[68Ga]‐HP‐DO3A‐nitroimidazole: a promising agent for PET detection of tumor hypoxia

The goal of this study is to evaluate a new ⁶⁸Ga‐based imaging agent for detecting tumor hypoxia using positron emission tomography (PET). The new hypoxia targeting agent reported here, [⁶⁸Ga]‐HP‐DO3A‐nitroimidazole ([⁶⁸Ga]‐HP‐DO3A‐NI), was constructed by linking a nitroimidazole moiety with the macrocyclic ligand component of ProHance®, HP‐DO3A. The hypoxia targeting capability of this agent was evaluated in A549 lung cancer cells in vitro and in SCID mice bearing subcutaneous A549 tumor xenografts. The cellular uptake assays showed that significantly more [⁶⁸Ga]‐HP‐DO3A‐NI accumulates in hypoxic tumor cells at 30, 60 and 120 min than in the same cells exposed to 21% O₂. The agent also accumulated in hypoxic tumors in vivo to give a tumor/muscle ratio (T/M) of 5.0 ± 1.2 (n = 3) as measured by PET at 2 h post‐injection (p.i.). This was further confirmed by ex vivo biodistribution data. In addition, [⁶⁸Ga]‐HP‐DO3A‐NI displayed very favorable pharmacokinetic properties, as it was cleared largely through the kidneys with little to no accumulation in liver, heart or lung (%ID/g < 0.5%) at 2 h p.i. The specificity of the agent for hypoxic tissues was further validated in a comparative study with a control compound, [⁶⁸Ga]‐HP‐DO3A, which lacks the nitroimidazole moiety, and by PET imaging of tumor‐bearing mice breathing air versus 100% O₂. Given the commercial availability of cGMP ⁶⁸Ge/⁶⁸Ga generators and the ease of ⁶⁸Ga labeling, the new agent could potentially be widely applied for imaging tumor hypoxia prior to radiation therapy. Copyright © 2015 John Wiley & Sons, Ltd.     

Gallium(III) complexes of NOTA‐bis (phosphonate) conjugates as PET radiotracers for bone imaging

Ligands with geminal bis(phosphonic acid) appended to 1,4,7‐triazacyclonone‐1,4‐diacetic acid fragment through acetamide (NOTAM^BP) or methylenephosphinate (NO2AP^BP) spacers designed for ⁶⁸Ga were prepared. Ga^III complexation is much faster for ligand with methylenephosphinate spacer than that with acetamide one, in both chemical (high reactant concentrations) and radiolabeling studies with no‐carrier‐added ⁶⁸Ga. For both ligands, formation of Ga^III complex was slower than that with NOTA owing to the strong out‐of‐cage binding of bis(phosphonate) group. Radiolabeling was efficient and fast only above 60 °C and in a narrow acidity region (pH ~3). At higher temperature, hydrolysis of amide bond of the carboxamide‐bis(phosphonate) conjugate was observed during complexation reaction leading to Ga–NOTA complex. In vitro sorption studies confirmed effective binding of the ⁶⁸Ga complexes to hydroxyapatite being comparable with that found for common bis(phosphonate) drugs such as pamindronate. Selective bone uptake was confirmed in healthy rats by biodistribution studies ex vivo and by positron emission tomography imaging in vivo. Bone uptake was very high, with SUV (standardized uptake value) of 6.19 ± 1.27 for [⁶⁸Ga]NO2AP^BP) at 60 min p.i., which is superior to uptake of ⁶⁸Ga–DOTA‐based bis(phosphonates) and [¹⁸F]NaF reported earlier (SUV of 4.63 ± 0.38 and SUV of 4.87 ± 0.32 for [⁶⁸Ga]DO3AP^BP and [¹⁸F]NaF, respectively, at 60 min p.i.). Coincidently, accumulation in soft tissue is generally low (e.g. for kidneys SUV of 0.26 ± 0.09 for [⁶⁸Ga]NO2AP^BP at 60 min p.i.), revealing the new ⁶⁸Ga complexes as ideal tracers for noninvasive, fast and quantitative imaging of calcified tissue and for metastatic lesions using PET or PET/CT. Copyright © 2014 John Wiley & Sons, Ltd.     

