SPECT imaging of lung ischemia-reperfusion injury using [99mTc]cFLFLF for molecular targeting of formyl peptide receptor 1.

Primary graft dysfunction after lung transplantation, a consequence of ischemia-reperfusion injury (IRI), is a major cause of morbidity and mortality. IRI involves acute inflammation and innate immune cell activation, leading to rapid infiltration of neutrophils. Formyl peptide receptor 1 (FPR1) expressed by phagocytic leukocytes plays an important role in neutrophil function. The cell surface expression of FPR1 is rapidly and robustly upregulated on neutrophils in response to inflammatory stimuli. Thus, we hypothesized that use of [99mTc]cFLFLF, a selective FPR1 peptide ligand, would permit in vivo neutrophil labeling and noninvasive imaging of IRI using single-photon emission computed tomography (SPECT). A murine model of left lung IRI was utilized. Lung function, neutrophil infiltration, and SPECT imaging were assessed after 1 h of ischemia and 2, 12, or 24 h of reperfusion. [99mTc]cFLFLF was injected 2 h before SPECT. Signal intensity by SPECT and total probe uptake by gamma counts were 3.9- and 2.3-fold higher, respectively, in left lungs after ischemia and 2 h of reperfusion versus sham. These values significantly decreased with longer reperfusion times, correlating with resolution of IRI as shown by improved lung function and decreased neutrophil infiltration. SPECT results were confirmed using Cy7-cFLFLF-based fluorescence imaging of lungs. Immunofluorescence microscopy confirmed cFLFLF binding primarily to activated neutrophils. These results demonstrate that [99mTc]cFLFLF SPECT enables noninvasive detection of lung IRI and permits monitoring of resolution of injury over time. Clinical application of [99mTc]cFLFLF SPECT may permit diagnosis of lung IRI for timely intervention to improve outcomes after transplantation.

Targeted interleukin-10 plasmid DNA therapy in the treatment of osteoarthritis: Toxicology and pain efficacy assessments.

Osteoarthritis results in chronic pain and loss of function. Proinflammatory cytokines create both osteoarthritis pathology and pain. Current treatments are poorly effective, have significant side effects, and have not targeted the cytokines central to osteoarthritis development and maintenance. Interleukin-10 is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. However, interleukin-10 protein has a short half-life in vivo and poor joint permeability. For sustained IL-10 activity, we developed a plasmid DNA-based therapy that expresses a long-acting human interleukin-10 variant (hIL-10var). Here, we describe the 6-month GLP toxicology study of this therapy. Intra-articular injections of hIL-10var pDNA into canine stifle joints up to 1.5 mg bilaterally were well-tolerated and without pathologic findings. This represents the first long-term toxicologic assessment of intra-articular pDNA therapy. We also report results of a small double-blind, placebo-controlled study of the effect of intra-articular hIL-10var pDNA on pain measures in companion (pet) dogs with naturally occurring osteoarthritis. This human IL-10-based targeted therapy reduced pain measures in the dogs, based on veterinary and owner ratings, without any adverse findings. These results with hIL-10var pDNA therapy, well-tolerated and without toxicologic effects, establish the basis for clinical trials of a new class of safe and effective therapies for OA.

COMPARATIVE RENAL DISPOSITION OF CREATINE, AND TECHNETIUM DIAGNOSTIC CONTRAST AGENTS IN THE PIGEON (COLUMBA LIVIA).

Clinical assessment of renal function in avian species often involves the measurement of plasma uric acid and blood urea nitrogen, relatively insensitive markers of renal dysfunction and dehydration. In mammals, endogenous creatinine is widely used as an indicator of renal glomerular dysfunction. However, avian species produce primarily creatine. Here, renal creatine, 99mTc99-DTPA (diethylenepentaacetic acid, DTPA) and 99mTc-MAG3 (mercaptoacetyl triglycine, MAG3) renal clearances are characterized in the pigeon avian model by infusing DTPA with inulin and creatine with each tracer and examining the slope of their blood disappearance curves. Clearance curves for inulin and DTPA were parallel, suggesting DTPA is cleared by renal filtration. MAG3 clearance (slope: -2.74 × 105, r2 = 0.97) had a slope almost 10-fold steeper than for DTPA (slope: -6.29 × 104, r2 = 0.90), and orders of magnitude steeper than for creatine (slope: -1.4, r2 = 1.0). These results suggest that DTPA is cleared by glomerular filtration like inulin, while MAG3 is filtered and actively excreted in a manner similar to mammals. In contrast, creatine is filtered and resorbed, has a larger volume of distribution (Vd), or exhibits a greater blood protein binding, making it more complex as a renal marker, when compared with creatinine handling in mammals. The two radiotracers can be readily adapted for use in birds, inviting both qualitative and semiquantitative functional evaluation of avian renal function for research and clinical purposes. The elimination of creatine appears to be more complex requiring further study.

