Myocardial kinetics of Tc-99m glucarate in low flow, hypoxia, and aglycemia.

BACKGROUND: Technetium-99m glucarate is a myocardial infarct-avid imaging agent. Recent conflicting and inconclusive reports have suggested that the agent may be taken up by ischemic but viable myocardium. The purposes of this study were (1) to determine conclusively whether there is Tc-99m glucarate uptake in ischemic viable myocardium and (2) to investigate the potential mechanisms for such uptake by studying components of ischemia, namely, low flow, hypoxia, and aglycemia. METHODS AND RESULTS: Rat hearts were isolated and perfused in a modified Langendorff preparation with a crytalloid perfusate. Tc-99m glucarate was studied in control (n = 6), low-flow (n = 5), hypoxic (n = 5), and aglycemic (n = 5) conditions. The experimental protocol consisted of 20-minute baseline (12 mL/min flow) and 20-minute treatment (low flow at 1 mL/min, hypoxia, or aglycemia), followed by tracer uptake (20 minute) and washout (20 minutes). Activity was monitored with a sodium iodide detector. The tracer was delivered continuously over a 20-minute uptake period. The injected dose was 150 micro Ci (5.6 MBq). Hemodynamics were monitored throughout. Triphenyltetrazolium chloride staining was used to assess myocardial viability. There was no evidence of myocardial necrosis. Low flow tended to delay tracer uptake compared with control for the first 10 minutes, but this did not reach statistical significance. Low flow increased end fractional retention significantly compared with control (mean +/- SEM, 59.0% +/- 0.9% peak vs 41.2% +/- 1.4%, respectively; P <.05). Hypoxia resulted in a trend toward increased uptake; however, this was significant only at one early time point during the uptake phase. Retention in the hypoxia group was similar to control. Tc-99m glucarate uptake was significantly increased in aglycemia from 16 minutes to peak compared with control (1.36% +/- 0.71% injected dose per gram vs 0.91% +/- 0.37% injected dose per gram, respectively; P <.05). Aglycemia produced significantly higher end fractional retention compared with control (51.6% +/- 1.8% peak vs 41.2% +/- 1.4%, respectively; P <.05). CONCLUSIONS: Tc-99m glucarate myocardial retention is increased in the setting of ischemia, even in the absence of necrosis. This increased retention is not due to hypoxia. Furthermore, the retention is only partially explained by tissue hypoglycemia. Thus low flow per se appears to have a role in this increased retention, probably as a result of delayed flow-dependent washout.

Intracytoplasmic gene delivery for in vitro transfection with cytoskeleton-specific immunoliposomes.

A novel and highly efficient method of in vitro gene transfection has been developed. This method employs direct intracytoplasmic gene delivery into embryonic cardiocytes using neutral cytoskeletal-antigen specific immunoliposomes (CSIL). These immunoliposomes target cardiocytes specifically under reversible hypoxic conditions. Two independent reporter genes, pGL2 and pSV-beta-galactosidase, were used to verify CSIL-transfection (CSIL-fection). The efficiency of CSIL-fection with firefly luciferase pGL2 vector was 30+ times greater than controls consisting of hypoxic cardiocytes treated with plain liposomes (PL) or normoxic cardiocytes treated with CSIL, PL or naked DNA. CSIL-fection was also compared to cationic liposome transfection. Net cationic liposome transfection appeared to be more efficient than CSIL-fection for pGL2 vectors. However, a smaller number of viable cells was observed in the cationic liposome treated cultures than in the CSIL treated cultures. Therefore, to determine whether more cells were transfected with cationic liposomes or CSIL, pSV-beta-galactosidase vector was used. CSIL-fection with pSV-beta-galactosidase vector produced at least 40 times more transfected cells than those transfected with cationic liposomes. No transfection with pSV-beta-galactosidase vectors was obtained with IgG-liposome, PL or naked DNA treatments. Targeted enhanced efficiency of transfection by this novel method could have practical therapeutic applications in the genetic modification of cells ex vivo that could then be reimplanted into patients for gene therapy.

p-Nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment of specific ligands, including monoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups.

