F-18 Labeled Vasoactive Intestinal Peptide Analogue in the PET Imaging of Colon Carcinoma in Nude Mice

As large amount of vasoactive intestinal peptide (VIP) receptors are expressed in various tumors and VIP-related diseases, radiolabeled VIP provides a potential PET imaging agent for VIP receptor. However, structural modification of VIP is required before being radiolabeled and used for VIP receptor imaging due to its poor in vivo stability. As a VIP analogue, [R8, 15, 21, L17]-VIP exhibited improved stability and receptor specificity in preliminary studies. In this study, F-18 labeled [R8,15,21, L17]-VIP was produced with the radiochemical yield being as high as 33.6% ± 3% (decay-for-corrected, n = 5) achieved within 100 min, a specific activity of 255 GBq/μmol, and a radiochemical purity as high as 99% as characterized by radioactive HPLC, TLC, and SDS-Page radioautography. A biodistribution study in normal mice also demonstrated fast elimination of F-18 labeled [R8,15,21, L17]-VIP in the blood, liver, and gastrointestinal tracts. A further micro-PET imaging study in C26 colon carcinoma bearing mice confirmed the high tumor specificity, with the tumor/muscle radioactivity uptake ratio being as high as 3.03 at 60 min following injection, and no apparent radioactivity concentration in the intestinal tracts. In addition, blocking experiment and Western Blot test further confirmed its potential in PET imaging of VIP receptor-positive tumor.


Introduction
As an advanced imaging technique, position emission tomography (PET) allows noninvasive detection and quantitative assay of the in vivo distribution profile of radioactive isotope-labeled probes and has been extensively used in the diagnosis and staging of various diseases [1,2]. Both advanced imaging devices and imaging agents are essential for such a functional imaging approach. It is exactly the desirable nuclide properties of the electronic nuclide F-18 and the characteristics of polypeptide molecules, for example, susceptibility to modification, high target specificity, and the in vivo pharmacokinetics suitable for imaging, that have attracted much attention to PET imaging of F-18 labeled polypeptide molecules [3,4]. Large number of preclinical and clinical PET imaging studies have been conducted for F-18 labeled polypeptides, suggesting promising clinical potential in both tumor and cardiovascular research areas [5][6][7][8].
As a neuroendocrine mediator, vasoactive intestinal peptide (VIP) provides various marked biological activities including vasodilation, respiratory stimulation, and increasing blood glucose level [9,10]. In 1970, Said et al. for the first time separated and purified this polypeptide from swine small intestine and determined its amino acid composition [11,12]. VIP exerts physiological functions mainly through its receptor, which can be generally classified as VIP1 receptor and VIP2 receptor based on the differences in receptor distribution and in the affinity between the receptor and its ligand [13]. Recent studies identified expression of highdensity and high-affinity VIP receptors in the cellular membranes of the tumors including intestinal adenocarcinomas, carcinoids, small cell lung cancer, mammary duct carcinoma, insulinoma, papillary thyroid carcinoma, melanoma, neuroblastoma, chromophil tumor, and pituitary adenoma secreting adrenocorticotropic hormone (ACTH). The maximum number of binding sites ( max ) of the high-affinity VIP receptors in tumor tissues can be as high as 22.5-57.9-fold higher than in normal tissues [14,15].

Characterization of [F-18]FB-[R 8,15,21 , L 17 ]-VIP by Radio-TLC, Radio-HPLC, and SDS-Page Electrophoresis Incorporating Radioautography
Radio-TLC Method. After sampling and spotting the silica plate, spread in methanol/water (V/V, 85/15), and the value of the characterization product was obtained by scanning with Radio-TLC Scanner (Bioscan, USA). Radio-HPLC was performed as described under 2.1. Mice in each group were immediately subject to autopsy, where the organs and tissues of interest, including brain, liver, kidneys, heart, lungs, spleen, stomach, small intestine, muscles, bones, and blood, were collected. All the tissue samples were weighed and their counting were measured by counter, so that the percent injected dose per each gram of tissue (% ID/g) was derived. The imaging experiment with blocking was performed 60 min following injection, using scanning for 10 min and the same procedures and data treatment process as above.

