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Ultrasound Contrast Agents For Ultrasound Molecular Imaging Ultraschall-Kontrastmittel für molekulare Bildgebung im Ultraschall Authors
F. Tranquart1, M. Arditi1, T. Bettinger1, P. Frinking1, J. M. Hyvelin1, A. Nunn2, S. Pochon1, I. Tardy1
Affiliations
1
Bracco Suisse SA, Plan Les Ouates, Switzerland Bracco Research US, Monroe, USA
Schlüsselwörter
Zusammenfassung
Abstract
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Die Sonografie ist eine weitverbreitete Bildgebungsmethode mit hoher örtlicher und zeitlicher Auflösung. Die Einführung von Ultraschallkontrastmitteln im Zusammenhang mit kontrastmittelspezifischer Software erlaubt die Analyse organspezifischer Durchblutungsmuster und Perfusion mit Verbesserung der diagnostischen Genauigkeit. Die weitere Entwicklung von mit biologischen Markern versehenen Ultraschallkontrastmitteln erlaubt eine molekulare Bildgebung. Die molekulare Bildgebung ermöglicht die Darstellbarkeit der Expression von intravaskulären Markern vor, während und nach medikamentöser Behandlung. Der Vorteil der Ultraschallkontrastmittel liegt in ihrer strikten intravaskulären Verteilung, sodass Neoangiogenese und Entzündungsvorgänge dargestellt werden können. Verschiedene Technologien wurden evaluiert, um das zielgerichtete Molekül an die Kontrastmittelhülle zu binden. Diese reichen von einfachen Biotin-Avidin-Konstrukten bis zu komplizierteren Anheftungen mittels PEG-Abkömmlingen. Aktuelle Fortschritte auf diesem Gebiet erlauben die Übertragung präklinischer Erfahrung für eine verbesserte Detektion und Charakterisierung von Neoplasien und Entzündungsprozessen. Die kombinierte Anwendung und Auswertung anatomischer, funktionsanalytischer und molekularer Informationen im Rahmen der Kontrastmittelsonografie ist vielversprechend steht aber noch in den Anfängen, da bisher nur wenige klinisch relevante Markermoleküle existieren. Die beschriebenen Vorteile der Sonografie kombiniert mit der molekularen Signatur von Neoplasien wird im Rahmen der personalisierten Medizin eine relevante Rolle spielen.
Ultrasound is a real-time imaging technique which is widely used in many clinical applications for its capacity to provide anatomic information with high spatial and temporal resolution. The advent of ultrasound contrast agents in combination with contrast-specific imaging modes has given access to perfusion assessments at an organ level, leading to an improved diagnostic accuracy. More recently, the development of biologically-targeted ultrasound contrast agents has expanded the role of ultrasound even further into molecular imaging applications. Ultrasound molecular imaging can be used to visualize the expression of intravascular markers, and to assess their local presence over time and/or during therapeutic treatment. Major applications are in the field of inflammation and neoangiogenesis due to the strictly intravascular presence of microbubbles. Various technologies have been investigated for attaching the targeting moiety to the shell from simple biotin-avidin constructs to more elaborated insertion within the shell through attachment to PEG residues. This important improvement has allowed a clinical translation of initial pre-clinical investigations, opening the way for an early detection and an accurate characterization of lesions in patients. The combination of anatomic, functional and molecular information/data provided by contrast ultrasound is a powerful tool which is still in its infancy due to the lack of agents suitable for clinical use. The advantages of ultrasound techniques combined with the molecular signature of lesions will represent a significant advance in imaging in the field of personalized medicine.
● Mikrobläschen ● kontrastverstärkter "
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Ultraschall molekulare Bildgebung VEGFR2 Selektine gastrointestinaler Stromatumor (GIST) kolorektales Karzinom gastro-entero-pankreatische Tumoren
Key words
● microbubbles ● contrast-enhanced " "
ultrasound
● molecular imaging ● VEGFR2 ● selectins ● gastrointestinal stromal " " " "
tumors (GIST)
● colorectal cancer ● gastro-entero-pancreatic " "
tumors
received accepted
27.5.2014 13.7.2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1384999 Z Gastroenterol 2014; 52: 1268–1276 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0044-2771 Correspondence Prof. Francois Tranquart Bracco Suisse SA Route de la Galaise, 31 1228 Plan Les Ouates Switzerland [email protected]
Tranquart F et al. Ultrasound Contrast Agents … Z Gastroenterol 2014; 52: 1268–1276
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Contrast agents are now extensively used in medical imaging to enhance visualization of particular body compartments or tissue perfusion. Even though computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT) and positron emission tomography (PET) have made extensive use of contrast agents since their initial development, the use of ultrasound contrast agents (UCA) is more recent and really emerged as a routine technique in the early 2000 s. Besides gross anatomy, lesion extent, and other morphological information provided by ultrasound (US) imaging, UCA are able, after intravenous administration, to provide valuable functional information about perfusion, which often represents a key factor for a definitive diagnosis or for therapeutic guidance. Since the first report made by Gramiak and Shah in 1968 [1], showing that air bubbles could be used in vivo as strong ultrasound scatterers, many improvements have been made in bubble formulations (bubble stabilization methods based on various shell components or fluorinated gas) and in ultrasound equipment, with the introduction of specific imaging sequences to exploit the acoustic properties of these agents in an optimal way. Nowadays, this new method, called “contrast-enhanced ultrasound” (CEUS), is becoming more and more popular in almost all possible indications and organs thanks to its performance ease of use, real-time capabilities and low cost [2, 3]. In the early 2000s, after the launch of clinically approved UCA and further to initial developments, a few teams became interested in the potential of CEUS for molecular imaging. Molecular imaging is generally defined as non-invasive imaging of biomarkers, implying the use of methods able to track events at a cellular level [4 – 8]. This could be achieved by preparing UCA functionalized with ligands specific for the biomarkers of interest. This requires methods able to (i) detect UCA distribution in the targeted tissue/organ, designed to image a biomarker’s expression and (ii) monitor biomarker expression changes over time according to the specific disease or process being followed. Therefore, there has been active effort in this field to generate new UCAs aimed at addressing this purpose, taking advantage of the unique properties of CEUS.
the added advantage that several consecutive injections can be performed during the same examination [3, 9 – 11]. Only minute amounts of gas and stabilizing material are administered in an imaging dose, in the range of a few µL of gas and a few tens of micrograms of phospholipids per injection per patient. These minute quantities reflect the remarkable potency of microbubble-based ultrasound contrast agents combined with the high sensitivity of the ultrasound contrast imaging mode [12], significantly higher than the sensitivity reported for X-ray and magnetic resonance methods.
Targeted UCA !
Ultrasound molecular imaging (US-MI) requires the use of targeted UCA, which differ from those initially developed for blood pool imaging by the presence of a targeting moiety able to link the microbubble to the selected cell biomarker. The general strategy is to link molecular entities to the phospholipid-stabilizing monolayer, allowing the microbubbles to remain attached to selected sites in the vascular compartment. Indeed, because of their relatively large diameter, UCA microbubbles cannot escape the vascular compartment. Once attached, these microbubbles are able to generate detectable echo signals similar to those obtained with blood pool agents, since the signal is generated by the microbubble itself and not the ligand [4 – 8, 13 – 19]. This raises important points for US-MI: (i) As the microbubbles remain strictly within the vascular compartment, targets must be selected that are accessible to these microbubbles, i. e., on the luminal side of the endothelial cells [4, 6 – 8, 14, 16, 19 – 21]. (ii) The attachment of microbubbles to the surface of endothelial cells must be strong enough for vascular areas where shear stress is high due to high blood velocity and viscosity [21]. (iii) As the detected signal is the combination of echoes from bound microbubbles and circulating microbubbles, there is a need to develop specific imaging strategies aiming at detecting the echoes from bound bubbles [8, 22, 23] in a distinct way. These issues will be addressed here below in addition to the concepts used for inserting the targeting moiety into the microbubble shell.
Specificities of Ultrasound Contrast Agents (UCA)
Selection of Targets [24]
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Microbubble diameter sizes are in the micron range. After intravenous administration, microbubbles larger than 6 ‑ 8 µm in diameter are eliminated at the pulmonary level and thus do not reach the left heart and general circulation. Microbubble sizes should therefore preferably be in the 2 to 5 µm range, i. e. close to or below the size of red blood cells. Microbubbles in this size range are able to move freely in the entire vasculature, including the smallest capillaries. They act as strong ultrasound scatterers due to the fortunate coincidence that their resonance frequencies match well the range of ultrasound diagnostic frequencies (2 − 20 MHz). Today, all commercially available ultrasound contrast agents contain high concentrations of micron-sized gas microbubbles (108 − 1010 bubbles per ml) stabilized by lipids or heat denatured albumin. The half-life of ultrasound contrast agents after intravenous administration is short, typically a few minutes. This duration is however in most cases sufficient for imaging purposes and has
Based on the features listed above, two application areas have clearly emerged for US-MI: inflammation and neoangiogenesis, " Fig. 1). since both involve endothelial cells (● In the course of the inflammatory process, various cell surface markers are expressed or up-regulated on the luminal side of endothelium and therefore are accessible to targeted microbubbles [21, 25 – 28]. The cellular inflammatory response, which involves leukocyte infiltration of tissue, relies on a timely sequence of events, the so-called inflammation cascade. First, leukocytes are recruited to the vessel wall (tethering) and roll along the endothelium, these first interactions being mediated by selectins. Then, leukocytes roll with increased interactions with selectins before slowing down and finally stop with a strong attachment to the vessel wall. This phase is mediated by integrins, interacting with their counter-receptors (such as intercellular cell adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]). Finally, leukocytes cross the vessel wall towards the interstitial space, triggering local effects such as edema. This
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Fig. 1 Targets eligible for ultrasound molecular imaging due to their intra-vascular expression.
makes these different inflammatory mediators appealing targets for US-MI. Inflammation plays a key role in the development and progression of cardiovascular diseases [29, 30], including atherosclerosis [31, 32], stroke, transplant rejection, inflammatory bowel disease [33], and myocardial infarct. In the latter case, a hallmark of ischemia-reperfusion (I/R) is the activation of inflammatory processes, leading to the expression of specific adhesion molecules at the surface of endothelial cells. Thanks to their location, these markers are accessible to targeted UCA and therefore can be detected using US-MI. So far, most of the studies have been performed using agents specific for either P- or E-selectins or both [27, 28, 34], or VCAM-1 [31]. The second important area in which US-MI has clearly emerged, is tumor detection [35] and more particularly tumoral neoangiogenesis [20, 36], i. e., the formation of new blood vessels, which is a fundamental process occurring during tumor progression. Sequential steps are involved in effective tumoral angiogenesis with an initial trigger of hypoxia. Under hypoxic conditions, expression of hypoxia-inducible factors is induced in endothelial vessels, resulting in vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor 2 (VEGFR2) expressions [37, 38]. Many other biomarkers have been identified in this complex process such as neuropilin 2, the αvβ3 integrin, and endoglin (CD105) [17, 20, 39 – 42]. In addition to endothelial receptors, site targeted microbubbles can also be used for the visualization of thrombi associated with stroke. Immunobubbles bearing Abciximab, a glycoprotein IIb/ IIIa receptor inhibitor, were shown to improve visualization of human clots both in vitro and in an in vivo model of acute arterial thrombotic occlusion [43, 44].
