Rapid hepatic clearance of 99mTc‐TMEOP: a new candidate for myocardial perfusion imaging

Background

^99mTc labeled radiotracers used in clinical practice lack the perfect characteristics for myocardial perfusion imaging. In particular, the high liver uptake can interfere in the interpretation of the inferior myocardial wall. Within the tricarbonyl approach, we used tris(pyrazolyl)methane ^99mTc organometallic complexes as a lead structure. Herein we present the production, in vivo and in vitro metabolic studies in rats and the first in vivo biodistribution in rats for tri‐methoxy‐tris‐pyrazolyl‐^99mTc‐(CO)₃ (^99mTc‐TMEOP), compared with ^99mTc‐sestamibi and ^99mTc‐tetrofosmin.

Methods

The chemical identity of ^99mTc‐TMEOP was characterized by RP‐HPLC. The octanol–water partition coefficient was determined under physiological conditions. In vitro stability and protein binding were determined using RP‐HPLC. In vivo stability was determined in blood, heart, liver and kidney homogenates, intestine and urine using RP‐HPLC. In vivo biodistribution was determined using dynamic planar acquisitions. Pinhole gated SPECT images were performed in other animals. Cardiac, liver and lung uptake were expressed as differential uptake ratios by drawing regions of interest in the organs of interest and around the total body. Heart–liver and heart–lung ratios were derived. Cardiac uptake was also expressed as percentage of injected activity. SPECT images were processed to determine the heart–liver ratio on SPECT images, to compare functional parameters between different tracers and to visualize homogeneous intracardiac tracer distribution.

Results

^99mTc‐TMEOP is a moderately lipophilic cation, is stable and does not undergo any transformation in vitro. ^99mTc‐TMEOP also shows a high in vivo stability. In vivo imaging shows liver kinetics faster than those of ^99mTc‐sestamibi and ^99mTc‐tetrofosmin. Cardiac uptake and functional analysis of pinhole gated SPECT data are comparable to those of ^99mTc‐sestamibi and ^99mTc‐tetrofosmin.

Conclusion

Although ^99mTc‐TMEOP shows a cardiac uptake between those of ^99mTc‐sestamibi and ^99mTc‐tetrofosmin, a better heart–liver ratio is obtained due to the faster liver washout. These results suggest possible faster cardiac perfusion imaging using ^99mTc‐TMEOP without liver activity interference. Copyright © 2010 John Wiley & Sons, Ltd.     

A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts

α_Vβ₃ Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near‐infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α_Vβ₃ integrin‐overexpressing tumors. DA364's binding affinity for α_Vβ₃ integrin was first evaluated in vitro. Human α_Vβ₃ integrin‐positive, U‐87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α_Vβ₃ integrin on U‐87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic‐RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α_Vβ₃ integrin‐overexpressing tumors. Copyright © 2011 John Wiley & Sons, Ltd.     

A radiolabeled nonapeptide probe targeting PC‐3 cells and bone metastases of prostate cancer in mice

Previous investigations showed that interleukin‐11 (IL‐11) and the IL‐11 receptor (IL‐11R) are correlated with regulation of tumor progression and may play significant roles in bone metastases. The nonapeptide structure c(CGRRAGGSC) is a phage‐display‐selected IL‐11 mimic that binds to IL‐11R. The aim of this study was to synthesize radiolabeled c(CGRRAGGSC) and to investigate the possible interaction between this radioactive probe and an IL‐11R‐positive bone metastasis model of PC‐3 prostate cancer. The molecular probe ^99mTc–DTPA–c(CGRRAGGSC) was radiolabeled with ^99mTc using the diethylenetriaminepentaacetic acid (DTPA) chelate. Counterstaining was performed with LSS670, a near‐infrared dye. The binding sites of the molecular probe in PC‐3 cells were observed under a fluorescence microscope. The binding characteristics of the labeled probe were analyzed using radioreceptor analysis. Single photon emission tomography imaging and biodistribution of the probe were investigated using xenografts of PC‐3 cells into tibias of nude mice. The labeled product, ^99mTc–DTPA–c(CGRRAGGSC), was obtained with high labeling efficiency, high radiochemical purity and good stability. The molecular probe was combined with the PC‐3 cell membrane and cytoplasm through fluorescence tracing. In the saturation and competitive inhibition experiments performed in vitro, the K_d value was 0.32 ± 0.02 n m and the B_max value was 754 ± 34 fmol mg^?1 pro. The probe exhibited a high tumor uptake in vivo. The radioactive molecular probe ^99mTc–DTPA–c(CGRRAGGSC) may be used as a specific molecular imaging agent for detecting IL‐11R overexpression in tumors and bone metastasis, such as prostate cancers. Copyright © 2012 John Wiley & Sons, Ltd.     

