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Hard ticks feed on their host for multiple days. To ensure firm attachment, they secrete a protein-rich saliva that eventually forms a solid cement cone. The underlying mechanism of this liquid-to-solid transition is not yet understood. This study focuses on the phase transitions of a disordered glycine-rich protein (GRP) that is prominent in tick saliva. We show that GRP undergoes liquid-liquid phase separation via simple coacervation to form biomolecular condensates in salty environments. Cation-pi and pi-pi interactions near the C-terminus promote coacervation while a negatively charged N-terminus prolongs its onset through electrostatic repulsion. Interestingly, GRP condensates exhibit ageing and undergo liquid-to-gel transition to form viscoelastic networks as well as solid-like condensates. Lastly, we provide evidence for protein-rich condensates in natural tick saliva. Our findings provide a starting point to gain insights into the bioadhesion of ticks, develop novel tick control strategies, and towards biomedical applications such as tissue sealants.

INTRODUCTION: Vitamin K antagonists (VKA) and non-vitamin K oral antagonist anticoagulants (NOAC) are used in the clinic to reduce risk of thrombosis. However, they also exhibit vascular off-target effects. The aim of this study is to compare VKA and NOAC on atherosclerosis progression and calcification in an experimental setup.MATERIAL AND METHODS: Female Apoe-/- mice (age 12 weeks) were fed Western-type diet as control or supplemented with dabigatran etexilate or warfarin for 6 or 18 weeks. Vascular calcification was measured in whole aortic arches using µCT and [18 F]-NaF. Atherosclerotic burden was assessed by (immuno)histochemistry. Additionally, in vitro effects of warfarin, thrombin, and dabigatran on primary vascular smooth muscle cells (VSMC) were assessed.RESULTS: Short-term treatment with warfarin promoted formation of atherosclerotic lesions with a pro-inflammatory phenotype, and more rapid plaque progression compared with control and dabigatran. In contrast, dabigatran significantly reduced plaque progression compared with control. Long-term warfarin treatment significantly increased both presence and activity of plaque calcification compared with control and dabigatran. Calcification induced by warfarin treatment was accompanied by increased presence of uncarboxylated matrix Gla protein. In vitro, both warfarin and thrombin significantly increased VSMC oxidative stress and extracellular vesicle release, which was prevented by dabigatran.CONCLUSION: Warfarin aggravates atherosclerotic disease activity, increasing plaque inflammation, active calcification, and plaque progression. Dabigatran lacks undesired vascular side effects and reveals beneficial effects on atherosclerosis progression and calcification. The choice of anticoagulation impacts atherosclerotic disease by differential off target effect. Future clinical studies should test whether this beneficial effect also applies to patients.

Background: Ticks puncture the skin of their hosts and secrete saliva, containing antiplatelet proteins, into the blood. Here, we studied disagregin, a potent platelet-inhibiting protein derived from the salivary glands of Ornithodoros moubata, an African soft tick. Whereas conventional alpha IIb beta 3 antagonists contain an Arg-Gly-Asp (RGD) sequence for platelet integrin binding, disagregin contains an Arg-Glu-Asp (RED) sequence, hypothesizing a different mode of inhibitory action.Objectives: We aimed to compare the inhibitory effects of disagregin and its RGD variant (RGD-disagregin) on platelet activation and to unravel the molecular basis of disagregin-alpha IIb beta 3 integrin interactions.Methods: Disagregin and RGD-disagregin were synthesized by tert-butyloxycarbonyl -based solid-phase peptide synthesis. Effects of both disagregins on platelet aggregation were assessed by light transmission aggregometry in human platelet-rich plasma. Whole-blood thrombus formation was investigated by perfusing blood over collagen I with and without tissue factor at a high wall-shear rate (1000 s(-1)) in the presence of disagregin, RGD-disagregin, or eptifibatide.Results: Disagregin showed inhibition of collagen- and ADP-induced platelet aggregation with half maximal inhibitory concentration values of 64 and 99 nM, respectively. This resembled the complete antiaggregatory effect of eptifibatide. Multiparameter assessment of thrombus formation showed highly suppressed platelet adhesion and aggregate formation with both disagregins, in contrast to eptifibatide treatment, which incompletely blocked aggregation under flow. Fibrin formation under flow was delayed by both disagregin and RGD-disagregin (P Conclusions: Both alpha IIb beta 3-blocking disagregins have a strong potential to suppress collagen-tissue factor-mediated platelet adhesion, thrombus formation, and fibrin formation. Both disagregins can be seen as potential new alpha IIb beta 3 inhibitors.

Glycoprotein (GP)VI and integrin αIIbβ3 are key signaling receptors in collagen-dependent platelet aggregation and in arterial thrombus formation under shear. The multiple downstream signaling pathways are still poorly understood. Here, we focused on disclosing the integrin-dependent roles of focal adhesion kinase (protein tyrosine kinase 2, PTK2), the shear-dependent collagen receptor GPR56 (ADGRG1 gene), and calcium and integrin-binding protein 1 (CIB1). We designed and synthetized peptides that interfered with integrin αIIb binding (pCIB and pCIBm) or mimicked the activation of GPR56 (pGRP). The results show that the combination of pGRP with PTK2 inhibition or of pGRP with pCIB > pCIBm in additive ways suppressed collagen- and GPVI-dependent platelet activation, thrombus buildup, and contraction. Microscopic thrombus formation was assessed by eight parameters (with script descriptions enclosed). The suppressive rather than activating effects of pGRP were confined to blood flow at a high shear rate. Blockage of PTK2 or interference of CIB1 no more than slightly affected thrombus formation at a low shear rate. Peptides did not influence GPVI-induced aggregation and Ca2+ signaling in the absence of shear. Together, these data reveal a shear-dependent signaling axis of PTK2, integrin αIIbβ3, and CIB1 in collagen- and GPVI-dependent thrombus formation, which is modulated by GPR56 and exclusively at high shear. This work thereby supports the role of PTK2 in integrin αIIbβ3 activation and signaling.