PET/CT to detect adverse reactions to metal debris in patients with metal‐on‐metal hip arthroplasty: an exploratory prospective study

Metal‐on‐metal (MoM) bearings in total hip arthroplasties and hip resurfacing arthroplasties have recently shown a new type of complication: adverse reactions to metal debris (ARMD). ARMD is characterized by local severe inflammation and tissue necrosis leading to implant failures. The gluteal muscle region is important for the patient outcome after revision surgery. This prospective positron emission tomography/computed tomography (PET/CT) study was undertaken to evaluate the characteristics of 2‐deoxy‐2‐[¹⁸F]fluoro‐d ‐glucose ([¹⁸F]FDG) and [⁶⁸Ga]Gallium citrate ([⁶⁸Ga]Citrate) PET/CT in ARMD patients. [¹⁸F]FDG and [⁶⁸Ga]Citrate PET/CT were performed in 18 hip arthroplasty patients: 12 ARMD patients (with 16 MoM hips) and six arthroplasty controls without ARMD. Tracer uptake was evaluated visually, and maximum standardized uptake (SUV_max) was measured in the gluteal muscle region. ARMD severity was graded by metal artefact reduction sequence‐magnetic resonance imaging (MARS‐MRI). Periprosthetic [¹⁸F]FDG uptake was observed in 15 of 16 hips, [⁶⁸Ga]Citrate uptake in three of 16 hips, respectively. The distribution of tracer uptake resembled infection in three hips. In the gluteal muscle region, the SUV_max of [¹⁸F]FDG was significantly greater in hips with moderate and severe ARMD compared with the controls (P = 0·009 for [¹⁸F]FDG and P = 0·217 for [⁶⁸Ga]Citrate). In patients who needed revision surgery, an intraoperative finding of gluteal muscle necrosis was associated with increased local SUV_max as detected by preoperative [¹⁸F]FDG (P = 0·039), but not by [⁶⁸Ga]Citrate (P = 0·301). In conclusion, the inflammatory reaction to metal debris in hip arthroplasty patients is best visualized with [¹⁸F]FDG.     

In vivo near‐infrared fluorescence targeting of T cells: comparison of nanobodies and conventional monoclonal antibodies

The large size of conventional antibodies impedes tissue penetration and renal elimination, resulting in suboptimal in vivo targeting. Here we assess the utility of nanobodies and nanobody‐Fc‐fusion proteins as alternatives to monoclonal antibodies as theranostics, using T cell ADP–ribosyltransferase 2 (ART2) as a model antigen for specific targeting of lymph nodes. ART2‐specific monovalent nanobody s + 16a (17 kDa), a bivalent Fc‐fusion protein of s + 16a (s + 16‐mFc, 82 kDa), and conventional antibody Nika102 (150 kDa) were labeled with AlexaFluor680. In vitro binding and inhibitory properties of the three AF680 conjugates were assessed by flow cytometry. For in vivo imaging experiments, AF680 conjugates were intravenously injected in mice lacking (KO) or overexpressing (TG) ART2. We monitored circulating and excreted AF680 conjugates in plasma and urine and performed in vivo near‐infrared fluorescence imaging. Nanobody s + 16a⁶⁸⁰ and s + 16mFc⁶⁸⁰ labeled and inhibited ART2 on T cells in lymph nodes within 10 min. In contrast, mAb Nika102⁶⁸⁰ required 2 h for maximal labeling without inhibition of ART2. In vivo imaging revealed specific labeling of ART2‐positive lymph nodes but not of ART2‐negative lymph nodes with all AF680 conjugates. Even though bivalent s + 16mFc⁶⁸⁰ showed the highest labeling efficiency in vitro, the best lymph node imaging in vivo was achieved with monovalent nanobody s + 16a⁶⁸⁰, since renal elimination of unbound s + 16a⁶⁸⁰ significantly reduced background signals. Our results indicate that small single‐domain nanobodies are best suited for short‐term uses, such as noninvasive imaging, whereas larger nanobody‐Fc‐fusion proteins are better suited for long‐term uses, such as therapy of inflammation and tumors. Copyright © 2014 John Wiley & Sons, Ltd.     

