Radiometal Complexes as Pharmacokinetic Modifiers: A Potent 68Ga-Labeled Gastrin-Releasing Peptide Receptor Antagonist Based on the Macrocyclic Metal Chelator NODIA-Me.

The gastrin-releasing peptide receptor (GRPr) is overexpressed in various cancer types including prostate and breast carcinomas, making it an attractive target for molecular imaging and therapy. In this work, we designed a novel GRPr antagonistic probe comprising metal chelator NODIA-Me. This 1,4,7-triazacyclononane-based chelator forms positively charged metal complexes due to its neutral methylimidazole arms. Because a positive charge at the N-terminus of GRPr conjugates is responsible for high receptor affinity as exemplified by the current gold standard DOTA-RM2, we investigated if a positively charged radiometal complex can be used as a pharmacokinetic modifier to also produce high-affinity GRPr conjugates. In this respect, the bioconjugate NODIA-Me-Ahx-JMV594 was prepared by a combination of solid-phase peptide synthesis and solution-based reactions in a 94% yield. Radiolabeling provided the 68Ga-labeled conjugate in radiochemical yields of >95% and radiochemical purities of >98% with mean molar activities of Am ∼17 MBq nmol-1. The competitive GRPr affinity of the metal-free and 69/71Ga-labeled conjugate was determined to be IC50 = 0.41 ± 0.06 and 1.45 ± 0.06 nM, respectively. The metal-free GRPr antagonist DOTA-RM2 and its corresponding 69/71Ga complex had IC50 values of 1.42 ± 0.07 and 0.98 ± 0.19 nM, respectively. Small-animal PET imaging of mice bearing GRPr(+) PC-3 tumors revealed high radioactivity accumulation in the tumors and in the pancreas as an organ with high levels of GRPr expression. These findings were corroborated by the corresponding ex vivo biodistribution data, in which the tumors and the pancreas exhibited the highest radioactivity accumulation. By coinjection of an excess of NODIA-Me-Ahx-JMV594, uptake in the tumors and GRPr(+) organs was significantly reduced, confirming specific receptor-mediated uptake. The estrogen receptor-positive tumor of a female breast cancer patient was clearly visualized by PET imaging using 68Ga-labeled NODIA-Me-Ahx-JMV594. To summarize, the positive charge at the N-terminus of the conjugate induced by the Ga(NODIA-Me) complex resulted in high GRPr affinity comparable to that of the potent antagonist DOTA-RM2. The conjugate NODIA-Me-Ahx-JMV594 is a promising probe for imaging of GRPr tumors that warrants further evaluation in larger patient cohorts as well as in combination with other radiometals.

Novel 64Cu Labeled RGD2-BBN Heterotrimers for PET Imaging of Prostate Cancer.

Bombesin receptor 2 (BB2) and integrin αvß3 receptor are privileged targets for molecular imaging of cancer because of their overexpression in a number of tumor tissues. The most recent developments in heterodimer-based radiopharmaceuticals concern BB2- and integrin αvß3-targeting compounds, consisting of bombesin (BBN) and cyclic arginine-glycine-aspartic acid peptides (RGD), connected through short length linkers. Molecular imaging probes based on RGD-BBN heterodimer design exhibit improved tumor targeting efficacy compared to the single-receptor targeting peptide monomers. However, their application in clinical study is restricted because of inefficient synthesis or unfavorable in vivo properties, which could depend on the short linker nature. Thus, the aim of the present study was to develop a RGD2-BBN heterotrimer, composed of (7-14)BBN-NH2 peptide (BBN) linked to the E[ c(RGDyK)]2 dimer peptide (RGD2), bearing the new linker type [Pro-Gly]12. The heterodimer E[c(RGDyK)]2-PEG3-Glu-(Pro-Gly)12-BBN(7-14)-NH2 (RGD2-PG12-BBN) was prepared through conventional solid phase synthesis, then conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODA-GA). In 64Cu labeling, the NODA-GA chelator showed superior radiochemical characteristics compared to DOTA (70% vs 40% yield, respectively). Both conjugates displayed dual targeting ability, showing good αvß3 affinities and high BB2 receptor affinities which, in the case of the NODA-GA conjugate, were in the same range as the best RGD-BBN heterodimer ligands reported to date ( Ki = 24 nM). 64Cu-DOTA and 64Cu-NODA-GA probes were also found to be stable after 1 h incubation in mouse serum (>90%). In a microPET study in prostate cancer PC-3 xenograft mice, both probes showed low tumor uptake, probably due to poor pharmacokinetic properties in vivo. Overall, our study demonstrates that novel RGD-BBN heterodimer with long linker can be prepared and they preserve high binding affinities to BB2 and integrin αvß3 receptor binding ability. The present study represents a step forward in the design of effective heterodimer or heterotrimer probes for dual targeting.

Rerouting the metabolic pathway of (18)F-labeled peptides: the influence of prosthetic groups.

