GC-C Agonists: Specific Probes for the Detection of Colorectal Tumors

描述(申请人提供)：尿鸟苷(UG)和鸟苷(GN)是鸟苷环化酶C(GC-C)受体的内源性激动剂，基于它们与导致旅行者腹泻的分泌肽-细菌肠毒素(ST)的结构相似性，被发现是利钠激素。这些肽激素与GC-C受体的结合刺激细胞内环鸟苷一磷酸(CGMP)的产生，cGMP反过来调节胃肠粘膜内离子和水的动态平衡。该小组首次报道了编码UG和GN的mRNAs在培养的人结肠癌细胞以及人结肠息肉和腺癌中的表达显著减少。相比之下，GC-C在人类结肠息肉和肿瘤组织中过度表达，可能是为了弥补UG和GN供应的减少。这项研究的假设是，近红外(NIR)荧光标记的SP-333，一种稳定的UG类似物，可以用作一种非侵入性探针，用于可靠地检测多发性肠道肿瘤小鼠的自发性大肠息肉，特别是缺乏升高生长成分的病变。初步数据表明，SP-333是UG的一个抗蛋白水解性类似物，1)与GC-C受体具有高结合亲和力；2)在模拟人的胃液和肠液中37℃下稳定；3)可以用近红外荧光标记；4)与小鼠组织体外孵育时，优先定位于结直肠息肉，而不是邻近的正常结肠粘膜。这些数据支持进一步鉴定和验证近红外荧光标记的SP-333作为检测Clapper博士品系(APC？fccc)中唯一发生多发性大肠息肉的结直肠肿瘤的特异性探针。具体目标1将集中于测定近红外荧光标记的SP-333在模拟肠液和胃液中的稳定性，以及它在体外与人癌细胞和切除的结肠组织(正常和肿瘤)结合的特异性。一旦确定了探针给药的最佳剂量和程序，将确定其在小鼠口服后检测自发性结直肠息肉的敏感性和特异性(特定目标2)。这项拟议的研究将与克拉珀博士的团队合作完成，克拉珀博士的团队最近建立了几种成像模式，用于检测APC？fccc小鼠的结直肠息肉。拟议研究的成功完成将证明，GC-C激动剂SP-333可以作为探针可靠地检测原发性结肠肿瘤，从而支持其进一步开发转化为临床环境。未来对该探头检测结直肠转移的有效性的评估也是有必要的。 公共卫生相关性：将SP-333用作检测结肠肿瘤细胞的荧光探针可导致结肠息肉和肿瘤的早期诊断，从而有可能降低与结肠癌相关的发病率和死亡率。

DESCRIPTION (provided by applicant): Uroguanylin (UG) and guanylin (GN) are endogenous agonists of the guanylate cyclase C (GC-C) receptor that were discovered as natriuretic hormones based on their structural similarity to bacterial enterotoxin (ST), the secreted peptide that is responsible for traveler's diarrhea. Binding of these peptide hormones to the GC-C receptor stimulates intracellular production of cyclic guanosine monophosphate (cGMP), which in turn regulates ion and water homeostasis within the gastrointestinal mucosa. This group was the first to report that the expression of mRNAs encoding UG and GN are markedly reduced in cultured human colon carcinoma cells as well as human colon polyps and adenocarcinomas. In contrast, GC-C is overexpressed in human colon polyps and tumor tissues, perhaps to compensate for the reduced supply of UG and GN. The hypothesis of the proposed study is that near-infrared (NIR) fluorochrome-tagged SP-333, a stable analog of UG, can be used as a noninvasive probe for the reliable detection of spontaneous colorectal polyps in multiple intestinal neoplasia mice, in particular lesions that lack an elevated growth component. Preliminary data indicate that SP-333, a proteolytically resistant analog of UG, 1) possesses a high binding affinity for GC-C receptors; 2) is stable in simulated human gastric and intestinal fluids at 37oC; 3) can be tagged with an NIR fluorochrome; and 4) localizes preferentially to colorectal polyps, as compared to adjacent normal colonic mucosa, when incubated with mouse tissue ex vivo. These data support the further characterization and validation of NIR fluorochrome-tagged SP-333 as a specific probe for the detection of colorectal tumors in Dr. Clapper's strain of mice (Apc?FCCC) that uniquely develops multiple colorectal polyps. Specific Aim 1 will focus on determining the stability of NIR fluorochrome-tagged SP-333 in simulated intestinal and gastric fluids as well as the specificity of its binding to human carcinoma cells in vitro and excised colon tissue (normal and tumor). Once the optimal dose and schedule for probe administration have been established, its sensitivity and specificity to detect spontaneous colorectal polyps following oral administration to mice will be determined (Specific Aim 2). The proposed research will be accomplished jointly in collaboration with Dr. Clapper's group, who has recently established several imaging modalities for the detection of colorectal polyps in Apc?FCCC mice. Successful completion of the proposed research will have demonstrated the proof-of-concept that the GC-C agonist SP-333 can be used as a probe to reliably detect primary colon tumors, thus supporting its further development for translation to a clinical setting. Future evaluation of the utility of the probe to detect colorectal metastases may also be warranted. PUBLIC HEALTH RELEVANCE: Relevance to Public Health Use of SP-333 as a fluorescent probe to detect colon tumor cells could lead to early diagnosis of colon polyps and tumors, potentially resulting reduced morbidity and mortality associated with colon cancer.