We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to make it applicable for single-step binding of a large variety of a primary amino group-containing substances, including proteins and small molecules. With this in mind, we have introduced a new amphiphilic PEG derivative, p-nitrophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG-DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcarbonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily incorporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and non-toxic urethane (carbamate) bonds. The reaction between the pNP group and the ligand amino group proceeds easily and quantitatively at pH around 8.0, and remaining free pNP groups are promptly eliminated by spontaneous hydrolysis. Therefore, pNP-PEG-DOPE could serve as a very convenient tool for protein attachment to the distal ends of liposome-grafted PEG chains. To investigate the applicability of the suggested protocol for the preparation of long-circulating targeted liposomes, we have coupled several proteins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), avidin, monoclonal antimyosin antibody 2G4 (mon2G4), and monoclonal antinucleosome antibody 2C5 (mon2C5) to PEG-liposomes via terminal pNP groups and studied whether the specific activity of these immobilized proteins is preserved. The method permits the binding of several dozens protein molecules per single 200 nm liposome. All bound proteins completely preserve their specific activity. Lectin-liposomes are agglutinated by the appropriate polyvalent substrates (mannan for ConA-liposomes and glycophorin for WGA-liposomes); avidin-liposomes specifically bind with biotin-agarose; antibody-liposomes demonstrate high specific binding to the substrate monolayer both in the direct binding assay and in ELISA. A comparison of the suggested method with the method of direct membrane incorporation was made. The effect of the concentration of liposome-grafted PEG on the preservation of specific protein activity in different coupling protocols was also investigated. It was also shown that pNP-PEG-DOPE-liposomes with and without attached ligands demonstrate increased stability in mouse serum.

In vivo uptake of radiolabeled antibody to proliferating smooth muscle cells in a swine model of coronary stent restenosis.

UNLABELLED: Z2D3 is a monoclonal chimeric antibody fragment that is directed against a protein expressed on the surface of proliferating smooth muscle cells. The purpose of this study was to investigate the uptake of 111In-labeled Z2D3 F(ab')2 in a swine model of coronary neointimal proliferation after overexpansion coronary stenting. METHODS: Twenty-two domestic swine underwent overexpansion coronary stenting of 2 vessels. Fifteen swine survived 2-4 wk, at which time they received an injection of 111In Z2D3 F(ab')2 and underwent planar imaging. After the swine were killed, the hearts were excised and imaged on the detector. The cross-sectional area of each stented vessel was measured with digital morphometry. RESULTS: Pathology could be correlated with imaging for 24 vessels. The cross-sectional area of stenosis comprising neointimal proliferation ranged from 8% to 95%, with a mean +/- SD of 41% +/- 21%. The maximal stenosis ranged from 13% to 95%, with a mean of 51% +/- 20%. Seventeen of 24 vessels (71%) showed focal uptake on in vivo imaging, and 7 of 24 (29%) did not. Twenty of 24 (83%) showed uptake on ex vivo imaging. Of 11 stented vessels with maximal vessel stenosis less than 50%, 7 (64%) showed uptake both in vivo and ex vivo, and of 13 stented vessels with maximal vessel stenosis greater than 50%, 10 (77%) showed uptake both in vivo and ex vivo. CONCLUSION: Uptake of a radiolabeled antibody directed against a component of proliferating neointimal tissue can be visualized in the coronary arteries on in vivo imaging using a scintillation gamma camera.

Technetium-99m glucarate uptake in a swine model of limited flow plus increased demand.

