Evaluation of Tumor-Targeting Properties of an Antagonistic Bombesin Analogue RM26 Conjugated with a Non-Residualizing Radioiodine Label Comparison with a Radiometal-Labelled Counterpart

Radiolabelled antagonistic bombesin analogues are successfully used for targeting of gastrin-releasing peptide receptors (GRPR) that are overexpressed in prostate cancer. Internalization of antagonistic bombesin analogues is slow. We hypothesized that the use of a non-residualizing radioiodine label might not affect the tumour uptake but would reduce the retention in normal organs, where radiopharmaceutical would be internalized. To test this hypothesis, tyrosine was conjugated via diethylene glycol linker to N-terminus of an antagonistic bombesin analogue RM26 to form Tyr-PEG2-RM26. [111In]In-DOTA-PEG2-RM26 was used as a control with a residualizing label. Tyr-PEG2-RM26 was labelled with 125I with 95% radiochemical purity and retained binding specificity to GRPR. The IC50 values for Tyr-PEG2-RM26 and DOTA-PEG2-RM26 were 1.7 ± 0.3 nM and 3.3 ± 0.5 nM, respectively. The cellular processing of [125I]I-Tyr-PEG2-RM26 by PC-3 cells showed unusually fast internalization. Biodistribution showed that uptake in pancreas and tumour was GRPR-specific for both radioconjugates. Blood clearance of [125I]I-Tyr-PEG2-RM26 was appreciably slower and activity accumulation in all organs was significantly higher than for [111In]In-DOTA-PEG2-RM26. Tumor uptake of [111In]In-DOTA-PEG2-RM26 was significantly higher than for [125I]I-Tyr-PEG2-RM26, resulting in higher tumour-to-organ ratio for [111In]In-DOTA-PEG2-RM26 at studied time points. Incorporation of amino acids with hydrophilic side-chains next to tyrosine might overcome the problems associated with the use of tyrosine as a prosthetic group for radioiodination.


Introduction
Prostate cancer (PC) is the second leading cause of cancer-related fatality in men [1]. The treatment of disseminated castration-resistant PC requires a systemic targeting approach. As the androgen receptor (AR) is the main oncogenic driver in PC, most drugs for its treatment are used to inhibit AR activity. However, since resistance to both first-and second-line androgen deprivation therapies (ADT) is a common problem, development of drugs towards other targets for visualization and treatment of PC is of utmost importance [2,3].
Targeting of prostate-specific membrane antigen (PSMA) is currently the major focus in development of a targeted radionuclide therapy of PC and its metastases [4,5]. There is no known binding of RM26 to the receptors [40]. For development of radioiodine labelled analogue, we have conjugated a tyrosine instead of chelator to the PEG 2 linker.
The goal of this study was to evaluate the tumour-targeting properties of an antagonistic bombesin analogue labelled with non-residualizing ( 125 I-Tyr) and labels residualizing ( 111 In-DOTA). For this purpose, the nine-amino-acid bombesin analogue RM26 was synthesized and N-terminus conjugated to tyrosine (Tyr) or DOTA via the diethylene glycol chain (PEG 2 ) to form Tyr-PEG 2 -RM26 and DOTA-PEG 2 -RM26, respectively ( Figure 1). Tyr-PEG 2 -RM26 was labelled with radiohalogen iodine-125 and in vitro and in vivo properties of [ 125 I]I-Tyr-PEG 2 -RM26 were studied and compared to properties of [ 111 In]In-DOTA-PEG 2 -RM26 having a residualizing label.
The labelling of DOTA-PEG 2 -RM26 with 111 In was performed based on previously developed protocol [41]. Briefly, 92 µL of 0.2 M ammonium acetate buffer pH 5.5 was added to aqueous solution of DOTA-PEG 2 -RM26 (2 µL, 2 nmol) followed by addition of 111 In-indium chloride solution (30-40 µL, 10~20 MBq). The reaction mixture was incubated at 85 • C for 30 min. The radiochemical yield and purity were analysed using instant thin-layer chromatography (ITLC) strips using citric acid (0.2 M, pH 2.0) as the running solution. In vitro stability of [ 125 I]I-Tyr-PEG 2 -RM26 was evaluated in PBS and in presence of 100-fold molar excess of sodium iodide (1 mM). After purification, samples of freshly labelled conjugate (10 µL, 0.01 nmol) were mixed with 1 µL of NaI and incubated at room temperature for 1 h. Control samples were mixed with equal volume of PBS. The experiment was performed in duplicates. The stability of [ 125 I]I-Tyr-PEG 2 -RM26 was analyzed by HPLC.

