Bis(Disulfide)-Bridged Somatostatin-14 Analogs and Their [111In]In-Radioligands: Synthesis and Preclinical Profile.

The overexpression of one or more somatostatin receptors (SST1-5R) in human tumors has provided an opportunity for diagnosis and therapy with somatostatin-like radionuclide carriers. The application of "pansomatostatin" analogs is expected to broaden the clinical indications and upgrade the diagnostic/therapeutic efficacy of currently applied SST2R-prefering radioligands. In pursuit of this goal, we now introduce two bicyclic somatostatin-14 (SS14) analogs, AT5S (DOTA-Ala1-Gly2-c[Cys3-Lys4-Asn5-c[Cys6-Phe7-DTrp8-Lys9-Thr10-Cys11]-Thr12-Ser13-Cys14]) and AT6S (DOTA-Ala1-Gly2-c[Cys3-Lys4-c[Cys5-Phe6-Phe7-DTrp8-Lys9-Thr10-Phe11-Cys12]-Ser13-Cys14]), suitable for labeling with trivalent radiometals and designed to sustain in vivo degradation. Both AT5S and AT6S and the respective [111In]In-AT5S and [111In]In-AT6S were evaluated in a series of in vitro assays, while radioligand stability and biodistribution were studied in mice. The 8/12-mer bicyclic AT6S showed expanded affinity for all SST1-5R and agonistic properties at the SST2R, whereas AT5S lost all affinity to SST1-5R. Both [111In]In-AT5S and [111In]In-AT6S remained stable in the peripheral blood of mice, while [111In]In-AT6S displayed low, but specific uptake in AR4-2J tumors and higher uptake in HEK293-SST3R tumors in mice. In summary, high radioligand stability was acquired by the two disulfide bridges introduced into the SS14 motif, but only the 8/12-mer ring AT6S retained a pansomatostatin profile. In consequence, [111In]In-AT6S targeted SST2R-/SST3R-positive xenografts in mice. These results call for further research on pansomatostatin-like radioligands for cancer theranostics.

[111In]In-CP04 as a novel cholecystokinin-2 receptor ligand with theranostic potential in patients with progressive or metastatic medullary thyroid cancer: final results of a GRAN-T-MTC Phase I clinical trial.

INTRODUCTION: Medullary thyroid cancer (MTC) is a rare malignant tumour of the parafollicular C-cells with an unpredictable clinical course and currently suboptimal diagnostic and therapeutic options, in particular in advanced disease. Overexpression of cholecystokinin-2 receptors (CCK2R) represents a promising avenue to diagnostic imaging and targeted therapy, ideally through a theranostic approach. MATERIALS AND METHODS: A translational study (GRAN-T-MTC) conducted through a Phase I multicentre clinical trial of the indium-111 labelled CP04 ([111In]In-CP04), a CCK2R-seeking ligand was initiated with the goal of developing a theranostic compound. Patients with proven advanced/metastatic MTC or short calcitonin doubling time were enrolled. A two-step concept was developed through the use of low- and high-peptide mass (10 and 50 µg, respectively) for safety assessment, with the higher peptide mass considered appropriate for therapeutic application. Gelofusine was co-infused in a randomized fashion in the second step for the evaluation of potential reduction of the absorbed dose to the kidneys. Imaging for the purpose of biodistribution, dosimetry evaluation, and diagnostic assessment were performed as well as pre-, peri-, and postprocedural clinical and biochemical assessment. RESULTS: Sixteen patients were enrolled. No serious adverse events after application of the compound at both peptide amounts were witnessed; transient tachycardia and flushing were observed in two patients. No changes in biochemistry and clinical status were observed on follow-up. Preliminary dosimetry assessment revealed the highest dose to urinary bladder, followed by the kidneys and stomach wall. The effective dose for 200 MBq of [111In]In-CP04 was estimated at 7±3 mSv and 7±1 mSv for 10 µg and 50 µg CP04, respectively. Administration of Gelofusine reduced the dose to the kidneys by 53%, resulting in the organ absorbed dose of 0.044±0.019 mSv/MBq. Projected absorbed dose to the kidneys with the use of [177Lu]Lu-CP04 was estimated at 0.9±0.4 Gy/7.4 GBq. [111In]In-CP04 scintigraphy was positive in 13 patients (detection rate of 81%) with superior diagnostic performance over conventional imaging. CONCLUSION: In the present study, [111In]In-CP04 was shown to be a safe and effective radiopharmaceutical with promising theranostic characteristics for patients with advanced MTC.

