CYCLIC CHOLECYSTOKININ ANALOGUES EXHIBIT HIGH BLOOD STABILITY AND BINDING AFFINITY WITH CHOLECYSTOKININ RECEPTOR

Recently, incidence of Cholecystokinin (CCK) receptor is recognized as a factor that determines the aggressive phenotype of pancreatic cancer. In this study, a novel Cholecystokinin (CCK) analogues; DOTA-Nle-cyclo (Glu-Trp-Met-Asp-Phe-Lys-NH2) (DOTA-cCCK) and DOTA-Nle-cyclo (Glu-Trp-NleAsp-Phe-Lys-NH2) (DOTA-[Nle]-cCCK) were synthesized and radiolabeled and the targeting abilities on the CCK receptor were evaluated for new CCK receptor targeting agents searching. Peptides were prepared through a solid phase synthesis method and their purity was over 98%. DOTA is the chelating agent for 68Ga-labelling, which the peptides were radiolabeled with 68Ga by a high radiolabeling yield (>98%). Peptides were stable over 98% by incubation in mouse blood at 37°C for 2 h. A competitive displacement of 125I-CCK8 on the AR42J human pancreatic carcinoma cells revealed that 50% inhibitory concentration value (IC50) were 12.31 nM of DOTA-cCCK and 1.69 nM of DOTA-[Nle]-cCCK. Stable in the blood of both DOTA-cCCK and DOTA-[Nle]-cCCK, but the binding rate with the CCK receptor on AR42J cells, DOTA-[Nle]-cCCK confirmed better than DOTA-cCCK. Therefore, it is concluded that 68Ga-DOTA[Nle]-cCCK can be potential candidate as a targeting modality for the CCK receptor over-expressing tumors and further studies to evaluate their biological characteristics are needed.


INTRODUCTION
A CCK receptor is known as a receptor that is over expressed in tumor cells (Chakravarty et al., 2013;Oikonomou et al., 2008). Its incidence is higher than other cancer in lung cancer and pancreatic cancer (Korner et al., 2010). The CCK receptor subtype can be distinguished pharmacologically by the affinity of gastrin (Sosabowski et al., 2009). The CCK2 receptor has a high affinity of gastrin, but the CCK1 receptor has a lower affinity to gastrin (Liu et al., 2011). Tyr residues of the receptor binding peptide have a critical role in receptor specificity (Stone et al., 2007). If this tyrosine residue is sulfated, the peptide shows a high affinity for both receptors. This is displayed in the cell types of small cell lung cancer rich in CCK2 receptor affinity of medullary thyroid cancer and some stromal ovarian cancers (Roosenburg et al., 2011).
It has been shown that the targeting moiety of CCK peptide is the C-terminal amino acid sequences Trp-Met-Asp-Phe-NH 2 for retaining the receptor binding affinity and preserving the biological activity of CCK-like peptides (Martin-Martinez, 2005). Hence, the N-terminal region of the peptide can be used for radiolabeling and a number of potent CCK analogues have been labeled with a lot of radionuclides such as 99m Tc, 111 In, 90 Y, 64 Cu, 177 Lu, 68 Ga, or 18 F for targeting CCK receptor-expressing cancer cells (D'Andrea et al., 2008;Heppeler et al., 2000).

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Among the radionuclides available, 68 Ga decays by a half-life of 1.13 h with the emission of beta-rays (1899 keV) and gamma-rays (511 keV) and it came into the spotlight for the imaging of tumors. Thus, 68 Ga can monitor the in vivo localization of injected therapeutic radiopharmaceuticals during treatment performing a dosimetric evaluation (Velikyan et al., 2005). The well-known chelating agent of radiolabeling peptides with 68 Ga is 1,4,7,10tetraazacyclododecane-1,4,7,10-Tetraacetic Acid (DOTA) (Beylergil et al., 2013). 68 Ga exists primarily in an oxidation state of 3+ and is stabilized by hard donor atoms such as nitrogen or oxygen.
Several methods have been studied to improve the stability of peptide in the blood (Ocak et al., 2011). A method for improving the reliability has been demonstrated in studies of the target peptide melanoma through cyclization of an internal peptide (Lim et al., 2012). Therefore, the stability and functionality of the cyclic structure of the CCK analogues are confirmed.
In this study, we employed cyclic CCK as a linker of DOTA to prepare radiolabeled candidates for CCK receptor targeting. A novel DOTA-cCCK and DOTA-[Nle]-cCCK were synthesized and radiolabeled and IC 50 of the peptides by 125 I-CCK8 on human pancreatic tumor cells were evaluated.