Targeted UCA Engineering !
The methods for making targeted microbubbles do not differ significantly from those used for conventional microbubbles. There is an additional step of attaching/inserting a site-specific ligand to the microbubble shell. Today, two packages are being used: the first one is a lyophilized product that is resuspended before injection and the second is one in which microbubble precursors are kept in solution before activation. The main objective is to get a regular and stable microbubble concentration and size distribution which are stable from vial to vial, allowing highly reprodu-
cible imaging acquisitions. The problems of storage and transportation must be addressed at very early stages of development to facilitate the clinical use of these agents. A common strategy makes use of a spacer arm like polyethylene glycol (PEG) to conjugate avidin or streptavidin to the surface of the microbubble, and then an avidin-biotin link is used to attach a pathology-specific ligand. The avidin-biotin interaction is among the strongest non-covalent bonds. The resulting avidin/ streptavidin-functionalized microbubbles are then incubated with a ligand bearing a biotin moiety. Thus, thanks to the multivalency of avidin/streptavidin molecules, biotinylated molecular entities can be bound to the avidin/streptavidin coated microbubbles. In this way, moieties may be attached to the microbubble shell through a biotin-avidin-biotin bridge. This procedure is very flexible and convenient for small-animal imaging, as it allows the preparation of microbubbles coated on their surface with a biotinylated molecule selected by the investigator [45]. Recently, commercial preparations have been designed for smallanimal imaging based on streptavidin-functionalized microbubbles (Target Ready MicroMarker™, Visualsonics Inc., Toronto, Canada; Targestar SA, Targeson Inc, San Diego, CA) [11]. The ligand can be a protein, an antibody or antibody fragment, an oligopeptide or even an organic small molecule, the only requirement being that a biotin residue be attached to the molecule selected. No separation stage is usually necessary since the amount of biotinylated ligand added can be adjusted in order to be captured entirely by the streptavidin bubbles. While advantageous for preclinical testing, this approach cannot be translated into clinical use due to the immunogenicity of avidin. Another strategy is the direct conjugation of the ligand on the PEG spacer. The specific issue of random coupling, which can jeopardize the interaction of the ligand with its target and can complicate significantly the analytical characterization of the agent, must be addressed during the development phase. It will be necessary to fully characterize the conjugation sites on the ligand and to identify any interference with the precise interaction mechanism between the ligand and the endothelial marker. Among the various methods available in bioconjugate chemistry, two methods have been proposed for covalent protein coupling. In the early reported methods, a carboxyl group on a UCA is activated with carbodiimide in the presence of N-hydroxysulfosuccinimide (NHS), forming active ester [46]. This NHS ester reacts with the protein amino group, forming an amide bond but
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with a relatively low coupling yield. Alternatively, a maleimide reactive group on the shell is coupled to a thiol group on the ligand, forming a thioether bond [47]. In the presence of a cysteine residue, the latter approach is preferred when clinical use is foreseen thanks to the site-selective coupling which favors the orientation of the ligand and therefore the attachment to the target [48]. Overall, region-selective coupling of a thiol protein with a maleimide-carrying UCA looks more attractive. While adaptable for clinical use, antibodies have to be humanized to avoid an immune response, while this is not needed for preclinical testing. However, these antibodies are not always available and the species specificity can be problematic when performing preclinical development. Indeed, the results obtained with one antibody in one species might differ significantly from another antibody in another species even if directed towards the same epitope found on the biomarker. This is of utmost importance when dealing with a targeted agent intended for clinical use. There is a need to first establish animal models using such agent formulations to validate the sensitivity as well as imaging conditions before initiating clinical dosing. This limitation must be carefully considered when proposing antibodies for clinical application, in addition to other constraints. The role of PEG spacer arms must also be carefully studied with regard to the binding capacity of the agent. It has been shown recently that PEGs with higher mass could impede adequate binding, by preventing the ligand at the microbubble surface from reaching its target [49]. Taking advantage of these new approaches, targeted contrast agents, whose outer shell is functionalized with the direct incorporation of specific ligands, have been made available for preclinical research (Visistar®-VEGFR2, Visistar®-VCAM1 and Visistar®Integrin Targeson Inc., San Diego, USA). Finally, small molecules [50] such as peptides can be conjugated to the shell-forming material before UCA generation. Targeted UCA BR55 (Bracco Suisse SA, Geneva, Switzerland) [37, 38], which has reached clinical trial stage, is using a pegylated lipopeptide construct that is inserted into the UCA shell [51]. BR55’s capacity to be used in many animal models and humans, provides a major " Fig. 2). advantage when developing a new agent (●
Contrast-Specific Imaging Modes !
Basically, the imaging modes used for imaging bound microbubbles are rather similar to those used for non-targeted agents: using specific pulse sequences, the non-linear components in microbubble echoes are selectively used for imaging, while cancelling all linear components from tissue echoes, collected using low acoustic pressure pulses. Microbubble-specific signals can thus be generated and are used in imaging methods called “coherent contrast imaging”, “contrast pulse sequencing” and such. The intensity of the detected signals is proportional to the local concentration of microbubbles interrogated by the acoustic beam [52, 53]. The simultaneous presence of bound and circulating microbubbles after injection has triggered the need to develop new imaging strategies for the specific detection of bound microbubbles, excluding those remaining in circulation. As a matter of fact, only a very small fraction of the injected site-specific microbubbles actually reach and stick to the endothelial receptors. The large majority will transit freely throughout the microcirculation for minutes after injection before complete clearance from the bloodstream. Therefore, one strategy could be to wait for a complete disappearance of circulating microbubbles from the vascular compartment but this would be time consuming. There might be a risk of acquiring images at time-points which are not optimal for an optimal detection of bound microbubbles, possibly leading to an underestimate of the number of microbubbles bound to the target. Therefore, the use of an alternative imaging sequence based on the destruction-replenishment concept has been proposed. Since these UCA are relatively fragile, they can be destroyed within the imaging field by applying ultrasound pulses at sufficiently high mechanical index (MI > 0.8) over five or more frames. Following the destruction within the imaging frame of both attached and circulating microbubbles, UCA still in circulation will replenish the scan plane with an unbound bubble concentration close to the one immediately before destruction. Therefore, by subtracting the signal measured after the destructive pulses from the signal measured before this destructive pulse, the relative amount of bound microbubbles within the area of interest can be estimated [22, 23, 54]. The name “differential targeted enhancement” was coined at Bracco Suisse for this
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Fig. 2 Differences in microbubble formulation between a blood pool agent for contrast-enhanced ultrasound imaging (SonoVue®) and a VEGFR2-targeted agent with a ligand inserted within the shell (BR55).
Übersicht
method, as used in the VevoCQTM software package (Visualsonics Inc., Toronto, Canada). Other approaches to selectively visualize and quantify bound targeted microbubbles are under investigation such as image processing or the use of new acoustic methods like plane waves [55].
Applications of Targeted Ultrasound Contrast Agents !
As said before, the main areas of use of these targeted UCA are for the detection of neoangiogenesis and inflammation. A large number of preclinical studies have been performed taking advantage of the commercially available biotinylated ligands and ready to use streptavidin bubbles. In vitro initial tests could not be easily translated into in vivo results due to the presence of additional factors such as shear stress which can be higher, tissue absorption of ultrasound waves and lung filtration of microbubbles following intravenous injection. It should be noted that the translation of animal results into clinical use is far from trivial, as it requires a specific validation process in humans beforehand. It is well-known that the kinetics, tissue distribution and level of expression of certain receptors could differ significantly between animals and patients (e. g., PSMA expression between rodent and human). In that perspective, performing an exploratory clinical trial is highly recommended to establish the proof of concept for the binding and detection of the targeted UCA in humans with a correlation to the receptor’s expression would serve as a proof of concept. In some cases, the absence of animal models or the inability to use the selected ligand in animal models, due to lack of cross-species reactivity, could require this clinical phase for agent validation. This exploratory phase, if positive, will precede the full clinical development as also required for therapeutic drugs. In the neoangiogenesis field, a biotinylated anti-mouse VEGFR2 antibody coupled to streptavidin microbubbles showed significantly higher signal intensity in two murine models of breast cancer compared to control microbubbles. In addition, the level of signal observed correlated well with the expression of VEGFR2 in these two tumor types [56]. Similar results were reported in two murine models of angiosarcoma and malignant glioma [23]. The same strategy has been used for the preparation of a whole
range of target-specific microbubbles for other neoangiogenesis receptors such as αv integrins [36, 41]. More recently, BR55, a clinical grade UCA targeted to VEGFR2 (Bracco Suisse SA, Geneva, Switzerland) has been evaluated in animal models [38, 57, 58] " Fig. 3 – 5) and also in man for the assessment of tumoral an(● giogenesis. BR55 has been tested in prostate cancer patients and preliminary results from this proof-of-concept trial confirmed the capacity of this targeted agent to reach detectable level of " Fig. 6). binding to VEGFR2 in patients (● Several receptors can also be imaged in the same animals during tumor growth. For instance, VEGFR2 and αvβ3 integrin or endoglin (CD105) can be monitored separately during treatment " Fig. 7). As shown in these studies, targeted microbub[60, 61] (● bles can be used not only to detect the presence of different markers expressed on endothelial cells, but also to monitor changes in receptor levels longitudinally in response to therapy. Treatment with antiangiogenic or cytotoxic therapy resulted in a reduction in binding of microbubbles targeted to these receptors. This reduction correlated with a reduction in tumor microvessel density. Changes in receptor levels might be very early signs of treatment efficacy, well before any morphological and/or func-
Fig. 3 Typical example of a good match between imaging results (good and specific binding of BR55 in a mouse model with an implant expressing human VEGFR2) and immunohistochemistry of hVEGFR2 in the same implant at the same level.