Fast clearing RGD‐based near‐infrared fluorescent probes for in vivo tumor diagnosis

A fast clearing hydrophilic near‐infrared (NIR) dye ICG‐Der‐02 was used to constitute tumor targeting contrast agents. Cell adhesion molecule integrin α_vβ₃ served as the target receptor because of its unique expression on almost all sprouting tumor vasculatures. The purpose of this study was to synthesize and compare the properties of integrin α_vβ₃‐targeted, fast clearing NIR probes both in vitro and in vivo for tumor diagnosis. ICG‐Der‐02 was covalently conjugated to three kinds of RGD peptide including linear, monoeric cyclic and dimeric RGD to form three RGD‐based NIR probes. The integrin receptor specificities of these probes were evaluated in vitro by confocal microscopy. The dynamic bio‐distribution and elimination ratse were in vivo real‐time monitored by a near‐infrared imaging system in normal mice. Further, the in vivo tumor targeting abilities of the RGD‐based NIR probes were compared in α_vβ₃‐positive MDA‐MB‐231, U87MG and α_vβ₃‐negtive MCF‐7 xenograft mice models. Three RGD‐based NIR probes were successfully synthesized with good optical properties. In vitro cellular experiments indicated that the probes have a clear binding affinity to α_υβ₃‐positive tumor cells, with a cyclic dimeric RGD probe owing the highest integrin affinity. Dynamic bio‐distributions of these probes showed a rapid clearing rate through the renal pathway. In vivo tumor targeting ability of the RGD‐based porbes was demonstrated on MDA‐MB‐231 and U87MG tumor models. As expected, the c(RGDyK)₂‐ICG‐Der‐02 probe displayed the highest tumor‐to‐normal tissue contrast. The in vitro and in vivo block experiments confirmed the receptor binding specificity of the probes. The hydrophilic dye‐labeled NIR probes exhibited a fast clearing rate and deep tissue penetration capability. Further, the α_υβ₃ receptor affinity of the three RGD‐based NIR probes followed the order of dimer cyclic > monomer cyclic > linear. The results demonstrate potent fast clearing probes for in vivo early tumor diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Targeted RGD nanoparticles for highly sensitive in vivo integrin receptor imaging

A new magnetic resonance imaging (MRI) contrast bearing RGD peptide is reported. In this study, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles with various sizes were prepared. Particles sizes between 6 and 13 nm were tuned by varying the stirring rate. Remarkable negative contrast was observed because USPIO nanoparticles (13.1 ± 2.1 nm) generated high transversal relaxivity r₂ (188 ± 3 m m ^?1 s^?1) and saturation magnetization (94 emu g^?1 Fe). The USPIO nanoparticles were coated with PDA [2‐(pyridyldithio)‐ethylamine; PDA nanoparticles] containing functional polymer, which can be readily synthesized by Michael addition. The PDA nanoparticles were conjugated with RGD peptide (RGD nanoparticles) for targeting the specific site. The target specificity and high relaxivity allowed RGD nanoparticles to differentiate the expression level of integrin receptor on several cell lines and tumors (MCF‐7, A‐549, HT‐29 and HT‐1080) by in vitro and in vivo MR imaging. Importantly, a remarkable negative contrast (?51.3 ± 6.7%) was observed for in vivo MR imaging of MCF‐7 tumor. This result implies that the RGD nanoparticles that greatly enhance the MR imaging are highly sensitive for early stage tumor detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Optical imaging detection of microscopic mammary cancer in ErbB‐2 transgenic mice through the DA364 probe binding αvβ3 integrins

