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A series of potent and selective β1-adrenoreceptor ligands were identified (IC50 range, 0.04-0.25 nM; β1/β2 selectivity range, 65-450-fold), labeled with the PET radioisotope fluorine-18 and evaluated in normal Sprague-Dawley rats. Tissue distribution studies demonstrated uptake of each radiotracers from the blood pool into the myocardium (0.48-0.62% ID/g), lung (0.63-0.97% ID/g), and liver (1.03-1.14% ID/g). Dynamic μPET imaging confirmed the in vivo dissection studies.

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.

Objective— Angiogenic expansion of the vasa vasorum is a well-known feature of progressive atherosclerosis, suggesting that antiangiogenic therapies may stabilize or regress plaques. α ν β 3 Integrin–targeted paramagnetic nanoparticles were prepared for noninvasive assessment of angiogenesis in early atherosclerosis, for site-specific delivery of antiangiogenic drug, and for quantitative follow-up of response. Methods and Results— Expression of α ν β 3 integrin by vasa vasorum was imaged at 1.5 T in cholesterol-fed rabbit aortas using integrin-targeted paramagnetic nanoparticles that incorporated fumagillin at 0 μg/kg or 30 μg/kg. Both formulations produced similar MRI signal enhancement (16.7%±1.1%) when integrated across all aortic slices from the renal arteries to the diaphragm. Seven days after this single treatment, integrin-targeted paramagnetic nanoparticles were readministered and showed decreased MRI enhancement among fumagillin-treated rabbits (2.9%±1.6%) but not in untreated rabbits (18.1%±2.1%). In a third group of rabbits, nontargeted fumagillin nanoparticles did not alter vascular α ν β 3 -integrin expression (12.4%±0.9%; P >0.05) versus the no-drug control. In a second study focused on microscopic changes, fewer microvessels in the fumagillin-treated rabbit aorta were counted compared with control rabbits. Conclusions— This study illustrates the potential of combined molecular imaging and drug delivery with targeted nanoparticles to noninvasively define atherosclerotic burden, to deliver effective targeted drug at a fraction of previous levels, and to quantify local response to treatment.

The integrin receptor alpha(v)beta(3) is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. Radiolabeled alpha(v)beta(3) antagonists have demonstrated potential application as tumor imaging agents and as radiotherapeutic agents. This report describes the total synthesis of eight new HYNIC and DOTA conjugates of receptor alpha(v)beta(3) antagonists belonging to the quinolin-4-one class of peptidomimetics, and their radiolabeling with (99m)Tc (for HYNIC) and (111)In (for DOTA). Tethering of the radionuclide-chelator complexes was achieved at two different sites on the quinolin-4-one molecule. All such derivatives maintained high affinity for receptor alpha(v)beta(3) and high selectivity versus receptors alpha(IIb)beta(3), alpha(v)beta(5), alpha(5)beta(1). Biodistribution of the radiolabeled compounds was evaluated in the c-neu Oncomouse mammary adenocarcinoma model. DOTA conjugate (111)In-TA138 presented the best biodistribution profile. Tumor uptake at 2 h postinjection was 9.39% of injected dose/g of tissue (%ID/g). Activity levels in selected organs was as follows: blood, 0.54% ID/g; liver, 1.94% ID/g; kidney, 2.33% ID/g; lung, 2.74% ID/g; bone, 1.56% ID/g. A complete biodistribution analysis of (111)In-TA138 and the other radiolabeled compounds of this study are presented and discussed. A scintigraphic imaging study with (111)In-TA138 showed a clear delineation of the tumors and rapid clearance of activity from nontarget tissues.

In this study, porcine carotid arteries were subjected to balloon overstretch injury followed by local delivery of paramagnetic nanoparticles targeted to alphavbeta3-integrin expressed by smooth muscle cells or collagen III within the extracellular matrix. Carotid T1-weighted angiography and vascular imaging was performed at 1.5T. While MR angiograms were indistinguishable between control and targeted vessel segments, alphavbeta3-integrin-and collagen Ill-targeted nanoparticles spatially delineated patterns and volumes of stretch injury. In conclusion, MR molecular imaging with alphavbeta3-integrin or collagen Ill-targeted nanoparticles enables the non-invasive, three-dimensional characterization of arterial pathology unanticipated from routine angiography.

Neovascularization is a critical component in the progression of malignant melanoma. The objective of this study was to determine whether alpha(nu)beta(3)-targeted paramagnetic nanoparticles can detect and characterize sparse alpha(nu)beta integrin expression on neovasculature induced by nascent melanoma xenografts ( approximately 30 mm(3)) at 1.5T. Athymic nude mice bearing human melanoma tumors were intravenously injected with alpha(v)beta(3)-integrin-targeted paramagnetic nanoparticles, nontargeted paramagnetic nanoparticles, or alpha(v)beta(3)-targeted-nonparamagnetic nanoparticles 2 hr before they were injected with alpha(v)beta(3)-integrin-targeted paramagnetic nanoparticles (i.e., in vivo competitive blockade) and imaged with MRI. Contrast enhancement of neovascularity in animals that received alpha(nu)beta(3)-targeted paramagnetic nanoparticles increased 173% by 120 min. Signal contrast with nontargeted paramagnetic nanoparticles was approximately 50% less than that in the targeted group (P < 0.05). Molecular MRI results were corroborated by histology. In a competitive cell adhesion assay, incubation of alpha(nu)beta(3)-expressing cells with targeted nanoparticles significantly inhibited binding to a vitronectin-coated surface, confirming the bioactivity of the targeted nanoparticles. The present study lowers the limit previously reported for detecting sparse biomarkers with molecular MRI in vivo. This technique may be employed to noninvasively detect very small regions of angiogenesis associated with nascent melanoma tumors, and to phenotype and stage early melanoma in a clinical setting.

