Preclinical evaluation of PAC1 targeting with radiolabeled Maxadilan

There is an ongoing search for new tracers to optimize imaging of beta cell-derived tumors (insulinomas). The PAC1 receptor, expressed by insulinomas, can be used for targeting of these tumors. Here, we investigated whether radiolabeled maxadilan could be used for insulinoma imaging. Maxadilan was C- or N-terminally conjugated with DTPA (termed maxadilan-DPTA or DTPA-maxadilan respectively). BALB/c nude mice bearing subcutaneous INS-1 tumors were injected with either In-111-labeled maxadilan-DTPA or In-111-DTPA-maxadilan. Biodistribution studies were carried out at 1, 2 and 4 hours after injection and SPECT/CT imaging 1 and 4 hours after injection of maxadilan-DTPA-111In. Radiolabeling of maxadilan-DTPA (680 MBq/nmol) was more efficient than of DTPA-maxadilan (55 MBq/nmol). Conjugation with DTPA slightly reduced receptor binding affinity in vitro: IC50 values were 3.2, 21.0 and 21.0 nM for maxadilan, natIn-DTPA-maxadilan and maxadilan-DTPA-natIn respectively. Upon i.v. injection maxadilan-DTPA-111In accumulated specifically in INS-1 tumors (7.30 ± 1.87%ID/g) and in the pancreas (3.82 ± 0.22%ID/g). INS-1 tumors were clearly visualized by small animal SPECT/CT. In conclusion, this study showed that the high affinity of maxadilan to the PAC1 receptor was maintained after DTPA conjugation. Furthermore, radiolabeled maxadilan-DTPA accumulated specifically in INS-1 tumors and, therefore, may qualify as a useful tracer to image insulinomas.

tumors, as was shown previously by Baumann et al. 5 . For example, malignant insulinomas have a low incidence and expression density of the GLP-1 receptor, but show high expression of the somatostatin receptor, where in benign insulinomas the expression profile is more favorable for the GLP-1R 4,18,19 . Therefore, to improve the diagnostic tool box, the search for new tracers for detection of insulinomas is ongoing.
Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating polypeptide (PACAP), and their receptors PAC1, VPAC1 and PAC2 have emerged as important factors in islet cell function (insulin secretion) and growth and differentiation of neuroendocrine tumors including insulinomas. As these receptors are highly expressed in insulinomas and beta cells, they are potential targets for beta cell and insulinoma imaging [20][21][22] .
Using in vitro receptor autoradiography with VPAC1 and VPAC2 subtype-selective ligands, respectively, insulinomas have been demonstrated to bind high levels of radiolabeled selective VPAC1 receptor ligands, although substantially less than GLP1 selective ligands 4 . As PAC1, VPAC1 and VPAC2 mRNAs were detected in a rat insulinoma cell line with PAC1 binding prevailing over that of VPAC1 and VPAC2 23 , the PAC1 receptor seemed to be a promising alternative for targeting insulinomas. The natural mammalian ligands at the PAC1 receptor are PACAP38 and the truncated form PACAP27. They bind to PAC1, VPAC1 and VPAC2, whereas VIP only binds to VPAC1 and VPAC2. Since PACAP38 and PACAP27 are non-selective PAC1/VPAC1/VPAC2 ligands and unstable in plasma 24 , they are not suited for in vivo targeting of PAC1 receptors. More than two decades ago it was demonstrated that the stable analogue maxadilan shared features with ligands of the PACAP family 25,26 . Maxadilan is a 61-amino acid vasoactive peptide, derived from sand flies 27,28 , and has been demonstrated to bind specifically and with high affinity to the PAC1 receptor but not to either VPAC1 or VPAC2 28 .
In the present study we have examined the ability of 111 In-labeled maxadilan to image rat insulinoma xenografts (INS-1) in a nude mouse model. For this purpose maxadilan was either C-or N-terminally conjugated with DTPA to allow labeling with 111 In. These compounds were evaluated for their radiolabeling properties and in vitro and in vivo binding characteristics.