VCAM‐1‐targeting gold nanoshell probe for photoacoustic imaging of atherosclerotic plaque in mice

The development of molecular probes and novel imaging modalities, allowing better resolution and specificity, is associated with an increased potential for molecular imaging of atherosclerotic plaques especially in basic and pre‐clinical research applications. In that context, a photoacoustic molecular probe based on gold nanoshells targeting VCAM‐1 in mice (immunonanoshells) was designed. The molecular probe was validated in vitro and in vivo, showing no noticeable acute toxic effects. We performed the conjugation of gold nanoshells displaying near‐infrared absorption properties with VCAM‐1 antibody molecules and PEG to increase their biocompatibility. The resulting immunonanoshells obtained under different conditions of conjugation were then assessed for specificity and sensitivity. Photoacoustic tomography was performed to determine the ability to distinguish gold nanoshells from blood both in phantoms and in vivo. Ex vivo optical projection tomography of hearts and aortas from atherosclerotic and control mice confirmed the selective accumulation of the immunonanoshells in atherosclerotic‐prone regions in mice, thus validating the utility of the probe in vivo in small animals for pre‐clinical research. These immunonanoshells represent an adequate mean to target atherosclerotic plaques in small animals, leading to new tools to follow the effect of therapies on the progression or regression of the disease. Copyright © 2012 John Wiley & Sons, Ltd.     

68Ga‐PET: a powerful generator‐based alternative to cyclotron‐based PET radiopharmaceuticals

PET (positron emission tomography) is a powerful diagnostic and imaging technique which requires short‐lived positron emitting isotopes. The most commonly used are accelerator‐produced ¹¹C and ¹⁸F. An alternative is the use of metallic positron emitters. Among them ⁶⁸Ga deserves special attention because of its availability from long‐lived ⁶⁸Ge/⁶⁸Ga generator systems which render ⁶⁸Ga radiopharmacy independent of an onsite cyclotron. The coordination chemistry of Ga³⁺ is dominated by its hard acid character. A variety of mono‐ and bifunctional chelators have been developed which allow the formation of stable ⁶⁸Ga³⁺complexes and convenient coupling to biomolecules. ⁶⁸Ga coupling to small biomolecules is potentially an alternative to ¹⁸F‐ and ¹¹C‐based radiopharmacy. In particular, peptides targeting G‐protein coupled receptors overexpressed on human tumour cells have shown preclinically and clinically high and specific tumour uptake. Kit‐formulated precursors along with the generator may be provided, similar to the ⁹⁹Mo/^99mTc‐based radiopharmacy, still the mainstay of nuclear medicine. Copyright © 2008 John Wiley & Sons, Ltd.     

Assessment of a training programme for the prevention of ventilator‐associated pneumonia

Background: Ventilator‐associated pneumonia (VAP) is the most frequent nosocomial infection in intensive care units (ICUs). Most published studies have analysed nurses' theoretical knowledge about a specific procedure; however, the transfer of this knowledge to the practice has received little attention. Aim: To assess the impact of training session on nurses' knowledge regarding VAP, compliance with VAP preventive measures, VAP incidence and determining whether nursing workload affects compliance. Method: A prospective, quasiexperimental, pre‐ and post‐study of the nursing team in a 16‐bed medical/surgical ICU. Pre‐intervention phase: a questionnaire to assess nurses' knowledge of VAP prevention measures, direct observation and review of clinical records to assess compliance. Intervention phase: eight training sessions for nurses. The post‐intervention phase mirrored the pre‐intervention phase. Findings: Nurses answered more questions correctly on the post‐intervention questionnaire than on the pre‐intervention (17·87 ± 2·69 versus 15·91 ± 2·68, p = 0·002). Compliance with the following measures was better during the post‐intervention period (p = 0·001): use of the smallest possible nasogastric tube, controlled aspiration of subglottic secretions and endotracheal tube cuff pressure, use of oral chlorhexidine and recording the endotracheal tube fixation number. VAP incidence remained unchanged throughout the study. However, a trend towards lower incidence of late (>4 days after intubation) VAP was observed (4·6 versus 3·1 episodes/1000 ventilation days, p = 0·37). Conclusion: The programme improved both knowledge of and compliance with VAP preventive measures, although improved knowledge did not always result in improved compliance.     

Assessing Glomerular Filtration in Small Animals Using [<Superscript>68</Superscript>Ga]DTPA and [<Superscript>68</Superscript>Ga]EDTA with PET Imaging

Purpose

Determining the glomerular filtration rate (GFR) is essential for clinical medicine but also for pre-clinical animal studies. Functional imaging using positron emission tomography (PET) allows repetitive almost non-invasive measurements. The aim of the study was the development and evaluation of easily synthesizable PET tracers for GFR measurements in small animals.