Background— The α v β 3 integrin plays a critical role in cell proliferation and migration. We hypothesized that vascular cell proliferation, a hallmark of injury-induced remodeling, can be tracked by targeting α v β 3 integrin expression in vivo. Methods and Results— RP748, a novel 111 In-labeled α v β 3 -specific radiotracer, was evaluated for its cell-binding characteristics and ability to track injury-induced vascular proliferation in vivo. Three groups of experiments were performed. In cultured endothelial cells (ECs), TA145, a cy3-labeled homologue of RP748, localized to α v β 3 at focal contacts. Activation of α v β 3 by Mn 2+ led to increased EC binding of TA145. Left common carotid artery wire injury in apolipoprotein E −/− mice led to vascular wall expansion over a period of 4 weeks. RP748 (7.4 MBq) was injected into groups of 9 mice at 1, 3, or 4 weeks after left carotid injury, and carotids were harvested for autoradiography. Relative autographic intensity, defined as counts/pixel of the injured left carotid area divided by counts/pixel of the uninjured right carotid area, was higher at 1 and 3 weeks (1.8±0.1 and 1.9±0.2, respectively) and decreased significantly by 4 weeks after injury (1.4±0.1, P v and β 3 integrin expression was maximal at 1 week and decreased by 4 weeks after injury. The proliferation index, as determined by Ki67 staining, followed a temporal pattern similar to that of RP748 uptake. Dynamic gamma imaging demonstrated rapid renal clearance of RP748. Conclusions— RP748 has preferential binding to activated α v β 3 integrin and can track the injury-induced vascular proliferative process in vivo.

90Y-TA138 is a 9 0 Y-labeled nonpeptide integrin α v β 3 receptor antagonist that binds with high affinity and specificity to integrin α v β 3 receptors overexpressed on both endothelial and tumor cells. 9 0 Y-TA138 has demonstrated significant therapeutic effects in several preclinical tumor-bearing animal models. Since 9 0 Y is a pure β-emitter, 1 1 1 In-TA138 has been chosen as the imaging surrogate for dosimetry determination of 9 0 Y-TA138. This report describes the synthesis of 1 1 1 In-TA138 and biological evaluations of both 1 1 1 In-TA138 and 9 0 Y-TA138 in the c-neu Oncomouse model. The HPLC data shows that 1 1 1 In-TA138 is more hydrophilic with the retention time ∼4.5 min shorter than that of 9 0 Y-TA138 under identical chromatographic conditions. Since the only difference between 1 1 1 In-TA138 and 9 0 Y-TA138 is the metal ion, the HPLC retention time difference strongly suggests that indium and yttrium chelates do not share the same coordination sphere in solution even though they are coordinated by the same DOTA conjugate. Despite their differences in lipophilicity and solution structure, biodistribution data in the c-neu Oncomouse model clearly showed that 1 1 1 In-TA138 and 9 0 Y-TA138 are biologically equivalent with respect to their uptake in tumors and other major organs. Therefore, 1 1 1 In-TA138 is useful as an imaging surrogate for 9 0 Y-TA138 and should be able to predict the radiation dosimetry of 9 0 Y-TA138, a therapeutic radiopharmaceutical for treatment of rapidly growing tumors.

The goal of this research is the development of tumor imaging and radiotherapeutic agents based on targeting of the integrin alpha(v)beta(3) (vitronectin receptor). Macrocyclic chelator DOTA has been conjugated to peptidomimetic vitronectin receptor antagonist SH066 to give TA138. TA138 and (89)Y-TA138 retain antagonist properties and high affinity for integrin alpha(v)beta(3) (IC(50) = 12 and 18 nM, respectively), and good selectivity versus integrin alpha(IIb)beta(3) (IC(50)10,000 nM). TA138 forms stable complexes with (111)In and (90)Y in95% RCP. (111)In-TA138 demonstrates high tumor uptake in the c-neu Oncomouse (Charles River Laboratories [Charles River, Canada]) mammary adenocarcinoma model (9.39% ID/g at 2 hours PI) and low background activity. Blood clearance is rapid and excretion is renal. Tumors are visible as early as 0.5 hours PI. Radiotherapy studies in the c-neu Oncomouse model demonstrated a slowing of tumor growth at a dose of 15 mCi/m(2), and a regression of tumors at a dose of 90 mCi/m(2).