Lipophilicity Assay: LogP Measurements
Lipophilicity was determined as the logarithm of partition coefficient, logP, of the radiolabelled compound between n-octanol and water [42]. To determine the lipophilicity of both radioconjugates, 500 µL of n-octanol was added to a tube containing the same volume of Milli-Q water. 1 pmol (10 kBq) of radioconjugate was added. The mixture was vigorously vortexed for 1-2 min and then centrifuged. Aliquots of 100 µL were taken from each phase and their radioactivity was measured by an automated gamma-counter. The partition coefficient was calculated as the average log of a ratio of the radioactivity in organic and aqueous fractions. Each measurement was repeated in triplicate.

In Vitro Studies
GRPR-expressing human PC cell line PC-3 and DU-145 cell lines (ATCC) were cultured in RPMI media complemented with 10% FBS, 2 mM l-glutamine, and PEST (penicillin 100 IU/mL) (all from Biochrom AG, Berlin, Germany). This media is referred to as complete media in the text. In all in vitro experiments, cells were incubated in complete media. During in vitro specificity experiments, cells were detached using trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer; Biochrom AG). All experiments were performed in triplicate and 1 × 10 6 cells/dish were seeded one day before the experiment.

In Vitro Binding Specificity Assay
The in vitro binding specificity test of [ 125 I]I-Tyr-PEG 2 -RM26 was performed on PC-3 and DU-145 cell lines. A set of three dishes containing approximately 10 6 cells/dish was seeded. The cells were incubated with 1 nM concentration of [ 125 I]I-Tyr-PEG 2 -RM26 solution for 1 h at 37 • C. To saturate GRPR, one set of dishes was treated with 200-fold molar excess of non-labelled peptide, NOTA-PEG 4 -RM26, 15 min before adding the radiolabelled compound. After washing with serum-free media, cells were detached by treatment with 1 mL trypsin-EDTA solution twice. Cell-associated radioactivity was measured in the gamma-counter and presented as percentage of cell-associated activity.  (1,2,4,8,24 h), incubation media was aspirated, cells were washed with serum-free media, and membrane-bound and internalized radioactivity were collected using the method described earlier [43]. The membrane-bound radiolabelled conjugate were removed from cells by treatment with 4 M urea solution in a 0.1 M glycine buffer, pH 2, for 5 min on ice. The cell debris containing the internalized conjugate was detached by treatment with 1 M NaOH. Radioactivity of samples was measured, and percentage of membrane-bound, internalized and total radioactivity was calculated. The experiments were performed in triplicate.
Additionally, PC-3 cells were incubated with 1 nM of [ 125 I]I-Tyr-PEG 2 -RM26 in presence of 20 mM sodium azide/10 mM 2-deoxyglucose at 37 • C for 8 h. Another set of dishes were incubated with 100 µM of chloroquine diphosphate and 20 nM of ammonium chloride in the presence of [ 125 I]I-Tyr-PEG 2 -RM26 (1 nM). At predetermined time points (2, 4, and 8 h), media from a set of three dishes was removed, cells were detached by trypsin-EDTA solution, re-suspended, and radioactivity in cells was measured.