Side-Chain Modified [99mTc]Tc-DT1 Mimics: A Comparative Study in NTS1R-Positive Models.

Radiolabeled neurotensin analogs have been developed as candidates for theranostic use against neurotensin subtype 1 receptor (NTS1R)-expressing cancer. However, their fast degradation by two major peptidases, neprilysin (NEP) and angiotensin-converting enzyme (ACE), has hitherto limited clinical success. We have recently shown that palmitoylation at the ε-amine of Lys7 in [99mTc]Tc-[Lys7]DT1 (DT1, N4-Gly-Arg-Arg-Pro-Tyr-Ile-Leu-OH, N4 = 6-(carboxy)-1,4,8,11-tetraazaundecane) led to the fully stabilized [99mTc]Tc-DT9 analog, displaying high uptake in human pancreatic cancer AsPC-1 xenografts but unfavorable pharmacokinetics in mice. Aiming to improve the in vivo stability of [99mTc]Tc-DT1 without compromising pharmacokinetics, we now introduce three new [99mTc]Tc-DT1 mimics, carrying different pendant groups at the ε-amine of Lys7: MPBA (4-(4-methylphenyl)butyric acid)-[99mTc]Tc-DT10; MPBA via a PEG4-linker-[99mTc]Tc-DT11; or a hydrophilic PEG6 chain-[99mTc]Tc-DT12. The impact of these modifications on receptor affinity and internalization was studied in NTS1R-positive cells. The effects on stability and AsPC-1 tumor uptake were assessed in mice without or during NEP/ACE inhibition. Unlike [99mTc]Tc-DT10, the longer-chain modified [99mTc]Tc-DT11 and [99mTc]Tc-DT12 were significantly stabilized in vivo, resulting in markedly improved tumor uptake compared to [99mTc]Tc-DT1. [99mTc]Tc-DT11 was found to achieve the highest AsPC-1 tumor values and good pharmacokinetics, either without or during NEP inhibition, qualifying for further validation in patients with NTS1R-positive tumors using SPECT/CT.

[68Ga]Ga-DATA5m-LM4, a PET Radiotracer in the Diagnosis of SST2R-Positive Tumors: Preclinical and First Clinical Results.

Radiolabeled somatostatin subtype 2 receptor (SST2R)-antagonists have shown advantageous profiles for cancer theranostics compared with agonists. On the other hand, the newly introduced hybrid chelator (6-pentanoic acid)-6-(amino)methyl-1,4-diazepinetriacetate (DATA5m) rapidly binds Ga-68 (t1/2: 67.7 min) at much lower temperature, thus allowing for quick access to "ready-for-injection" [68Ga]Ga-tracers in hospitals. We herein introduce [68Ga]Ga-DATA5m-LM4 for PET/CT imaging of SST2R-positive human tumors. LM4 was obtained by 4Pal3/Tyr3-substitution in the known SST2R antagonist LM3 (H-DPhe-c[DCys-Tyr-DAph(Cbm)-Lys-Thr-Cys]-DTyr-NH2) and DATA5m was coupled at the N-terminus for labeling with radiogallium (Ga-67/68). [67Ga]Ga-DATA5m-LM4 was evaluated in HEK293-SST2R cells and mice models in a head-to-head comparison with [67Ga]Ga-DOTA-LM3. Clinical grade [68Ga]Ga-DATA5m-LM4 was prepared and injected in a neuroendocrine tumor (NET) patient for PET/CT imaging. DATA5m-LM4 displayed high SST2R binding affinity. [67Ga]Ga-DATA5m-LM4 showed markedly higher uptake in HEK293-SST2R cells versus [67Ga]Ga-DOTA-LM3 and was stable in vivo. In HEK293-SST2R xenograft-bearing mice, it achieved longer tumor retention and less kidney uptake than [67Ga]Ga-DOTA-LM3. [68Ga]Ga-DATA5m-LM4 accurately visualized tumor lesions with high contrast on PET/CT. In short, [68Ga]Ga-DATA5m-LM4 has shown excellent prospects for the PET/CT diagnosis of SST2R-positive tumors, further highlighting the benefits of Ga-68 labeling in a hospital environment via the DATA5m-chelator route.

Imaging of inflammatory cellular protagonists in human atherosclerosis: a dual-isotope SPECT approach.

