Fibrin-targeted PET probes for the detection of thrombi.

There is an ongoing effort to develop better methods for noninvasive detection and characterization of thrombi. Here we describe the synthesis and evaluation of three new fibrin-targeted positron emission tomography (PET) probes (FBP1, FBP2, FBP3). Three fibrin-specific peptides were conjugated as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-monoamides at the C- and N-termini and chelated with (64)CuCl2. Probes were prepared with a specific activity ranging from 10 to 130 µCi/nmol. Both the peptides and the probes exhibited nanomolar dissociation constants (Kd) for the soluble fibrin fragment DD(E), although the Cu-DOTA derivatization resulted in a 2-3 fold loss in affinity relative to the parent peptide. Biodistribution and imaging studies were performed in a rat model of carotid artery thrombosis. For FBP1 and FBP2 at 120 min post injection, the vessel containing the thrombus showed the highest concentration of radioactivity after the excretory organs, that is, the liver and kidneys. This was confirmed ex vivo by autoradiography, which showed >4-fold activity in the thrombus-containing artery compared to the contralateral artery. FBP3 showed much lower thrombus uptake, and the difference was traced to greater metabolism of this probe. Hybrid MR-PET imaging with FBP1 or FBP2 confirmed that these probes were effective for the detection of an arterial thrombus in this rat model. A thrombus was visible on PET images as a region of high activity that corresponded to a region of arterial occlusion identified by simultaneous MR angiography. FBP1 and FBP2 represent promising new probes for the molecular imaging of thrombi.

Keys for unlocking photolabile metal-containing cages.

Photolabile metal-containing cages are metal complexes that undergo a change in coordination environment upon exposure to light of an appropriate wavelength. The light-responsive functionality can either be a component of the encapsulating ligand or a property of the metal complex itself. The altered coordination properties of light-responsive complexes can result in release of the coordinated metal ion into its surroundings, a differential reactivity of the metal center, or the liberation of a reactive molecule that had been passivated by binding to the metal center. These triggerable agents can be useful tools for manipulating the bioavailability of metals or their coordinating ligands in order to study biological pathways or for potential therapeutic purposes.

Development of next-generation photolabile copper cages with improved copper binding properties.

Seven new nitrogen-donor ligands that contain a photoactive nitrophenyl group within the ligand backbone have been prepared and evaluated for their binding affinity for copper(ii) and zinc(ii). Among this series, the ligand 3Gcage (pyridine-2-carboxylic acid {1-(2-nitro-phenyl)-3-[(pyridin-2-ylmethyl)-amino]-propyl}-amide) has the best affinity for copper(ii), with an apparent dissociation constant at pH 7.4 of 0.18 fM. Exposure of buffered aqueous solutions of 3Gcage or Cu(ii)-bound 3Gcage to UV light induces bond cleavage in the ligand backbone, which reduces the denticity of the ligands. The quantum yields of photolysis for 3Gcage in the absence and presence of Cu(ii) are 0.66 and 0.43, respectively. Prior to photolysis, the 3Gcage ligand inhibits copper from generating hydroxyl radicals in the presence of hydrogen peroxide and ascorbic acid; however, hydroxyl radical formation increases by more than 300% following light activation, showing that the reactivity of the copper center can be triggered by light.

Molecular MRI of Thrombosis.

This review focuses on recent approaches in using targeted MRI probes for noninvasive molecular imaging of thrombosis. Probe design strategies are discussed: choice of molecular target; nanoparticle versus small-molecule probe; and gadolinium versus iron oxide imaging reporter. Examples of these different design strategies are chosen from the recent literature. Novel contrast agents used to image direct and indirect binding to fibrin have been described as well as direct binding to activated platelets. Emphasis is placed on probes where utility has been demonstrated in animal models or in human clinical trials.

A photolabile ligand for light-activated release of caged copper.

A photosensitive caged copper complex has been prepared from a tetradentate ligand (H2cage) composed of two pyridyl-amide arms connected by a photoreactive nitrophenyl group. H2cage binds Cu2+ in aqueous solution with a stability constant (log beta) of 10.8, which corresponds to a KD of 16 pM at pH 7.4. The neutral Cu2+ complex, [Cu(OH2)(cage)], crystallizes as a distorted trigonal bipyramid coordinated by two amide and two pyridyl N atoms, with a water molecule bound in the trigonal plane. Photolysis with 350 nm UV light cleaves the ligand backbone to release photoproducts with significantly diminished affinity for Cu2+, thereby uncaging the metal ion. When coordinated as the caged complex, copper has diminished reactivity to produce hydroxyl radicals from Fenton-like reaction mixtures containing hydrogen peroxide and ascorbic acid. Postphotolysis, uncaged copper promotes hydroxyl radical formation under the same conditions. The strategy of caging copper is promising for applications where light could be used to trigger release of copper as a pro-oxidant to increase oxidative stress or as a tool to release copper intracellularly to study mechanisms of copper trafficking.