In Vivo Studies
Animal studies were planned and performed in agreement with EU Directive 2010/63/EU for animal experiments and Swedish national legislation concerning protection of laboratory animals. Experiments were approved by the Ethics Committee for Animal Research in Uppsala (C4/16, 26 February 2016). Biodistribution studies were performed in female BALB/C nu/nu mice purchased from Scanbur A/S (Karlslunde, Denmark). GRPR-expressing xenografts were established by subcutaneous injection of 5 × 10 6 PC-3 cells/mouse in the right hind leg of mice 3 weeks before the experiment. The animals were randomized into groups of four mice for each data point. At the time of the experiment, the average animal weight was 16.3 ± 0.9 g. Average tumour weight was 0.09 ± 0.07 g. To study tumour targeting, female BALB/c nu/nu mice bearing PC-3 xenografts (4 mice per data point) were intravenously co-injected into the tail vein with totally 40 pmol of [ 125 I]I-Tyr-PEG 2 -RM26 and [ 111 In]In-DOTA-PEG 2 -RM26 mixture (30 kBq, 100 µL in PBS). Mice were euthanized followed by dislocation of their neck at 0.5 h after injection and blood samples were collected by heart puncture. At 3 and 24 h after injection, mice were euthanized by an intraperitoneal injection of anaesthetic solution (20 µL of solution per gram of body weight: Ketalar, 10 mg/mL; Rompun, 1 mg/mL) followed by heart puncture. Salivary glands, thyroid, lung, liver, spleen, pancreas, stomach, small intestine, kidneys, tumour, muscle, and bone were collected and weighed. The organ radioactivity was measured in a gamma-counter and uptake values of organs were calculated as percentage of injected dose per gram tissue (% ID/g). To test the in vivo binding specificity, a group of mice was co-injected with 7.5 nmol of non-labelled peptide (NOTA-PEG 4 -RM26), and biodistribution was measured at 0.5 h after injection. Another group of mice was injected with same activity by co-injection of PA (15 µL of 20 mg/mL stock solution, 300 µg). After 0.5 h, mice were euthanized by dislocation of their neck, and blood samples were collected. Salivary glands, thyroid, lung, liver, spleen, pancreas, stomach, small intestine, kidneys, tumour, muscle, and bone were collected and weighed. The organ radioactivity was measured in a gamma-counter and uptake values of organs were calculated as percentage injected dose per gram tissue (% ID/g).

Statistical Analysis
Statistical treatment and linear regression analysis were performed using GraphPad Prism software version 5.00 for Windows, GraphPad Software, San Diego California. A two-tailed unpaired t-test was used for comparison of the two sets of data. The difference was considered as significant when p value was less than 0.05.

Peptide Synthesis and Characterization
Tyr-PEG 2 -RM26 was synthesized as previously described and purified by preparative HPLC, generating in 1.7 mg of desired product, corresponding to a total yield of 6.0% (based on the initial loading of the resin). After LC-MS analysis, purity was determined, based on the 280 nm trace, to be over 95% (Figure 2a  t-test was used for comparison of the two sets of data. The difference was considered as significant when p value was less than 0.05.

Peptide Synthesis and Characterization
Tyr-PEG2-RM26 was synthesized as previously described and purified by preparative HPLC, generating in 1.7 mg of desired product, corresponding to a total yield of 6.0% (based on the initial loading of the resin). After LC-MS analysis, purity was determined, based on the 280 nm trace, to be over 95% (Figure 2a

Radiolabelling and In Vitro Stability Test
The radiolabelling of Tyr-PEG2-RM26 with 125 I was performed at room temperature followed by HPLC analysis and preparative separation. The radiochemical yield was 77 ± 7% as determined using radio-HPLC. After purification using Sep-Pak C8 cartridge, the radiochemical purity was 95 ± 2%. The results of the in vitro stability test demonstrated that the radiolabelled conjugate was stable in PBS (95 ± 1%) and also in presence of excess amount of 1 mM NaI (92 ± 3%) after 1 h incubation at room temperature.
Labelling of DOTA-PEG2-RM26 with 111 In resulted in radiochemical purity of 100%, and [ 111 In]In-DOTA-PEG2-RM26 was used in biological experiments without additional purification.