Current translational cancer research is directed to the development of high affinity peptide ligands for targeting neuropeptide receptors overexpressed in different types of cancer. Besides their desired high binding affinity to the receptor, the suitability of radiolabeled peptides as targeting vectors for molecular imaging and therapy depends on additional aspects such as high tumor-to-background ratio, favorable clearance pattern from nontarget tissue, and sufficient metabolic stability in vivo. This study reports how a switch from the prosthetic group, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB), to 2-deoxy-2-[(18)F]fluoro-d-glucose ([(18)F]FDG) effects the metabolic pathway of an (18)F-labeled bombesin derivative, QWAV-Sar-H-FA01010-Tle-NH2. (18)F-Labeled bombesin derivatives represent potent peptide ligands for selective targeting of gastrin-releasing peptide (GRP) receptor-expressing prostate cancer. Radiosynthesis of (18)F-labeled bombesin analogues [(18)F]FBz-Ava-BBN2 and [(18)F]FDG-AOAc-BBN2 was achieved in good radiochemical yields of ~50% at a specific activity exceeding 40 GBq/µmol. Both nonradioactive compounds FBz-Ava-BBN2 and FDG-AOAc-BBN2 inhibited binding of [(125)I]Tyr(4)-bombesin(1-14) in PC3 cells with IC50 values of 9 and 16 nM, respectively, indicating high inhibitory potency. Influence of each prosthetic group was further investigated in PC3 mouse xenografts using dynamic small animal PET imaging. In comparison to [(18)F]FBz-Ava-BBN2, total tumor uptake levels were doubled after injection of [(18)F]FDG-AOAc-BBN2 while renal elimination was increased. Blood clearance and in vivo metabolic stability were similar for both compounds. The switch from [(18)F]SFB to [(18)F]FDG as the prosthetic group led to a significant reduction in lipophilicity which resulted in more favorable renal clearance and increased tumor uptake. The presented single step radiolabeling-glycosylation approach represents an innovative strategy for site-directed peptide labeling with the short-lived positron emitter (18)F while providing a favorable pharmacokinetic profile of (18)F-labeled peptides.

The diagnostic role of 99mTc-dual receptor targeted probe and targeted peptide bombesin (RGD-BBN) SPET/CT in the detection of malignant and benign breast tumors and axillary lymph nodes compared to ultrasound.

OBJECTIVE: This study aimed to explore the diagnostic role of a new dual receptor-targeted probe, integrin ανß3 and gastrin releasing peptide receptor (GRPR) targeted peptide Glu-c(RGDyK)-bombesin (RGD-BBN) labeled with technetium-99m ((99m)Tc-RGD-BBN), using single photon emission tomography/computed tomography (SPET/CT) in the detection of breast tumor in comparison to ultrasound (US). SUBJECTS AND METHODS: One hundred and twenty six female patients with suspicious breast lesions who had already been scheduled for biopsy or surgery were enrolled in this study. All patients had previously underwent breast US and (99m)Tc-RGD-BBN SPET/CT. The US findings were evaluated according to the breast imaging report and the data system (BI-RADS). Technetium-99m-RGD-BBN SPET/CT images were interpreted independently by two experienced nuclear medicine physicians. A final diagnosis was made by histopathology of the specimens. A total of 130 lesions, 77 malignant and 53 benign lesions were ascertained. One hundred and twelve breast lesions, 69 malignant and 43 benign lesions were above 10mm in diameter and 18 breast lesions (8 malignant lesions and 10 benign lesions) were below 10mm. The overall sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of (99m)Tc-RGD-BBN SPET/CT and US for breast lesions were 93.5% vs. 81.8% (P<0.05), 79.2% vs. 75.5% (P>0.05), 86.7% vs. 82.9% (P>0.05), 89.4% vs. 74.1% (P<0.05) and 87.7% vs. 79.2% (P>0.05). Technetium-99m-RGD-BBN SPET/CT detected all lesions ≥10mm and US only detected 57 (P<0.05). In malignant lesions <10mm, US was superior than (99m)Tc-RGD-BBN SPET/CT (75.0% vs. 37.5%, P<0.05). There was no significant difference between the two methods no matter the size of the benign lesions. The overall sensitivity and specificity of (99m)Tc-RGD-BBN SPET/CT and US for axillae lymph nodes were 87.5% vs. 71.9% (P<0.05) and 77.6% vs. 68.9% (P>0.05), respectively. For the metastatic lymph nodes of <10mm, the sensitivity of (99m)Tc-RGD-BBN SPET/CT and of US was 88.5% and 72.1% respectively (P<0.05). Statistical analysis was not performed due to the small number of metastatic lesions of <10mm. The specificity of (99m)Tc-RGD-BBN SPET/CT and of US was not different, no matter the size of the axilla lymph nodes that had no metastases (P>0.05). Technetium-99m-RGD-BBN SPET/CT had higher sensitivity and NPV than US in detecting primary breast tumors and axilla lymph nodes and it also showed an advantage in distance metastatic lesions detection. On the contrary, specificity and PPV of the two methods were not different. CONCLUSION: Technetium-99m-RGD-BBN SPET/CT cannot totally replace US in the detection of primary breast cancer and axillary lymph nodes metastases. It can be used as an additional imaging tool of eliminating the necessity of surgical biopsy and histopathologic examination because of its high NPV.