Intravenously Delivered Mesenchymal Stem Cells: Systemic Anti-Inflammatory Effects Improve Left Ventricular Dysfunction in Acute Myocardial Infarction and Ischemic Cardiomyopathy.

RATIONALE: Virtually all mesenchymal stem cell (MSC) studies assume that therapeutic effects accrue from local myocardial effects of engrafted MSCs. Because few intravenously administered MSCs engraft in the myocardium, studies have mainly utilized direct myocardial delivery. We adopted a different paradigm. OBJECTIVE: To test whether intravenously administered MSCs reduce left ventricular (LV) dysfunction both post-acute myocardial infarction and in ischemic cardiomyopathy and that these effects are caused, at least partly, by systemic anti-inflammatory activities. METHODS AND RESULTS: Mice underwent 45 minutes of left anterior descending artery occlusion. Human MSCs, grown chronically at 5% O2, were administered intravenously. LV function was assessed by serial echocardiography, 2,3,5-triphenyltetrazolium chloride staining determined infarct size, and fluorescence-activated cell sorting assessed cell composition. Fluorescent and radiolabeled MSCs (1×106) were injected 24 hours post-myocardial infarction and homed to regions of myocardial injury; however, the myocardium contained only a small proportion of total MSCs. Mice received 2×106 MSCs or saline intravenously 24 hours post-myocardial infarction (n=16 per group). At day 21, we harvested blood and spleens for fluorescence-activated cell sorting and hearts for 2,3,5-triphenyltetrazolium chloride staining. Adverse LV remodeling and deteriorating LV ejection fraction occurred in control mice with large infarcts (≥25% LV). Intravenous MSCs eliminated the progressive deterioration in LV end-diastolic volume and LV end-systolic volume. MSCs significantly decreased natural killer cells in the heart and spleen and neutrophils in the heart. Specific natural killer cell depletion 24 hours pre-acute myocardial infarction significantly improved infarct size, LV ejection fraction, and adverse LV remodeling, changes associated with decreased neutrophils in the heart. In an ischemic cardiomyopathy model, mice 4 weeks post-myocardial infarction were randomized to tail-vein injection of 2×106 MSCs, with injection repeated at week 3 (n=16) versus PBS control (n=16). MSCs significantly increased LV ejection fraction and decreased LV end-systolic volume. CONCLUSIONS: Intravenously administered MSCs for acute myocardial infarction attenuate the progressive deterioration in LV function and adverse remodeling in mice with large infarcts, and in ischemic cardiomyopathy, they improve LV function, effects apparently modulated in part by systemic anti-inflammatory activities.

Comparison of quantitative wall-motion analysis and strain for detection of coronary stenosis with three-dimensional dobutamine stress echocardiography.

BACKGROUND: Quantitative analysis of wall motion from three-dimensional (3D) dobutamine stress echocardiography (DSE) could provide additional diagnostic information not available from qualitative analysis. In this study, we compare the effectiveness of 3D fractional shortening (3DFS), a measure of wall motion computed from 3D echocardiography (3DE), to strain and strain rate measured with sonomicrometry for detecting critical stenoses during DSE. METHODS: Eleven open-chest dogs underwent DSE both with and without a critical stenosis. 3DFS was measured from 3DE images acquired at peak stress. 3DFS was normalized by subtracting average 3DFS during control peak stress (∆3DFS). Strains in the perfusion defect (PD) were measured from sonomicrometry, and PD size and location were measured with microspheres. RESULTS: A ∆3DFS abnormality indicated the presence of a critical stenosis with high sensitivity and specificity (88% and 100%, respectively), and ∆3DFS abnormality size correlated with PD size (R(2) = 0.54). The sensitivity and specificity for ∆3DFS were similar to that for area strain (88%, 100%) and circumferential strain and strain rate (88%, 92% and 88%, 86%, respectively), while longitudinal strain and strain rate were less specific. ∆3DFS correlated significantly with both coronary flow reserve (R(2) = 0.71) and PD size (R(2) = 0.97), while area strain correlated with PD size only (R(2) = 0.67), and other measures were not significantly correlated with flow reserve or PD size. CONCLUSION: Quantitative wall-motion analysis using ∆3DFS is effective for detecting critical stenoses during DSE, performing similar to 3D strain, and provides potentially useful information on the size and location of a perfusion defect.