In Vitro Binding Specificity Assay
In vitro binding specificity test demonstrated that binding of [ 125 I]I-Tyr-PEG2-RM26 to PC-3 and DU-145 cells is receptor mediated. Pre-saturation of the cells with non-labelled NOTA-PEG4-RM26 significantly (p < 0.05) decreased the cell binding of the radiolabelled compounds ( Figure 3). Specific binding to DU-145 cells was much lower than specific binding to PC-3 cells.

Radiolabelling and In Vitro Stability Test
The radiolabelling of Tyr-PEG 2 -RM26 with 125 I was performed at room temperature followed by HPLC analysis and preparative separation. The radiochemical yield was 77 ± 7% as determined using radio-HPLC. After purification using Sep-Pak C8 cartridge, the radiochemical purity was 95 ± 2%. The results of the in vitro stability test demonstrated that the radiolabelled conjugate was stable in PBS (95 ± 1%) and also in presence of excess amount of 1 mM NaI (92 ± 3%) after 1 h incubation at room temperature.
Labelling of DOTA-PEG 2 -RM26 with 111 In resulted in radiochemical purity of 100%, and [ 111 In]In-DOTA-PEG 2 -RM26 was used in biological experiments without additional purification.

In Vitro Binding Specificity Assay
In vitro binding specificity test demonstrated that binding of [ 125 I]I-Tyr-PEG 2 -RM26 to PC-3 and DU-145 cells is receptor mediated. Pre-saturation of the cells with non-labelled NOTA-PEG 4 -RM26 significantly (p < 0.05) decreased the cell binding of the radiolabelled compounds ( Figure 3). Specific binding to DU-145 cells was much lower than specific binding to PC-3 cells.        Figure 6 shows the effect of the lysosomotropic weak bases, chloroquine, and ammonium chloride, on amount of cell-bound activity of [ 125 I]I-Tyr-PEG2-RM26 over time. Cell-bound activity and retention of [ 125 I]I-Tyr-PEG2-RM26 on PC-3 cells in the presence of these two weak bases were considerably higher compared to control. Cell-associated activity for the cell treated with sodium azid, that should inhibit synthesis of the new receptors, was similar to the controls.          (Table 2). Moreover, the washout rate of [ 111 In]In-DOTA-PEG2-RM26 from the target-expressing normal tissues was higher than that from the tumour, leading to increasing tumour-to-organ ratios over time.  (Table 2). Moreover, the washout rate of [ 111 In]In-DOTA-PEG 2 -RM26 from the target-expressing normal tissues was higher than that from the tumour, leading to increasing tumour-to-organ ratios over time.

Biodistribution of [ 125 I]I-Tyr-PEG 2 -RM26 in NMRI Mice by Co-Injection of Phosphoramidon (PA) as an In Vivo Stabilizer
Influence of co-injection of the enzyme inhibitor phosphoramidon (PA) on in vivo stabilization of the radiopeptide [ 125 I]I-Tyr-PEG 2 -RM26 in non-tumour bearing mice was investigated. This strategy led to a remarkable enhanced uptake of the radiopeptides in tumour and receptor-expressing tissues, whereas uptake in most non-target organs and tissues was not affected. Results from the biodistribution study of [ 125 I]I-Tyr-PEG 2 -RM26 are summarized in Figure 8. No significant increase of uptake in GRPR-positive organs (pancreas, small intestines, and stomach) was observed at 0.5 h after co-injection of PA. Moreover, co-injection of PA did not show any significant influence on blood clearance rate or uptake in normal non-targeted organs and tissues. Influence of co-injection of the enzyme inhibitor phosphoramidon (PA) on in vivo stabilization of the radiopeptide [ 125 I]I-Tyr-PEG2-RM26 in non-tumour bearing mice was investigated. This strategy led to a remarkable enhanced uptake of the radiopeptides in tumour and receptor-expressing tissues, whereas uptake in most non-target organs and tissues was not affected. Results from the biodistribution study of [ 125 I]I-Tyr-PEG2-RM26 are summarized in Figure 8. No significant increase of uptake in GRPR-positive organs (pancreas, small intestines, and stomach) was observed at 0.5 h after co-injection of PA. Moreover, co-injection of PA did not show any significant influence on blood clearance rate or uptake in normal non-targeted organs and tissues.