Results
Radiolabeling. 111 In-DTPA-maxadilan and maxadilan-DTPA-111 In could be labeled with a specific activity of 55 and 680 MBq/nmol, respectively. The radiochemical purity after purification exceeded 95%. Serum stability. The stability of maxadilan-DTPA-111 In was analyzed in human serum. The radiochemical purity directly after labeling was >95% (Fig. 1A). The radiolabeled peptide remained intact up to 24 hrs after incubation in human serum, as is shown in Fig. 1B. 111 In-EDTA and radiolabeled maxadilan have a retention time of 3-4 min and 14.6 min, respectively.
Competitive binding assay. The results of the IC 50 determination of labeled and unlabeled maxadilan analogues are summarized in Fig. 2 and Table 1. Unlabeled maxadilan had the highest affinity (IC 50 value: 3.2 nM) for the receptor. DTPA conjugation resulted in a somewhat lower affinity as indicated by slightly higher IC 50 values: 18.3 and 13.2 nM (p < 0.001 and p < 0.001) for DTPA-maxadilan and maxadilan-DTPA, respectively. Labeling of DTPA-maxadilan or maxadilan-DTPA with 115 In did not significantly reduce the affinity (p = 0.07 and p = 0.12 respectively) compared to the unlabeled compounds.
Biodistribution studies. The results of the in vivo PAC1 receptor targeting study with 111 In-labeled maxadilan in BALB/c nude mice bearing subcutaneous INS-1 tumors are summarized in Fig. 3. Uptake    of maxadilan-DTPA-111 In in the tumor was 7.76 ± 1.37%ID/g, which was significantly higher than that of 111 In-DTPA-maxadilan (4.82 ± 0.87%ID/g) (p = 0.0037). Also, pancreatic uptake was significantly higher for maxadilan-DTPA-111 In compared to 111 In-DTPA-maxadilan, (3.87 ± 0.56%ID/g and 2.73 ± 0.35%ID/g respectively, p = 0.0048). Accumulation in both tumor and pancreas could be blocked by an excess of unlabeled maxadilan, demonstrating specific uptake of the peptides via the PAC1 receptor (1.54 ± 0.49%ID/g and 1.24 ± 0.48%ID/g for maxadilan-DTPA-111 In and 111 In-DTPA-maxadilan respectively in tumor tissue and 0.59 ± 0.07%ID/g and 0.43 ± 0.04%ID/g respectively for pancreatic uptake). Renal uptake was very high and similar for both peptides (148 ± 3 and 145 ± 19%ID/g for 111 In-DTPA-maxadilan and maxadilan-DTPA-111 In, respectively), which could not be blocked with an excess of unlabeled maxadilan. Furthermore, in all other dissected organs, except for blood and spleen, receptor-mediated uptake of maxadilan was observed. Since these results showed that maxadilan-DTPA-111 In has higher accumulation in tumor and pancreatic tissue than 111 In-DTPA-maxadilan, this peptide was used to study the pharmacokinetics. Figure 4 shows the fast clearance of the peptide from the blood. Furthermore, tumor accumulation peaked at one hour after injection (7.30 ± 1.87%ID/g) and decreased slightly over time (5.85 ± 1.17%ID/g and 5.49 ± 0.66%ID/g after 2 and 4 hours (not significant)). A similar trend was observed for the uptake in the pancreas: 3.82 ± 0.22%ID/g at 1 h p.i., 2.71 ± 0.36%ID/g at 2 h p.i. and 2.55 ± 0.07%ID/g at 4 h p.i.. Renal uptake increased over time, while the specific accumulation in the liver decreased. Table 2 gives an overview of the tumor-to-normal-organ ratios for 111 In-labeled maxadilan-DTPA.

SPECT of BALB/c nude mice bearing subcutaneous INS-1 tumors. SPECT/CT images of BALB/c
nude mice bearing subcutaneous INS-1 tumors are shown in Fig. 5. Images were acquired one and four hours after injection of 111 In-labeled maxadilan-DTPA and INS-1 tumors in the right shoulder were clearly visualized. In addition to tumor and kidney, accumulation of the tracer in the liver was observed. Figure 5C shows the image of a mouse which was co-injected with an excess of unlabeled maxadilan. The tumor on the right shoulder was not visualized with SPECT/CT, demonstrating specific uptake via the PAC1 receptor.