Despite spontaneous tumor growth in genetically engineered mice being one of the most recognized tools for the in vivo evaluation of novel diagnostic and therapeutic anticancer compounds, monitoring early stage lesions in live animals is a goal that has yet to be achieved. A large number of targets for the molecular imaging of various diseases have been identified and many imaging technologies, including optical techniques are emerging. One of the most commonly exploited targets in tumor imaging is α_vβ₃ integrin, which plays an important role in the expansion, invasiveness and metastatic capability of a number of cancers, including breast cancer. The aim of this study was to set up an optical imaging method for the early detection of autochthonous mammary cancer in female BALB/c mice transgenic for the rat‐ErbB‐2 oncogene (BALB‐neuT). We show that DA364, a near‐infrared fluorescence arginine–glycine–aspartic acid cyclic probe, was taken up by neoplastic mammary glands and that its uptake increased with cancer progression. By contrast, the nonaccumulation of DA364 in the healthy mammary glands of virgin and lactating wild‐type mice suggests that the probe specifically targets breast cancers. Comparisons of optical imaging with whole‐mount and histological findings showed that DA364 allows the noninvasive visualization of atypical hyperplasia and microscopic foci of in situ carcinoma 2 months before mammary lesions become detectable by palpation. Moreover, DA364 was successfully used to monitor the outcome of anticancer vaccination. Therefore, it can be considered a promising early detection tool in near‐infrared noninvasive optical imaging for the early diagnosis of breast cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

Improvements in endotoxemic syndromes using a disintegrin,rhodostomin, through integrin αvβ3‐dependent pathway

Summary. Background and objectives: Septic shock is a major cause of morbidity and mortality in intensive care units, but there is still no effective therapy for the patients. We evaluated the effects of rhodostomin (Rn), an Arg‐Gly‐Asp‐containing snake venom disintegrin, on lipopolysaccharide (LPS)‐activated phagocytes in vitro and LPS‐induced endotoxemia in vivo. Methods and results: Rn inhibited adhesion, migration, cytokine production and mitogen‐activated protein kinase (MAPK) activation of macrophage induced by LPS. Flow cytometric analysis revealed that Rn specifically blocked anti‐αv mAb binding to RAW264.7. Besides inhibiting MAPK activation of THP‐1, Rn bound to LPS‐activated THP‐1 and specifically blocked anti‐αvβ3 mAb binding to THP‐1. Binding assays proved that integrin αvβ3 was the binding site for rhodostomin on phagocytes. Rn reversed the enhancement of fibronectin and vitronectin on LPS‐induced monocyte adhesion and cytokine release. Transfection of integrin αv siRNA also inhibited LPS‐induced activation of monocyte, and Rn exerted no further inhibitory effect. Furthermore, Rn significantly decreased the production of tumor necrosis factor‐α (TNF‐a), interleukin (IL)‐6, ‐1β and ‐10 and attenuated cardiovascular dysfunction, including blood pressure and heart pulse, and thrombocytopenia in LPS‐induced endotoxemic mice. Rn also protected against tissue inflammation as evidenced by histological examination. Conclusions: Rn may interact with αvβ3 integrin of monocytes/macrophages leading to interfere with the activation of phagocytes triggered by LPS. These results suggest that the protective function of Rn in LPS‐induced endotoxemia may be attributed to its anti‐inflammation activities in vivo.     

Evaluation of Two Optical Probes for Imaging the Integrin αvβ6− In Vitro and In Vivo in Tumor-Bearing Mice

Purpose

The purpose of this study was to develop and evaluate two α_vβ₆-targeted fluorescent imaging agents. The integrin subtype α_vβ₆ is significantly upregulated in a wide range of epithelial derived cancers, plays a key role in invasion and metastasis, and expression is often located at the invasive edge of tumors. α_vβ₆-targeted fluorescent imaging agents have the potential to guide surgical resection leading to improved patient outcomes. Both imaging agents were based on the bi-PEGylated peptide NH₂-PEG₂₈-A20FMDV2-K16R-PEG₂₈ (1), a peptide that has high affinity and selectivity for the integrin α_vβ₆: (a) 5-FAM-X-PEG₂₈-A20FMDV2-K16R-PEG₂₈ (2), and (b) IRDye800-PEG₂₈-A20FMDV2-K16R-PEG₂₈ (3).