Tumor Western Blot Test after Completion of Imaging.
Upon completion of PET imaging, C26 colon carcinoma bearing mice were sacrificed. Tumor tissues were extracted and about 1 g was weighed. After chopping on ice, they were homogenized with 10 mL of Western Blot tissue lysis solution in a glass homogenizer. The homogenized mix solution was added into several 1 mL cylindrical tubes, which were centrifuged at 14000 g for 5 min. The supernatant was extracted and a small amount was subject to BCA protein assay. A final concentration of 10 g/ L was produced to be used in Western Blot test, following the specific procedures in reference to literature [28].  Through radioactive TLC, we noted that the of both products was around 0.1, which was consistent with the TLC chromatographic characteristics of polypeptides, and no impurity peaks were identified. In the HPLC chromatograph, the retention time of [F-18]FB-[R 8,15,21 , L 17 ]-VIP was 11.23 min, which was similar to that of the [R 8,15,21 , L 17 ]-VIP UV absorption peak (about 11 min). As the stoichiometric amount of the radioactive product was too small (at approximately nanomole scale) to be characterized by 1 H-NMR or MS, we further selected SDS-Page gel electrophoresis and radioautography for analysis. It was noted that the electrophoresis band of the radioactive product corresponded to the band of [R 8,15,21 , L 17 ]-VIP and the 3313 Dalton band in marker (Figure 1), further suggesting that this was our target product. In this study, confirmation of purity and target product of F-18 labeled polypeptide by several characterization methods was reported for the first time, which will provide methodology guidance for the characterization of radiolabeled products of other polypeptides and antibodies.

Tissue Distribution Test in Normal Mice.
Results of the tissue distribution test (Figure 2) suggested low bone absorption, indicating good defluorination stability, which is a mandatory consideration in selecting F-18 PET imaging agent. In addition, data showed that, 5 min following injection, radioactivity had been quickly distributed in various organs, mostly in kidneys and liver tissues. This was primarily due to the fact that, after metabolizing in the blood and liver, polypeptide was quickly eliminated in the kidneys. At 30 min, the radioactivity concentration in the VIP expressing tissues, for example, lungs, liver, and intestines, was slightly higher than in other tissues but was by far lower than in the kidneys. By 120 min following injection, absorption by each organ had markedly reduced, indicating that the target product was consistent with the fast elimination characteristic of polypeptides in the organism.

Micro-PET Images and Results
Analysis. Given the in vivo imaging advantage of PET imaging and based on our preliminary animal evaluation results, micro-PET imaging test for [F-18]FB-[R 8,15,21 , L 17 ]-VIP in C26 tumor bearing mice was conducted and produced the imaging outcomes as shown in Figure 3. As can be seen from the images, the molecular probe maintained good in vivo defluorination stability, with no marked bone absorption observed up until 120 min. Furthermore, as it could not pass through the blood-brain barrier, very small amount entered the brain; absorption in the lungs was not marked, either. Radioactivity had been systemically distributed in the mice by 5 min and had achieved certain concentration at the tumor site, with the tumor-to-muscle ratio (T/M) being as high as 1.84. At 60 min following injection, with the exception of kidneys and liver, radioactivity had been eliminated from the majority of tissues. At that time the T/M could be as high as 3.03, and no marked radioactivity concentration was observed in the intestinal tracts. At 120 min following injection, the T/M had reached 3.74, the S/N ratio had further increased, and the radioactivity concentration in the liver had markedly reduced. In addition, it was noted in the blocking experiment that, at 60 min following injection, the radioactivity concentrations in the tumors of blocked mice had markedly reduced from those in the tumors of unblocked mice, with the T/M reduced to 1.01.

Western Blot Analysis of VIP Receptor Expression in the
Tumor. The purpose of this test was to further confirm that the micro-PET imaging outcome was specific radioactivity concentration, proving that the concentration signals were the result of [F-18]FB-[R 8,15,21 , L 17 ]-VIP and the VIP receptors positively expressed in C26 colon carcinoma tissues. We noted from the test results that positively expressed VIP receptors presented in the tumor tissues (Figure 4).