Discussion
The goal of this study was to test the hypothesis that use of non-residualizing radioiodine might offer advantages in radionuclide therapy when antagonistic, slowly internalizing bombesin analogues are used for targeting. For this purpose, bombesin analogue Tyr-PEG2-RM26 was synthesized, purified, and characterized ( Figure 2). Tyr-PEG2-RM26 was successfully radiolabelled with 125 I with good radiochemical yield and about 95% purity. The label was stable in PBS and under challenge with large molar excess of sodium iodide.
The lipophilicity test showed that [ 125 I]I-Tyr-PEG2-RM26 is appreciably more lipophilic than

Discussion
The goal of this study was to test the hypothesis that use of non-residualizing radioiodine might offer advantages in radionuclide therapy when antagonistic, slowly internalizing bombesin analogues are used for targeting. For this purpose, bombesin analogue Tyr-PEG 2 -RM26 was synthesized, purified, and characterized ( Figure 2). Tyr-PEG 2 -RM26 was successfully radiolabelled with 125 I with good radiochemical yield and about 95% purity. The label was stable in PBS and under challenge with large molar excess of sodium iodide.
The lipophilicity test showed that [ 125 I]I-Tyr-PEG 2 -RM26 is appreciably more lipophilic than This is in agreement with previous data showing that a negative charge on N-terminus is unfavourable for binding of bombesin analogues to GRPR [31,33].
The cellular processing of [ 111 In]In-DOTA-PEG 2 -RM26 ( Figure 5) was characteristic for radiometal-labelled antagonistic analogues of bombesin [32][33][34][35][36][37]. The internalization was slow reaching 14% of cell-associated radioactivity after 24 h incubation. Cell-associated activity of [ 125 I]I-Tyr-PEG 2 -RM26 showed rapid increase during the first hour followed by decrease over time. Such type of cellular processing resembles the behaviour of radioiodinated GRPR-binding agonists, when rapid internalization is followed by intracellular degradation and leakage of radiometabolites. However, the internalization and efflux rates of [ 125 I]I-Tyr-PEG 2 -RM26 were much slower than the rates for GRPR-binding agonists. Internalization of agonistic [ 125 I]I-GRP and [ 125 I]I-Tyr 4 -BBN was 75-80% within 20-40 min [19,45]. The decrease in cell-associated activity may be explained by lysosomal degradation of internalized radioiodinated conjugate followed by excretion of radiocatabolites and recycling of GRPR membrane surface [46,47]. These recycled GRPR can further bind and internalize [ 125 I]I-Tyr-PEG 2 -RM26 existing in media. Although retention of cell-associated activity for [ 125 I]I-Tyr-PEG 2 -RM26 was much worse than for the metal labelled RM26, it was better for this antagonistic agent than should be expected for agonistic ones. Efflux of 45-65% of cell associated activity was reported both for residualizing ( 111 In-DOTA and 99m Tc((CO) 3 ) and non-residualizing labels ( 18 F-benzoate and 125 I-Tyr) attached to agonists [19,48,49].
Previous studies have shown that use of lysosomotropic bases can reduce lysosomal degradation of radiohalogenated targeting agents and increase intercellular retention and uptake [50].  15) linked to DOTA as the chelator switched its pharmacological function to an agonist [51]. In that study, authors concluded that prediction of the influence of a peptide modification on its functional profile by adding a chelator is not possible and properties has to be tested experimentally. This study demonstrates for the first time that the internalization pattern of bombesin analogues might be changed by modification of the N-terminus. This may be an important information to consider in molecular design of radiolabelled peptides.
We have used a dual label approach for comparison of [ 125 I]I-Tyr-PEG 2 -RM26 and [ 111 In]In-DOTA-PEG 2 -RM26, when two labelled conjugates are co-injected in the same mice, and the uptake of different labels in tissue is determined by gamma-spectroscopy. In this way, all factors related to an animal (e.g., hormonal status, heart rate, renal function) and to a tumour (e.g., vascularization, vascular permeability, and a target expression level) influence biodistribution of both compounds in the same way. This permits to reveal a significant diffidence in biodistribution with relatively low number of animals. The in vivo specificity study (Figure 7) showed that uptake of both [ 125 I]I-Tyr-PEG 2 -RM26 and [ 111 In]In-DOTA-PEG 2 -RM26 in tumours and GRPR-expressing tissues was specific. By co-injection with an excess of non-labelled peptide, tumour uptake was significantly (p < 0.05) reduced from 13 ± 5% ID/g to 1 ± 0% ID/g for [ 111 In]In-DOTA-PEG 2 -RM26 and from 7 ± 1% ID/g to 5 ± 1% ID/g for [ 125 I]I-Tyr-PEG 2 -RM26, indicating specific GRPR-mediated uptake. There was a significant (p < 0.05) reduction of the uptake of both tracers in GRPR-expressing pancreatic tissue.
