H. J. K. Ananias

Expression of the Gastrin-Releasing Peptide Receptor, the Prostate Stem Cell Antigen and the Prostate-Specific Membrane Antigen in Lymph Node and Bone Metastases of Prostate Cancer

Cell membrane antigens like the gastrin‐releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate‐specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we investigated in this study whether these antigens are expressed in metastasized prostate cancer.

Nuclear Imaging of Prostate Cancer with Gastrin-Releasing-Peptide-Receptor Targeted Radiopharmaceuticals

Prostate cancer is one of the most common causes of cancer in men. Evaluating the different stages of prostate cancer with conventional imaging techniques still proves difficult. Nuclear imaging might provide a technique that is able to evaluate prostate cancer, but clinical application has been limited due to lack of accuracy of current radiopharmaceuticals. The development of radiopharmaceuticals that can be targeted to specific antigens, overexpressed in prostate cancer, but sparse in normal tissue, is crucial. Peptides are of particular interest because of their favourable characteristics, leading to increased attention for nuclear imaging of the gastrin-releasing-peptide-receptor (GRPR) with radiolabelled bombesin-like peptides. Several derivatives of bombesin and its truncated form have been prepared for imaging with single photon emission computed tomography (SPECT) or positron emission tomography (PET), thereby delivering potent candidates for further clinical evaluation. This article provides an overview of the development and preclinical evaluation of radiolabelled bombesin analogues for in vivo targeting of GRPR in prostate cancer. The effect of the radionuclide, chelator, spacer and unnatural amino acids on affinity, metabolic stability and image quality are discussed, as well as agonistic or antagonistic properties. Potent candidates are proposed based on these selection criteria: (I) high affinity for GRPR, with rapid and specific tumour uptake (II) high hydrophilicity resulting in the preferred renal-urinary mode of excretion and low hepatobiliary excretion, (III) high stability, but relatively rapid clearance from blood. Also, a summary is made of clinical studies that report on the detection of prostate cancer with GRPR targeted radiopharmaceuticals.

PSMA, EpCAM, VEGF and GRPR as Imaging Targets in Locally Recurrent Prostate Cancer after Radiotherapy

In this retrospective pilot study, the expression of the prostate-specific membrane antigen (PSMA), the epithelial cell adhesion molecule (EpCAM), the vascular endothelial growth factor (VEGF) and the gastrin-releasing peptide receptor (GRPR) in locally recurrent prostate cancer after brachytherapy or external beam radiotherapy (EBRT) was investigated, and their adequacy for targeted imaging was analyzed. Prostate cancer specimens were collected of 17 patients who underwent salvage prostatectomy because of locally recurrent prostate cancer after brachytherapy or EBRT. Immunohistochemistry was performed. A pathologist scored the immunoreactivity in prostate cancer and stroma. Staining for PSMA was seen in 100% (17/17), EpCAM in 82.3% (14/17), VEGF in 82.3% (14/17) and GRPR in 100% (17/17) of prostate cancer specimens. Staining for PSMA, EpCAM and VEGF was seen in 0% (0/17) and for GRPR in 100% (17/17) of the specimens’ stromal compartments. In 11.8% (2/17) of cases, the GRPR staining intensity of prostate cancer was higher than stroma, while in 88.2% (15/17), the staining was equal. Based on the absence of stromal staining, PSMA, EpCAM and VEGF show high tumor distinctiveness. Therefore, PSMA, EpCAM and VEGF can be used as targets for the bioimaging of recurrent prostate cancer after EBRT to exclude metastatic disease and/or to plan local salvage therapy.