Although anatomical imaging modalities (X-ray, computed tomography (CT), magnetic resonance imaging (MRI)) still have a higher spatial resolution (0.1–1 mm) than molecular imaging modalities (single-photon emission computed tomography (SPECT), positron emission tomography (PET), optical imaging (OI)), the advantage of molecular imaging is that it can detect molecular and cellular changes at the onset of a disease before it leads to morphological tissue changes, which can be detected by anatomical imaging. During the last decades, noninvasive diagnostic imaging has encountered a rapid growth due to the development of dedicated imaging equipment for preclinical animal studies. In addition, the introduction of multimodality imaging (PET/CT, SPECT/CT, PET/MRI) which combines high-resolution conventional anatomical imaging with high sensitivity of tracer-based molecular imaging techniques has led to successful accomplishments in this exciting field. In this book chapter, we will focus on chemical synthesis techniques for site-specific incorporation of radionuclide chelators. Subsequently, radiolabeling based on complexation of a radionuclide with a chelator will be discussed, with focus on: diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-triacetic acid (NOTA), hexa-histidine (His-tag), and 6-hydrazinonicotinic acid (HYNIC) that allow the production of peptides labeled with 18F, 68Ga, 99mTc, and 111In – the currently most widely used isotopes.

Atherosclerosis is one of the leading causes of mortality in developed and developing countries. The onset of atherosclerosis development is accompanied by overexpression of several inflammatory chemokines. Neutralization of these chemokines by chemokine-binding agents attenuates atherosclerosis progression. Here, we studied structural binding features of the tick protein Evasin-3 to chemokine (C-X-C motif) ligand 1 (CXCL1). We showed that Evasin-3-bound CXCL1 is unable to activate the CXCR2 receptor, but retains affinity to glycosamino-glycans. This observation was exploited to detect inflammation by visualizing a group of closely related CXC-type chemokines deposited on cell walls in human endothelial cells and murine carotid arteries by a fluorescent Evasin-3 conjugate. This work highlights the applicability of tick-derived chemokine-binding conjugates as a platform for the development of new agents for inflammation imaging.

Ticks can seriously affect human and animal health around the globe, causing significant economic losses each year. Chemical acaricides are widely used to control ticks, which negatively impact the environment and result in the emergence of acaricide-resistant tick populations. A vaccine is considered as one of the best alternative approaches to control ticks and tick-borne diseases, as it is less expensive and more effective than chemical controls. Many antigen-based vaccines have been developed as a result of current advances in transcriptomics, genomics, and proteomic techniques. A few of these (e.g., Gavac® and TickGARD®) are commercially available and are commonly used in different countries. Furthermore, a significant number of novel antigens are being investigated with the perspective of developing new anti-tick vaccines. However, more research is required to develop new and more efficient antigen-based vaccines, including on assessing the efficiency of various epitopes against different tick species to confirm their cross-reactivity and their high immunogenicity. In this review, we discuss the recent advancements in the development of antigen-based vaccines (traditional and RNA-based) and provide a brief overview of recent discoveries of novel antigens, along with their sources, characteristics, and the methods used to test their efficiency.

To feed successfully, ticks must bypass or suppress the host’s defense mechanisms, particularly the immune system. To accomplish this, ticks secrete specialized immunomodulatory proteins into their saliva, just like many other blood-sucking parasites. However, the strategy of ticks is rather unique compared to their counterparts. Ticks’ tendency for gene duplication has led to a diverse arsenal of dozens of closely related proteins from several classes to modulate the immune system’s response. Among these are chemokine-binding proteins, complement pathways inhibitors, ion channels modulators, and numerous poorly characterized proteins whose functions are yet to be uncovered. Studying tick immunomodulatory proteins would not only help to elucidate tick-host relationships but would also provide a rich pool of potential candidates for the development of immunomodulatory intervention drugs and potentially new vaccines. In the present review, we will attempt to summarize novel findings on the salivary immunomodulatory proteins of ticks, focusing on biomolecular targets, structure-activity relationships, and the perspective of their development into therapeutics.

1380 Objectives: The purpose of this study was to investigate how gender, BMI, patient cross-sectional area and heart size affect the influence of time-of-flight (TOF) and point spread function (PSF) modeling in image reconstruction on regional absolute myocardial blood flow (MBF) measured with 82Rb PET in an obese population. Methods: Dynamic rest/stress 82Rb cardiac PET studies were performed for 30 obese subjects on a TOF PET scanner. Subject characteristics were (mean ± SD for continuous variables): gender 9 male, 21 female; weight 138.9 ± 32.8 kg; BMI 47.8 ± 9.5 kg/m2; heart size 92.4 ± 55.3 ml; cross-sectional area in a transaxial slice containing the heart 1133 ± 233 cm2. Images were reconstructed in four ways: (1) OSEM without TOF or PSF modeling (OSEM), (2) OSEM with TOF (TOF), (3) OSEM with PSF modeling (PSF) and (4) OSEM with TOF and PSF modeling (PSFTOF). All were with 3 iterations, 8 subsets and with a post-reconstruction 8mm 3D Gaussian filter. A one tissue compartmental model was used to compute MBF. Average rest and stress flow and myocardial flow reserve were computed in five regions: anterior, septal, inferior, lateral and apical. Results from TOF, PSF and PSFTOF were compared with those from OSEM, with subjects grouped by gender, and by tertiles (10 subjects each) for BMI (I: 32.5-40.5, II: 41.1-52.3, III: 53.3-75.6 (kg/m2)), cross-sectional area (I: 532 - 1086, II: 1089 - 1157, III: 1187 - 1662 (cm2)) and heart size (I: 19 -56, II: 66 - 93, III: 97 - 238 (ml)). The MBF or myocardial flow reserve (MFR) in a given region was compared with the corresponding OSEM value and the change was expressed as a percent. Results: The general trends for all subjects with TOF were similar MBF increases in the apical (+22.0%) and septal walls (+19.4%), and a slightly lower increase in the anterior wall (+11.3 %). Increases were small in the inferior (+4.6%) and lateral (+4.9%) walls. There was little effect of PSF modeling in any region. MBF trends with PSFTOF were similar to those for TOF alone. Rest and stress increases with TOF tended to be matched, so that MFR was little changed. Trends were examined for the groupings by patient characteristics. (1) Gender. MBF showed a greater increase with TOF for females (mean +20.9%) compared to males (mean +14.9%) in the apical, septal and anterior walls. Results with PSFTOF were similar. (2) BMI. The MBF increase with TOF in the apical, anterior and septal walls was greatest for tertile II (mean +26.9%) compared with tertile I (mean +13.6%) and tertile III (mean +16.7%). (3) Cross-sectional area. The MBF increase with TOF in the apical, anterior and septal walls was similar for tertiles II (mean +22.1%) and III (mean +22.6%), and was greater than that for tertile I (mean +12.6%). (4) Heart size. The MBF increase with TOF in the apical, anterior and septal walls was greater for smaller heart sizes: tertile I (mean +21.2%), tertile II (+20.7%) and tertile III (+15.3%). These results are for our patient population and particular hardware, software and processing implementation. Conclusions: Patient gender, BMI, cross-sectional area and heart size affect the degree to which MBF values change with TOF. The largest changes are observed for females, patients with larger BMI, larger cross-sectional area and smaller hearts. The greatest changes are found in the apical, septal and lateral walls. The studied patient characteristics have little effect on flow reserve. It is important to consider these patient-dependent effects when evaluating absolute MBF in a clinical environment, and to recognize that equipment or protocol changes may affect MBF results.

