Imaging the Distribution of Gastrin-Releasing Peptide Receptors in Cancer.

Targeting tumor-expressed receptors using selective molecules for diagnostic, therapeutic, or both diagnostic and therapeutic (theragnostic) purposes is a promising approach in oncologic applications. Such approaches have increased significantly over the past decade. Peptides such as gastrin-releasing peptide receptors targeting radiopharmaceuticals are small molecules with fast blood clearance and urinary excretion. They demonstrate good tissue diffusion, low immunogenicity, and highly selective binding to their target cell-surface receptors. They are also easily produced. Gastrin-releasing peptide receptors, part of the bombesin family, are overexpressed in many tumors, including breast and prostate cancer, and therefore represent an attractive target for future development.

Radiometal-Dependent Biological Profile of the Radiolabeled Gastrin-Releasing Peptide Receptor Antagonist SB3 in Cancer Theranostics: Metabolic and Biodistribution Patterns Defined by Neprilysin.

Recent advances in oncology involve the use of diagnostic/therapeutic radionuclide-carrier pairs that target cancer cells, offering exciting opportunities for personalized patient treatment. Theranostic gastrin-releasing peptide receptor (GRPR)-directed radiopeptides have been proposed for the management of GRPR-expressing prostate and breast cancers. We have recently introduced the PET tracer 68Ga-SB3 (SB3, DOTA- p-aminomethylaniline-diglycolic acid-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt), a receptor-radioantagonist that enables the visualization of GRPR-positive lesions in humans. Aiming to fully assess the theranostic potential of SB3, we herein report on the impact of switching 68Ga to 111In/177Lu-label on the biological properties of resulting radiopeptides. Notably, the bioavailability of 111In/177Lu-SB3 in mice drastically deteriorated compared with metabolically robust 68Ga-SB3, and as a result led to poorer 111In/177Lu-SB3 uptake in GRPR-positive PC-3 xenografts. The peptide cleavage sites were identified by chromatographic comparison of blood samples from mice intravenously receiving 111In/177Lu-SB3 with each of newly synthesized 111In/177Lu-SB3-fragments. Coinjection of the radioconjugates with the neprilysin (NEP)-inhibitor phosphoramidon led to full stabilization of 111In/177Lu-SB3 in peripheral mouse blood and resulted in markedly enhanced radiolabel uptake in the PC-3 tumors. In conclusion, in situ NEP-inhibition led to indistinguishable 68Ga/111In/177Lu-SB3 profiles in mice emphasizing the theranostic prospects of SB3 for clinical use.

Gastrin-Releasing Peptide Receptor- and Prostate-Specific Membrane Antigen-Specific Ultrasmall Gold Nanoparticles for Characterization and Diagnosis of Prostate Carcinoma via Fluorescence Imaging.

Gold nanoparticles (AuNPs) have widely been used for 70 years in cancer treatment, but only in the last 15 years has the focus been on specific AuNPs with homogeneous size and shape for various areas in science. They constitute a perfect platform for multifunctionalization and therefore enable the enhancement of target affinity. Here we report on the development of tumor specific AuNPs as diagnostic tools intended for the detection of prostate cancer via fluorescence imaging and positron emission tomography (PET). The AuNPs were further evaluated in vitro and in vivo and exhibited favorable diagnostic properties concerning tumor cell uptake, biodistribution, clearance, and tumor retention.

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.

68Ga-BBN-RGD PET/CT for GRPR and Integrin αvß3 Imaging in Patients with Breast Cancer.

