Tilby M J, McCartney H, Gould K A, O'Hare C C, Hartley J A, Hall A G, Golding B T, Lawley P D
Leukaemia Research Fund Laboratory and Department of Chemistry, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K.
Chem Res Toxicol. 1998 Oct;11(10):1162-8. doi: 10.1021/tx980129a.
Bifunctional alkylating agents, such as those based on nitrogen mustard, form important parts of many anti-cancer chemotherapy protocols and are responsible for increased incidences of secondary tumors in successfully treated patients. These drugs generally form a majority of monofunctional DNA adducts, although the bifunctional adducts appear to be necessary for their powerful cytotoxic and antitumor effects. The relative importance of bifunctional as opposed to monofunctional adducts in the varied biological consequences of drug exposure has not been studied in detail, particularly in relation to the role and specificity of biochemical responses to therapy-related DNA damage. A simple method is described for the preparation of useful quantities of a pure monofunctional derivative of the nitrogen mustard-based drug melphalan. Monohydroxymelphalan was prepared by partial hydrolysis, purified by reversed phase chromatography, and characterized by MS, NMR, and HPLC. Contamination with melphalan was </=0.2%. The heat labile DNA base adducts formed by monohydroxymelphalan were shown to contain undetectable levels of cross-linked species. The ratio of adenine to guanine adducts was 0.62, similar to the equivalent ratio for melphalan. The sequence-dependent pattern of alkylation of purified DNA was indistinguishable from that of melphalan, but required a higher dose to achieve comparable extents of reaction. The specificities of two monoclonal antibodies that recognize melphalan-DNA adducts were investigated using DNA alkylated with [3H]monohydroxymelphalan. Adducts on this DNA showed similar immunoreactivities to adducts formed by melphalan. This shows clearly that neither antibody was specific for cross-linked adducts and that it is therefore possible to quantify adducts formed by both monohydroxymelphalan and melphalan with high sensitivities. The availability of monohydroxymelphalan in addition to melphalan, together with sensitive immunoassays for adducts on extracted DNA and in individual cells, constitutes a useful system for investigating cellular responses to the DNA modifications formed by a clinically relevant drug.
双功能烷基化剂，如基于氮芥的那些，是许多抗癌化疗方案的重要组成部分，并且是成功治疗患者中继发性肿瘤发病率增加的原因。这些药物通常形成大多数单功能DNA加合物，尽管双功能加合物似乎对其强大的细胞毒性和抗肿瘤作用是必需的。双功能加合物与单功能加合物在药物暴露的各种生物学后果中的相对重要性尚未得到详细研究，特别是与对治疗相关DNA损伤的生化反应的作用和特异性有关。本文描述了一种简单的方法，用于制备有用量的基于氮芥的药物美法仑的纯单功能衍生物。单羟基美法仑通过部分水解制备，通过反相色谱法纯化，并通过质谱、核磁共振和高效液相色谱进行表征。美法仑的污染率≤0.2%。单羟基美法仑形成的热不稳定DNA碱基加合物显示含有不可检测水平的交联物种。腺嘌呤与鸟嘌呤加合物的比例为0.62，与美法仑的等效比例相似。纯化DNA的烷基化序列依赖性模式与美法仑的无法区分，但需要更高的剂量才能达到可比的反应程度。使用用[3H]单羟基美法仑烷基化的DNA研究了两种识别美法仑-DNA加合物的单克隆抗体的特异性。该DNA上的加合物与美法仑形成的加合物显示出相似的免疫反应性。这清楚地表明，两种抗体都不是交联加合物的特异性抗体，因此可以高灵敏度地定量单羟基美法仑和美法仑形成的加合物。除了美法仑之外，单羟基美法仑的可用性，以及对提取的DNA和单个细胞中加合物的灵敏免疫测定，构成了一个有用的系统，用于研究细胞对临床相关药物形成的DNA修饰的反应。