Chemokines are a group of chemotaxis proteins that regulate cell trafficking and play important roles in immune responses and inflammation. Ticks are blood-sucking parasites that secrete numerous immune-modulatory agents in their saliva to evade host immune responses. Evasin-3 is a small salivary protein that belongs to a class of chemokine-binding proteins isolated from the brown dog tick, Rhipicephalus sanguineus. Evasin-3 has been shown to have a high affinity for chemokines CXCL1 and CXCL8 and to diminish inflammation in mice. In the present study, solution NMR spectroscopy was used to investigate the structure of Evasin-3 and its CXCL8-Evasin-3 complex. Evasin-3 is found to disrupt the glycosaminoglycan-binding site of CXCL8 and inhibit the interaction of CXCL8 with CXCR2. Structural data were used to design two novel CXCL8-binding peptides. The linear tEv3 17-56 and cyclic tcEv3 16-56 dPG Evasin-3 variants were chemically synthesized by solid-phase peptide synthesis. The affinity of these newly synthesized variants to CXCL8 was measured by surface plasmon resonance biosensor analysis. The K-d values of tEv3 17-56 and tcEv3 16-56 dPG were 27 and 13 nm, respectively. Both compounds effectively inhibited CXCL8-induced migration of polymorphonuclear neutrophils. The present results suggest utility of synthetic Evasin-3 variants as scaffolds for designing and fine-tuning new chemokine-binding agents that suppress immune responses and inflammation.

Selenocysteine scanning (SecScan) is a novel technique to map disulfide networks in proteins independent of structure-based distance information and mass spectrometry. SecScan applies systematic substitution of single Cys by Sec in combination with NMR spectroscopy for reliable and unambiguous determination of disulfide bond networks.

Cardiovascular disease is the major cause of morbidity and mortality in developed countries and atherosclerosis is the major cause of cardiovascular disease. Atherosclerotic lesions obstruct blood flow in the arterial vessel wall and can rupture leading to the formation of occlusive thrombi. Conventional diagnostic tools are still of limited value for identifying the vulnerable arterial plaque and for predicting its risk of rupture and of releasing thromboembolic material. Knowledge of the molecular and biological processes implicated in the process of atherosclerosis will advance the development of imaging probes to differentiate the vulnerable plaque. The development of imaging probes with high sensitivity and specificity in identifying high-risk atherosclerotic vessel wall changes and plaques is crucial for improving knowledge-based decisions and tailored individual interventions. Arterial PET imaging with 18F-FDG has shown promising results in identifying inflammatory vessel wall changes in numerous studies and clinical trials. However, due to its limited specificity in general and its intense physiological uptake in the left ventricular myocardium that impair imaging of the coronary arteries, different PET tracers for the molecular imaging of atherosclerosis have been evaluated. This review describes biological, chemical and medical expertise supporting a translational approach that will enable the development of new or the evaluation of existing PET tracers for the identification of vulnerable atherosclerotic plaques for better risk prediction and benefit to patients.

The integrin alpha IIb beta 3 is the most abundant integrin on platelets. Upon platelet activation, the integrin changes its conformation (inside-out signalling) and outside-in signalling takes place leading to platelet spreading, platelet aggregation and thrombus formation. Bloodsucking parasites such as mosquitoes, leeches and ticks express anticoagulant and antiplatelet proteins, which represent major sources of lead compounds for the development of useful therapeutic agents for the treatment of haemostatic disorders or cardiovascular diseases. In addition to hematophagous parasites, snakes also possess anticoagulant and antiplatelet proteins in their salivary glands. Two snake venom proteins have been developed into two antiplatelet drugs that are currently used in the clinic. The group of proteins discussed in this review are disintegrins, low molecular weight integrin-binding cysteine-rich proteins, found in snakes, ticks, leeches, worms and horseflies. Finally, we highlight various oral antagonists, which have been tested in clinical trials but were discontinued due to an increase in mortality. No new alpha IIb beta 3 inhibitors are developed since the approval of current platelet antagonists, and structure-function analysis of exogenous disintegrins could help find platelet antagonists with fewer adverse side effects.

During feeding, a tick's mouthpart penetrates the host's skin and damages tissues and small blood vessels, triggering the extrinsic coagulation and lectin complement pathways. To elude these defense mechanisms, ticks secrete multiple anticoagulant proteins and complement system inhibitors in their saliva. Here, we characterized the inhibitory activities of the homologous tick salivary proteins tick salivary lectin pathway inhibitor, Salp14, and Salp9Pac from Ixodes scapularis in the coagulation cascade and the lectin complement pathway. All three proteins inhibited binding ofmannan-binding lectin to the polysaccharide mannan, preventing the activation of the lectin complement pathway. In contrast, only Salp14 showed an appreciable effect on coagulation by prolonging the lag timeof thrombin generation. We found that the anticoagulant properties of Salp14 are governed by its basic tail region, which resembles the C terminus of tissue factor pathway inhibitor alpha and blocks the assembly and/or activity of the prothrombinase complex in the same way. Moreover, the Salp14 protein tail contributes to the inhibition of the lectin complement pathway via interaction with mannan binding lectin-associated serine proteases. Furthermore, we identified BaSO4-adsorbing protein 1 isolated from the tick Ornithodoros savignyi as a distant homolog of tick salivary lectin pathway inhibitor/Salp14 proteins and showed that it inhibits the lectin complement pathway but not coagulation. The structure of BaSO4-adsorbing protein 1, solved here usingNMR spectroscopy, indicated that this protein adopts a noncanonical epidermal growth factor domain-like structural fold, the first such report for tick salivary proteins. These data support a mechanism by which tick saliva proteins simultaneously inhibit both the host coagulation cascade and the lectin complement pathway.

Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin‐1 and galectin‐3, we identified several interacting pairs, such as CXCL12 and galectin‐3. Based on NMR and MD studies of the CXCL12/galectin‐3 heterodimer, we identified contact sites between CXCL12 β‐strand 1 and Gal‐3 F‐face residues. Mutagenesis of galectin‐3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin‐3, but not its mutants, inhibited CXCL12‐induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin‐3 attenuated CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted., Chemokines and galectins are simultaneously upregulated during inflammation and mediate leukocyte recruitment. A systematic screen now demonstrates their physical interaction as heterodimers, identifying several novel interacting pairs.