B-cell aortic homing and atheroprotection depend on Id3.

RATIONALE: B cells are abundant in the adventitia of normal and diseased vessels. Yet, the molecular and cellular mechanisms mediating homing of B cells to the vessel wall and B-cell effects on atherosclerosis are poorly understood. Inhibitor of differentiation-3 (Id3) is important for atheroprotection in mice and polymorphism in the human ID3 gene has been implicated as a potential risk marker of atherosclerosis in humans. Yet, the role of Id3 in B-cell regulation of atherosclerosis is unknown. OBJECTIVE: To determine if Id3 regulates B-cell homing to the aorta and atheroprotection and identify molecular and cellular mechanisms mediating this effect. METHODS AND RESULTS: Loss of Id3 in Apoe(-/-) mice resulted in early and increased atherosclerosis. Flow cytometry revealed a defect in Id3(-/-) Apoe(-/-) mice in the number of B cells in the aorta but not the spleen, lymph nodes, and circulation. Similarly, B cells transferred from Id3(-/-) Apoe(-/-) mice into B-cell-deficient mice reconstituted spleen, lymph node, and blood similarly to B cells from Id3(+/+) Apoe(-/-) mice, but aortic reconstitution and B-cell-mediated inhibition of diet-induced atherosclerosis was significantly impaired. In addition to retarding initiation of atherosclerosis, B cells homed to regions of existing atherosclerosis, reduced macrophage content in plaque, and attenuated progression of disease. The chemokine receptor CCR6 was identified as an important Id3 target mediating aortic homing and atheroprotection. CONCLUSIONS: Together, these results are the first to identify the Id3-CCR6 pathway in B cells and demonstrate its role in aortic B-cell homing and B-cell-mediated protection from early atherosclerosis.

COVE-1: A Phase 2, Open-Label Study to Evaluate Efficacy and Safety and the Optimal Regimen of VP-102, a Proprietary Drug-Device Product Containing Topical Cantharidin (0.7% w/v) Under Occlusion for the Treatment of Common Warts.

INTRODUCTION: Verrucae vulgaris, or common warts, is a common skin condition for which there is no US Food and Drug Administration-approved treatment. Compounded cantharidin has been used to treat warts for years but lacks a controlled formulation, consistent application schedule and methods, and robust safety and efficacy studies. VP-102 is a proprietary drug-device combination product containing a topical formulation of 0.7% (w/v) cantharidin in a single-use delivery device. This objective of the phase 2 study was to evaluate the efficacy, safety, tolerability, and optimal regimen of VP-102 in the treatment of common warts. METHODS: In this open-label trial, participants aged ≥ 2 years with one to six common warts were administered VP-102 topically to treatable common warts once every 14 days (Cohort 1) or once every 21 days in conjunction with paring (Cohort 2), for up to four treatments. Participants were evaluated through to day 84 (Cohort 1) or day 147 (Cohort 2). The primary endpoint was the percentage of participants with complete clearance of all treatable common warts (baseline and new) at day 84. Secondary endpoints included percentage of participants achieving complete clearance of all treatable common warts at other visits. Safety assessments included treatment-emergent adverse events (TEAEs), including local skin reactions (LSRs). RESULTS: A total of 21 and 35 participants were enrolled in Cohort 1 and Cohort 2, respectively. Complete clearance at day 84 was seen in 19.0% of participants in Cohort 1 and 51.4% of those in Cohort 2. The most common TEAEs were expected LSRs and included application site vesicles, pain, pruritus, erythema, and scab. Most LSRs were mild or moderate in severity. CONCLUSION: VP-102 showed efficacy in complete clearance of common warts from baseline to day 84, as well as at follow-up visits. Due to the higher percentage of patients exhibiting complete clearance in Cohort 2, the treatment regimen of Cohort 2 will be pursued in future studies. TEAEs were expected due to the pharmacodynamic action of cantharidin, a vesicant. Clinical Trials ID: NCT03487549.

Phase II, Double-Blind, Vehicle-Controlled Study to Determine the Cantharidin Dose Regimen, Efficacy, Safety, and Tolerability of VP-102 in Subjects with External Genital Warts.