Cell Culture
The AR42J pancreatic carcinoma cells were obtained from the American Type Culture Collection (ATCC) and grown in 100 mm culture dishes (Corning, Corning, NY, USA). The cells were cultured in RPMI-1640 (LONZA, Walkersville, MD, USA), supplemented with 10% fetal bovine serum, 100 units mL −1 penicillin and 100 g mL −1 streptomycin (Sigma, Milan, Italy) in an atmosphere of 5% CO 2 in air at 37°C for up to approximately a 90% confluence.

Radiolabelling
Peptides were dissolved in a 50 mM sodium acetate buffer (pH = 5.5) to give a concentration of 10 −6 mole mL −1 . 37 MBq of a 68 GaCl 3 solution diluted in a 5.5M HCl was injected into 1×10 −8 mole of a peptide solution vial to give a final volume of 1 mL and heated at 110°C for 7 min. The radiolabeling yield and radiochemical purity/stability of the radiolabeled compound were analyzed by a Waters Chromatograph equipped with an X-Terra C-18 column. The column was eluted with a binary gradient system with a flow rate of 1.0 mL min −1 using an elution solvent of 0.1% TFA in 5% acetonitrile and 0.1% TFA in 95% acetonitrile. The gradient elution profile based on the solution of 0.1% TFA in 95% acetonitrile is as follows: 0%, 5 min; 0-100%, 9 min; 100%, 6 min; 100% and 2 min with 100% of 0.1% TFA in 5% acetonitrile.

68
Ga-labeled peptides were added to 200 µL of mouse blood and incubated at 37°C for 1 or 2 h. About 100 µL aliquots of the incubations were mixed with 500 µL of MeOH and acetonitrile mixed solution (1:1) and incubated at RT for at least 5 min to remove the blood proteins. The aliquots were centrifuged at 13,000 rpm for 10 min and analyzed through an HPLC analysis.

Receptor Binding Study
The in vitro receptor binding of the 68 Ga labelled peptides was studied on AR42J cells. Cells were cultured to confluency in 12-well plates (Corning Life Sciences, Corning, NY, USA). Cells were incubated with 800 to 10,000 cpm 125 I-CCK8 in a binding buffer (DMEM with 0.1% w/v bovine serum albumin). After incubation at RT for 1 h, the cells were washed the times with ice-cold PBS and counted using a gammacounter (Perkin-Elmer, Boston, MA, USA) to determine the cell associated radioactivity.

IC 50 Determination
The 50% Inhibitory Concentration (IC 50 ) for binding the CCKR of peptides was determined on the AR42J cell line in a competitive binding assay. AR42J cells were grown to confluency in 12-well plates. Cells were washed three times with a binding buffer. After 10 min incubation at RT with a binding buffer, unlabelled peptides were added in a range of 0.001 to 1000 nM together with a trace amount of 125 I-CCK8 (10,000 cpm mL −1 , Perkin-Elmer, USA). After incubation at RT for 1 h, the medium was collected and cells were washed three times with ice-cold PBS. The cells were solubilized with 1 N NaOH for 5 min and the cellassociated radioactivity was determined using a gamma-counter. IC 50 was defined as the peptide concentration at which 50% of binding without a competitor was reached. IC 50 values were calculated using GraphPad Prism software (Version 5.00 for Windows, GraphPad Software, San Diego CA, USA).

Statistical Analysis
All mean values are given as mean±Standard Deviation (SD). A statistical analysis was performed using a Welch's corrected unpaired Student's t test or one-way analysis of variance using GraphPad Prism computer fitting program. The level of significance was set at p<0.05.

