Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability.

INTRODUCTION: Atherosclerotic plaque rupture is the primary cause for myocardial infarction and stroke. During plaque progression macrophages and mast cells secrete matrix-degrading proteolytic enzymes, such as matrix metalloproteinases (MMPs). We studied levels of MMPs and tissue inhibitor of metalloproteinases-3 (TIMP-3) in relation to the characteristics of carotid plaques. We evaluated in vitro two radiolabeled probes targeting active MMPs towards non-invasive imaging of rupture-prone plaques. METHODS: Human carotid plaques obtained from endarterectomy were classified into stable and vulnerable by visual and histological analysis. MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-14, TIMP-3, and CD68 levels were investigated by quantitative polymerase chain reaction. Immunohistochemistry was used to localize MMP-2 and MMP-9 with respect to CD68-expressing macrophages. Western blotting was applied to detect their active forms. A fluorine-18-labeled MMP-2/MMP-9 inhibitor and a tritiated selective MMP-9 inhibitor were evaluated by in vitro autoradiography as potential lead structures for non-invasive imaging. RESULTS: Gene expression levels of all MMPs and CD68 were elevated in plaques. MMP-1, MMP-9, MMP-12 and MMP-14 were significantly higher in vulnerable than stable plaques. TIMP-3 expression was highest in stable and low in vulnerable plaques. Immunohistochemistry revealed intensive staining of MMP-9 in vulnerable plaques. Western blotting confirmed presence of the active form in plaque lysates. In vitro autoradiography showed binding of both inhibitors to stable and vulnerable plaques. CONCLUSIONS: MMPs differed in their expression patterns among plaque phenotypes, providing possible imaging targets. The two tested MMP-2/MMP-9 and MMP-9 inhibitors may be useful to detect atherosclerotic plaques, but not the vulnerable lesions selectively.

Design, synthesis, and initial evaluation of a high affinity positron emission tomography probe for imaging matrix metalloproteinases 2 and 9.

The activity of matrix metalloproteinases (MMPs) is elevated locally under many pathological conditions. Gelatinases MMP2 and MMP9 are of particular interest because of their implication in angiogenesis, cancer cell proliferation and metastasis, and atherosclerotic plaque rupture. The aim of this study was to identify and develop a selective gelatinase inhibitor for imaging active MMP2/MMP9 in vivo. We synthesized a series of N-sulfonylamino acid derivatives with low to high nanomolar inhibitory potencies. (R)-2-(4-(4-Fluorobenzamido)phenylsulfonamido)-3-(1H-indol-3-yl)propanoic acid (7) exhibited the best in vitro binding properties: MMP2 IC50 = 1.8 nM, MMP9 IC50 = 7.2 nM. Radiolabeling of 7 with no carrier added (18)F-radioisotope was accomplished starting from iodonium salts as precursors. The radiochemical yield strongly depended on the iodonium counteranion (ClO4(-) > Br(-) > TFA(-) > tosylate). (18)F-7 was obtained in up to 20% radiochemical yield (decay corrected), high radiochemical purity, and >90 GBq/µmol specific radioactivity. The radiolabeled compound showed excellent stability in vitro and in mice in vivo.

Biodistribution and stability studies of [18F]fluoroethylrhodamine B, a potential PET myocardial perfusion agent.

INTRODUCTION: Fluorine-18-labeled rhodamine B was developed as a potential positron emission tomography (PET) tracer for the evaluation of myocardial perfusion, but preliminary studies in mice showed no accumulation in the heart suggesting that it was rapidly hydrolyzed in vivo in mice. A study was therefore undertaken to further evaluate this hypothesis. METHODS: [(18)F]Fluoroethylrhodamine B was equilibrated for 2 h at 37 degrees C in human, rat and mouse serum and in phosphate-buffered saline. Samples were removed periodically and assayed by high-performance liquid chromatography. Based on the results of the stability study, microPET imaging and a biodistribution study were carried out in rats. RESULTS: In vitro stability studies demonstrated that [(18)F]fluoroethylrhodamine B much more stable in rat and human sera than in mouse serum. After 2 h, the compound was >80% intact in rat serum but <30% intact in mouse serum. The microPET imaging and biodistribution studies in rats confirmed this result showing high and persistent tracer accumulation in the myocardium compared with the absence of uptake by the myocardium in mice thereby validating our original hypothesis that (18)F-labeled rhodamines should accumulate in the heart. CONCLUSIONS: [(18)F]Fluoroethylrhodamine B is more stable in rat and human sera than it is in mouse serum. This improved stability is demonstrated by the high uptake of the tracer in the rat heart in comparison to the absence of visible uptake in the mouse heart. These observations suggest that (18)F-labeled rhodamines are promising candidates for more extensive evaluation as PET tracers for the evaluation of myocardial perfusion.

