CN1213406A - 抑制甲硫腺苷磷酸化酶缺陷细胞的腺苷酸琥珀酸合成酶活性的方法 - Google Patents
抑制甲硫腺苷磷酸化酶缺陷细胞的腺苷酸琥珀酸合成酶活性的方法 Download PDFInfo
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Abstract
本发明提供了一种可耗尽哺乳动物细胞中的腺苷－5′－磷酸(AMP)的体内方法,以用于治疗某些癌症。根据此方法,从宿主中获取一组细胞并检测其甲硫腺苷磷酸化酶(MTAse)活性。MTAse将甲硫腺苷分解代谢产生腺嘌呤用于内源性补救胞内AMP库。优选的检测MTAse活性丧失的方法是一种杂交技术,它用以检测编码该活性的基因的纯合缺失,该活性的丧失可与治疗有效量的抑制将肌苷5′－磷酸转化为AMP的腺苷酸琥珀酸合成酶活性的药剂一起,从而耗尽肿瘤细胞用于AMP从头产生的底物。L－亚硝基羟基丙氨酸是用于本发明方法中的优选ASS抑制剂。此图是基因组MTAse核苷酸序列(序列1),其中外显子下面划线。
Description
与相关申请的相互参照
这是1993年12月29日归档，现在还待定的美国专利申请编号为No.08/176,855的延续部分。
本发明的背景
本发明涉及用于化学治疗癌症中的药物和方法。更具体地，本发明涉及鉴别不能代谢甲硫腺苷磷酸化酶产生腺嘌呤用于腺嘌呤核苷酸补救合成的癌细胞，以及使用L-亚硝基羟基丙氨酸抑制此类癌细胞中腺苷-5＇-一磷酸(AMP)的从头合成。
本发明的历史
甲硫腺苷(MTA)在健康哺乳动物细胞中被甲硫腺苷磷酸化酶(MTAse)分解为腺嘌呤和甲硫核糖-1-磷酸，后者是代谢合成甲硫氨酸的一种底物。腺嘌呤补救进入腺苷5＇-一磷酸(AMP)的细胞库中，细胞从中可衍生出用作代谢能的腺苷5＇-三磷酸(ATP)和用于DNA合成的2＇-脱氧腺苷-5＇-三磷酸(dATP)。
基于早期的体外研究，一种细菌性抗生素亚硝基羟基丙氨酸的L异构体(从丙氨菌素链霉菌(Streptomyces alanosinicus)中获得；以下称为“L-亚硝基羟基丙氨酸”)看来有希望用作一种抗病毒和抗肿瘤药物。特别是，据信L-亚硝基羟基丙氨酸抑制腺苷酸琥珀酸合成酶(ASS)将肌苷5＇-磷酸(IMP)转化成AMP，从而耗尽靶细胞的AMP和ATP(在缺乏腺嘌呤的条件下)。然而，L-亚硝基羟基丙氨酸对癌症病人的治疗效应的临床研究是令人失望的(参看，例如，收集在Tyagi和Cooney，药理学、化学治疗进展(Adv.Pharmacol.Chemotherapy,20:69-120,1984中的资料[迄今为止的关于L-亚硝基羟基丙氨酸在治疗人类癌症中的效用的结果“几乎未提供有意义的进展”]；Creagan，等，癌(Cancer),52:615-618,1993[Ⅱ期研究的总体效应比率只有4％]；Creagan，等，美国临床肿瘤学期刊(Am.J.Clin.Oncol.),7:543-544,1984[黑素瘤患者的Ⅱ期研究；几乎观察不到治疗效应]；VouHoff等，新药研究(Invest.New Drugs),9:87-88,1991[在乳腺癌病人中未观察到真实效应])。逐渐地，将L-亚硝基羟基丙氨酸用于治疗癌症的所有临床试验都被放弃了。
另一个已知ASS活性的抑制剂是N-羟-N甲酰甘氨酸。但是，据信N-羟-N-甲酰甘氨酸对人的毒性比L-亚硝基羟基丙氨酸强的多。此外，其它能够阻碍IMP合成(从而理论上消除作为AMP产生的一种来源的IMP)的嘌呤从头合成的抑制剂(例如氨甲蝶呤，6-巯基嘌呤，6-硫代鸟嘌呤和双脱吖四氢叶酸(dideazatetrahydrofolate))的活性在体内被在原生质中富含的可用作产生IMP的底物的次黄嘌呤的补救作用阻止。因而，迄今为止，用来在体内阻碍细胞内产生AMP的腺嘌呤代谢途径的试剂的表现是相当令人沮丧的。
然而，随着具有足够灵敏度的鉴定在某些人癌细胞中编码MTAse的基因的纯合缺失的检测技术的发展(参看，一般指定为父专利的美国专利申请编号No.08/176,855)，用L-亚硝基羟基丙氨酸进行临床实验治疗的肿瘤能产生MTAse，因而尽管有从IMP产生AMP的抑制，它还能提供足够的腺嘌呤以维持AMP库。本发明从而提供了一种鉴定缺失MTAse的细胞的方法并通过耗尽其AMP处理这类细胞。
本发明的总结
已经发现在体内与治疗有效剂量的诸如L-亚硝基羟基丙氨酸等AMP从头合成的抑制剂接触可选择性杀死已缺失了编码MTAse蛋白的基因因而不能代谢MTA以产生腺嘌呤的细胞(“MTAse缺陷细胞”)。因而，虽然L-亚硝基羟基丙氨酸并非对所有癌细胞都有效治疗，但它对MTAse缺陷细胞是能够有效治疗的。
一方面，通过提供确定细胞是否缺少MTAse蛋白的检测，本发明提供了一种确定特定癌细胞是否为MTAse缺陷的方法。在这一方面使用的优选检测方法是一种检定细胞中编码MTAse蛋白基因的纯合缺失的方法。
另一方面，本发明提供了一种通过将MTAse缺陷细胞与治疗有效量的能抑制ASS活性的嘌呤从头合成抑制剂，优选地是L-亚硝基羟基丙氨酸相接触来治疗MTAse缺陷癌症的方法。本发明的ASS抑制试剂可使用任何临床可接受方法给药，但优选地通过在低于最大可忍受剂量浓度下连续输液，以延长所需的抑制活性以及减小对病人组织的毒性。
同时提供了用于本发明的方法中的试剂盒，包括用于本发明进行MTAse缺陷检测的试剂以及一种ASS抑制剂，优选地是L-亚硝基羟基丙氨酸和/或其活性代谢物，L-亚硝基羟基丙氨酰AICOR的药物组合物。
附图的简要描述
图1是基因组MTAse的核苷酸序列(序列1)，其中的外显子下面划线。
图2是连续输液L-亚硝基羟基丙氨酸对裸鼠中建立的人的具MTAse能力和MTAse缺陷的非小细胞肺癌异种移植肿瘤的体内效应曲线。
图3是连续输液L-亚硝基羟基丙氨酸对裸鼠中建立的人的MTAse缺陷急性淋巴母细胞白血病异种移植肿瘤的体内效应曲线。
图4和5(A-B)是在用L-亚硝基羟基丙氨酸处理后向具MTAse能力或MTAse缺陷细胞系中使用不同的外源MTAse底物的效应曲线。
图6是胞内产生AMP的代谢途径的示意图。
图7是每天注射L-亚硝基羟基丙氨酸对裸鼠中建立的人的MTAse缺陷非小细胞肺癌异种移植肿瘤的体内效应曲线。
优选实施例的描述Ⅰ．胞内产生AMP的代谢途径
为了助于对本发明的理解，图6中提供了描述产生AMP的胞内代谢途径的示意图。总体来说，主要有三种用于胞内AMP产生的底物来源。第一种是MTAse催化甲硫腺苷产生腺嘌呤。此途径在MTAse缺陷细胞中被阻断。
第二种是通过ASS或腺苷酸琥珀酸裂解酶(ASL)活性将IMP转化为AMP。现在没有已知的ASL活性抑制剂。但是，只要丧失ASS活性，IMPAMP转化可基本消除。