Fig. 4 Preclinical ultrasound molecular imaging of a Dunning prostate tumor with BR55 (ultrasound targeted agent for VEGFR2): Top panel: left, Bmode image of the rat prostate with the tumoral part in green and the healthy part in pink. Middle: 20 seconds after BR55 injection the enhancement was slightly higher on the tumor side, indicating a higher vascularity. Right: 10 minutes after BR55 injection, only the tumor remains strongly enhanced. Lower panel: Immunohistochemistry confirmed the strong expression of VEGFR2 in the tumor vessels (identified by CD31 staining) compared to the basal level of VEGFR2 in the healthy prostate.
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Fig. 5 Summary of ultrasound molecular imaging signals obtained from intra-animal comparison after intravenous administration of MBVEGFR2, MBControl, and MBVEGFR2 after blocking with anti-VEGFR2 antibodies p < 0.001). Representative transverse B-mode images with overlaid VEGFR2-targeted ultrasound molecular imaging signals are shown from one mammary gland harboring invasive breast cancer. Green line represents ROIs. Scale bar, 1 mm. From [57]
tional change is detected and might therefore be used as surrogate markers and good predictors of response to antiangiogenic or cytotoxic chemotherapy. Several studies have demonstrated the feasibility of imaging inflammation (targeting ICAM-1, P-selectin and VCAM-1) in various cardiovascular diseases, including heart transplant rejection, atherosclerosis [30], kidney I/R and cardiac I/R [25, 28], inflam" Fig. 8) and more recently in inmatory bowel disease [33] (● flamed endothelium of obese macaques [62]. For instance, postischemic injury is known to result in the expression of P-selectin. Microbubbles targeted to P-selectin have been prepared using an anti-P-selectin monoclonal antibody and showed a much stronger contrast signal in a mouse kidney I/R model compared to that observed with similar microbubbles bearing a control antibody [26]. More recently, in a rat model of transient ischemia, we showed that US-MI using a targeted UCA specific for selectins based on rPSGL-Ig ligand (MBselectin) allows the detection of myocardial I/R events. More importantly, the detected area of the post ischemic myocardium matched the ischemic territory as identified by the absence of contrast enhancement during coronary ligation. We showed that US-MI using MBselectin detected an ischemic event up to 24 h after ischemia was resolved, suggesting that US-MI of selectins might offer a large diagnostic time window. We demonstrated that early (2 h and 5 h after reperfusion)
Fig. 6 Clinical examples obtained with BR55: for these two patients with increased PSA level, left panel is a transverse scan of the prostate at 11 minutes after BR55 injection demonstrating broad areas with diffuse enhancement due to both fixed and circulating bubbles while post-processing of images (middle) identified clearly areas of increased binding matching well with the location of prostate cancer lesions (right panel).
Fig. 7 Ultrasound molecular imaging assessment of various markers in the same animal model (here MATBIII tumor model) suggestive of a difference in the expression and accessibility of VEGFR2, VCAM, integrin α5 and P-selectin. Top line images show Peak enhancement, about 20 s after injection. Bottom line images show bound microbubbles (10 min after injection) color coded after subtraction of circulating bubbles and overlaid on the corresponding background contrast image.
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detection of the ischemic event relied on both P- and E-selectin detection, whereas after 24 h, detection of the transient ischemic event relied on the detection of E-selectin only. Finally, we showed that binding of targeted UCA within the ischemic myo-
cardium correlated with the temporal and spatial accessibility of P- and E-selectin, i. e., the expression of these selectins on the lu" Fig. 9). minal side of endothelial cells [28] (●
The Importance of Quantification !
Fig. 8 Selectin-targeted US imaging and MB localization in the colon of mice after induction of acute colitis. A Schematic representation of MBSelectin binding to the inflamed capillaries within the colon after TNBS-induced colitis. B Representative transverse US images of the colon in a control mouse (left) and experimental mice at day 1 (middle) and day 5 (right) after TNBS administration with MBSelectin (top) and MBControl (bottom). Green ring corresponds to region of interest (scale bar = 1 mm). From [33].
The advent of contrast-specific imaging methods has dramatically improved the detection sensitivity of microbubbles. This is of utmost importance for US-MI since a relatively low number of microbubbles are expected to bind to cellular biomarkers and so it is important to be able to detect these few microbubbles with high sensitivity. The situation is more complex than for perfusion quantification [53, 63] in the sense that there is no strict proportionality between the detected signal and the absolute level of expression of a given marker. As such, it is recommended to rely more on relative measurements than on absolute values but nowadays, no straightforward method is available for that purpose. Nevertheless, some groups have already reported changes in the amount of microbubbles bound in a selected area which matched nicely the level of expression of a given receptor as assessed by immunohistochemistry. This means that US-MI might be used for treatment monitoring in order to assess treatment efficacy or to identify possible mechanisms underlying therapeutic effects " Fig. 10). [60, 61] (●
Fig. 9 Typical examples of US-MI of a transient ischemic event in rat subjected to 20-minute LAD occlusion followed by 2-hour reperfusion. A During LAD ligation, injection of BR38 allowed imaging of the ischemic territory within the anterolateral and free walls of the LV. B Late phase enhancement of BR38, 2 hours after reperfusion, revealed uniform enhancement in the left myocardium, suggesting no binding of BR38. C Image obtained with a postprocessing US-MI method was overlaid on the corresponding B mode (anatomical) image and revealed no bound BR38 MB. D The same rat, US-MI using MBselectin, 2 hours after reperfusion, revealed higher late phase enhancement whose location matched the ischemic area as determined with BR38 during LAD ligation. E Image obtained with a postprocessing US-MI method, identifying bound MBselectin in the myocardium previously ischemic, was overlaid on the corresponding B mode (anatomic) image. From [28].
Fig. 10 BR55 US-MI imaging in a rat tumor model (breast cancer induced by NMU) under treatment with Sunitinib at a dose of 20 mg/kg/day. A Representative images of the subcutaneous tumour in Bmode (left upper), before injection of BR55 (right upper), at peak intensity (left lower) and 10 minutes after BR55 injection (right lower). Β Follow-up of these tumors undergoing Sunitinib treatment demonstrated an early and rapid decrease in BR55 binding followed by a decrease in vascularity before an ultimate volume decrease.
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Discussion and Conclusion
References
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01 Gramiak R, Shah PM, Kramer DH. Ultrasound cardiography: contrast studies in anatomy and function. Radiology 1969: 939 – 948 02 Claudon M, Dietrich CF, Choi BI et al. Guidelines and good clinical practice recommendations for contrast enhanced ultrasound (CEUS) in the liver – update 2012: a WFUMB-EFSUMB initiative in cooperation with representatives of AFSUMB, AIUM, ASUM, FLAUS and ICUS. Ultraschall in Med 2013; 34: 11 – 29 03 Piscaglia F, Nolsoe C, Dietrich CF et al. The EFSUMB guidelines and recommendations on the clinical practice of contrast enhanced ultrasound (CEUS): update 2011 on non-hepatic applications. Ultraschall in Med 2012; 33: 33 – 59 04 Deshpande N, Needles A, Willmann JK. Molecular ultrasound imaging: current status and future directions. Clin Radiol 2010; 65: 567 – 581 05 Hwang M, Lyshchik A, Fleischer AC. Molecular sonography with targeted microbubbles: current investigations and potential applications. Ultrasound Q 2010; 26: 75 – 82 06 Inaba Y, Lindner JR. Molecular imaging of disease with targeted contrast ultrasound imaging. Transl Res 2012; 159: 140 – 148 07 Kiessling F, Huppert J, Palmowski M. Functional and molecular ultrasound imaging: concepts and contrast agents. Curr Med Chem 2009; 16: 627 – 642 08 Willmann JK, van Bruggen N, Dinkelborg LM et al. Molecular imaging in drug development. Nat Rev Drug Disc 2008; 7: 591 – 607 09 Correas JM, Claudon M, Tranquart F et al. The kidney: imaging with microbubble contrast agents. Ultrasound Q 2006; 22: 53 – 66 10 Claudon M, Cosgrove D, Albrecht T et al. Guidelines and good clinical practice recommendations for contrast enhanced ultrasound (CEUS) – Update 2008. Ultraschall in Med 2008; 29: 28 – 44 11 Hyvelin JM, Tardy I, Arbogast C et al. Use of ultrasound contrast agent microbubbles in preclinical research: recommendations for small animal imaging. Invest Radiol 2013; 8: 570 – 583 12 Klibanov AL, Rasche PT, Hughes MS et al. Detection of individual microbubbles of an ultrasound contrast agent: fundamental and pulse inversion imaging. Acad Radiol 2002; 9 (Suppl 2): S279 – S281 13 Dayton PA, Rychak JJ. Molecular ultrasound imaging using microbubble contrast agents. Frontiers Biosci 2007; 12: 5124 – 5142 14 Kiessling F, Fokong S, Koczera P et al. Ultrasound microbubbles for molecular diagnosis, therapy, and theranostics. J Nucl Med 2012; 53: 345 – 348 15 Kiessling F, Bzyl J, Fokong S et al. Targeted ultrasound imaging of cancer: an emerging technology on its way to clinics. Curr Pharm Des 2012; 18: 2184 – 2199 16 Kircher MF, Willmann JK. Molecular body imaging: MR imaging, CT, and US. part I. principles. Radiology 2012; 263: 633 – 643 17 Klibanov AL. Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging. Adv Drug Deliv Rev 1999; 37: 139 – 157 18 Pysz MA, Willmann JK. Targeted contrast-enhanced ultrasound: an emerging technology in abdominal and pelvic imaging. Gastroenterology 2011; 140: 785 – 790 19 Pysz MA, Gambhir SS, Willmann JK. Molecular imaging: current status and emerging strategies. Clin Radiol 2010; 65: 500 – 516 20 Deshpande N, Pysz MA, Willmann JK. Molecular ultrasound assessment of tumor angiogenesis. Angiogenesis 2010; 13: 175 – 188 21 Takalkar AM, Klibanov AL, Rychak JJ et al. Binding and detachment dynamics of microbubbles targeted to P-selectin under controlled shear flow. J Control Release 2004; 96: 473 – 482 22 Pysz MA, Guracar I, Foygel K et al. Quantitative assessment of tumor angiogenesis using real-time motion-compensated contrast-enhanced ultrasound imaging. Angiogenesis 2012; 15: 433 – 442 23 Willmann JK, Paulmurugan R, Chen K et al. US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice. Radiology 2008; 246: 508 – 518 24 Moestue SA, Gribbestad IS, Hansen R. Intravascular targets for molecular contrast-enhanced ultrasound imaging. Int J Mol Sci 2012; 13: 6679 – 6697 25 Davidson BP, Kaufmann BA, Belcik JT et al. Detection of antecedent myocardial ischemia with multiselectin molecular imaging. J Am Coll Cardiol 2012; 60: 1690 – 1697 26 Lindner JR, Song J, Christiansen J et al. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation 2001; 104: 2107 – 2112 27 Bettinger T, Bussat P, Tardy I et al. Ultrasound molecular imaging contrast agent binding to both E- and P-selectin in different species. Invest Radiol 2012; 47: 516 – 523