Purpose: This study was to assess a gastrin-releasing peptide receptor (GRPR) and integrin αvß3 dual targeting tracer 68Ga-BBN-RGD for positron emission tomography (PET)/computed tomography (CT) imaging of breast cancer and metastasis. Materials and Methods: Twenty-two female patients were recruited either with suspected breast cancer on screening mammography (n = 16) or underwent breast cancer radical mastectomy (n = 6). All the 22 patients underwent PET/CT at 30-45 min after intravenous injection of 68Ga-BBN-RGD. Eleven out of 22 patients also accepted 68Ga-BBN PET/CT within 2 weeks for comparison. A final diagnosis was made based on the histopathologic examination of surgical excision or biopsy. Results: Both the primary cancer and metastases showed positive 68Ga-BBN-RGD accumulation. The T/B ratios of 68Ga-BBN-RGD accumulation were 2.10 to 9.44 in primary cancer and 1.10 to 3.71 in axillary lymph node metastasis, 3.80 to 10.7 in distant lymph nodes, 2.70 to 5.35 in lung metastasis and 3.17 to 22.8 in bone metastasis, respectively. For primary lesions, the SUVmax from 68Ga-BBN-RGD PET in ER positive group was higher than that in ER negative group (P < 0.01). For both primary and metastatic lesions, SUVmean quantified from 68Ga-BBN-RGD PET correlated well with both GRPR expression and integrin αvß3 expression. Conclusion: This study demonstrated significant uptake of a new type of dual integrin αvß3 and GRPR targeting radiotracer in both the primary lesion and the metastases of breast cancer. 68Ga-BBN-RGD PET/CT may be of great value in discerning both primary breast cancers, axillary lymph node metastasis and distant metastases.

High Contrast PET Imaging of GRPR Expression in Prostate Cancer Using Cobalt-Labeled Bombesin Antagonist RM26.

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer. The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with 57Co and 55Co, stability, binding specificity, and in vitro and in vivo characteristics of 57Co-NOTA-PEG2-RM26 were studied. NOTA-PEG2-RM26 was successfully radiolabeled with 57Co and 55Co with high yields and demonstrated high stability. The radiopeptide showed retained binding specificity to GRPR in vitro and in vivo. 57Co-NOTA-PEG2-RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressing organs and low hepatic uptake. The clearance was predominantly renal with a low degree of radioactivity reabsorption. Tumor-to-blood ratios were approximately 200 (3 h pi) and 1000 (24 h pi). The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26 translated into high contrast preclinical PET/CT (using 55Co) and SPECT/CT (using 57Co) images of PC-3 xenografts. The initial biological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors.

Gastrin-releasing Peptide Receptor Imaging in Breast Cancer Using the Receptor Antagonist (68)Ga-RM2 And PET.

INTRODUCTION: The gastrin-releasing peptide receptor (GRPR) is overexpressed in breast cancer. The present study evaluates GRPR imaging as a novel imaging modality in breast cancer by employing positron emission tomography (PET) and the GRPR antagonist (68)Ga-RM2. METHODS: Fifteen female patients with biopsy confirmed primary breast carcinoma (3 bilateral tumors; median clinical stage IIB) underwent (68)Ga-RM2-PET/CT for pretreatment staging. In vivo tumor uptake of (68)Ga-RM2 was correlated with estrogen (ER) and progesterone (PR) receptor expression, HER2/neu status and MIB-1 proliferation index in breast core biopsy specimens. RESULTS: 13/18 tumors demonstrated strongly increased (68)Ga-RM2 uptake compared to normal breast tissue (defined as PET-positive). All PET-positive primary tumors were ER- and PR-positive (13/13) in contrast to only 1/5 PET-negative tumors. Mean SUVMAX of ER-positive tumors was 10.6±6.0 compared to 2.3±1.0 in ER-negative tumors (p=0.016). In a multivariate analysis including ER, PR, HER2/neu and MIB-1, only ER expression predicted (68)Ga-RM2 uptake (model: r(2) =0.55, p=0.025). Normal breast tissue showed inter- and intraindividually variable, moderate GRPR binding (SUVMAX 2.3±1.0), while physiological uptake of other organs was considerably less except pancreas. Of note, (68)Ga-RM2-PET/CT detected internal mammary lymph nodes with high (68)Ga-RM2 uptake (n=8), a contralateral axillary lymph node metastasis (verified by biopsy) and bone metastases (n=1; not detected by bone scan and CT). CONCLUSION: Our study demonstrates that (68)Ga-RM2-PET/CT is a promising imaging method in ER-positive breast cancer. In vivo GRPR binding assessed by (68)Ga-RM2-PET/CT correlated with ER expression in primary tumors of untreated patients.

Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs.

Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs.

In Vivo Stabilization of a Gastrin-Releasing Peptide Receptor Antagonist Enhances PET Imaging and Radionuclide Therapy of Prostate Cancer in Preclinical Studies.