As CD13 is selectively expressed in angiogenesis, it can serve as a target for molecular imaging tracers to noninvasively visualize angiogenic processes in vivo. The CD13-targeting moiety NGR was synthesized and cyclized by native chemical ligation (NCL) instead of disulfide bridging, leading to a cyclic peptide backbone: cyclo(Cys-Asn-Gly-Arg-Gly) (coNGR). Beside this new monomeric coNGR, a tetrameric NGR peptide co(NGR)4 was designed and synthesized. After radiolabeling, their in vitro and in vivo characteristics were determined. Both coNGR-based imaging agents displayed considerably higher standardized uptake values (SUVs) at infarcted areas compared to the previously reported disulfide-cyclized cNGR imaging agent. Uptake patterns of 111In-coNGR and 111In-co(NGR)4 coincided with CD13 immunohistochemistry on excised hearts. Blood stability tests indicated better stability for both novel imaging agents after 50 min blood incubation compared to the disulfide-cyclized cNGR imaging agent. In mice, both coNGR peptides cleared rapidly from the blood mainly via the kidneys. In addition, co(NGR)4 showed a significantly higher specific uptake in infarcted myocardium compared to coNGR and thus is a promising sensitive imaging agent for detection of angiogenesis in infarcted myocardium.

Integrin αv β3 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 αv β3 , but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of (18)F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with (18)F 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 (18)F at a specific activity of 1.8 MBq nmol(-1) and a radiochemical purity of 100% could be achieved. The logP value of (18)F-labeled NODAGA-E-[c(RGDfK)]2 was -4.26 ± 0.02. In biodistribution studies, (18)F-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 (18)F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID g(-1), 2 h p.i.) was significantly lower than that of reference compounds (68) Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID g(-1) ; p

After arterial occlusion, diametrical growth of pre-existing natural bypasses around the obstruction, i.e. arteriogenesis, is the body's main coping mechanism. We have shown before that continuous infusion of chemokine (C-X-C motif) ligand 1 (CXCL1) promotes arteriogenesis in a rodent hind limb ischemia model.For clinical translation of these positive results, we developed a new administration strategy of local and sustained delivery. Here, we investigate the therapeutic potential of CXCL1 in a drug delivery system based on microspheres.We generated poly(ester amide) (PEA) microspheres loaded with CXCL1 and evaluated them in vitro for cellular toxicity and chemokine release characteristics. In vivo, murine femoral arteries were ligated and CXCL1 was administered either intra-arterially via osmopump or intramuscularly encapsulated in biodegradable microspheres. Perfusion recovery was measured with Laser-Doppler.The developed microspheres were not cytotoxic and displayed a sustained chemokine release up to 28 d in vitro. The amount of released CXCL1 was 100-fold higher than levels in native ligated hind limb. Also, the CXCL1-loaded microspheres significantly enhanced perfusion recovery at day 7 after ligation compared with both saline and non-loaded conditions (55.4 ± 5.0% CXCL1-loaded microspheres versus 43.1 ± 4.5% non-loaded microspheres; n = 8-9; p 0.05). On day 21 after ligation, the CXCL1-loaded microspheres performed even better than continuous CXCL1 administration (102.1 ± 4.4% CXCL1-loaded microspheres versus 85.7 ± 4.8% CXCL1 osmopump; n = 9; p 0.05).Our results demonstrate a proof of concept that sustained, local delivery of CXCL1 encapsulated in PEA microspheres provides a new tool to stimulate arteriogenesis in vivo.

Item does not contain fulltext The overexpression of the chemokine receptor CXCR4 plays an important role in oncology, since together with its endogenous ligand, the stromal cell-derived factor (SDF1-alpha), CXCR4 is involved in tumor development, growth, and organ-specific metastasis. As part of our ongoing efforts to develop highly specific CXCR4-targeted imaging probes and with the aim to assess the suitability of this ligand for first proof-of-concept studies in humans, we further evaluated the new (68)Ga-labeled high-affinity cyclic CXCR4 ligand, (68)Ga-CPCR4-2 (cyclo(D-Tyr(1)-[NMe]-D-Orn(2)-[4-(aminomethyl) benzoic acid,(68)Ga-DOTA]-Arg(3)-2-Nal(4)-Gly(5))). METHODS: Additional biodistribution and competitions studies in vivo, dynamic PET studies, and investigations on the metabolic stability and plasma protein binding were performed in nude mice bearing metastasizing OH1 human small cell lung cancer xenografts. CXCR4 expression on OH1 tumor sections was determined by immunohistochemical staining. RESULTS: (nat)Ga-CPCR4-2 exhibits high CXCR4 affinity with a half maximum inhibitory concentration of 4.99 +/- 0.72 nM. (68)Ga-CPCR4-2 showed high in vivo stability and high and specific tumor accumulation, which was reduced by approximately 80% in competition studies with AMD3100. High CXCR4 expression in tumors was confirmed by immunohistochemical staining. (68)Ga-CPCR4-2 showed low uptake in nontumor tissue and particularly low kidney accumulation despite predominant renal excretion, leading to high-contrast delineation of tumors in small-animal PET studies. CONCLUSION: The small and optimized cyclic peptide CPCR4-2 labeled with (68)Ga is a suitable tracer for targeting and imaging of human CXCR4 receptor expression in vivo. The high affinity for CXCR4, its in vivo stability, and the excellent pharmacokinetics recommend the further evaluation of (68)Ga-CPCR4-2 in a proof-of-concept study in humans. 01 november 2011

Contains fulltext : 89131.pdf (Publisher’s version ) (Closed access) Single-photon emission computed tomography (SPECT) and position emission tomography (PET) are the two main imaging modalities in nuclear medicine. SPECT imaging is more widely available than PET imaging and the radionuclides used for SPECT are easier to prepare and usually have a longer half-life than those used for PET. In addition, SPECT is a less expensive technique than PET. Commonly used gamma emitters are: (99m)Tc (E(max) 141 keV, T (1/2) 6.02 h), (123)I (E(max) 529 keV, T (1/2) 13.0 h) and (111)In (E(max) 245 keV, T (1/2) 67.2 h). Compared to clinical SPECT, PET has a higher spatial resolution and the possibility to more accurately estimate the in vivo concentration of a tracer. In preclinical imaging, the situation is quite different. The resolution of microSPECT cameras (1.5 mm). In this report, studies on new radiolabelled tracers for SPECT imaging of angiogenesis in tumours are reviewed. 01 augustus 2010

EJNMMI Research 5, 10 Seiten (2015). doi:10.1186/s13550-015-0081-7, Published by Springer, Berlin

Purpose The integrin αvβ3 is expressed on sprouting endothelial cells and on various tumour cell types. Due to the restricted expression of αvβ3 in tumours, αvβ3 is considered a suitable receptor for tumour targeting. In this study the αvβ3 binding characteristics of an 111In-labelled monomeric, dimeric and tetrameric RGD analogue were compared.

