SF0166, a small-molecule αvβ3 antagonist, has physiochemical properties that allow distribution to the posterior segment of the eye after topical administration in an ophthalmic solution. The pharmacodynamics and ocular distribution of SF0166 were evaluated in several cell lines, chick chorioallantoic membrane assays, and models of ocular neovascularization in mice and pigmented rabbits. SF0166 inhibited cellular adhesion to vitronectin across human, rat, rabbit, and dog cell lines with IC50 values of 7.6 pM to 76 nM. SF0166 inhibited integrin–ligand interactions at IC50 values of 0.6–13 nM for human αvβ3, αvβ6, and αvβ8. SF0166 significantly decreased neovascularization in the oxygen-induced retinopathy mouse model. SF0166 distributed to the choroid and retina after topical ocular administration in amounts that substantially exceeded the cellular IC50 for adhesion to vitronectin; drug concentrations were maintained for >12 hours. In the laser-induced choroidal neovascularization model, topical ocular administration of SF0166 decreased lesion area compared with vehicle and was comparable to a bevacizumab injection. In the vascular endothelial growth factor–induced early neovascularization and vascular leakage model, topical ocular application of SF0166 resulted in a dose-dependent reduction in vascular leakage; the highest ocular doses tested showed comparable activity to a bevacizumab injection.

analysis, and TL and MIBI integrated defect magnitude (% left ventricle (LV)) calculated. Hearts were sliced (5 mm thick), and each slice divided into 8 segments (segs). Myocardial tracer activity and mSPH flow were determined by gamma well counting. lschemic (IS) segs wers defined by a coronary flow reserve (CFR) of 2.5. IS/N1 tracer and flow ratios were calculated. Results: In IS segs. FL (mllminlg) at rest was not reduced (IS: 0.62+0.04; NI: 0.61*0.04: p=ns), although CVT stress FL in IS segs was significantly reduced compared to NI (IS: 0.70*0.06; NI: 1.90+0.21; pcO.001). CVT stress IS/N1 FL ratio (0.38t0.02) was significantly reduced compared to rest (p~O.001). TL and MlBl uptake ratios both under estimated the FL deficit (TL: 0.54*0.02, p<O.OOl vs Stress FL; MIBI: 0.69*0.05, p<O.OOl vs Stress FL). The TL IS/N1 ratio provided a better index of the flow deficit than MIBI during CVT stress (p<O.OOl). The SPECT TL defects (22*3.1 %LV) were also significantly larger than MIBI (16.4+2.1 %LV, p<O.O5). Conclusions: CVT administered as a bolus created marked flow heterogsnerty. and significant TL and MIBI perfusion defects in the presence of a critical coronary stenosis. However, dunng CVT stress, TL tracked flow better than MIBI, and produced larger SPECT defects.

Objective— To establish and validate early noninvasive imaging of matrix metalloproteinase (MMP) activation for monitoring the progression of vascular remodeling and response to dietary modification. Methods and Results— Apolipoprotein E −/− mice that were fed a high-fat diet underwent left common carotid artery wire injury. One week after surgery, a group of animals were withdrawn from the high-fat diet. The other group of animals continued that diet throughout the study. Micro single-photon emission computed tomographic (microSPECT)/CT imaging with RP805 (a 99m Tc-labeled tracer targeting activated MMPs) was repeatedly performed at 2 and 4 weeks after surgery. Histological analysis at 4 weeks showed significant left carotid neointima formation, monocyte/macrophage infiltration, and upregulation of several MMPs, which were ameliorated by withdrawal from the high-fat diet. In vivo microSPECT/CT images visualized significant RP805 uptake, reflecting MMP activation, in the injured carotid arteries. MMP activation was reduced as early as 1 week after withdrawal from the high-fat diet and significantly correlated with neointimal area at 4 weeks after surgery. Conclusion— MMP activation predicts the progression of vascular remodeling and can track the effect of dietary modification after vascular injury.