An Update of Radiolabeled Bombesin Analogs for Gastrin-Releasing Peptide Receptor Targeting

Prostate cancer is a critical public health problem in USA and Europe. New non-invasive imaging methods are urgently needed, due to the low accuracy and specificity of current screen methods and the desire of localizing primary prostate cancer and bone metastasis. Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) are the non-invasive and sensitive imaging methods which have been widely used for diagnosing diseases in the clinic. Lack of suitable radiotracers is the major issue for nuclear imaging of prostate cancer, although radiolabeled bombesin (BN) peptides targeting the Gastrin-Releasing Peptide Receptor (GRPR) on tumor cells are widely investigated. In this review we discuss the recent trends in the development of GRPR-targeted radiopharmaceuticals based on BN analogs with regard to their potential for imaging and therapy of GRPR-expressing malignancies. Following a brief introduction of GRPR and bombesin peptides, we summarize the properties of prostate cancer specific radiolabeled bombesins. New bombesin tracers published in the last five years are reviewed and compared according to their novelties in biomolecules, radionuclides, labeling methods, bifunctional chelators and linkers. Hot topics such as multimerization, application of agonists and antagonists are highlighted in the review. Lastly, a few clinical trials of cancer nuclear imaging with radiolabeled bombesin have been discussed.

(99m)technetium-HYNIC(tricine/TPPTS)-Aca-bombesin(7-14) as a targeted imaging agent with microSPECT in a PC-3 prostate cancer xenograft model.

The peptide bombesin (BN) and derivates thereof show high binding affinity for the gastrin-releasing peptide receptor (GRPR), which is highly expressed in primary and metastasized prostate cancer. We have synthesized a new BN-based radiopharmaceutical (99m)technetium-HYNIC(tricine/TPPTS)-Aca-BN(7-14) ((99m)Tc-HABN) and evaluated its GRPR targeting properties in vitro and in a xenograft tumor model for human prostate cancer in athymic mice. (99m)Tc-HABN was synthesized, and its lipophilicity and stability were investigated. The IC(50), internalization and efflux properties were determined in vitro using the GRPR expressing human prostate cancer cell line PC-3. (99m)Tc-HABN biodistribution and microSPECT imaging were performed in PC-3 tumor-bearing athymic mice. (99m)Tc-HABN was prepared with high labeling yield (>90%), high radiochemical purity (>95%) and a specific activity of ~19.8 MBq/nmol. The partition coefficient log D value was -1.60 ± 0.06. (99m)Tc-HABN proved to be stable in human serum for 6 h. The IC50 of HYNIC-Aca-BN(7-14) was 12.81 ± 0.14 nM. Incubation of PC-3 cells with (99m)Tc-HABN demonstrated rapid cellular internalization and a long intracellular retention time. When mice were injected with (99m)Tc-HABN, the activity was predominantly cleared via the kidneys. Uptake in the tumor was 2.24 ± 0.64% ID/g after 30 min, with a steady decrease during the 4 h study period. In vivo experiments with a blocking agent showed GRPR mediated uptake. (99m)Tc-HABN microSPECT imaging resulted in clear delineation of the tumor. (99m)Tc-HABN is a novel BN-based radiopharmaceutical that proved to be suitable for targeted imaging of prostate cancer with microSPECT using the human prostate cancer cell line PC-3 in a xenograft mouse model.

Application of (99m)Technetium-HYNIC(tricine/TPPTS)-Aca-Bombesin(7-14) SPECT/CT in prostate cancer patients A first-in-man study