Procedures

Peptides were synthesized using solid-phase peptide synthesis and standard Fmoc chemistry. Affinity for α_vβ₆ was evaluated by ELISA. In vitro binding, internalization, and localization of 2 was monitored using confocal microscopy in DX3puroβ₆ (α_vβ₆+) and DX3puro (α_vβ₆?) cells. The in vivo imaging and ex vivo biodistribution of 3 was evaluated in three preclinical mouse models, DX3puroβ₆/DX3puro and BxPC-3 (α_vβ₆+) tumor xenografts and a BxPC-3 orthotopic pancreatic tumor model.

Results

Peptides were obtained in >?99% purity. IC₅₀ values were 28 nM (2) and 39 nM (3). Rapid α_vβ₆-selective binding and internalization of 2 was observed. Fluorescent intensity (FLI) measurements extracted from the in vivo images and ex vivo biodistribution confirmed uptake and retention of 3 in the α_vβ₆ positive subcutaneous and orthotopic tumors, with negligible uptake in the α_vβ₆-negative tumor. Blocking studies with a known α_vβ₆-targeting peptide demonstrated α_vβ₆-specific binding of 3.

Conclusion

Two fluorescence imaging agents were developed. The α_vβ₆-specific uptake, internalization, and endosomal localization of the fluorescence agent 2 demonstrates potential for targeted therapy. The selective uptake and retention of 3 in the α_vβ₆-positive tumors enabled clear delineation of the tumors and surgical resection indicating 3 has the potential to be utilized during image-guided surgery.

     

Technetium-99m-labelled HL91 and technetium-99m-labelled MIBI SPECT imaging for the detection of ischaemic viable myocardium: a preliminary study

Purpose: The assessment of myocardial viability has become an important aspect of the diagnostic and prognostic work‐up of patients with coronary artery disease. Technetium‐99m labelled sestamibi (^99mTc‐MIBI) myocardial perfusion imaging may underestimate the viability of ischaemic myocardium. Technetium‐99m labelled 4,9‐diaza‐3,3,10,10‐tetramethyldodecan‐2,11‐dione dioxime (^99mTc‐HL91) is a hypoxia‐avid agent which can identify acutely ischaemic viable myocardium in a canine model using a standard gamma camera. The aim of this study was to evaluate uptake character of ischaemic viable myocardium and diagnostic performance of single‐photon emission computed tomography (SPECT) imaging by ^99mTc‐HL91 and ^99mTc‐MIBI in detecting ischaemic viable myocardium in coronary heart disease. Methods: A total of 41 patients with coronary artery disease were recruited from March 2008 to May 2009. For detecting ischaemic viable myocardium, SPECT imaging by ^99mTc‐HL91 and ^99mTc‐MIBI were performed in all patients before coronary revascularization. Six patients with single ischaemic myocardial segment received a 2‐day SEPCT/CT imaging protocol and the uptake of ^99mTc‐HL91 in ischaemic myocardium was quantitatively analysed. The remaining 35 patients received a 1‐day ^99mTc‐HL91 and ^99mTc‐MIBI SPECT imaging protocol. Resting ^99mTc‐MIBI myocardial perfusion imaging in 3–18 months after revascularization was used as the standard methodology to evaluate the myocardial viability. Results: In 41 patients, 66 ischaemic myocardial segments were proven to be viable and 12 to be necrotic by resting ^99mTc‐MIBI myocardial perfusion imaging after coronary revascularization. Furthermore, 60 viable segments with negative uptake of ^99mTc‐MIBI showed positive uptake of ^99mTc‐HL91. The remaining six viable segments and 12 necrotic segments showed both negative uptake of ^99mTc‐HL91 and ^99mTc‐MIBI. The sensitivity, specificity, accuracy, Younden Index, positive predictive value and negative predictive value for evaluating ischaemic viable myocardium were 90·9%, 100%, 92·3%, 90·9%, 100% and 66·7%, respectively. Ischaemic viable myocardium had the negative ^99mTc‐MIBI uptake and positive ^99mTc‐HL91 uptake, which demonstrated a mismatched uptake character. Quantitative analysis indicated the uptake of ^99mTc‐HL91 in viable myocardium was increasing in the first 1–3 h and remained stable at the 3–4 h after injection. Conclusion: Functional SPECT imaging with ^99mTc‐HL91 and ^99mTc‐MIBI can be used to detect the seriously ischaemic but viable myocardium with a mismatched uptake character. The uptake of ^99mTc‐HL91 in the viable myocardium reached a stable level at 3–4 h after injection.     

