂和载体指本领域技术人员公知是与M4N,G4N和其它类似衍生物相容的并且适合根据本发明对人或其它哺乳动物局部施用的那些的化合物。尽管下面描述的实施例通过局部注射描述了给药,也可以使用例如对肿瘤部位局部(topical)施用或靶向送递这样的局部(local)给药方式。
获得期望的治疗效果的所施用的化合物的量将是不同的,但是本领域技术人员能够容易地确定。剂量,给药频率,治疗时间长短取决于情况的不同而不同,主要取决于肿瘤的大小和类型。但是,为了举例说明的目的,可以提到对于每克重量肿瘤单独使用10毫克至20毫克M4N或者与类似量的G4N一起的剂量,以每天至每周间隔或更低的频率给药。可预计对于1-1.5cm3肿瘤,在很多情况下施用50微升至100微升溶解于DMSO中的浓度是200mg/ml的M4N,或者单独地施用或者与G4N联合施用,是有效的。
附图简要说明
图1.M4N和G4N的结构
图2A.显示E6/E7启动子(pPV16P97)的区和对Sp1蛋白的结合位点的HPV-16 LCR。
图2B.M4N对C-33A细胞中E6/E7启动子活性的影响。(不同浓度的M4N对E6/E7启动子驱动的萤光素酶基因转录的抑制)。
图3A-3C.40μM M4N对病毒E6和E7RNA转录物的抑制。使从在生长培养基中用40μM M4N或单独DMSO处理71小时的C3细胞分离的总RNA进行相对(relative)RTPCR。提高扩增的循环之后取出RTPCR样品并且在琼脂糖凝胶上分离。凝胶照片(3A和3B)表示这些循环,生长培养基中M4N存在(+)或不存在(-),以及用作大小标记物的两种pGMT载体的消化物。扩增图谱(2C)表示两种期望大小的扩增产物,其从早期病毒RNA转录物的可变剪接得到。
图4A.M4N对C3细胞生长的抑制。
图4B.M4N去除之后C3细胞生长的抑制。
图5A-5B.通过基因测试分析检测M4N对C3细胞中基因表达的影响。5A.DMSA处理>2小时之后C3细胞中表达的基因(C3 DMSO)。5B.在使用DMSO作为溶剂的M4N处理>2小时之后C3细胞中表达的基因(C3 M4N)。
图6A-6B.M4N处理之后对携带肿瘤的小鼠的目测观察。
6A.原位注射DMSO(#3)或M4N(#7)治疗带一个肿瘤的小鼠。也对长有两个肿瘤的小鼠对其中一处肿瘤原位注射M4N #9。6B.同一只小鼠M4N处理的肿瘤(白色疤痕)和未处理的肿瘤,#9,如表2所示。
图7.M4N和M4N/G4N对小鼠肿瘤生长的组织病理学作用。
图片第一个纵柱代表与小鼠#12,10,27和20(M4N)相对小的药物处理(M4N或M4N/G4N)的损伤相比DMSO处理(CON)之后#4,10,12号小鼠的大的肿瘤尺寸。随后的照片是放大100倍下观察的这些肿瘤的例子(A,B,C,DMSO处理的,D未处理的,E,F,G,H,M4N或M4N/G4N处理的)小鼠(表1和表2)。
图8.在没有药物存在下(HSV-C,HSV-SC),在无效药物存在下(ABDS1[″HSV-ABDS1″],ABDS2[″HSV-ABDS2″]),和有效药物存在下(M4N[″HSV-4N″]和ACV[″HSV-ACV″]),HSV-1的复制。
图9.M4N引起哺乳动物细胞生长停滞。
(a-d)用不同浓度的M4N处理C3,CEM-T4,C33a和TC-1细胞。将实验开始时存在的细胞数指定为第0天。三天之后,计数成活细胞数并且对M4N浓度作图。(e)将C3细胞分装入T-25烧瓶,每个烧瓶5×103个细胞,并且加入培养基中在1%DMSO中的M4N或者只加入培养基中1%DMSO(第一次培养基更换)。三天之后,对M4N处理的细胞的一半加入只含有1%DMSO(M-D)的新鲜培养基,而对其余细胞加入具有相同条件的新鲜培养基(第二次培养基更换)。每天计数细胞数并且对处理时间作图。
图10.用M4N处理的细胞在G2/M停滞。
在含有1%DMSO或者1%DMSO和M4N(M4N)的培养基中将C3细胞(a),C33a细胞(b),CEM-T4细胞(c)和TC-1细胞(d)培养三天。将细胞用胰蛋白酶消化,用乙醇固定,用碘化丙锭染色,接着通过流式细胞仪分析。该数据以对碘化丙锭染色强度的细胞数表示(3-5×104总细胞)。标记细胞周期的指定阶段并且与通过染色强度测定的相对DNAcompliment对应。
图11.用40μM M4N处理C3细胞证明G2细胞结构。
在含有1%DMSO(对照)或者1%DMSO和40μM M4N(M4N)的培养基中C3细胞在盖玻片上生长3天。样品用乙醇固定并且与抗α(绿色)和γ(橙色)微管蛋白(a)的抗体或者DAPI DNA染色剂(b)一起培养。通过荧光显微镜观察细胞。
图12.通过M4N减少CDC2和病毒癌基因。
C3细胞在含有1%DMSO(D)或者1%DMSO和40μM M4N(M4N)的培养基中生长不同时间(数量以小时计)。特定时间之后,从细胞分离全蛋白质和总RNA。用相同的硝化纤维素滤膜用抗CDC2或细胞周期蛋白B抗体进行Western印迹(a-顶部两张图片)。用抗细胞周期蛋白B的抗体进行免疫沉淀后,接着通过与γ-32P ATP和组蛋白H1温育进行激酶分析(a-底部两张图片)。包括PAGE凝胶的考马斯染色剂作为加载对照。分别进行24小时和72小时药物处理的激酶分析。
对总RNA提取物进行RNA印迹分析(b)。对于CDC2或GAPDH,滤膜与随机引发的32P-标记的DNA一起温育过夜,洗涤,并且将其曝光到胶片三天。使用相同的滤膜测试CDC2和GAPDH RNA。
用与或者HPV-16 E7或者GAPDH中的区杂交的引物对总RNA提取物进行rtPCR分析(c)。在相同反应中使用两种引物对,并且通过琼脂糖凝胶电泳分析产物。
图13.G4N与HIV Sp1-结合位点(-87至-49)的相互作用的电泳迁移率变动分析。(A)Sp1-167D与32P标记的HIV Sp1 DNA模板结合的G4N抑制作用。泳道1,只有模板;泳道2,模板加0.1微克Sp1-167D;泳道3-9,在加入0.1微克Sp1-167D之前与递增浓度的G4N(0.25-1.75mM)温育的模板。(B)G4N置换与HIV模板结合的Sp1-167D。泳道1,只有模板;泳道2,模板加0.1微克Sp1-167D加100倍过量的没有标记的模板;泳道3,模板加0.1微克Sp1-167D;泳道4-10,用递增浓度的G4N(0.25-1.75mM)攻击的Sp1/DNA复合物;泳道11,在含有1.75mM G4N的反应缓冲液中温育的模板。(C)Sp1-167D置换与模板结合的G4N。泳道1,只有模板;泳道2-4,模板加递增量的Sp1-167D(0.075,0.150,0.300微克);泳道5-8,在含有1.2mM G4N的反应缓冲液中温育后用递增量的Sp1-167D(0.075,0.150,0.300微克)攻击的模板,泳道8没有接受Sp1-167D。(D)与(A)-●-和(B)-●-中使用的递增浓度的G4N响应的减小的Sp1-167D/DNA复合物泳带强度的图。使用的凝胶是5%没有变性的聚丙烯酰胺,每条泳道接受5微升的各反应体积,如实验部分和参考文献[1]所示。
图14.G4N在COS细胞内对HIV Tat-调节的反式激活的抑制。
图15.G4N存在下SIV产生。37℃下混合107 174X细胞和SIV mac239(4ng p27)的24小时收获的原液2小时。将细胞再次悬浮并且向三个96孔板的每一个孔加入100微升培养基中1×105细胞。从新鲜制备的原液制备各种浓度的G4N并且加给6个设计的孔的每一个中。4和8天之后,收集培养上清夜用于病毒产生分析。通过改进的p27衣壳蛋白抗原捕捉ELISA分析病毒产生,如实验部分所述。
图16.G4N对H9细胞中HIV p24抗原产生的抑制。
用AZT抗性HIV株,HIV-1RTMF病毒感染后9天,通过比较G4N处理过的和没有处理的H9细胞的两份重复的培养物的平均的p24水平计算抑制百分率。
本发明的详细描述
实验方法
化学合成NDGA衍生物(15)。细胞系C3是HPV16E+C57BL/6kh源的L加激活的Ras转化的细胞系,由Loyola University Medical Center,Chicago,Illinois,U.S.A的W.Martin Kast提供。根据Greenstone等人(21)和Feltkamp等人(22,23)所述进行保持和培养细胞。
G4N的合成
制备内消旋(meso)-1,4-二[3,4-(二甲基氨基乙酰氧基)苯基]-(2R,3S)-二甲基丁烷盐酸盐四甘氨酰基(Tetraglycinyl)NDGA,G4N的标准方法。向含有NDGA(12.8g,42.3mmol,1.0当量)和N,N,-二甲基甘氨酸(26.2g,254mmol,6.0当量)的二氯甲烷(250ml)溶液加入DCC(52.4g,254mmol,6.0当量)和DMAP(2.32g,18.9mmol,1.0当量)。将该反应混合物在氮气下室温下搅拌24小时。过滤反应混合物之后,减压浓缩溶液。然后向反应瓶中加入丙酮(250ml)并且鼓泡通入该溶液过量HCl(g)。将水溶性沉淀物溶解于水并且在室温下从丙酮中再沉淀两次,得到(1)(29,2g,36.8mmol),为白色固体,产率87%。在Varian Unity-400(400MHz)光谱计上通过使用D2O溶剂和TSP作为内标,获得质子NMR谱。使用D2O作为溶剂,在Varian Unity-400(400MHz)光谱计上获得碳-13NMR谱。碳-13化学位移参比TSP单峰(δ0.0ppm)。
合成如合成路线1所示。合成路线1
一般方法。除非另有说明,所有的反应都在烘箱干燥过的玻璃器具中在氮气氛下进行(120℃)。从Mallinckrodt Chemical Co.购买丙酮,二氯甲烷,1,4-二噁烷,乙酸乙酯,己烷和四氢呋喃。用4埃分子筛干燥丙酮并且蒸馏。干燥二氯甲烷,乙酸乙酯,和己烷并且用CaH2蒸馏。通过在氮气下用钠和二苯甲酮蒸馏来干燥1,4-二噁烷和四氢呋喃。从FlukaChemical Co.购买去甲二氢愈创木酸。从Merck Inc.购买N,N′-二环己基碳化二亚胺(DCC),4-二甲基氨基吡啶(DMAP),吗啉,三乙胺,和碳酸钾。从Aldrich Chemical Co.购买1-溴-3-氯丙烷,N,N-二甲基甘氨酸,和二氯磷酸甲酯。