BACKGROUND: External genital warts are caused by various subtypes of the human papilloma virus and spread through direct skin-to-skin contact. Approximately 1% of the US population have external genital warts. Although cantharidin has been used to treat external genital warts for decades, there are no US Food and Drug Administration-approved cantharidin products and no reliable or controlled sources of cantharidin available. VP-102 is a drug-device combination product containing cantharidin (0.7% w/v) in a single-use shelf-stable applicator. OBJECTIVE: The objective of this randomized, double-blind, vehicle-controlled, phase II clinical trial was to determine the optimal regimen for the treatment, safety, and efficacy of VP-102 in external genital warts. METHODS: The study was conducted in two parts. Part A was dose finding and Part B was performed following the completion of Part A for a safety and efficacy evaluation. Following completion of Part A, 6-h and 24-h VP-102 regimens under occlusion were selected to be evaluated in Part B. RESULTS: Pooled results from Part B and Part A of the 6-h and 24-h VP-102 treatment regimens showed that 36.7% and 33.3% of participants achieved complete clearance of all treatable external genital warts at the end of treatment vs 4.2% (p < 0.0048) and 0% (p < 0.0075) with the vehicle. Adverse events experienced by the VP-102-treated participants were consistent with the pharmacodynamic action of cantharidin as a vesicant and were primarily mild to moderate in severity. The most common adverse events included application-site vesicles, pain, and erythema. No participants discontinued the study because of adverse events and no serious adverse events were deemed treatment related. CONCLUSIONS: The adverse event profile and efficacy of VP-102 under occlusion demonstrated in this study support the conclusion that a 6-h or up to 24-h exposure regimen represents an acceptable risk:benefit profile and justifies the conduct of a larger vehicle-controlled phase III study in external genital warts. CLINICAL TRIAL REGISTRATION: NCT03981822, actual study start date: 25 June, 2019; actual primary completion date: 21 May, 2020; actual study completion date: 8 July, 2020.

Reduction in myocardial infarct size at 48 hours after brief intravenous infusion of ATL-146e, a highly selective adenosine A2A receptor agonist.

This study was undertaken to determine whether the myocardial infarct-sparing effect of ATL-146e, a selective adenosine A(2A) receptor agonist, persists without a rebound effect for at least 48 h and to determine the optimal duration of ATL-146e treatment in anesthetized dogs. Reperfusion injury after myocardial infarction (MI) is associated with inflammation lasting 24-48 h that contributes to ongoing myocyte injury. We previously showed that an ATL-146e infusion, starting just before reperfusion, decreased inflammation and infarct size in dogs examined 2 h after MI without increasing coronary blood flow. In the present study, adult dogs underwent 90 min of left anterior descending coronary artery occlusion. Thirty minutes before reperfusion, ATL-146e (0.01 microg x kg(-1) x min(-1); n = 21) or vehicle (n = 12) was intravenously infused and continued for 2.5 h (protocol 1) or 24 h (protocol 2). At 48 h after reperfusion hearts were excised and assessed for histological risk area and infarct size. Infarct size based on triphenyltetrazolium chloride (TTC) staining as a percentage of risk area was significantly smaller in ATL-146e-treated vs. control dogs (16.7 +/- 3.7% vs. 33.3 +/- 6.2%, P < 0.05; protocol 1). ATL-146e reduced neutrophil accumulation into infarcted myocardium of ATL-146e-treated vs. control dogs (30 +/- 7 vs. 88 +/- 16 cells/high-power field, P < 0.002). ATL-146e infusion for 24 h (protocol 2) conferred no significant additional infarct size reduction compared with 2.5 h of infusion. A 2.5-h ATL-146e infusion initiated 30 min before reperfusion results in marked, persistent (48 h) reduction in infarct size as a percentage of risk area in dogs with a reduction in infarct zone neutrophil infiltration. No significant further benefit was seen with a 24-h infusion.

Tc-99m-N-MPO: novel cationic Tc-99m radiotracer for myocardial perfusion imaging.