Procedures

Diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) were labeled with Ga-68. The binding to blood cells and plasma proteins was tested in vitro. The distribution of the tracers in rats was analyzed by PET imaging and ex vivo measurements. From the time-activity-curve of the blood compartment (heart) and the total tracer mass excreted by the kidney, the GFR was calculated. These values were compared directly with the inulin clearance in the same animals.

Results

Both tracers did not bind to blood cells. [⁶⁸Ga]DPTA but not [⁶⁸Ga]EDTA showed strong binding to plasma proteins. For this reason, [⁶⁸Ga]DPTA stayed much longer in the blood and only 30 % of the injected dose was eliminated by the kidney within 60 min whereas the excretion of [⁶⁸Ga]EDTA was 89 ± 1 %. The calculated GFR using [⁶⁸Ga]EDTA was comparable to the measured inulin clearance in the same animal. Using [⁶⁸Ga]-DPTA, the measurements led to values which were 80 % below the normal GFR. The results also revealed that definition of the volume of interest for the blood compartment affects the calculation and may lead to a slight overestimation of the GFR.

Conclusions

[⁶⁸Ga]EDTA is a suitable tracer for GFR calculation from PET imaging in small animals. It is easy to be labeled, and the results are in good accordance with the inulin clearance. [⁶⁸Ga]DTPA led to a marked underestimation of GFR due to its strong binding to plasma proteins and is therefore not an appropriate tracer for GFR measurements.

     

Targeted ultrasound contrast agent for molecular imaging of inflammation in high‐shear flow

Targeted ultrasound contrast materials (gas‐filled microbubbles carrying ligands to endothelial selectins or integrins) have been investigated as potential molecular imaging agents. Such microbubbles normally exhibit good targeting capability at the slower flow conditions. However, in the conditions of vigorous flow, binding may be limited. Here, we describe a microbubble capable of efficient binding to targets both in slow and fast flow (exceeding 4 dyne/cm² wall shear stress) using a clustered polymeric form of the fast‐binding selectin ligand sialyl Lewis^X. Microbubbles were prepared from decafluorobutane gas and stabilized with a monolayer of phosphatidylcholine, PEG stearate and biotin‐PEG‐lipid. Biotinylated PSLe^x (sialyl Lewis^X polyacrylamide) or biotinylated anti‐P‐selectin antibody (RB40.34) was attached to microbubbles via a streptavidin bridge. In a parallel plate flow chamber targeted adhesion model, PSLe^x bubbles demonstrated specific adhesion, retention and slow rolling on P‐selectin‐coated plates. Efficiency of firm targeted adhesion to a P‐selectin surface (140 molecules/µm²) was comparable for antibody‐carrying bubbles and PSLe^x‐targeted bubbles at 0.68 dyne/cm² shear stress. At fast flow (4.45 dyne/cm²), PSLe^x‐targeted bubbles maintained their ability to bind, while antibody‐mediated targeting dropped more than 20‐fold. At lower surface density of P‐selectin (7 molecules/µm²), targeting via PSLe^x was more efficient than via antibody under all the flow conditions tested. Negative control casein‐coated plates did not retain bubbles in the range of flow conditions studied. To confirm echogenicity, targeted PSLe^x‐bubbles were visualized on P‐selectin‐coated polystyrene plates by ultrasound imaging with a clinical scanner operated in pulse inversion mode; control plates lacking targeted bubbles did not show significant acoustic backscatter. In vivo, in a murine model of inflammation in the femoral vein setting, targeting efficacy of intravenously administered PSLe^x‐microbubbles was comparable with targeting mediated by anti‐P‐selectin antibody, and significantly exceeded the accumulation of non‐targeted control bubbles. In the inflamed femoral artery setting, PSLe^x‐mediated microbubble targeting was superior to antibody‐mediated targeting. Copyright © 2006 John Wiley & Sons, Ltd.     