在从Merck Inc.购买的预先涂布的平板(硅胶60 F-254)上进行分析性薄层色谱(TLC)。在装有25-m交联甲基硅氧烷树胶毛细管柱(0.32mmi.d.)的Hewlett-Packard 5890 Series II仪器上进行气相色谱分析。将氮气用作载体气体并且将流速保持在14.0毫升/分钟不变。在下面的条件下测定保留时间TR:注射器温度260℃,等温柱温度280℃。在装有Hewlett-Packard 5971A质量选择性检测器和毛细管HP-1柱的Hewlett-Packard5890系列II仪器上进行气相色谱和低分辨率质谱分析。使用Jasco Model880-PU智能HPLC泵以120毫升/小时的流速进行中压液相色谱(MPLC)分离。MPLC填充材料,反相硅胶C18(颗粒度0.035-0.070mm),从Knauer Co.购买。通过使用Merek试剂硅胶60(颗粒度0.063-0.200mm,70-230目ASTM)进行通过重力柱色谱的纯化。
在Bomem Michelson系列FT-IR光谱计上测定红外光谱(JR)。报导的波数参比聚苯乙烯1601cm~1吸收。用下面的缩写记录吸收强度:s,强;m,中;w,弱。在Varian Unity-400(400 MHz)光谱计上利用D2O作为溶剂,3-(三甲基甲硅烷基)丙酸钠盐为内标,获得质子NMR谱。使用D2O作为溶剂,在Varian Unity-400(400MHz)光谱计上获得碳-13 NMR谱。碳-13化学位移参比3-(三甲基甲硅烷基)丙酸钠盐单峰中心(60.0ppm)。用下面缩写记录多重性:s,单峰;d,双重峰;t,三重峰;q,四重峰;m,多重峰;J.偶合常数(赫兹)。利用JEOL JMS-HX110质谱仪获得高分辨率质谱。
内消旋-1,4-二[3,4(二甲基氨基乙酰氧基)苯基]-2R,3S-二甲基丁烷盐酸盐(2)。向NDGA(1,12.81g,42.37mmol,1.0当量)和N,N-二甲基甘氨酸(26.21g,254.2mmol,6.0当量)的二氯甲烷(250mL)溶液中加入DCC(52.45g,254.2mmol,6.0当量)和DMAP(5.176g,42.37mmol,1.0当量)。该反应混合物在室温下在氮气中被搅拌24小时。过滤出反应混合物中的二环己基脲之后,减压浓缩得到的溶液。然后,向残余物加入丙酮(250mL)并且向得到的溶液鼓泡通入过量HCl(g)。沉淀物溶解于水并且在室温下用丙酮再沉淀两次,得到2(28.97g,36.86mmol)为白色固体,产率87%: 1HNMR(D2O,400MHz)δ0.78(d,J=6.0Hz,6H.2×CH3),1.73(m,2H.2×CH),2.38(dd,J=13.2,9.6Hz,2H.2×ArCH),2.78(dd,J=13.2,4.4Hz,2H.2×ArCH),3.03(s,24H.8×CH3N),4.53(s,8H,4×CH2N),7.22(m,4H.4×ArH),7.29(d,J=8.4Hz,2H.2×ArH);13C NMR(D2O,100MHz)δ18.11,40.82,41.73,46.75,59.59,125.79,126.58,131.63,140.66,142.47,146.11,167.84;IR(KBr)3461(br),2963(m),1777(s,C=O),1620(m),1478(m),1377(m),1210(m),1106(m),961(w),852(w)cm-1;MS(FAB)of(2-4HCl)m/z(相对强度)643(M+,30),600(20),558(43),515(20),473(42),430(13),388(26),185(18),93(38),58(100),44(22);HRMS(FAB)of(2-4 HCl) 计算值C34H50N4O8 642.3628,实测值642.3614;计算值C34H54N4O8Cl4:C,51.78;H.6.90;N.7.10;O.16.23.实测值:C,51.70;H.6.85;N.7.05;O.16.21.
要明白通过其它N,N-二甲基取代的氨基酸的合适的取代能够合成本发明另外的氨基酸取代的化合物。
实施例1
M4N和几种其它NDGA衍生物的SP1-调节的HPV E6/E7启动子活性的作用
利用荧光素酶作为报道因子检测M4N和几种其它NDGA衍生物的SP1-调节的HPV E6/E7启动子活性的作用。该项测定的基础是通过磷酸钙方法将与荧光素酶报道基因融合的HPV16 LCR(P97启动子)到C33A细胞中的DNA转染。C33A是宫颈肿瘤细胞系(ATCC保藏号.HTB-31),其不包含任何整合的HPV DNA,但是具有HPV早期基因启动子的强表达所需的转录因子。DNA转染之后的一天,对细胞加给借助于二甲亚砜(DMSO)溶解的各种浓度的药物。药物处理之后30小时(这样使得对于瞬时转染实验在标准的48小时之内完成检测),裂解细胞并且测定荧光素酶比活性(荧光素酶测定系统,Promega,美国专利5,283,179)。随着M4N药物浓度的提高,荧光素酶比活降低。
结果(图2所示)证明在荧光素酶分析中M4N显著地降低Sp1调节的HPV E6/E7启动子的转录起始。
实施例2
M4N处理之后E6/E7 mRNA合成的抑制
通过RT-PCR测定宫颈细胞系C3中M4N处理之后E6/E7 mRNA合成的抑制。用对计数的细胞数标准化的定量总细胞RNA进行相对RT-PCR。在2%琼脂糖凝胶上分析RT-PCR产物。图3给出了结果。RT-PCR结果表明在扩增第22个循环这样早时在DMSO处理的细胞中检测到了对于E7(321 bp)和E6(204 bp)预期大小的扩增的cDNA。在30循环扩增之后,在药物处理过的RNA提取物中几乎检测不到这些相同的产物。对于没有模板的PCR对照物或者从HPV16-阴性C33a细胞系的总RNA提取物检测不到扩增产物。
实施例3
M4N处理对宫颈C3细胞生长的抑制
以每管105个细胞的密度平板培养HPV-16转化的无限增殖的小鼠上皮细胞(C3细胞)。24小时之后,对1/2的小管加给含有溶解于1%DMSO的40μM M4N的生长培养基,而对另一半给予只含有1%DMSO的生长培养基。结果如图4A所示。24小时之内,观察药物处理和对照C3细胞之间的细胞形态学差异。与未处理的对照物相比,药物处理过的细胞的生长和分裂大大降低,而活细胞与总细胞数之比对于药物处理过的细胞和只用DMSO处理的对照细胞都保持恒定。这表明M4N显著地减少了细胞分裂。
也检测了从培养基中去除M4N之后对C3生长的影响。以每管104个细胞的密度平板培养C3细胞。在时间=0时,对2/3的小管给予补充有溶解于1%DMSO的40μM M4N的生长培养基。剩下的小管给予只含有1%DMSO的生长培养基。73小时之后,洗涤在其培养基中接受了M4N的1/2的小管并且加入只含有1%DMSO的培养基。洗涤另外2/3的细胞小管并且用先前施用的相同培养基置换。如图4B所示,这些结果表明,变成没有药物的培养基之后M4N处理过的样品中细胞生长的速度没有显著增加,表明即使从胞外环境去除之后,M4N还持续地显著减少细胞分裂。
实施例4
药物处理之前和72小时之后对C3细胞中细胞基因表达的分析
研究了9600个基因阵列基因表达(图5)。根据基因组(Genomics)51,313-324 1998中描述的方法,在一对人9600个基因阵列杂交研究中使用来自72小时M4N(40μM)处理过的(C3 M4N)和未处理过的(C3DMSO)的5微克各种poly A+RNA。用带有Nikon 55 mm AF micro Niko镜头的彩色视频摄象机拍摄杂交成像,并且用Macintosh LC630计算机数字化处理。这样的单色或双色模式的通过形成颜色的酶的酶底物反应的检测是可重复的并且是十分敏感的(能检测每个细胞<5个拷贝的转录物,RNA来自107个细胞)。
排列显示示差地表达的基因(C3 M4N/C3 DMSO>10和C3 DMSO/C3M4N>10)的计算机打印结果进行检查。在ZIP磁盘上保存TIFF格式图象文件和MS excel格式数字文件。保留获得的成像克隆的基因名称和克隆ID号用于将来RNA印迹确认。
M4N处理之后72小时正调节或负调节的基因的组中,下面那些特别地与细胞分裂和编程性细胞死亡相关。几种其他细胞周期相关的基因也响应M4N而大大地正调节。除了依赖细胞周期蛋白的激酶CDC2(实施例11)之外,例如:
提高
依赖细胞周期蛋白的激酶抑制剂 (100倍)
编程性细胞死亡(APO-1)抗原 (100倍)
死亡区三DR3 (100倍)
Ras-相关的蛋白质RAP-1 (60倍)
人Map激酶 (40倍)
下面细胞周期相关的基因响应于M4N而大大地负调节:
处理过的 没有处理的
依赖细胞周期蛋白的激酶7 (5%) 100%
人细胞因子受体 (2%) 100%
增殖细胞核抗原,PCNA (1%) 100%
人TNF-相关的编程性细胞死亡APO2 (3%) 100%
半胱氨酸蛋白酶 (7%) 100%
在较早时间点,例如1小时药物处理之后,发现E6/E7水平与对照细胞中的那些接近,而4.5小时之后,RT-PCR不再能检测出E6/E7(39)。为了进一步追踪该药物的最初的细胞作用,用从这些短期处理过的细胞(1小时和5小时)分离到的RNA重复9600个基因阵列的基因表达。
实施例5
在小鼠中局部注射M4N靶向C3肿瘤生长
在小鼠后背的肩之间用5×105个C3细胞对36只C57b1-16 NCR小鼠注射。20天之内,其中24只小鼠发生肿瘤。每天注射(50微升-100微升的M4N或M4N/G4N)(200mg/ml M4N的DMSO溶液,200mg/ml G4N的PBS溶液)表明对动物肿瘤生长的极深的影响,如表1和2,图6和7所示。
表1
M4N和G4N对小鼠体内发生的单一肿瘤的生长的作用
小鼠# | 处理时间1-16天 | 病灶大小(m)第1天 第7天 第21天 | 切下的病灶的重量(g)第16天 第24天 | 体重(g)第1天 第24天 |
1234 | DMSO*DMSODMSODMSO | 3×8×3.3 - 5×7×44.4×6×3.5 10×12×8 -0.8×0.8×1 - 10.5×11×92.8×3.8×2.5 - 18×11×9 | - 0.31.56 -- 1.14- 2.9 | 18.8 - 20.219.6 20.5 -18.2 - 16.117.6 - 20.2 |