BACKGROUND: Technetium 99m-N-MPO ([Tc-99m-N(mpo)(PNP5)](+)) is a cationic Tc-99m nitrido complex. The objective of this study is to evaluate its potential as a new radiotracer for myocardial perfusion imaging. METHODS AND RESULTS: Biodistribution studies were performed in Sprague-Dawley rats and guinea pigs to compare the myocardial uptake and excretion kinetics of Tc-99m-N-MPO from noncardiac organs, such as the liver and lungs, with those of the known cationic Tc-99m radiotracers: Tc-99m-N-DBODC5 and Tc-99m-sestamibi. Planar imaging was performed in Sprague-Dawley rats to evaluate the utility of Tc-99m-N-MPO as a myocardial perfusion imaging agent. Metabolism studies were carried out by use of both Sprague-Dawley rats and guinea pigs. In general, the heart uptake of Tc-99m-N-MPO was between that of Tc-99m-sestamibi and Tc-99m-N-DBODC5 over the 2-hour study period. However, the heart-liver ratio of Tc-99m-N-MPO (12.75 +/- 3.34) at 30 minutes after injection was more than twice that of Tc-99m-N-DBODC5 (6.01 +/- 1.45) and approximately 4 times higher than that of Tc-99m-sestamibi (2.90 +/- 0.22). The heart uptake and heart-liver ratio of Tc-99m-N-MPO and Tc-99m-sestamibi in guinea pigs were significantly lower than those obtained in Sprague-Dawley rats. The metabolism studies demonstrated no detectable Tc-99m-N-MPO metabolites in the urine and feces samples of the Sprague-Dawley rats at 120 minutes after injection. In guinea pigs no Tc-99m-N-MPO metabolites were detected in the urine at 120 minutes, but only approximately 60% of Tc-99m-N-MPO remained intact in the feces samples. In contrast, there was no intact Tc-99m-sestamibi detected in urine samples, and less than 15% of Tc-99m-sestamibi remained intact in the feces samples. Planar imaging studies indicated that clinically useful images of the heart may be obtained as early as 15 minutes after injection of Tc-99m-N-MPO. CONCLUSION: The combination of favorable organ biodistribution and myocardial uptake with rapid liver clearance makes Tc-99m-N-MPO a very promising myocardial perfusion radiotracer worthy of further evaluation in various preclinical animal models.

Organ biodistribution and myocardial uptake, washout, and redistribution kinetics of Tc-99m N-DBODC5 when injected during vasodilator stress in canine models of coronary stenoses.

BACKGROUND: Technetium 99m N-DBODC5 is a new myocardial perfusion tracer shown to exhibit high heart uptake and rapid liver clearance in normal rats. The objectives of this canine study were (1) to compare the organ biodistribution and myocardial uptake, washout, and redistribution kinetics of Tc-99m N-DBODC5 with Tc-99m sestamibi over a period of 3 hours in a more clinically relevant large animal species and (2) to compare the myocardial uptake of Tc-99m N-DBODC5 with thallium 201 when co-injected during vasodilator stress in dogs with coronary stenoses. METHODS AND RESULTS: At peak adenosine-induced hyperemia, 10 dogs with critical left anterior descending artery stenoses received either Tc-99m N-DBODC5 (n = 6) or Tc-99m sestamibi (n = 4) and microspheres, followed by serial imaging and blood sampling over a period of 3 hours. Another 14 dogs with either critical (n = 7) or mild (n = 7) left anterior descending artery stenoses underwent simultaneous injection of Tc-99m N-DBODC5, Tl-201, and microspheres during peak vasodilator stress. Like sestamibi, Tc-99m N-DBODC5 showed good myocardial uptake with slow washout and minimal redistribution over a period of 3 hours (P = not significant); however, Tc-99m N-DBODC5 cleared more rapidly from the liver (heart-lung ratio at 30 minutes, 0.92+/-0.11 versus 0.51 +/- 0.05; P < .05). When injected during hyperemic flow, the myocardial extraction plateau for Tc-99m N-DBODC5 was lower than that for Tl-201 and was intermediate between Tc-99m sestamibi and Tc-99m tetrofosmin. CONCLUSIONS: Excellent organ biodistribution and myocardial uptake and clearance kinetic properties, combined with rapid liver clearance and a favorable flow-extraction relationship, make Tc-99m N-DBODC5 a very promising new myocardial perfusion imaging agent.

Assessment of myocardial inflammation produced by experimental coronary occlusion and reperfusion with 99mTc-RP517, a new leukotriene B4 receptor antagonist that preferentially labels neutrophils in vivo.

BACKGROUND: 99mTc-RP517 is a new leukotriene B4 (LTB4) receptor antagonist developed for imaging acute inflammation or infection. A unique property of 99mTc-RP517 is its ability to label white blood cells in vivo after intravenous injection. The goals of this study were to determine relative 99mTc-RP517 binding to human leukocyte subtypes and the 99mTc-RP517 uptake pattern in canine myocardium where inflammation was induced by either coronary occlusion and reperfusion or tumor necrosis factor alpha (TNFalpha) 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 99mTc-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. 99mTc-RP517 was injected intravenously in anesthetized, open-chest dogs before coronary occlusion (90 minutes) and reperfusion (120 minutes) (n=9) or before intramyocardial TNFalpha injection (n=3). Ex vivo images of heart slices were acquired. The left ventricle was divided into 72 segments for flow and 99mTc-RP517 uptake analysis. There was an inverse exponential relationship between 99mTc-RP517 uptake and occlusion flow (r=0.73). In the same 15 segments, 99mTc-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, 99mTc-RP517 uptake correlated with myeloperoxidase and was observed on ex vivo images, indicating that this tracer may have potential as an inflammation-imaging agent.