Synthesis and in vitro and in vivo evaluation of SiFA-tagged bombesin and RGD peptides as tumor imaging probes for positron emission tomography.

Gastrin-releasing-peptide (GRP)-receptors and αvß3-integrins are widely discussed as potential target structures for oncological imaging with positron emission tomography (PET). Favored by the overexpression of receptors on the surface of tumor cells good imaging characteristics can be achieved with highly specific radiolabeled receptor ligands. PEGylated bombesin (PESIN) derivatives as specific GRP receptor ligands and RGD (one-letter codes for arginine-glycine-aspartic acid) peptides as specific αvß3 binders were synthesized and tagged with a silicon-fluorine-acceptor (SiFA) moiety. The SiFA synthon allows for a fast and highly efficient isotopic exchange reaction at room temperature giving the [(18)F]fluoride labeled peptides in up to 62% radiochemical yields (d.c.) and ≥99% radiochemical purity in a total synthesis time of less than 20 min. Using nanomolar quantities of precursor high specific activities of up to 60 GBq µmol(-1) were obtained. To compensate the high lipophilicity of the SiFA moiety various hydrophilic structure modifications were introduced leading to significantly reduced logD values. Competitive displacement experiments with the PESIN derivatives showed a 32 to 6 nM affinity to the GRP receptor on PC3 cells, and with the RGD peptides a 7 to 3 µM affinity to the αvß3 integrins on U87MG cells. All derivatives proved to be stable in human plasma over at least 120 min. Small animal PET measurements and biodistribution studies revealed an enhanced and specific accumulation of the RGD peptide (18)F-SiFA-LysMe3-γ-carboxy-d-Glu-RGD (17) in the tumor tissue of U87MG tumor-bearing mice of 5.3% ID/g whereas the PESIN derivatives showed a high liver uptake and only a low accumulation in the tumor tissue of PC3 xenografts. Stability studies with compound 17 provided further information on its metabolism in vivo. These results altogether demonstrate that the reduction of the overall lipophilicity of SiFA tagged RGD peptides is a promising approach for the generation of novel potent (18)F-labeled imaging agents.

The effect of mini-PEG-based spacer length on binding and pharmacokinetic properties of a 68Ga-labeled NOTA-conjugated antagonistic analog of bombesin.

The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide-receptor mediated radionuclide imaging and therapy. We have previously shown that an antagonist analog of bombesin RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethyleneglycol (PEG2) spacer (NOTA-PEG2-RM26) and labeled with 68Ga can be used for imaging of GRPR-expressing tumors. In this study, we evaluated if a variation of mini-PEG spacer length can be used for optimization of targeting properties of the NOTA-conjugated RM26. A series of analogs with different PEG-length (n = 2, 3, 4, 6) was synthesized, radiolabeled and evaluated in vitro and in vivo. The IC50 values of natGa-NOTA-PEGn-RM26 (n = 2, 3, 4, 6) were 3.1 ± 0.2, 3.9 ± 0.3, 5.4 ± 0.4 and 5.8 ± 0.3 nM, respectively. In normal mice all conjugates demonstrated similar biodistribution pattern, however 68Ga-NOTA-PEG3-RM26 showed lower liver uptake. Biodistribution of 68Ga-NOTA-PEG3-RM26 was evaluated in nude mice bearing PC-3 (prostate cancer) and BT-474 (breast cancer) xenografts. High uptake in tumors (4.6 ± 0.6%ID/g and 2.8 ± 0.4%ID/g for PC-3 and BT-474 xenografts, respectively) and high tumor-to-background ratios (tumor/blood of 44 ± 12 and 42 ± 5 for PC-3 and BT-474 xenografts, respectively) were found already at 2 h p.i. of 68Ga-NOTA-PEG3-RM26. Results of this study suggest that variation in the length of the PEG spacer can be used for optimization of targeting properties of peptide-chelator conjugates. However, the influence of the mini-PEG length on biodistribution is minor when di-, tri-, tetra- and hexaethylene glycol are compared.

Hybrid bombesin analogues: combining an agonist and an antagonist in defined distances for optimized tumor targeting.

Radiolabeled hybrid ligands with defined distances between an agonist and an antagonist for the gastrin-releasing peptide receptor were found to have excellent tumor-targeting properties. Oligoprolines served as rigid scaffolds that allowed for tailoring distances of 10, 20, and 30 Å between the recognition elements. In vitro and in vivo studies revealed that the hybrid ligand with a distance of 20 Å between the recognition elements exhibits the highest yet observed tumor cell uptake and retention time in prostate cancer cells.