This study describes the discovery and development of an anaerobic formulation for the routine preparation of (90)Y and (177)Lu complexes ((90)Y-TA138 and (177)Lu-TA138) of a DOTA-conjugated nonpeptide vitronectin receptor antagonist (TA138: 3-sulfon-N-[[4,7,10-tris(carboxymethyl)1,4,7,10-tetraazacyclododec-1-yl]acetyl]-l-alanyl-N-[2-[4-[[[(1S)-1-carboxy-2[[[1,4-dihydro-7-[(1H-imidazol-2-ylamino]meth-yl]-1-methyl-4-oxo-3-quinolinyl]carbonyl]amino]ethyl]amino]-sulfonyl]-3,5-dimethylphenoxy]-1-oxobutyl]amino]ethyl]-3-sulfo-l-alaninamide). Since (90)Y-TA138 and (177)Lu-TA138 are very sensitive to radiolytic degradation, exclusion of oxygen is necessary during the radiolabeling. Using the anaerobic formulation, (90)Y-TA138 and (177)Lu-TA138 can be prepared in high yield and high specific activity. The anaerobic formulation described in this study is particularly useful for (90)Y- and (177)Lu-labeling of DOTA-conjugated small biomolecules, which are sensitive to the radiolytic degradation during radiolabeling.

A cyclic peptide GP IIb/IIIa receptor antagonist containing the N-Me-Arg-Gly-Asp motif has been derivatized with the technetium chelating hydrazinonicotinyl group (Hynic). The Hynic derivative, and the Tc-99 diazenido complex, retain the high receptor affinity of the parent peptide. The Tc-99m complex shows high thrombus uptake, and rapid clearance of background, producing excellent images in under 1 h.

Tc-99m labeled derivatives of N-Me-Arg-Gly-Asp containing cyclic peptide GP IIb/IIIa receptor antagonists are potential radiopharmaceuticals for the diagnosis of thrombosis. The design and synthesis of these peptides are described. These compounds are incorporated in rapidly growing thrombi under both arterial and venous conditions in canine models. Thrombi are clearly visible in images acquired at 50 min.

Indazole-based vitronectin receptor antagonists have been conjugated to chelators DOTA and Hynic using a variety of tethers and pharmacokinetic modifying groups. All modifications resulted in potent αvβ3 antagonists in an Elisa assay. The chelator conjugates were radiolabeled with 111In (DOTA) and 99mTc (Hynic) in >90% RCP. Biodistribution studies in the c-Neu Oncomouse tumor model have shown high tumor uptake and primarily renal excretion for these agents. The potential use of these agents in cancer diagnosis will be discussed.

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.

Studies have demonstrated that the bivalent (111)In-labeled leukotriene B4 (LTB4) antagonist DPC11870 reveals infectious and inflammatory lesions in various rabbit models. The radioactive tracer accumulates quickly at the site of infection and clears rapidly from the circulation, resulting in high-quality images. In this study, 2 new hydrazinonicotinamide (HYNIC)-conjugated compounds that are structurally related to DPC11870 were studied to further improve image quality.A bivalent HYNIC-conjugated LTB4 antagonist (MB81) and a monovalent one (MB88) were labeled with (99m)Tc. The radiolabeled compounds were intravenously injected into New Zealand White rabbits with E. coli infection in the left thigh muscle. The imaging characteristics of both compounds were compared with those of the bivalent (111)In-labeled LTB4 antagonist.Both (99m)Tc-labeled LTB4 antagonists revealed the abscess from 2 h after injection onward. Abscess uptake at 8 h after injection was similar for both compounds (0.22 +/- 0.08 percentage injected dose per gram [%ID/g] and 0.36 +/- 0.13%ID/g for the bivalent and monovalent compounds, respectively). However, visualization of the abscess and the quality of the images were better after injection of MB88 than after injection of either of the bivalent LTB4 antagonists. The excellent delineation of the abscess by MB88 was mainly due to the more rapid clearance of this compound from nontarget organs.The (99m)Tc-labeled HYNIC conjugated LTB4 antagonists MB88 and MB81 revealed infectious foci in rabbits within a few hours after injection. Imaging characteristics of monovalent (99m)Tc-MB88 were superior to those of the bivalent LTB4 antagonists DPC11870 and MB81. Therefore, of the 3 LTB4 antagonists, the monovalent LTB4 antagonist MB88 is the most potent and promising agent for visualizing and evaluating infection and inflammation in patients.