第三种是次黄嘌呤补救成为AMP。然而，由于IMP AMP转化发生在次黄嘌呤补救的远端，对ASS催化IMP AMP的抑制阻断了次黄嘌呤补救途径。Ⅱ．用于确定取自哺乳动物宿主的可检测样品中的肿瘤细胞是否为MTAse缺陷的方法
A．用干确定MTAse缺陷细胞的多聚核苷酸
用来确定特定种群细胞是否为MTAse缺陷的优选方法是通过杂交检测测定细胞中编码MTAse基因的纯合缺失。只要存在合适的探针，依赖于核酸杂交的筛选方法可在任何生物中检测出任何多聚核苷酸序列(以及，推论及由这些多聚核苷酸序列编码的任何蛋白)。
在同时待定的，一般指定为美国专利申请编号No.08/176,855(1993年12月29日归档)中有适用于本发明的杂交技术的完整描述以及编码MTAse基因的描述，其公开内容通过此文献与可能的其它补充并入本专利。为了易于查询，将编码MTAse基因的多聚核苷酸序列描述于附录序列表中的序列1以及图1中，其中基因的编码区用下划线标出。基因组MTAse多聚核苷酸位于第9号染色体的p21区。有趣的是，很高比例的具有编码肿瘤抑制因子p16基因纯合缺失的细胞也具有编码MTAse基因的纯合缺失。因此，一种检测后一个基因(编码MTAse)的纯合缺失的替代方法是通过检测前一个基因(编码p16)的纯合缺失来进行。有关的进一步参考，参看一般指定的同时待定的美国专利申请编号No.08/227,800，其公开部分通过文献并于此处。
通过邮寄在1993年12月29日以前将一株含有大鼠MTAse全长基因组DNA的大肠杆菌(E.coli)菌株保藏于美国典型培养物保藏中心(American TypeCulture Collection),Rockville,MD并一致地，共同地，命名为55536,55537,55538,55539和55540。并未表明或试图表明认为此保藏物对实施本发明是必需的。不过，将保藏物保存为任何时期的存活形式是或可能是适用于本公开文的专利法所需的。
为了确定所研究的细胞中的MTAse基因是否纯合缺失，从宿主中获取细胞的可检测样品。例如，样品可含有来自例如宿主组织或肿瘤的体液或细胞。这类样品可使用临床学科中已知的方法获得，例如，可通过活体解剖或外科手术切除得到肿瘤细胞。优选地，细胞基本不含“杂质”；即可能导致检测结果出错的细胞、蛋白和类似组分。例如，当固相肿瘤是可检测细胞样品的来源时，正常的非恶性细胞及在获取生物样品过程中可能从正常细胞中释放的MTAse都认为是杂质。这些杂质可通过常规纯化除去；例如用一种抗MTAse抗体进行亲合层析。
因为本发明涉及检测怀疑为MTAse阴性的细胞样品的该基因的存在与否，所以优选地将样品中的核酸扩增以增强检测方法的灵敏度。这种扩增优选地通过使用聚合酶链式反应(PCR)进行，但在聚合步骤中使用链式反应并非是绝对必要的。
如果样品中存在，样品中要扩增的核酸将含有通常预期为编码MTAse的基因组或野生型DNA(参看序列1)。MTAse编码DNA(此后称为“靶DNA”)据信存在于所有的正常哺乳动物细胞，包括人细胞中。
为了用作对照或寡核苷酸探针和引物的来源，可根据本技术领域已知的方法例如Maniatis等在分子克隆：实验室手册(Molecular Cloning:ALaboratory Manual)(冷泉港实验室(Cold Spring Harbor Loboratory),1982)中所述的方法分离基因组MTAse编码DNA。此处提供了分离人MTAse基因的一个基因组克隆的实施例，其中使用一种MTAse cDNA寡核苷酸探针筛选粘粒基因文库(参看，实施例Ⅲ)。然而，那些本技术领域熟练技术人员会认识到也可使用其它合适的方法获得MTAse编码DNA。
例如，可通过将来自cDNAs的不同mRNA注射到卵母细胞中来筛选据信含有所研究的多聚核苷酸的cDNA文库，提供充分的时间使cDNA基因产物发生表达，并检测期望cDNA表达产物的存在，例如，通过使用所研究的多聚核苷酸编码肽的特异性抗体或通过使用所研究的多聚核苷酸编码肽的重复模式和组织表达模式特征的探针检测。此外，还可使用肽特异性抗体检测至少含有一个抗原决定簇的所研究的肽的表达(例如，MTAse蛋白)间接筛选cDNA文库。这些抗体可以是多克隆的或单克隆的并用于检测指示所研究的cDNA存在的表达产物(作为进一步参考，参看Maniatis等，分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)(冷泉港实验室(ColdSPring Harbor Lab)，纽约，1982)。
本发明中用作对照的多聚核苷酸、探针或引物也可使用本技术领域熟知的技术和核酸合成仪器进行合成。此处作为参考，参看Ausubel等，现代分子生物学方法(Current Protocols in Molecular Biology)，第2和4章(WileyInterscience,1989)。
B．MTAse编码基因组DNA的扩增及其杂交检测
为了增强本发明杂交检测的灵敏度，优选地将要检测的细胞样品置于适合靶核酸选择性扩增的条件下。优选地，靶核酸是编码MTAse基因的多聚核苷酸部分(即“靶多聚核苷酸”)。扩增靶多聚核苷酸的优选方法是PCR。
PCR是一种酶法合成特定DNA或RNA序列的体外方法，使用与特定核酸序列杂交并位于靶核酸所研究的区段的侧翼区的寡核苷酸引物。一系列重复的模板变性，引物退火和退火引物酶延伸的循环使得由引物5′端限定其末端的特定核酸片段呈指数积累。一个循环中产生的产物(PCR产物)作为模板进入下一循环；从而，每一循环后的靶核酸拷贝大致加倍。
基本的PCR技术描述于Mullis等的美国专利4,683,195和4,683,202中，其公开部分作为进行PCR的常规技术的实施例并于此处。然而，本发明并非要局限于Mullis等的′202专利中所讲的PCR技术的应用。由于Mullis等的技术的发展，许多基于PCR的检测使用改进的技术得以发展。因此，这些本技术领域熟知的改进将不详细描述于此处。然而，为了说明此领域技术的范围，下面描述几种这些改进。
在美国国家科学院院报(Proc.Natl.Acad.Sci.)美国(1990)87:2725-2729(Gilliland等，作者)中描述了一种能提供内部扩增标准的使用与靶核酸在顺序和大小上有差别的竞争模板的PCR技术。另一种使用在序列上而不是大小上有差别的模板进行“竞争性”PCR的技术描述于核酸研究(Nuc.Acids.Res.),21:3469-3472,(1993),(Kohsaka等，作者)。这种技术由于在酶联免疫吸附测定(ELISA)技术用于检测扩增核酸中的应用而成为特别优选的技术。另一种可能用于本发明方法的使用位点特异寡核苷酸检测基因的突变或多态性的非竞争性PCR技术描述于美国国家科学院院报中(Proc.Natl.Acad.Sci.)美国(1990)86:6230-6234(Saiki等，作者)。以上每种技术都具有使用杂交探针的优点，该探针有助于消除在PCR中可能发生的非特异性扩增造成的假阳性结果。