The biodistribution of [ 125 I]I-Tyr-PEG 2 -RM26 and [ 111 In]In-DOTA-PEG 2 -RM26 in mice was quite different. Biodistribution of [ 111 In]In-DOTA-PEG 2 -RM26 was characterized by a rapid clearance of radioactivity from blood, GRPR-expressing organs, and other healthy organs and tissues, and the excretion of radioactivity was predominantly via kidney filtration. Clearance of activity from blood and other tissues after injection of [ 125 I]I-Tyr-PEG 2 -RM26 was noticeably slower and there was an appreciable hepatic uptake. The high accumulation of activity in organs with expression of Na/I-symporters (thyroid, salivary glands and stomach) was observed for [ 125 I]I-Tyr-PEG 2 -RM26, which indicates a re-distribution of radiometabolites. Tumor uptake for [ 111 In]In-DOTA-PEG 2 -RM26 (13 ± 5% ID/g) was significantly (p < 0.05) higher than for [ 125 I]I-Tyr-PEG 2 -RM26 (7 ± 1% ID/g) at 0.5 h after injection.
Tumor uptake of [ 125 I]I-Tyr-PEG 2 -RM26 decreased more than 3-fold at 3 h compared to 0.5 h after injection, while decrease of tumour uptake was not so dramatic for [ 111 In]In-DOTA-PEG 2 -RM26. The clearance of [ 111 In]In-DOTA-PEG 2 -RM26 from non-targeted organs was very fast, which resulted in a significant (p < 0.05) higher tumour-to-organ ratio at 3 h after injection.
Due to the relatively small size of the peptides, modification of their structure can influence their affinity and selectivity for the targeted receptors. Previous studies of X-PEG 2 -RM26 (X = NOTA, NODAGA, DOTA, and DOTAGA) containing different chelators labelled with 111 In and 68 Ga showed that changing the radiometal and chelator can substantially influence the affinity and biodistribution profile [31,41]. All analogues showed antagonistic properties confirmed by the slow internalization rate after binding to the GRPR.
Degradation of radiopeptides, including bombesin-like peptides, by proteolytic enzymes present in blood, vasculature walls, liver, lungs, kidney, and gastrointestinal tract limits their successful application as theranostic probes [52,53]. These enzymes can potentially hamper radiopeptide-based imaging and therapy by cleaving radiopeptides into inactive radiometabolites. Previous studies have shown that co-injection of the enzyme inhibitor phosphoramidon (PA), can stabilize radiopeptides in vivo, resulting in a remarkable increase of tumour uptake, whereas uptake in non-target healthy organs and tissues is not affected [54]. The results of biodistribution of [ 125 I]I-Tyr-PEG2-RM26 in non-tumourbearing mice demonstrated that co-injection of PA could not improve blood clearance rate or uptake in healthy organs and tissues. Thereby, degradation of radiolabelled conjugate cannot be the main reason for the high uptake in non-targeted organs and slow blood clearance rate.
Taken together, in vitro and in vivo data suggest that conjugation of tyrosine at N-terminus of PEG 2 -RM26 results in an appreciably more rapid internalization compared to the internalization of PEG 2 -RM26 with DOTA conjugated at N-terminus. The use of a non-residualizing radiohalogen label causes a poor retention of activity by malignant cells both in vitro and in vivo. Furthermore, blood clearance of [ 125 I]I-Tyr-PEG 2 -RM26 is slower comparing to [ 111 In]In-DOTA-PEG 2 -RM26. Studies with other peptides [55,56] suggest that slow clearance might be due to binding of blood proteins caused by an elevated lipophilicity. Overall, such features are undesirable for radionuclide therapy.
The undesirable features of [ 125 I]I-Tyr-PEG 2 -RM26 are highly likely associated with the presence of lipophilic side-chain of tyrosine at N-terminus. At the same time, the presence of phenolic moiety is critical for radioiodination and endowing of non-residualizing property of the label. Obviously, the improved molecular design of a GRPR antagonist with non-residualizing label should keep the presence of a tyrosine at N-terminus but foresee a compensation of its lipophilicity. Earlier studies have shown that increase of overall and local hydrophilicity of targeted polypeptides by incorporation of amino acids with polar or charged side-chain in a proximity to a label facilitates their blood clearance [57,58] and reduces hepatic uptake [57][58][59][60]. An incorporation of a glutamate had the strongest benign affect in these studies. However, both this and previous studies [33,34] show that a placement of negatively charged moiety decreases affinity of RM26 binding to GRPR. Therefore, we consider the use of amino acids with neutral polar or positively charged side-chains, i.e., glutamine and lysine. For preliminary assessment of hydrophilicity of the combination of these amino acids with tyrosine, logP of the oligopeptides was calculated using ChemDraw Ultra assuming amidated C-tremini (CambfidgeSoft, Cambridge, MA, USA) and compared with logP of tyrosine ( Figure 9). Apparently, addition of glutamine and lysine to tyrosine would enhance hydrophilicity of the N-terminus of tyrosine-containing RM26 analogue. Particularly a Tyr-Gln-Gln-combination looks promising. The obvious next step should be preparation of a small library of RM26 analogues containing both tyrosine and glutamine and their comparative evaluation.