A single tool for early detection, accurate staging, and personalized treatment of prostate cancer (PCa) would be a major breakthrough in the field of PCa. Gastrin-releasing peptide receptor (GRPR) targeting peptides are promising probes for a theranostic approach for PCa overexpressing GRPR. However, the successful application of small peptides in a theranostic approach is often hampered by their fast in vivo degradation by proteolytic enzymes, such as neutral endopeptidase (NEP). Here we show for the first time that co-injection of a NEP inhibitor (phosphoramidon (PA)) can lead to an impressive enhancement of diagnostic sensitivity and therapeutic efficacy of the theranostic (68)Ga-/(177)Lu-JMV4168 GRPR-antagonist. Co-injection of PA (300 µg) led to stabilization of (177)Lu-JMV4168 in murine peripheral blood. In PC-3 tumor-bearing mice, PA co-injection led to a two-fold increase in tumor uptake of (68)Ga-/(177)Lu-JMV4168, 1 h after injection. In positron emission tomography (PET) imaging with (68)Ga-JMV4168, PA co-injection substantially enhanced PC-3 tumor signal intensity. Radionuclide therapy with (177)Lu-JMV4168 resulted in significant regression of PC-3 tumor size. Radionuclide therapy efficacy was confirmed by production of DNA double strand breaks, decreased cell proliferation and increased apoptosis. Increased survival rates were observed in mice treated with (177)Lu-JMV4168 plus PA as compared to those without PA. This data shows that co-injection of the enzyme inhibitor PA greatly enhances the theranostic potential of GRPR-radioantagonists for future application in PCa patients.

GLP1- and GIP-producing cells rarely overlap and differ by bombesin receptor-2 expression and responsiveness.

The incretin hormones glucagon-like peptide-1 (GLP1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted from intestinal endocrine cells, the so-called L- and K-cells. The cells are derived from a common precursor and are highly related, and co-expression of the two hormones in so-called L/K-cells has been reported. To investigate the relationship between the GLP1- and GIP-producing cells more closely, we generated a transgenic mouse model expressing a fluorescent marker in GIP-positive cells. In combination with a mouse strain with fluorescent GLP1 cells, we were able to estimate the overlap between the two cell types. Furthermore, we used primary cultured intestinal cells and isolated perfused mouse intestine to measure the secretion of GIP and GLP1 in response to different stimuli. Overlapping GLP1 and GIP cells were rare (∼5%). KCl, glucose and forskolin+IBMX increased the secretion of both GLP1 and GIP, whereas bombesin/neuromedin C only stimulated GLP1 secretion. Expression analysis showed high expression of the bombesin 2 receptor in GLP1 positive cells, but no expression in GIP-positive cells. These data indicate both expressional and functional differences between the GLP1-producing 'L-cell' and the GIP-producing 'K-cell'.

Evaluation of three different families of bombesin receptor radioantagonists for targeted imaging and therapy of gastrin releasing peptide receptor (GRP-R) positive tumors.

Two new classes of radiolabeled GRP receptor antagonists are studied and compared with the well-established statine-based receptor antagonist DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM2, 1; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Sta:(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid). The bombesin-based pseudopeptide DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (RM7, 2), and the methyl ester DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-OCH3 (ARBA05, 3) analogues are labeled with (111)In and evaluated in vitro in PC-3 cell line and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was assessed by immunofluorescence-based receptor internalization and Ca(2+) mobilization assays. The conjugates showed good binding affinity, the IC50 value of 2 (3.2 ± 1.8 nM) being 2 and 10 times lower than 1 and 3. Compared to (111)In-1, (111)In-2 showed higher uptake in target tissues such as pancreas (1.5 ± 0.5%IA/g and 39.8 ± 9.3%IA/g at 4 h, respectively), whereas the compounds had similar tumor uptake (11.5 ± 2.4%IA/g and 11.8 ± 3.9%IA/g at 4h, respectively). The displacement of the radioligand in vivo was different in different receptor positive organs and depended on the displacing peptide.

Gastrin-releasing peptide receptor expression in lung cancer.