PURPOSE: Atherosclerotic plaque development and progression signifies a complex inflammatory disease mediated by a multitude of proinflammatory leukocyte subsets. Using single photon emission computed tomography (SPECT) coupled with computed tomography (CT), this study tested a new dual-isotope acquisition protocol to assess each radiotracer's capability to identify plaque phenotype and inflammation levels pertaining to leukocytes expressing leukocyte function-associated antigen-1 (LFA-1) and the leukocyte subset of proinflammatory macrophages expressing somatostatin receptor subtype-2 (SST2). Individual radiotracer uptake was quantified and the presence of corresponding immunohistological cell markers was assessed. METHODS: Human symptomatic carotid plaque segments were obtained from endarterectomy. Segments were incubated in dual-isotope radiotracers [111In]In-DOTA-butylamino-NorBIRT ([111In]In-Danbirt) and [99mTc]Tc-[N0-14,Asp0,Tyr3]-octreotate ([99mTc]Tc-Demotate 2) before scanning with SPECT/CT. Plaque phenotype was classified as pathological intimal thickening, fibrous cap atheroma or fibrocalcific using histology sections based on distinct morphological characteristics. Plaque segments were subsequently immuno-stained with LFA-1 and SST2 and quantified in terms of positive area fraction and compared against the corresponding SPECT images. RESULTS: Focal uptake of co-localising dual-radiotracers identified the heterogeneous distribution of inflamed regions in the plaques which co-localised with positive immuno-stained regions of LFA-1 and SST2. [111In]In-Danbirt and [99mTc]Tc-Demotate 2 uptake demonstrated a significant positive correlation (r = 0.651; p = 0.001). Fibrous cap atheroma plaque phenotype correlated with the highest [111In]In-Danbirt and [99mTc]Tc-Demotate 2 uptake compared with fibrocalcific plaques and pathological intimal thickening phenotypes, in line with the immunohistological analyses. CONCLUSION: A dual-isotope acquisition protocol permits the imaging of multiple leukocyte subsets and the pro-inflammatory macrophages simultaneously in atherosclerotic plaque tissue. [111In]In-Danbirt may have added value for assessing the total inflammation levels in atherosclerotic plaques in addition to classifying plaque phenotype.

[99mTc]Tc-DGA1, a Promising CCK2R-Antagonist-Based Tracer for Tumor Diagnosis with Single-Photon Emission Computed Tomography.

Radiolabeled gastrin analogues have been proposed for theranostics of cholecystokinin subtype 2 receptor (CCK2R)-positive cancer. Peptide radioligands based on other receptor antagonists have displayed superior pharmacokinetics and higher biosafety than agonists. Here, we present DGA1, a derivative of the nonpeptidic CCK2R antagonist Z-360 carrying an acyclic tetraamine, for [99mTc]Tc labeling. Preclinical comparison of [99mTc]Tc-DGA1 with [99mTc]Tc-DG2 (CCK2R-agonist reference) was conducted in HEK293-CCK2R/CCK2i4svR cells and mice models, qualifying [99mTc]Tc-DGA1 for further study in patients with CCK2R-positive tumors and single-photon emission computed tomography/CT.

Optimizing the Profile of [99mTc]Tc-NT(7-13) Tracers in Pancreatic Cancer Models by Means of Protease Inhibitors.

BACKGROUND: The overexpression of neurotensin subtype 1 receptors (NTS1Rs) in human tumors may be elegantly exploited for directing neurotensin (NT)-based radionuclide carriers specifically to cancer sites for theranostic purposes. We have recently shown that [99mTc]Tc-DT1 ([99mTc]Tc-[N4-Gly7]NT(7-13)) and [99mTc]Tc-DT5 ([99mTc]Tc-[N4-ßAla7,Dab9]NT(7-13)) show notably improved uptake in human colon adenocarcinoma WiDr xenografts in mice treated with neprilysin (NEP) inhibitors and/or angiotensin-converting enzyme (ACE) inhibitors compared with untreated controls. Aiming toward translation of this promising approach in NTS1R-positive pancreatic ductal adenocarcinoma (PDAC) patients, we now report on the impact of registered NEP/ACE inhibitors on the performance of [99mTc]Tc-DT1 and [99mTc]Tc-DT5 in pancreatic cancer models. METHODS: The cellular uptake of [99mTc]Tc-DT1 and [99mTc]Tc-DT5 was tested in a panel of pancreatic cell lines, and their stability was assessed in mice treated or not treated with Entresto, lisinopril, or their combinations. Biodistribution was conducted in severe combined immunodeficiency (SCID) mice bearing pancreatic AsPC-1 xenografts. RESULTS: The Entresto + lisinopril combination maximized the metabolic stability of the fast-internalizing [99mTc]Tc-DT1 in mice, resulting in notably enhanced tumor uptake (7.05 ± 0.80% injected activity (IA)/g vs. 1.25 ± 0.80% IA/g in non-treated controls at 4 h post-injection; p < 0.0001). CONCLUSIONS: This study has shown the feasibility of optimizing the uptake of [99mTc]Tc-DT1 in pancreatic cancer models with the aid of clinically established NEP/ACE inhibitors, in favor of clinical translation prospects.