Radiolabelled peptides are used for specific targeting of receptors (over-)expressed by tumour cells. Dependent on the kind of labelling and the radionuclide used, these compounds may be utilised for imaging or for therapy. A concise overview is provided on basic principles of designing and developing radiopeptides for these applications. Furthermore, clinical application of these compounds for imaging and therapy is described. Advantages of the method compared to other techniques (such as the use of radiolabelled antibodies or antibody fragments) are discussed as well as pitfalls and limitations.

We evaluated a dimeric RGD-peptide, [Ga-68]DOTA-E-[c(RGDfK)](2), for positron emission tomography (PET) imaging of myocardial integrin expression associated with extracellular matrix remodeling after myocardial infarction (MI) in rat. Male Sprague-Dawley rats were studied at 7 days and 4 weeks after MI induced by permanent ligation of the left coronary artery and compared with sham-operated controls. In vivo imaging revealed higher tracer uptake in the infarcted area than in the remote non-infarcted myocardium of the same rats both at 7 days (MI/remote ratio, 2.25 +/- 0.24) and 4 weeks (MI/remote ratio, 2.13 +/- 0.37) post-MI. Compared with sham-operated rats, tracer uptake was higher also in the remote, non-infarcted myocardium of MI rats both at 7 days and 4 weeks where it coincided with an increased interstitial fibrosis. Standardized uptake values correlated well with the results of tracer kinetic modeling. Autoradiography confirmed the imaging results showing 5.1 times higher tracer uptake in the infarcted than remote area. Tracer uptake correlated with the amount of beta(3) integrin subunits in the infarcted area. Our results show that integrin-targeting [Ga-68]DOTA-E-[c(RGDfK)](2) is a potential tracer for monitoring of myocardial extracellular matrix remodeling after MI using PET.

Integrin αv β3 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 αv β3 , but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of (18)F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with (18)F 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 (18)F at a specific activity of 1.8 MBq nmol(-1) and a radiochemical purity of 100% could be achieved. The logP value of (18)F-labeled NODAGA-E-[c(RGDfK)]2 was -4.26 ± 0.02. In biodistribution studies, (18)F-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 (18)F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID g(-1), 2 h p.i.) was significantly lower than that of reference compounds (68) Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID g(-1) ; p0.001) and (111) In-labeled NODAGA-E-[c(RGDfK)]2 (4.99 ± 0.64 %ID g(-1) ; p0.01). Co-injection of an excess of unlabeled NODAGA-E-[c(RGDfK)]2 along with (18)F-NODAGA-E-[c(RGDfK)]2 resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g(-1)). The αv β3 integrin-expressing SK-RC-52 tumor could be successfully visualized by microPET with (18)F-labeled NODAGA-E-[c(RGDfK)]2 . In conclusion, NODAGA-E-[c(RGDfK)]2 could be labeled rapidly with (18)F using a direct aqueous, one-pot method and it accumulated specifically in αv β3 integrin-expressing SK-RC-52 tumors, allowing for visualization by microPET.

The bioconjugation of organometallic complexes with peptides has proven to be a novel approach for drug discovery. We report the facile and chemoselective reaction of tyrosine-containing G-protein-coupled receptor (GPCR) peptides with [Cp*Rh(H(2)O)(3)](OTf)(2), in water, at room temperature, and at pH 5-6. We have focused on three important GPCR peptides; namely, [Tyr(1)]-leu-enkephalin, [Tyr(4)]-neurotensin(8-13), and [Tyr(3)]-octreotide, each of which has a different position for the tyrosine residue, together with competing functionalities. Importantly, all other functional groups present, i.e., amino, carboxyl, disulfide, phenyl, and indole, were not prominent sites of reactivity by the Cp*Rh tris aqua complex. Furthermore, the influence of the Cp*Rh moiety on the structure of [Tyr(3)]-octreotide was characterized by 2D NMR, resulting in the first representative structure of an organometallic-peptide complex. The biological consequences of these Cp*Rh-peptide complexes, with respect to GPCR binding and growth inhibition of MCF7 and HT29 cancer cells, will be presented for [(η(6)-Cp*Rh-Tyr(1))-leu-enkephalin](OTf)(2) and [(η(6)-Cp*Rh-Tyr(3))-octreotide](OTf)(2).

Item does not contain fulltext The gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer. Bombesin (BBN) is a neurotransmitter of 14 amino acids and binds with selectivity and with high affinity to GRPRs. We have synthesized a NOTA-conjugated bombesin derivative, NOTA-8-Aoc-BBN(7-14)NH(2), to label this analog with (18)F using the new Al(18)F method. In this study, the GRPR-targeting potential of (18)F-labeled NOTA-8-Aoc-BBN(7-14)NH(2) was studied using (68)Ga-NOTA-8-Aoc-BBN(7-14)NH(2) as a reference. METHODS: The NOTA-conjugated bombesin analog was synthesized and radiolabeled with (68)Ga or (18)F. For (18)F labeling, we used our new 1-pot, 1-step method. The labeled product was purified by reversed-phase high-performance liquid chromatography. The log P values of the radiotracers were determined. The tumor-targeting characteristics of the compounds were assessed in mice with subcutaneously growing PC-3 xenografts. GRPR-binding specificity was studied by coinjection of an excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH(2). Small-animal PET/CT images were acquired. RESULTS: NOTA-8-Aoc-BBN(7-14)NH(2) could be efficiently labeled with (18)F or with (68)Ga. NOTA-8-Aoc-BBN(7-14)NH(2) was labeled with (18)F in a single step, with 50%-90% yield. Radiolabeling, including purification, was performed in 45 min and resulted in a specific activity of greater than 10 GBq/mumol. The log P values of (18)F- and (68)Ga-labeled NOTA-8-Aoc-BBN(7-14)NH(2) were -1.47 +/- 0.05 and -1.98 +/- 0.03, respectively. In mice, both radiolabeled compounds cleared rapidly from the blood (

The chemokine receptor CXCR4 is a critical regulator of inflammation and immune surveillance, and it is specifically implicated in cancer metastasis and HIV-1 infection. On the basis of the observation that several of the known antagonists remarkably share a C 2 symmetry element, we constructed symmetric dimers with excellent antagonistic activity using a derivative of a cyclic pentapeptide as monomer. To optimize the binding affinity, we investigated the influence of the distance between the monomers and the pharmacophoric sites in the synthesized constructs. The affinity studies in combination with docking computations support a two-site binding model. In a final step, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was introduced as chelator for (radio-)metals, thus allowing to exploit these compounds as a new group of CXCR4-binding peptidic probes for molecular imaging and endoradiotherapeutic purposes. Both the DOTA conjugates and some of their corresponding metal complexes retain good CXCR4 affinity, and one 68Ga labeled compound was studied as PET tracer. © 2011 American Chemical Society.