Ultrasound is a real-time imaging modality ideally suited for medical imaging, thanks to its very broad range of applications, its high spatial and temporal resolution and low cost. With the addition of a contrast agent, ultrasound imaging provides additional functional information on blood flow and tissue perfusion [3, 10, 63]. The development of targeted UCA has expanded the role of ultrasound and contrast-enhanced ultrasound beyond perfusion measurements into molecular imaging applications. Microbubbles targeted to specific markers present at the surface of the endothelial layer or on intravascular elements can be detected due to the very high sensitivity of contrast-specific imaging modes capable of detecting even individual bubbles [12]. Ultrasound molecular imaging (USMI) allows the confirmation of the presence of a receptor, and the assessment of relative receptor density over time and/or during treatment with drugs. It can be used to detect early response to therapy or to provide surrogate end points for measuring drug efficacy or therapy success [7, 14, 15]. Compared to methods based on the use of radiolabeled agents, molecular ultrasound imaging shows much higher spatial resolution and therefore anatomic information without ionizing radiation. US-MI has already been used by scientists involved in inflammation and/or angiogenesis research in animal models, and importantly, the recent clinical availability of anti-angiogenic or anti-inflammatory agents is enlarging significantly its potential. This will be part of the ‘tool box’ for clinicians aiming at precisely identifying biomarkers for detecting and characterizing as well as phenotyping lesions. This will help clinicians in the selection of drugs to be used as well as monitoring their impact on well-defined targets. This might replace more invasive tissue analyses and the use of radioactive tracers for the diagnosis or during therapy follow-up. This exciting new area requires a multidisciplinary approach involving biologists, physiologists, physicists, engineers and clinicians to identify the right targets and to develop the various elements needed to pave the way for clinical use of this unique modality. However, additional work must be done before a complete approval of these agents which are considered as drugs and thus require a full clinical development. As such, it remains difficult to envision this approval in the next 5 years. The strict intravascular localization of microbubbles might be seen as a limiting factor in view of the small number of targets which can be imaged, but this can also be seen as a strength for selected indications where the role of vessels is clearly predominant, such as neo-angiogenesis and inflammation. Another clear advantage is the absence of extravasation, even at sites of inflammation or formation, thus limiting the non-specific accumulation of the contrast agent in tissues. Ultrasound molecular imaging although widely used at the preclinical level is still in its infancy at the clinical level due to the limited number of agents available for this purpose. There is no doubt that the recent agent developments together with improved scanner performance will offer great opportunities in the near future.
Acknowledgements !
The authors thank Christoph Dietrich for translating the abstract into German language and for revising the text before publication. Conflict of Interest: We are all Bracco employees.
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28 Hyvelin JM, Tardy I, Bettinger T et al. Ultrasound molecular imaging of transient acute myocardial ischemia with a clinically translatable pand e-selectin targeted contrast agent: correlation with the expression of selectins. Invest Radiol 2014; 49: 224 – 235 29 Villanueva FS, Lu E, Bowry S et al. Myocardial ischemic memory imaging with molecular echocardiography. Circulation 2007; 115: 345 – 352 30 Weller GE, Villanueva FS, Tom EM et al. Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx. Biotechnol Bioeng 2005; 92: 780 – 788 31 Kaufmann BA, Sanders JM, Davis C et al. Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1. Circulation 2007; 116: 276 – 284 32 Leong-Poi H, Christiansen J, Heppner P et al. Assessment of endogenous and therapeutic arteriogenesis by contrast ultrasound molecular imaging of integrin expression. Circulation 2005; 111: 3248 – 3254 33 Wang H, Machtaler S, Bettinger T et al. Molecular imaging of inflammation in inflammatory bowel disease with a clinically translatable dualselectin-targeted US contrast agent: comparison with FDG PET/CT in a mouse model. Radiology 2013; 267: 818 – 829 34 Kaufmann BA, Lewis C, Xie A et al. Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography. Eur Heart J 2007; 28: 2011 – 2017 35 Foygel K, Wang H, Machtaler S et al. Detection of pancreatic ductal adenocarcinoma in mice by ultrasound imaging of thymocyte differentiation antigen 1. Gastroenterology 2013; 145: 885 – 894 36 Willmann JK, Kimura RH, Deshpande N et al. Targeted Contrast-enhanced ultrasound imaging of tumor angiogenesis with contrast microbubbles conjugated to integrin-binding knottin peptides. J Nucl Med 2010; 51: 433 – 440 37 Pochon S, Tardy I, Bussat P et al. BR55: a lipopeptide-based VEGFR2targeted ultrasound contrast agent for molecular imaging of angiogenesis. Invest Radiol 2010; 45: 89 – 95 38 Tardy I, Pochon S, Theraulaz M et al. Ultrasound molecular imaging of VEGFR2 in a rat prostate tumor model using BR55. Invest Radiol 2010; 45: 573 – 578 39 Ellegala DB, Leong-Poi H, Carpenter JE et al. Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha (v)beta3. Circulation 2003; 108: 336 – 341 40 Kiessling F, Gaetjens J, Palmowski M. Application of molecular ultrasound for imaging integrin expression. Theranostics 2011: 1127 – 1134 41 Weller GE, Wong MK, Modzelewski RA et al. Ultrasonic imaging of tumor angiogenesis using contrast microbubbles targeted via the tumorbinding peptide arginine-arginine-leucine. Cancer Res 2005; 65: 533 – 539 42 Leong-Poi H, Christiansen J, Klibanov AL et al. Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted to alpha(v)integrins. Circulation 2003; 107: 455 – 460 43 Alonso A, Della MA, Stroick M et al. Molecular imaging of human thrombus with novel abciximab immunobubbles and ultrasound. Stroke 2007; 38: 1508 – 1514 44 Della MA, Allemann E, Bettinger T et al. Grafting of abciximab to a microbubble-based ultrasound contrast agent for targeting to platelets expressing GP IIb/IIIa – characterization and in vitro testing. Eur J Pharm Biopharm 2008; 68: 555 – 564 45 Warram JM, Sorace AG, Saini R et al. A triple-targeted ultrasound contrast agent provides improved localization to tumor vasculature. J Ultrasound Med 2011; 30: 921 – 931
46 Villanueva FS, Jankowski RJ, Klibanov S et al. Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial cells. Circulation 1998; 98: 1 – 5 47 Klibanov AL. Ligand-carrying gas-filled microbubbles: ultrasound contrast agents for targeted molecular imaging. Bioconjug Chem 2005; 16: 9 – 17 48 Unnikrishnan S, Klibanov AL. Microbubbles as ultrasound contrast agents for molecular imaging: preparation and application. Am J Roentgenol Am J Roentgenol 2012; 199: 292 – 299 49 Chen CC, Sirsi SR, Homma S et al. Effect of surface architecture on in vivo ultrasound contrast persistence of targeted size-selected microbubbles. Ultrasound Med Biol 2012; 38: 492 – 503 50 Anderson CR, Rychak JJ, Backer M et al. scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis. Invest Radiol 2010; 45: 579 – 585 51 Pillai R, Marinelli ER, Fan H et al. A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis. Bioconjug Chem 2010; 21: 556 – 562 52 Rafter P, Phillips P, Vannan MA. Imaging technologies and techniques. Cardiol Clin 2004; 22: 181 – 197 53 Dietrich CF, Averkiou MA, Correas JM et al. An EFSUMB introduction into dynamic contrast-enhanced ultrasound (DCE-US) for quantification of tumour perfusion. Ultraschall in Med 2012; 33: 344 – 351 54 Deshpande N, Lutz AM, Ren Y et al. Quantification and monitoring of inflammation in murine inflammatory bowel disease with targeted contrast-enhanced US. Radiology 2012; 262: 172 – 180 55 Couture O, Fink M, Tanter M. Ultrasound contrast plane wave imaging. IEEE Trans Ultrason Ferroelectr Freq Control 2012; 59: 2676 – 2683 56 Lyshchik A, Fleischer AC, Huamani J et al. Molecular imaging of vascular endothelial growth factor receptor 2 expression using targeted contrast-enhanced high-frequency ultrasonography. J Ultrasound Med 2007; 26: 1575 – 1586 57 Bachawal SV, Jensen KC, Lutz AM et al. Earlier detection of breast cancer with ultrasound molecular imaging in a transgenic mouse model. Cancer Res 2013; 73: 1689 – 1698 58 Bzyl J, Lederle W, Rix A et al. Molecular and functional ultrasound imaging in differently aggressive breast cancer xenografts using two novel ultrasound contrast agents (BR55 and BR38). Eur Radiol 2011; 21: 1988 – 1995 59 Bzyl J, Palmowski M, Rix A et al. The high angiogenic activity in very early breast cancer enables reliable imaging with VEGFR2-targeted microbubbles (BR55). Eur Radiol 2013; 23: 468 – 475 60 Korpanty G, Carbon JG, Grayburn PA et al. Monitoring response to anticancer therapy by targeting microbubbles to tumor vasculature. Clin Cancer Res 2007; 13: 323 – 330 61 Palmowski M, Huppert J, Ladewig G et al. Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects. Mol Cancer Therap 2008; 7: 101 – 109 62 Chadderdon SM, Belcik JT, Bader L et al. Proinflammatory endothelial activation detected by molecular imaging in obese nonhuman primates coincides with onset of insulin resistance and progressively increases with duration of insulin resistance. Circulation 2014; 129: 471 – 478 63 Tranquart F, Mercier L, Frinking P et al. Perfusion quantification in contrast-enhanced ultrasound (CEUS) – ready for research projects and routine clinical use. Ultraschall in Med 2012; 33 (Suppl 1): S31 – S38
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Ultrasound Contrast Agents For Ultrasound Molecular Imaging Ultraschall-Kontrastmittel für molekulare Bildgebung im Ultraschall Authors
F. Tranquart1, M. Arditi1, T. Bettinger1, P. Frinking1, J. M. Hyvelin1, A. Nunn2, S. Pochon1, I. Tardy1
Affiliations
1
Bracco Suisse SA, Plan Les Ouates, Switzerland Bracco Research US, Monroe, USA
Schlüsselwörter
Zusammenfassung
Abstract
"
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Die Sonografie ist eine weitverbreitete Bildgebungsmethode mit hoher örtlicher und zeitlicher Auflösung. Die Einführung von Ultraschallkontrastmitteln im Zusammenhang mit kontrastmittelspezifischer Software erlaubt die Analyse organspezifischer Durchblutungsmuster und Perfusion mit Verbesserung der diagnostischen Genauigkeit. Die weitere Entwicklung von mit biologischen Markern versehenen Ultraschallkontrastmitteln erlaubt eine molekulare Bildgebung. Die molekulare Bildgebung ermöglicht die Darstellbarkeit der Expression von intravaskulären Markern vor, während und nach medikamentöser Behandlung. Der Vorteil der Ultraschallkontrastmittel liegt in ihrer strikten intravaskulären Verteilung, sodass Neoangiogenese und Entzündungsvorgänge dargestellt werden können. Verschiedene Technologien wurden evaluiert, um das zielgerichtete Molekül an die Kontrastmittelhülle zu binden. Diese reichen von einfachen Biotin-Avidin-Konstrukten bis zu komplizierteren Anheftungen mittels PEG-Abkömmlingen. Aktuelle Fortschritte auf diesem Gebiet erlauben die Übertragung präklinischer Erfahrung für eine verbesserte Detektion und Charakterisierung von Neoplasien und Entzündungsprozessen. Die kombinierte Anwendung und Auswertung anatomischer, funktionsanalytischer und molekularer Informationen im Rahmen der Kontrastmittelsonografie ist vielversprechend steht aber noch in den Anfängen, da bisher nur wenige klinisch relevante Markermoleküle existieren. Die beschriebenen Vorteile der Sonografie kombiniert mit der molekularen Signatur von Neoplasien wird im Rahmen der personalisierten Medizin eine relevante Rolle spielen.