A novel PET tracer for the imaging of αvβ3 and αvβ5 integrins in experimental breast cancer bone metastases

The aim of this study was the evaluation of ⁶⁸Ga‐DOTA‐E‐[c(RGDfK)]₂ as a novel PET tracer to image αvβ3 and αvβ5 integrins. For this purpose, DOTA‐E‐[c(RGDfK)]₂ was labeled with ⁶⁸Ga, which was obtained from a ⁶⁸Ge/⁶⁸Ga generator, purified by solid‐phase extraction and the radiochemical purity analyzed by radio‐RP‐HPLC. ⁶⁸Ga‐DOTA‐E‐[c(RGDfK)]₂ was obtained reproducibly in radiochemical yields of 60 ± 6% and with an excellent radiochemical purity of >99%. In nude rats bearing bone metastases after injection of MDA‐MB‐231 human breast cancer cells, biodistribution studies were performed to evaluate the accumulation of the radiotracer in selected organs, blood and bone metastases 0.5, 1, 2 and 3 h post injection. A rapid uptake into the bone metastases and rapid blood clearance was observed, resulting in tumor–blood ratios of up to 26.6 (3 h post injection) and tumor–muscle ratios of up to 7.9 (3 h post injection). A blocking experiment with coinjected αvβ3/αvβ5 antagonist showed the tumor uptake to be receptor‐specific. In an initial in vivo micro PET evaluation of the tracer using the same animal model, the bone metastasis was clearly visualized. These results suggest that ⁶⁸Ga‐DOTA‐E‐[c(RGDfK)]₂ is a promising PET tracer suitable for the imaging of αvβ3 and αvβ5 integrins in bone metastases. This novel PET tracer should be further evaluated concerning its usefulness for early detection of bone metastases and monitoring treatment response of these lesions. Copyright © 2011 John Wiley & Sons, Ltd.     

MR molecular imaging of HER‐2 in a murine tumor xenograft by SPIO labeling of anti‐HER‐2 affibody

In vivo molecular imaging is a rapidly growing research area both for basic and clinical science. Non‐invasive imaging of in vivo conditions at the molecular level increases understanding of the biological characteristics of normal and diseased tissues without the need for invasive surgical procedures. Among the various imaging modalities, magnetic resonance imaging (MRI) has garnered interest as a molecular imaging modality due to its high spatial resolution. Here, we have demonstrated that the combined use of HER‐2 targeting affibody, a small 7 kDa molecule that behaves similarly to antibodies, and superparamagnetic iron oxide (SPIO) can non‐invasively image HER‐2 expressing cells or tissues both in vitro and in vivo by MRI. This preliminary study demonstrates that affibody‐SPIO is a feasible, target‐specific contrast agent for in vivo MR molecular imaging. Copyright © 2010 John Wiley & Sons, Ltd.     

Incorporation of an apoE‐derived lipopeptide in high‐density lipoprotein MRI contrast agents for enhanced imaging of macrophages in atherosclerosis

Magnetic resonance (MR) imaging is becoming a pivotal diagnostic method to identify and characterize vulnerable atherosclerotic plaques. We previously reported a reconstituted high‐density lipoprotein (rHDL) nanoparticle platform enriched with Gd‐based amphiphiles as a plaque‐specific MR imaging contrast agent. Further modification can be accomplished by inserting targeting moieties into this platform to potentially allow for improved intraplaque macrophage uptake. Since studies have indicated that intraplaque macrophage density is directly correlated to plaque vulnerability, modification of the rHDL platform may allow for better detection of vulnerable plaques. In the current study we incorporated a carboxyfluoresceine‐labeled apolipoprotein E‐derived lipopeptide, P2fA2, into rHDL. The in vitro macrophage uptake and in vivo MR efficacy were demonstrated using murine J774A.1 macrophages and the apolipoprotein E knock‐out (apoE^?/?) mouse model of atherosclerosis. The in vitro studies indicated enhanced association of murine macrophages to P2fA2 enriched rHDL (rHDL–P2A2) nanoparticles, relative to rHDL, using optical techniques and MR imaging. The in vivo studies showed a more pronounced and significantly higher signal enhancement of the atherosclerotic wall 24 h after the 50 µmol Gd/kg injection of rHDL–P2A2 relative to administration of rHDL. The normalized enhancement ratio for atherosclerotic wall of rHDL–P2A2 contrast agent injection was 90%, while that of rHDL was 53% 24 h post‐injection. Confocal laser scanning microscopy revealed that rHDL–P2A2 nanoparticles co‐localized primarily with intraplaque macrophages. The results of the current study confirm the hypothesis that intraplaque macrophage uptake of rHDL may be enhanced by the incorporation of the P2fA2 peptide into the modified HDL particle. Copyright © 2008 John Wiley & Sons, Ltd.     

