Rosalba Mansi

Exendin-4–Based Radiopharmaceuticals for Glucagonlike Peptide-1 Receptor PET/CT and SPECT/CT

Strong overexpression of glucagonlike peptide-1 (GLP-1) receptors in human insulinoma provides an attractive target for imaging. The first clinical trials demonstrated that GLP-1 receptor SPECT/CT using [Lys40(Ahx [6-aminohexanoic acid]-DOTA-111In)NH2]-exendin-4 can localize hardly detectable insulinomas. However, [Lys40(Ahx-DOTA-111In)NH2]-exendin-4 imaging has drawbacks related to the use of 111In in that it is costly and carries a relatively high radiation burden for the patient. The aim of this study was the preclinical evaluation of [Lys40(Ahx-DOTA-68Ga)NH2]-exendin-4 for PET/CT and [Lys40(Ahx-hydrazinonicotinamide [HYNIC]-99mTc)NH2]-exendin-4 for SPECT/CT. Methods: Internalization, biodistribution, dosimetry, and imaging studies were performed in the Rip1Tag2 mouse model of pancreatic β-cell carcinogenesis and compared with our gold standard [Lys40(Ahx-DOTA-111In)NH2]-exendin-4. Poly-glutamic acid and Gelofusine, a gelatin-based plasma expander, were used for renal uptake reduction studies. Results: The tumor uptake of [Lys40(Ahx-DOTA-68Ga)NH2]-exendin-4 was 205 ± 59 percentage injected activity per gram of tissue at 4 h. Other GLP-1 receptor–positive organs showed more than 4.8 times lower radioactivity uptake. [Lys40(Ahx-HYNIC-99mTc/ethylenediaminediacetic acid [EDDA])NH2]-exendin-4, compared with its 111In- and 68Ga-labeled sister compounds, showed significantly less tumor and organ uptake. The significantly lower tumor and organ uptake of [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4 did not result in inferior tumor-to-organ ratios or reduced image quality. All radiopeptides tested showed a high tumor-to-background ratio, resulting in the visualization of small tumors (maximum diameter between 1.0 and 3.2 mm) by SPECT and PET. The only exception was the kidneys, which also showed high uptake. This uptake could be reduced by 49%−78% using poly-glutamic acid, Gelofusine, or a combination of the 2. The estimated effective radiation dose was 3.7 μSv/MBq for [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4, which was 8 times less than that for [Lys40(Ahx-DOTA-68Ga)NH2]-exendin-4 and 43 times less than that for [Lys40(Ahx-DOTA-111In)NH2]-exendin-4. Conclusion: These promising pharmacokinetic and imaging data show that [Lys40(Ahx-DOTA-68Ga)NH2]-exendin-4 and [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4 are suitable candidates for clinical GLP-1 receptor imaging studies.

Evaluation of a 1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid–Conjugated Bombesin-Based Radioantagonist for the Labeling with Single-Photon Emission Computed Tomography, Positron Emission Tomography, and Therapeutic Radionuclides

Purpose: G protein–coupled receptor agonists are being used as radiolabeled vectors for in vivo localization and therapy of tumors. Recently, somatostatin-based antagonists were shown to be superior to agonists. Here, we compare the new [111In/68Ga]-labeled bombesin-based antagonist RM1 with the agonist [111In]-AMBA for targeting the gastrin-releasing peptide receptor (GRPR). Experimental Design: IC50, Kd values, and antagonist potency were determined using PC-3 and HEK-GRPR cells. Biodistribution and imaging studies were done in nude mice transplanted with the PC-3 tumor. The antagonist potency was assessed by evaluating the effects on calcium release and on receptor internalization monitored by immunofluorescence microscopy. Results: The IC50 value of [natIn]-RM1 was 14 ± 3.4 nmol/L. [nat/111In]-RM1 was found to bind to the GRPR with a Kd of 8.5 ± 2.7 nmol/L compared with a Kd of 0.6 ± 0.3 nmol/L of [111In]-AMBA. A higher maximum number of binding site value was observed for [111In]-RM1 (2.4 ± 0.2 nmol/L) compared with [111In]-AMBA (0.7 ± 0.1 nmol/L). [natLu]-AMBA is a potent agonist in the immunofluorescence-based internalization assay, whereas [natIn]-RM1 is inactive alone but efficiently antagonizes the bombesin effect. These data are confirmed by the calcium release assay. The pharmacokinetics showed a superiority of the radioantagonist with regard to the high tumor uptake (13.4 ± 0.8% IA/g versus 3.69 ± 0.75% IA/g at 4 hours after injection. as well as to all tumor-to-normal tissue ratios. Conclusion: Despite their relatively low GRPR affinity, the antagonists [111In/68Ga]-RM1 showed superior targeting properties compared with [111In]-AMBA. As found for somatostatin receptor–targeting radiopeptides, GRP-based radioantagonists seem to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of GRPR-positive tumors. (Clin Cancer Res 2009;15(16):5240–9)