Item does not contain fulltext We report on the SSTR2-binding properties of a series of four dimeric [Tyr3]octreotate analogues with different spacer lengths (nine, 19, 41, and 57 atoms) between the peptides. Two analogues (9 and 57 atoms) were selected as precursors for the design, synthesis, and biological evaluation of DOTA-conjugated dimeric [Tyr3]octreotate analogues for tumor targeting. These compounds were synthesized by using a two-stage click ligation procedure: a Cu(I) -catalyzed 1,3-dipolar cycloaddition ("copper-click" reaction) and a thio acid/sulfonyl azide amidation ("sulfo-click" reaction). The IC(50) values of these DOTA-conjugated [Tyr3]octreotate analogues were comparable, and internalization studies showed that the nine-atom (111) In-DOTA-labeled [Tyr3]octreotate dimer had rapid and high receptor binding. Biodistribution studies with BALB/c nude mice bearing subcutaneous AR42J tumors showed that the (111) In-labeled [Tyr3]octreotate dimer (nine atoms) had a high tumor uptake at 1 h p.i. (38.8 +/- 8.3 % ID g(-1) ), and excellent tumor retention at 4 h p.i. (40.9 +/- 2.5 % ID g(-1) ). However, the introduction of the extended hydrophilic 57 atoms spacer led to rapid clearance from the circulation; this limited tumor accumulation of the radiotracer (21.4 +/- 4.9 % ID g(-1) at 1 h p.i.). These findings provide important insight on dimerization and spacer effects on the in vivo properties of DOTA-conjugated [Tyr3]octreotate dimers.

The sweet, minty-lemony leaves of costmary ( Chrysanthemum balsamita ) are used for salads and tea, and as flavourings in meats, sausages, cakes and ale. In this study, the extracts isolated from costmary aerial parts were investigated as antioxidants in rapeseed oil and as free radical-scavengers in DPPH and ABTS + assays. It was found that costmary extracts and their fractions were weak antioxidants in rapeseed oil; however, some fractions were active in scavenging synthetic free radicals. Crude methanol–water extract, its tert -butyl methyl ether and butanol fractions were the most effective in DPPH assay by scavenging 87.0%, 86.9% and 86.4% of radicals present in the reaction, respectively. Several active compounds were detected in these fractions, using HPLC with on-line radical-scavenging detection. After multi-step fractionation of these fractions, four radical-scavenging constituents were isolated and their properties were assessed by DPPH (antiradical power, ARP, calculated as an inverse value of the effective concentration, 1/EC 50 ) and ABTS + (Trolox equivalent antioxidant capacity, TEAC 6min ) free radical-scavenging assays. The following structures were elucidated by NMR and MS: 5- O -caffeoylquinic acid (ARP = 3.85; TEAC 6min = 0.60), 3,5- O -dicaffeoylquinic acid (ARP = 6.25; TEAC 6min = 1.16), 5,7,4′-trihydroxy-3′,8-dimethoxyflavone (ARP = 0.03) and 5,7,3′,4′-tetrahydroxy-3,8-dimethoxyflavonol (ARP = 3.79; TEAC 6min = 1.50).

Contains fulltext : 81773.pdf (Publisher’s version ) (Open Access) Angiogenesis is a multistep process regulated by pro- and antiangiogenic factors. In order to grow and metastasize, tumors need a constant supply of oxygen and nutrients. For growth beyond 1-2 mm in size, tumors are dependent on angiogenesis. Inhibition of angiogenesis is a new cancer treatment strategy that is now widely investigated clinically. Researchers have begun to search for objective measures that indicate pharmacologic responses to antiangiogenic drugs. Therefore, there is a great interest in techniques to visualize angiogenesis in growing tumors noninvasively. Several markers have been described that are preferentially expressed on newly formed blood vessels in tumors (alpha(v)beta(3) integrin, vascular endothelial growth factor, and its receptor, prostate-specific membrane antigen) and in the extracellular matrix surrounding newly formed blood vessels (extra domain B of fibronectin, Tenascin-C, matrix metalloproteinases, and Robo-4). Several ligands targeting these markers have been tested as a radiotracer for imaging angiogenesis in tumors. The potential of some of these tracers, such as radiolabeled cyclic RGD peptides and radiolabeled anti-PSMA antibodies, has already been tested in cancer patients, while for markers such as Robo-4, the ligand has not yet been identified. In this review, an overview on the currently used nuclear imaging probes for noninvasive visualization of tumor angiogenesis is given.

An optimized protocol for the mild and selective Fukuyama-Mitsunobu reaction was used for mono- and di- N-alkylation on solid support. Thereby, nonfunctionalized aliphatic and aromatic residues are quickly introduced into transiently protected, primary amines of a linear peptide. N-Alkylation can also be used to implement alkyl chains carrying (protected) functionalities suited for subsequent modification. Applicability of this method is demonstrated by various N-alkylated analogues of a cyclic CXCR4 receptor antagonist originally developed by Fujii et. al.

Contains fulltext : 51419.pdf (Publisher’s version ) (Closed access) Ovarian cancer is the fourth most common cause of cancer deaths among females in the western world after cancer of the breast, colon and lung. The inability to control the disease within the peritoneal cavity is the major cause of treatment failure in patients with ovarian cancer. The majority of ovarian carcinomas express the alpha(v)beta(3) integrin. Here we studied the tumor targeting potential of an (111)In-labeled cyclic RGD peptide in athymic BALB/c mice with intraperitoneally (i.p.) growing NIH:OVCAR-3 human ovarian carcinoma tumors. The cyclic RGD peptide, c(RGDfK)E, was synthesized, conjugated with DOTA and radiolabeled with (111)In. The targeting potential of (111)In-DOTA-E-c(RGDfK) was studied in athymic mice with i.p. growing NIH:OVCAR-3 xenografts and the optimal dose of this compound was determined (0.01 microg up to 10 microg). The biodistribution at optimal peptide dose was determined at various time points (0.5 up to 72 hr). Furthermore, the therapeutic potential of (177)Lu-DOTA-E-c(RGDfK) was studied in this model. Two hours after i.p. administration, (111)In-DOTA-E-c(RGDfK) showed high and specific uptake in the i.p. growing tumors. Optimal uptake in the i.p. growing tumors was observed at a 0.03-0.1 microg dose range. Tumor uptake of (111)In-DOTA-E-c(RGDfK) peaked 4 hr p.i. [(38.8 +/- 2.7)% ID/g], gradually decreasing at later time points [(24.0 +/- 4.1)% ID/g at 48 hr p.i.]. Intraperitoneal growth of OVCAR-3 could be significantly delayed by injecting 37 MBq (177)Lu-labeled peptide i.p. Radiolabeled DOTA-E-c(RGDfK) is suitable for targeting of i.p. growing tumors and potentially can be used for peptide receptor radionuclide therapy of these tumors.