CONTEXT: Gastrin-releasing peptide receptors (GRPRs) activate mitogen-activated protein kinase signaling pathway primarily through epidermal growth factor receptor activation and are under investigation as a molecular target because they are overexpressed in several solid tumors. OBJECTIVE: To determine GRPR expression in both non-small cell lung carcinoma and small cell lung carcinoma, comparing results with clinical stages and demographic data. DESIGN: We analyzed the immunohistochemical expression of GRPR in 200 non-small cell lung carcinoma and 38 small cell lung carcinoma archival cases from 2004 to 2008. RESULTS: Non-small cell lung carcinoma cases tended to be higher GRPR expressers at a rate of 62.5% (weak, moderate, and strong expression in 41.5%, 13.5%, and 7.5%, respectively), compared with 52.62% in small cell lung carcinoma cases (weak, moderate, and strong expression in 34.21%, 15.78%, and 2.63%, respectively; P = .30). In non-small cell lung carcinoma there was a trend for higher percentages of strong expression in adenocarcinoma cases (10%; P = .67), and in patients with advanced stages (III and IV; 9.43% and 6.9%; P = .01). CONCLUSIONS: To the best of our knowledge, this is the first study to demonstrate GRPR tissue expression in a large population of patients with lung cancer. Although GRPR expression was similar in small cell and non-small cell carcinoma, the expression was more pronounced in an advanced-stage lung cancer, particularly in adenocarcinoma cases, and may represent a potential target for the development of new treatment approaches in this population.

miR-335 and miR-363 regulation of neuroblastoma tumorigenesis and metastasis.

BACKGROUND: microRNA (miRNA) functions broadly as post-transcriptional regulators of gene expression, and disproportionate miRNAs can result in dysregulation of oncogenes in cancer cells. We have previously shown that gastrin-releasing peptide receptor (GRP-R) signaling regulates tumorigenicity of neuroblastoma cells. Herein, we sought to characterize miRNA profile in GRP-R silenced neuroblastoma cells, and to determine the role of miRNAs on tumorigenicity and metastatic potential. METHODS: Human neuroblastoma cell lines, BE(2)-C and SK-N-SH, were used for our study. Stably transfected GRP-R silenced cells were assessed for miRNA profiles. Cells were transfected with miR-335, miR-363, or miR-CON, a nontargeting control, and in vitro assays were performed. In vivo functions of miR-335 and miR-363 were also assessed in a spleen-liver metastasis murine model. RESULTS: GRP-R silencing significantly increased expression of miR-335 and miR-363 in BE(2)-C cells. Overexpression of miR-335 and miR-363 decreased tumorigenicity as measured by clonogenicity, anchorage-independent growth, and metastasis determined by cell invasion assay and liver metastasis in vivo. CONCLUSION: We report, for the first time, that GRP-R-mediated tumorigenicity and increased metastatic potential in neuroblastoma are regulated, in part, by miR-335 and miR-363. A better understanding of the anti-tumor functions of miRNAs could provide valuable insights to discerning molecular mechanisms responsible for neuroblastoma metastasis.

2-Fluoropyridine prosthetic compounds for the 18F labeling of bombesin analogues.

Acetylene-bearing 2-[(18)F]fluoropyridines [(18)F]FPy5yne and PEG-[(18)F]FPyKYNE were prepared via efficient nucleophilic heteroaromatic [(18)F]fluorination of their corresponding 2-trimethylammoniumpyrdinyl precursors. The prosthetic groups were conjugated to azide- and PEG3-modified bombesin(6-14) analogues via copper-catalyzed azide-alkyne cycloaddition couplings to yield mono- and di-mini-PEGylated ligands for PET imaging of the gastrin- releasing peptide receptor. The PEG3- and PEG2/PEG3-bearing (18)F peptides showed decreased lipophilicity relative to an analogous non-mini-PEGylated (18)F peptide. Assessment of water-soluble peptide pharmacokinetics and tumour-targeting capabilities in a mouse model of prostate cancer is currently underway.

(68)Ga-labeled bombesin analogs for receptor-mediated imaging.