BACKGROUND: Glucarate is a 6-carbon dicarboxylic acid shown to be taken up by necrotic myocytes, binding to nuclear histones in animal models of coronary occlusion, resulting in infarction. This study investigated glucarate uptake in a model of severe ischemia. METHODS AND RESULTS: Thirty-five experiments were performed, in which a catheter-mounted stenosis (reducing lumen dimensions by 80%) was placed in the left anterior descending coronary artery (LAD) of an anesthetized, instrumented domestic swine and technetium-99m glucarate (GLU) was injected during the last minute of 5 minutes of pacing. Hemodynamic and blood flow measurements were performed at control, during pacing, and during recovery. The animals were killed; their hearts were stained with fluorescein dye and triphenyl tetrazolium chloride (TTC). Electron micography (EM; n = 6) and cell centrifugation (n = 7) were also performed. On the basis of net lactate production and severe blood flow reduction in the risk region (RR), ischemia with pacing developed in 25 animals. Fifteen of 25 animals showed tracer uptake in the RR on in vivo and ex vivo imaging (scan positive), and 10 were scan negative in the RR. Endocardial blood flow in the RR during pacing was 0.28+/-0.16 mL/g/min for scan-positive and 0.30+/-0.17 mL/g/min for scan-negative experiments (P = not significant [NS]). Transmyocardial net lactate extraction during pacing was -63%+/-44% for scan-positive and -53%+/-60% for scan-negative experiments (P = NS). Control and recovery heart rates were higher in scan-positive experiments (108+/-14 vs. 92+/-17, and 125+/-24 vs. 104+/-18, P<.02). Lactate extraction was lower during control and recovery in scan-positive animals (2+/-29 vs. 30+/-19, P = .03). Scan-positive animals had a more proximal stenosis position. Minimal necrosis was documented by means of TTC negative staining in 8 of 15 scan-positive experiments (comprising 10%+/-4.3% of RR area). EM or cell fractionation was performed in 5 of the 7 remaining scan-positive and TTC-positive hearts, and in those 5 experiments, necrosis was documented by means of EM in 2 and by means of cell fractionation in 3. CONCLUSIONS: Uptake of Tc-99m glucarate was seen in the RR in a swine model of ischemia severe enough to produce myocyte injury and early cell death.

10 targeting in myocardial infarction.

Targeting pharmaceuticals to the infarcted myocardium has two primary objectives: the diagnostic imaging of the infarcted myocardium and the delivery of therapeutic agents to compromised myocardial areas. Various diagnostic and therapeutic agents (such as radiolabeled compounds, thrombolytic enzymes, proteolytic drugs, and antioxidants) have been proposed for visualization or treatment of the infarcted myocardium. However, none of these reagents has the specificity for targeting the compromised myocardium. Therefore, the availability of a target-specific delivery system should increase the efficacy of diagnosis and therapy. Furthermore, the existence of such a targeted delivery system may pave the way for the use of new pharmaceuticals that by themselves can be harmful to normal tissues.

Detection of acute myocardial infarction by 99mTc-labeled D-glucaric acid imaging in patients with acute chest pain.

UNLABELLED: Definitive diagnosis of acute myocardial infarction early in the process is often difficult. An imaging agent that localized quickly and specifically in areas of acute necrosis could provide this critical diagnostic information. To determine whether imaging with 99mTc-labeled D-glucaric acid (GLA) could provide this information, we imaged a group of patients presenting with symptoms suggestive of acute infarction. METHODS: Twenty-eight patients presenting to the emergency department with symptoms highly suggestive of acute infarction were injected with 99mTC-GLA and imaged about 3 h later. RESULTS: The sensitivity of lesion detection was remarkably time dependent. Fourteen patients with acute infarction injected within 9 h of onset of chest pain had positive scans, even in the presence of persistent occlusion. The remaining 14 patients had negative scans. Nine patients with negative scans had acute infarction but were injected more than 9 h after onset of chest pain. The final diagnosis in the remaining 5 patients was unstable angina (3 injected <9 h and 2 injected >9 h after onset of chest pain). Six patients were reinjected with 99mTc-GLA 4-6 wk after their initial study to determine whether persistent positive scans occurred with this agent. All 6 had negative scans. CONCLUSION: This study suggests that 99mTc-GLA localizes in zones of acute myocardial necrosis when injected within 9 h of onset of infarction.

Usefulness of antimyosin antibody imaging for the detection of active rheumatic myocarditis.

Myocarditis constitutes an important component of rheumatic carditis. Antimyosin scintigraphy, which allows noninvasive assessment of myocyte damage, can be used for documentation of cardiac involvement in patients with rheumatic fever where clinical diagnosis is not unequivocal.

The current role of infarct avid imaging.