671114151617 | 1-16天M4NM4NM4NM4NM4NM4NM4N | - 9×8×5 -- 6×7×7 -1×1.3×1 9.5×10×9 -3.8×3.8×3.5 8×9×6 -- 5×4×4 -2.8×2.8×2.8 9×6×4 -2.3×2.3×2.3 6×6×4 - | 0.2 -- 0.1- 00.4 -0.1 -0 -0.2** - | 19 19.2 -18.2 - 20.419.5 - 20.217 17.6 -18.9 20.0 -17.2 17.6 -17.3 - - |
1819212227 | 天 天1-10 9-17M4N G4NM4N G4NM4N G4NM4N G4NM4N G4N | 3×2.8×3 8×7×5 -- 5×5×5 -1.8×1.8×1.8 9×10×5 -- 7×7×5 -2.5×5×2.5 9×6×6 - | - 1.0***0.2 -0.2 -- 0- 1.8*** | 18.8 - 21.118.2 19.9 -17.3 19.2 -17.9 - 19.520 - 20.7 |
2829 | M4N G4NM4N G4N | 2.8×2.3×2.8 5×5×4 -2.8×2.5×2.8 5×6×4 - | 0.17 -- 0.2 | 18.1 19.8 -18.8 - 19.6 |
*DMSO=药物的赋形剂
**在第15天操作
***病灶大多数含有坏死细胞,这正如也在第6,7,11,14,15,17,19,21,28,19号小鼠病灶中发现(图6,7)。药物处理之后的#11和#22小鼠没有病灶。对照小鼠#1,2,3,4中发现的肿瘤含有生长着的细胞(图2)。
实验方法:
用5×105个C3细胞/小鼠对36只C57b1-16 NCR小鼠注射。在小鼠后背的肩之间皮下注射100微升。细胞悬浮于低盐HBSS中,并且通过轻轻涡旋保持悬浮液均匀。
24只小鼠发生肿瘤。用刻度卡钳测量病灶大小。将小鼠剃毛,称重并且开始治疗(第1天)。隔离4只小鼠作为对照。对照小鼠每天肿瘤内注射50微升DMSO。实验小鼠(10)接受50微升溶解于DMSO的M4N(200mg/ml)。另外10只小鼠接受M4N处理8天,接着每天接受G4N处理(50微升,200mg/ml,于PBS中),接受8天。对肿瘤的几个区域进行注射。在注射之前用乙醚或metaphane将小鼠麻醉。
表2
M4N和G4N对携带多个肿瘤的小鼠处理过的病灶的生长的作用
小鼠# | 处理时间1-16天 | 病灶大小(mm)第1天 第7天 | 切下的病灶的总量(g)处理过* 没有处理** | 体重(g)第1天 第24天 |
91012 | M4NM4NM4N | 1.3×5×0.75 7×9×82.3×2.5×2.3 9.5×10×92.5×2.5×2.5 8×9×6 | 0.25 0.60.1 2.90.11 1.82 | 20.2 17.917.5 22.117.8 20.0 |
202426 | 天 天1-9 10-18M4N G4NM4N G4NM4N G4N | 1.8×1.8×1.8 9×10×5- 7×9×65×3.3×2.5 7×7×7 | 0.1 0.20 1.70.2 1.9 | 17 20.217.2 20.819.3 20.6 |
*直接对肿瘤区域注射DMSO中的药物
**来自没有给药的临近肿瘤
表3
G4N对小鼠的毒性研究
组 | 小鼠编号# | 途径 | 每天处理 | 注射天数 | 死亡率 | |
1234 | 187.5mg/kg375mg/kg750mg/kg375mg/kg | 3342 | 皮下皮下皮下IV | 2X1X1X2X | 6666 | 0/30/31/40/2 |
在该项研究中使用来NCI的C57BL-16NCR雌性小鼠。每天在PBS中新鲜制备Tetraglycinal NDGA(G4N),浓度75mg/ml。每次处理,对第1组注射0.05ml,对第2和4组注射0.1ml,对第3组注射0.2ml,为期6天。实验持续7天。在注射之前和之后6天测量体重。在实验期间没有观察到明显的体重变化。
所有的处理过的小鼠,对照小鼠(小鼠编号1-4)和实验小鼠(小鼠编号6,7,9,10,11,12,14,15,16,17 M4N编号18-22,24,26-29 M4N/G4N)表现出肿胀。用刻度卡钳测量病灶大小。一些小鼠由于注射而轻度出血。治疗方案和结果如下:
第10天:对小鼠再次称重。所有的小鼠都表现出增重2克。
第12天:没有进行处理。
第13天:所有的小鼠皮肤隆起,但是程度非常不同。一只M4N处理过的小鼠(#7)皮肤裂开,通过该裂开的皮肤脱落出″干涸的肿瘤(dried-out tumor)″。
第14天:注射体积升高到100微升。
第15天:一只M4N处理过的小鼠(#17)死于过剂量麻醉/操作。#17病灶处皮肤破裂现出″干涸的肿瘤″。将其解剖,并且切除病灶并且称重。
第16天:将4只以上M4N处理过的小鼠(#6,14,15,16),3只M4N/G4N处理过的小鼠(#19,21,28)和一只对照小鼠(#2)实施安死术,解剖并且称重。对其余对照小鼠(#1,3,4)非入侵性检查并且正带有肿瘤。
第21天:用刻度卡钳测量来自对照小鼠的肿瘤大小。观察结果:小鼠#10和#12(M4N处理过的区域)病灶处的皮肤裂开现出″干涸的肿瘤″。
第24天:小鼠#7皮肤完全复原。实验在这一天终止。所有剩下的小鼠,M4N处理过的(#7,9,10,11,12)和M4N/G4N处理过的(#18,20,24,26,29)被实施安乐死,解剖,检查并且称重。
表1和2和图5和6总结了M4N和M4N/G4N对小鼠C3肿瘤生长的作用。表1给出药物对携带一个肿瘤的小鼠的C3细胞生长的作用。对照组的4个切除的肿瘤的平均重量是1.48g,而来自M4N处理过的和M4N/G4N处理过的病灶的重量分别是0.142和0.51g。药物处理过的病灶主要由干涸的坏死细胞组成(图6)。来自对照组的肿瘤表现出是同质的并且含有活跃生长细胞。表2给出药物对携带多个肿瘤的小鼠的C3肿瘤生长的作用。在该项研究中,将药物注射给这些肿瘤中的一个。未处理过的肿瘤的平均重量是1.77g,而M4N处理过的病灶的平均重量是0.15g。M4N/G4N注射后获得相似结果-未处理肿瘤的平均重量是1.27g,而药物处理过的病灶的平均重量只有0.103g。
在整个实验期间,所有小鼠的体重变化似乎不明显(表1和2)。
实施例6
制备来自两组小鼠的药物处理的(M4N)和DMSO赋形剂-处理的或者未处理的肿瘤(CON)用于组织病理学检查。立即固定切除的肿瘤,然后保存于4%甲醛磷酸盐缓冲盐水中。然后通过一系列等级的醇和二甲苯将固定的组织脱水并且包在石蜡中。将石蜡组织块切成薄片并用苏木紫和曙红染色用于显微镜观察。组织病理学研究表明对照肿瘤不受DMSO处理的影响并且继续生长。它们表现出高的细胞核/胞质比,多形核变化,高有丝分裂形状,纺锤样肉瘤形状,并且渗入周围组织,其是癌细胞的特征。
相反,接受M4N治疗的那些肿瘤在开始治疗之后很快不继续生长。它们证明显著坏死并且不再存活。在更高放大倍数下可见少量药物沉淀,并且病灶区显现慢性炎症和纤维变性。该治疗作用导致这些减少的肿瘤细胞从该区的脱落。用M4N/G4N治疗可以见到和单独用M4N治疗一样的结果。但是,因为G4N是水溶性的,它可以扩散到比M4N更大的肿瘤区。预计当与M4N协同使用G4N时,可以更有效治疗大体积的肿瘤(即大于2cm3)。
实施例7
M4N对豚鼠HSV-1皮肤感染的作用
也测定了药物M4N对豚鼠皮肤感染HSV-1复制的抑制。用针刺豚鼠皮肤,并且局部施用HSV-1抑制以感染每一个穿刺的区域。感染之后,每天对该穿刺的感染区应用M4N,进行6天。
用5=DIN针头对豚鼠裸背的六个区无菌穿刺。两个区感染HSV-1(HSV-C,培养上清液,或者在盐水中的分离的HSV,HSV-SC)。另外4个区感染HSV-SC。感染15分钟之后,对一个区的每个穿刺的感染的部分施用60mg/ml的DMSO中的30微升的试验化合物(ABDS1,ABDS2,ACV和M4N(4N),每天5次,持续6天。ABDS1和ABDS2为阴性对照。在第6天照下图8中的照片,并且其给出了在不存在药物(HSV-C,HSV-SC)的条件下,在存在无效药物(HSV-ABDS1,HSV-ABDS2)的条件下和在存在有效药物(HSV-M4N和HSV-ACV)的条件下,HSV-1复制的程度。可以看到,在HSV-C,HSV-SC,HSV-ABDS1,HSV-ABDS2处理区出现6个大的汇合疱,而M4N(4N)和ACV处理之后,感染的区域没有发现任何疱。
如药物处理之后4天没有出现皮肤病灶和没有病毒脱落所证明的,在该模型系统中获得了M4N能够阻断HSV复制这样的准确无误的结果。最初的动物研究也表明,当腹膜内给药时,以高达300mg/kg这样的浓度,和皮下或者静脉内给药时,以高达375mg/kg这样的浓度,M4N对小鼠没有毒性(表3)(6)。
实施例8
利用原位注射将M4N用于临床治疗
作为药物送递途径直接对肿瘤施用M4N提供了几个有特色的优点。1)M4N是疏水性化合物并且极易溶解于DMSO(200mg/ml)。因此,为了达到有效剂量的药物,对于注射只需要小体积的药物溶液。在上面实施例5所述的小鼠研究中,每天注射50微升至100微升,注射几天,足以完全阻止小鼠肿瘤生长。对于大剂量(每次治疗30ml IV)DMSO治疗疾病的应用,先前也有一些研究(24)。这些结果没有最后确定(25)。但是,因为过去在全世界范围用大剂量DMSO已经在数千万人群中经过了安全测试,证明当只使用小体积时,DMSO作为药物送递的赋形剂应该是安全的(26)。2)通过原位注射,大多数药物残留物在肿瘤区保持不溶和浓缩,并且不进入循环系统,这样避免了对全身的毒性。另外,因为肿瘤中保留足够的药物来抑制肿瘤生长,相对较少次数的处理之后,不再需要继续注射药物。在实施例5的小鼠研究中,即使在中断M4N注射之后,肿瘤细胞也持续死亡。因此,当直接靶向施用药物时,肿瘤大小变成了所施用的药物的所需要的量的决定因素。人与小鼠的整个体重之间的差异变得无关紧要。在小鼠肿瘤研究中,20mg/天,持续10天远远地足以除去肿瘤。没有理由使用比该剂量高的剂量来治疗人的相当大小的肿瘤(1-1.5cm3)。这应该大大地减少人试验的危险。