Ultrasound is a real-time imaging technique which is widely used in many clinical applications for its capacity to provide anatomic information with high spatial and temporal resolution. The advent of ultrasound contrast agents in combination with contrast-specific imaging modes has given access to perfusion assessments at an organ level, leading to an improved diagnostic accuracy. More recently, the development of biologically-targeted ultrasound contrast agents has expanded the role of ultrasound even further into molecular imaging applications. Ultrasound molecular imaging can be used to visualize the expression of intravascular markers, and to assess their local presence over time and/or during therapeutic treatment. Major applications are in the field of inflammation and neoangiogenesis due to the strictly intravascular presence of microbubbles. Various technologies have been investigated for attaching the targeting moiety to the shell from simple biotin-avidin constructs to more elaborated insertion within the shell through attachment to PEG residues. This important improvement has allowed a clinical translation of initial pre-clinical investigations, opening the way for an early detection and an accurate characterization of lesions in patients. The combination of anatomic, functional and molecular information/data provided by contrast ultrasound is a powerful tool which is still in its infancy due to the lack of agents suitable for clinical use. The advantages of ultrasound techniques combined with the molecular signature of lesions will represent a significant advance in imaging in the field of personalized medicine.
● Mikrobläschen ● kontrastverstärkter "
● ● ● ● " " " "
● ● " "
Ultraschall molekulare Bildgebung VEGFR2 Selektine gastrointestinaler Stromatumor (GIST) kolorektales Karzinom gastro-entero-pankreatische Tumoren
Key words
● microbubbles ● contrast-enhanced " "
ultrasound
● molecular imaging ● VEGFR2 ● selectins ● gastrointestinal stromal " " " "
tumors (GIST)
● colorectal cancer ● gastro-entero-pancreatic " "
tumors
received accepted
27.5.2014 13.7.2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1384999 Z Gastroenterol 2014; 52: 1268–1276 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0044-2771 Correspondence Prof. Francois Tranquart Bracco Suisse SA Route de la Galaise, 31 1228 Plan Les Ouates Switzerland [email protected]
Tranquart F et al. Ultrasound Contrast Agents … Z Gastroenterol 2014; 52: 1268–1276
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Contrast agents are now extensively used in medical imaging to enhance visualization of particular body compartments or tissue perfusion. Even though computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT) and positron emission tomography (PET) have made extensive use of contrast agents since their initial development, the use of ultrasound contrast agents (UCA) is more recent and really emerged as a routine technique in the early 2000 s. Besides gross anatomy, lesion extent, and other morphological information provided by ultrasound (US) imaging, UCA are able, after intravenous administration, to provide valuable functional information about perfusion, which often represents a key factor for a definitive diagnosis or for therapeutic guidance. Since the first report made by Gramiak and Shah in 1968 [1], showing that air bubbles could be used in vivo as strong ultrasound scatterers, many improvements have been made in bubble formulations (bubble stabilization methods based on various shell components or fluorinated gas) and in ultrasound equipment, with the introduction of specific imaging sequences to exploit the acoustic properties of these agents in an optimal way. Nowadays, this new method, called “contrast-enhanced ultrasound” (CEUS), is becoming more and more popular in almost all possible indications and organs thanks to its performance ease of use, real-time capabilities and low cost [2, 3]. In the early 2000s, after the launch of clinically approved UCA and further to initial developments, a few teams became interested in the potential of CEUS for molecular imaging. Molecular imaging is generally defined as non-invasive imaging of biomarkers, implying the use of methods able to track events at a cellular level [4 – 8]. This could be achieved by preparing UCA functionalized with ligands specific for the biomarkers of interest. This requires methods able to (i) detect UCA distribution in the targeted tissue/organ, designed to image a biomarker’s expression and (ii) monitor biomarker expression changes over time according to the specific disease or process being followed. Therefore, there has been active effort in this field to generate new UCAs aimed at addressing this purpose, taking advantage of the unique properties of CEUS.
the added advantage that several consecutive injections can be performed during the same examination [3, 9 – 11]. Only minute amounts of gas and stabilizing material are administered in an imaging dose, in the range of a few µL of gas and a few tens of micrograms of phospholipids per injection per patient. These minute quantities reflect the remarkable potency of microbubble-based ultrasound contrast agents combined with the high sensitivity of the ultrasound contrast imaging mode [12], significantly higher than the sensitivity reported for X-ray and magnetic resonance methods.
Targeted UCA !
Ultrasound molecular imaging (US-MI) requires the use of targeted UCA, which differ from those initially developed for blood pool imaging by the presence of a targeting moiety able to link the microbubble to the selected cell biomarker. The general strategy is to link molecular entities to the phospholipid-stabilizing monolayer, allowing the microbubbles to remain attached to selected sites in the vascular compartment. Indeed, because of their relatively large diameter, UCA microbubbles cannot escape the vascular compartment. Once attached, these microbubbles are able to generate detectable echo signals similar to those obtained with blood pool agents, since the signal is generated by the microbubble itself and not the ligand [4 – 8, 13 – 19]. This raises important points for US-MI: (i) As the microbubbles remain strictly within the vascular compartment, targets must be selected that are accessible to these microbubbles, i. e., on the luminal side of the endothelial cells [4, 6 – 8, 14, 16, 19 – 21]. (ii) The attachment of microbubbles to the surface of endothelial cells must be strong enough for vascular areas where shear stress is high due to high blood velocity and viscosity [21]. (iii) As the detected signal is the combination of echoes from bound microbubbles and circulating microbubbles, there is a need to develop specific imaging strategies aiming at detecting the echoes from bound bubbles [8, 22, 23] in a distinct way. These issues will be addressed here below in addition to the concepts used for inserting the targeting moiety into the microbubble shell.
Specificities of Ultrasound Contrast Agents (UCA)
Selection of Targets [24]
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Microbubble diameter sizes are in the micron range. After intravenous administration, microbubbles larger than 6 ‑ 8 µm in diameter are eliminated at the pulmonary level and thus do not reach the left heart and general circulation. Microbubble sizes should therefore preferably be in the 2 to 5 µm range, i. e. close to or below the size of red blood cells. Microbubbles in this size range are able to move freely in the entire vasculature, including the smallest capillaries. They act as strong ultrasound scatterers due to the fortunate coincidence that their resonance frequencies match well the range of ultrasound diagnostic frequencies (2 − 20 MHz). Today, all commercially available ultrasound contrast agents contain high concentrations of micron-sized gas microbubbles (108 − 1010 bubbles per ml) stabilized by lipids or heat denatured albumin. The half-life of ultrasound contrast agents after intravenous administration is short, typically a few minutes. This duration is however in most cases sufficient for imaging purposes and has
Based on the features listed above, two application areas have clearly emerged for US-MI: inflammation and neoangiogenesis, " Fig. 1). since both involve endothelial cells (● In the course of the inflammatory process, various cell surface markers are expressed or up-regulated on the luminal side of endothelium and therefore are accessible to targeted microbubbles [21, 25 – 28]. The cellular inflammatory response, which involves leukocyte infiltration of tissue, relies on a timely sequence of events, the so-called inflammation cascade. First, leukocytes are recruited to the vessel wall (tethering) and roll along the endothelium, these first interactions being mediated by selectins. Then, leukocytes roll with increased interactions with selectins before slowing down and finally stop with a strong attachment to the vessel wall. This phase is mediated by integrins, interacting with their counter-receptors (such as intercellular cell adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]). Finally, leukocytes cross the vessel wall towards the interstitial space, triggering local effects such as edema. This
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Fig. 1 Targets eligible for ultrasound molecular imaging due to their intra-vascular expression.
makes these different inflammatory mediators appealing targets for US-MI. Inflammation plays a key role in the development and progression of cardiovascular diseases [29, 30], including atherosclerosis [31, 32], stroke, transplant rejection, inflammatory bowel disease [33], and myocardial infarct. In the latter case, a hallmark of ischemia-reperfusion (I/R) is the activation of inflammatory processes, leading to the expression of specific adhesion molecules at the surface of endothelial cells. Thanks to their location, these markers are accessible to targeted UCA and therefore can be detected using US-MI. So far, most of the studies have been performed using agents specific for either P- or E-selectins or both [27, 28, 34], or VCAM-1 [31]. The second important area in which US-MI has clearly emerged, is tumor detection [35] and more particularly tumoral neoangiogenesis [20, 36], i. e., the formation of new blood vessels, which is a fundamental process occurring during tumor progression. Sequential steps are involved in effective tumoral angiogenesis with an initial trigger of hypoxia. Under hypoxic conditions, expression of hypoxia-inducible factors is induced in endothelial vessels, resulting in vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor 2 (VEGFR2) expressions [37, 38]. Many other biomarkers have been identified in this complex process such as neuropilin 2, the αvβ3 integrin, and endoglin (CD105) [17, 20, 39 – 42]. In addition to endothelial receptors, site targeted microbubbles can also be used for the visualization of thrombi associated with stroke. Immunobubbles bearing Abciximab, a glycoprotein IIb/ IIIa receptor inhibitor, were shown to improve visualization of human clots both in vitro and in an in vivo model of acute arterial thrombotic occlusion [43, 44].