Matrix metalloproteinases (MMPs) play a key role in the development of atherosclerosis and its complications. In vivo detection and quantification of MMP activation can help track the propensity to complications and response to therapy. We sought to establish an in vivo imaging approach for monitoring MMP activation in atherosclerotic mouse aorta and use it to assess the response to dietary modification. Method: Apolipoprotein-deficient mice were fed normal chow or a high-fat diet (HFD) for up to 3 mo or a HFD for 2 mo, followed by 1 mo on normal chow. Then they underwent micro-SPECT/CT, along with autoradiography and oil red O staining of tissues. Results: After 3 mo of HFD, there was considerable atherosclerosis in the aorta. In vivo micro-SPECT/CT using RP782 (an 111In-labeled tracer targeting activated MMPs) showed a heterogeneous pattern of tracer uptake along the aorta. Heterogeneity of RP782 uptake was confirmed by autoradiography, and specificity was demonstrated using excess unlabeled precursor. Tracer uptake quantified by micro-SPECT significantly correlated with uptake quantified by autoradiography. Comparison of oil red O staining with autoradiography demonstrated areas of discordance between plaque presence and tracer uptake. HFD withdrawal led to significant reduction in RP782 uptake beyond the effect on plaque area. MMP expression and macrophage infiltration were similarly heterogeneous along the aorta and significantly reduced after withdrawal from the HFD. Finally, RP782 uptake significantly correlated with aortic macrophage content. Conclusion: Molecular imaging of MMP activation reveals the heterogeneity of atherosclerotic plaques and is a useful tool for tracking plaque biology and response to therapy.

OBJECTIVES:Technetium-99m-labeled matrix metalloproteinase inhibitor (MPI) was used for the noninvasive assessment of matrix metalloproteinase (MMP) activity in atherosclerotic plaques after minocycline (MC) intervention. BACKGROUND:MMP activity in atherosclerosis contributes to plaque instability. Some antimicrobial agents may attenuate MMP activity. METHODS:Atherosclerotic lesions were produced in 38 rabbits with a high cholesterol diet for 4 months; 5 groups of rabbits, in the fourth month, received fluvastatin (FS) (n = 6), low-dose MC (n = 7), high-dose MC (n = 7), a combination of low-dose MC and FS (n = 6), or no intervention (n = 12); 8 unmanipulated rabbits were used as disease controls. Micro-single-photon emission computed tomography imaging was performed in all animals after intravenous MPI administration, followed by pathologic characterization of the aorta. A cell culture study evaluated the effect of MC on MMP production by activated human monocytes. RESULTS:MPI uptake was visualized best in untreated atherosclerotic animals (percent injected dose per gram MPI uptake, 0.11 +/- 0.04%). MPI uptake was reduced in the FS (0.06 +/- 0.01%; p < 0.0001), high-dose MC (0.05 +/- 0.01%; p < 0.0001), and MC-FS (0.05 +/- 0.005%; p < 0.0001) groups. Low-dose MC did not resolve MPI uptake significantly (0.08 +/- 0.02; p = 0.167). There was no incremental benefit of the combination of MC and FS. MPI uptake showed a significant correlation with plaque MMP-2, and MMP-9 activity. MMP-9 release from tumor necrosis factor-alpha-activated macrophages was abrogated by incubation with MC. CONCLUSIONS:Molecular imaging of MMP activity in atherosclerotic plaque allows for the study of the efficacy of therapeutic interventions. MC administration resulted in substantial reduction in plaque MMP activity and histologically verified plaque stabilization. MC was found to be equally effective as FS.

Matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques and play an important role in plaque instability. Methods: Using Tc-99m-labeled broad-spectrum MMP inhibitor (MPI), we performed noninvasive imaging of MMP expression with micro-SPECT/micro-CT in mice deficient in apolipoprotein E (ApoE(-/-), n = 14), mice deficient in low-density-lipoprotein receptor (LDLR-/-, n = 14), and C57/BL6 mice as controls (n = 7). Seven ApoE(-/-) and 7 LDLR-/- received a high-cholesterol diet. After in vivo imaging, aortas were explanted, ex vivo images acquired, and the percent injected dose of MPI per gram (%ID/g) determined, followed by histologic characterization of atherosclerotic lesions. Results: MPI uptake was noninvasively visualized in atherosclerotic lesions by micro-SPECT, with confirmation by micro-CT of anatomic location and aortic calcification. %ID/g in each part of the aorta was highest in ApoE(-/-) that were fed a high-cholesterol diet, followed by LDLR-/- that were fed a high-cholesterol diet, ApoE(-/-) that were fed normal chow, and LDLR-/- that were fed normal chow. The control mice had minimal MPI uptake. A significant correlation was noted between %ID/g and % area positive for macrophages (r = 0.81, P = 0.009), MMP-2 (r = 0.65, P = 0.013), and MMP-9 (r = 0.62, P = 0.008). Conclusion: This study demonstrates the usefulness of molecular imaging for noninvasive assessment of the extent of MMP expression in various transgenic mouse models of atherosclerosis receiving a normal or hyperlipidemic diet. It is conceivable that such a strategy may be translationally developed for identification of unstable atherosclerotic plaques.