Discussion
We demonstrate here for the first time that indeed radiolabeled maxadilan is a promising new tool to image subcutaneously transplanted insulinomas in mice.
We were able to radiolabel maxadilan with 111 In with high specific activity while preserving the affinity for the PAC1 receptor. High and specific uptake of this tracer in both subcutaneous INS-1 tumors and pancreas was found. SPECT imaging clearly visualized the s.c. INS-1 xenografts. Both, pancreatic as well as tumor uptake were receptor mediated, as was demonstrated by blocking experiments with unlabeled maxadilan.
Since the conjugation of a DTPA moiety can affect the affinity of the peptide for the receptor, the peptide was conjugated with DPTA at either the C-or the N-terminus and the molecules were compared. The maximum specific activity of maxadilan-DTPA-111 In was at least twelve times higher than that of 111 In-DTPA-maxadilan. This could possibly be explained by hindrance of the radiometal incorporation into the DTPA molecule, due to either the secondary or tertiary structure of maxadilan or a conformational change in the peptide as a result of the conjugation.  Conjugation of DTPA to either the C-or N-terminus of maxadilan influenced the receptor binding affinity significantly. Maxadilan contains four cysteine residues, which form two disulfide bridges, one of which is located close to the N-terminus. Furthermore, it is known that the N-terminal residues of maxadilan are important for receptor binding 23,29 . Therefore, it was thought likely that conjugation of DTPA to the N-terminus could influence the binding affinity. However, it has also been shown in previous studies that when the disulfide bridge at the N-terminal part of maxadilan is removed or changed, the receptor binding capacity of maxadilan is preserved 29,30 . Nevertheless, as was determined in our in vivo biodistribution studies, the C-terminally modified maxadilan-DTPA-111 In analogue showed higher uptake in the tumor, therefore this compound was used for further characterization.
The biodistribution shows specific accumulation of maxadilan in most organs. This is in line with the widespread PAC1 receptor expression and function in the pituitary, adrenal medulla, pancreas, stomach, colon, lung, and heart 31,32 . As demonstrated in the SPECT images, maxadilan-DTPA-111 In clearly visualized the subcutaneous INS-1 tumors and they were easily distinguished from the specific accumulation of radiolabeled maxadilan in various other tissues.
Other preclinical studies using INS-1 tumour-bearing mice have shown that GLP-1 receptor targeting ligands, labeled either with 111 In, 68 Ga or 18 F, show a more favorable biodistribution compared to this maxadilan tracer. Although those previously-studied tracers show high tumor and kidney uptake similar to maxadilan, the tumor-to-normal-organ ratios are more optimal, which can be correlated with expression levels of the different receptors that are targeted 9,33,34 . Future studies need to be conducted to compare radiolabeled maxadilan with existing relevant PET agents in animals, before moving to clinical trials.
Recently, clinical imaging techniques such as 68 Ga-labeled somatostatin receptor imaging and radiolabeled GLP-1 receptor imaging were introduced for diagnosis of insulinomas. These tracers would be useful for differentiating benign from malignant tumors or in diagnosis of metastatic NETS. However, the sensitivity is not optimal and false positive or false negative cases continue to be described using somatostatin receptor targeting 8,19,35 . False negative-SSTR PET/CT could be explained by small lesions (missed because the resolution of the scanner is not sufficient to detect them) or low somatostatin receptor expression (leading to a low signal which cannot be detected as the sensitivity of the scanner is not sufficient). A solution to the latter problem would be the use of a combination of different radiotracers, thus targeting alternative receptors. This was also suggested by Reubi et al., who conducted an elegant in vitro study proving that a cocktail of three different ligands was able to detect all tested NETs 36 . Tumors with no or low receptor density, which would be missed in vivo when using only one radiotracer, have a higher probability of being detected when multiple receptors are targeted. In addition, if one of the tracers had a very high uptake in the tumor, this high uptake could help to overcome the limited spatial resolution and also visualization of small lesions.
In addition to diagnostic imaging, some somatostatin analogues, when radiolabeled with a beta emitter, are used as therapeutic agents 37 . Due to the high accumulation of radiolabeled maxadilan in the kidneys, and to a lesser extent in other organs, the feasibility of peptide receptor radionuclide therapy with maxadilan would be questionable. Insulinomas may not be the only NETs in which radiolabeled maxadilan could play a diagnostic role. In a study carried out by Pisegna et al. high expression levels of the PAC1 receptor in rat gastric ECL (enterochromaffin-like) cells were observed 38,39 . Currently, 111 In-DTPA-octreotide or 68 Ga-DOTATOC/ 68 Ga-DOTA-octreotide are used for the detection of gastric neuroendocrine tumors, which originate from ECL cells 40 . Furthermore, PAC1 receptor expression has been demonstrated in Lewis lung tumour transplants 41 . Radiolabeled maxadilan could therefore be a possible candidate for detecting both ECL derived tumors and a subtype of small cell lung cancer, in addition to somatostatin receptor tomography. Moreover, there is evidence that there is high expression of PAC1 in human tumors such as paragangliomas, neuroblastomas, pituitary adenomas and endometrial cancers [42][43][44][45] with expression found also in human lung cancers 46 . Importantly, most of these studies were conducted in vitro, so in vivo studies are needed to explore the ratio between peptide uptake in PAC1 receptor positive tumors and that in normal tissues where PAC1 is widely expressed.
In conclusion, radiolabeled maxadilan accumulates efficiently and specifically in INS-1 tumors and could potentially be used for in vivo PAC1 targeting in patients with insulinoma. Radiolabeled maxadilan, therefore, represents a new tracer to image insulinomas using SPECT in addition to or in combination with octreotide and exendin for the identification of benign or malignant insulinoma. Furthermore, the expression of PAC1 in other human tumors indicates a broader application for the usage of radiolabeled maxadilan. Thus, the newly generated PAC1 selective high affinity radiopeptide maxadilan can be employed for multireceptor tumour targeting in vivo.