RATIONALE The peptide bombesin (BBN) and its derivatives exhibit high binding affinity for the gastrin-releasing peptide receptor (GRPR), which is highly expressed in prostate cancer. We used the BBN-based radiopharmaceutical (99m)Technetium-HYNIC(tricine/TPPTS)-Aca-Bombesin(7-14) ((99m)Tc-HABBN) to perform a first-in-man clinical pilot study to evaluate the feasibility of (99m)Tc-HABBN SPECT/CT for detection of prostate cancer in patients. METHODS Eight patients with biopsy-proven prostate cancer who were scheduled for either radical prostatectomy or external beam radiotherapy underwent (99m)Tc-HABBN scintigraphy and SPECT/CT prior to treatment. Serial blood samples were taken to assess blood radioactivity and to determine in vivo metabolic stability. Clinical parameters were measured and reported side effects, if present, were recorded. Prostate cancer specimens of all patients were immunohistochemically stained for GRPR. RESULTS (99m)Tc-HABBN was synthesized with high radiochemical yield, purity and specific activity. There were no significant changes in clinical parameters, and there were no adverse or subjective side effects. Low metabolic stability was observed, as less than 20% of (99m)Tc-HABBN was intact after 30 min. Immunohistochemical staining for GRPR was observed in the prostate cancer specimens in all patients. (99m)Tc-HABBN scintigraphy and SPECT/CT did not detect prostate cancer in patients with proven disease. CONCLUSIONS (99m)Tc-HABBN SPECT/CT for visualization of prostate cancer is safe but hampered by an unexpected low in vivo metabolic stability in man. The difference between the excellent in vitro stability of (99m)Tc-HABBN in human serum samples determined in our previous study regarding (99m)Tc-HABBN and the low in vivo metabolic stability determined in this study, is striking. This issue warrants further study of peptide-based radiopharmaceuticals.

Preclinical Evaluation of a Novel In-111-Labeled Bombesin Homodimer for Improved Imaging of GRPR-Positive Prostate Cancer

Rational-designed multimerization of targeting ligands can be used to improve kinetic and thermodynamic properties. Multimeric targeting ligands may be produced by tethering multiple identical or two or more monomeric ligands of different binding specificities. Consequently, multimeric ligands may simultaneously bind to multiple receptor molecules. Previously, multimerization has been successfully applied on radiolabeled RGD peptides, which resulted in an improved tumor targeting activity in animal models. Multimerization of peptide-based ligands may improve the binding characteristics by increasing local ligand concentration and by improving dissociation kinetics. Here, we present a preclinical study on a novel radiolabeled bombesin (BN) homodimer, designated (111)In-DOTA-[(Aca-BN(7-14)]2, that was designed for enhanced targeting of gastrin-releasing peptide receptor (GRPR)-positive prostate cancer cells. A BN homodimer was conjugated with DOTA-NHS and labeled with (111)In. After HPLC purification, the GRPR targeting ability of (111)In-DOTA-[Aca-BN(7-14)]2 was assessed by microSPECT imaging in SCID mice xenografted with the human prostate cancer cell line PC-3. (111)In labeling of DOTA-[(Aca-BN(7-14)]2 was achieved within 30 min at 85 °C with a labeling yield of >40%. High radiochemical purity (>95%) was achieved by HPLC purification. (111)InDOTA-[Aca-BN(7-14)]2 specifically bound to GRPR-positive PC-3 prostate cancer cells with favorable binding characteristics because uptake of 111In-DOTA-[Aca-BN(7-14)]2 in GRPR-positive PC-3 cells increased over time. A maximum peak with 30% radioactivity was observed after 2 h of incubation. The log D value was -1.8 ± 0.1. (111)In-DOTA-[Aca-BN(7-14)]2 was stable in vitro both in PBS and human serum for at least 4 days. In vivo biodistribution analysis and microSPECT/CT scans performed after 1, 4, and 24 h of injection showed favorable binding characteristics and tumor-to-normal tissue ratios. This study identifies (111)In-DOTA-[(Aca-BN(7-14)]2 as a promising radiotracer for nuclear imaging of GRPR in prostate cancer.

Multimerization Improves Targeting of Peptide Radio-Pharmaceuticals

Multimerization offers unique kinetic and thermodynamic properties to molecules. Multimeric ligands, characterized by multiple similar or different monomeric molecules tethered together, can bind several receptors simultaneously. Multimerization occurs also in nature. This process can be used to develop molecules with high diagnostic and therapeutic value. By altering parameters as linkers` length and flexibility, scaffold and backbones insertion, and ligands-receptors recognition, it is possible to provide high selectivity and binding affinity. The resultant multimeric ligand has a more favorable binding affinity than corresponding monomeric ligands.