Graft arteriopathy (GA), characterized by diffuse concentric narrowing of coronary arteries, is the major cause of late graft failure in cardiac transplantation. alphavbeta3 Integrin is up-regulated in proliferating vascular cells and may constitute an appropriate target for imaging GA. We used a human/mouse chimeric model of GA, in which segments of human coronary artery were transplanted to severe combined immunodeficiency mice, followed by reconstitution with allogeneic human peripheral blood mononuclear cells (PBMC). This led to vascular remodeling characterized by neointima formation over a period of 4 wk. alphavbeta3 expression in the graft was minimal in animals without PBMC, considerably increased by 2 wk, and decreased toward baseline by 4 wk after PBMC reconstitution. Cell proliferation was maximal at 2 wk, correlating with peak alphavbeta3 expression. RP748, an 111In-labeled alphavbeta3 (active conformation)-targeted radiotracer was injected into groups of 5 recipients at 0, 2, and 4 wk after PBMC reconstitution. Relative uptakes, defined as autoradiographic intensity in the graft/native aortas closely tracked the proliferative process. Specificity of uptake was demonstrated using excess nonlabeled tracer. In conclusion, alphavbeta3 integrin is transiently up-regulated (and activated) in GA and may be targeted by RP748 for detection of the proliferative process in early GA.

Radiolabeled leukotriene B4 (LTB4) antagonist DPC11870 is able to reveal infectious and inflammatory foci in distinct animal models. Because previous studies showed that accumulation of (111)In-DPC11870 in the abscess continued although the tracer had cleared from the circulation, we decided to investigate the pharmacodynamics of (111)In-DPC11870 and determine the mechanism of accumulation of the radiolabeled LTB4 antagonist in the abscess.(111)In-DPC11870 was intravenously injected in healthy New Zealand White rabbits and rabbits with intramuscular Escherichia coli infection. Pharmacodynamics were studied by serial imaging and by ex vivo counting of dissected tissues. The mechanism of visualization of the abscess was investigated in rabbits with intramuscular infection that was induced 16 h after intravenous administration of (111)In-DPC11870. In addition, heterologous leukocytes and bone marrow cells of a donor rabbit were labeled with (111)In-DPC11870 in vitro and the biodistribution of these in vitro radiolabeled cells was compared with that of (111)In-DPC11870 in rabbits with an infection.The LTB4 antagonist (111)In-DPC11870 revealed the intramuscular abscess in rabbits only a few hours after injection. Quantitative analysis of the images confirmed accumulation of (111)In-DPC11870 in the abscess although the compound had cleared almost completely from the circulation. Radioactivity concentration in the bone marrow decreased more rapidly in infected animals than in healthy animals. Therefore, we hypothesized that (111)In-DPC11870 associates with receptor-positive (bone marrow) cells and accumulated in the abscess because of subsequent migration from the bone marrow to the abscess. Accumulation of radioactivity in the abscess induced 16 h after (111)In-DPC11870 injection was similar to that in animals intravenously injected with the tracer 24 h after induction of the abscess (0.37 +/- 0.16 percentage injected dose [%ID]/g). Moreover, differences in radioactivity concentration in the bone marrow of healthy and infected animals (0.67 +/- 0.29 %ID/g and 0.15 +/- 0.03 %ID/g at 24 h, respectively, after injection) supported our hypothesis. Additional studies with peripheral blood leukocytes and bone marrow cells that were labeled ex vivo with (111)In-DPC11870 showed the ability of these cells to migrate to the abscess (0.40 %ID/g and 0.52 %ID/g for (111)In-DPC11870 bone marrow cells and (111)In-DPC11870 peripheral blood leukocytes, respectively, 24 h after injection).The (111)In-labeled LTB4 antagonist DPC11870 accumulates in infectious and inflammatory foci because of binding to LTB4 receptors expressed on activated hematopoietic cells that subsequently migrate to the site of infection, which leads to visualization of the infectious lesions.

Neovascularization is a critical component in the progression of malignant melanoma. The objective of this study was to determine whether alpha(nu)beta(3)-targeted paramagnetic nanoparticles can detect and characterize sparse alpha(nu)beta integrin expression on neovasculature induced by nascent melanoma xenografts ( approximately 30 mm(3)) at 1.5T. Athymic nude mice bearing human melanoma tumors were intravenously injected with alpha(v)beta(3)-integrin-targeted paramagnetic nanoparticles, nontargeted paramagnetic nanoparticles, or alpha(v)beta(3)-targeted-nonparamagnetic nanoparticles 2 hr before they were injected with alpha(v)beta(3)-integrin-targeted paramagnetic nanoparticles (i.e., in vivo competitive blockade) and imaged with MRI. Contrast enhancement of neovascularity in animals that received alpha(nu)beta(3)-targeted paramagnetic nanoparticles increased 173% by 120 min. Signal contrast with nontargeted paramagnetic nanoparticles was approximately 50% less than that in the targeted group (P0.05). Molecular MRI results were corroborated by histology. In a competitive cell adhesion assay, incubation of alpha(nu)beta(3)-expressing cells with targeted nanoparticles significantly inhibited binding to a vitronectin-coated surface, confirming the bioactivity of the targeted nanoparticles. The present study lowers the limit previously reported for detecting sparse biomarkers with molecular MRI in vivo. This technique may be employed to noninvasively detect very small regions of angiogenesis associated with nascent melanoma tumors, and to phenotype and stage early melanoma in a clinical setting.