Influence of propranolol, enalaprilat, verapamil, and caffeine on adenosine A(2A)-receptor-mediated coronary vasodilation.

OBJECTIVES: The study was done to determine the effects of propranolol, enalaprilat, verapamil, and caffeine on the vasodilatory properties of the adenosine A(2A)-receptor agonist ATL-146e (ATL). BACKGROUND: ATL is a new adenosine A(2A)-receptor agonist proposed as a vasodilator for myocardial stress perfusion imaging. Beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, and calcium blockers are commonly used for the treatment of coronary artery disease (CAD), and their effect on ATL-mediated vasodilation is unknown. Dietary intake of caffeine is also common. METHODS: In 19 anesthetized, open-chest dogs, hemodynamic responses to bolus injections of ATL (1.0 microg/kg) and adenosine (60 microg/kg) were recorded before and after administration of propranolol (1.0 mg/kg, ATL only), enalaprilat (0.3 mg/kg, ATL only), caffeine (5.0 mg/kg, ATL only), and verapamil (0.2 mg/kg bolus, ATL and adenosine). RESULTS: Neither propranolol nor enalaprilat attenuated the ATL-mediated vasodilation (225 +/- 86% and 237 +/- 67% increase, respectively, p = NS vs. control). Caffeine had an inhibitory effect (97 +/- 28% increase, p < 0.05 vs. control). Verapamil blunted both ATL- and adenosine-induced vasodilation (63 +/- 20% and 35 +/- 7%, respectively, p < 0.05 vs. baseline), and also inhibited the vasodilation induced by the adenosine triphosphate-sensitive potassium (K(ATP)) channel activator pinacidil. CONCLUSIONS: Beta-blockers and ACE inhibitors do not reduce the maximal coronary flow response to adenosine A(2A)-agonists, whereas verapamil attenuated this vasodilation through inhibition of K(ATP) channels. The inhibitory effect of verapamil and K(ATP) channel inhibitors like glybenclamide on pharmacologic stress using adenosine or adenosine A(2A)-receptor agonists should be evaluated in the clinical setting to determine their potential for reducing the sensitivity of CAD detection with perfusion imaging.

Arbutamine stress perfusion imaging in dogs with critical coronary artery stenoses: (99m)Tc-sestamibi versus (201)Tl.

UNLABELLED: Having previously shown that dobutamine reduces (99m)Tc-methoxyisobutylisonitrile (sestamibi [MIBI]) uptake in normal myocardium by elevating intracellular calcium, we hypothesized that arbutamine, which has less inotropic effect than dobutamine, might cause less reduction in MIBI uptake, thereby improving defect contrast. In this study using a canine model, we compared the effects of arbutamine stress on myocardial blood flow, myocardial MIBI uptake, and systolic thickening in the presence of a coronary artery stenosis. METHODS: Arbutamine was infused (0.5-250 ng/kg/min) in 8 open-chest dogs with critical coronary stenoses that abolished flow reserve. At the time of peak arbutamine effect, MIBI (296 MBq), (201)Tl (27.75 MBq), and microspheres were coinjected. The dogs were killed 5 min later, and myocardial tracer activities and flow were quantified by well counting. Ex vivo imaging of heart slices was also performed. RESULTS: Arbutamine increased mean heart rate, peak positive left ventricular pressure and its first time-derivative, and normal-zone myocardial thickening. Stenotic zone flow and thickening did not increase during arbutamine infusion. MIBI uptake versus flow was significantly lower than (201)Tl uptake at the same flow values. By imaging, defect magnitude (stenotic/normal) was greater for (201)Tl than MIBI (0.57 vs. 0.77; P < 0.001) [corrected]. CONCLUSION: In the presence of coronary stenoses that abolished regional flow reserve, myocardial uptake of MIBI, compared with (201)Tl, significantly underestimated the arbutamine-induced flow heterogeneity. The attenuation of MIBI uptake and diminished defect contrast during arbutamine stress were comparable with those previously reported for dobutamine stress.

Molecular imaging identifies regions with microthromboemboli during primary angioplasty in acute coronary thrombosis.