Comparison of [<Superscript>99m</Superscript>Tc]3PRGD<Subscript>2</Subscript> Imaging and [<Superscript>18</Superscript>F]FDG PET/CT in Breast Cancer and Expression of Integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> in Breast Cancer Vascular Endothelial Cells

Purpose

This study aimed to investigate the value of ^99mtechnetium-three polyethylene glycol spacers-arginine-glycine-aspartic acid ([^99mTc]3PRGD₂) imaging in diagnosis and staging of breast cancer compared with 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) imaging, and to explore the expression of integrin α_vβ₃ in tumor vascular endothelial cells.

Procedures

Forty-two women with suspected breast cancer underwent both [^99mTc]3PRGD₂ imaging and [¹⁸F]FDG imaging. Visual analysis was used to assess primary breast lesion, axillary lymph node, and distant metastasis. The tumor-blood (T/B) ratios from [^99mTc]3PRGD₂ imaging and the maximum standardized uptake value (SUVmax) from [¹⁸F]FDG imaging were analyzed for breast lesions. Integrin α_vβ₃ was analyzed through immunohistochemistry.

Results

Forty-five breast lesions were found (malignant, n?=?38; benign, n?=?7). The sensitivity, specificity, and accuracy of [^99mTc]3PRGD₂ and [¹⁸F]FDG imaging in visual analysis for the breast lesion were 97.4, 87.5, and 95.6 % and 97.4, 71.4, and 93.3 %, respectively (P?>?0.05). For semi-quantitative analysis, no significant difference of the area under the curves (AUC) was found in the imaging using the two radiopharmaceuticals (0.880 and 0.955; Z?=?0.88, P?>?0.05). The sensitivity, specificity, and accuracy for axillary lymph node metastasis with [^99mTc]3PRGD₂ and [¹⁸F]FDG were 78.05, 99.36, and 94.92 % and 85.37, 98.72, and 95.64 %, respectively (P?>?0.05). Nine patients with distant metastases were all detected with the two radiopharmaceuticals. The expression of integrin α_vβ₃ was correlated with [^99mTc]3PRGD₂ uptake (r?=?0.582, P?=?0.001), which were significantly higher in the HER2-positive and stage III–IV patients (P?<?0.05).

Conclusions

The prospective study demonstrated that [^99mTc]3PRGD₂ imaging seems to be valuable for diagnosis of breast cancer and its staging. It may be less sensitive for detecting small lymph node metastatic lesions when compared with [¹⁸F]FDG imaging. Integrin α_vβ₃ in tumor microvessels was associated with the breast cancer subtype and its staging.

     

Localization,mechanism and reduction of renal retention of technetium‐99m labeled epidermal growth factor receptor‐specific nanobody in mice

Background

Nanobodies are single‐domain antigen binding fragments derived from functional heavy‐chain antibodies elicited in Camelidae. They are powerful probes for radioimmunoimaging, but their renal uptake is relatively high. In this study we have evaluated the role of megalin on the renal uptake of anti‐EGFR ^99mTc‐7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of ^99mTc‐7C12.

Methods

First we compared the renal uptake of ^99mTc‐7C12 in megalin‐deficient and megalin‐wild‐type mice using pinhole SPECT/microCT and ex vivo analysis. The effect of gelofusine and lysine administration on renal accumulation of ^99mTc‐7C12 was analyzed in CD‐1 mice divided into lysine preload at 30 min before tracer injection (LysPreload), LysPreload + gelofusine coadministration (LysPreload + GeloCoad), lysine coadministration (LysCoad), gelofusine coadministration (GeloCoad) and LysCoad + GeloCoad. The combined effect of gelofusine and lysine on tumor uptake was tested in mice xenografts.