RESULTS
Nonradioactive peptides were synthesized by solid phase peptide synthesis following the standard Fmoc strategies, as shown in Fig. 1. The retention time of the analytical HPLC for DOTA-cCCK was found to be 6.80 and 7.19 min for DOTA-[Nle]-cCCK, resepectively and the chemical purities were over 98% ( Fig. 2A-D The new conjugates, 68 Ga-DOTA-cCCK and 68 Ga-DOTA-[Nle]-cCCK were routinely prepared in a high yield (>98%) by adding 68 GaCl 3 to an aqueous solution (pH 5.5 ammonium acetate) of the peptides at 110°C for 7 min. The HPLC chromatogram of 68 Ga-DOTA-cCCK (Fig. 3A) and 68 Ga-DOTA-[Nle]-cCCK (Fig. 3B) showed a retention time of 12.12 min and 12.29 min, respectively. As shown in Fig. 4, 68 Ga-DOTA-cCCK and 68 Ga-DOTA-[Nle]-cCCK were stable in mouse blood for 2 h. In contrast, 41% of noncyclic CCK peptide, 68 Ga-DOTA-HHHHHH-Gly-Trp-Nle-Asp-Phe-NH 2 was degraded for 2 h (data not shown). Because the difference between cyclic and noncyclic peptide is cyclization, it seems that the structure using Lys as a cyclic agent is more stable than noncyclic 68 Ga-DOTA-HHHHHH-Gly-Trp-Nle-Asp-Phe-NH 2. This might be caused by the cyclization.
The in vitro CCK receptor binding affinities and specificities of DOTA-cCCK and DOTA-[Nle]-cCCK were assessed through a competitive displacement assay using 125 I-CCK8 as the radioligand. It was found that DOTA-cCCK and DOTA-[Nle]-cCCK were able to compete with 125 I-CCK8 bound to AR42J pancreatic carcinoma cells (Fig. 5). In other peptide of CCK, IC 50 value of DOTA-sCCK8, sulfated cholecystokinin fragment 26-33, is 11.6 nM (Roosenburg et al., 2011). However, IC 50 values of new cyclic peptides were 12.31 and 1.69 nM for DOTA-cCCK and DOTA-[Nle]-cCCK, respectively. It seems that the structure using Nle instead of Met in a CCK analogue is a higher binding affinity than the original CCK analogue in cyclic structures.

DISCUSSION
The incidence of the CCK receptor is higher in lung cancer and pancreatic cancer than other cancers (Brabez et al., 2013;Korner et al., 2010). Studies of tumor-associated via CCK peptides are actively performed, has been recognized possible. However, it has emerged as a point through the research conducted, improved stability in blood and excretion rate on kidney (Breeman et al., 2008).
Several methods have been studied to improve the stability of the peptide in blood. There is a way to improve the stability through the cyclization of the peptide therein, which has been demonstrated in studies of the targeting peptide of melanoma. Thus, by the cyclic structure of the peptide CCK, it was confirmed targeting functionality and stability of this material (Lim et al., 2012).
Several methods have been studied to improve the stability of the peptide in blood. There is a way to Science Publications OJBS improve the reliability through the cyclization of the peptide therein, which has been demonstrated in studies on the target peptide of melanoma. Thus, based on the cyclic structure of the peptide CCK, the target functionality and stability of this material were confirmed.
Bifunctional Chelating Agents (BFCA) are used for the preparation of many radiolabeled compounds. In particular, DOTA is able to strongly chelate many radionuclides such as 177 Lu, 111 In, 149 Pm, 212 Pb, 90 Y and 68 Ga. Additionally, 68 Ga emits strong energy β-rays (1899 keV) and γ-rays (511 keV) and its half life is short (1.13 h). Thus, 68 Ga is considered a suitable radionuclide for performing dosimetry and the imaging of tumors or metastatic deposits. DOTA-cCCK and DOTA-[Nle]-cCCK were also easily labeled with 68 Ga and an imaging study might be possible in the next investigations.
Two peptides showed a high stability. However, DOTA-[Nle]-cCCK showed a higher binding affinity than DOTA-cCCK on the CCK receptor. Therefore, the pharmacokinetic characteristics and therapeutic efficacy of 68 Ga-DOTA-[Nle]-cCCK should be evaluated in the next investigation. If 68 Ga-DOTA-[Nle]-cCCK shows fast excretion in the kidney it can be expected to be a good pharmaceutical agent in further in vivo studies.

CONCLUSION
The newly synthesized cyclic CCK peptides are stable in the blood. In particular, DOTA-[Nle]-cCCK showed high binding affinity with CCK receptor on AR42J cells. In further studies, in vivo metabolism and bio-distribution researches of 68 Ga-DOTA-[Nle]-cCCK are necessary and if studies with longer half-life therapeutic radioisotopes than 68 Ga have been performed, DOTA-[Nle]-cCCK can be expected as therapeutic agents.

ACKNOWLEDGMENT
This study was supported by the KAERI Major Project, Development of Radioisotope Production and Application Technology based on Research Reactor (525140-13).