实施例9
对细胞的M4N处理阻断细胞增殖
我们先前对M4N的研究表明通过将依赖Sp1的启动子灭活能抑制病毒转录。很多哺乳动物细胞周期基因也包含基本的Sp1启动子,并且因此,M4N可以阻断它们的转录。通过检测M4N对多种不同的细胞系的抗增殖作用来验证该假设。先前已经证明低浓度(10μM)母体化合物,NDGA,诱导哺乳动物细胞编程性细胞死亡(27)。但是,通过封备邻苯二酚氧中的一个或者对NDGA加入一个亲水基团,可以回避该作用(28)。对HPV-16/ras转化的C3细胞系的培养物测定增加量的NDGA衍生物M4N(29)以便测定抑制增殖所需要的最佳的浓度(图9a)。该细胞对M4N响应很好,在40-60μM浓度范围内在72小时之后停止分裂。在这些浓度下在3天之后,细胞数保持和处理开始时(第0天,图9)的数量相等。在较低的药物浓度时,观察到细胞生长中更适度的减少,并且在高于60μM的浓度下观察到一些细胞死亡。
M4N对C3细胞系的抗增殖作用不只是完全由于药物使依赖Sp1的HPV-16 E6/E7致癌基因启动子失活的能力,因为在其E6/E7致癌基因受非-Sp1依赖性逆转录病毒启动子控制下的HPV-16转化的TC-1细胞系中也观察到类似的生长抑制(30),图9d)。另外,用M4N处理也阻断C33a细胞系(图9c),HPV-阴性人子宫颈癌细胞系,和CEM-T4细胞系(图9b),人白血病细胞系的生长31)。在用该药物处理的四个细胞系中,在M4N浓度超过“阈”值(对于C3细胞是60μM,对于TC-1细胞是40μM,等)之前,几乎所有的(>95%)的停滞细胞是成活的。这些浓度之上,成活细胞百分比急剧降低。令人感兴趣的是,即使在延长接触药物的时间之后,停滞的细胞(arrested cell)保持>95%的成活率。用40μMM4N处理8天之后,C3细胞表现出细胞死亡没有增加(图9e)。
实施例10
用M4N处理的细胞停滞在G2期
一旦确定了用M4N处理的细胞停止增殖但是仍然保持成活,利用细胞DNA含量分析和细胞结构的荧光检查来确定细胞停滞的细胞周期中的点。证明接触M4N 72小时的细胞G2/M DNA含量相对对照增加(图10a-d)。对C3和CEMT4细胞系见到最极端的响应,其中>90%的细胞表现G2/M DNA的含量。
为了区别G2期停滞或有丝分裂的阻断,利用抗α微管蛋白(绿色)和γ微管蛋白(红色)抗体来确定M4N处理72小时之后C3细胞中中心体的状态。如图11a所示,M4N处理过的细胞的中心体是双份的,但是仍然在细胞核中彼此相邻地定位。因为在早前期中心体分开,可以得出结论,即这些细胞还没有开始有丝分裂。相反,对照细胞的γ微管蛋白染色呈G1或S期的扩散的模式特征(32)。用DAPI染色也观察到M4N处理过的细胞中没有染色质凝聚(图12b),细胞没有脱离G2期的附加证据(33)。
实施例11
40μM M4N抑制CDC2的产生
因为细胞发育脱离G2取决于MPF的产生,在用40μM M4N处理过的C3细胞中检查其蛋白质成分的状态。在含有在1%DMSO的M4N,或者只含有1%DMSO的培养基中异步的细胞生长24或72小时。收获细胞,通过蛋白质印迹分析相等量的总细胞蛋白质。用M4N处理72小时之后,发现CDC2的量显著减少(图12a)。但是,通过剥离和重新探测该相同的膜检测细胞周期蛋白B的水平,发现没有变化。这些结果表明,在这些条件下,停滞不可能是对p53的响应,因为已经证明p53的的过量表达导致细胞周期蛋白B的减少(34,35)。与蛋白质印迹分析的结果相吻合,M4N处理72小时除去CDC2激酶活性(图12a)。这些实验支持这样的观点,即药物通过抑制CDC2蛋白质的产生,导致MPF活性降低,而起作用。
我们先前证明M4N阻断依赖Sp1的病毒转录的能力的研究提示CDC2 mRNA水平的降低可能是CDC2蛋白质减少的机理。这与这样的发现相吻合,即其基因的表达不需要Sp1的细胞周期蛋白B蛋白以正常水平产生,而其基因在其启动子中有两个必需的Sp1位点的CDC2蛋白质的量大大减少。为了验证该假设,对从用40μM M4N处理5-72小时的C3细胞收获的RNA进行RNA印迹分析。如图12b所示,CDC2 mRNA的量只有用M4N处理24小时之后才降低并且在72小时之后基本消除。非-Sp1调节的管家基因GAPDH的产生用作RNA加载对照,并且其水平不受40μM M4N的影响。
C3细胞系的使用对我们提供了分析M4N介导的细胞周期停滞的机理的另外的对照,因为其它依赖Sp1的基因启动子也可能受到M4N处理的抑制。通过分析M4N对自依赖Sp1的HPV-16 E6/E7启动子的转录的作用检测C3细胞中的这种可能性。从用40μM M4N处理5-72小时的C3细胞分离的RNA的rtPCR分析证明E7转录物的水平清楚地降低(图12c)。在该项实验中GAPDH又被用作内部对照,并且其水平不受药物处理的影响。这些结果提供了M4N减少Sp1调节的启动子的转录物的另外的证据。
实施例12
在凝胶迁移率变动分析中G4N对Sp1-结合活性的抑制
Sp1家族蛋白质基于结合诱导转向DNA的大沟(36)。Sp1蛋白质的锌指区域负责GC盒序列5’-GGGGCGGGG-3’的结合。从计算分析,确定G4N,NDGA的氨基酯衍生物,能够与该大沟中这样一种序列形成稳定复合物。为了确定G4N是否能够作为Sp1封阻剂以及Sp1置换剂,我们通过凝胶迁移率变动分析在存在或不存在G4N下进行了Sp1/增强子相互作用研究,只使用Sp1的DNA结合区域用于试验。在该封闭实验中,首先,在25℃下将不同浓度的G4N与32P-标记的DNA在结合缓冲液中温育30分钟。接着,加入重组Sp1蛋白质(Sp1-167D)的DNA结合域。在该置换研究中,首先使重组Sp1-167D结合DNA,然后,在温育的第二步加入G4N。G4N和Sp1-167D的浓度,温育和凝胶电泳条件在这两项研究中相同(实验部分)。如图13所示,在各种情况下,发现G4N能保持DNA不与Sp1-167D蛋白质相互作用。当只单独测试Sp1的DNA结合域时,如凝胶迁移率变动分析所示,G4N表现出比阻断Sp1不结合增强子还有效的结合的Sp1的置换作用(图13,A,B,D)。我们也检验了被结合的G4N是否能被Sp1-167D置换。在该项研究中,首先通过迁移率变动分析确定G4N对Sp1-167D结合的抑制(图13C,第2和5道)。当用另外的Sp1-167D攻击结合了G4N的模板时,我们发现Sp1-167D/DNA复合物的泳带强度的剂量依赖性的增强(图6C,第6,7道),表明Sp1-167D从模板置换了G4N。
实施例13
G4N对HIV启动子活性的Sp1调节的Tat-反式激活的抑制
如先前报道的,甲基化的NDGA衍生物能够阻断Sp1结合到各种病毒启动子的增强子位点,所述各种病毒启动子包括HIV,HSV的ICP4,HPV的E6/E7基因(37,38,39)。我们通过先前描述的SEAP分析进一步测定了G4N对Cos细胞中HIV启动子活性的Tat-反式激活的作用。先前发现HIV LTR驱动的SEAP表达的基础水平在Cos细胞中几乎检测不到。当用CMV启动子驱动的Tat基因共转染Cos细胞时,SEAP表达增强60倍或更高(37)。先前证明HIV LTR启动子活性的这样的Tat-驱动的反式激活受Sp1调节(37,40)。在G4N存在下,我们发现HIV反式激活的抑制是依赖于剂量的方式(图14)。对于G4N,IC50值的平均值36μM可与3-O-甲基NDGA Mal.4的(IC50 25μM)相比较,并且一定程度上高于四甲基NDGA,M4N的(IC50 11μM)。差异可能是由于影响细胞吸收药物的试验化合物的化学性质。
实施例14
G4N对细胞培养物中SIV-1和HIV-1产生的抑制
HIV-1和SIV都是逆转录病毒,其需要整合到宿主基因组中完成它们的复制。两者都依赖于它们前病毒转录的宿主转录因子。在共同有着几乎相同的转录调节的模式的这两种病毒中,Sp1起着为这样的表达的中心作用。在预计使用SIV感染的猕猴作为测试G4N的抗病毒作用的动物模型中,我们研究并比较了在174×CEM细胞SIV的抑制作用中与H9细胞HIV的抑制作用中G4N的作用。也检查了在这两种细胞系中G4N的细胞毒性。对于SIV抑制研究,107 174×CEM细胞与高滴度的SIVmac239原液在37℃下混合2小时,然后用冷的PBS缓冲液洗涤两次去除没有吸收的病毒。将细胞悬浮液分成等份到三个96孔板的每一个孔中。从新鲜制备的贮存液制备各种浓度的G4N溶液并且分成等份并且每份加给一个96孔板的一列6个孔中。注射后(P.I.)每四天收集培养上清液并且在收集上清液之后,加给该培养物含有适当浓度的新鲜培养基。如图15所示,通过修饰的p27核心抗原捕获ELISA测定病毒产生。使用5μM以上浓度的G4N检测不到SIV产生。G4N浓度低于2.5μM时,与不存在药物的病毒产生相比较,在感染后第4和8天的培养物的培养上清液中检测到SIV产生(图15)。如MTT测试测定的(41),G4N(250μM或更低)对没有感染的174×CEM细胞没有毒性作用。对于H9细胞中G4N对HIV-1的抑制的研究也进行了相似实验。H9细胞以1×105/ml传代培养并且用HIV-1的AZT抗性株(HIV-1RTMF)感染。感染后2小时加入不同浓度的G4N。每四天更换新鲜培养基。在9天实验期间小心监测G4N存在下的细胞生长。通过p23核心抗原捕获ELISA测定病毒产生。如图所示(图16),80μM的G4N浓度完全抑制H9细胞中的HIV复制。发现抑制HIV-1RTMF的G4N的IC50是12μM。同样,在测试范围内(并且低于250μM)没有检测到对没有感染的H9细胞的毒性。
