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

描述（由申请人提供）：尿鸟苷素（UG）和鸟苷素（GN）是鸟苷酸环化酶C（GC-C）受体的内源性激动剂，基于其与细菌肠毒素（ST）（导致旅行者腹泻的分泌肽）的结构相似性，发现其为利钠激素。这些肽激素与GC-C受体的结合刺激细胞内环磷酸鸟苷（cGMP）的产生，其进而调节胃肠粘膜内的离子和水的稳态。该小组首次报道了编码UG和GN的mRNA的表达在培养的人结肠癌细胞以及人结肠息肉和腺癌中显著降低。相反，GC-C在人类结肠息肉和肿瘤组织中过表达，可能是为了补偿UG和GN供应的减少。该研究的假设是，近红外（NIR）荧光染料标记的SP-333，一种稳定的UG类似物，可用作可靠检测多发性肠肿瘤小鼠自发性结直肠息肉的非侵入性探针，特别是缺乏升高的生长成分的病变。初步数据表明，SP-333，一种具有蛋白水解抗性的UG类似物，1）对GC-C受体具有高结合亲和力; 2）在37 ℃下在模拟的人胃液和肠液中稳定; 3）可以用NIR荧光染料标记; 4）与离体小鼠组织孵育时，与邻近的正常结肠粘膜相比，优先定位于结肠直肠息肉。这些数据支持进一步表征和验证NIR荧光染料标记的SP-333作为用于检测Clapper博士品系小鼠（Apc？FCCC），独特的发展多结直肠息肉。具体目标1将侧重于确定NIR荧光染料标记的SP-333在模拟肠液和胃液中的稳定性，以及其与体外人类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时间表，将确定其对小鼠经口给药后检测自发性结肠直肠息肉的灵敏度和特异性（具体目标2）。这项拟议的研究将与克拉珀博士的小组合作完成，克拉珀博士最近建立了几种用于检测APC中结肠直肠息肉的成像模式。FCCC小鼠。拟议研究的成功完成将证明GC-C激动剂SP-333可用作可靠检测原发性结肠肿瘤的探针的概念验证，从而支持其进一步开发以转化为临床环境。未来评估的实用性探针检测结直肠转移也可能是必要的。

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.