对进一步的背景，那些本技术领域熟练人员可能会参考Innis等，“PCR的最优化(Optimization of PCR＇s),PCR方法：方法和应用指南(PCRProtocols:A Guide to Methods and Applications)(Acad.Press,1990)。该出版物总结了影响所需PCR产物的特异性，忠实性和产率的技术。
选择能与MTAse靶多聚核苷酸任一端(即5′和3′端)的一小段碱基对(即，“侧翼序列”)特异性杂交的寡核苷酸引物(至少一对引物)。那些本技术领域熟练人员不需多余的实验即可根据在附录序列表中序列1和图1中列出的多聚核苷酸序列很容易地选出合适的引物。
对于引物设计，重要的是引物不含互补碱基以防止它们自身杂交。为了消除样品中可能存在的任何杂质的扩增，引物优选地设计为跨越外显子(对于MTAse基因，是在图1中的划线部分)。
上面指出，在此聚合步骤中使用链式反应来充分扩增样品中的核酸并非必需。例如，使用Kohsaka等，Supra中所述的技术，在固相载体支持物上进行聚合步骤，然后与靶多聚核苷酸特异性的探针杂交，所需的也许只是使检测的灵敏度达到靶多聚核苷酸的单一聚合就行了。
一旦完成扩增步骤，检测PCR产物以确定样品中是否有编码MTAse基因的存在。优选地，基本如Kohsaka等Supra中所述，可将双链PCR产物结合于固相，这样可通过变性分离其双链，从而使序列特异性探针与结合的PCR产物的反义链杂交以检测基因。此外，可在加入探针与双链PCR产物杂交之前将PCR产物从反应环境中移出并从扩增混合物中分离。在后一种方法中，在考虑到精选的特定检测方法，依据本技术领域已知的方法从扩增混合物中分离PCR产物；例如，通过凝胶排阻，电泳或亲合层析。
对扩增产物的检测，可以通过使用与可检测标记稳定相连的杂交探针实现。标记是一种能共价吸附于或稳固结合到核酸探针的基质，导致能检测到探针的能力。例如，其水平可是一种放射性同位素，一种酶底物或抑制剂，一种酶，一种不透射线基质(包括胶体金属)，一种荧光物质，一种化学发光分子，含由上述任何标记的脂质体，或特异成对结合物质的一员。一种合适的标记在扩增中将不会丧失其检测灵敏度的能力。
那些检定领域熟练人员会对可用于体外检测的合适的可检测标记非常熟悉。例如，用于体外检测的合适放射性同位素包括3H,125I,131I,32P,14C,35S。使用放射性同位素标记的扩增片段可通过γ计数器直接检定或将扩增片段的Southern印迹与光密度测定法相结合通过放射自显影的光密度测定法检定。合适的化学发光分子的例子是吖啶或鲁米诺。与使用吖啶酯衍生的探针杂交的靶序列通过插层反应保护不被水解。合适的荧光物质的例子是荧光素，藻胆蛋白，稀土螯合物，丹酰或罗丹明。
合适的酶底物或抑制剂的例子是能与辣根过氧化物酶，葡糖氧化酶，葡糖-6-磷酸脱氢酶，β一半乳糖苷酶，丙酮酸激酶或碱性磷酸酶乙酰胆碱酯酶特异性结合的组合物。不透射线基质的例子是胶体金或磁性颗粒。
特异的结合对包括两种不同分子，其中一个分子表面或空穴的某区域可特异地与另一个分子的特定空间和极性结构结合。特异结合对的成员常指配体和受体或配体和抗配体。例如，如果受体是一种抗体，配体则是相应的抗原。其它特异结合对包括激素受体对，酶底物对，生物素-抗生物素蛋白对和糖蛋白-受体对。还包括保留结合特异性的特异结合对的片段和部分，这类片段如免疫球蛋白片段，包括Fab片段及其类似片段。抗体可以是单克隆的或多克隆的。如果特异结合对的一员用作标记，优选的分离步骤为亲合层析。
如果上述检测未能测到扩增产物，则表明样品中细胞的MTAse缺陷。由于正常(即非恶性)细胞中通常可预期存在可检测量的(甚至丢失一等位基因后也是如此)MTAse编码基因，发现细胞没有MTAse编码基因(即具有基因的纯合缺失)即意味着检测的细胞同时缺乏催化活性和非催化活性的MTAse。
然而，如果需要，可使用Seidenfeld等在生化生物物理研究通讯(Biochem.Biophys.Res.Commun.),95:1861-1866(1980)中所述的方法预筛选样品的MTAse催化活性；亦可参看、实施例Ⅰ,infra)。然后使用本发明检测方法测定样品中细胞是否存在MTAse编码基因。为了筛查要检测样品中的细胞污染物，即非恶性细胞，还需测定样品中催化活性或非活性蛋白的存在。这方面替代使用的一种合适的免疫测定(即，代替杂交测定)描述于Nobori等，癌研究(Cancer Res.)53:1098-1101(1991)和待定的1993年12月29日归档的通常指定为美国专利申请编号No.08/177,855中，其公开部分并于此处。
C．MTAse缺陷细胞
使用上述的检测方法，确定下述的人原发肿瘤是MTAse缺陷的。当然此列表是代表的而不是穷举使用所述检测方法可测定为MTAse缺陷的所有癌症类型。·急性淋巴母细胞白血病(大致80％出现率)·神经胶质瘤(大致67％出现率)·非小细胞肺癌(大致18％出现率)·膀胱上皮肿瘤(例如膀胱癌；发病率随肿瘤类型不同而变化)
基于上述资料，感染这些病症的病人具有MTAse缺陷的强烈嫌疑。因而，应当常规地对这类病人的细胞样品进行MTAse缺陷的检测以确定病人是否可从本发明治疗方法受益。
作为临床需要，应当检测从其它癌症病人中取到的细胞样品的MTAse缺陷。作为参考，从患有下列病症的患者中得到的原发癌细胞未发现MTAse缺陷(即，癌症是“具MTAse能的”)：·乳腺癌·结肠癌·头颈癌·黑素瘤·肾癌·成人非淋巴母细胞白血病·某些急性白血病(成人和青年)使用L-亚硝基羟基丙氨酸处理上列具MTAse能的癌症进行了临床实验，未取得明显的成功。
Ⅱ．处理MTAse缺陷细胞的方法
A.L-亚硝基羟基丙氨酸的药理和毒性参数
基本上，在大约24小时内大致75％的L-亚硝基羟基丙氨酸从尿中排泄出，主要是以L-亚硝基羟基丙氨酰-IMP和L-亚硝基羟基丙氨酰-AICOR的核苷形式。人静脉给药后从血浆中的清除是双相的，t1/2α=14分钟和t1/2β=99分钟(其中“t1/2”是半寿期，时间(t)为约数)。
在以往的临床实验中，毒性是有剂量限制的，包括肾毒性，口炎，食管炎以及发生率较低的骨髓抑制，头痛，恶心和低或高血压。大约4克/米2体重的单一丸剂可导致肾毒性。另外，每天分数次摄入高达约350毫克/米2体重的剂量的两位儿科病人出现肝功能障碍。多次服用丸剂后出现口炎和食管炎。发现的其它反应是病人特异的。
在大约125毫克/米2体重剂量下共5天对患有急性非淋巴母细胞白血病成年病人连续输液进行一次Ⅱ期临床试验。剂量限制毒性是粘膜炎。
B．将ASS抑制剂向宿主给药