[99mTc]Tc-DB1 Mimics with Different-Length PEG Spacers: Preclinical Comparison in GRPR-Positive Models.

Background: The frequent overexpression of gastrin-releasing peptide receptors (GRPRs) in human cancers provides the rationale for delivering clinically useful radionuclides to tumor sites using peptide carriers. Radiolabeled GRPR antagonists, besides being safer for human use, have often shown higher tumor uptake and faster background clearance than agonists. We herein compared the biological profiles of the GRPR-antagonist-based radiotracers [99mTc]Tc-[N4-PEGx-DPhe6,Leu-NHEt13]BBN(6-13) (N4: 6-(carboxy)-1,4,8,11-tetraazaundecane; PEG: polyethyleneglycol): (i) [99mTc]Tc-DB7 (x = 2), (ii) [99mTc]Tc-DB13 (x = 3), and (iii) [99mTc]Tc-DB14 (x = 4), in GRPR-positive cells and animal models. The impact of in situ neprilysin (NEP)-inhibition on in vivo stability and tumor uptake was also assessed by treatment of mice with phosphoramidon (PA). Methods: The GRPR affinity of DB7/DB13/DB14 was determined in PC-3 cell membranes, and cell binding of the respective [99mTc]Tc-radioligands was assessed in PC-3 cells. Each of [99mTc]Tc-DB7, [99mTc]Tc-DB13, and [99mTc]Tc-DB14 was injected into mice without or with PA coinjection and 5 min blood samples were analyzed by HPLC. Biodistribution was conducted at 4 h postinjection (pi) in severe combined immunodeficiency disease (SCID) mice bearing PC-3 xenografts without or with PA coinjection. Results: DB7, -13, and -14 displayed single-digit nanomolar affinities for GRPR. The uptake rates of [99mTc]Tc-DB7, [99mTc]Tc-DB13, and [99mTc]Tc-DB14 in PC-3 cells was comparable and consistent with a radioantagonist profile. The radiotracers were found to be ≈70% intact in mouse blood and >94% intact after coinjection of PA. Treatment of mice with PA enhanced tumor uptake. Conclusions: The present study showed that increase of PEG-spacer length in the [99mTc]Tc-DB7-[99mTc]Tc-DB13-[99mTc]Tc-DB14 series had little effect on GRPR affinity, specific uptake in PC-3 cells, in vivo stability, or tumor uptake. A significant change in in vivo stability and tumor uptake was observed only after treatment of mice with PA, without compromising the favorably low background radioactivity levels.

Trastuzumab cotreatment improves survival of mice with PC-3 prostate cancer xenografts treated with the GRPR antagonist 177 Lu-DOTAGA-PEG2 -RM26.

Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer and are suitable for targeted radionuclide therapy (TRT). We optimized the bombesin-derived GRPR-antagonist PEG2 -RM26 for labeling with 177 Lu and further determined the effect of treatment with 177 Lu-labeled peptide alone or in combination with the anti-HER2 antibody trastuzumab in a murine model. The PEG2 -RM26 analog was coupled to NOTA, NODAGA, DOTA and DOTAGA chelators. The peptide-chelator conjugates were labeled with 177 Lu and characterized in vitro and in vivo. A preclinical therapeutic study was performed in PC-3 xenografted mice. Mice were treated with intravenous injections (6 cycles) of (A) PBS, (B) DOTAGA-PEG2 -RM26, (C) 177 Lu-DOTAGA-PEG2 -RM26, (D) trastuzumab or (E) 177 Lu-DOTAGA-PEG2 -RM26 in combination with trastuzumab. 177 Lu-DOTAGA-PEG2 -RM26 demonstrated quantitative labeling yield at high molar activity (450 GBq/µmol), high in vivo stability (5 min pi >98% of radioligand remained when coinjected with phosphoramidon), high affinity to GRPR (KD = 0.4 ± 0.2 nM), and favorable biodistribution (1 hr pi tumor uptake was higher than in healthy tissues, including the kidneys). Therapy with 177 Lu-DOTAGA-PEG2 -RM26 induced a significant inhibition of tumor growth. The median survival for control groups was significantly shorter than for treated groups (Group C 66 days, Group E 74 days). Trastuzumab together with radionuclide therapy significantly improved survival. No treatment-related toxicity was observed. In conclusion, based on in vitro and in vivo characterization of the four 177 Lu-labeled PEG2 -RM26 analogs, we concluded that 177 Lu-DOTAGA-PEG2 -RM26 was the most promising analog for TRT. Radiotherapy using 177 Lu-DOTAGA-PEG2 -RM26 effectively inhibited tumor growth in vivo in a murine prostate cancer model. Anti-HER2 therapy additionally improved survival.