Tc-99m pyrophosphate is the grandfather of infarct avid agents. Its value is its clinical availability and ease of use. However, its shortcomings are the delay of 2 to 3 days for reliable interpretation in nonreperfused myocardial infarction (MI) and the overarching bone activity. Antimyosin provides exquisite specificity for the detection of myocardial necrosis irrespective of the cause of the injury. Therefore, diagnosis of equivocal MI or confirmation of diffuse myocardial necrosis would benefit from the availability of In-111 labeled antimyosin Fab. The drawback of antimyosin, like that of Tc-99m pyrophosphate, is the delay, in this case because of the protracted blood clearance of the antibody protein macromolecules. Tc-99m glucaric acid, on the other hand, may fulfill the original role envisioned for antimyosin, which was to enable early, rapid diagnosis of acute MI. However, the window for the use of Tc-99m glucaric acid appears to be limited to within the first day of the acute event. Therefore, there is a potential use of both Tc-99m glucaric acid and In-111 antimyosin in tandem with Tc-99m glucaric acid, which would not only facilitate early detection and diagnosis of acute MI and diagnosis of equivocal MI, but also may permit stratification of the infarct age.

Complementary roles of antibody affinity and specificity for in vivo diagnostic cardiovascular targeting: how specific is antimyosin for irreversible myocardial damage?

BACKGROUND: Identification of irreversible myocyte injury with antimyosin antibody imaging depends on both antibody specificity and affinity. To characterize the role of antibody affinity, we performed studies in dogs with acute coronary occlusion followed by reperfusion using 3 monoclonal antimyosin antibodies with different affinities. METHODS AND RESULTS: Dogs with experimental reperfused acute myocardial infarction were injected with 2 high-affinity radiolabeled monoclonal antimyosin Fab fragments (R11D10 and 2G42D7), 1 low-affinity antimyosin Fab (3H31E6), and a nonspecific Fab. The left lateral gamma images at 5 H were used to assess the infarct (I) to blood (B) region of interest (ROI) count density ratios by computer planimetry. All infarcts were confirmed by in vivo imaging with 201Tl for perfusion defects as well as by postmortem histochemical staining. The mean I/B ROI (+/-standard deviation [SD]) for R11D10 (1.701+/-0.376) was not significantly different from that of 2G42D7 (1.501+/-0.267, P = NS), but both were significantly greater than that of 3H31E6 Fab (0.85+/-0.12, P = .0001 and .0012, respectively). The I/B ROI of 3H31E6 Fab was similar to that of nonspecific Fab (0.75 to 0.77 range). Radiolabeled R11D10 and 2G42D7 were unequivocally positive by gamma imaging in all infarcts by 5 H. No infarcts were visualized with 3H31E6 or nonspecific Fab. CONCLUSIONS: The low-affinity antibody, despite its specificity for cardiac myosin, cannot be used to image the infarct zone. Therefore immunoscintigraphic diagnosis of irreversible myocardial injury with radiolabeled antimyosin Fab is doubly specific because in vivo visualization required both specificity and high enough affinity of the antibody.

Noninvasive localization of human atherosclerotic lesions with indium 111-labeled monoclonal Z2D3 antibody specific for proliferating smooth muscle cells.

BACKGROUND: Targeting exclusive antigens in atherosclerotic plaques with antibodies may provide a noninvasive means to detect rapidly proliferative atherosclerotic lesions. 111In-labeled negative charge-modified Z2D3 F(ab')2 (Z2D3) specific for an antigen expressed exclusively by proliferating smooth muscle cells has been shown to accumulate in rabbit atherosclerotic plaques. METHODS: The safety, biodistribution, accumulation, and elimination of Z2D3 were assessed in 11 patients who were candidates for carotid endarterectomy. The presence of atheromas in these patients was confirmed by angiography and Doppler ultrasound. Z2D3 (250 microg) labeled with 5 mCi of 111In was administered by slow intravenous injection. Planar and single photon emission computed tomography (SPECT) images were obtained 4, 24, 48, and 72 hours later. Carotid endarterectomy was performed and the surgical specimens were imaged, weighed, gamma-counted, and analyzed by immunostaining. RESULTS: Uptake of Z2D3 at the site of the carotid plaques was observed in the planar and SPECT views at 4 hours in all subjects. In addition, antibody uptake was noted in the contralateral vessel in 5 subjects. SPECT images identified the atherosclerotic plaques with focal uptake. The antibody uptake corresponded with the angiographic location of the disease. Immunohistochemical studies of the endarterectomy specimens confirmed the localization of Z2D3 into the plaque areas containing smooth muscle cells. Adverse drug reactions were not observed. CONCLUSION: This study demonstrates the feasibility of targeting atherosclerotic lesions with negative charge-modified antibody. It also proposes the possibility of selective identification of various components of atherosclerotic plaque, which may contribute to determining strategies of intervention in future.