Conclusions
The results showed that use of [ 125 I]I-Tyr-PEG2-RM26 was not advantageously for radionuclide targeting of PC compared with [ 111 In]In-DOTA-PEG2-RM26 as hypothesized. An introduction of a hydrophobic tyrosine group at N-terminus of RM26 changed cellular processing and biodistribution patterns of antagonistic bombesin analogue making them unfavourable for radionuclide therapy. Incorporation of amino acids with hydrophilic side-chains in the proximity to tyrosine might improve the biodistribution pattern of a radioiodinated tyrosine-containing RM26 analogue.   Apparently, addition of glutamine and lysine to tyrosine would enhance hydrophilicity of the N-terminus of tyrosine-containing RM26 analogue. Particularly a Tyr-Gln-Gln-combination looks promising. The obvious next step should be preparation of a small library of RM26 analogues containing both tyrosine and glutamine and their comparative evaluation.

Conclusions
The results showed that use of [ 125 I]I-Tyr-PEG 2 -RM26 was not advantageously for radionuclide targeting of PC compared with [ 111 In]In-DOTA-PEG 2 -RM26 as hypothesized. An introduction of a hydrophobic tyrosine group at N-terminus of RM26 changed cellular processing and biodistribution patterns of antagonistic bombesin analogue making them unfavourable for radionuclide therapy. Incorporation of amino acids with hydrophilic side-chains in the proximity to tyrosine might improve the biodistribution pattern of a radioiodinated tyrosine-containing RM26 analogue.