Results

Renal uptake of ^99mTc‐7C12 was 44.22 ± 3.46% lower in megalin‐deficient compared with megalin‐wild‐type mice. In CD‐1 mice, lysine preload had no effect on the renal retention whereas coinjection of lysine or gelofusine with the tracer resulted in 25.12 ± 2.99 and 36.22 ± 3.07% reduction, respectively. The combined effect of gelofusine and lysine was the most effective, namely a reduction of renal retention of 45.24 ± 2.09%. Gelofusine and lysine coadministration improved tumor uptake.

Conclusion

Megalin contributes to the renal accumulation of ^99mTc‐7C12. Gelofusine and lysine coinjection with the tracer reduces the renal uptake while tumor uptake is improved. Although this methodology allows for optimization of imaging protocol using nanobodies, further improvements are needed before using these molecules for radionuclide therapy. Copyright © 2010 John Wiley & Sons, Ltd.     

Dual In Vivo Quantification of Integrin-targeted and Protease-activated Agents in Cancer Using Fluorescence Molecular Tomography (FMT)

Purpose

Integrins, especially α_vβ₃ and α_vβ₅, are upregulated in tumor cells and activated endothelial cells and as such, serve as cancer biomarkers. We developed a novel near-infrared-labeled optical agent for the in vivo detection and quantification of α_vβ₃/α_vβ₅.

Procedures

A small peptidomimetic α_vβ₃ antagonist was synthesized, coupled to a near-infrared fluorescent (NIRF) dye, and tested for binding specificity using integrin-overexpressing cells, inhibition of vitronectin-mediated cell attachment, binding to tumor and endothelial cells in vitro, and competition studies. Pharmacokinetics, biodistribution, specificity of tumor targeting, and the effect of an antiangiogenic treatment were assessed in vivo.

Results

The integrin NIRF agent showed strong selectivity towards α_vβ_3/α_vβ₅ in vitro and predominant tumor distribution in vivo, allowing noninvasive and real-time quantification of integrin signal in tumors. Antiangiogenic treatment significantly inhibited integrin signal in vivo but had no effect on a cathepsin-cleavable NIR agent. Simultaneous imaging revealed different patterns of distribution reflecting the underlying differences in integrin and cathepsin biology during tumor progression.

Conclusions

NIRF-labeled integrin antagonists allow noninvasive molecular fluorescent imaging and quantification of tumors in vivo, improving and providing more refined approaches for cancer detection and treatment monitoring.     

RGD‐ligand mimetic antagonists of integrin αIIbβ3 paradoxically enhance GPVI‐induced human platelet activation

Summary. Background: The integrin α_IIbβ₃ is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of α_IIbβ₃, for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD‐ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. Objectives: To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. Methods: We assessed the effect of lotrafiban, and other α_IIbβ₃ antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro‐coagulant activity) and dense granule release. Results: In all cases, no effect of α_IIbβ₃ antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)‐associated FcR γ‐chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y₁₂ antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. Conclusions: These data provide a mechanism for enhancement of platelet activity by α_IIbβ₃ inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.     

Imaging VEGF Receptors and α<Subscript>v</Subscript>β<Subscript>3</Subscript> Integrins in a Mouse Hindlimb Ischemia Model of Peripheral Arterial Disease

Purpose

To compare targeted imaging of vascular endothelial growth factor (VEGF) receptors vs. α_vβ₃ integrins in a mouse hindlimb ischemia model of peripheral artery disease.