Comparative evaluation of the new GRPR-antagonist 111 In-SB9 and 111 In-AMBA in prostate cancer models: Implications of in vivo stability.

Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer, representing attractive targets for diagnosis and therapy with bombesin (BBN)-like radioligands. GRPR-antagonists have lately attracted much attention owing to inherent biosafety and favorable pharmacokinetics. We herein present the GRPR-antagonist SB9 structurally resembling the known BBN-based agonist AMBA (SB9 = [Leu13 NHEt-desMet14 ]AMBA). The profiles of 111 In-SB9 and 111 In-AMBA were directly compared in PC-3 cells and tumor-bearing mice. SB9 and AMBA displayed high GRPR affinities. 111 In-AMBA strongly internalized in PC-3 cells, while 111 In-SB9 remained bound on the cell surface showing a typical GRPR-radioantagonist profile. 111 In-SB9 was more stable than 111 In-AMBA, but coinjection of the neprilysin (NEP) inhibitor phosphoramidon (PA) stabilized both in vivo. The radioligands displayed high tumor uptake (20.23 ± 3.41 %ID/g and 18.53 ± 1.54 %ID/g, respectively, at 4 hours pi), but 111 In-SB9 washed faster from background. PA coinjection led to significant increase of tumor uptake, combined with better clearance for 111 In-SB9. In short, this study has revealed superior pharmacokinetics and higher stability for the GRPR-antagonist 111 In-SB9 vs the corresponding agonist 111 In-AMBA consolidating previous evidence that GRPR antagonists are preferable to agonists for tumor imaging and therapy. It has also demonstrated that further pharmacokinetic improvements were feasible by in situ metabolic radioligand stabilization using PA.

Clinical translation of theranostic radiopharmaceuticals: Current regulatory status and recent examples.

With the development of ever more radiopharmaceuticals suitable for theranostic applications, translation of novel compounds from the preclinical stage towards clinical application becomes a bottleneck for the advances in Nuclear Medicine. This review article summarizes the current regulatory framework for clinical trials with radiopharmaceuticals in the European Union, provides a general overview of the documentation required, and addresses quality, safety, and clinical aspects to be considered. By using a recent successful example of translating a theranostic peptide radioligand, namely 111 In-CP04, which targets receptors expressed in medullary thyroid carcinoma, the pathway from the preclinical development over establishing the required pharmaceutical documentation to designing and submitting a clinical trial is reviewed. Details regarding preclinical data, generation of the documentation, and final successful application are described. This article should provide an insight in an ever more complex process to bring innovations in the field of radiopharmaceuticals into patients.

Comparing Gly11/dAla11-Replacement vs. the in-Situ Neprilysin-Inhibition Approach on the Tumor-targeting Efficacy of the 111In-SB3/111In-SB4 Radiotracer Pair.

Background: The GRPR-antagonist 68Ga-SB3 visualized prostate cancer lesions in animal models and in patients. Switching radiometal from 68Ga to 111In impaired tumor targeting in mice, but coinjection of the neprilysin (NEP)-inhibitor phosphoramidon (PA) stabilized 111In-SB3 in circulation and remarkably increased tumor uptake. We herein report on the biological profile of 111In-SB4: 111In-[dAla11]SB3. Methods: The biological responses of 111In-SB3/SB4 were compared in PC-3 cells and animal models. Results: Gly11/dAla11-replacement deteriorated GRPR-affinity (SB4 IC50: 10.7 ± 0.9 nM vs. SB3 IC50: 4.6 ± 0.3 nM) and uptake in PC-3 cells (111In-SB4: 1.3 ± 0.4% vs. 111In-SB3 16.2 ± 0.8% at 1 h). 111In-SB4 was more stable than 111In-SB3, but PA-coinjection stabilized both radiotracers in peripheral mice blood. Unmodified 111In-SB3 showed higher uptake in PC-3 xenografts (8.8 ± 3.0%ID/g) vs. 111In-SB4 (3.1 ± 1.1%ID/g) at 4 h pi. PA-coinjection improved tumor uptake, with 111In-SB3 still showing superior tumor targeting (38.3 ± 7.9%ID/g vs. 7.4 ± 0.3%ID/g for 111In-SB4). Conclusions: Replacement of Gly11 by dAla11 improved in vivo stability, however, at the cost of GRPR-affinity and cell uptake, eventually translating into inferior tumor uptake of 111In-SB4 vs. unmodified 111In-SB3. On the other hand, in-situ NEP-inhibition turned out to be a more efficient and direct strategy to optimize the in vivo profile of 111In-SB3, and potentially other peptide radiotracers.