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity.

Development of cancer receptor-specific gold nanoparticles will allow efficient targeting/optimum retention of engineered gold nanoparticles within tumors and thus provide synergistic advantages in oncology as it relates to molecular imaging and therapy. Bombesin (BBN) peptides have demonstrated high affinity toward gastrin-releasing peptide (GRP) receptors in vivo that are overexpressed in prostate, breast, and small-cell lung carcinoma. We have synthesized a library of GRP receptor-avid nanoplatforms by conjugating gold nanoparticles (AuNPs) with BBN peptides. Cellular interactions and binding affinities (IC(50)) of AuNP-BBN conjugates toward GRP receptors on human prostate cancer cells have been investigated in detail. In vivo studies using AuNP-BBN and its radiolabeled surrogate (198)AuNP-BBN, exhibiting high binding affinity (IC(50) in microgram ranges), provide unequivocal evidence that AuNP-BBN constructs are GRP-receptor-specific showing accumulation with high selectivity in GRP-receptor-rich pancreatic acne in normal mice and also in tumors in prostate-tumor-bearing, severe combined immunodeficient mice. The i.p. mode of delivery has been found to be efficient as AuNP-BBN conjugates showed reduced RES organ uptake with concomitant increase in uptake at tumor targets. The selective uptake of this new generation of GRP-receptor-specific AuNP-BBN peptide analogs has demonstrated realistic clinical potential in molecular imaging via x-ray computed tomography techniques as the contrast numbers in prostate tumor sites are severalfold higher as compared to the pretreatment group (Hounsfield unit = 150).

Novel radiolabeled peptides for breast and prostate tumor PET imaging: (64)Cu/and (68)Ga/NOTA-PEG-[D-Tyr(6),ßAla(11),Thi(13),Nle(14)]BBN(6-14).

Bombesin (BBN)-based radiolabeled peptides exhibit promising properties for targeted imaging of gastrin-releasing peptide receptors (GRPR)-positive tumors. The aim of this study was to evaluate with positron emission tomography (PET) the pharmacokinetic and imaging properties of two novel BBN-based radiolabeled peptides, (64)Cu/and (68)Ga/NOTA-PEG-BBN(6-14), for diagnosis of breast and prostate cancers using small animal models. Competitive binding assays on T47D breast and PC3 prostate cancer cells showed that the affinity for GRPR depends on the complexed metal and can vary up to a factor of about 3; (64)Cu/NOTA-PEG-BBN(6-14) was found to have the lowest inhibition constant (1.60 ± 0.59 nM). (64)Cu/and (68)Ga/NOTA-PEG-BBN(6-14) presented similar cell uptake on T47D and PC3 cells and were stable in vivo. Biodistribution studies of radiolabeled peptides carried out in Balb/c and tumor-bearing Balb/c nude mice showed that (64)Cu/NOTA-PEG-BBN(6-14) presented higher GRPR-mediated uptake in pancreas and adrenal glands, but comparable PC3 tumor uptake as (68)Ga/NOTA-PEG-BBN(6-14). Finally, receptor-dependent responses were observed during blocking studies with unlabeled peptide in both biodistribution and small-animal PET imaging studies. Our results confirmed the dependence of the affinity and pharmacokinetics of BBN-based radiopeptides on the complexed radiometal. Interspecies differences between mouse and human GRPR binding properties were also noted in these preclinical studies. Considering their good imaging characteristics, both (64)Cu/NOTA-PEG-BBN(6-14) and (68)Ga/NOTA-PEG-BBN(6-14) are promising candidates for GRPR-targeted PET imaging of breast and prostate cancers.