Positron Emission Tomography (PET) Imaging of Prostate Cancer with a Gastrin Releasing Peptide Receptor Antagonist - from Mice to Men

Ex vivo studies have shown that the gastrin releasing peptide receptor (GRPr) is overexpressed on almost all primary prostate cancers, making it a promising target for prostate cancer imaging and targeted radiotherapy. Methods: Biodistribution, dosimetry and tumor uptake of the GRPr antagonist 64Cu-CB-TE2A-AR06 [(64Cu-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane)-PEG4-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-LeuNH2] were studied by PET/CT in four patients with newly diagnosed prostate cancer (T1c-T2b, Gleason 6-7). Results: No adverse events were observed after injection of 64Cu-CB-TE2A-AR06. Three of four tumors were visualized with high contrast [tumor-to-prostate ratio > 4 at 4 hours (h) post injection (p.i.)], one small tumor (T1c, < 5% tumor on biopsy specimens) showed moderate contrast (tumor-to-prostate ratio at 4 h: 1.9). Radioactivity was cleared by the kidneys and only the pancreas demonstrated significant accumulation of radioactivity, which rapidly decreased over time. Conclusion: 64Cu-CB-TE2A-AR06 shows very favorable characteristics for imaging prostate cancer. Future studies evaluating 64Cu-CB-TE2A-AR06 PET/CT for prostate cancer detection, staging, active surveillance, and radiation treatment planning are necessary.

Bombesin Antagonist–Based Radioligands for Translational Nuclear Imaging of Gastrin-Releasing Peptide Receptor–Positive Tumors

Bombesin receptors are overexpressed on a variety of human tumors. In particular, the gastrin-releasing peptide receptor (GRPr) has been identified on prostate and breast cancers and on gastrointestinal stromal tumors. The current study aims at developing clinically translatable bombesin antagonist–based radioligands for SPECT and PET of GRPr-positive tumors. Methods: A potent bombesin antagonist (PEG4-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 [AR]) was synthesized; conjugated to the chelators DOTA, 6-carboxy-1,4,7,11-tetraazaundecane (N4), 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A); and radiolabeled with 111In, 99mTc, 68Ga, and 64Cu, respectively. The radioconjugates were evaluated in vitro and in vivo in PC-3 tumor–bearing nude mice. Antagonist potency was determined by Ca2+-flux measurements and immunofluorescence. Results: All the conjugates showed high binding affinity to GRPr (inhibitory concentration of 50% [IC50], 2.5–25 nmol/L). The immunofluorescence and Ca2+-flux assays confirmed the antagonist properties of the conjugates. Biodistribution revealed high and specific uptake in PC-3 tumor and in GRPr-positive tissues. Tumor uptake of 64Cu-CB-TE2A-AR (31.02 ± 3.35 percentage injected activity per gram [%IA/g]) was higher than 99mTc-N4-AR (24.98 ± 5.22 %IA/g), 111In-DOTA-AR (10.56 ± 0.70 %IA/g), and 68Ga-NODAGA-AR (7.11 ± 3.26 %IA/g) at 1 h after injection. Biodistribution at later time points showed high tumor-to-background ratios because of the fast washout of the radioligand from normal organs, compared with tumor. High tumor-to-background ratios were further illustrated by PET and SPECT images of PC-3 tumor–bearing nude mice acquired at 12 h after injection showing high tumor uptake, clear background, and negligible or no radioactivity in the abdomen. Conclusion: The chelators do influence the affinity, antagonistic potency, and pharmacokinetics of the conjugates. The promising preclinical results warrant clinical translation of these probes for SPECT and PET.

Targeting GRPR in urological cancers--from basic research to clinical application.