Synthesis of fluorine-18 labeled rhodamine B: A potential PET myocardial perfusion imaging agent.

There is considerable interest in developing an (18)F-labeled PET myocardial perfusion agent. Rhodamine dyes share several properties with (99m)Tc-MIBI, the most commonly used single-photon myocardial perfusion agent, suggesting that an (18)F-labeled rhodamine dye might prove useful for this application. In addition to being lipophilic cations, like (99m)Tc-MIBI, rhodamine dyes are known to accumulate in the myocardium and are substrates for Pgp, the protein implicated in MDR1 multidrug resistance. As the first step in determining whether (18)F-labeled rhodamines might be useful as myocardial perfusion agents for PET, our objective was to develop synthetic methods for preparing the (18)F-labeled compounds so that they could be evaluated in vivo. Rhodamine B was chosen as the prototype compound for development of the synthesis because the ethyl substituents on the amine moieties of rhodamine B protect them from side reactions, thus eliminating the need to include (and subsequently remove) protecting groups. The 2'-[(18)F]fluoroethyl ester of rhodamine B was synthesized by heating rhodamine B lactone with [(18)F]fluoroethyltosylate in acetonitrile at 165 degrees C for 30min using [(18)F]fluoroethyl tosylate, which was prepared by the reaction of ethyleneglycol ditosylate with Kryptofix 2.2.2, K(2)CO(3), and [(18)F]NaF in acetonitrile for 10min at 90 degrees C. The product was purified by semi-preparative HPLC to produce the 2'-[(18)F]fluoroethylester in >97% radiochemical purity with a specific activity of 1.3GBq/mumol, an isolated decay corrected yield of 35%, and a total synthesis time of 90min.

Novel rhenium chelate system derived from dimercaptosuccinic acid for the selective labeling of biomolecules.

This work is part of an effort to develop chelating agents for stable binding and easy conjugation of Re-188 to biologically interesting structures. Starting from the well-known in vivo stability of [(188)ReO(DMSA)(2)](-), we want to exploit this coordination system for the design of (188)ReO(V) chelates, which are stable toward reoxidation to perrhenate and toward ligand exchange under all conditions of radiopharmaceutical development. Therefore, a new type of tetradentate ligand has been synthesized by bridging two molecules of N,N'-diisobutyl-2,3-dimercaptosuccinamide with N-(3-aminopropyl)propane-1,3-diamine. The resulting stereoisomeric tetrathiolato S(4) ligand of composition ((i)()Bu)(2)N(O)C-C(SH)-C(SH)-C(O)NH-(CH(2))(3)-NH-(CH(2))(3)-NHC(O)-C(SH)-C(SH)-C(O)N((i)Bu)(2) forms anionic five-coordinate oxorhenium(V) complexes by a ligand-exchange reaction of NBu(4)[ReOCl(4)] in methanol. In the absence of a base, the compounds were isolated as "betaine", [ReO(S(4))], with the protonated nitrogen of the bridge serving as an internal "counterion". Two representatives have been fully characterized in both the solid and solution states and found to adopt the expected square-pyramidal coordination geometry. The equatorial plane is formed by four thiolate sulfur atoms, whereas the oxygen occupies the apical position. The orientation of the metal oxo group is exo in relation to the carbamido groups in both isomers. Both complexes are stereoisomeric regarding the junction of the triamine chain.