UNLABELLED: Microthromboemboli (MTE) may contribute to the no-reflow phenomenon in acute myocardial infarction (AMI) either spontaneously or after primary percutaneous transluminal coronary angioplasty (PTCA). We hypothesized that myocardial MTE in acute coronary syndromes can be identified on imaging by in vivo (99m)Tc labeling of the coronary thrombus with a compound that binds to the glycoprotein IIb/IIIa present on activated platelets (DMP-444). METHODS: Fifteen dogs underwent left anterior descending coronary artery (LAD) injury in to produce thrombus, whereas 5 control dogs had LAD ligation. Before recanalization, the risk area (RA) and myocardial blood flow (MBF) were measured, and in vivo thrombus labeling was performed using (99m)Tc-labeled DMP-444. Nine of the 15 LAD injury dogs had occlusive thrombus on angiography and underwent PTCA. MBF measurements were repeated 30 and 60 min after recanalization, and (99m)Tc autoradiography (hot spot imaging) was performed ex vivo to determine the extent and magnitude of MTE. RESULTS: The ratio of hot spot size to RA size was higher in the 9 LAD injury dogs with thrombus compared with the 6 dogs with no thrombus (90% +/- 22% vs. 42% +/- 16%; P = 0.005). In control dogs, this ratio was significantly lower (29% +/- 11%; P = 0.05). (99m)Tc activity within the RA was higher in 8 of the 15 coronary injury dogs with AMI compared with those without AMI (1.8 +/- 0.48 vs. 1.24 +/- 0.22; P = 0.02). CONCLUSION: MTE can be detected and quantified after primary PTCA. The infarct size is proportional to the magnitude and extent of MTE, indicating that MTE may contribute to the AMI. Thus, in vivo thrombus labeling during reperfusion may provide important information in patients with AMI that may lead to better adjuvant therapy during PTCA.

Effects of increased lipid concentration and hyperemic blood flow on the intrinsic myocardial washout kinetics of (99m)TcN-NOET.

UNLABELLED: Bis(N-ethoxy,N-ethyldithiocarbamato)nitrido technetium (V) ((99m)Tc) ((99m)TcN-NOET) is a myocardial perfusion imaging agent demonstrating significant redistribution and currently in phase III clinical trials. Previous studies have suggested that (99m)TcN-NOET is bound intravascularly. Therefore, we sought to determine whether modifications in the vascular compartment would provide further insights into the mechanisms of (99m)TcN-NOET myocardial washout and redistribution. METHODS: (99m)TcN-NOET cardiac washout was studied ex vivo in 15 isolated perfused rat hearts after bolus injection (1.5 MBq) in the absence (n = 6) or presence of bovine serum albumin ([BSA] 0.03%) with (n = 5) or without (n = 4) bound lipids. The intrinsic myocardial washout of the tracer was also studied in vivo in 6 dogs after intracoronary bolus injection of the tracer (0.75 MBq) before and after hyperlipidemia induced by intravenous administration of 300 mL of 20% intralipids (n = 3) or hyperemia induced by intravenous infusion of the adenosine A(2A) receptor agonist ATL-146e (0.3 micro g/kg/min; n = 6). RESULTS: On isolated hearts, there was no significant myocardial washout of (99m)TcN-NOET with Krebs-Henseleit buffer. Addition of BSA without bound lipids resulted in a significant cardiac washout of the tracer (P < 0.001 by repeated measures ANOVA). The presence of lipids bound to BSA further accelerated the washout rate of (99m)TcN-NOET (half-life [t(1/2)], 431.5 +/- 23.2 min vs. 242.9 +/- 63.2 min; P < 0.05). In vivo in dogs, intralipid administration significantly increased the intrinsic washout rate of (99m)TcN-NOET (t(1/2), 108.0 +/- 23.9 min vs. 51.8 +/- 11.8 min; P < 0.05). In addition, vasodilatation with ATL-146e resulted in a 4.9-fold increase in coronary flow (P < 0.05 vs. baseline) and a significantly faster intrinsic (99m)TcN-NOET myocardial washout (t(1/2), 81.1 +/- 12.1 min vs. 40.7 +/- 7.3 min; P < 0.05). CONCLUSION: The myocardial washout kinetics of (99m)TcN-NOET are affected by a variety of intravascular factors, supporting the hypothesis that the tracer is most likely localized on the vascular endothelium. The potential impact of variations in circulating lipid levels among patients on clinical imaging with (99m)TcN-NOET requires further investigation.

99mTc-N-DBODC5, a new myocardial perfusion imaging agent with rapid liver clearance: comparison with 99mTc-sestamibi and 99mTc-tetrofosmin in rats.