Contains fulltext : 48443.pdf (Publisher’s version ) (Closed access) In previous studies we demonstrated that lipophilic (99m)Tc-labeled LTB4 antagonist 1 (RP517) accumulated in infectious foci in rabbits, but hepatobiliary clearance hampered imaging of abdominal lesions. We now report the use of cysteic acid as a pharmacokinetic modifier to improve the water solubility and renal clearance of three hydrophilic analogues of 1. Divalent LTB4 antagonist 17 (DPC11870-11) is a DTPA conjugate for radiolabeling with In-111. Monovalent LTB4 antagonists 15 (BMS57868-88) and divalent LTB4 antagonist 18 (BMS57868-81) are conjugated to bifunctional chelator HYNIC for radiolabeling with (99m)Tc. The three compounds labeled efficiently with 111In or (99m)Tc with high radiochemical purity and specific activities. Scintigraphic images obtained in New Zealand White rabbits having acute intramuscular E. coli infection demonstrated that all agents were able to clearly visualize the abscess, and clearance was exclusively renal. The biodistribution of the (99m)Tc-labeled LTB4 antagonists was affected by the coligands used with the HYNIC chelator and by the monovalent or divalent nature of the receptor binding moiety. The best scintigraphic images were obtained with monovalent HYNIC conjugate 15 using tricine and isonicotinic acid as coligands with HYNIC for coordination with (99m)Tc.

Radiolabeled chemotactic peptides have been studied for their applicability to the visualization of infectious and inflammatory foci. Because a radiolabeled leukotriene B4 (LTB4) antagonist allowed visualization of intramuscular E. coli abscesses in rabbits within a few hours after injection, we decided to test the imaging characteristics of this agent in a more clinically relevant model of pulmonary aspergillosis. The pharmacokinetics and imaging characteristics of the 111In-labeled LTB4 antagonist DPC11870 were studied in New Zealand White rabbits with experimental pulmonary aspergillosis infection. The imaging characteristics of 111In-DPC11870 were compared with those of 67Ga-citrate, a radiopharmaceutical commonly used to detect pulmonary infections in patients.Pulmonary aspergillosis was induced in the left lung of rabbits by intratracheal inoculation of 1 x 10(8) conidia of Aspergillus fumigatus. Three days after the inoculation, the rabbits received 111In-DPC11870 or 67Ga-citrate intravenously. Images were acquired at several time points up to 24 h after injection.Pulmonary aspergillosis was visualized with both agents. Images acquired after injection of 111In-DPC11870 showed uptake in the pulmonary lesions from 6 h after injection. Because of accumulation at the site of infection and clearance from the background, the images improved with time. Region-of-interest analysis at 24 h after injection revealed infected lung-to-normal lung ratios of 5.0 +/- 1.5 for 111In-DPC11870 and 2.9 +/- 0.6 for 67Ga-citrate.The radiolabeled LTB4 antagonist DPC11870 clearly delineated experimentally induced pulmonary aspergillosis in rabbits. Images acquired at 24 h after injection of 111In-DPC11870 were superior to those obtained after injection of 67Ga-citrate.

The use of radiolabeled leukocytes is considered the gold standard for scintigraphic imaging of inflammatory bowel disease. The disadvantages of (99m)Tc-hexamethylpropyleneamine oxime (HMPAO)-leukocytes, however, encourage the search for new imaging agents with at least similar diagnostic accuracy but without the laborious preparation and subsequent risk of contamination. In this study we investigated the imaging characteristics of a new imaging agent that specifically binds to the leukotriene B(4) (LTB(4)) receptors expressed on neutrophils. Imaging characteristics of the (111)In-labeled LTB(4) antagonist (DPC11870) were compared with those of (18)F-FDG and (99m)Tc-HMPAO-granulocytes in a rabbit model of experimental colitis.Acute colitis was induced in New Zealand White (NZW) rabbits by infusion of trinitrobenzene sulfonic acid in the descending colon. Forty-eight hours after induction of colitis, all animals were injected intravenously with (99m)Tc-granulocytes, (18)F-FDG, or (111)In-DPC11870. The pharmacokinetics and biodistribution were studied by serial scintigraphic imaging and by ex vivo counting of dissected tissues.All 3 radiopharmaceuticals showed the inflamed colon as early as 1 h after injection. However, compared with (99m)Tc-granulocytes, both (111)In-DPC11870 and (18)F-FDG were superior in revealing the inflamed lesions. The biodistribution data showed that uptake of (111)In-DPC11870 in the inflamed colon was highest (0.72 +/- 0.18 percentage injected dose per gram [%ID/g]), followed by uptake of (99m)Tc-granulocytes (0.40 +/- 0.11 %ID/g) and of (18)F-FDG (0.16 +/- 0.04 %ID/g). Because of low activity concentrations in the noninflamed colon, the radiolabeled LTB(4) antagonist also revealed the highest ratio of affected colon to unaffected colon (11.6 for (111)In-DPC11870, 5.5 for (99m)Tc-granulocytes, and 4.1 for (18)F-FDG).The radiolabeled LTB(4) antagonist DPC11870 clearly delineated acute colitis lesions in NZW rabbits within 1 h after injection. Because of high uptake in the inflamed lesions and a low activity concentration in the noninflamed colon, images acquired with (111)In-DPC11870 were better than those acquired with (99m)Tc-granulocytes or (18)F-FDG.