为了方便起见,下面列出这里引用的参考文献,并且它们引入本文作参考。1.Nurse,P.,调节M-期的起始的普遍的控制机理(Universal ControlMechanism Regulating Onset of M-Phase),自然(Nature),344,503-508.(1990)2.Fang,F.和J.W.Newport,高级真核生物中不同cdc2蛋白质控制G1-S和G2-M转换的证据(Evidence That the G1-S and G2-Mtransitions AreControlled by Different cdc2 Proteins in High Eukaryotes),细胞(Cell),66,731-742(1991)3.Dalton,S.,人cdc2基因的细胞周期调节(Cell Cycle Regulation of theHuman cdc2 Gene),The EMBO Journal,11,1797-1804(1992)4.Morgan,D.O.,CDK调节的原理(Principles of CDK Regulation),自然(Nature),374,131-134(1995)5.Murray,A.W.,创造性阻断:细胞周期关卡和反馈控制(Creative Blocks:Cell-cycle Checkpoins and Feedback Controls),自然(Nature),359,599-604(1992)6.Kao,G.D.,M.W.G.,和R.J.Muschel,Hela细胞中放射-诱导的G(2)停滞期间从细胞核排除p34(Cdc2)激酶活性(p34(Cdc2)Kinase Activity IsExcluded From the Nucleus During the Radiation-induced G(2)Arrest inHela Cells),生物化学杂志(J.Biol.Chem.),274,34779-34784(1999)7.Hwu,J.R.,Tseng,W.N.,Gnabre,J.,Giza,P.和Huang,R.C.C.(1998),甲基化去甲二氢愈创木酸(I)的抗病毒活性,合成,结构鉴定和抑制Tat调节的HIV反式激活(Antiviral Activities of MethlatedNordihydroguaiaretic Acids(I),Synthesis,Structure Identification andInhibition of Tat Regulated HIV Transactivation),药物化学杂志(J.Med.Chem.)41,2994-30008.Gnabre,J.N.,等,DNA-序列选择性植物木脂体对1型人免疫缺陷病毒转录和复制的抑制(Inhibition of Human Immunodeficiency Virus Type 1Transcription and Replication by Sequence-Selective Plant Lignans),美国国家科学院院刊(Proc.Natl.Acad.Sci.U.S.A.)92,11239-11243(1995)9.Chen,H.,等,甲基化去甲二氢愈创木酸(II)的抗病毒活性,2.突变不敏感转录抑制剂四-O-甲基-NDGA靶向单纯疱疹病毒复制(AntiviralActivities of Methylated Nordihydroguaiaretic Acids.2.Targeting HerpesSimplex Virus replication by the Mutation Insensitive TranscriptionInhibitor Tetra-O-methyl-NDGA.),药物化学杂志(J.Med.Chem.)41:3001-3007(1998).10.Craigol,J.,等,去甲二氢愈创木酸植物木脂体衍生物对人16型乳头瘤病毒基因表达的抑制(Inhibition of Human Papillomavirus Type 16 GeneExpression by Nordihydroguaiaretic Acids Plant Lignan Derivatives),抗病毒研究(Antiviral Research),47,19-28(2000)11.Baba,M.(1997)微型综述,作为抑制HIV-1的可供选择的靶物的细胞因子(Mini Review.Cellular Factors as Alternative Targets for inhibition ofHIV-1)。抗病毒研究(Antiviral Res.)33,144 i-1452.12.Gnabre,J.N.,Brady,J.N.,Clanton,D.J.,Ito,Y.,Dittmer,J.,Bates,R.B.和Huang,R.C.(1995),DNA-序列选择性植物木脂体对1型人免疫缺陷病毒转录和复制的抑制(Inhibition of Human Immunodeficiency VirusType 1 Transcription and Replication by Sequence-Selective Plant Lignans),美国国家科学院院刊(Proe.Natl.Acad.Sci.U.S.A.)92,11239-1124313.Gnabre,J.N.,Ito,Y.,Ma.Y.和Huang,R.C.(1996)通过逆流层析从Larrea Tridentata分离抗-HIV-1木脂体(Isolation of Anti-HIV-1 Lignansfrom Larrea Tridentata by Counter-Current Chromatography).层析杂志(J.Chromatogr.)A 719,353。14.Gnabre,J.N.,Huang,R.C.,Bates,R.B.,Bums,J.J.,Calder,S.,Malcomson,M.E.和McClure,K.J.(1995),来自Larrea Tridentata的抗-HIV木脂体的表征(Characterization of Anti-HIV Lignans from LarreaTridentata),四面体(Tetrahedron)51,12203。15.Hwu,J.R.,Tseng,W.N.,Gnabre,J.,Giza,P.和Huang,R.C.C.(1998),甲基化去甲二氢愈创木酸(I)的抗病毒活性,合成,结构鉴定和抑制Tat调节的HIV反式激活(Antiviral Activities of MethlatedNordihydroguaiaretic Acids(I),Synthesis,Strycture Identification and Inhibition of Tat Regulated HIV Transactivation),药物化学杂志(J.Med.Chem.)41,2994-3000。16.Chen,H.,Teng,L.,Li,J-N.,Park,R.,Mold,D.E.,Gnabre,J.,Hwu,J.R.,Tseng,W. N.和Huang,R.C.C.(1998)甲基化去甲二氢愈创木酸的抗病毒活性(II),突变不敏感转录抑制剂四-O-甲基-NDGA靶向单纯疱疹病毒复制(Antiviral Activities of Methylated Nordihydroguaiaretic Acids(II)Targeting Herpes Simplex Virus replication by the Mutation InsensitiveTranscription Inhibitor Tetra-O-methyl-NDGA.),药物化学杂志(J.Med.Chem.)41:3001-3007。17.Honess,R.W.,和Roizman,B.(1988)疱疹病毒大分子合成的调节.1.三组病毒蛋白质的合成的级联调节(Regulation fo Herpes VirusMacromolecular Synthesis.1.Cascade Regulation of Synthesis of ThreeGroups of Viral Proteins).病毒杂志(J.Virol.)1974.14,8。18.Courey,A.J.,和Tjian.R.(1988)体内Sp1分析表明多个转录区域。包括新的富含谷氨酰胺活性基序(Analysis of Sp1 in vivo RevealsMultiple Transcription Domains.Including a Novel Glutamine-richActivation Motif).细胞(Cell)55,887。19.一些Sp1-调节的细胞基因:Sartorelli,V.;Webster,K.A.;Kedes,L.心脏α-肌动蛋白基因的肌肉特异性表达需要myoD1,CarG-hox结合因子和Sp1(Muscle-specif~c expresison of the cardiac alpha-actin generequires myoD1,CarG-hox binding factor and Sp1)。基因发展(Gene Dev.)1990,4,1811。Dailey,L.;Roberts,S.B.;Heintz,N.组蛋白H4基因特异性转录因子H4TF-1和H4TF-2的纯化(Purification of the histone H4gene-specific transcription factors,H4TF-1 and H4TF-2).基因发展(GeneDev.)1988,2,1700。Means,A.L.;Famham,P.J.起始位点HIP-I结合定位转录起始形式二氢叶酸还原酶启动子(Transcription initiation formthe dihydrogolate reductase promoter is positioned by HIP-I binding at theinitiation site).分子细胞生物学(Mol Cell Biol.)1990,10,653。Abravaya,K.;Phillips,B.;Morimoto,R.I.体内足迹蛋白法检查热激诱导的相互作用和热激转录因子和人hsp70启动子(Heat shock-induced interaction so heat shock transcrlption tactor and human hsp70 promoter examined by invivo footprinting).分子细胞生物学(Mol.Cell Biol.)1991,11,586。Leask,A.;Rosenberg,M.;Vassar,R.;Fuchs,E.