Contains fulltext : 52270.pdf (Publisher’s version ) (Open Access) This report describes the design and synthesis of a series of alpha(V)beta(3) integrin-directed monomeric, dimeric and tetrameric cyclo[Arg-Gly-Asp-d-Phe-Lys] dendrimers using "click chemistry". It was found that the unprotected N-epsilon-azido derivative of cyclo[Arg-Gly-Asp-d-Phe-Lys] underwent a highly chemoselective conjugation to amino acid-based dendrimers bearing terminal alkynes using a microwave-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition. The alpha(V)beta(3) binding characteristics of the dendrimers were determined in vitro and their in vivoalpha(V)beta(3) targeting properties were assessed in nude mice with subcutaneously growing human SK-RC-52 tumors. The multivalent RGD-dendrimers were found to have enhanced affinity toward the alpha(V)beta(3) integrin receptor as compared to the monomeric derivative as determined in an in vitro binding assay. In case of the DOTA-conjugated (111)In-labeled RGD-dendrimers, it was found that the radiolabeled multimeric dendrimers showed specifically enhanced uptake in alpha(V)beta(3) integrin expressing tumors in vivo. These studies showed that the tetrameric RGD-dendrimer had better tumor targeting properties than its dimeric and monomeric congeners.

The integrin alpha(v)beta(3) is expressed on sprouting endothelial cells and on various tumour cell types. Due to the restricted expression of alpha(v)beta(3) in tumours, alpha(v)beta(3) is considered a suitable receptor for tumour targeting. In this study the alpha(v)beta(3) binding characteristics of an (111)In-labelled monomeric, dimeric and tetrameric RGD analogue were compared.A monomeric (E-c(RGDfK)), dimeric (E-[c(RGDfK)](2)), and tetrameric (E{E[c(RGDfK)](2)}(2)) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with (111)In. In vitro alpha(v)beta(3) binding characteristics were determined in a competitive binding assay. In vivo alpha(v)beta(3) targeting characteristics of the compounds were assessed in mice with SK-RC-52 xenografts.The IC(50) values for DOTA-E-c(RGDfK), DOTA-E-[c(RGDfK)](2), and DOTA-E{E[c(RGDfK)](2)}(2)were 120 nM, 69.9 nM and 19.6 nM, respectively. At all time points, the tumour uptake of the dimer was significantly higher as compared to that of the monomer. At 8 h p.i., tumour uptake of the tetramer (7.40+/-1.12%ID/g) was significantly higher than that of the monomer (2.30+/-0.34%ID/g), p0.001, and the dimer (5.17+/-1.22%ID/g), p0.05. At 24 h p.i., the tumour uptake was significantly higher for the tetramer (6.82+/-1.41%ID/g) than for the dimer (4.22+/-0.96%ID/g), p0.01, and the monomer (1.90+/-0.29%ID/g), p0.001.Multimerisation of c(RGDfK) resulted in enhanced affinity for alpha(v)beta(3) as determined in vitro. Tumour uptake of a tetrameric RGD peptide was significantly higher than that of the monomeric and dimeric analogues, presumably owing to the enhanced statistical likelihood for rebinding to alpha(v)beta(3).

Contains fulltext : 51403.pdf (Publisher’s version ) (Closed access) INTRODUCTION: alpha(v)beta(3) Integrin is expressed in sprouting endothelial cells in growing tumors, whereas it is absent in quiescent blood vessels. In addition, various tumor cell types express alpha(v)beta(3) integrin. alpha(v)beta(3) Integrin, a transmembrane heterodimeric protein, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin and plays a pivotal role in invasion, proliferation and metastasis. Due to the selective expression of alpha(v)beta(3) integrin in tumors, radiolabeled RGD peptides and peptidomimetics are attractive candidates for tumor targeting. METHODS: A cyclic RGD peptide, a peptoid-peptide hybrid, an all-peptoid and a peptidomimetic compound were synthesized, conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and radiolabeled with (111)In. Their in vitro and in vivo alpha(v)beta(3)-binding characteristics were determined. RESULTS: IC(50) values were 236 nM for DOTA-E-c(RGDfK), 219 nM for DOTA-peptidomimetic, >10 mM for DOTA-all-peptoid and 9.25 mM for the peptoid-peptide hybrid DOTA-E-c(nRGDfK). (111)In-labeled compounds, except for [(111)In]DOTA-all-peptoid, showed specific uptake in human alpha(v)beta(3)-expressing tumors xenografted in athymic mice. Tumor uptake for [(111)In]DOTA-E-c(RGDfK) was 1.73+/-0.4% ID/g (2 h postinjection) and that of [(111)In]DOTA-peptidomimetic was 2.04+/-0.3% ID/g. Tumor uptake for the peptoid-peptide hybrid [(111)In]DOTA-E-c(nRGDfK) was markedly lower (0.45+/-0.07% ID/g). The all-peptoid [(111)In]DOTA-E-c(nRGnDnFnK) did not show specific uptake in tumors (0.11+/-0.04% ID/g). CONCLUSIONS: The peptidomimetic compound and the cyclic RGD peptide have a high affinity for alpha(v)beta(3) integrin, and these compounds have better tumor-targeting characteristics than the peptoid-peptide hybrid and the all-peptoid.