使用医疗有效剂量的一种ASS抑制剂例如L-亚硝基羟基丙氨酸，L-亚硝基羟基丙氨酰-AICOR或N-羟-N-甲酰甘氨酸，优选地是前者处理患有根据本发明MTAse缺陷检测方法确定为MTAse缺陷癌症的哺乳动物宿主(例如人)。此处“治疗有效量”是指在可评价患者中产生的客观的肿瘤应答的剂量，其中肿瘤应答是由临床可接受标准确定的生长中止或减退(参看，例如，Eagan等，癌(Cancer),44:1125-1128,1979[其公开部分并于此处]以及据IND#14,247(国家食品和药物管理局(Food and Drug Administration))进行的临床实验中所应用的(基本依据Eagan等)可公开获得的报告中的参数)。根据对这些标准的参考，那些在癌症学领域的普通技术人员可很容易地确定本发明中使用ASS抑制剂耗尽胞内AMP的医疗有效剂量。
概括来说，在低于已知产生毒性剂量下每天给药或连续输液ASS抑制剂将是优选的增强药物抗代谢物活性的治疗方法。由于MTAse缺陷细胞在这种治疗方法中的独特敏感性，预期需要低于在用L-亚硝基羟基丙氨酸处理具MTAse能的细胞的临床实验中测得的剂量，从而降低对非增殖细胞的毒性。
也可通过施用用于腺嘌呤合成的MTA或一种合适的底物类似物来保护非恶性具MTAse能力细胞免除暴露于ASS抑制剂的伤害。适用于这方面的组合物包括MTA,2′-5′双脱氧腺苷，5′-脱氧腺苷，2＇-脱氧-5-脱氧-5′甲硫腺苷(参看，例如，实施例Ⅱ)。然而有一点是令人满意的，即具MTAse能力的细胞能够由甲硫腺苷的代谢产生腺嘌呤来补充AMP细胞库从而预期不会象MTAse缺陷细胞那样耗尽AMP。
本发明已完整描述，其应用由下面陈述的实施例说明。当然，这样实施例并非限制本发明的范围，它由附录的权利要求确定。在实施例中使用标准的缩写，例如“ml”表示毫升，“h”表示小时以及“mg”表示毫克。
实施例Ⅰ
连续输液后L-亚硝基羟基丙氨酸对MTAse缺陷和
具MTAse能力的人肿瘤异种移植体的体内效应
为了评价连续输液后L-亚硝基羟基丙氨酸对已知人MTAse缺陷肿瘤的体内效应并将该效应与药物对已知人具MTAse能力肿瘤的效应相比较，将2×106MTAse缺陷的H292NSCLC细胞和具MTAse能力的Calu-6肿瘤细胞(以0.3毫升同50％MATRIGELTM载体一起)分别注射到8个Balb/C无胸腺裸鼠的右和左肋部。MATRIGELTM开始在体内形成一固体基质，在输液第一天在每头鼠中对其大小进行测量，然后作为对照肿瘤大小(它在14天时间里重吸收)。为了进一步比较，将50％MATRIGELTM载体中的107CEM T-ALL细胞注射到8头其它裸鼠的右肋部。许多这类细胞获自ATCC的商品化细胞系，ATCC登记号分别为CRL-1848和HTB-56。
在第二天，将ALZETTM 1007DM渗透输液泵在距肿瘤细胞接种部位一定距离的背部皮下植入到4只两侧NSCLC接种和4只T-ALL接种的鼠中。泵中储有L-亚硝基羟基丙氨酸，其浓度调节至连续输液时每天释放60毫克/公斤。由于在60毫克/公斤剂量7天后没有明显毒性，除去该泵并以含有每天可释放90毫克/公斤的L-亚硝基羟基丙氨酸的泵代替，再持续7天。
如图2和图3所示，与处理的具MTAse能的NSCLC细胞和未处理的MTAse缺陷细胞相比，施用L-亚硝基羟基丙氨酸使得免疫缺陷鼠宿主中形成的MTAse缺陷NSCLC(“H292 L-亚硝基羟基丙氨酸时距曲线”；图2)和T-ALL(“处理鼠”；图3)异种移植接种物萎缩并防止了免疫缺陷鼠宿主中新的MTAse缺陷NSCLC或T-ALL异种移植接种物的生长。特别是，尽管存在L-亚硝基羟基丙氨酸，已形成的具MTAse能的肿瘤快速生长，未处理的MTAP-缺陷NSCLC或T-ALL异种移植也是如此。
实施例Ⅱ
使用MTAse底物保护具MTAse能力的健康细胞
两个细胞系，具MTAse能力的Calu-6和MTAse缺陷的H292(图4)的比较证实了尽管每一培养物中添加了MTAse底物甲硫腺苷(MTA),L-亚硝基羟基丙氨酸的存在(40μM)使MTAse缺陷NSCLC细胞选择性失去增殖能力。尽管有L-亚硝基羟基丙氨酸，含有腺嘌呤(tAde)的对照培养物可增殖，证实选择性毒性是由于MTAse缺陷细胞不能代谢MTA产生腺嘌呤而引起的。
作为进一步的比较，添加MTA或MTA底物类似物5＇-脱氧腺苷只使具MTAse能的A427细胞和MOLT-4细胞恢复生长，而MTAse缺陷A549和CEM细胞的生长仍受抑制(图5A和B)。因为MTA是精胺合成酶的反馈抑制剂，所以一些细胞系例如MOIT-4在高浓度下被抑制。这形成了伴随MTA浓度增长的双相生长恢复曲线(图5A)。
实施例Ⅲ
MTAse基因组克隆的克隆和部分表征
如下分离了人MTAse的基因组克隆。使用MTAse cDNA基因探针(来自亚克隆MTAP-7的Not I/EcoRi片段)筛查从人胎盘DNA构建的粘粒基因文库(Clontech)。将来自文库的转化大肠杆菌(E.coli)细胞在含有氨苄青霉素(50毫克/升)的LB平板中铺平板，菌落密度为1-2×104/135×15毫米平板。
进行下列步骤。从50万菌落中，分离到一个命名为MTAP-10的阳性克隆并用PCR检测和直接测序得到部分特征。合成了两个引物，一个为位于终止密码子上游120碱基对(bp)的有义寡核苷酸和一个为位于终止密码子下游20bp的反义寡核苷酸，并用于PCR检测。进行了25循环PCR，每一循环包括变性(92℃,1分钟)，退火(55℃,2分钟)和延伸(72℃,5分钟)。在0.8％琼脂糖凝胶上分离PCR产物。
迄今为止使用上述技术在MTAse基因中确定的外显子的位置通过下划线描绘于图1中。
实施例Ⅳ
将MTAse序列特异性寡核苷酸用于恶性
细胞系以检测其中MTAse的存在与否
为了检验从如实施例Ⅳ(序列1)中所述的已确定的MTAse基因组克隆中设计的寡核苷酸探针的有效性，将PCR用于几种含有已知具有MTAse能的(Calu-6；ATCC命名No.HTB-185)和缺陷细胞(A549；ATCC命名NO.CCL-185)的人肺癌细胞系。如实施例Ⅲ所述分离基因组DNA并将其1毫克用于PCR。
如上所述进行了40循环扩增，每一循环包括变性(92℃,1分钟)，退火(55℃,1分钟)，和延伸(72℃,1/2分钟)。在1.5％琼脂糖凝胶上检测PCR产物。在已知为MTAse缺陷的细胞系中未检测到MTAse，而在具MTAse能的细胞系中检测到了MTAse。
实施例Ⅴ
每天向建立有肿瘤异种移植体的裸鼠注射L-
亚硝基羟基丙氨酸对MTAP-缺陷肿瘤的抑制
将107A549或A427NSCLC细胞皮下注射到六周龄Balb/c无胸腺裸小鼠中。当肿瘤达到大约0.4厘米直径时，通过每12小时腹膜内注射L-亚硝基羟基丙氨酸或盐水开始处理。
向小鼠每次注射15毫克/公斤连续7天然后22.5毫克/公斤连续5天。肿瘤大小以垂直直径量度。处理小鼠(每种细胞类型n=6[样品大小])对对照小鼠(每种细胞类型n=4[样品大小]的结果显示于图7。
