Development of Radiopharmaceuticals for NPY Receptor-5 (Y5) Nuclear Imaging in Tumors by Synthesis of Specific Agonists and Investigation of Their Binding Mode.

The neuropeptide-Y (NPY) family acts through four G protein-coupled receptor subtypes in humans, namely, Y1, Y2, Y4, and Y5. A growing body of evidence suggest the involvement of the NPY system in several cancers, notably the Y5 subtype, thus acting as a relevant target for the development of radiopharmaceuticals for imaging or targeted radionuclide therapy (TRT). Here, the [cPP(1-7),NPY(19-23),Ala31,Aib32,Gln34]hPP scaffold, further referred to as sY5ago, was modified with a DOTA chelator and radiolabeled with 68Ga and 111In and investigated in vitro and in vivo using the MCF-7 model. For in vivo studies, MCF-7 cells were orthotopically implanted in female nude mice and imaging with small animal positron emission tomography/computed tomography (µPET/CT) was performed. At the end of imaging, the mice were sacrificed. A scrambled version of sY5ago, which was also modified with a DOTA chelator, served as a negative control (DOTA-[Nle]sY5ago_scrambled). sY5ago and DOTA-sY5ago showed subnanomolar affinity toward the Y5 (0.9 ± 0.1 and 0.8 ± 0.1 nM, respectively) and a single binding site at the Y5 was identified. [68Ga]Ga-DOTA-sY5ago and [111In]In-DOTA-sY5ago were hydrophilic and showed high specific internalization (1.61 ± 0.75%/106 cells at 1 h) and moderate efflux (55% of total binding externalized at 45 min). On µPET/CT images, most of the signal was depicted in the kidneys and the liver. MCF-7 tumors were clearly visualized. On biodistribution studies, [68Ga]Ga-DOTA-sY5ago was eliminated by the kidneys (∼60 %ID/g). The kidney uptake is Y5-mediated. A specific uptake was also noted in the liver (5.09 ± 1.15 %ID/g vs 1.13 ± 0.21 %ID/g for [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled, p < 0.05), the lungs (1.03 ± 0.34 %ID/g vs 0.20 %ID/g, p < 0.05), and the spleen (0.85 ± 0.09%ID/g vs 0.16 ± 0.16%ID/g, p < 0.05). In MCF-7 tumors, [68Ga]Ga-DOTA-sY5ago showed 12-fold higher uptake than [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled (3.43 ± 2.32 vs 0.27 ± 0.15 %ID/g, respectively, p = 0.0008) at 1 h post-injection. Finally, a proof-of-principle tissular micro-imaging study on a human primary cancer sample showed weak binding of [111In]In-DOTA-sY5ago in prostatic intra-neoplasia and high binding in the ISUP1 lesion while normal prostate was free of signal.

Design, Synthesis, and Biological Evaluation of the First Radio-Metalated Neurotensin Analogue Targeting Neurotensin Receptor 2.

Neurotensin receptor 2 (NTS2) is a well-known mediator of central opioid-independent analgesia. Seminal studies have highlighted NTS2 overexpression in a variety of tumors including prostate cancer, pancreas adenocarcinoma, and breast cancer. Herein, we describe the first radiometalated neurotensin analogue targeting NTS2. JMV 7488 (DOTA-(ßAla)2-Lys-Lys-Pro-(D)Trp-Ile-TMSAla-OH) was prepared using solid-phase peptide synthesis, then purified, radiolabeled with 68Ga and 111In, and investigated in vitro on HT-29 cells and MCF-7 cells, respectively, and in vivo on HT-29 xenografts. [68Ga]Ga-JMV 7488 and [111In]In-JMV 7488 were quite hydrophilic (logD7.4 = -3.1 ± 0.2 and -2.7 ± 0.2, respectively, p < 0.0001). Saturation binding studies showed good affinity toward NTS2 (K D = 38 ± 17 nM for [68Ga]Ga-JMV 7488 on HT-29 and 36 ± 10 nM on MCF-7 cells; K D = 36 ± 4 nM for [111In]In-JMV 7488 on HT-29 and 46 ± 1 nM on MCF-7 cells) and good selectivity (no NTS1 binding up to 500 nM). On cell-based evaluation, [68Ga]Ga-JMV 7488 and [111In]In-JMV 7488 showed high and fast NTS2-mediated internalization of 24 ± 5 and 25 ± 11% at 1 h for [111In]In-JMV 7488, respectively, along with low NTS2-membrane binding (<8%). Efflux was as high as 66 ± 9% at 45 min for [68Ga]Ga-JMV 7488 on HT-29 and increased for [111In]In-JMV 7488 up to 73 ± 16% on HT-29 and 78 ± 9% on MCF-7 cells at 2 h. Maximum intracellular calcium mobilization of JMV 7488 was 91 ± 11% to that of levocabastine, a known NTS2 agonist on HT-29 cells demonstrating the agonist behavior of JMV 7488. In nude mice bearing HT-29 xenograft, [68Ga]Ga-JMV 7488 showed a moderate but promising significant tumor uptake in biodistribution studies that competes well with other nonmetalated radiotracers targeting NTS2. Significant uptake was also depicted in lungs. Interestingly, mice prostate also demonstrated [68Ga]Ga-JMV 7488 uptake although the mechanism was not NTS2-mediated.