Targeted receptor-mediated imaging techniques have become crucial tools in present targeted diagnosis and radiotherapy as they provide accurate and specific diagnosis of disease information. Peptide-based pharmaceuticals are gaining popularity, and there has been vast interest in developing (68)Ga-labeled bombesin (Bn) analogs. The gastrin-releasing peptide (GRP) family and its Bn analog have been implicated in the biology of several human cancers. The three bombesin receptors GRP, NMB, and BRS-3 receptor are most frequently ectopically expressed by common, important malignancies. The low expression of Bn/GRP receptors in normal tissue and relatively high expression in a variety of human tumors can be of biological importance and form a molecular basis for Bn/GRP receptor-mediated imaging. To develop a Bn-like peptide with favorable tumor targeting and pharmacokinetic characteristics for possible clinical use, several modifications in the Bn-like peptides, such as the use of a variety of chelating agents, i.e., acyclic and macrocyclic agents with different spacer groups and with different metal ions (gallium), have been performed in recent years without significant disturbance of the vital binding scaffold. The favorable physical properties of (68)Ga, i.e., short half-life, and the fast localization of small peptides make this an ideal combination to study receptor-mediated imaging in patients.

Old and new peptide receptor targets in cancer: future directions.

A precise definition of the tumor tissue targets to be selected for in vivo peptide receptor targeting, namely to know which peptide receptor is expressed in which type of cancer, is an important prerequisite for successful clinical application of this technology. In this short review, I give three selected examples of new and promising peptide receptor targets. In the somatostatin receptor field, based on in vitro receptor autoradiography experiments showing that much more sst(2) binding sites are detected in tumors using a (177)Lu-labeled sst(2) antagonist than a (177)Lu-labeled agonist, it can be proposed that, in addition to neuroendocrine tumors, nonneuroendocrine tumors with lower sst(2) levels such as breast carcinomas, renal cell carcinomas, and non-Hodgkin lymphomas may become potential candidates for sst(2) antagonist targeting. In the gastrin-releasing peptide receptor field, recent in vitro data show that not only tumor cells may overexpress gastrin-releasing peptide receptors but also neoangiogenic tumoral vessels, making tumors expressing high levels of gastrin-releasing peptide receptors in tumor vessels, such as ovarian or urinary tract cancers, attractive new candidates for gastrin-releasing peptide receptor targeting. In the incretin receptor field, it was found in vitro that, apart from glucagon-like peptide 1 receptors overexpressed in benign insulinomas, incretin receptors, especially the glucose-dependent insulinotropic polypeptide receptors, can be overexpressed in medullary thyroid cancers, an unexpected finding making also these tumors potential novel candidates for incretin receptor targeting. Due to the abundance of peptide receptors in various cancers, it may be possible in the future to define for each tumor type a corresponding overexpressed peptide receptor suitable for targeting.

PET of tumors expressing gastrin-releasing peptide receptor with an 18F-labeled bombesin analog.

UNLABELLED: The gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer. Bombesin (BBN) is a neurotransmitter of 14 amino acids and binds with selectivity and with high affinity to GRPRs. We have synthesized a NOTA-conjugated bombesin derivative, NOTA-8-Aoc-BBN(7-14)NH(2), to label this analog with (18)F using the new Al(18)F method. In this study, the GRPR-targeting potential of (18)F-labeled NOTA-8-Aoc-BBN(7-14)NH(2) was studied using (68)Ga-NOTA-8-Aoc-BBN(7-14)NH(2) as a reference. METHODS: The NOTA-conjugated bombesin analog was synthesized and radiolabeled with (68)Ga or (18)F. For (18)F labeling, we used our new 1-pot, 1-step method. The labeled product was purified by reversed-phase high-performance liquid chromatography. The log P values of the radiotracers were determined. The tumor-targeting characteristics of the compounds were assessed in mice with subcutaneously growing PC-3 xenografts. GRPR-binding specificity was studied by coinjection of an excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH(2). Small-animal PET/CT images were acquired. RESULTS: NOTA-8-Aoc-BBN(7-14)NH(2) could be efficiently labeled with (18)F or with (68)Ga. NOTA-8-Aoc-BBN(7-14)NH(2) was labeled with (18)F in a single step, with 50%-90% yield. Radiolabeling, including purification, was performed in 45 min and resulted in a specific activity of greater than 10 GBq/µmol. The log P values of (18)F- and (68)Ga-labeled NOTA-8-Aoc-BBN(7-14)NH(2) were -1.47 ± 0.05 and -1.98 ± 0.03, respectively. In mice, both radiolabeled compounds cleared rapidly from the blood (<0.07 percentage injected dose per gram at 1 h after injection), mainly via the kidneys. At 1 h after injection, the uptake of (18)F- and (68)Ga-labeled NOTA-8-Aoc-BBN(7-14)NH(2) in the PC-3 tumors was 2.15 ± 0.55 and 1.24 ± 0.26 percentage injected dose per gram, respectively. GRPR-binding specificity was demonstrated by reduced tumor uptake of radiolabeled NOTA-8-Aoc-BBN(7-14)NH(2) after coinjection of a 100-fold excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH(2) peptide. The accumulation of (18)F-NOTA-8-Aoc-BBN(7-14)NH(2) in the subcutaneous PC-3 tumors could be visualized via small-animal PET. CONCLUSION: NOTA-8-Aoc-BBN(7-14)NH(2) could be labeled rapidly and efficiently with (18)F using a 1-pot, 1-step method. Radiolabeled NOTA-8-Aoc-BBN(7-14)NH(2) specifically accumulated in the GRPR-expressing PC-3 tumors and should be evaluated clinically.