在检测的每一剂量水平都没有明显的毒性(小鼠在检测中体重稳定)。每天两次丸剂注射施用L-亚硝基羟基丙氨酸导致非常确实的MTAP缺陷肿瘤的大小减小，但对MTAP阳性细胞的生长没有影响。
序列总结序列1是甲硫腺苷磷酸化酶(MTAse)的基因组克隆。
序列表(1)基本资料
(ⅰ)申请人：加利福尼亚大学董事会
(ⅱ)发明名称：抑制恶性甲硫腺苷磷酸化酶缺陷细胞中腺苷酸琥珀酸合
成酶活性的方法。
(ⅲ)序列数目：1
(ⅳ)通讯地址
(A)收信人：Robbins,Berliner&Carson
(B)街道：201 N.Figueroa街，5楼
(C)城市：洛杉矶
(D)州：加利福尼亚
(E)国家：美国
(F)邮编：90012-2628
(V)计算机可读形式：
(A)媒介类型：软盘
(B)计算机：IBM兼容PC
(C)操作系统：PC-DOS/MS-DOS
(D)软件：PatentIn Relase#1.0,#1.25版本
(ⅵ)当前申请数据：
(A)申请号：
(B)填表日期：
(C)分类：
(ⅷ)律师/代理人资料
(A)姓名：Berliner,Robert
(B)注册号：20,121
(C)参考/概要号数：5555-423
(ⅸ)电信资料：
(A)电话：(213)977-1001
(B)传真：(213)977-1003
(2)序列1(SEQID NO:1:)资料
(ⅰ)序列特征：
(A)长度：2763碱基对
(B)类型：核酸
(C)链型：单链
(D)拓扑结构：线性
(ⅱ)分子类型：DNA(基因组)
(ⅶ)直接来源：
(B)克隆：甲基腺苷磷酸酶
(ⅸ)特征：
(A)名字/关键词：CDS
(B)位置：1..2763
(ⅹⅰ)序列描述：系列1：(SEQ ID NO:1:)TTTATACAGA GCATGACAGT GGGGTCCTCA CTAGGGTCTG TCTGCCACTC TACATATTTG 60AAACAGGAGT GGCTTCTCAG AATCCAGTGA ACCTAAATTT TAGTTTTAGT TGCTCACTGG 120ACTGGGTTCT AGGAGACCCC CTGTGTTAGT CTGTGGTCAT TGCTAGSAGA ATCACTTAAT 180TTTTTCTAGA CTCTAGGAGA AAACAGTTGG TGGTGTACTC ATCACGGGTT AACAATTTCT 240TCTCTCCTTC CATAGGCATG GAAGGCAGCA CACCATCATG CCTTCAAAGG TCAACTACCA 300 GGCGAACATC TGGGCTTTGA AGGAAGAGGG CTGTACACAT GTCATAGTGA CCACAGCTTG 360TGGCTCCTTG AGGGAGGAGA TTCAGCCCGG CGATATTGTC ATTATTGATC AGTTCATTGA 420CANNNNNNNN NNNNNNNNNN GAGGTCGACG GTATCGATAA GCTTTGTAAA CAATTGTCTT 480TAGCTTATCC AGAGGAATTG AGTCTGGAGT AAAGACCCAA ATATTGACCT AGATAAAGTT 540GACTCACCAG CCCTCGGAGG ATGGAAAGAT GGCCTTAAAA TAAAACAAAC AAAAACCTTT 600TTTGCTTTAT TTTGTAGGAC CACTATGAGA CCTCAGTCCT TCTATGATGG AAGTCATTCT 660TGTGCCAGAG GAGTGTGCCA TATTCCAATG GCTGAGCCGT TTTGCCCCAA AACGAGAGAG 720GTGTGTAGTC TTTCTGGAAG GTGTACCAGA ATAAATCATG TGGGCTTGGG GTGGCATCTG 780GCATTTGGTT AATTGGCAGA CGGAGTGGCC CCATACCCTC ACTCAAGTTT GCTTTGTATT 840ATGCAAGTTT ATGGAGAGTT ATTTCCTGTT GCTAATAATT TNNNNNNNNN NNNNNNNNNN 900AAGTGCAGCC TTAAGTTGTG CATGTGCTAG TATGTTTTGA AGTTTCTGGT TTTTCTTTTC 960TAGGTTCTTA TAGAGACTGC TAAGAAGCTA GGACTCCGGT GCCACTCAAA GGGGACAATG 1020GTCACAATCG AGGGACCTCG TTTTAGCTCC CGGGCAGAAA GCTTCATGTT CCGCACCTGG 1080GGGGCGGATG TTATCAACAT GACCACAGTT CCAGAGGTGG TTCTTGCTAA GGAGGCTGGA 1140ATTTGTTACG CAAGTATCGC CATGGGCACA GATTATGACT GCTGGAAGGA GCACGAGGAA 1200GCAGTAGGTG GAATTCTTTT CTAAGCACAT ATAGCATGGG TTTCTGGGTG CCAATAGGGT 1260GTCTTAACTG TTTGTTTCTA TTACGTTAGT TTCAGAAAGT GCCTTTCTAC AAGGTTTTGA 1320AGTTGTTAAT ATTTTCTGTA GTTCCATTGG AAGGTAAGAA CAAAGATCAA AAGAAAGAAA 1380GAGACACTTT TACCCAAGGA TCAGTAGTGA AAATAGTACA TTGTAGGCAT GTAGATGTGT 1440TGAGAATCAT ACTAAGACTT GGGCCTTANN NNNNNNNNNN NNNNNNNNNN NNTACCCTAC 1500ATTGAGGATT CGGTTTCAGC AGATAAATTT GAGGGACACA AACATTTAGG CTGTAGCAAG 1560GCTGGAGCTC AGAAAAATGT TTTATGACAA GCAGTGGAAT TTTAAGTTCT AGTAACCTCC 1620AGTGCTATTG TTTCTCTAGG TTTCGGTGGA CCGGGTCTTA AAGACCCTGA AAGAAAACGC 1680TAATAAAGCC AAAAGCTTAC TGCTCACTAC CATACCTCAG ATAGGGTCCA CAGAATGGTC 1740AGAAACCCTC CATAACCTGA AGGTAAGTGC AGCCATGGAC AATCAGGCAT GTCTGTAGAC 1800TCTCTATTGT CTTCTTTTCT TACTTGCATT TCACCTTTGG TCCTCATGTA