Contains fulltext : 58153.pdf (Publisher’s version ) (Open Access) Arginine-glycine-aspartic acid (RGD) peptides preferentially bind to alphavbeta3 integrin, an integrin expressed on newly formed endothelial cells and on various tumor cells. When labeled with beta-emitting radionuclides, these peptides can be used for peptide-receptor radionuclide therapy of malignant tumors. These studies aimed to investigate whether tumor targeting and tumor therapy could be optimized by dose fractionation. The RGD-peptide DOTA-E-[c(RGDfK)]2 was labeled with 111In for biodistribution experiments and with 90Y for therapy experiments. In mice with NIH:OVCAR-3 ovarian carcinoma xenografts, optimal tumor uptake was obtained at peptide doses up to 1.0 microg (4.8 %ID/g). A peptide dose of 5 microg, required to administer the maximum tolerable dose (MTD) 90Y-DOTA-E-[c(RGDfK)]2, was administered as 5 portions of 1.0 microg. Tumor uptake of the fifth portion was significantly higher than that of the single 5.0 microg portion (3.3 %ID/g versus 2.1 %ID/g). The therapeutic efficacy of 37 MBq 90Y-DOTA-E-[c(RGDfK)]2 (1 x 5.0 microg) was compared with that of 37 MBq administered in five equal portions (5 x 1.0 microg). No difference in tumor growth between the fractionated and the nonfractionated therapy was observed. In conclusion, dose fractionation resulted in higher radiation doses. However, therapeutic efficacy of the radiolabeled peptide was not significantly improved by dose fractionation.

The alpha(v)beta(3) integrin is expressed on proliferating endothelial cells such as those present in growing tumors, as well as on tumor cells of various origin. Tumor-induced angiogenesis can be blocked in vivo by antagonizing the alpha(v)beta(3) integrin with small peptides containing the Arg-Gly-Asp (RGD) amino acid sequence. This tripeptidic sequence, naturally present in extracellular matrix proteins, is the primary binding site of the alpha(v)beta(3) integrin. Because of selective expression of alpha(v)beta(3) integrin in tumors, radiolabeled RGD peptides are attractive candidates for alpha(v)beta(3) integrin targeting in tumors. We studied the in vivo behavior of the radiolabeled dimeric RGD peptide E-[c(RGDfK)](2) in the NIH:OVCAR-3 s.c. ovarian carcinoma xenograft model in BALB/c nude mice. Conjugation of the 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) and hydrazinonicotinamide (HYNIC) chelators enabled efficient radiolabeling with (111)In/(90)Y and (99m)Tc, respectively. The radiolabeled peptide was rapidly excreted renally. Uptake in nontarget organs such as liver and spleen was considerable. Tumor uptake peaked at 7.5% injected dose (ID)/g ((111)In-DOTA-E-[c(RGDfK)](2)) or 6.0%ID/g ((99m)Tc-HYNIC-E-[c(RGDfK)](2)) at 2 and 1 h postinjection, respectively. Integrin alpha(v)beta(3) receptor binding specificity was demonstrated by reduced tumor uptake after injection of the scrambled control peptide (111)In-DOTA-E-[c(RDKfD)](2) (0.28%ID/g at 2 h p.i.) and after coinjection of excess nonradioactive (115)In-DOTA-E-[c(RGDfK)](2) (0.22%ID/g at 2 h p.i.). A single injection of (90)Y-DOTA-E-[c(RGDfK)](2) at the maximum-tolerated dose (37 MBq) in mice with small s.c. tumors caused a significant growth delay as compared with mice treated with 37 MBq (90)Y-labeled scrambled peptide or untreated mice (median survival of 54 versus 33.5 versus 19 days, respectively). In conclusion, the radiolabeled RGD peptides (111)In-DOTA-E-[c(RGDfK)](2) and (99m)Tc-HYNIC-E-[c(RGDfK)](2) demonstrated high and specific tumor uptake in a human tumor xenograft. Injection of (90)Y-DOTA-E-[c(RGDfK)](2) induced a significant delay in tumor growth. Potentially, these peptides can be used for peptide receptor radionuclide imaging as well as therapy.

Contains fulltext : 186517.pdf (Publisher’s version ) (Closed access) The alpha v beta 3 integrin, a transmembrane heterodimeric protein expressed on sprouting endothelial cells, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin. Growing malignant tumors continuously require angiogenesis. As a result, alpha v beta 3 is preferentially expressed in growing tumors and is a potential target for radiolabeled RGD-peptides. In this study we compared the tumor targeting characteristics of a monomeric radiolabeled RGD-peptide with those of a dimeric analogue. Both peptides were radiolabeled with 99mTc via the hydrazinoni-cotinamid (= HYNIC) moiety to form 99mTc-HYNIC-c(RGDfK) and 99mTc-HYNIC-E-[c(RGDfK)]2. In vitro, the IC50 showed a 10-fold higher affinity of the dimer for the alpha v beta 3 integrin as compared to the monomer (0.1 vs. 1.0 nM). In athymic female BALB/c mice with subcutaneously growing OVCAR-3 ovarian carcinoma xenografts, tumor uptake peaked at 5.8 +/- 0.7% ID/g and 5.2 +/- 0.6% ID/g for the dimer and the monomer, respectively. At 1, 2, and 4 h postinjection (p.i.) uptake of the dimer in the tumor was significantly higher than that of the monomeric analogue. Tumor-to-blood ratios were highest at 24 h p.i. at a value of 63 for both compounds. At all timepoints kidney retention of the dimer was significantly higher as compared to kidney retention of the monomer. In conclusion, in this mouse model the dimeric RGD-peptide showed better retention in the tumor than the monomeric analogue, most likely due to the bivalent interaction with the target cell. Furthermore, kidney retention of the dimeric peptide was higher than that of the monomeric peptide.

Contains fulltext : 80162.pdf (Publisher’s version ) (Open Access) Integrin alphavbeta3 plays a pivotale role in tumor angiogenesis and is a receptor for the extracellular matrix proteins with the exposed arginine-glysine-aspartic acid (RGD) tripeptide sequence (e.g. vitronectin, fibronectin). Alphavbeta3 is overexpressed on activated endothelial cells during tumor-induced angiogenesis, whereas it is absent on quiescent endothelial cells and normal tissues. Furthermore, alphavbeta3 is expressed on various tumor cell lines. Due to this restricted expression of alphavbeta3 in tumors, alphavbeta3 is considered a suitable receptor for tumor targeting. In the past decade, several RGD-containing peptide antagonists have been evaluated for monitoring alphavbeta3 expression using SPECT, PET, MRI, OI and US. Molecular imaging tracers for this integrin receptor could be used to noninvasively visualize alphavbeta3 expression in tumors. Noninvasive determination of alphavbeta3 expression potentially can be used to monitor treatment response to antiangiogenic drugs or even to select patients likely to respond to treatment with antiangiogenic drugs. In this review a brief overview on the currently used RGD-containing peptides as imaging probes for noninvasive visualization of alphavbeta3 expression using PET, SPECT, MRI, OI and US is given.