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.

Electrospray ionization study of tricarbonyl fac-[Re(CO)3 (PO)(X)]-type complexes: influence of ancillary co-ligands in the release of carbon monoxide.

RATIONALE: fac-[Re(CO)3 (PO)(X)]-type complexes (PO = chelated bidentate tertiary phosphine (1-), X = various neutral, mono-dentate ligands) represent a class of compounds that meets the synthetic criteria for the preparation of potential carbon monoxide (CO) release molecules (CORMs) for medicinal application. The aim of our investigation was to achieve qualitative information whether the nature of the ancillary X ligand might influence the release of CO. METHODS: The release of CO has been investigated by means of product ion spectrometry of electrospray ionization (ESI)-generated [M + H]+ species, produced by multiple collisional experiments, using an ion trap mass spectrometer. RESULTS: Tandem mass spectrometry applied to the protonated species [Re(CO)3 (PO)(X) + H]+ of seven complexes (those including X = OH2 (1), isonitrile (2, 3), imidazole (4), pyridine (5) and phosphine (6, 7)) shows initial loss of coordinated water (1) or pyridine (5), whereas the majority of investigated entries display initial, sequential release of CO groups. The energetics of CO release have been investigated by breakdown curves for selected collisionally activated decomposition processes involving CO, and compared with those involving X groups. CONCLUSIONS: The nature of the co-ligand X drives the primary loss in the MSn processes of [Re(CO)3 (PO)(X) + H]+ compounds. When X = solvent, the energetics of these decompositions follow the trend H2 O < MeOH < CO. In each case, loss of CO is a favored fragmentation route with associated energies following the trend: N-py ≤ P-phosphine < C-isonitrile. Overall, MSn pathways indicate that [Re(PO)] (Re with chelated PO phosphine) constitutes the residual moiety. This behavior indicates that the presence of a functionalized phosphine is essential for a sequential, controlled release of CO.

Preclinical in vivo cancer, straightway to patients?

Detection of useful cellular targets has strongly stimulated personalized tumor-targeted imaging and therapy approaches, also involving synthesis and evaluation of nuclear imaging probes with potential for clinical applications. Reviews of preclinical and translational studies concerning such probes, including radiolabeled antibodies, nanobodies, affibodies, peptides, small molecule inhibitors, and nanoparticles, are presented in this issue. As most tracers described in these articles have been developed for the field of cancer imaging and radionuclide therapy, the current article on preclinical studies will focus on cancer research as well. The main steps in developing a nuclear probe for clinical application for radionuclide imaging and therapy, after identification of a suitable molecular target on tumor cells, comprise: 1) synthesis and radiolabeling of the probe; 2) in vitro characterization, such as the evaluation of target binding affinity; 3) in vivo evaluation to assess the biodistribution and tumor targeting capability, for radionuclide therapy purposes also dosimetry studies to determine the absorbed doses and efficacy; 4) radiolabeled probes that successfully pass such tests as well as toxicological studies may enter clinical evaluation. For preclinical testing of radiolabeled probes various relevant in vitro and in vivo models dedicated to oncological research have been developed along with preclinical imaging platforms, including positron emission tomography (PET) and single photon emission computed tomography (SPECT) systems, in combination with magnetic resonance imaging (MRI) or computed tomography (CT). These developments hold great promise for fast translation of new candidate probes from preclinical validation into the clinic. This overview article describes preclinical studies typically being performed to bring a new radiopharmaceutical into clinical oncology practice. It also aims to raise awareness of confounding factors during translation of preclinical studies and ways to overcome them.

Rhenium(I) Tricarbonyl Complexes with (2-Hydroxyphenyl)diphenylphosphine as PO Bidentate Ligand.