Background— Matrix metalloproteinase (MMP) activation plays a key role in vascular remodeling. RP782 is a novel indium 111 In–labeled tracer with specificity for activated MMPs. We hypothesized that RP782 can detect injury-induced vascular remodeling in vivo. Methods and Results— Left common carotid artery injury was induced with a guidewire in apolipoprotein E −/− mice. Sham surgery was performed on the contralateral artery, which served as control for imaging experiments. Carotid wire injury led to significant hyperplasia and expansive remodeling over a period of 4 weeks. MMP activity, detected by in situ zymography, increased in response to injury and was maximal by 3 to 4 weeks after injury. RP782 (11.1 MBq) was injected intravenously into apolipoprotein E −/− mice at 1, 2, 3, and 4 weeks after left carotid injury. MicroSPECT imaging was performed at 2 hours and was followed by CT angiography to localize the carotid arteries. In vivo images revealed focal uptake of RP782 in the injured carotid artery at 2, 3, and 4 weeks. Increased tracer uptake in the injured artery was confirmed by quantitative autoradiography. Pretreatment with 50-fold excess nonlabeled tracer significantly reduced RP782 uptake in injured carotids, thus demonstrating uptake specificity. Weekly changes in the vessel-wall area closely paralleled and correlated with RP782 uptake (Spearman r =0.95, P =0.001). Conclusions— Injury-induced MMP activation in the vessel wall can be detected by RP782 microSPECT/CT imaging in vivo. RP782 uptake tracks the hyperplastic process in vascular remodeling and provides an opportunity to track the remodeling process in vivo.

Objectives This study sought to evaluate the feasibility of noninvasive detection of matrix metalloproteinase (MMP) activity in experimental atherosclerosis using technetium-99m-labeled broad matrix metalloproteinase inhibitor (MPI) and to determine the effect of dietary modification and statin treatment on MMP activity.Background The MMP activity in atherosclerotic lesions contributes to the vulnerability of atherosclerotic plaques to rupture.Methods Atherosclerosis was produced in 34 New Zealand White rabbits by balloon de-endotheliazation of the abdominal aorta and a high-cholesterol diet. In addition, 12 unmanipulated rabbits were used as controls and 3 for blood clearance characteristics. In vivo micro-single-photon emission computed tomography (SPECT) imaging was performed after radiolabeled MPI administration. Subsequently, aortas were explanted to quantitatively measure percent injected dose per gram (%ID/g) MPI uptake. Histological and immunohistochemical characterization was performed and the extent of MMP activity was determined by gel zymography or enzyme-linked immunosorbent assays.Results The MPI uptake in atherosclerotic lesions (n=18) was clearly visualized by micro-SPECT imaging; MPI uptake was markedly reduced by administration of unlabeled MPI before the radiotracer (n=4). The MPI uptake was also significantly reduced after diet withdrawal (n=6) and fluvastatin treatment (n=6); no uptake was observed in normal control rabbits ( n=12). The %ID/g MPI uptake (0.10 +/- 0.03%) in the atherosclerotic lesions was significantly higher than the uptake in control aorta (0.016 +/- 0.004%, p < 0.0001). Uptake in fluvastatin (0.056 +/- 0.011%, p < 0.0005) and diet withdrawal groups (0.043 +/- 0.011%, p < 0.0001) was lower than the untreated group. The MPI uptake correlated with immunohistochemically verified macrophage infiltration (r = 0.643, p < 0.0001), and MMP-2 (r = 0.542, p < 0.0001) or MMP-9 (r = 0.578, p < 0.0001) expression in plaques.Conclusions The present data show the feasibility of noninvasive detection of MMP activity in atherosclerotic plaques, and confirm that dietary modification and statin therapy reduce MMP activity. (J Am Coll Cardiol 2008; 52: 1847-57) (c) 2008 by the American College of Cardiology Foundation