TTTTTTGCCA 1860GCCTAGATGT TTTCAACAAG TTTTTGTGAC ATCTACTACT ACCATACCAA CCACTTGTGA 1920AACTGAGTAG TCTTATTTTC TTGGCTGGTA GTGCAGANNN NNNNNNNNNN NNAATAAACA 1980ATAATCCAGG CTGGGCTGGT ATGGCAATAA GTGATTATCA GAACAATGCT CTGAGATAAG 2040CATTATTAAC CTCACTTTAC AGGAAAGGGA GGTGAGGAAC CAAGAGTTTA GAGTACCCGA 2100AGTTCCACAT CTGGTTAGTG AACTTGAAAA TTTTCTGTAG AATTTATTTA AAGTGTATGT 2160TTCCTGCGTC CTCACTTTGA TCTAGAAAAT CAAAATCTGT TTTTTTTTTT AACAAACATC 2220TCAGTAATTA CGCCAACATG TGAATATCAC TGCCTCCTTT CTTCCTTTCA GAATATGGCC 2280CAGTTTTCTG TTTTATTACC AAGACATTAA AGTAGCATGG CTGCCCAGGA GAAAAGAAGA 2340CATTCTAATT CCAGTCATTT TGGGAATTCC TGCTTAACTT GAAAAAAATA TGGGAAAGAC 2400ATGCAGCTTT CATGCCCTTG CCTATCAAAG AGTATGTTGT AAGAAAGACA AGACATTGTG 2460TGTATAGAGA CTCCTCAATG ATTTAGACAA CTTCAAAATA CAGAAGAAAA GCAAATGACT 2520AGTAACATGT GGGAAAAAAT ATTACATTTT AAGGGGGAAA AAAAACCCCA CCATTCTCTT 2580CTCCCCCTAT TAAATTTGCA ACAATAAAGG GTGGAGGGTA ATCTCTACTT TCCTATACTG 2640CCAAAGAATG TGAGGAAGAA ATGGGACTCT TTGGTTATTT ATTGATGCGA CTGTAAATTG 2700GTACAGTATT TCTGGAGGGC AATTTGGTAA AATGCATCAA AAGACTTAAA AATACGGACG 2760TAC 2763
Claims (13)
1．一种抑制甲硫腺苷磷酸化酶(MTAse)缺陷细胞中腺苷酸琥珀酸合成酶(ASS)活性的方法，包括：
(a)确定从哺乳动物宿主中获得的某种群细胞是MTAse缺陷的；以及，
(b)向宿主施用治疗有效量的ASS抑制剂，其中上述接触具有耗尽MTAse缺陷宿主细胞中的腺苷5＇-磷酸的效应。
2．根据权利要求1的方法，其中步骤(a)按下面进行：
(a)获取推测为MTAse缺陷的细胞的可检测样品；
(b)将能够特异地与样品中存在的任何MTAse编码核酸杂交的寡核苷酸探针在能够使探针可检测地与样品中存在的任何此类核酸杂交的条件下加入样品中；以及，
(c)检测样品中是否存在MTAse编码核酸，其中所述核酸的存在表示细胞样品中有MTAse蛋白质的存在。
3．根据权利要求1的方法，其中的ASS抑制剂是L-亚硝基羟基丙氨酸。
4．根据权利要求1的方法，其中的宿主是人。
5．根据权利要求1的方法，其中的MTAse缺陷宿主细胞是选自包括非小细胞肺癌细胞，急性淋巴母细胞白血病细胞，神经胶质瘤细胞和泌尿道上皮肿瘤的组中的原发肿瘤细胞。
6．根据权利要求1的方法，其中的ASS抑制剂每天或连续向宿主给药。
7．根据权利要求1的方法，其中用于胞内产生AMP的底物是在根据步骤(b)处理宿主后施用到宿主的。
8．一种抑制已知为甲硫腺苷磷酸化酶(MTAse)缺陷的哺乳动物宿主细胞中腺苷酸琥珀酸合成酶(ASS)活性的方法，包括向宿主施用治疗有效量的ASS抑制剂，其中所述接触具有耗尽MTAse缺陷宿主细胞中的腺苷5＇-磷酸的效应。
9．根据权利要求8的方法，其中的ASS抑制剂是L-亚硝基羟基丙氨酸。
10．根据权利要求8的方法，其中的宿主是人。
11．根据权利要求8的方法，其中的MTAse缺陷宿主细胞是选自包括非小细胞肺癌，急性淋巴母细胞白血病细胞和神经胶质瘤细胞的组中的原发肿瘤细胞。
12．根据权利要求8的方法，其中的ASS抑制剂每天或连续向宿主给药。
13。根据权利要求8的方法，其中用于胞内产生AMP的底物是在将ASS抑制剂施用到宿主后才施用到宿主的。
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|---|---|---|---|
| US08/612,542 US5840505A (en) | 1993-12-29 | 1996-03-08 | Method for inhibiting adenylosuccinate synthetase activity in methylthioadenosine phosphorylase deficient cells |
| US08/612,542 | 1996-03-08 |
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| Publication Number | Publication Date |
|---|---|
| CN1213406A true CN1213406A (zh) | 1999-04-07 |
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| CN97192895A Pending CN1213406A (zh) | 1996-03-08 | 1997-01-27 | 抑制甲硫腺苷磷酸化酶缺陷细胞的腺苷酸琥珀酸合成酶活性的方法 |
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| US (1) | US5840505A (zh) |
| EP (1) | EP0902839B1 (zh) |
| JP (1) | JP2000509247A (zh) |
| KR (1) | KR19990087627A (zh) |
| CN (1) | CN1213406A (zh) |
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| BR (1) | BR9708002A (zh) |
| CA (1) | CA2244733A1 (zh) |
| CZ (1) | CZ286698A3 (zh) |