In the present work, we investigated potential means to obtain neutral tricarbonyl mixed-ligand fac-[M(CO)3L1L2] complexes (M = Re, 99mTc) containing the (2-hydroxyphenyl)diphenylphosphine (POH) bidentate ligand (L1H) and a series of monodentate ligands (L2). First, fac-[Re(CO)3(PO)(H2O)], 1, was synthesized by reaction of POH and [Et4N]2[Re(CO)3Br3] in equimolar amounts in MeOH at room temperature. Interestingly, with excess of POH this reaction afforded fac-[Re(CO)3(PO)(POH)], 2, with POH operating both as a bidentate and as a monodentate ligand. Owing to the presence of the labile aqua ligand, which can be readily replaced by various monodentate ligands, 1 was further used as a precursor to generate a small library of the desired fac-[M(CO)3L1L2] complexes. Specifically, by reaction of triphenylphosphine (PPh3), imidazole (im), pyridine (py), cyclohexyl isocyanide (cisc), and tert-butyl isocyanide (tbi), the following products were readily obtained in excellent yields (92%-95%): fac-[Re(CO)3(PO)(PPh3)], 3, fac-[Re(CO)3(PO)(im)], 4, fac-[Re(CO)3(PO)(py)], 5, fac-[Re(CO)3(PO)(cisc)], 6, and fac-[Re(CO)3(PO)(tbi)], 7. All compounds were fully characterized by elemental analysis, IR and NMR spectroscopies, and electrospray ionization(+) mass spectrometry. Their solid-state structure was elucidated by X-ray crystallography. Of considerable interest is the fact that the corresponding 2'-7' were easily accessible at the 99mTc-tracer level in quantitative yields after reaction of POH and the respective monodentate ligand L2 with fac-[99mTc(CO)3(H2O)3]+ in aqueous MeOH, as verified by comparative chromatographic methods adopting dual photo- and radiometric detection modes. The high stability displayed by all 99mTc complexes during histidine and cysteine challenge assays underscored the suitability of the fac-[M(CO)3(PO)L2] system for radiopharmaceutical development purposes.

NeoBOMB1, a GRPR-Antagonist for Breast Cancer Theragnostics: First Results of a Preclinical Study with [67Ga]NeoBOMB1 in T-47D Cells and Tumor-Bearing Mice.

BACKGROUND: The GRPR-antagonist-based radioligands [67/68Ga/111In/177Lu]NeoBOMB1 have shown excellent theragnostic profiles in preclinical prostate cancer models, while [68Ga]NeoBOMB1 effectively visualized prostate cancer lesions in patients. We were further interested to explore the theragnostic potential of NeoBOMB1 in GRPR-positive mammary carcinoma, by first studying [67Ga]NeoBOMB1 in breast cancer models; Methods: We investigated the profile of [67Ga]NeoBOMB1, a [68Ga]NeoBOMB1 surrogate, in GRPR-expressing T-47D cells and animal models; Results: NeoBOMB1 (IC50s of 2.2 ± 0.2 nM) and [natGa]NeoBOMB1 (IC50s of 2.5 ± 0.2 nM) exhibited high affinity for the GRPR. At 37 °C [67Ga]NeoBOMB1 strongly bound to the T-47D cell-membrane (45.8 ± 0.4% at 2 h), internalizing poorly, as was expected for a radioantagonist. [67Ga]NeoBOMB1 was detected >90% intact in peripheral mouse blood at 30 min pi. In mice bearing T-47D xenografts, [67Ga]NeoBOMB1 specifically localized in the tumor (8.68 ± 2.9% ID/g vs. 0.6 ± 0.1% ID/g during GRPR-blockade at 4 h pi). The unfavorably high pancreatic uptake could be considerably reduced (206.29 ± 17.35% ID/g to 42.46 ± 1.31% ID/g at 4 h pi) by increasing the NeoBOMB1 dose from 10 pmol to 200 pmol, whereas tumor uptake remained unaffected. Notably, tumor values did not decline from 1 to 24 h pi; Conclusions: [67Ga]NeoBOMB1 can successfully target GRPR-positive breast cancer in animals with excellent prospects for clinical translation.

Preclinical and first clinical experience with the gastrin-releasing peptide receptor-antagonist [68Ga]SB3 and PET/CT.