Radiolabelled GLP‐1 analogues for in vivo targeting of insulinomas

Internalizing agonists are usually selected for peptide receptor targeting. There is increasing evidence that non‐internalizing receptor antagonists can be used for this purpose. We investigated whether the glucagon‐like peptide‐1 receptor (GLP‐1R) antagonist exendin(9–39) can be used for in vivo targeting of GLP‐1R expressing tumours and compared the in vitro and in vivo characteristics with the GLP‐1R agonists exendin‐3 and exendin‐4. The binding and internalization kinetics of labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) were determined in vitro using INS‐1 cells. The in vivo targeting properties of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) were examined in BALB/c nude mice with subcutaneous INS‐1 tumours. ^natIn‐labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) exhibited similar IC₅₀ values (13.5, 14.4 and 13.4 n m , respectively) and bound to 26 × 10³, 41 × 10³ and 37 × 10³ receptors per cell, respectively. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed rapid in vitro binding and internalization kinetics, whereas [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed lower binding and minimal internalization in vitro. In mice, high specific uptake of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 [25.0 ± 6.0% injected dose (ID) g^?1] in the tumour was observed at 0.5 h post‐injection (p.i.) with similar uptake up to 4 h p.i. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed higher tumour uptake at 1 and 4 h p.i. (40.8 ± 7.0 and 41.9 ± 7.2% ID g^?1, respectively). Remarkably, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed only low specific uptake in the tumour at 0.5 h p.i. (3.2 ± 0.7% ID g^?1), rapidly decreasing over time. In conclusion, the GLP‐1R agonists [Lys⁴⁰(DTPA)]exendin‐3 and [Lys⁴⁰(DTPA)]exendin‐4 labelled with ¹¹¹In could be useful for in vivo GLP‐1R targeting, whereas [Lys⁴⁰(DTPA)]exendin(9–39) is not suited for in vivo targeting of the GLP‐1R. Copyright © 2012 John Wiley & Sons, Ltd.     

Spectroscopic,radiochemical, and theoretical studies of the Ga3+‐N‐2‐hydroxyethyl piperazine‐N′‐2‐ethanesulfonic acid (HEPES buffer) system: evidence for the formation of Ga3+‐ HEPES complexes in 68 Ga labeling reactions

Recent reports have claimed a superior performance of HEPES buffer in comparison to alternative buffer systems for ^67/68 Ga labeling in aqueous media. In this paper we report spectroscopic (¹H and ⁷¹ Ga NMR), radiochemical, mass spectrometry and theoretical modeling studies on the Ga³⁺/HEPES system (HEPES = N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid) performed with the aim of elucidating a potential contribution of HEPES in the ^68/67 Ga radiolabeling process. Our results demonstrate that HEPES acts as a weakly but competitive chelator of Ga³⁺ and that this interaction depends on the relative Ga³⁺: HEPES concentration. A by‐product formed in the labeling mixture has been identified as a [⁶⁸ Ga]Ga(HEPES) complex via chromatographic comparison with the nonradioactive analog. The formation of this complex was verified to compete with [⁶⁸ Ga]Ga(NOTA) complexation at low NOTA concentration. Putative chelation of Ga³⁺ by the hydroxyl and adjacent ring nitrogen of HEPES is proposed on the basis of ¹H NMR shifts induced by Ga³⁺ and theoretical modeling studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Tumor Targeting <Emphasis Type="Italic">via</Emphasis> Sialic Acid: [<Superscript>68</Superscript>Ga]DOTA-en-pba as a New Tool for Molecular Imaging of Cancer with PET

Purpose

The aim of this study was to demonstrate the potential of Ga-68-labeled macrocycle (DOTA-en-pba) conjugated with phenylboronic vector for tumor recognition by positron emission tomography (PET), based on targeting of the overexpressed sialic acid (Sia).

Procedures

The imaging reporter DOTA-en-pba was synthesized and labeled with Ga-68 at high efficiency. Cell binding assay on Mel-C and B16-F10 melanoma cells was used to evaluate melanin production and Sia overexpression to determine the best model for demonstrating the capability of [⁶⁸Ga]DOTA-en-pba to recognize tumors. The in vivo PET imaging was done with B16-F10 tumor-bearing SCID mice injected with [⁶⁸Ga]DOTA-en-pba intravenously. Tumor, blood, and urine metabolites were assessed to evaluate the presence of a targeting agent.