Discussion
Like other neuropeptides, VIP is quickly metabolized in liver and excreted from kidneys. Given this, when VIP is to be used as an imaging agent, chemical modification of its structure is mandatory to extend the biological half-life in order to meet the imaging requirement. The method by Virgolini et al. and Hessenius et al. using I-123 labeling nonmodified VIP for the diagnosis of intestinal adenoma and endocrine tumor remains significantly controversial [16][17][18][19]. Pallela et al. labeled VIP analogue (TP3654) by 99m Tc and conducted animal distribution and imaging tests in nude mice transplanted with colon carcinoma cells LS174T [20]. It was observed that, despite the relatively depressed radioactivity concentration in tumor tissues with the absorption of 0.24 ± 0.08 (%ID/g) by tumor tissues at 4 h after injection, the retention time was relatively long, contributing to an absorption maintained at 0.23 ± 0.13 (%ID/g) at 24 h; in addition, radioactivity was quickly eliminated in blood and other nontarget organs, with the elimination phase half-life of 120 min. Therefore, at 4 h after injection, the tumor/muscle ratio (T/M) could be as high as 2.73 ± 1.09 and the tumor/blood ratio (T/B) could be as high as 1.16 ± 0.29; at 24 h, the T/M and T/B could be as high as 6.28 ± 3.09 and 1.98 ± 1.44, respectively. The results of the distribution test were compared with the test results on the same animal model: at 24 h after injection, in comparison to the T/M (0.38 ± 0.56) and T/B (0.88 ± 0.16) with 125 I-VIP, 99m Tc-TP3654 resulted in substantial improvement.
While 99m Tc labeled VIP analogues have successfully undergone large number of preclinical human imaging studies, PET features ever-increasing application and the advantages in sensibility and definition, and so forth. PET imaging studies of VIP will be studied by different study teams. Tharkur et al. also synthesized a new VIP analogue TP3982, which was successfully labeled by 64 Cu using (N 2 S-benzoyl) 2 as the bifunctional chelating agent for animal distribution and micro-PET imaging studies in T47D breast tumor bearing mice [25]. The study confirmed that 64 Cu-TP3982 not only maintained the biological activities of endogenous VIP, but also presented better protein degradation stability. Animal distribution study data showed that the absorption of 64 Cu-TP3982 (17.04 ± 0.73 ID%/g) in tumor tissues was 74fold higher than that of 99m Tc-TP3982 (0.23 ± 0.13 ID%/g), which was possibly the result of the very good in vivo stability of 64 Cu-TP3982. In contrast, the chelate 99m Tc was likely to be oxidized into 99m Tc c 7+ and thus quickly eliminated from the organism, leading to relatively low absorption in tumor and other tissues. In vivo PET imaging results showed very marked absorption of 64 Cu-TP3982 in tumor tissues. ROI analysis showed that the intensity of radioactivity in tumor tissues at 4 h following injection was 9.15 ± 0.5 higher than in muscles and was even 13.9±0.7-fold higher at 24 h, indicating a significantly superior imaging quality than with 99m Tc-TP3654 and 99m Tc-TP3982. A series of studies suggested 64 Cu-TP3982 to be a promising PET imaging agent for tumors with high VIP receptor expression. However, while emitting + (655 keV), 64 Cu also emits − (573 keV); the + required for PET imaging only accounts for a small portion of 19% [1]. From the perspective of PET imaging, F-18 features desirable nuclide properties. Despite its short half-life relative to 64 Cu, for a polypeptide with quick pharmacokinetics, the physical half-life of F-18 is more suitable for labeling polypeptide to meet the requirement of radiolabeling of polypeptide agents in PET [1]. However, in a PET imaging study of an F-18 labeled VIP analogue (Arg 15 , Arg 21 ) VIP on T-47D nude mice model of human breast cancer, despite the T/M was as high as 3.4, the T/B was only 0.94. As can be seen from the study results, even with T/M being greater than 1, elevated blood background would also interfere with imaging quality, making it difficult to identify the lesion sites [24]. In summary, despite the limited reports of tumor VIP receptor imaging studies, a promising prospect of application has been preliminarily revealed: (1) VIP receptor imaging provides a highly sensitive and highly specific qualitative niveau diagnostic method for VIP receptor-positive tumors, gastrointestinal adenomas in particular, with better pharmacokinetics than in monoclonal antibody immune-imaging; (2) VIP receptor imaging is expected to be used for predicting the efficacy of VIP, VIP analogues, or VIP receptor antagonists on different tumors, helping the patients to select the treatment regimen; (3) VIP receptor imaging provides a noninvasive study approach for in-vitro revelation of the histological distribution and density of VIP receptors in the organism in physiological settings, which is vital for the study of the physiological, pathological, and pharmacological effects on certain tissues and organs.

Conclusions
In the present study, a novel VIP analogue was successfully labeled by F-18 and characterized by various methods, providing a detailed characterization methodology study of F-18 labeling polypeptides and antibodies. In addition, several key messages were obtained by micro-PET imaging. Firstly, the lung absorption significantly lower than tumor absorption level suggested a significant improvement from 123 I-VIP, which was possibly the favorable result of improved stability. Secondly, the target specificity of [F-18]FB-[R 8,15,21 , L 17 ]-VIP for colon carcinoma, a VIP receptor-positive tumor, was confirmed: high tumor absorption had been shown by 60 min, with the T/M up to 3.03. Over time, T/M had increased to 3.74 by 120 min. This further increase of T/M over time also predicts the target specificity of this molecular probe. In addition, the lowered liver background at 120 min was favorable for the imaging of liver metastasis of colon carcinoma. Furthermore, the blocking experiment at 60 min following injection and the Western Blot test of tumor tissues affirmed the specificity of [F-18]FB-[R 8,15,21 , L 17 ]-VIP for VIP receptor-positive tumors from different perspectives, indicating a further improved imaging accuracy than in F-18-FDG PET imaging.