PURPOSE: Gastrin-releasing peptide receptors (GRPR) represent attractive targets for tumor diagnosis and therapy because of their overexpression in major human cancers. Internalizing GRPR agonists were initially proposed for prolonged lesion retention, but a shift of paradigm to GRPR antagonists has recently been made. Surprisingly, radioantagonists, such as [(99m)Tc]DB1 ((99m)Tc-N4'-DPhe(6),Leu-NHEt(13)]BBN(6-13)), displayed better pharmacokinetics than radioagonists, in addition to their higher inherent biosafety. We introduce here [(68)Ga]SB3, a [(99m)Tc]DB1 mimic-carrying, instead of the (99m)Tc-binding tetraamine, the chelator DOTA for labeling with the PET radiometal (68)Ga. METHODS: Competition binding assays of SB3 and [(nat)Ga]SB3 were conducted against [(125)I-Tyr(4)]BBN in PC-3 cell membranes. Blood samples collected 5 min postinjection (pi) of the [(67)Ga]SB3 surrogate in mice were analyzed using high-performance liquid chromatography (HPLC) for degradation products. Likewise, biodistribution was performed after injection of [(67)Ga]SB3 (37 kBq, 100 µL, 10 pmol peptide) in severe combined immunodeficiency (SCID) mice bearing PC-3 xenografts. Eventually, [(68)Ga]SB3 (283 ± 91 MBq, 23 ± 7 nmol) was injected into 17 patients with breast (8) and prostate (9) cancer. All patients had disseminated disease and had received previous therapies. PET/CT fusion images were acquired 60-115 min pi. RESULTS: SB3 and [(nat)Ga]SB3 bound to the human GRPR with high affinity (IC50: 4.6 ± 0.5 nM and 1.5 ± 0.3 nM, respectively). [(67)Ga]SB3 displayed good in vivo stability (>85 % intact at 5 min pi). [(67)Ga]SB3 showed high, GRPR-specific and prolonged retention in PC-3 xenografts (33.1 ± 3.9%ID/g at 1 h pi - 27.0 ± 0.9%ID/g at 24 h pi), but much faster clearance from the GRPR-rich pancreas (≈160%ID/g at 1 h pi to <17%ID/g at 24 h pi) in mice. In patients, [(68)Ga]SB3 elicited no adverse effects and clearly visualized cancer lesions. Thus, 4 out of 8 (50 %) breast cancer and 5 out of 9 (55 %) prostate cancer patients showed pathological uptake on PET/CT with [(68)Ga]SB3. CONCLUSION: [(67)Ga]SB3 showed excellent pharmacokinetics in PC-3 tumor-bearing mice, while [(68)Ga]SB3 PET/CT visualized lesions in about 50 % of patients with advanced and metastasized prostate and breast cancer. We expect imaging with [(68)Ga]SB3 to be superior in patients with primary breast or prostate cancer.

Improved Quantification of the Beta Cell Mass after Pancreas Visualization with 99mTc-demobesin-4 and Beta Cell Imaging with 111In-exendin-3 in Rodents.

OBJECTIVE: Accurate assessment of the 111In-exendin-3 uptake within the pancreas requires exact delineation of the pancreas, which is highly challenging by MRI and CT in rodents. In this study, the pancreatic tracer 99mTc-demobesin-4 was evaluated for accurate delineation of the pancreas to be able to accurately quantify 111In-exendin-3 uptake within the pancreas. METHODS: Healthy and alloxan-induced diabetic Brown Norway rats were injected with the pancreatic tracer 99mTc-demobesin-4 ([99mTc-N4-Pro1,Tyr4,Nle14]bombesin) and the beta cell tracer 111In-exendin-3 ([111In-DTPA-Lys40]exendin-3). After dual isotope acquisition of SPECT images, 99mTc-demobesin-4 was used to define a volume of interest for the pancreas in SPECT images subsequently the 111In-exendin-3 uptake within this region was quantified. Furthermore, biodistribution and autoradiography were performed in order to gain insight in the distribution of both tracers in the animals. RESULTS: 99mTc-demobesin-4 showed high accumulation in the pancreas. The uptake was highly homogeneous throughout the pancreas, independent of diabetic status, as demonstrated by autoradiography, whereas 111In-exendin-3 only accumulates in the islets of Langerhans. Quantification of both ex vivo and in vivo SPECT images resulted in an excellent linear correlation between the pancreatic uptake, determined with ex vivo counting and 111In-exendin-3 uptake, determined from the quantitative analysis of the SPECT images (Pearson r = 0.97, Pearson r = 0.92). CONCLUSION: 99mTc-demobesin-4 shows high accumulation in the pancreas of rats. It is a suitable tracer for accurate delineation of the pancreas and can be conveniently used for simultaneous acquisition with 111In labeled exendin-3. This method provides a straightforward, reliable, and objective method for preclinical beta cell mass (BCM) quantification with 111In-exendin-3.