We evaluated the correlation between matrix metalloproteinase (MMP) and apoptotic activities in atherosclerosis lesions, and the impact of lipid lowering by dietary modification and statin therapy employing molecular imaging with Tc-99m broad MMP inhibitor (MPI) and indium-111 annexin A5 (AA5). Atherosclerosis was produced in 13 New Zealand White rabbits by balloon deendothelialization and hypercholesterolemic diet for 4 months; 6 unmanipulated rabbits on normal chow were used as controls. In the last month, 3 of the 13 atherosclerotic rabbits were changed to normal chow, and 4 received fluvastatin (1mg/kg) once a day. MPI and AA5 imaging were performed using micro-SPECT/micro-CT. After in vivo imaging, aortas were explanted to acquire ex vivo images and calculate percent injected dose per gram (%ID/g) uptake. Histological and immunohistochemical characterization and extent of MMP and apoptosis were evaluated in representative aortic samples. Both MPI and Annexin uptake were clearly visualized in the atherosclerotic lesions by noninvasive imaging; No uptake were observed after diet withdrawal, fluvastatin, and in normal control groups. %ID/g MPI and AA5 uptakes in the atherosclerotic lesions (0.09 ± 0.02%, 0.03 ± 0.01%) were significantly higher than the uptake in control abdominal aorta (0.014 ± 0.004%, 0.0007 ± 0.0002%; p<0.0001). The quantitative uptake in fluvastatin (0.053 ± 0.013%, 0.02 ± 0.01%; p<0.05) and diet withdrawal (0.047 ± 0.005%, 0.02 ± 0.004%; p<0.05) groups were statistically significantly lower compared to diet uninterrupted group. There were significant direct relationship betweem MPI and AA5 uptake in all experimental group (r=0.62, p<0.0001). The decrease in the tracer uptake maintained the relationship in the fluvastatin group (r=0.62, p<0.0001) but became less apparent in the diet withdrawal (r=0.24, p<0.05). Histopathological and immunohisto-chemical studies revealed a significant correlation of both MPI and Annexin uptake with macrophage infiltration, MMP-2, 9 expression and the extent of apoptosis. Dual imaging with MPI and AA5 showed good correlation between apoptosis and MMP expression except diet withdrawal group.

The integrin receptor alphavbeta3 is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. A radiolabeled alphavbeta3 antagonists belonging to the quinolin-4-one class of peptidomimetics (TA138) was previously shown to exhibit high affinity for integrin alphavbeta3 and high selectivity versus other integrin receptors. 111In-TA138 exhibited high tumor uptake in the c-neu Oncomouse mammary adenocarcinoma model and produced excellent scintigraphic images. This study describes the synthesis of eight divalent versions of TA138 and their evaluation as potential tumor radiotherapeutic agents. The two main variables in this study were the length of the spacer bridging the biotargeting moieties and the total negative charge of the molecules imparted by the cysteic acid pharmacokinetic modifiers. Receptor affinity was evaluated in a panel of integrin receptor affinity assays, and biodistribution studies using the 111In-labeled derivatives were carried out in the c-neu Oncomouse model. All divalent agents maintained the high receptor affinity and selectivity of TA138, and six of the eight 111In derivatives exhibited blood clearance that was faster than 111In-TA138 at 24 h postinjection (PI). All divalent agents exhibited tumor uptake and retention at 24 h PI that was higher than 111In-TA138. Tumor/organ ratios were improved for most of the divalent agents at 24 h PI in critical nontarget organs marrow, kidney, and liver, with the agents having intermediate-length spacers (29-43 A) showing the largest improvement. As an example, 111In-15 showed tumor uptake of 14.3% ID/g at 24 h PI and tumor/organ ratios as follows: marrow, 3.24; kidney, 7.29; liver, 8.51. A comparison of therapeutic indices for 90Y-TA138 and 177Lu-15 indicate an improved therapeutic index for the divalent agent. The implications for radiotherapeutic applications and the mechanism of this multivalent effect are discussed.