Targeted UCA Engineering !
The methods for making targeted microbubbles do not differ significantly from those used for conventional microbubbles. There is an additional step of attaching/inserting a site-specific ligand to the microbubble shell. Today, two packages are being used: the first one is a lyophilized product that is resuspended before injection and the second is one in which microbubble precursors are kept in solution before activation. The main objective is to get a regular and stable microbubble concentration and size distribution which are stable from vial to vial, allowing highly reprodu-
cible imaging acquisitions. The problems of storage and transportation must be addressed at very early stages of development to facilitate the clinical use of these agents. A common strategy makes use of a spacer arm like polyethylene glycol (PEG) to conjugate avidin or streptavidin to the surface of the microbubble, and then an avidin-biotin link is used to attach a pathology-specific ligand. The avidin-biotin interaction is among the strongest non-covalent bonds. The resulting avidin/ streptavidin-functionalized microbubbles are then incubated with a ligand bearing a biotin moiety. Thus, thanks to the multivalency of avidin/streptavidin molecules, biotinylated molecular entities can be bound to the avidin/streptavidin coated microbubbles. In this way, moieties may be attached to the microbubble shell through a biotin-avidin-biotin bridge. This procedure is very flexible and convenient for small-animal imaging, as it allows the preparation of microbubbles coated on their surface with a biotinylated molecule selected by the investigator [45]. Recently, commercial preparations have been designed for smallanimal imaging based on streptavidin-functionalized microbubbles (Target Ready MicroMarker™, Visualsonics Inc., Toronto, Canada; Targestar SA, Targeson Inc, San Diego, CA) [11]. The ligand can be a protein, an antibody or antibody fragment, an oligopeptide or even an organic small molecule, the only requirement being that a biotin residue be attached to the molecule selected. No separation stage is usually necessary since the amount of biotinylated ligand added can be adjusted in order to be captured entirely by the streptavidin bubbles. While advantageous for preclinical testing, this approach cannot be translated into clinical use due to the immunogenicity of avidin. Another strategy is the direct conjugation of the ligand on the PEG spacer. The specific issue of random coupling, which can jeopardize the interaction of the ligand with its target and can complicate significantly the analytical characterization of the agent, must be addressed during the development phase. It will be necessary to fully characterize the conjugation sites on the ligand and to identify any interference with the precise interaction mechanism between the ligand and the endothelial marker. Among the various methods available in bioconjugate chemistry, two methods have been proposed for covalent protein coupling. In the early reported methods, a carboxyl group on a UCA is activated with carbodiimide in the presence of N-hydroxysulfosuccinimide (NHS), forming active ester [46]. This NHS ester reacts with the protein amino group, forming an amide bond but
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with a relatively low coupling yield. Alternatively, a maleimide reactive group on the shell is coupled to a thiol group on the ligand, forming a thioether bond [47]. In the presence of a cysteine residue, the latter approach is preferred when clinical use is foreseen thanks to the site-selective coupling which favors the orientation of the ligand and therefore the attachment to the target [48]. Overall, region-selective coupling of a thiol protein with a maleimide-carrying UCA looks more attractive. While adaptable for clinical use, antibodies have to be humanized to avoid an immune response, while this is not needed for preclinical testing. However, these antibodies are not always available and the species specificity can be problematic when performing preclinical development. Indeed, the results obtained with one antibody in one species might differ significantly from another antibody in another species even if directed towards the same epitope found on the biomarker. This is of utmost importance when dealing with a targeted agent intended for clinical use. There is a need to first establish animal models using such agent formulations to validate the sensitivity as well as imaging conditions before initiating clinical dosing. This limitation must be carefully considered when proposing antibodies for clinical application, in addition to other constraints. The role of PEG spacer arms must also be carefully studied with regard to the binding capacity of the agent. It has been shown recently that PEGs with higher mass could impede adequate binding, by preventing the ligand at the microbubble surface from reaching its target [49]. Taking advantage of these new approaches, targeted contrast agents, whose outer shell is functionalized with the direct incorporation of specific ligands, have been made available for preclinical research (Visistar®-VEGFR2, Visistar®-VCAM1 and Visistar®Integrin Targeson Inc., San Diego, USA). Finally, small molecules [50] such as peptides can be conjugated to the shell-forming material before UCA generation. Targeted UCA BR55 (Bracco Suisse SA, Geneva, Switzerland) [37, 38], which has reached clinical trial stage, is using a pegylated lipopeptide construct that is inserted into the UCA shell [51]. BR55’s capacity to be used in many animal models and humans, provides a major " Fig. 2). advantage when developing a new agent (●
Contrast-Specific Imaging Modes !
Basically, the imaging modes used for imaging bound microbubbles are rather similar to those used for non-targeted agents: using specific pulse sequences, the non-linear components in microbubble echoes are selectively used for imaging, while cancelling all linear components from tissue echoes, collected using low acoustic pressure pulses. Microbubble-specific signals can thus be generated and are used in imaging methods called “coherent contrast imaging”, “contrast pulse sequencing” and such. The intensity of the detected signals is proportional to the local concentration of microbubbles interrogated by the acoustic beam [52, 53]. The simultaneous presence of bound and circulating microbubbles after injection has triggered the need to develop new imaging strategies for the specific detection of bound microbubbles, excluding those remaining in circulation. As a matter of fact, only a very small fraction of the injected site-specific microbubbles actually reach and stick to the endothelial receptors. The large majority will transit freely throughout the microcirculation for minutes after injection before complete clearance from the bloodstream. Therefore, one strategy could be to wait for a complete disappearance of circulating microbubbles from the vascular compartment but this would be time consuming. There might be a risk of acquiring images at time-points which are not optimal for an optimal detection of bound microbubbles, possibly leading to an underestimate of the number of microbubbles bound to the target. Therefore, the use of an alternative imaging sequence based on the destruction-replenishment concept has been proposed. Since these UCA are relatively fragile, they can be destroyed within the imaging field by applying ultrasound pulses at sufficiently high mechanical index (MI > 0.8) over five or more frames. Following the destruction within the imaging frame of both attached and circulating microbubbles, UCA still in circulation will replenish the scan plane with an unbound bubble concentration close to the one immediately before destruction. Therefore, by subtracting the signal measured after the destructive pulses from the signal measured before this destructive pulse, the relative amount of bound microbubbles within the area of interest can be estimated [22, 23, 54]. The name “differential targeted enhancement” was coined at Bracco Suisse for this
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Fig. 2 Differences in microbubble formulation between a blood pool agent for contrast-enhanced ultrasound imaging (SonoVue®) and a VEGFR2-targeted agent with a ligand inserted within the shell (BR55).
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method, as used in the VevoCQTM software package (Visualsonics Inc., Toronto, Canada). Other approaches to selectively visualize and quantify bound targeted microbubbles are under investigation such as image processing or the use of new acoustic methods like plane waves [55].
Applications of Targeted Ultrasound Contrast Agents !
As said before, the main areas of use of these targeted UCA are for the detection of neoangiogenesis and inflammation. A large number of preclinical studies have been performed taking advantage of the commercially available biotinylated ligands and ready to use streptavidin bubbles. In vitro initial tests could not be easily translated into in vivo results due to the presence of additional factors such as shear stress which can be higher, tissue absorption of ultrasound waves and lung filtration of microbubbles following intravenous injection. It should be noted that the translation of animal results into clinical use is far from trivial, as it requires a specific validation process in humans beforehand. It is well-known that the kinetics, tissue distribution and level of expression of certain receptors could differ significantly between animals and patients (e. g., PSMA expression between rodent and human). In that perspective, performing an exploratory clinical trial is highly recommended to establish the proof of concept for the binding and detection of the targeted UCA in humans with a correlation to the receptor’s expression would serve as a proof of concept. In some cases, the absence of animal models or the inability to use the selected ligand in animal models, due to lack of cross-species reactivity, could require this clinical phase for agent validation. This exploratory phase, if positive, will precede the full clinical development as also required for therapeutic drugs. In the neoangiogenesis field, a biotinylated anti-mouse VEGFR2 antibody coupled to streptavidin microbubbles showed significantly higher signal intensity in two murine models of breast cancer compared to control microbubbles. In addition, the level of signal observed correlated well with the expression of VEGFR2 in these two tumor types [56]. Similar results were reported in two murine models of angiosarcoma and malignant glioma [23]. The same strategy has been used for the preparation of a whole
range of target-specific microbubbles for other neoangiogenesis receptors such as αv integrins [36, 41]. More recently, BR55, a clinical grade UCA targeted to VEGFR2 (Bracco Suisse SA, Geneva, Switzerland) has been evaluated in animal models [38, 57, 58] " Fig. 3 – 5) and also in man for the assessment of tumoral an(● giogenesis. BR55 has been tested in prostate cancer patients and preliminary results from this proof-of-concept trial confirmed the capacity of this targeted agent to reach detectable level of " Fig. 6). binding to VEGFR2 in patients (● Several receptors can also be imaged in the same animals during tumor growth. For instance, VEGFR2 and αvβ3 integrin or endoglin (CD105) can be monitored separately during treatment " Fig. 7). As shown in these studies, targeted microbub[60, 61] (● bles can be used not only to detect the presence of different markers expressed on endothelial cells, but also to monitor changes in receptor levels longitudinally in response to therapy. Treatment with antiangiogenic or cytotoxic therapy resulted in a reduction in binding of microbubbles targeted to these receptors. This reduction correlated with a reduction in tumor microvessel density. Changes in receptor levels might be very early signs of treatment efficacy, well before any morphological and/or func-
Fig. 3 Typical example of a good match between imaging results (good and specific binding of BR55 in a mouse model with an implant expressing human VEGFR2) and immunohistochemistry of hVEGFR2 in the same implant at the same level.
