Imaging of O6-Alkylguanine-DNA Alkyltransferase

描述（申请人提供）：化疗，尤其是烷基化化疗，是治疗癌症的主要方式之一；然而，回复率往往很低。许多患者预后不良的一个主要原因是耐药性的发展，导致肿瘤再生和最终患者死亡。耐药是由于烷基化剂通过修复蛋白O6烷基-DNA烷基转移酶（AGT）将鸟嘌呤和胸腺嘧啶残基烷基化而产生的DNA损伤进行修复。AGT通过将修饰DNA上的烷基转移到活性部位的半胱氨酸上来修复损伤。这是一个自杀过程，因为没有再生蛋白质的机制，因此耐药性或治疗效果取决于肿瘤中AGT含量的多少。一种无创的肿瘤AGT定量检测方法，对于规划烷基化化疗方案，监测治疗过程中药物对AGT水平的影响，以及开发新的烷基化化疗药物都有很大的帮助。苄基鸟嘌呤（BG）已被证明是一种很好的AGT灭活剂，有几个临床试验正在进行中，评估在化疗前使用BG来消耗AGT。本提案的长期目标是开发放射性碘或18f标记的BG类似物，用于通过科学成像无创定位AGT。苯基4位含氟的BG （4-FBG）已被证明是BG的极好类似物。将开发4-FBG以及放射性BG衍生物的18f标记方法。虽然有可能使用这些化合物来证明原理，但也将努力开发更好的类似物，这些类似物更容易制备，更有效，并且具有更高的代谢稳定性和更长的肿瘤滞留时间。考虑的化合物包括4-苄基氧基-2,6-二氨基-5-硝基/亚硝基嘧啶的衍生物，以及在其C-8和N-9位置上具有取代基的各种BG衍生物。新的未标记化合物将首先用转染AGT的中国仓鼠卵巢（CHO）细胞来评估其AGT灭活能力。标记的化合物将评估其与纯AGT的结合，以及它们在含AGT细胞中的摄取和保留。并对其体外代谢进行了研究。将建立肿瘤AGT含量与标记BG类似物的肿瘤摄取的相关性。首先，te - 671人类髓母细胞瘤异种移植物将用于这些研究。肿瘤摄取作为药物浓度和时间的函数将被研究。潜在的代谢物，特别是在肿瘤中，将被确定。同样，肿瘤摄取和肿瘤AGT含量之间的相关性也将在异种移植模型中进行研究。综上所述，本研究旨在确定一种适合肿瘤AGT成像的药物是否有助于化疗计划，从而改善烷基化剂化疗的结果。随后，这些示踪剂将在携带异种移植物的胸腺小鼠中进行评估。首先，TE-671人髓母细胞瘤异种移植物将用于这些研究。肿瘤摄取作为药物浓度和时间的函数将被研究。潜在的代谢物，特别是在肿瘤中，将被确定。同样，肿瘤摄取和肿瘤AGT含量之间的相关性也将在异种移植模型中进行研究。综上所述，本研究旨在确定一种适合肿瘤AGT成像的药物是否有助于化疗计划，从而改善烷基化剂化疗的结果。

DESCRIPTION (provided by applicant): Chemotherapy, especially alkylator chemotherapy, is one of the primary modes for the treatment of cancer; however, the response rate is often low. A major reason for poor prognosis in many patients is the development of drug resistance, resulting in tumor regrowth and eventual patient death. The drug resistance is due to the repair of DNA lesions, generated by alkylation of guanine and thymine residues by the alkylating agents, by the repair protein O6 alkyl-DNA alkyltrasferase (AGT). AGT repairs the lesions by transferring the alkyl groups from the modified DNA to a cysteine in its active site. This is a suicidal process as there is no mechanism to regenerate the protein and hence the resistance, or alternatively the therapeutic efficacy, depends on the amount of AGT content in the tumor. A means to noninvasively quantitate tumor AGT will go a long way in the planning of alkylator chemotherapy, monitoring the effect of drugs on AGT levels during the therapeutic course, and in the development of newer alkylator chemotherapeutic agents. Benzylguanine (BG) has been shown to be an excellent inactivator of AGT and there are several clinical trials in progress evaluating the use of BG to deplete AGT prior to chemotherapy. The long-term objective of this proposal is to develop radioiodine- or 18F-labeled BG analogues useful in the noninvasive mapping of AGT by scintigraphic imaging. BG containing fluorine at the 4-position of its benzyl group (4-FBG) has already been shown to be an excellent analogue of BG. Methods will be developed for the 18F-labeling of 4-FBG as well as radioiodinated BG derivatives. While it may be possible to demonstrate the proof of principle using these compounds, efforts will also be directed in the development of better analogues that are easier to prepare, more potent, and will have higher metabolic stability, and longer tumor retention. Among the compounds considered are the derivatives of 4-benzyloxy-2,6-diamino-5-nitro/nitroso-pyrimidine, and various BG derivatives with substituents at its C-8 and N-9 positions. Novel unlabeled compounds will be first evaluated for their AGT inactivating ability using Chinese hamster ovary (CHO) cells transfected with AGT. Labeled compounds will be evaluated for their binding with pure AGT and for their uptake and retention in AGT-containing cells. Their in vitro metabolism also will be studied. Correlativity of tumor AGT content and tumor uptake of labeled BG analogues will be Established. Primarily, TE-67 1 human medulloblastoma xenografts will be utilized for these studies. Tumor uptake as a function of drug concentration and time will be studied. Potential metabolites, especially in the tumor, will be determined. Again, the correlativity between tumor uptake and tumor AGT content will be investigated in xenograft models as well. In summary, this proposal seeks to determine whether the development of a suitable agent for the imaging of tumor AGT will facilitate chemotherapy planning, thereby improving the outcome of alkylator chemotherapy. Subsequently these tracers will be evaluated in athymic mice hosting xenografts. Primarily, TE-671 human medulloblastoma xenografts will be utilized for these studies. Tumor uptake as a function of drug concentration and time will be studied. Potential metabolites, especially in the tumor, will be determined. Again, the correlativity between tumor uptake and tumor AGT content will be investigated in xenograft models as well. In summary, this proposal seeks to determine whether the development of a suitable agent for the imaging of tumor AGT will facilitate chemotherapy planning, thereby improving the outcome of alkylator chemotherapy.