Results

The affinity of [⁶⁸Ga]DOTA-en-pba to Sia was demonstrated on B16-F10 melanoma cells, after the production of melanin as well as Sia overexpression was proved to be up to four times higher in this cell line compared to that in Mel-C cells. Biodistribution studies in B16-F10 tumor-bearing SCID mice showed blood clearance at the time points studied, while uptake in the tumor peaked at 60 min post-injection (6.36?±?2.41 % ID/g). The acquired PET images were in accordance with the ex vivo biodistribution results. Metabolite assessment on tumor, blood, and urine samples showed that [⁶⁸Ga]DOTA-en-pba remains unmetabolized up to at least 60 min post-injection.

Conclusions

Our work is the first attempt for in vivo imaging of cancer by targeting overexpression of sialic acid on cancer cells with a radiotracer in PET.

     

Preclinical evaluation of [111In]MICA‐401, an activity‐based probe for SPECT imaging of in vivo uPA activity

Urokinase‐type plasminogen activator (uPA) and its inhibitor PAI‐1 are key players in cancer invasion and metastasis. Both uPA and PAI‐1 have been described as prognostic biomarkers; however, non‐invasive methods measuring uPA activity are lacking. We developed an indium‐111 (¹¹¹In)‐labelled activity‐based probe to image uPA activity in vivo by single photon emission computed tomography (SPECT). A DOTA‐conjugated uPA inhibitor was synthesized and radiolabelled with ¹¹¹In ([¹¹¹In]MICA‐401), together with its inactive, hydrolysed form ([¹¹¹In]MICA‐402). A biodistribution study was performed in mice (healthy and tumour‐bearing), and tumour‐targeting properties were evaluated in two different cancer xenografts (MDA‐MB‐231 and HT29) with respectively high and low levels of uPA expression in vitro, with either the active or hydrolysed radiotracer. MicroSPECT was performed 95 h post injection followed by ex vivo biodistribution. Tumour uptake was correlated with human and murine uPA expression determined by ELISA and immunohistochemistry (IHC). Biodistribution data with the hydrolysed probe [¹¹¹In]MICA‐402 showed almost complete clearance 95 h post injection. The ex vivo biodistribution and SPECT data with [¹¹¹In]MICA‐401 demonstrated similar tumour uptakes in the two models: ex vivo 5.68 ± 1.41%ID/g versus 5.43 ± 1.29%ID/g and in vivo 4.33 ± 0.80 versus 4.86 ± 1.18 for MDA‐MB‐231 and HT‐29 respectively. Human uPA ELISA and IHC showed significantly higher uPA expression in the MDA‐MB‐231 tumours, while mouse uPA staining revealed similar staining intensities of the two tumours. Our data demonstrate non‐invasive imaging of uPA activity in vivo, although the moderate tumour uptake and hence potential clinical translation of the radiotracer warrants further investigation. Copyright © 2016 John Wiley & Sons, Ltd.     

SPECT imaging of fibrin using fibrin‐binding peptides

Noninvasive detection of fibrin in vivo using diagnostic imaging modalities may improve clinical decision‐making on possible therapeutic options in atherosclerosis, cancer and thrombus‐related pathologies such as pulmonary embolism and deep venous thrombosis. The aim of this study was to assess the potential of a novel ¹¹¹In‐labeled fibrin‐binding peptide (FibPep) to visualize thrombi in mice noninvasively using single‐photon emission computed tomography (SPECT). FibPep and a negative control peptide (NCFibPep) were synthesized and their fibrin‐binding properties were assessed in vitro. FibPep showed enhanced binding compared with NCFibPep to both fibrin and blood clots. FibPep bound to fibrin with a dissociation constant (K_d) of 0.8 μ m , whereas NCFibPep displayed at least a 100‐fold lower affinity towards fibrin. A FeCl₃‐injury carotid artery thrombosis mouse model was used to evaluate the peptides in vivo. FibPep and NCFibPep displayed rapid blood clearance and were eliminated via the renal pathway. In vivo SPECT imaging using FibPep allowed clear visualization of thrombi. Ex vivo biodistribution showed significantly increased uptake of FibPep in the thrombus‐containing carotid in comparison to the noninjured carotid (5.7 ± 0.7 and 0.6 ± 0.4% injected dose per gram (%ID g^?1), respectively; p < 0.01; n = 4), whereas nonspecific NCFibPep did not (0.4 ± 0.2 and 0.3 ± 0.0%ID g^?1, respectively; n = 4). In conclusion, FibPep displayed high affinity towards fibrin in vitro and rapid blood clearance in vivo, and allowed sensitive detection of thrombi using SPECT imaging. Therefore, this particular imaging approach may provide a new tool to diagnose and monitor diseases such as atherosclerosis and cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