| DE (1) | DE69737499D1 (zh) |
| EA (1) | EA001988B1 (zh) |
| HU (1) | HUP9901858A3 (zh) |
| IL (1) | IL126114A0 (zh) |
| NO (1) | NO984124D0 (zh) |
| NZ (1) | NZ331088A (zh) |
| PL (1) | PL328810A1 (zh) |
| RO (1) | RO118210B1 (zh) |
| UA (1) | UA47465C2 (zh) |
| WO (1) | WO1997032994A1 (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110124038A (zh) * | 2019-05-08 | 2019-08-16 | 山东大学齐鲁医院 | 腺苷酸琥珀酸合成酶基因和/或其编码的蛋白的新应用 |
| CN110225983A (zh) * | 2016-12-01 | 2019-09-10 | 葛兰素史密斯克莱知识产权发展有限公司 | 治疗癌症的方法 |
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| US6210917B1 (en) * | 1993-12-29 | 2001-04-03 | The Regents Of The University Of California | Method for suppressing multiple drug resistance in cancer cells |
| US6214571B1 (en) * | 1993-12-29 | 2001-04-10 | The Regents Of The University Of California | Method for inhibiting adenylosuccinate synthetase activity in malignant methylthioadenosine phosphorylase deficient cells |
| US6911309B2 (en) * | 1993-12-29 | 2005-06-28 | The Regents Of The University Of Californnia | Nucleic acids encoding MTAse |
| AU2001297765A1 (en) * | 2000-12-05 | 2002-10-21 | Incyte Genomics, Inc. | Ligases |
| US20040043959A1 (en) * | 2002-03-04 | 2004-03-04 | Bloom Laura A. | Combination therapies for treating methylthioadenosine phosphorylase deficient cells |
| US20040127435A1 (en) * | 2002-08-02 | 2004-07-01 | Regents Of The University Of California | Uses for inhibitors of inosine monophosphate dehydrogenase |
| EP1594900A2 (en) * | 2003-02-14 | 2005-11-16 | Salmedix, Inc. | Compositions and methods for the detection and treatment of methylthioadenosine phosphorylase deficient cancers |
| US20060041013A1 (en) * | 2004-08-18 | 2006-02-23 | Brittain Jason E | Alanosine formulations and methods of use |
| WO2009014642A1 (en) * | 2007-07-19 | 2009-01-29 | Amgen Inc. | Combination of a de novo purine biosynthesis inhibitor and a cyclin dependent kinase inhibitor for the treatment of cancer |
| JP7016958B2 (ja) * | 2017-12-21 | 2022-02-07 | ボード オブ リージェンツ，ザ ユニバーシティ オブ テキサス システム | アデノシンおよび／またはメチルチオアデノシンの酵素媒介枯渇方法 |
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| US4792520A (en) * | 1984-02-16 | 1988-12-20 | University Of Cincinnati | Methods and kits for identifying mutagenic agents and molecular mutations in DNA in mammalian cells |
| US4822736A (en) * | 1984-07-05 | 1989-04-18 | Baylor College Of Medicine | Amplification of adenosine deaminase genes in mammalian cells using adenosine, alanosine, uridine, and deoxycoformycin |
| US5030623A (en) * | 1986-03-27 | 1991-07-09 | The Regents Of The University Of California | Methods for increasing extracellular adenosine and for stabilizing mast cells |
| US5118601A (en) * | 1986-03-27 | 1992-06-02 | The Regents Of The University Of California | Method of screening purine nucleoside compounds or analogs for the ability to enhance the cellular synthesis and release of adenosine |