A Radiotracer for Molecular Imaging and Therapy of Gastrin-Releasing Peptide Receptor-Positive Prostate Cancer.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in many solid malignancies, particularly in prostate and breast cancers, among others. We synthesized ProBOMB2, a novel bombesin derivative radiolabeled with 68Ga and 177Lu, and evaluated its ability to target GRPR in a preclinical model of human prostate cancer. Methods: ProBOMB2 was synthesized in solid phase using fluorenylmethoxycarbonyl chemistry. The chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid was coupled to the N terminus and separated from the GRPR-targeting sequence by a cationic 4-amino-(1-carboxymethyl)-piperidine spacer. Binding affinity for both human and murine GRPR was determined using a cell-based competition assay, whereas a calcium efflux assay was used to measure the agonist and antagonist properties of the derivatives. ProBOMB2 was radiolabeled with 177Lu and 68Ga. SPECT and PET imaging and biodistribution studies were conducted using male immunocompromised mice bearing GRPR-positive PC-3 human prostate cancer xenografts. Results: Ga-ProBOMB2 and Lu-ProBOMB2 bound to PC-3 cells with an inhibition constant of 4.58 ± 0.67 and 7.29 ± 1.73 nM, respectively. 68Ga-ProBOMB2 and 177Lu-ProBOMB2 were radiolabeled with a radiochemical purity greater than 95%. Both radiotracers were excreted primarily via the renal pathway. PET images of PC-3 tumor xenografts were visualized with excellent contrast at 1 and 2 h after injection with 68Ga-ProBOMB2, and there was very low off-target organ accumulation. 177Lu-ProBOMB2 enabled clear visualization of PC-3 tumor xenografts by SPECT imaging at 1, 4, and 24 h after injection 177Lu-ProBOMB2 displayed higher tumor uptake than 68Ga-ProBOMB2 at 1 h after injection. 177Lu-ProBOMB2 tumor uptake at 1, 4, and 24 h after injection was 14.9 ± 3.1, 4.8 ± 2.1, and 1.7 ± 0.3 percentage injected dose per gram of tissue, respectively. Conclusion: 68Ga-ProBOMB2 and 177Lu-ProBOMB2 are promising radiotracers with limited pancreas uptake, good tumor uptake, and favorable pharmacokinetics for imaging and therapy of GRPR-expressing tumors.

Gastrin-releasing peptide receptor-based targeting using bombesin analogues is superior to metabolism-based targeting using choline for in vivo imaging of human prostate cancer xenografts.

PURPOSE: Prostate cancer (PC) is a major health problem. Overexpression of the gastrin-releasing peptide receptor (GRPR) in PC, but not in the hyperplastic prostate, provides a promising target for staging and monitoring of PC. Based on the assumption that cancer cells have increased metabolic activity, metabolism-based tracers are also being used for PC imaging. We compared GRPR-based targeting using the (68)Ga-labelled bombesin analogue AMBA with metabolism-based targeting using (18)F-methylcholine ((18)F-FCH) in nude mice bearing human prostate VCaP xenografts. METHODS: PET and biodistribution studies were performed with both (68)Ga-AMBA and (18)F-FCH in all VCaP tumour-bearing mice, with PC-3 tumour-bearing mice as reference. Scanning started immediately after injection. Dynamic PET scans were reconstructed and analysed quantitatively. Biodistribution of tracers and tissue uptake was expressed as percent of injected dose per gram tissue (%ID/g). RESULTS: All tumours were clearly visualized using (68)Ga-AMBA. (18)F-FCH showed significantly less contrast due to poor tumour-to-background ratios. Quantitative PET analyses showed fast tumour uptake and high retention for both tracers. VCaP tumour uptake values determined from PET at steady-state were 6.7 ± 1.4%ID/g (20-30 min after injection, N = 8) for (68)Ga-AMBA and 1.6 ± 0.5%ID/g (10-20 min after injection, N = 8) for (18)F-FCH, which were significantly different (p <0.001). The results in PC-3 tumour-bearing mice were comparable. Biodistribution data were in accordance with the PET results showing VCaP tumour uptake values of 9.5 ± 4.8%ID/g (N = 8) for (68)Ga-AMBA and 2.1 ± 0.4%ID/g (N = 8) for (18)F-FCH. Apart from the GRPR-expressing organs, uptake in all organs was lower for (68)Ga-AMBA than for (18)F-FCH. CONCLUSION: Tumour uptake of (68)Ga-AMBA was higher while overall background activity was lower than observed for (18)F-FCH in the same PC-bearing mice. These results suggest that peptide receptor-based targeting using the bombesin analogue AMBA is superior to metabolism-based targeting using choline for scintigraphy of PC.

A new 18F-labeled BBN-RGD peptide heterodimer with a symmetric linker for prostate cancer imaging.

A peptide heterodimer comprises two different receptor-targeting peptide ligands. Molecular imaging probes based on dual-receptor targeting peptide heterodimers exhibit improved tumor targeting efficacy for multi-receptor expressing tumors compared with their parent single-receptor targeting peptide monomers. Previously we have developed bombesin (BBN)-RGD (Arg-Gly-Asp) peptide heterodimers, in which BBN and RGD are covalently connected with an asymmetric glutamate linker (J Med Chem 52:425-432, 2009). Although (18)F-labeled heterodimers showed significantly better microPET imaging quality than (18)F-labeled RGD and BBN monomers in a PC-3 xenograft model which co-expresses gastrin-releasing peptide receptor (GRPR) and integrin αvß3, tedious heterodimer synthesis due to the asymmetric nature of glutamate linker restricts their clinical applications. In this study, we report the use of a symmetric linker AEADP [AEADP = 3,3'-(2-aminoethylazanediyl)dipropanoic acid] for the synthesis of BBN-RGD peptide heterodimer. The (18)F-labeled heterodimer ((18)F-FB-AEADP-BBN-RGD) showed comparable microPET imaging results with glutamate linked BBN-RGD heterodimers, indicating that the replacement of glutamate linker with AEADP linker did not affect the biological activities of BBN-RGD heterodimer. The heterodimer synthesis is rather easy and straightforward. Because tumors often co-express multiple receptors, the use of a symmetric linker provides a general method of fast assembly of various peptide heterodimers for imaging multi-receptor expressing tumors.