Procedures

Male wild-type (WT) C57BL/6 mice (8- to 10-week old) (n?=?24) underwent left femoral artery ligation. The right leg served as control. Five days later, mice were injected with either VEGF receptor targeting [^99mTc]DOTA-PEG-scVEGF ([^99mTc]scV) (n?=?8) or with α_vβ₃-targeting tracer [^99mTc]HYNIC-cycloRGD ([^99mTc]RGD) (n?=?8) and underwent single photon emission computed tomography (SPECT) x-ray computed tomography imaging. To assess non-specific [^99mTc]scV uptake, six additional mice received a mixture of [^99mTc]scV and 30-fold excess of targeting protein, scVEGF. Tracer uptake as %ID was measured using volumetric regions encompassing the hindlimb muscles and as %ID/g from harvested limb muscles. Double and triple immunofluorescent analysis on tissue sections established localization of α_vβ₃, VEGFR-1, VEGFR-2, as well as certain cell lineage markers.

Results

Tracer uptake, as %ID/g, was higher in ligated limbs of mice injected with [^99mTc]scV compared to ligated hindlimbs in mice injected with [^99mTc]RGD (p?=?0.02). The ratio of tracer uptake for ligated/control hindlimb was borderline higher for [^99mTc]scV than for [^99mTc]RGD (p?=?0.06). Immunofluorescent analysis showed higher prevalence of VEGFR-1, VEGFR-2, and α_vβ₃, in damaged vs. undamaged hindlimb tissue, but with little co-localization of these markers. Double immunofluorescent staining showed partial co-localization of VEGFR-1, VEGFR-2, and α_vβ_3, with endothelial cell marker FVIII, but not with CD31. Immunostaining for VEGFR-1 and VEGFR-2 additionally co-localized with lineage markers for endothelial progenitor cell and monocytes/macrophages, with a more diverse pattern of co-localization for VEGFR-2.

Conclusion

In a mouse hindlimb ischemia model of peripheral artery disease, [^99mTc]scV SPECT tracer-targeting VEGF receptors showed a more robust signal than [^99mTc]RGD tracer-targeting α_vβ₃. Immunofluorescent analysis suggests that uptake of [^99mTc]scV and [^99mTc]RGD in damaged tissue is due to non-overlapping cell populations and reflects different dynamic processes and that enhanced uptake of [^99mTc]scV may be due to the presence of VEGF receptors on additional cell types.

     

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

Summary. Background: β₃‐Integrins are involved in platelet aggregation via α_IIbβ₃ [glycoprotein (GP)IIb–GPIIIa], and in angiogenesis via endothelial α_Vβ₃. Cross‐reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. Objectives: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on β₃‐integrins. Methods: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC₅₀ were determined by immunodot assays, enzyme‐linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb–GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. Results: We observed specific and high‐affinity binding to an intact GPIIb–GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose‐dependent fibrinogen binding to GPIIb–GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross‐reactivity with α_Vβ₃, and also demonstrated proangiogenic effects in tube formation and CAM assays. Conclusions: These Fab fragments are the first entirely human anti‐GPIIb–GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual‐specificity anti‐β₃‐integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.     

In vivo biodistribution of radiolabeled MMP‐2/9 activatable cell‐penetrating peptide probes in tumor‐bearing mice

Matrix metalloproteinases (MMPs) play a pivotal role in cancer progression and present therefore an interesting biomarker for early diagnosis, staging and therapy evaluation. Consequently, MMP‐specific molecular imaging probes have been proposed for noninvasive visualization and quantification of MMP activity. An interesting approach is MMP‐2/9 activatable cell‐penetrating peptides (ACPP) that accumulate in the tumor tissue after activation. However, a recent study revealed that probe activation occurred already in the vasculature followed by nonspecific tumor targeting. In the latter study, biodistribution was determined 6 and 24 h post‐ACPP injection. An alternative explanation could still be that the kinetics of tumor‐specific activation is faster than that of blood activation plus subsequent nonspecific uptake in tumor. The aim of this study was to assess if tumor‐specific ACPP activation occurs in mice with MMP‐2/9 positive subcutaneous HT‐1080 tumors at 3 h post‐injection. As control, we studied the MMP‐2/9 sensitive ACPP in mice bearing subcutaneous BT‐20 tumors with low MMP‐2/9 expression to test if probe cleavage correlates with tumoral MMP expression. Ex vivo biodistribution showed no improved tumoral ACPP activation in HT‐1080 tumor‐bearing mice at 3 h post‐injection compared with previous reported data collected at 24 h post‐injection. Furthermore, tumoral uptake and relative tumoral activation for ACPP were similar in both BT‐20 and HT‐1080 tumor‐bearing mice. In conclusion, this study suggests that tumoral ACPP uptake in these tumor models originates from probe activation in the vasculature instead of tumor‐specific MMP activation. Novel ACPPs that target tissue‐specific proteases without nonspecific activation may unleash the full potential of the elegant ACPP concept. Copyright © 2014 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.     