Nature‐inspired nanoformulations for contrast‐enhanced in vivo MR imaging of macrophages

Magnetic resonance imaging (MRI) of macrophages in atherosclerosis requires the use of contrast‐enhancing agents. Reconstituted lipoprotein particles that mimic native high‐density lipoproteins (HDL) are a versatile delivery platform for Gd‐based contrast agents (GBCA) but require targeting moieties to direct the particles to macrophages. In this study, a naturally occurring methionine oxidation in the major HDL protein, apolipoprotein (apo) A‐I, was exploited as a novel way to target HDL to macrophages. We also tested if fully functional GBCA–HDL can be generated using synthetic apo A‐I peptides. The fluorescence and MRI studies reveal that specific oxidation of apo A‐I or its peptides increases the in vitro macrophage uptake of GBCA–HDL by 2–3 times. The in vivo imaging studies using an apo E‐deficient mouse model of atherosclerosis and a 3.0 T MRI system demonstrate that this modification significantly improves atherosclerotic plaque detection using GBCA–HDL. At 24 h post‐injection of 0.05 mmol Gd kg^?1 GBCA–HDL containing oxidized apo A‐I or its peptides, the atherosclerotic wall/muscle normalized enhancement ratios were 90 and 120%, respectively, while those of GBCA–HDL containing their unmodified counterparts were 35 and 45%, respectively. Confocal fluorescence microscopy confirms the accumulation of GBCA–HDL containing oxidized apo A‐I or its peptides in intraplaque macrophages. Together, the results of this study confirm the hypothesis that specific oxidation of apo A‐I targets GBCA–HDL to macrophages in vitro and in vivo. Furthermore, our observation that synthetic peptides can functionally replace the native apo A‐I protein in HDL further encourages the development of these contrast agents for macrophage imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

Phage Display Selection, <Emphasis Type="Italic">In Vitro</Emphasis> Characterization,and Correlative PET Imaging of a Novel HER3 Peptide

Purpose

HER3 (ERBB3) is a receptor tyrosine kinase that is implicated in treatment resistance across multiple cancers, including those of the breast, lung, and prostate. Overexpression of HER3 following targeted therapy can occur rapidly and heterogeneously both within a single lesion and across sites of metastasis, making protein quantification by biopsy highly challenging. A global, non-invasive methodology such as positron emission tomography (PET) imaging can permit serial quantification of HER3, providing a useful approach to monitor HER3 expression across the entire tumor burden both prior to and following treatment. PET imaging of HER3 expression may permit a more personalized approach to targeted therapy by allowing for detection of HER3-mediated resistance, in addition to informing clinical trial patient selection for novel therapies targeting HER3.

Procedures

Phage display selection targeting the HER3 extracellular domain was performed in order to develop a peptide with optimal blood clearance and highly accurate HER3 quantification.

Results

The selection converged to a consensus peptide sequence that was subsequently found to bind HER3 with an affinity of 270 ± 151 nM. The peptide, termed HER3P1, was bound with high selectivity to HER3 over other similar receptor tyrosine kinases such as EGFR and HER2. Furthermore, HER3P1 was able to distinguish between high and low HER3-expressing cells in vitro. The peptide was radiolabeled with Ga-68 and demonstrated to specifically bind HER3 by in vivo PET imaging. Uptake of [⁶⁸Ga]HER3P1 was highly specific for HER3-positive tumors, with tumor-to-background ratios ranging from 1.59–3.32, compared to those of HER3-negative tumors, ranging from 0.84–0.93. The uptake of [⁶⁸Ga]HER3P1 also demonstrated high (P < 0.001) correlation with protein expression as quantified by Western blot and confirmed by biodistribution.

Conclusions

HER3P1 accurately quantifies expression of HER3 by PET imaging and has potential utility as a clinical imaging agent.