Fig. 4 Preclinical ultrasound molecular imaging of a Dunning prostate tumor with BR55 (ultrasound targeted agent for VEGFR2): Top panel: left, Bmode image of the rat prostate with the tumoral part in green and the healthy part in pink. Middle: 20 seconds after BR55 injection the enhancement was slightly higher on the tumor side, indicating a higher vascularity. Right: 10 minutes after BR55 injection, only the tumor remains strongly enhanced. Lower panel: Immunohistochemistry confirmed the strong expression of VEGFR2 in the tumor vessels (identified by CD31 staining) compared to the basal level of VEGFR2 in the healthy prostate.
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Fig. 5 Summary of ultrasound molecular imaging signals obtained from intra-animal comparison after intravenous administration of MBVEGFR2, MBControl, and MBVEGFR2 after blocking with anti-VEGFR2 antibodies p < 0.001). Representative transverse B-mode images with overlaid VEGFR2-targeted ultrasound molecular imaging signals are shown from one mammary gland harboring invasive breast cancer. Green line represents ROIs. Scale bar, 1 mm. From [57]
tional change is detected and might therefore be used as surrogate markers and good predictors of response to antiangiogenic or cytotoxic chemotherapy. Several studies have demonstrated the feasibility of imaging inflammation (targeting ICAM-1, P-selectin and VCAM-1) in various cardiovascular diseases, including heart transplant rejection, atherosclerosis [30], kidney I/R and cardiac I/R [25, 28], inflam" Fig. 8) and more recently in inmatory bowel disease [33] (● flamed endothelium of obese macaques [62]. For instance, postischemic injury is known to result in the expression of P-selectin. Microbubbles targeted to P-selectin have been prepared using an anti-P-selectin monoclonal antibody and showed a much stronger contrast signal in a mouse kidney I/R model compared to that observed with similar microbubbles bearing a control antibody [26]. More recently, in a rat model of transient ischemia, we showed that US-MI using a targeted UCA specific for selectins based on rPSGL-Ig ligand (MBselectin) allows the detection of myocardial I/R events. More importantly, the detected area of the post ischemic myocardium matched the ischemic territory as identified by the absence of contrast enhancement during coronary ligation. We showed that US-MI using MBselectin detected an ischemic event up to 24 h after ischemia was resolved, suggesting that US-MI of selectins might offer a large diagnostic time window. We demonstrated that early (2 h and 5 h after reperfusion)
Fig. 6 Clinical examples obtained with BR55: for these two patients with increased PSA level, left panel is a transverse scan of the prostate at 11 minutes after BR55 injection demonstrating broad areas with diffuse enhancement due to both fixed and circulating bubbles while post-processing of images (middle) identified clearly areas of increased binding matching well with the location of prostate cancer lesions (right panel).
Fig. 7 Ultrasound molecular imaging assessment of various markers in the same animal model (here MATBIII tumor model) suggestive of a difference in the expression and accessibility of VEGFR2, VCAM, integrin α5 and P-selectin. Top line images show Peak enhancement, about 20 s after injection. Bottom line images show bound microbubbles (10 min after injection) color coded after subtraction of circulating bubbles and overlaid on the corresponding background contrast image.
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detection of the ischemic event relied on both P- and E-selectin detection, whereas after 24 h, detection of the transient ischemic event relied on the detection of E-selectin only. Finally, we showed that binding of targeted UCA within the ischemic myo-
cardium correlated with the temporal and spatial accessibility of P- and E-selectin, i. e., the expression of these selectins on the lu" Fig. 9). minal side of endothelial cells [28] (●
The Importance of Quantification !
Fig. 8 Selectin-targeted US imaging and MB localization in the colon of mice after induction of acute colitis. A Schematic representation of MBSelectin binding to the inflamed capillaries within the colon after TNBS-induced colitis. B Representative transverse US images of the colon in a control mouse (left) and experimental mice at day 1 (middle) and day 5 (right) after TNBS administration with MBSelectin (top) and MBControl (bottom). Green ring corresponds to region of interest (scale bar = 1 mm). From [33].
The advent of contrast-specific imaging methods has dramatically improved the detection sensitivity of microbubbles. This is of utmost importance for US-MI since a relatively low number of microbubbles are expected to bind to cellular biomarkers and so it is important to be able to detect these few microbubbles with high sensitivity. The situation is more complex than for perfusion quantification [53, 63] in the sense that there is no strict proportionality between the detected signal and the absolute level of expression of a given marker. As such, it is recommended to rely more on relative measurements than on absolute values but nowadays, no straightforward method is available for that purpose. Nevertheless, some groups have already reported changes in the amount of microbubbles bound in a selected area which matched nicely the level of expression of a given receptor as assessed by immunohistochemistry. This means that US-MI might be used for treatment monitoring in order to assess treatment efficacy or to identify possible mechanisms underlying therapeutic effects " Fig. 10). [60, 61] (●
Fig. 9 Typical examples of US-MI of a transient ischemic event in rat subjected to 20-minute LAD occlusion followed by 2-hour reperfusion. A During LAD ligation, injection of BR38 allowed imaging of the ischemic territory within the anterolateral and free walls of the LV. B Late phase enhancement of BR38, 2 hours after reperfusion, revealed uniform enhancement in the left myocardium, suggesting no binding of BR38. C Image obtained with a postprocessing US-MI method was overlaid on the corresponding B mode (anatomical) image and revealed no bound BR38 MB. D The same rat, US-MI using MBselectin, 2 hours after reperfusion, revealed higher late phase enhancement whose location matched the ischemic area as determined with BR38 during LAD ligation. E Image obtained with a postprocessing US-MI method, identifying bound MBselectin in the myocardium previously ischemic, was overlaid on the corresponding B mode (anatomic) image. From [28].
Fig. 10 BR55 US-MI imaging in a rat tumor model (breast cancer induced by NMU) under treatment with Sunitinib at a dose of 20 mg/kg/day. A Representative images of the subcutaneous tumour in Bmode (left upper), before injection of BR55 (right upper), at peak intensity (left lower) and 10 minutes after BR55 injection (right lower). Β Follow-up of these tumors undergoing Sunitinib treatment demonstrated an early and rapid decrease in BR55 binding followed by a decrease in vascularity before an ultimate volume decrease.
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Discussion and Conclusion
References
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01 Gramiak R, Shah PM, Kramer DH. Ultrasound cardiography: contrast studies in anatomy and function. Radiology 1969: 939 – 948 02 Claudon M, Dietrich CF, Choi BI et al. Guidelines and good clinical practice recommendations for contrast enhanced ultrasound (CEUS) in the liver – update 2012: a WFUMB-EFSUMB initiative in cooperation with representatives of AFSUMB, AIUM, ASUM, FLAUS and ICUS. Ultraschall in Med 2013; 34: 11 – 29 03 Piscaglia F, Nolsoe C, Dietrich CF et al. The EFSUMB guidelines and recommendations on the clinical practice of contrast enhanced ultrasound (CEUS): update 2011 on non-hepatic applications. Ultraschall in Med 2012; 33: 33 – 59 04 Deshpande N, Needles A, Willmann JK. Molecular ultrasound imaging: current status and future directions. Clin Radiol 2010; 65: 567 – 581 05 Hwang M, Lyshchik A, Fleischer AC. Molecular sonography with targeted microbubbles: current investigations and potential applications. Ultrasound Q 2010; 26: 75 – 82 06 Inaba Y, Lindner JR. Molecular imaging of disease with targeted contrast ultrasound imaging. Transl Res 2012; 159: 140 – 148 07 Kiessling F, Huppert J, Palmowski M. Functional and molecular ultrasound imaging: concepts and contrast agents. Curr Med Chem 2009; 16: 627 – 642 08 Willmann JK, van Bruggen N, Dinkelborg LM et al. Molecular imaging in drug development. Nat Rev Drug Disc 2008; 7: 591 – 607 09 Correas JM, Claudon M, Tranquart F et al. The kidney: imaging with microbubble contrast agents. Ultrasound Q 2006; 22: 53 – 66 10 Claudon M, Cosgrove D, Albrecht T et al. Guidelines and good clinical practice recommendations for contrast enhanced ultrasound (CEUS) – Update 2008. Ultraschall in Med 2008; 29: 28 – 44 11 Hyvelin JM, Tardy I, Arbogast C et al. Use of ultrasound contrast agent microbubbles in preclinical research: recommendations for small animal imaging. Invest Radiol 2013; 8: 570 – 583 12 Klibanov AL, Rasche PT, Hughes MS et al. Detection of individual microbubbles of an ultrasound contrast agent: fundamental and pulse inversion imaging. Acad Radiol 2002; 9 (Suppl 2): S279 – S281 13 Dayton PA, Rychak JJ. Molecular ultrasound imaging using microbubble contrast agents. Frontiers Biosci 2007; 12: 5124 – 5142 14 Kiessling F, Fokong S, Koczera P et al. Ultrasound microbubbles for molecular diagnosis, therapy, and theranostics. J Nucl Med 2012; 53: 345 – 348 15 Kiessling F, Bzyl J, Fokong S et al. Targeted ultrasound imaging of cancer: an emerging technology on its way to clinics. Curr Pharm Des 2012; 18: 2184 – 2199 16 Kircher MF, Willmann JK. Molecular body imaging: MR imaging, CT, and US. part I. principles. Radiology 2012; 263: 633 – 643 17 Klibanov AL. Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging. Adv Drug Deliv Rev 1999; 37: 139 – 157 18 Pysz MA, Willmann JK. Targeted contrast-enhanced ultrasound: an emerging technology in abdominal and pelvic imaging. Gastroenterology 2011; 140: 785 – 790 19 Pysz MA, Gambhir SS, Willmann JK. Molecular imaging: current status and emerging strategies. Clin Radiol 2010; 65: 500 – 516 20 Deshpande N, Pysz MA, Willmann JK. Molecular ultrasound assessment of tumor angiogenesis. Angiogenesis 2010; 13: 175 – 188 21 Takalkar AM, Klibanov AL, Rychak JJ et al. Binding and detachment dynamics of microbubbles targeted to P-selectin under controlled shear flow. J Control Release 2004; 96: 473 – 482 22 Pysz MA, Guracar I, Foygel K et al. Quantitative assessment of tumor angiogenesis using real-time motion-compensated contrast-enhanced ultrasound imaging. Angiogenesis 2012; 15: 433 – 442 23 Willmann JK, Paulmurugan R, Chen K et al. US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice. Radiology 2008; 246: 508 – 518 24 Moestue SA, Gribbestad IS, Hansen R. Intravascular targets for molecular contrast-enhanced ultrasound imaging. Int J Mol Sci 2012; 13: 6679 – 6697 25 Davidson BP, Kaufmann BA, Belcik JT et al. Detection of antecedent myocardial ischemia with multiselectin molecular imaging. J Am Coll Cardiol 2012; 60: 1690 – 1697 26 Lindner JR, Song J, Christiansen J et al. Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation 2001; 104: 2107 – 2112 27 Bettinger T, Bussat P, Tardy I et al. Ultrasound molecular imaging contrast agent binding to both E- and P-selectin in different species. Invest Radiol 2012; 47: 516 – 523
