Brown Adipose Tissue Response Dynamics: In Vivo Insights with the Voltage Sensor 18F-Fluorobenzyl Triphenyl Phosphonium.

Brown adipose tissue (BAT) thermogenesis is an emerging target for prevention and treatment of obesity. Mitochondria are the heat generators of BAT. Yet, there is no noninvasive means to image the temporal dynamics of the mitochondrial activity in BAT in vivo. Here, we report a technology for quantitative monitoring of principal kinetic components of BAT adaptive thermogenesis in the living animal, using the PET imaging voltage sensor 18F-fluorobenzyltriphenylphosphonium (18F-FBnTP). 18F-FBnTP targets the mitochondrial membrane potential (ΔΨm)--the voltage analog of heat produced by mitochondria. Dynamic 18F-FBnTP PET imaging of rat's BAT was acquired just before and during localized skin cooling or systemic pharmacologic stimulation, with and without administration of propranolol. At ambient temperature, 18F-FBnTP demonstrated rapid uptake and prolonged steady-state retention in BAT. Conversely, cold-induced mitochondrial uncoupling resulted in an immediate washout of 18F-FBnTP from BAT, which was blocked by propranolol. Specific variables of BAT evoked activity were identified and quantified, including response latency, magnitude and kinetics. Cold stimulation resulted in partial washout of 18F-FBnTP (39.1%±14.4% of basal activity). The bulk of 18F-FBnTP washout response occurred within the first minutes of the cold stimulation, while colonic temperature remained nearly intact. Drop of colonic temperature to shivering zone did not have an additive effect. The ß3-adrenergic agonist CL-316,243 elicited 18F-FBnTP washout from BAT of kinetics similar to those caused by cold stimulation. Thus, monitoring ΔΨm in vivo using 18F-FBnTP PET provides insights into the kinetic physiology of BAT. 18F-FBnTP PET depicts BAT as a highly sensitive and rapidly responsive organ, emitting heat in short burst during the first minutes of stimulation, and preceding change in core temperature. 18F-FBnTP PET provides a novel set of quantitative metrics highly important for identifying novel therapeutic targets at the mitochondrial level, for developing means to maximize BAT mass and activity, and assessing intervention efficacy.

Compensation for spill-in and spill-out partial volume effects in cardiac PET imaging.

BACKGROUND: Partial volume effects (PVEs) in PET imaging result in incorrect regional activity estimates due to both spill-out and spill-in from activity in neighboring regions. It is important to compensate for both effects to achieve accurate quantification. In this study, an image-based partial volume compensation (PVC) method was developed and validated for cardiac PET. METHODS AND RESULTS: The method uses volume-of-interest (VOI) maps segmented from contrast-enhanced CTA images to compensate for both spill-in and spill-out in each VOI. The PVC method was validated with simulation studies and also applied to images of dog cardiac perfusion PET data. The PV effects resulting from cardiac motion and myocardial uptake defects were investigated and the efficacy of the proposed PVC method in compensating for these effects was evaluated. RESULTS: Results indicate that the magnitude and the direction of PVEs in cardiac imaging change over time. This affects the accuracy of activity distributions estimates obtained during dynamic studies. The defect regions have different PVEs as compared to the normal myocardium. Cardiac motion contributes around 10% to the PVEs. PVC effectively removed both spill-in and spill-out in cardiac imaging. CONCLUSIONS: PVC improved left ventricular wall uniformity and quantitative accuracy. The best strategy for PVC was to compensate for the PVEs in each cardiac phase independently and treat severe uptake defects as independent regions from the normal myocardium.

Stable delineation of the ischemic area by the PET perfusion tracer 18F-fluorobenzyl triphenyl phosphonium after transient coronary occlusion.