Disulfide cross-linked Fab-aggregates: preparation and biodistribution.

The high-molecular-weight soluble aggregates of Fab fragments of murine antibodies against cardiac myosin were prepared as a potential long-circulating and low immunogenic pharmaceutical carriers by conjugation of thiolated Fab and Fab modified with succinimidyl 3-(2-pyridyldithio)propionate. The clearance time and biodistribution of 111In-radiolabeled aggregates were studied in normal and nude-mice bearing human breast tumor implant and in rabbits with experimental myocardial infarction. The aggregates had a prolonged circulation time (half clearance time ca. 3-5 h) and ability to concentrate in the tumor and in the necrotic area of infarcted myocardium. Similar tumor-to-normal and infarct-to-normal accumulation ratios (ca. 3 h in both cases) suggest that combination of long circulation with impaired filtration in necrotic tissues is responsible for this accumulation rather than a specific interaction. The aggregates prepared may serve as long-circulating drug carriers able to deliver pharmaceuticals into areas with affected and leaky vasculature.

Rapid noninvasive detection of experimental atherosclerotic lesions with novel 99mTc-labeled diadenosine tetraphosphates.

The development of a noninvasive imaging procedure for identifying atherosclerotic lesions is extremely important for the clinical management of patients with coronary artery and peripheral vascular disease. Although numerous radiopharmaceuticals have been proposed for this purpose, none has demonstrated the diagnostic accuracy required to replace invasive angiography. In this report, we used the radiolabeled purine analog, 99mTc diadenosine tetraphosphate (Ap4A; AppppA, P1,P4-di(adenosine-5')-tetraphosphate) and its analogue 99mTc AppCHClppA for imaging experimental atherosclerotic lesions in New Zealand White rabbits. Serial gamma camera images were obtained after intravenous injection of the radiolabeled dinucleotides. After acquiring the final images, the animals were sacrificed, ex vivo images of the aortas were recorded, and biodistribution was measured. 99mTc-Ap4A and 99mTc AppCHClppA accumulated rapidly in atherosclerotic abdominal aorta, and lesions were clearly visible within 30 min after injection in all animals that were studied. Both radiopharmaceuticals were retained in the lesions for 3 hr, and the peak lesion to normal vessel ratio was 7.4 to 1. Neither of the purine analogs showed significant accumulation in the abdominal aorta of normal (control) rabbits. The excised aortas showed lesion patterns that were highly correlated with the in vivo and ex vivo imaging results. The present study demonstrates that purine receptors are up-regulated in experimental atherosclerotic lesions and 99mTc-labeled purine analogs have potential for rapid noninvasive detection of plaque formation.

Avidity of technetium 99m glucarate for the necrotic myocardium: in vivo and in vitro assessment.

BACKGROUND: Similar to other 99mTc-based infarct-avid agents, 99mTc-glucarate localizes in myocardial infarcts. Whether severely ischemic viable myocytes sequester 99mTc-glucarate is uncertain. To assess the infarct specificity, in vitro and in vivo studies were performed. METHODS AND RESULTS: H9C2 embryonic rat cardiocytes cultured under normoxia (N) or hypoxia (H) for 24 hours in 7.5 muCi 99mTc-glucarate were compared with necrotic cardiocytes. Mean H/N ratio (3.0 +/- 0.004, mean +/- SD) was significantly less than that of the necrotic/N ratio (39.9 +/- 6.5, p < 0.01). Reperfused myocardial infarction (MI) in 4 dogs confirmed by 201Tl (0.5 to 1.0 mCi) scintigraphy were imaged serially with simultaneously injected mixture of 99mTc-glucarate and 111In-antimyosin Fab. Infarcts were detected scintigraphically within 4 to 10 minutes with 99mTc-glucarate. 111In-antimyosin required more than 1 hour. Myocardial distribution at 5 hours showed a direct correlation between 99mTc-glucarate and 111In-antimyosin uptake (r = 0.99, p < 0.0001). Both 99mTc-glucarate (r = -0.777, p < 0.0001) and 111In-antimyosin (r = -0.775, p < 0.0001) were inversely related to 201Tl distribution. CONCLUSIONS: The near perfect correlation between 99mTc-glucarate and 111In-antimyosin uptake (r = 0.99) in reperfused canine MI and the insignificant glucarate uptake by viable cardiocytes in vitro attest to the avidity of 99mTc-glucarate for the necrotic myocardium and favor its use as a specific early marker of myocyte necrosis in acute MI.