人上皮角蛋白基因的调节:角质细胞特异性转录中涉及的序列和核因子(Regulation fo a humanepidermal keratin gene:Sequences and nuclear factors involved inkeratinocyte-specific transcription).基因发展(Gene Dev.)1990,4,1985。Desjardins,E.;Hay,N.锌指蛋白结合的重复的CT元件控制两个主要人cmyc启动子的绝对和相对活性(Repeated CT elements bound by zincfinger proteins control the absolute and relative activities of the twoprincipal huyman cmyc promoter).分子细胞生物学(Mol.Cell Biol.)1993,13,5710。Sanchez,H.B.;Yieh,L.;Osbome,T.F.甾醇调节元件-结合蛋白质和Sp1在低密度脂蛋白受体基因的甾醇调节中的共同作用(Cooperation by sterol regulatory element-binding proteins and Sp1 insterol regulation of low-density lipoprotein receptor gene).生物化学杂志(J.Biol Chem.)1995,270,1161。Lemaigre,F.P.;Lafontaine,D.A.;Courtois,S.J.;Durviaux,S.M.;Rousseau,G.G.Sp1能够从其远的结合位点置换GHF-1并且刺激转录形成生长激素基因启动子(Sp1 candisplace GHF-1 from its distal binding site and stimulate transcription formgrowth hormone gene promoter)。分子细胞生物学(Mol.l Cell.Biol.)1990,10,1811。20.Phelps,W.C.,Yee,C.L.,Munger,K.和Howley,P.M.(1988)人16型乳头瘤病毒E7基因编码类似于腺病毒E/A的反式激活和转化功能(TheHuman Papilloma Virus Type 16 E7 Gene Encodes Transactivation andTransformation Functions Similar to Those of Adenovirus E/A)。细胞(Cell)53,539-547。21.Greenstone,H.L.Nieland,J.D.,DeVisser,K.E.,DeBruijn,M.L.,Kimbauer,R.,Roden,R.B.,Lowy,D.R.,Kast,W.M.和Schiller,J.T.(1998)在HPV16肿瘤模型中嵌合乳头瘤病毒样颗粒激发抗E7致癌蛋白的抗肿瘤免疫性(Chimeric Papillomavirus Virus-Like particles ElicitAntitumor Immunity Against the E7 Oncoprotein in an HPV 16 Tumor Model).PNAS 95,1800-1805。22.Feltkamp,M.C.,Vreugdenhil,G.R.,Vierboom,M.P.,Ras,E.,Van derBurg,S.H.,Schegget,J.Ter,Melief,C.J.M.和Kast,W.M.(1995)作为合成肽提供抗显性表位产生的CTL根除人16型乳头瘤病毒诱导的肿瘤(CTL Raised Against a Subdominant Epitope Offered as a SyntheticPeptide Eradicate Human Papillonavirus Type 16-Induced Tumors).欧洲免疫学杂志(European Journal of Immunology)25,2638-2642。23.Feltkamp,M.C.,Smits,H.L.,Vierboom,M.P.,Minaar,R.P.,B.M.Drijfhout,J.W.,Schegget,J.,Melief,C.和Kast,W.M.(1993)用含有细胞毒性T淋巴细胞表位的肽接种免疫预防人16型乳头瘤病毒转化细胞诱导的肿瘤(Vaccination with Cytotoxic T Lymphocyte Epitope-Containing Peptide Protects Against a Tumor Induced by HumanPapillomavirus Type 16-Transformed Cells).欧洲免疫学杂志Eur.JImmunol.23,2242-2249。24.Jacob,S.W.和Herschler(1986)DMSO药理学(Pharmacology ofDMSO)。科学出版社(Academic Press,Inc.)。25.Jack,C.,和Torre,de la(1983)二甲亚砜的生物学作用和医学应用(Biological Actions and Medical Applications of Dimethyl Sulfoxide).NewYork Academy of Sciences,New York,N.Y.26.Spruance,S.L.,McKeough.M.B.和Cardinal,J.R.(1983)用于局部抗病毒化学治疗的作为赋形剂的二甲亚砜(Dimethyl Sulfoxide as a vehiclefor topical antiviral chemotherapy).Ann.N.Y.Acad.Sci.411,28-33.27.Biswal,S.S.,等,谷胱甘肽氧化和线粒体去极化作为脂肪氧化酶-缺陷FI5.12细胞中去甲二氢愈创木酸-诱导的编程性细胞死亡的机理(Glutathione Oxidation and Mitochondrial Depolarization as Mechanisms ofNordihydroguaiaretic Acid-induced Apoptosis in Lipoxygenase-deficientFI5.12Cell),毒理学(Toxicol.Sci.)53,77-83(2000)。28.Schegg,K.M.和W.J.Welch,去甲二氢愈创木酸和相关的木脂体对甲酰基四氢叶酸合成酶和Carbodylesterase的作用(The Effect ofNordihydroguaiaretic Acid and Related Lignans on Formyltetrahydrofolate Synthetase and Carbodylesterase),生物化学.生物物理学报(Biochim.Biophys.Acta.)788,167-180(1984)。29.Feltkamp,M.C.W.,等,用含有细胞毒性T淋巴细胞表位的肽接种免疫预防人16型乳头瘤病毒转化的细胞诱导的肿瘤(Vaccination withCytotoxic T Lymphocyte Epitope-Containing Peptide Protects Against aTumor Induced by Human Papillomavirus Type 16-Transformed Cells).欧洲免疫学杂志(Eur.J Immunol.)23,2242-2249.(1993)。30.Lin,K.,等,用增强肿瘤抗原的II类主要组织相容性呈递的新疫苗治疗确立的肿瘤(Treatment of Established Tumors With a Novel Vaccine ThatEnhances Major Histocompatiblity Class II Presentation of Tumor Antigen),癌症研究(Cancer Res.,)56,21-26(1996)。31.Foley,G.E.,等,急性白血病儿童外周血中人原淋巴细胞的连续培养(Continuous Culture of Human Lymphoblasts From Peripheral Blood of aChild With Acute Leukemia),癌症(Cancer),18,522-529(1965)。32.Shiebel,E.,γ-微管蛋白复合物:结合到中心体,调节和微管成核作用(Gamma-Tubulin Complexes:Binding to the Centrosome,Regulation andMicrotubule Nucleation)细胞生物学最新观点(Current Opinion CelluarBiology)12,113-118(2000)。33.Marsden,M.P.F.,Laemmli,U.K.,中期染色体结构:径向状环模式的证据(Metaphase Chromosome Structure:Evidence for a Radial LoopModel)细胞(Cell)17,849-858(1979)。34.Taylor,W.R.,等,响应于p53过量表达的G2停滞的机理(Mechanisms ofG2 Arrest in Respanse to Overexpreeion of p53)细胞分子生物学(Molecular Biology of the Cell)10,3607-3622(1999)。35.Innocente,S.A.,等,p53调节通过细胞周期蛋白B1的G2关卡(p53Regulates a G2 Checkpoin Through Cyclin B1)美国国家科学院院刊(Proc.Natl.Acad.Sci.U.S.A.)96,2147-2152(1999)。36.sjottem,E.,;Anderson,C.;Johansen,T.Sp1家族转录因子诱导的DNA弯曲的结构和功能分析(Structural and Functional Analyses of DNABending Induced by Sp1 Family Transcription Factors),分子生物学杂志 (J.Mol.Bio.)1997,267,490-504。