UNLABELLED: (99m)Tc-[bis (dimethoxypropylphosphinoethyl)-ethoxyethylamine (PNP5)]-[bis (N-ethoxyethyl)-dithiocarbamato (DBODC)] nitride (N-PNP5-DBODC or N-DBODC5) is a new monocationic myocardial perfusion tracer. We sought to compare the myocardial uptake and clearance kinetics and organ biodistribution of (99m)Tc-N-DBODC5 with (99m)Tc-sestamibi and (99m)Tc-tetrofosmin. METHODS: Seventy-five anesthetized Sprague-Dawley rats were injected intravenously with 22.2-29.6 MBq (99m)Tc-N-DBODC5 (n = 25), (99m)Tc-sestamibi (n = 25), or (99m)Tc-tetrofosmin (n = 25). Rats were euthanized at either 2, 10, 20, 30, or 60 min after injection and gamma-well counting was performed on excised organ (heart, lung, and liver) and blood samples. In 3 additional rats, serial in vivo whole-body gamma-camera imaging with each tracer was performed. RESULTS: (99m)Tc-N-DBODC5 cleared rapidly from the blood pool. At 2 min after injection, (99m)Tc-N-DBODC5 blood activity was significantly lower than either (99m)Tc-sestamibi or (99m)Tc-tetrofosmin (P < 0.01) and remained lower over 60 min. Myocardial (99m)Tc-N-DBODC5 uptake was rapid (2.9% +/- 0.1% injected dose/g at 2 min), and there was no significant clearance over 60 min, similar to (99m)Tc-sestamibi and (99m)Tc-tetrofosmin. All 3 tracers exhibited rapid lung clearance. Importantly, (99m)Tc-N-DBODC5 cleared more rapidly from the liver than either (99m)Tc-sestamibi or (99m)Tc-tetrofosmin. As early as 30 min after injection, (99m)Tc-N-DBODC5 heart-to-liver ratio was 5.7 +/- 1.0 versus 1.6 +/- 0.1 and 2.9 +/- 0.3 for (99m)Tc-sestamibi and (99m)Tc-tetrofosmin (P < 0.05). By 60 min, (99m)Tc-N-DBODC5 heart-to-liver ratio further increased to 18.4 +/- 2.0 compared with 2.6 +/- 0.2 and 5.8 +/- 0.7 for (99m)Tc-sestamibi and (99m)Tc-tetrofosmin (P < 0.001). The rapid blood pool, lung, and liver clearance of (99m)Tc-N-DBODC5 resulted in excellent-quality myocardial images within 30 min after injection. CONCLUSION: (99m)Tc-N-DBODC5 is a promising new myocardial perfusion tracer with superior biodistribution properties. The rapid (99m)Tc-N-DBODC5 liver clearance may shorten the duration of imaging protocols by allowing earlier image acquisition and may markedly reduce the problem of photon scatter from the liver into the inferoapical wall on myocardial images.

Glucose regulation of load-induced mTOR signaling and ER stress in mammalian heart.

BACKGROUND: Changes in energy substrate metabolism are first responders to hemodynamic stress in the heart. We have previously shown that hexose-6-phosphate levels regulate mammalian target of rapamycin (mTOR) activation in response to insulin. We now tested the hypothesis that inotropic stimulation and increased afterload also regulate mTOR activation via glucose 6-phosphate (G6P) accumulation. METHODS AND RESULTS: We subjected the working rat heart ex vivo to a high workload in the presence of different energy-providing substrates including glucose, glucose analogues, and noncarbohydrate substrates. We observed an association between G6P accumulation, mTOR activation, endoplasmic reticulum (ER) stress, and impaired contractile function, all of which were prevented by pretreating animals with rapamycin (mTOR inhibition) or metformin (AMPK activation). The histone deacetylase inhibitor 4-phenylbutyrate, which relieves ER stress, also improved contractile function. In contrast, adding the glucose analogue 2-deoxy-d-glucose, which is phosphorylated but not further metabolized, to the perfusate resulted in mTOR activation and contractile dysfunction. Next we tested our hypothesis in vivo by transverse aortic constriction in mice. Using a micro-PET system, we observed enhanced glucose tracer analog uptake and contractile dysfunction preceding dilatation of the left ventricle. In contrast, in hearts overexpressing SERCA2a, ER stress was reduced and contractile function was preserved with hypertrophy. Finally, we examined failing human hearts and found that mechanical unloading decreased G6P levels and ER stress markers. CONCLUSIONS: We propose that glucose metabolic changes precede and regulate functional (and possibly also structural) remodeling of the heart. We implicate a critical role for G6P in load-induced mTOR activation and ER stress.