Early noninvasive detection and characterization of solid tumors and their supporting neovasculature is a fundamental prerequisite for effective therapeutic intervention, particularly antiangiogenic treatment regimens. Emerging molecular imaging techniques now allow recognition of early biochemical, physiological, and anatomical changes before manifestation of gross pathological changes. Although new tumor, vascular, extracellular matrix, and lymphatic biomarkers continue to be discovered, the alpha(nu)beta(3)-integrin remains an attractive biochemical epitope that is highly expressed on activated neovascular endothelial cells and essentially absent on mature quiescent cells. In this study, we report the first in vivo use of a magnetic resonance (MR) molecular imaging nanoparticle to sensitively detect and spatially characterize neovascularity induced by implantation of the rabbit Vx-2 tumor using a common clinical field strength (1.5T). New Zealand White rabbits (2 kg) 12 days after implantation of fresh Vx-2 tumors (2 x 2 x 2 mm(3)) were randomized into one of three treatment groups: (a) alpha(nu)beta(3)-targeted, paramagnetic formulation; (b) nontargeted, paramagnetic formulation; and (c) alpha(nu)beta(3)-targeted nonparamagnetic nanoparticles followed by (2 h) the alpha(nu)beta(3)-targeted, paramagnetic formulation to competitively block magnetic resonance imaging (MRI) signal enhancement. After i.v. systemic injection (0.5 ml of nanoparticles/kg), dynamic T(1)-weighted MRI was used to spatially and temporally determine nanoparticle deposition in the tumor and adjacent tissues, including skeletal muscle. At 2-h postinjection, alpha(nu)beta(3)-targeted paramagnetic nanoparticles increased MRI signal by 126% in asymmetrically distributed regions primarily in the periphery of the tumor. Similar increases in MR contrast were also observed within the walls of some vessels proximate to the tumor. Despite their relatively large size, nanoparticles penetrated into the leaky tumor neovasculature but did not appreciably migrate into the interstitium, leading to a 56% increase in MR signal at 2 h. Pretargeting of the alpha(nu)beta(3)-integrin with nonparamagnetic nanoparticles competitively blocked the specific binding of alpha(nu)beta(3)-targeted paramagnetic nanoparticles, decreasing the MR signal enhancement (50%) to a level attributable to local extravasation. The MR signal of adjacent hindlimb muscle or contralateral control tissues was unchanged by either the alpha(nu)beta(3)-targeted or control paramagnetic agents. Immunohistochemistry of alpha(nu)beta(3)-integrin corroborated the extent and asymmetric distribution of neovascularity observed by MRI. These studies demonstrate the potential of this targeted molecular imaging agent to detect and characterize (both biochemically and morphologically) early angiogenesis induced by minute solid tumors with a clinical 1.5 Tesla MRI scanner, facilitating the localization of nascent cancers or metastases, as well as providing tools to phenotypically categorize and segment patient populations for therapy and to longitudinally follow the effectiveness of antitumor treatment regimens.

Several radiolabeled chemotactic peptides have been tested for their suitability to show infection and inflammation. Leukotriene B(4) (LTB(4)) receptor-binding ligands could be useful agents for revealing neutrophilic infiltrations because the LTB(4) receptor is abundantly expressed on neutrophils after an inflammatory stimulus. In this study, we investigated the in vivo and in vitro characteristics of a new hydrophilic (111)In-labeled LTB(4) antagonist.The LTB(4) antagonist DPC11870-11 was labeled with (111)In and intravenously injected into New Zealand White rabbits with Escherichia coli infection in the left thigh muscle. The pharmacokinetics and biodistribution were studied by serial scintigraphic imaging (0-24 h after injection) and by ex vivo counting of dissected tissues (6 and 24 h after injection). The receptor-mediated in vivo localization of the compound was investigated in 3 rabbits that received an excess of nonradioactive indium-labeled agent 2 min before the administration of the (111)In-labeled LTB(4) antagonist.In rabbits with intramuscular E. coli infection, the abscess was visualized as early as 2 h after injection. Accumulation in the abscess increased with time, resulting in excellent images at 6 h after injection. Blood clearance was rapid in the first hours after injection (alpha-half-life = 30 +/- 6 min, 85%; beta-half-life = 25.7 +/- 0.8 h, 15%). Abscess-to-background ratios, as derived from the region-of-interest analysis, increased to 34 +/- 7 at 24 h after injection. The images of both groups showed moderate uptake in the liver, spleen, kidneys, and bone marrow. No activity was seen in the bladder, indicating almost complete retention in the kidneys. The uptake in the abscess could be blocked completely by injection of an excess of nonradioactive agent, indicating a specific receptor-ligand interaction of the radiolabeled agent in the infected tissue. Biodistribution data showed that after saturation of the LTB(4) receptor, the abscess uptake, in percentage injected dose per gram, was significantly reduced (0.03 +/- 0.02 vs. 0.24 +/- 0.06, P = 0.008).The modified LTB(4) antagonist showed infectious foci rapidly after injection because of specific receptor-ligand interaction. Because of the high abscess-to-background ratios that were obtained and the fact that no accumulation of radioactivity was observed in the gastrointestinal tract, this compound has excellent characteristics for revealing infectious and inflammatory foci.