Tumor bombesin analog loaded long-circulating and pH-sensitive liposomes as tool for tumor identification.

Long-circulating and pH-sensitive liposomes trapping (99m)Tc-HYNIC-ßAla-bombesin((7-14)) (aSpHL-(99m)Tc-BBN((7-14))) were successfully prepared. Biodistribution studies and scintigraphic images were performed in Ehrlich tumor-bearing Swiss mice. This system showed high accumulation in tumor tissue with high tumor-to-muscle ratio. Therefore, aSpHL-(99m)Tc-BBN((7-14)) could be considered as a potential agent for tumor diagnosis.

In vivo MR/optical imaging for gastrin releasing peptide receptor of prostate cancer tumor using Gd-TTDA-NP-BN-Cy5.5.

Magnetic resonance imaging (MRI) has become the leading imaging tool for providing fine anatomical and physiology details. Optical imaging is offering a sensitive and specific method for in vivo molecular imaging of targeting molecules. The goal of this study is to design, synthesize, and characterize a new target-specific dual contrast agent for MR and optical imaging. Hence, [Gd(TTDA-NP)(H(2)O)](2-) was prepared and characterized. In addition, an 8-amino acid Bombesin analogue (BN) peptide substrate, which can target prostate, breast, and colon cancer, was synthesized by solid-phase peptide synthesis and subsequently conjugated with [Gd(TTDA-NP)(H(2)O)](2-) to form BN conjugated Gd-TTDA-NP-BN. The water-exchange rate (k(ex)(298)) for [Gd(TTDA-NP)(H(2)O)](2-) (110×10(6)s(-1)) is significantly higher than that of [Gd(DTPA)(H(2)O)](2-) complex and the rotational correlation time (τ(R)) for [Gd(TTDA-NP)(H(2)O)](2-) (145ps) is also higher than those of [Gd(TTDA)(H(2)O)](2-) (104ps) and [Gd(DTPA)(H(2)O)](2-) (103ps). The Gd-TTDA-NP-BN shows remarkable high relaxivity (7.12mM(-1)s(-1)) comparing to that of [Gd(TTDA-NP)(H(2)O)](2-). The fluorescence studies showed that the Gd-TTDA-NP-BN could efficiently enter PC-3 cells. Additionally, the human cancer cells xenografts using Gd-TTDA-NP-BN-Cy5.5 as an optical imaging probe clearly visualized subcutaneous PC-3 tumor and demonstrated its targeting ability to the gastrin releasing peptide (GRP) receptor overexpression. Furthermore, the biodistribution studies demonstrated significantly high tumor uptake (25.97±1.07% ID/g) and high tumor-to-normal tissue ratios at one hour post-injection of Gd-TTDA-NP-BN-Cy5.5 in the animal model. These results suggest that the Gd-TTDA-NP-BN-Cy5.5 is a superior probe for in vivo optical imaging. Importantly, the MR imaging studies showed notable signal enhancement (44.9±4.2%) on the tumor, indicating a high level accumulation of the contrast agent within the PC-3 tumor sites. Hence, targeting of prostate cancer cells was observed under in vitro and in vivo MR imaging studies using Gd-TTDA-NP-BN contrast agent. We conclude that Gd-TTDA-NP-BN and Gd-TTDA-NP-BN-Cy5.5 can be potentially used as the contrast agents for targeting GRP receptor overexpressing cells and tumors.

Androgen-regulated gastrin-releasing peptide receptor expression in androgen-dependent human prostate tumor xenografts.

Human prostate cancer (PC) overexpresses the gastrin-releasing peptide receptor (GRPR). Radiolabeled GRPR-targeting analogs of bombesin (BN) have successfully been introduced as potential tracers for visualization and treatment of GRPR-overexpressing tumors. A previous study showed GRPR-mediated binding of radiolabeled BN analogs in androgen-dependent but not in androgen-independent xenografts representing the more advanced stages of PC. We have further investigated the effect of androgen modulation on GRPR-expression in three androgen-dependent human PC-bearing xenografts: PC295, PC310 and PC82 using the androgen-independent PC3-model as a reference. Effects of androgen regulation on GRPR expression were initially studied on tumors obtained from our biorepository of xenograft tissues performing reverse transcriptase polymerase chain reaction (RT-PCR) and autoradiography ((125)I-universal-BN). A prospective biodistribution study ((111)In-MP2653) and subsequent autoradiography ((125)I-GRP and (111)In-MP2248) was than performed in castrated and testosterone resupplemented tumor-bearing mice. For all androgen-dependent xenografts, tumor uptake and binding decreased drastically after 7 days of castration. Resupplementation of testosterone to castrated animals restored GRPR expression extensively. Similar findings were concluded from the initial autoradiography and RT-PCR studies. Results from RT-PCR, for which human specific primers are used, indicate that variations in GRPR expression can be ascribed to mRNA downregulation and not to castration-induced reduction in the epithelial fraction of the xenograft tumor tissue. In conclusion, expression of human GRPR in androgen-dependent PC xenografts is reduced by androgen ablation and is reversed by restoring the hormonal status of the animals. This knowledge suggests that hormonal therapy may affect GRPR expression in PC tissue making GRPR-based imaging and therapy especially suitable for non-hormonally treated PC patients.