Materials and Methods
Peptides. DTPA-conjugated and native maxadilan were purchased from Think Peptides (ProImmune In-EDTA = 1). The reaction mixture was purified on a disposable PD-10 desalting column (GE Life Sciences, Diegem, Belgium), which was eluted with 10 ml PBS, 0.5% (v/w) bovine serum albumin (BSA) and fractions containing radiolabeled maxadilan were pooled. Serum stability. Maxadilan-DTPA was labeled with 5 MBq 111 InCl 3 as previously described and incubated with human serum (1:10) at 37 °C. Before addition of human serum and 1, 2, 4 and 24 hrs after incubation with human serum, samples were taken, mixed with acetonitrile (1:1) and centrifuged for 5 min at 5000 g to precipitate serum proteins. The supernatant was analyzed using RP-HPLC on a C 18 reversed-phase column (Alltima; 4.6 mm × 25 cm; Grace, Breda, The Netherlands) and ITLC. The column was eluted with a linear gradient of 0.1% TFA (trifluoroacetic acid, Lab-Scan, Analytical Sciences, Brussels, Belgium) in acetonitrile (3% to 100% over 10 min) with a flow rate of 1 ml/min. ITLC was performed as described above.
Scientific RepoRts | 7: 1751 | DOI:10.1038/s41598-017-01852-8 of INS-1 cells. Labeling of DTPA-maxadilan and maxadilan-DTPA with nat In was performed as previously described 9,48 . Unlabeled and 115 In-labeled peptides were added to the cells (approximately 10 × 10 6 cells in a final volume of 0.5 mL) in eppendorf tubes to final concentrations ranging from 0.1 to 300 nmol (n = 3) together with 1,000 Bq maxadilan-DTPA-111 In. After 4 h incubation on ice, the cells were centrifuged at 3,000 × g, the supernatant was removed and the cells were washed with 1 ml ice-cold PBS and the radioactivity in the cell pellet was determined in a well-type gamma counter (Wallac 1480-Wizard, Perkin-Elmer, Boston, MA, USA). The IC 50 value was calculated by one-site competition analysis with Graphpad Prism (version 5.03, GraphPad Software, San Diego, CA USA).
Biodistribution studies. All experiments were performed in accordance with Radboud University guidelines. Animal experiments were approved by the Animal Ethical Committee of the Radboud University, Nijmegen, The Netherlands.
In order to assess the feasibility of targeting insulinomas with radiolabeled maxadilan, female BALB/c nude mice (6-8 weeks old) were injected subcutaneously with INS-1 cells (1 × 10 7 cells in 200 µl). When the tumors had grown to approximately 5 mm in diameter, groups of five mice were injected with either 370 kBq 111 In-DTPA-maxadilan or maxadilan-DTPA-111 In (peptide dose: 13 pmol). For both peptides, an additional group of 5 mice was co-injected with an excess (1,300 pmol) of unlabelled maxadilan to determine the nonspecific binding of the peptides to the cells. Mice were euthanized 2 h p.i. and blood, muscle, tumor, heart, lung, spleen, pancreas, stomach, intestine, adrenals, kidney and liver were dissected, weighed and the radioactivity concentration was determined.
To examine the pharmacokinetics of maxadilan-DTPA-111 In, BALB/c nude mice (n = 4/group) were injected with 370 kBq maxadilan-DTPA-111 In and mice were euthanized at 1, 2 or 4 h p.i. The radioactivity concentration in the organs was measured in a gamma counter.

SPECT of BALB/c nude mice bearing subcutaneous INS-1 tumors. BALB/c nude mice bearing sub-
cutaneous INS-1 tumors were injected intravenously with 9 MBq maxadilan-DTPA-111 In (13 pmol). A separate group of tumor-bearing mice was co-injected with an excess of unlabelled maxadilan (1,300 pmol). One and four h p.i. SPECT/CT images were acquired using a dedicated small animal SPECT scanner (U-SPECT-II, MILabs, Utrecht, The Netherlands). SPECT images were acquired with a 0.6 mm pinhole mouse collimator with an acquisition time of 50 min. The images were reconstructed with OSEM (3 iterations, 16 subsets, voxel size 0.375) using the U-SPECT-Rec software (MILabs, Utrecht, The Netherlands). The settings for the CT were as follows: spatial resolution, 160 μm; 40 kV; 612 μA. Statistical analysis. Statistical analysis was done using GraphPad Prism version 5.03 for Windows. The unpaired t test was used for determination of significance. A p-value below 0.05 was considered as significant. For the competitive binding assay the F-test was used to manually calculate significance.