Noninvasive detection of atherosclerotic lesions by 99mTc-based immunoscintigraphic targeting of proliferating smooth muscle cells.

BACKGROUND: Mouse/human chimeric antibody Z2D3 identifies an antigen produced exclusively by proliferating smooth muscle cells of human atheroma, and also cross reacts with experimentally induced atherosclerotic lesions in rabbits. Fab' fragments of Z2D3 antibody were labeled with (99m)Tc using glucaric acid as a weak transchelator. The potential role of (99m)Tc-labeled Z2D3 scintigraphy was explored for noninvasive imaging of experimental atherosclerotic lesions. METHODS AND RESULTS: (99m)Tc-Z2D3 Fab' was utilized for noninvasive imaging in four rabbits with experimentally induced atherosclerotic lesions and in one control rabbit. In addition, (99m)Tc-labeled nonspecific 103D2 Fab' was used for comparison in four other rabbits with atherosclerotic lesions. The atherosclerotic lesions were induced by balloon de-endothelialization of the infradiaphragmatic abdominal aorta and 12 weeks of hyperlipidemic diet. An aliquot of 15 mCi (550 mBq) of (99m)Tc pertechnetate was incubated with 6.25 mg of glucaric acid for 30 min followed by incubation of (99m)Tc glucarate with 375 microg of Z2D3 Fab' or 103D2 Fab' for an additional 30 min. Instant thin-layer chromatography demonstrated almost complete radiolabeling. (99m)Tc-Z2D3 was administered IV and gamma imaging was performed at the time of injection, 3, 6, 9, and 12 h, followed by ex vivo imaging of the excised aorta, and biodistribution was performed. Unequivocal visualization of atherosclerotic lesions was possible in all four animals at 9 to 12 h with Z2D3 Fab'. Quantitative uptake, as represented by mean lesion-to-liver count density ratio, was 0.6+/-0.05. Imaging with nonspecific 103D2 Fab' did not show any localization in the abdominal aorta (lesion-to-liver ratio, 0.45+/-0.02, p=0.02). Ex vivo lesion-to-normal aortic segment ratio was 4.3+/-0.9 for Z2D3 and 1.04+/-0.08 for nonspecific 103D2 Fab' (p=0.01). Biodistribution studies demonstrated 0.03+/-0.003% injected Z2D3 dose per gram in the atherosclerotic lesions as compared with 0.01+/-0.003% in the nondenuded thoracic aorta of atherosclerotic rabbits (p=0.008). However, only 0.008+/-0.002% of the mean injected dose per gram was obtained in the atherosclerotic lesions (p=0.001) as compared with 0.005+/-0.003% in the normal aortic segments with 103D2. No Z2D3 uptake in normal rabbits was observed on either the in vivo or ex vivo images. CONCLUSIONS: The present study demonstrates that (99m)Tc-based immunoimaging of the vascular lesions may be feasible by the use of smaller antibody fragments. Earlier visualization is possible at the expense of a lower absolute antibody uptake in the lesions as compared to the use of intact antibody or larger fragments with longer circulating time.

Maximizing radiotracer delivery to experimental atherosclerotic lesions with high-dose, negative charge-modified Z2D3 antibody for immunoscintigraphic targeting.