In vitro and in vivo characterization of novel 18F-labeled bombesin analogues for targeting GRPR-positive tumors.

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with (18)F, and characterized in vitro and in vivo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K(i) = 0.7 nM) and 7b (K(i) = 0.1 nM) were chosen for further in vivo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [(18)F]6b, compound [(18)F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In vivo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [(18)F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.

A standardised study to compare prostate cancer targeting efficacy of five radiolabelled bombesin analogues.

PURPOSE: Prostate-specific antigen (PSA)-based screening for prostate cancer (PC) has dramatically increased early diagnosis. Current imaging techniques are not optimal to stage early PC adequately. A promising alternative to PC imaging is peptide-based scintigraphy using radiolabelled bombesin (BN) analogues that bind to gastrin-releasing peptide receptors (GRPR) being overexpressed in PC. When labelled to appropriate radionuclides BN targeting of GRPRs may also provide applications for peptide radionuclide receptor therapy (PRRT). Assessment studies under identical experimental conditions allowing a reliable comparison of the potential of such analogues are lacking. This study was performed to evaluate and directly compare five promising radiolabelled BN analogues for their targeting efficacy for PC under standardised conditions. METHODS: The BN agonists [(111)In]DOTA-PESIN, [(111)In]AMBA, [(111)In]MP2346 and [(111)In]MP2653 and one antagonist [(99m)Tc]Demobesin-1 were evaluated in GRPR-overexpressing human PC-3 tumour-bearing mice to determine peptide stability in vivo, biodistribution and GRPR targeting potential by animal SPECT/CT imaging and ex vivo autoradiography. RESULTS: HPLC analysis of blood showed intact Demobesin-1 at 5 and 15 min after injection (64.1 +/- 1.6% and 41.0 +/- 01%, respectively) being much less for the other compounds. AMBA, the second most stable analogue, showed 36.1 +/- 2.7% and 9.8 +/- 1.1% intact peptide after 5 and 15 min. PC-3 tumour uptake at 1 h was comparable for Demobesin-1, AMBA, PESIN and MP2346 (3.0 +/- 0.4, 2.7 +/- 0.5, 2.3 +/- 0.5 and 2.1 +/- 0.9%ID/g, respectively), but very low for MP2653 (0.9 +/- 0.2%ID/g). In addition, MP2346 showed undesirably high uptake in the kidneys (7.9 +/- 1.9%ID/g) being significantly less for the other analogues. AMBA, MP2346 and PESIN revealed favourable increases in tumour to blood ratios over time while changes in tumour to kidney and pancreas ratios for Demobesin-1 from 1 to 24 h after injection were significantly better than for the other analogues. All analogues visualised PC-3 tumours by SPECT/CT and autoradiography. CONCLUSION: In the present study the BN antagonist Demobesin-1 was the best performing analogue showing superior in vivo stability, highest tumour uptake and retention while pancreatic and renal clearance were rapid. PESIN and AMBA were the best GRP agonists with sufficient in vivo stabilities as well as high tumour uptake and retention. Based on these results all three analogues deserve further evaluation for clinical use in PC patients.

Development of a new bombesin analog radiolabeled with lutetium-177: in vivo evaluation of the biological properties in Balb-C mice.

In this work we describe the first results of radiolabeling with lutetium-177 ((177)Lu) and in vivo biodistribution and pharmacokinetics studies in normal Balb-c mice of a new bombesin analog (BEFG2)--DOTA-Phe-X-BBN(6-14), where X is a spacer of two aminoacids. Bombesin (BBN) is an amphibian analog of human gastrin releasing peptide (GRP). Development of radiolabeled BBN derivatives as agents for diagnostic imaging and systemic radiotherapy has increased considerable because of the observation that GRP receptors (GRPr) are over-expressed in a variety of human tumor cells, such as prostate tumor cells. (177)Lu-labeled peptides are attractive due to the excellent radiophysical properties and commercial availability of the radiometal. BEFG2 was successfully labeled with high yield and kept stable for more than 96 hours at 2-8 degrees C and 1 hour in human plasma. Data analysis obtained from the in vivo studies showed that the amount of BEFG2 present in plasma decreased rapidly and became almost undetectable at 60 min p.i., indicating rapid peptide excretion, which is performed mainly by renal pathway. In addition, biodistribution and single photon emission tomography showed low abdominal accumulation of (177)Lu-DOTA- Phe-X-BBN(6-14), indicating that this analog is a potential candidate for tumors target therapy.