Synthesis and Automated Labeling of [18F]Darapladib, a Lp-PLA2 Ligand, as Potential PET Imaging Tool of Atherosclerosis.

Atherosclerosis and its associated clinical complications are major health issues in industrialized countries. Lipoprotein-associated phospholipase A2 (Lp-PLA2) was demonstrated to play an important role in atherogenesis and to be a potential risk prediction factor of plaque rupture. Darapladib is one of the most potent Lp-PLA2 inhibitors with an IC50 of 0.25 nM. Using its affinity for Lp-PLA2, we describe herein the total synthesis of darapladib radiolabeling precursor and the automated radiolabeling process for positron emission tomography (PET) imaging via an arylboronate moiety. The tracer thus obtained was tested in a mouse model of atherosclerosis (ApoE KO) and compared with the widely used [18F]fluorodeoxyglucose ([18F]FDG) PET tracer, known to label metabolically active cells. [18F]Darapladib showed a significant accumulation within mice aortic atheromatous plaques dissected out ex vivo compared to [18F]FDG. Incubation of the radiotracer with human carotid samples showed a strong accumulation within the atherosclerotic plaques and supports its potential for use in PET imaging.

Development, synthesis, and 68Ga-Labeling of a Lipophilic complexing agent for atherosclerosis PET imaging.

Cardiovascular disease is the leading cause of mortality and morbidity worldwide. Atherosclerosis accounts for 50% of deaths in western countries. This multifactorial pathology is characterized by the accumulation of lipids and inflammatory cells within the vascular wall, leading to plaque formation. We describe herein the synthesis of a PCTA-based 68Ga3+ chelator coupled to a phospholipid biovector 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), which is the main constituent of the phospholipid moiety of High-Density Lipoprotein (HDL) phospholipid moiety. The resulting 68Ga-PCTA-DSPE inserted into HDL particles was compared to 18F-FDG as a PET agent to visualize atherosclerotic plaques. Our agent markedly accumulated within mouse atheromatous aortas and more interestingly in human endarterectomy carotid samples. These results support the potential use of 68Ga-PCTA-DSPE-HDL for atherosclerosis PET imaging.

Regioselectivity of thiouracil alkylation: Application to optimization of Darapladib synthesis.

Darapladib is one of the most potent Lp-PLA2 (Lipoprotein-associated phospholipase A2) inhibitor with an IC50 of 0.25 nM. We demonstrate that a crucial step of Darapladib synthesis was not correctly described in the literature, leading to the production of wrong regioisomers. Moreover we show that the inhibitory activity is directly linked to the position on N1 since compounds bearing alkylation on different sites have potentially less interaction within the active site of Lp-PLA2.

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules.

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

Evaluation of an anti-p185(HER2) (scFv-C(H)2-C(H)3)2 fragment following radioiodination using two different residualizing labels: SGMIB and IB-Mal-D-GEEEK.