37.Gnabre,J.N.,Brady,J.N.,Clanton,D.J.,Ito,Y.,Dittmer,J.,Bates,R.B.和Huang,R.C.,DNA-序列选择性植物木脂体对1型人免疫缺陷病毒转录和复制的抑制(Inhibition of Human Immunodeficiency Virus Type 1Transcription and Replication by Sequence-Selective Plant Lignans),美国国家科学院院刊(Proc.Natl.Acad.Sci.U.S.A.)(1995)92,11239-1124338.Chen,H.,Teng,L.,Li,J-N.,Park,R.,Mold,D.E.,Gnabre,J.,HWu,J.R.,Tseng,W.N.和Huang,R.C.C.甲基化去甲二氢愈创木酸的抗病毒活性,2.通过突变不敏感转录抑制剂四-O-甲基-NDGA靶向单纯疱疹病毒复制(Antiviral Activities of Methylated Nordihydroguaiaretic Acids(II)Targeting Herpes Simplex Virus replication by the Mutation InsensitiveTranscription Inhibitor Tetra-O-methyl-NDGA.),药物化学杂志(J.Med.Chem.)(1998),41:3001-3007。39.Craigol,J.;Callaham,M.;Huang,R.C.;Delucia,A.L.,去甲二氢愈创木酸植物木脂体衍生物对人16型乳头瘤病毒基因表达的抑制(Inhibitionof Human Papillomavirus Type Gene Expression by NordihydroguaiareticAcids Plant Lignan Derivatives),抗病毒研究(Antiviral Research),2000,印刷中。40.Hwu,J.R.,Tseng,W.N.,Gnabre,J.,Giza,P.和Huang,R.C.C.,甲基化去甲二氢愈创木酸的抗病毒活性,1.合成,结构鉴定和抑制Tat调节的HIV反式激活(Antiviral Activities of Methlated Nordihydroguaiaretic Acids.1.Synthesis,Structure Identification and Inhibition of Tat Regulated HIVTransactivation),药物化学杂志(J.Med.Chem.)(1998),41(16),2994-3000。41.Weislow,O.S.;Kiser,R.;Fine,D.L.;Bader,J.;Shoemaker,R.H.;Boyd,M.R.HIV-1细胞病变作用的新的可溶性-Formazan分析:用于爱滋病-抗病毒活性的合成和天然产物的高通量筛选的应用(New Soluble-Formazan Assay for HIV-1 Cytopathic Effects:Application to High-FluxScreening of Synthetic and Natural Products for Aids-Antiviral Activity).国家癌症研究所杂志(J.Natl.Cancer Inst.),1989,81(8),557-586。
Claims (30)
2.权利要求1的方法,其中所述肿瘤是HPV诱导的。
3.权利要求2的方法,其中所述肿瘤是子宫颈癌和口腔癌。
4.权利要求1的方法,其中所述肿瘤存在于哺乳动物中。
5.权利要求4的方法,其中所述肿瘤是恶性的。
6.权利要求5的方法,其中所述肿瘤选自鳞状细胞癌,腺癌和成神经管细胞瘤。
7.权利要求4的方法,其中所述肿瘤是良性的。
8.权利要求7的方法,其中所述肿瘤选自乳头瘤,畸胎瘤和腺瘤。
9.权利要求4的方法,其中所述肿瘤是实体肿瘤。
10.权利要求4的方法,其中所述哺乳动物是人。
11.权利要求4的方法,其中所述肿瘤自转化的细胞产生。
12.权利要求11的方法,其中所述细胞是C3细胞。
13.权利要求1的方法,其中所述去甲二氢愈创木酸衍生物与至少一种药学可接受赋形剂或载体一起施用。
14.权利要求13的方法,其中所述赋形剂或载体是二甲亚砜(DMSO)。
15.权利要求1的方法,其中所述衍生物是四-O-甲基去甲二氢愈创木酸(M4N)或四甘氨酰基去甲二氢愈创木酸(G4N)。
16.权利要求1的方法,其中所述四-O-甲基去甲二氢愈创木酸与至少一种药学可接受赋形剂或载体一起施用。
17.一种药物组合物,其含有四-O-甲基去甲二氢愈创木酸和至少一种药学可接受赋形剂或载体。
18.权利要求17的组合物,其中所述赋形剂或载体是DMSO。
19.下式的化合物
其中R1,R2,R3和R4是相同的并且代表一个氨基酸残基。
20.如示意图1所示的合成根据权利要求19的化合物的方法。
21.合成根据权利要求19的化合物的方法,包括步骤:
在二氯甲烷溶液中化合NDGA和一种氨基酸的N,N-二甲基衍生物;
向所述溶液中加入DCC和DMAP;
加入丙酮并且向所述溶液鼓泡通入过量氯化氢来获得所述化合物。
22.一种药物组合物,其含有四甘氨酰基去甲二氢愈创木酸和至少一种药学可接受赋形剂或载体。
23.权利要求22的组合物,其中所述赋形剂或载体是生理盐水。
24.四甘氨酰基去甲二氢愈创木酸。
26.权利要求25的方法,其中所述细胞是动物细胞。
27.权利要求26的方法,其中所述细胞是哺乳动物细胞。
28.权利要求27的方法,其中所述细胞是人细胞。
29.根据权利要求19的化合物作为药物的用途。
30.根据权利要求19的化合物治疗肿瘤的用途。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6365787B1 (en) * | 1994-09-30 | 2002-04-02 | The Johns Hopkins University | Compounds for the suppression of HIV TAT transactivation |
US6608108B2 (en) * | 1999-10-15 | 2003-08-19 | Johns Hopkins University | Method for treatment of tumors using nordihydroguaiaretic acid derivatives |
US6214874B1 (en) * | 1999-10-15 | 2001-04-10 | John Hopkins University | Treatment of HPV induced cancer using in situ application of two nordihydroguiaretic acid derivatives, tetramethyl NDGA M4N and tetraglycinal NDGA G4N |
US20060073530A1 (en) * | 2001-08-15 | 2006-04-06 | Olaf Schneewind | Methods and compositions involving sortase B |
WO2004112695A2 (en) * | 2003-05-20 | 2004-12-29 | Erimos Pharmaceutical Llc | Methods and compositions for delivery of catecholic butanes for treatment of obesity |
US7728036B2 (en) * | 2003-05-20 | 2010-06-01 | Erimos Pharmaceuticals, Llc | Methods for delivery of catecholic butanes for treatment of tumors |
US20060141029A1 (en) * | 2003-05-20 | 2006-06-29 | Erimos Pharmaceuticals Llc | Methods and compositions for delivery of catecholic butanes for treatment of diseases |
CN100440843C (zh) * | 2004-05-12 | 2008-12-03 | 华为技术有限公司 | 一种环网及其业务实现方法 |
EP1748767B1 (en) | 2004-05-28 | 2011-12-28 | Unigen, Inc. | 1-(3-methyl-2,4-dimethoxyphenyl)-3-(2',4'-dihydroxyphenyl)-propane as a potent tyrosinase inhibitor |
WO2006014669A2 (en) * | 2004-07-20 | 2006-02-09 | Erimos Pharmaceuticals Llc | Methods and compositions for treatment of intraepithelial neoplasia |
US8440648B2 (en) * | 2004-07-20 | 2013-05-14 | Erimos Pharmaceuticals Llc | Methods and compositions for treatment of intraepithelial neoplasia |
CA2583336A1 (en) * | 2004-10-06 | 2006-04-20 | Johns Hopkins University | Use of nordihydroguaiaretic acid derivatives in the treatment of drug resistant cancer, viral and microbial infection |