Estudio comparativo de la biodistribución de 99mTc-HYNIC-Lys3-bombesina obtenido con los coligandos EDDA/tricina y AN/tricina / Comparative study of the biodistribution of 99mTc-HYNIC-Lys3-Bombesin obtained with the EDDA/tricine and NA/tricine as coligands

El propósito de la presente investigación fue evaluar la biodistribución en animales sanos y en modelos de tumores de los radiofármacos 99mTc-EDDA/tricina-HYNIC-Lys3-bombesina (HYNIC-Lys3-BN) y 99mTc-AN/tricina-HYNIC-Lys3-BN. La biodistribución y la farmacocinética fueron realizados durante 24 horas para lo cual se utilizaron 24 ratas wistar sanas a las cuales se les administró 37,0±0,8 MBq/rata de cada radiofármaco y para el estudio de modelo de tumor fueron utilizados 20 ratones desnudos CD-1 a los que se les injertaron tumores de próstata (PC3). Diez días después fueron calculados los volúmenes tumorales y aplicados 40,00±0,04 MBq/ratón de cada radiofármaco. Ambos mostraron alta pureza radioquímica con valores de 98,08±0,25% para el compuesto con EDDA/tricina y de 95,1±0,3% para el conjugado con AN/tricina. La captación del radiofármaco con AN/tricina fue significativamente mayor en órganos del sistema retículo endotelial de ratas Wistar sanas durante 24h, específicamente en hígado y bazo. No se encontraron diferencias significativas entre los tiempos medios de eliminación de la sangre para ambos compuestos. El volumen promedio de crecimiento tumoral fue 0,93±0,02cm3 y la afinidad por tumores mostró una unión creciente y específica de ambos radiofármacos, siendo significativamente mayor para el conjugado con EDDA/tricina. Este resultado permitió corroborar la relación directa que existe entre la densidad de receptores del péptido liberador de la gastrina (PLGr) y la variación de la acumulación de los radiofármacos en el tumor. El uso de EDDA/tricina como coligando para marcar HYNIC-Lys3-BN con 99mTc, es más apropiado que AN/tricina(AU)