Background — 99m Tc-RP517 is a new leukotriene B 4 (LTB4) receptor antagonist developed for imaging acute inflammation or infection. A unique property of 99m Tc-RP517 is its ability to label white blood cells in vivo after intravenous injection. The goals of this study were to determine relative 99m Tc-RP517 binding to human leukocyte subtypes and the 99m Tc-RP517 uptake pattern in canine myocardium where inflammation was induced by either coronary occlusion and reperfusion or tumor necrosis factor α (TNFα) injection. Methods and Results — Fluorescence-activated cell sorter analysis was performed on whole human blood (n=2) and isolated neutrophils (n= 4) with a fluorescent analog of 99m Tc-RP517, [F]-RP517. In whole blood, [F]-RP517 (500 nmol/L) preferentially labeled neutrophils. On isolated neutrophils, [F]-RP517 (10 nmol/L) binding was inhibited by 44% when LTB4 (400 nmol/L) was added. 99m Tc-RP517 was injected intravenously in anesthetized, open-chest dogs before coronary occlusion (90 minutes) and reperfusion (120 minutes) (n=9) or before intramyocardial TNFα injection (n=3). Ex vivo images of heart slices were acquired. The left ventricle was divided into 72 segments for flow and 99m Tc-RP517 uptake analysis. There was an inverse exponential relationship between 99m Tc-RP517 uptake and occlusion flow ( r =0.73). In the same 15 segments, 99m Tc-RP517 uptake was highly correlated with the neutrophil enzyme myeloperoxidase ( r =0.91). Ex vivo images revealed tracer uptake in the reperfused area (ischemic to normal count ratio=2.7±0.2). Conclusions — RP517 binds to the neutrophil LTB4 receptor after intravenous injection. After reperfusion, 99m Tc-RP517 uptake correlated with myeloperoxidase and was observed on ex vivo images, indicating that this tracer may have potential as an inflammation-imaging agent.

In a search for a rapid and accurate imaging agent for scintigraphic detection of infection and inflammation, an LTB4 receptor antagonist, 99Tcm-RP517, which contains the hydrazino nicotinamide moiety, has been developed recently. To study the in vivo behaviour of 99Tcm-RP517, rabbits with Escherichia coli infection were injected intravenously with 99Tcm-RP517. Gamma camera images were obtained and ex vivo bio-distribution was determined at several hours post-injection (p.i.). In a separate set of rabbits the choledochal duct was cannulated to quantitatively monitor the hepatobiliary clearance of the radiopharmaceutical. The receptor binding fraction of the radiolabelled RP517 exceeded 70%. Accumulation of 99Tcm-RP517 in the abscess was visualized as early as 1 h p.i. Due to rapid blood clearance (t1/2 alpha=18+/-0.6 min, t1/2 beta=6.5+/-0.4 h) and high abscess uptake, the abscess-to-muscle ratios increased with time from 7.0+/-2.3 at 1 h p.i. to 44.3+/-4.6 at 20 h p.i. The agent mainly cleared via the hepatobiliary route: 50% of the radiolabel was recovered in the small bowel at 1 h p.i., whereas 85% was found in cecum and sigmoid at 20 h p.i. In conclusion, 99Tcm-RP517 rapidly visualized E. coli abscesses in rabbits. The agent rapidly cleared from the blood, mainly via the hepatobiliary route. High abscess-to-background ratios were achieved. The accumulation in the intestines could limit the applicability of this agent for detecting infectious processes in the abdominal area. The development of a more hydrophilic analogue of 99Tcm-RP517 could improve the clinical applicability of this agent.

Hydrazones of a 6-hydrazinonicotinyl-modified cyclic peptide IIb/IIIa receptor antagonist were prepared in order to protect the hydrazine moiety from reaction with trace aldehyde and ketone impurities encountered during the process of manufacturing and compounding lyophilized kits used in radiolabeling with (99m)Tc. Hydrazones were prepared by either a direct reaction of the 6-hydrazinonicotinyl-modified cyclic peptide with carbonyl compounds or by conjugation of the cyclic peptide with hydrazones of succinimidyl 6-hydrazinonicotinate. Stability of the hydrazones was evaluated by treatment with formaldehyde. Hydrazones derived from simple aliphatic aldehydes underwent an exchange reaction with formaldehyde, while hydrazones of aromatic aldehydes and ketones provided the greatest level of stability when challenged with formaldehyde. We have been successful in protecting 6-hydrazinonicotinyl-modified cyclic peptides from reacting with formaldehyde, while still allowing sufficient reactivity for radiolabeling with (99m)Tc. The hydrazones of succinimidyl 6-hydrazinonicotinate are convenient and general reagents for forming 6-hydrazinonicotinyl conjugates with amino-functionalized bioactive molecules.