INTRODUCTION: A 105-kDa double mutant single-chain Fv-Fc fragment (scFv-Fc DM) derived from the anti-p185(HER2) hu4D5v8 antibody (trastuzumab; Herceptin) has been described recently. The goal of this study was to investigate whether improved tumor targeting could be achieved with this fragment through the use of residualizing radioiodination methods. METHODS: The scFv-Fc DM fragment was radioiodinated using N-succinimidyl 4-guanidinomethyl 3-[(131)I]iodobenzoate ([(131)I]SGMIB) and N(epsilon)-(3-[(131)I]iodobenzoyl)-Lys(5)-N(alpha)- maleimido-Gly(1)-GEEEK ([(131)I]IB-Mal-D-GEEEK), two residualizing radioiodination agents that have been used successfully with intact antibodies. Paired-label internalization assays of the labeled fragments were performed in vitro using MCF7 human breast cancer cells transfected to express HER2 (MCF7-HER2); comparisons were made to scFv-Fc DM directly radioiodinated using Iodogen. The tissue distribution of the scFv-Fc DM labeled with [(125)I]IB-Mal-d-GEEEK and [(131)I]SGMIB was compared in athymic mice bearing MCF7-HER2 xenografts. RESULTS: The scFv-Fc DM fragment was labeled with [(131)I]SGMIB and [(131)I]IB-Mal-d-GEEEK in conjugation yields of 53% and 25%, respectively, with preservation of immunoreactivity for HER2. Internalization assays indicated that labeling via SGMIB resulted in a 1.6- to 3.5-fold higher (P<.05) retention of radioactivity, compared to that from the directly labeled fragment, in HER2-expressing cells during a 24-h observation period. Likewise, the amount of radioactivity retained in cells from the IB-Mal-d-GEEEK-labeled fragment was 1.4- to 3.3-fold higher (P<.05). Tumor uptake of radioiodine activity in athymic mice bearing MCF7-HER2 xenografts in vivo was significantly higher for the [(125)I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment compared with that of the [(131)I]SGMIB-labeled fragment, particularly at later time points. The uptake of (125)I was threefold (3.6+/-1.1 %ID/g vs. 1.2+/-0.4 %ID/g) and fourfold (3.1+/-1.7 %ID/g vs. 0.8+/-0.4 %ID/g) higher than that for (131)I at 24 and 48 h, respectively. However, the [(125)I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment also exhibited considerably higher levels of radioiodine activity in liver, spleen and kidney. CONCLUSIONS: The overall results further demonstrate the potential utility of these two prosthetic groups for the radiohalogenation of internalizing monoclonal antibodies and their fragments. Specifically, the trastuzumab-derived double mutant fragment in combination with these residualizing agents warrants further evaluation for imaging and possibly treatment of HER2 expressing malignancies.

High-activity radio-iodine labeling of conventional and stealth liposomes.

A new method to label preformed liposomes with high activities of radiohalogenated compounds has been developed. It uses activated esters of simple synthetic molecules that may be readily halogenated, such as Bolton-Hunter reagent (BH), and arginine-containing liposomes. BH, in the form of an activated ester, crosses the liposome membrane to react with arginine inside the liposomes, as demonstrated by thin-layer chromatography and by the fact that saline-containing liposomes, or hydrolyzed BH or the water soluble sulfo-BH afforded only marginal encapsulation yields. Under optimized conditions, between 37 and 55 degrees C, 62 +/- 4% (mean +/- SD) of radiolabeled BH were consistently encapsulated in the liposomes within 30 min. In molar amounts, this corresponds to a mean of 56 nmol of BH per micromol of lipids. Based on achievable specific activity, up to 2.8 GBq of iodine-131 could be entrapped per micromol of lipids. Leakage of radioactivity was very low, with less than 5% of the encapsulated activity released within 6 days at 4 degrees C in phosphate-buffered saline and less than 50% within 24 h in human serum at 37 degrees C. The labeling stability, and the fact that both conventional and PEGylated liposomes can be readily labeled with high doses of radioactivity, will make this technique useful for in vivo targeting applications, such as tumor detection (using iodine-123 or iodine-124) or therapy (with iodine-131 or astatine-211).