Ultrasound is a real-time imaging modality ideally suited for medical imaging, thanks to its very broad range of applications, its high spatial and temporal resolution and low cost. With the addition of a contrast agent, ultrasound imaging provides additional functional information on blood flow and tissue perfusion [3, 10, 63]. The development of targeted UCA has expanded the role of ultrasound and contrast-enhanced ultrasound beyond perfusion measurements into molecular imaging applications. Microbubbles targeted to specific markers present at the surface of the endothelial layer or on intravascular elements can be detected due to the very high sensitivity of contrast-specific imaging modes capable of detecting even individual bubbles [12]. Ultrasound molecular imaging (USMI) allows the confirmation of the presence of a receptor, and the assessment of relative receptor density over time and/or during treatment with drugs. It can be used to detect early response to therapy or to provide surrogate end points for measuring drug efficacy or therapy success [7, 14, 15]. Compared to methods based on the use of radiolabeled agents, molecular ultrasound imaging shows much higher spatial resolution and therefore anatomic information without ionizing radiation. US-MI has already been used by scientists involved in inflammation and/or angiogenesis research in animal models, and importantly, the recent clinical availability of anti-angiogenic or anti-inflammatory agents is enlarging significantly its potential. This will be part of the ‘tool box’ for clinicians aiming at precisely identifying biomarkers for detecting and characterizing as well as phenotyping lesions. This will help clinicians in the selection of drugs to be used as well as monitoring their impact on well-defined targets. This might replace more invasive tissue analyses and the use of radioactive tracers for the diagnosis or during therapy follow-up. This exciting new area requires a multidisciplinary approach involving biologists, physiologists, physicists, engineers and clinicians to identify the right targets and to develop the various elements needed to pave the way for clinical use of this unique modality. However, additional work must be done before a complete approval of these agents which are considered as drugs and thus require a full clinical development. As such, it remains difficult to envision this approval in the next 5 years. The strict intravascular localization of microbubbles might be seen as a limiting factor in view of the small number of targets which can be imaged, but this can also be seen as a strength for selected indications where the role of vessels is clearly predominant, such as neo-angiogenesis and inflammation. Another clear advantage is the absence of extravasation, even at sites of inflammation or formation, thus limiting the non-specific accumulation of the contrast agent in tissues. Ultrasound molecular imaging although widely used at the preclinical level is still in its infancy at the clinical level due to the limited number of agents available for this purpose. There is no doubt that the recent agent developments together with improved scanner performance will offer great opportunities in the near future.
Acknowledgements !
The authors thank Christoph Dietrich for translating the abstract into German language and for revising the text before publication. Conflict of Interest: We are all Bracco employees.
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28 Hyvelin JM, Tardy I, Bettinger T et al. Ultrasound molecular imaging of transient acute myocardial ischemia with a clinically translatable pand e-selectin targeted contrast agent: correlation with the expression of selectins. Invest Radiol 2014; 49: 224 – 235 29 Villanueva FS, Lu E, Bowry S et al. Myocardial ischemic memory imaging with molecular echocardiography. Circulation 2007; 115: 345 – 352 30 Weller GE, Villanueva FS, Tom EM et al. Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx. Biotechnol Bioeng 2005; 92: 780 – 788 31 Kaufmann BA, Sanders JM, Davis C et al. Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1. Circulation 2007; 116: 276 – 284 32 Leong-Poi H, Christiansen J, Heppner P et al. Assessment of endogenous and therapeutic arteriogenesis by contrast ultrasound molecular imaging of integrin expression. Circulation 2005; 111: 3248 – 3254 33 Wang H, Machtaler S, Bettinger T et al. Molecular imaging of inflammation in inflammatory bowel disease with a clinically translatable dualselectin-targeted US contrast agent: comparison with FDG PET/CT in a mouse model. Radiology 2013; 267: 818 – 829 34 Kaufmann BA, Lewis C, Xie A et al. Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography. Eur Heart J 2007; 28: 2011 – 2017 35 Foygel K, Wang H, Machtaler S et al. Detection of pancreatic ductal adenocarcinoma in mice by ultrasound imaging of thymocyte differentiation antigen 1. Gastroenterology 2013; 145: 885 – 894 36 Willmann JK, Kimura RH, Deshpande N et al. Targeted Contrast-enhanced ultrasound imaging of tumor angiogenesis with contrast microbubbles conjugated to integrin-binding knottin peptides. J Nucl Med 2010; 51: 433 – 440 37 Pochon S, Tardy I, Bussat P et al. BR55: a lipopeptide-based VEGFR2targeted ultrasound contrast agent for molecular imaging of angiogenesis. Invest Radiol 2010; 45: 89 – 95 38 Tardy I, Pochon S, Theraulaz M et al. Ultrasound molecular imaging of VEGFR2 in a rat prostate tumor model using BR55. Invest Radiol 2010; 45: 573 – 578 39 Ellegala DB, Leong-Poi H, Carpenter JE et al. Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to alpha (v)beta3. Circulation 2003; 108: 336 – 341 40 Kiessling F, Gaetjens J, Palmowski M. Application of molecular ultrasound for imaging integrin expression. Theranostics 2011: 1127 – 1134 41 Weller GE, Wong MK, Modzelewski RA et al. Ultrasonic imaging of tumor angiogenesis using contrast microbubbles targeted via the tumorbinding peptide arginine-arginine-leucine. Cancer Res 2005; 65: 533 – 539 42 Leong-Poi H, Christiansen J, Klibanov AL et al. Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted to alpha(v)integrins. Circulation 2003; 107: 455 – 460 43 Alonso A, Della MA, Stroick M et al. Molecular imaging of human thrombus with novel abciximab immunobubbles and ultrasound. Stroke 2007; 38: 1508 – 1514 44 Della MA, Allemann E, Bettinger T et al. Grafting of abciximab to a microbubble-based ultrasound contrast agent for targeting to platelets expressing GP IIb/IIIa – characterization and in vitro testing. Eur J Pharm Biopharm 2008; 68: 555 – 564 45 Warram JM, Sorace AG, Saini R et al. A triple-targeted ultrasound contrast agent provides improved localization to tumor vasculature. J Ultrasound Med 2011; 30: 921 – 931
46 Villanueva FS, Jankowski RJ, Klibanov S et al. Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial cells. Circulation 1998; 98: 1 – 5 47 Klibanov AL. Ligand-carrying gas-filled microbubbles: ultrasound contrast agents for targeted molecular imaging. Bioconjug Chem 2005; 16: 9 – 17 48 Unnikrishnan S, Klibanov AL. Microbubbles as ultrasound contrast agents for molecular imaging: preparation and application. Am J Roentgenol Am J Roentgenol 2012; 199: 292 – 299 49 Chen CC, Sirsi SR, Homma S et al. Effect of surface architecture on in vivo ultrasound contrast persistence of targeted size-selected microbubbles. Ultrasound Med Biol 2012; 38: 492 – 503 50 Anderson CR, Rychak JJ, Backer M et al. scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis. Invest Radiol 2010; 45: 579 – 585 51 Pillai R, Marinelli ER, Fan H et al. A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis. Bioconjug Chem 2010; 21: 556 – 562 52 Rafter P, Phillips P, Vannan MA. Imaging technologies and techniques. Cardiol Clin 2004; 22: 181 – 197 53 Dietrich CF, Averkiou MA, Correas JM et al. An EFSUMB introduction into dynamic contrast-enhanced ultrasound (DCE-US) for quantification of tumour perfusion. Ultraschall in Med 2012; 33: 344 – 351 54 Deshpande N, Lutz AM, Ren Y et al. Quantification and monitoring of inflammation in murine inflammatory bowel disease with targeted contrast-enhanced US. Radiology 2012; 262: 172 – 180 55 Couture O, Fink M, Tanter M. Ultrasound contrast plane wave imaging. IEEE Trans Ultrason Ferroelectr Freq Control 2012; 59: 2676 – 2683 56 Lyshchik A, Fleischer AC, Huamani J et al. Molecular imaging of vascular endothelial growth factor receptor 2 expression using targeted contrast-enhanced high-frequency ultrasonography. J Ultrasound Med 2007; 26: 1575 – 1586 57 Bachawal SV, Jensen KC, Lutz AM et al. Earlier detection of breast cancer with ultrasound molecular imaging in a transgenic mouse model. Cancer Res 2013; 73: 1689 – 1698 58 Bzyl J, Lederle W, Rix A et al. Molecular and functional ultrasound imaging in differently aggressive breast cancer xenografts using two novel ultrasound contrast agents (BR55 and BR38). Eur Radiol 2011; 21: 1988 – 1995 59 Bzyl J, Palmowski M, Rix A et al. The high angiogenic activity in very early breast cancer enables reliable imaging with VEGFR2-targeted microbubbles (BR55). Eur Radiol 2013; 23: 468 – 475 60 Korpanty G, Carbon JG, Grayburn PA et al. Monitoring response to anticancer therapy by targeting microbubbles to tumor vasculature. Clin Cancer Res 2007; 13: 323 – 330 61 Palmowski M, Huppert J, Ladewig G et al. Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects. Mol Cancer Therap 2008; 7: 101 – 109 62 Chadderdon SM, Belcik JT, Bader L et al. Proinflammatory endothelial activation detected by molecular imaging in obese nonhuman primates coincides with onset of insulin resistance and progressively increases with duration of insulin resistance. Circulation 2014; 129: 471 – 478 63 Tranquart F, Mercier L, Frinking P et al. Perfusion quantification in contrast-enhanced ultrasound (CEUS) – ready for research projects and routine clinical use. Ultraschall in Med 2012; 33 (Suppl 1): S31 – S38
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