EP1848278B1 (en) * | 2005-01-27 | 2016-09-07 | Erimos Pharmaceuticals LLC | Oral formulations for delivery of catecholic butanes including ndga compounds |
WO2007101111A2 (en) * | 2006-02-23 | 2007-09-07 | Erimos Pharmaceuticals Llc | Methods of treating influenza viral infections |
ES2476249T3 (es) * | 2006-10-02 | 2014-07-14 | Erimos Pharmaceuticals Llc | Derivados del NDGA tetrasustituidos por medio de enlaces éter y enlaces carbamatos y sus síntesis y usos farmacéuticos |
US9067875B2 (en) | 2006-10-02 | 2015-06-30 | Erimos Pharmaceuticals Llc | Tetra-substituted NDGA derivatives via ether bonds and carbamate bonds and their synthesis and pharmaceutical use |
WO2008088806A1 (en) * | 2007-01-16 | 2008-07-24 | Johns Hopkins University | Combinational paradigm combating hiv, hiv/hsv, or hiv/hpv infections in humans using small molecular weight compounds from plants |
HUE029409T2 (en) * | 2007-05-11 | 2017-02-28 | Convatec Technologies Inc | Osmotic device |
BRPI0916188A2 (pt) | 2008-07-21 | 2017-08-29 | Unigen Inc | Série de composto para clareamento da pele (iluminação) |
KR20100011963A (ko) * | 2008-07-25 | 2010-02-03 | 국립암센터 | Ndga를 포함하는 트란스글루타미나제 억제용 조성물 |
US20100093872A1 (en) * | 2008-10-15 | 2010-04-15 | Erimos Pharmaceuticals Llc | Stable aqueous formulations of water insoluble or poorly soluble drugs |
WO2010054264A1 (en) * | 2008-11-07 | 2010-05-14 | Triact Therapeutics, Inc. | Use of catecholic butane derivatives in cancer therapy |
KR101976642B1 (ko) | 2011-03-24 | 2019-05-09 | 유니젠, 인크. | 디아릴프로판의 제조를 위한 화합물 및 방법 |
US9084779B2 (en) | 2011-05-31 | 2015-07-21 | The Johns Hopkins University | Conjugates of nitroimidazoles and their use as chemotherapeutic agents |
US9456995B2 (en) | 2012-07-18 | 2016-10-04 | The Johns Hopkins University | Methods for inhibition of BNIP3 and prevention and treatment of ischemia reperfusion injury by tetra-O-methyl nordihydroguaiaretic acid |
EP2961412A4 (en) | 2013-02-26 | 2016-11-09 | Triact Therapeutics Inc | CANCER THERAPY |
CA2923667A1 (en) * | 2013-09-09 | 2015-03-12 | Triact Therapeutics, Inc. | Cancer therapy |
MX2017002227A (es) | 2017-02-17 | 2018-08-16 | Promotora Tecnica Ind S A De C V | Composicion mejorada a base de acido norhidroguayaretico. |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5276060A (en) | 1979-06-19 | 1994-01-04 | Block/Chemex, G.P. | Methods of treating tumors with compositions of catecholic butanes |
US5008294A (en) * | 1985-02-11 | 1991-04-16 | Chemex Pharmaceuticals, Inc. | Methods of treating tumors with compositions of catecholic butanes |
DE3009542A1 (de) * | 1980-03-13 | 1981-09-24 | Henkel KGaA, 4000 Düsseldorf | Desodorierende kosmetische zusammensetzungen |
US4774229A (en) | 1982-04-05 | 1988-09-27 | Chemex Pharmaceuticals, Inc. | Modification of plant extracts from zygophyllaceae and pharmaceutical use therefor |
US4880637A (en) * | 1985-02-11 | 1989-11-14 | Chemex Pharmaceuticals, Inc. | Compositions of catecholic butanes with zinc |
US5559149A (en) * | 1990-01-29 | 1996-09-24 | Johnson & Johnson Consumer Products, Inc. | Skin care compositions containing retinoids |
US5646186A (en) * | 1994-05-17 | 1997-07-08 | Johnson & Johnson Consumer Products, Inc. | Retinoid composition |
US6071949A (en) | 1995-03-14 | 2000-06-06 | The United States Of America As Represented By The Department Of Health And Human Services | Use of lipoxygenase inhibitors as anti-cancer therapeutic and intervention agents |
US5837252A (en) | 1996-07-01 | 1998-11-17 | Larreacorp, Ltd. | Nontoxic extract of Larrea tridentata and method of making same |
US5827898A (en) * | 1996-10-07 | 1998-10-27 | Shaman Pharmaceuticals, Inc. | Use of bisphenolic compounds to treat type II diabetes |
US6214874B1 (en) * | 1999-10-15 | 2001-04-10 | John Hopkins University | Treatment of HPV induced cancer using in situ application of two nordihydroguiaretic acid derivatives, tetramethyl NDGA M4N and tetraglycinal NDGA G4N |
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SG144712A1 (en) | 2008-08-28 |
CA2387873C (en) | 2008-12-30 |
WO2001028494A2 (en) | 2001-04-26 |
ATE350030T1 (de) | 2007-01-15 |
US6214874B1 (en) | 2001-04-10 |
CA2387873A1 (en) | 2001-04-26 |
US20050267208A1 (en) | 2005-12-01 |
JP4062664B2 (ja) | 2008-03-19 |
WO2001028494A9 (en) | 2002-08-01 |
EP1231914A2 (en) | 2002-08-21 |
AU1207501A (en) | 2001-04-30 |
US6958411B2 (en) | 2005-10-25 |
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