Gastrin releasing peptide (GRP) is a regulatory peptide that acts through its receptor (GRPR) to regulate physiological functions in various organs. GRPR is overexpressed in neoplastic cells of most prostate cancers and some renal cell cancers and in the tumoral vessels of urinary tract cancers. Thus, targeting these tumours with specifically designed GRP analogues has potential clinical application. Potent and specific radioactive, cytotoxic or nonradioactive GRP analogues have been designed and tested in various animal tumour models with the aim of receptor targeting for tumour diagnosis or therapy. All three categories of compound were found suitable for tumour targeting in animal models. The cytotoxic and nonradioactive GRP analogues have not yet shown convincing tumour-reducing effects in human trials; however, the first clinical studies of radioactive GRP analogues—both agonists and antagonists—suggest promising opportunities for both diagnostic tumour imaging and radiotherapy of prostate and other GRPR-expressing cancers.

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.

Targeted Radiotherapy of Prostate Cancer with a Gastrin-Releasing Peptide Receptor Antagonist Is Effective as Monotherapy and in Combination with Rapamycin

The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. Methods: To determine the effect of treatment with rapamycin and radiotherapy with a novel 177Lu-labeled GRPr antagonist (177Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and 177Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on 177Lu-RM2 tumor uptake, in vivo small-animal PET studies with 68Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of 177Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with 177Lu-RM2 alone or after pretreatment with rapamycin. Results: Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with 177Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of 177Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. Conclusion: Radiotherapy using a 177Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

N-Terminal Modifications Improve the Receptor Affinity and Pharmacokinetics of Radiolabeled Peptidic Gastrin-Releasing Peptide Receptor Antagonists: Examples of 68Ga- and 64Cu-Labeled Peptides for PET Imaging

Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. Methods: The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with 68Ga and 64Cu. The GRPr affinity of the corresponding metalloconjugates was determined using 125I-Tyr4-BN as a radioligand. The labeling efficiency of 68Ga3+ was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The 68Ga and 64Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. Results: The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar–low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates 68Ga3+ nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10−6 M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for 68Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for 68Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the 64Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. Conclusion: We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.

Peptide modified nanocarriers for selective targeting of bombesin receptors

The present work describes new supramolecular aggregates obtained by co-assembling two different amphiphilic molecules, one containing the bioactive bombesin peptide (BN), or a scramble sequence, and the other, the DOTA chelating agent, (C18)(2)DOTA, capable of forming stable complexes with the radioactive (111)In(III) isotope. The peptide in the amphiphilic monomer is spaced by the lipophilic moiety through ethoxylic spacers of different length: a shorter spacer with five units of dioxoethylene moieties in (C18)(2)L5-peptide, or a longer spacer consisting of a Peg3000 residue in (C18)(2)Peg3000-peptide. Structural characterization by SANS and DLS techniques indicates that, independently from the presence of the peptide containing monomer in the final composition, the predominant aggregates are liposomes of similar shape and size with a hydrodynamic radius R(h) around 200 nm and bilayer thickness, d, of 4 nm. In vitro data show specific binding of the (111)In-(C18)(2)DOTA/(C18)(2)L5-[7-14]BN 90:10 liposomes in receptor expressing cells. However, the presence of the Peg3000 unit on the external liposomal surface, could hide the peptide and prevent the receptor binding. In vivo experiments using (111)In-(C18)(2)DOTA/(C18)(2)L5-[7-14]BN show the expected biological behavior of aggregates of such size and molecular composition, moreover there is an increase in concentration of the GRPR targeting aggregate in the tumors compared to control at the 48 h time point evaluated (2.4% ID/g versus 1.6% ID/g).

Highly improved metabolic stability and pharmacokinetics of indium-111-DOTA-gastrin conjugates for targeting of the gastrin receptor

The development of metabolically stable radiolabeled gastrin analogues with suitable pharmacokinetics is a topic of recent research activity. These imaging vectors are of interest because the gastrin/CCK2 receptor is highly overexpressed in different tumors such as medullary thyroid cancer, neuroendocrine tumors, and SCLC. The drawback of current targeting agents is either their metabolic instability or their high kidney uptake. We present the synthesis and in vitro and in vivo evaluation of 11 (111)In-labeled DOTA-conjugated peptides that differ by their spacer between the peptide and the chelate. We introduced uncharged but hydrophilic spacers such as oligoethyleneglycol, serine, and glutamine. The affinity of all radiopeptides was high with IC(50) values between 0.5 and 4.8 nM. The improvement of human serum stability is 500-fold within this series of compounds. In addition the kidney uptake could be lowered distinctly and the tumor-to-kidney ratio improved almost 60-fold if compared with radiotracers having charged spacers such as glutamic acid.