The aim of present investigation was to evaluate biodistribution in healthy animals and in tumor models of the radiopharmaceuticals 99mTc-EDDA/tricine-HYNIC-Lys3-Bombesin (HYNIC-Lys3-BN) and 99mTc-NA/tricine-HYNIC-Lys3-BN. Biodistribution and pharmacokinetics were carried out over 24hours. To do so, 24 healthy Wistar rats were used and were administered 37.0±0.8 MBq/rat of each radiopharmaceutical. For the tumor model study, 20 CD-1 nude mice were used and prostate tumors (PC3) were implanted in all the mice. Ten days later, tumor volumes were calculated and 40.00±0.04 MBq/mice of each radiopharmaceutical were injected. Both showed high radiochemical purity: 98.08±0.25% for EDDA/tricine product and 95.1±0.3% for the conjugate with NA/tricine. Uptake of the radiopharmaceutical with NA/tricine was significantly higher in organs of the reticulo-endothelial system of healthy Wistar rats during 24h, specifically in the liver and spleen. Both labeled compounds showed no significant differences between their blood elimination half lives. Average of tumor growth was 0.93± 0.02cm3 and affinity for tumors showed a growing and specific binding of both radiopharmaceuticals, although it was significantly higher for the EDDA/tricine conjugate. This outcome made it possible to corroborate the direct relationship between the density of gastrin releasing peptide and its receptors (GRPr) and the variation of the accumulation of the radiopharmaceuticals in the tumor. Use of EDDA/tricine as coligand is more appropriate than NA/tricine for labeling of HYNIC-Lys3-BN with 99mTc(AU)

Comparison of two peptide radiotracers for prostate carcinoma targeting.

OBJECTIVES: Scintigraphy is generally not the first choice treatment for prostate cancer, although successful studies using bombesin analog radiopeptides have been performed. Recently, a novel peptide obtained using a phage display library demonstrated an affinity for prostate tumor cells. The aim of this study was to compare the use of a bombesin analog to that of a phage display library peptide (DUP-1) radiolabeled with technetium-99m for the treatment of prostate carcinoma. The peptides were first conjugated to S-acetyl-MAG3 with a 6-carbon spacer, namely aminohexanoic acid. METHODS: The technetium-99m labeling required a sodium tartrate buffer. Radiochemical evaluation was performed using ITLC and was confirmed by high-performance liquid chromatography. The coefficient partition was determined, and in vitro studies were performed using human prostate tumor cells. Biodistribution was evaluated in healthy animals at various time points and also in mice bearing tumors. RESULTS: The radiochemical purity of both radiotracers was greater than 95%. The DUP-1 tracer was more hydrophilic (log P = -2.41) than the bombesin tracer (log P = -0.39). The biodistribution evaluation confirmed this hydrophilicity by revealing the greater kidney uptake of DUP-1. The bombesin concentration in the pancreas was greater than that of DUP-1 due to specific gastrin-releasing peptide receptors. Bombesin internalization occurred for 78.32% of the total binding in tumor cells. The DUP-1 tracer showed very low binding to tumor cells during the in vitro evaluation, although tumor uptake for both tracers was similar. The tumors were primarily blocked by DUP1 and the bombesin radiotracer primarily targeted the pancreas. CONCLUSION: Further studies with the radiolabeled DUP-1 peptide are recommended. With further structural changes, this molecule could become an efficient alternative tracer for prostate tumor diagnosis.

The gastrin-releasing peptide receptor as a marker of dysplastic alterations in cervical epithelial cells.

BACKGROUND: Cervical cancer is a leading cancer in women worldwide. The Papanicolaou test (Pap test) remains the main screening tool; however, it produces high rates of false-negative and false-positive results. Gastrin-releasing peptide is a growth factor that has been implicated in many cancers, and its main receptor, the gastrin-releasing peptide receptor (GRPR), is nearly always expressed in cervical dysplasias and invasive carcinomas. The aim of this study was to evaluate the diagnostic potential of GRPR immunocytochemistry in detecting cervical dysplasia and invasive cancer. METHODS: Cervical smears were collected from 66 women in Brazil and subjected to GRPR immunocytochemistry and the Pap test. GRPR and p16 immunohistochemistry were performed in biopsies if abnormalities were detected. RESULTS: GRPR immunostaining sensitivity in detecting cervical lesions was 87.5% and its specificity was 76.7%. GRPR immunostaining showed 80% accuracy in identifying atypical squamous cells of undetermined significance (ASCUS), with 88% sensitivity and 71% specificity. CONCLUSION: This is the first immunocytochemical evaluation of GRPR expression in cervical epithelial cells. This biomarker was strongly associated with cervical dysplasia and invasive cancers. GRPR immunosignaling showed high accuracy in detecting dysplasias in cells classified as ASCUS by Pap tests. Based on these results, immunocytochemistry for GRPR may be regarded as a valuable method for early detection of cervical intraepithelial neoplasia.