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N‐terminus of blade 1 of the β‐propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Summary. Background: Studies of Glanzmann thrombasthenia (GT)‐causing mutations has generated invaluable information on the formation and function of integrin αIIbβ₃. Objective: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Methods and Results: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β‐propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ₃ was found in patients’ platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β₃. Although the αIIbβ₃ was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ₃ crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ₃. Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ₃ surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ₃, αVβ₃ harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ₃ to calcium chelation compared with αIIbβ₃. Conclusion: The new GT causing mutation highlights the importance of calcium binding domains in the β‐propeller for intracellular trafficking of αIIbβ₃. The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.     

The accuracy of quantitative parameters in 99mTc‐MAG3 dynamic renography: a national audit based on virtual image data

Assessment of image analysis methods and computer software used in ^99mTc‐MAG3 dynamic renography is important to ensure reliable study results and ultimately the best possible care for patients. In this work, we present a national multicentre study of the quantification accuracy in ^99mTc‐MAG3 renography, utilizing virtual dynamic scintigraphic data obtained by Monte Carlo‐simulated scintillation camera imaging of digital phantoms with time‐varying activity distributions. Three digital phantom studies were distributed to the participating departments, and quantitative evaluation was performed with standard clinical software according to local routines. The differential renal function (DRF) and time to maximum renal activity (T_max) were reported by 21 of the 28 Swedish departments performing ^99mTc‐MAG3 studies as of 2012. The reported DRF estimates showed a significantly lower precision for the phantom with impaired renal uptake than for the phantom with normal uptake. The T_max estimates showed a similar trend, but the difference was only significant for the right kidney. There was a significant bias in the measured DRF for all phantoms caused by different positions of the left and right kidney in the anterior–posterior direction. In conclusion, this study shows that virtual scintigraphic studies are applicable for quality assurance and that there is a considerable uncertainty associated with standard quantitative parameters in dynamic ^99mTc‐MAG3 renography, especially for patients with impaired renal function.     

Imaging integrin alpha‐v‐beta‐3 expression in tumors with an 18F‐labeled dimeric RGD peptide

Integrin α_vβ₃ receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine–glycine–aspartatic acid (RGD) peptide and integrin α_vβ₃, but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of ¹⁸F. In this study, the dimeric RGD peptide, E‐[c(RGDfK)]₂, was conjugated with NODAGA and radiolabeled with ¹⁸F in a simple one‐pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA‐E‐[c(RGDfK)]₂ labeled with ¹⁸F at a specific activity of 1.8 MBq nmol^?1 and a radiochemical purity of 100% could be achieved. The logP value of ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂ was ?4.26 ± 0.02. In biodistribution studies, ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g^?1) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ (3.44 ± 0.20 %ID g^?1, 2 h p.i.) was significantly lower than that of reference compounds ⁶⁸Ga‐labeled NODAGA‐E‐[c(RGDfK)]₂ (6.26 ± 0.76 %ID g^?1; p <0.001) and ¹¹¹In‐labeled NODAGA‐E‐[c(RGDfK)]₂ (4.99 ± 0.64 %ID g^?1; p < 0.01). Co‐injection of an excess of unlabeled NODAGA‐E‐[c(RGDfK)]₂ along with ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g^?1). The α_vβ₃ integrin‐expressing SK‐RC‐52 tumor could be successfully visualized by microPET with ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂. In conclusion, NODAGA‐E‐[c(RGDfK)]₂ could be labeled rapidly with ¹⁸F using a direct aqueous, one‐pot method and it accumulated specifically in α_vβ₃ integrin‐expressing SK‐RC‐52 tumors, allowing for visualization by microPET. Copyright © 2013 John Wiley & Sons, Ltd.