v 3-Integrin is a well-recognized biomarker of angiogenesis expressed on activated endothelial cells. It is a critical element in vascular restenosis where its upregulation by balloon overstretch injury promotes cell adhesion to the damaged extracellular matrix, induces extracellular metalloproteinase expression, and facilitates smooth muscle cell migration. We have conducted a series of in vivo studies in which v 3-integrin was detected by MRI (1.5T) as an early biomarker of nascent tumor growth (Vx-2 in rabbit and melanoma mice), early atherosclerosis in cholesterol-fed rabbits, and balloon overstretch-injury patterns in pigs. Paramagnetic nanoparticles covalentlycomplexed to a peptidomimetic v 3-integrin ligand were produced by microfluidization. Nontargeted paramagnetic nanoparticles and v 3-targeted nonparamagnetic nanoparticles were produced identically for control and competition studies. New Zealand White (NZW) rabbits implanted with nascent Vx-2 tumors and mice implanted with C-32 melanoma were treated systemically with v 3-integrin targeted paramagnetic nanoparticles that specifically bound and significantly enhanced (p 0.05) the MRI signal in the developing neovasculature after 2 hours (125% and 177%, respectively). Competitive blockage of v 3-integrin sites with v 3 targeted nonparamagnetic nanoparticles greatly ( 50%) diminished contrast signal enhancement (p 0.05) in both model systems. In cholesterol-fed NZW rabbits, black-blood MRI of the abdominal aorta v 3-targeted nanoparticles localized to angiogenic vessels in the expanding vaso-vasorum and produced enhanced signal (p 0.05) compared with nontargeted nanoparticles or control diet animals. In competition studies MRI enhancement was blunted, which confirmed the specificity of v 3-targeted nanoparticles. Furthermore, delayed enhancement (20 min) after injection of Gd-DTPA could not identify these early atherosclerotic changes. Finally, carotid arteries of pigs subjected to balloonoverstretch injury were treated locally with v 3 targeted, paramagnetic nanoparticles, saline or the nontargeted control. Contrast enhancement with v 3-targeted paramagnetic nanoparticles was dramatic and revealed asymmetrical patterns of balloon injury that were 50% greater than the actual balloon length and allowed the volume of vascular injury to be computed (955 234 mm2). No enhancement was discernible for the injured control vessels. v 3-Targeted paramagnetic nanoparticles provide a versatile molecular imaging platform useful for delineating vascular injury, diagnosing early atherosclerosis, and detecting nascent cancers with clinically relevant MRI systems (1.5 T). Early detection, effective therapy and sensitive longitudinal follow-up of these major disease processes with new molecular techniques could serve to noninvasively characterize and ameliorate their morbidity and mortality.

A new synthesis is described for the preparation of tri-o-thymotide ( 1 , TOT) and some TOT analogues. The methodology is based on the sequential coupling of appropriately substituted and protected salicylic acid monomers followed by cyclization of the deprotected open-chain trimers. A variety of protecting methodologies and coupling sequences are discussed. The procedure seems generally applicable for the preparation of salicylides and has been used to prepare TOT in 25% overall yield (for the coupling-deprotection-coupling-deprotection-cyclization sequence). In addition, two new modified TOT-analogues 9 (25%, and 10 (14% were prepared in which the isopropyl group(s) ortho to the phenolic units is TOT is replaced by one ( 9 ) or two ( 10 ) ethyl groups. A third analogue 66 . where the methyl group ortho to the carboxyl group is removed in one of the salicylic acid monomer units of TOT, requires only the last cyclization step for completion of its synthesis. This methodology represents an important breakthrough for the controlled preparation of selected thymotide (salicylide) trimers and allows easy access to a variety of modified thymotides (salicylides) for structural, conformational and host-guest studies.

Symmetrical disilthianes of the type M(C6H5)2SiSSi(C6H502M(M  CH3; CH2  CH3, C6H4X; X  H, F, Cl, Br and CH3) has been prepared by reaction of the appropriate chlorosilane in the presence of triethylamine with hydrogen sulfide. Selected alcoholysis attempts indicate that these symmetrical aromatic disilthianes are resistant to electrophylic cleavage of the SiSSi linkage. Physical properties of the new disithianes as well as their IR and PMR spectra are discussed.

A novel signal amplification method, catalyzed reporter deposition (CARD), and its application to immunoassays is described. The method involves utilizing an analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter on the surface in a solid-phase immunoassay. In the examples described, deposition of reporter is facilitated by using a horseradish peroxidase (HRP) ADRE to catalyze the deposition of biotin labeled phenols. The deposited biotins are then reacted with streptavidin-labeled enzyme, thereby resulting in deposition of enzyme. Using the ADRE to catalyze the deposition of additional enzyme results in an amplification of the signal of the ADRE alone and improves the detection limit of the assay. The method is highly sensitive, simple, flexible, and easy to implement.

A new synthesis of 1-methyl-2,5-piperazinedione in three steps starting from sarcosine is described. This method proceeds in higher overall yield (49%) than previous methods.