BACKGROUND: Two factors that directly affect target/background ratio in immunoscintigraphy are the concentration of the antibody bound to the target and the concentration of the antibody in the circulation. High dosages of monoclonal antibody have been reported to be more efficacious in visualization of tumors. Although administration of a higher dosage of antibody increases the absolute target accumulation of the radiotracer, it also increases the background activity, which may offset this advantage. Negative charge-modified antibodies carry high specific radioactivity to the target sites without significantly increasing the background activity. Therefore we investigated whether higher dosages of negative charge-modified antibody can be used to improve imaging of experimental atherosclerotic lesions. METHODS AND RESULTS: Experimental atherosclerotic lesions were produced in 16 New Zealand White rabbits by balloon deendothelialization of the infradiaphragmatic aorta and hyperlipidemic diet for 12 weeks. Negative charge-modified Z2D3 antibody F(ab')2 specific for an antigen on proliferating smooth muscle cells of human atheroma labeled with (111)In was used for imaging experimental atherosclerotic lesions either at high (100 to 125 microg) or low (25 to 50 microg) dosages. A lower dosage of Z2D3 was labeled with 507 +/- 29.5 microCi (25 to 50 microg) (111)In label, compared with 2.9 +/- 0.24 mCi (100 to 125 microg) for the higher dosage. Although noninvasive visualization of atherosclerotic lesions was possible in all animals at 24 hours, high antibody dose allowed unequivocal visualization of the lesion as early as 3 hours after intravenous administration of the antibody. Eight animals were killed at 24 hours and the remaining eight animals at 48 hours. Mean radioactivity dose delivered per gram of lesion with the low-dose protocol at 24 hours was 0.46 +/- 0.09 microCi, which remained essentially unchanged at 48 hours (0.37 +/- 0.09 microCi; p = 0.51). With the high-dosage protocol, the total radioactivity (dose) per gram uptake in the lesion increased by about eightfold (3.49 +/- 0.58 microCi; p = 0.002) at 24 hours and was sixfold higher at 48 hours (2.21 +/- 0.45 microCi; p < 0.02). CONCLUSIONS: The study demonstrated that the increase in the dosage of negatively charge-modified antibody allows a very high delivery of specific radioactivity to the target, which in turn enables early visualization of experimental atherosclerotic lesions.

Targeting human breast tumour in xeno-grafted SCID mice with 99Tcm-glucarate.

99Tcm-glucarate accumulation in human mammary BT-20 tumours hosted in severe combined immune deficiency (SCID) mice was compared to 111In-monoclonal antibody 103D2-F(ab')2, 99Tcm-methoxyisobutyl isonitrile (99Tcm-MIBI) and 99Tcm-diethylenetriamine pentaacetate (99Tcm-DTPA). The intracellular tumour distribution of 99Tcm-glucarate was also determined. SCID mice injected with a mixture of 99Tcm-glucarate and 111In-103D2-F(ab')2 were imaged serially up until 24 h. Computer planimetered tumour-to-blood activity (in the heart) ratios (T/BH) to 8 h were significantly greater for 99Tcm-glucarate than 111In-103D2. The mean (+/-S.D.) tumour-to-blood ratio (T/B) from biodistribution was 1.21 +/- 0.31 and 0.35 +/- 0.06 (P < 0.0001) at 5 h, and 1.526 +/- 0.29 and 0.75 +/- 0.2 (P < 0.0001) at 8 h, for 99Tcm-glucarate and 111In-103D2 respectively. At 24 h, T/B for 111In-103D2 (1.76 +/- 0.22) exceeded that of 99Tcm-glucarate (1.44 +/- 0.2, P = 0.01). 99Tcm-glucarate uptake in the tumours at 5 h (1.133 +/- 0.25 %ID g-1) and 8 h (1.213 +/- 0.23 %ID g-1) was significantly greater than that of 99Tcm-MIBI (0.340 +/- 0.09, P = 0.0002; 0.220 +/- 0.04, P = 0.0001) and 99Tcm-DTPA (0.091 +/- 0.03, P < 0.0002; 0.016 +/- 0.01, P < 0.0001) respectively. Intracellular tumour distribution of 99Tcm-glucarate was 50.91 +/- 6.55% in the nuclear fraction, 34.34 +/- 2.88% in the cytoplasmic fraction and 14.75 +/- 7.66% in the mitochondrial fraction. Thus glucarate may provide a 99Tcm-based mammary tumour imaging modality for visualization of tumours very quickly after tracer administration with maximal targeting in the nuclei of the tumour cells.