| PL181667B1 (pl) * | 1993-12-29 | 2001-08-31 | Univ California | Sposób wykrywania obecnosci katalitycznie aktywneji katalitycznie nieaktywnej fosforylazy metylotioadenozynowej (MTAzy), wyizolowany polinukleotyd oraz zrekombinowany wektor ekspresyjny PL |
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1996
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-
1997
- 1997-01-27 EA EA199800806A patent/EA001988B1/ru not_active IP Right Cessation
- 1997-01-27 CZ CZ982866A patent/CZ286698A3/cs unknown
- 1997-01-27 UA UA98105274A patent/UA47465C2/uk unknown
- 1997-01-27 CA CA002244733A patent/CA2244733A1/en not_active Abandoned
- 1997-01-27 IL IL12611497A patent/IL126114A0/xx unknown
- 1997-01-27 RO RO98-01362A patent/RO118210B1/ro unknown
- 1997-01-27 EP EP97903962A patent/EP0902839B1/en not_active Expired - Lifetime
- 1997-01-27 CN CN97192895A patent/CN1213406A/zh active Pending
- 1997-01-27 HU HU9901858A patent/HUP9901858A3/hu unknown
- 1997-01-27 KR KR1019980707079A patent/KR19990087627A/ko not_active Application Discontinuation
- 1997-01-27 AT AT97903962T patent/ATE357223T1/de not_active IP Right Cessation
- 1997-01-27 PL PL97328810A patent/PL328810A1/xx unknown
- 1997-01-27 DE DE69737499T patent/DE69737499D1/de not_active Expired - Lifetime
- 1997-01-27 JP JP9531750A patent/JP2000509247A/ja not_active Ceased
- 1997-01-27 WO PCT/US1997/001193 patent/WO1997032994A1/en active IP Right Grant
- 1997-01-27 NZ NZ331088A patent/NZ331088A/xx unknown
- 1997-01-27 AU AU18388/97A patent/AU722813B2/en not_active Ceased
- 1997-01-27 BR BR9708002A patent/BR9708002A/pt not_active Application Discontinuation
-
1998
- 1998-09-07 NO NO984124A patent/NO984124D0/no not_active Application Discontinuation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110225983A (zh) * | 2016-12-01 | 2019-09-10 | 葛兰素史密斯克莱知识产权发展有限公司 | 治疗癌症的方法 |
| CN110124038A (zh) * | 2019-05-08 | 2019-08-16 | 山东大学齐鲁医院 | 腺苷酸琥珀酸合成酶基因和/或其编码的蛋白的新应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| RO118210B1 (ro) | 2003-03-28 |
| EA001988B1 (ru) | 2001-10-22 |
| NO984124L (no) | 1998-09-07 |
| EA199800806A1 (ru) | 1999-02-25 |
| IL126114A0 (en) | 1999-05-09 |
| CA2244733A1 (en) | 1997-09-12 |
| HUP9901858A3 (en) | 1999-12-28 |
| KR19990087627A (ko) | 1999-12-27 |
| EP0902839A1 (en) | 1999-03-24 |
| EP0902839A4 (en) | 2004-06-30 |
| DE69737499D1 (de) | 2007-05-03 |
| JP2000509247A (ja) | 2000-07-25 |
| HUP9901858A2 (hu) | 1999-09-28 |
| BR9708002A (pt) | 1999-07-27 |
| UA47465C2 (uk) | 2002-07-15 |
| CZ286698A3 (cs) | 1999-03-17 |
| AU1838897A (en) | 1997-09-22 |
| NZ331088A (en) | 2000-08-25 |
| PL328810A1 (en) | 1999-02-15 |
| US5840505A (en) | 1998-11-24 |
| AU722813B2 (en) | 2000-08-10 |
| ATE357223T1 (de) | 2007-04-15 |
| EP0902839B1 (en) | 2007-03-21 |
| WO1997032994A1 (en) | 1997-09-12 |
| NO984124D0 (no) | 1998-09-07 |
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