The Effects of the Spacer on Radiochemical and Biological Properties of New Radiolabeled Bombesin(7-14) Derivative.

OBJECTIVE: The aim of this study was to develop 99mTc-[HYNIC-X-D-Phe13]-BBN(7-14)NH2 derivatives using two different tripeptidic spacer groups (X=GGG and X=SSS) in order to improve its pharmacokinetics, in vitro stability, specific binding, and affinity. BACKGROUND: Bombesin (BBN), a 14-aminoacid amphibian peptide homolog of mammalian gastrinreleasing peptide (GRP), has demonstrated the ability to bind with high affinity and specificity to GRP receptor, which is overexpressed on a variety of human cancers. METHODS: Peptide conjugates labeled with 99mTc using tricine-EDDA and radiochemical purity was assessed by TLC and HPLC. The stability of radio conjugates was evaluated in the presence of saline and human serum. Affinity, internalization, and also dissociation Constant was evaluated using MDAMB- 231 and PC-3 cell line. Biodistribution study was performed in BALB/C mice. RESULTS: Labeling yield of ˃95% was obtained. The change introduced in the BBN sequence increased plasma stability. In vitro blocking studies showed that binding and internalization of both radiolabeled peptides are mediated by their receptors on the surface of MDA-MB-231 and PC-3 cells. Biodistribution results demonstrated a rapid blood clearance, with predominantly renal excretion. Specific binding in GRP receptor-positive tissues, such as pancreas was confirmed with a blocking study. CONCLUSION: The introduction of the spacer sequence between chelator and BBN(7-14) led to improved bidistribution. Analog with tri-Gly spacer is the more promising radiopeptide for targeting GRP receptors than Ser conjugates. Therefore, these analogs can be considered as a candidate for the identification of bombesin-positive tumors.

MITIGATE-NeoBOMB1, a Phase I/IIa Study to Evaluate Safety, Pharmacokinetics, and Preliminary Imaging of 68Ga-NeoBOMB1, a Gastrin-Releasing Peptide Receptor Antagonist, in GIST Patients.

Gastrin-releasing peptide receptors (GRPRs) are potential molecular imaging targets in a variety of tumors. Recently, a 68Ga-labeled antagonist to GRPRs, NeoBOMB1, was developed for PET. We report on the outcome of a phase I/IIa clinical trial (EudraCT 2016-002053-38) within the EU-FP7 project Closed-loop Molecular Environment for Minimally Invasive Treatment of Patients with Metastatic Gastrointestinal Stromal Tumors ('MITIGATE') (grant agreement no. 602306) in patients with oligometastatic gastrointestinal stromal tumors (GIST). Methods: The main objectives were evaluation of safety, biodistribution, dosimetry, and preliminary tumor targeting of 68Ga-NeoBOMB1 in patients with advanced tyrosine-kinase inhibitors-treated GIST using PET/CT. Six patients with histologically confirmed GIST and unresectable primary lesion or metastases undergoing an extended protocol for detailed pharmacokinetic analysis were included. 68Ga-NeoBOMB1 was prepared using a kit procedure with a licensed 68Ge/68Ga generator. 68Ga-NeoBOMB1 (3 MBq/kg of body weight) was injected intravenously, and safety parameters were assessed. PET/CT included dynamic imaging at 5, 11, and 19 min as well as static imaging at 1, 2, and 3-4 h after injection for dosimetry calculations. Venous blood samples and urine were collected for pharmacokinetic analysis. Tumor targeting was assessed on a per-lesion and per-patient basis. Results:68Ga-NeoBOMB1 (50 µg) was prepared with high radiochemical purity (yield > 97%). Patients received 174 ± 28 MBq of the radiotracer, which was well tolerated in all patients over a follow-up period of 4 wk. Dosimetry calculations revealed a mean effective dose of 0.029 ± 0.06 mSv/MBq, with the highest organ dose to the pancreas (0.274 ± 0.099 mSv/MBq). Mean plasma half-life was 27.3 min with primarily renal clearance (mean 25.7% ± 5.4% of injected dose 4 h after injection). Plasma metabolite analyses revealed high stability; metabolites were detected only in the urine. In 3 patients, a significant uptake with increasing maximum SUVs (SUVmax at 2 h after injection: 4.3-25.9) over time was found in tumor lesions. Conclusion: This phase I/IIa study provides safety data for 68Ga-NeoBOMB1, a promising radiopharmaceutical for targeting GRPR-expressing tumors. Safety profiles and pharmacokinetics are suitable for PET imaging, and absorbed dose estimates are comparable to those of other 68Ga-labeled radiopharmaceuticals used in clinical routine.