Very early noninvasive detection of acute experimental nonreperfused myocardial infarction with 99mTc-labeled glucarate.

BACKGROUND: 99mTc glucarate has recently been reported to be an infarct-avid agent. The feasibility of imaging with 99mTc glucarate was evaluated for the early diagnosis of nonreperfused and reperfused myocardial infarction and compared with localization of simultaneously administered 111In anti-myosin. METHODS AND RESULTS: Four groups of six rabbits each were studied. The left anterior descending coronary artery (LAD) was kept persistently occluded (n = 6) or reperfused after 40 minutes (n = 6) in rabbits. After confirmation of LAD occlusion by 201Tl scintigraphy, a mixture of 99mTc glucarate (15.7 +/- 1.6 mCi) and 111In anti-myosin (0.53 +/- 0.03 mCi) was administered intravenously. Another group of rabbits (n = 6) with 5 or 15 minutes of LAD occlusion were used to assess the affinity of 99mTc glucarate for the ischemic myocardium. The remaining 6 rabbits with reperfused myocardial infarction were used for the assessment of subcellular localization of 99mTc glucarate. 99mTc glucarate cleared rapidly from circulation (elimination t1/2, 36 minutes). Infarcts were visualized within 10 minutes in reperfused and within 30 minutes in nonreperfused coronary territories after intravenous administration. 111In anti-myosin delineated reperfused infarcts within 1 to 3 hours, but no uptake was seen in persistently occluded rabbits. 99mTc glucarate uptake in reperfused and nonreperfused infarct centers was 28 and 12 times greater, respectively, than that in normal myocardium (P = .0001). A direct correlation between glucarate and anti-myosin localization (r = .60 for nonreperfused; 0.76 for reperfused; P < .0001) was observed. Ischemic hearts showed no glucarate uptake. Subcellularly, 99mTc glucarate localized predominantly in the nuclear fraction of the infarct, with lesser extents in the mitochondrial and cytoplasmic fractions. CONCLUSIONS: Noninvasive imaging of myocardial infarcts with 99mTc glucarate is possible within minutes in persistently occluded or reperfused myocardial infarcts. Early detectability results from the rapid blood clearance and high avidity of glucarate for the acutely necrotic myocardial tissue.

Apoptosis in myocytes in end-stage heart failure.

BACKGROUND: Heart failure can result from a variety of causes, including ischemic, hypertensive, toxic, and inflammatory heart disease. However, the cellular mechanisms responsible for the progressive deterioration of myocardial function observed in heart failure remain unclear and may result from apoptosis (programmed cell death). METHODS: We examined seven explanted hearts obtained during cardiac transplantation for evidence of apoptosis. All seven patients had severe chronic heart failure: four had idiopathic dilated cardiomyopathy, and three had ischemic cardiomyopathy. DNA fragmentation (an indicator of apoptosis) was identified histochemically by in situ end-labeling as well as by agarose-gel electrophoresis of end-labeled DNA. Myocardial tissues obtained from four patients who had had a myocardial infarction one to two days previously were used as positive controls, and heart tissues obtained from four persons who died in motor vehicle accidents were used as negative controls for the end-labeling studies. RESULTS: Hearts from all four patients with idiopathic dilated cardiomyopathy and from one of the three patients with ischemic cardiomyopathy had histochemical evidence of DNA fragmentation. All four myocardial samples from patients with dilated cardiomyopathy also demonstrated DNA laddering, a characteristic of apoptosis, whereas this was not seen in any of the samples from patients with ischemic cardiomyopathy. Histological evidence of apoptosis was also observed in the central necrotic zone of acute myocardial infarcts, but not in myocardium remote from the infarcted zone. Rare isolated apoptotic myocytes were seen in the myocardium from the four persons who died in motor vehicle accidents. CONCLUSIONS: Loss of myocytes due to apoptosis occurs in patients with end-stage cardiomyopathy and may contribute to progressive myocardial dysfunction.