UNLABELLED: (18)F-fluorobenzyl triphenyl phosphonium (FBnTP) has recently been introduced as a myocardial perfusion PET agent. We used a rat model of transient coronary occlusion to determine the stability of the perfusion defect size over time and the magnitude of redistribution. METHODS: Wistar rats (n = 15) underwent thoracotomy and 2-min occlusion of the left coronary artery (LCA), followed by reperfusion. During occlusion, (18)F-FBnTP (92.5 MBq) and (201)Tl-thallium chloride (0.74 MBq) were injected intravenously. One minute before the animals were sacrificed at 5, 45, and 120 min after reperfusion, the LCA was occluded again and 2% Evans blue was injected intravenously to determine the ischemic territory. The hearts were excised, frozen, and sliced for serial dual-tracer autoradiography and histology. Dynamic in vivo (18)F-FBnTP PET was performed on a subgroup of animals (n = 4). RESULTS: (18)F-FBnTP showed stable ischemic defects at all time points after tracer injection and reperfusion. The defects matched the blue dye defect (y = 0.97x+1.5, R(2) = 0.94, y = blue-dye defect, x = (18)F-FBnTP defect). Count density analysis showed no defect fill-in at 45 min but slightly increased activity at 120 min (LCA/remote uptake ratio = 0.19 ± 0.02, 0.19 ± 0.05, and 0.34 ± 0.06 at 5, 45, and 120 min, respectively, P < 0.05). For comparison, (201)Tl showed complete redistribution at 120 min (LCA/remote = 0.42 ± 0.04, 0.72 ± 0.03, and 0.97 ± 0.05 at 5, 45, and 120 min, respectively, P < 0.001). Persistence of the (18)F-FBnTP defect over time was confirmed by in vivo dynamic small-animal PET. CONCLUSION: In a transient coronary occlusion model, perfusion defect size using the new PET agent (18)F-FBnTP remained stable for at least 45 min and matched the histologically defined ischemic area. This lack of significant redistribution suggests a sufficient time window for future clinical protocols with tracer injection remote from the scanner, such as in a stress testing laboratory or chest pain unit.

18F-fluorobenzyl triphenyl phosphonium: a noninvasive sensor of brown adipose tissue thermogenesis.

UNLABELLED: Recent studies have proposed activation of brown adipose tissue (BAT) thermogenesis as a new strategy to combat obesity. Currently, there is no effective noninvasive imaging agent to directly detect unstimulated BAT and quantify the core mechanism of mitochondrial thermogenesis. We investigated an approach to detect BAT depots and monitor thermogenesis using the mitochondria-targeting voltage sensor radiolabeled fluorobenzyltriphenyl phosphonium (FBnTP). METHODS: (18)F-FBnTP, (14)C-FBnTP, (18)F-FDG, and (99m)Tc-sestamibi uptake in BAT at room temperature (n = 8) and cold-treated (n = 8) Lewis rats was assayed. The effect of the cold condition on (18)F-FBnTP retention in BAT was assessed in 8 treated and 16 control rats. The effect of the noradrenergic inhibitor propranolol on (14)C-FBnTP response to cold stimulation was investigated in an additional 8 treated and 8 control mice. RESULTS: At room temperature, (18)F-FBnTP accumulated in BAT to an extent similar to that in the heart, second only to the kidney and twice as much as (99m)Tc-sestamibi. Prior exposure to cold (4°C) for 4 h resulted in an 82% decrease of (14)C-FBnTP uptake and an 813% increase of (18)F-FDG uptake in BAT. (99m)Tc-sestamibi uptake was not affected by cold. Administration of (18)F-FBnTP at room temperature 60 min before 120 and 240 min of exposure to cold resulted in marked washout of the tracer from BAT. Propranolol significantly diminished the effect of cold on (14)C-FBnTP and (18)F-FDG uptake into BAT. CONCLUSION: The intense uptake of (18)F-FBnTP into BAT at room temperature and the response to cold stimulation suggest the unique potential advantage of (18)F-FBnTP not only in detecting unstimulated BAT at high contrast but also in quantifying the mitochondrial thermogenic activity. (18)F-FBnTP PET may serve as a useful technique to assess BAT volume and function.

The role of P-glycoprotein in intestinal transport versus the BBB transport of tetraphenylphosphonium.

Tetraphenylphosphonium (TPP), a phosphonium cation, is a promising means for tumor imaging. A major contributor to the pharmacokinetics of phosphonium cations is the efflux transporter P-glycoprotein (P-gp). For this application it is important to ascertain the influence of the multidrug resistance system on TPP. Therefore, our aim was to characterize the interaction of TPP with P-gp, in vitro and in in vivo models. P-gp-mediated transport of [3H]-TPP was assessed in Caco-2 cells and ex vivo in rat intestinal wall by the use of a diffusion cell system. The distribution of [3H]-TPP across the blood-brain barrier (BBB) was studied in rats and mice treated with P-gp modulators and in Mdr-1a/b((-/-)) knockout mice. The in vitro permeability coefficient of basolateral-to-apical transfer (PappB-A) of [3H]-TPP was 8-fold greater than apical-to-basolateral (PappA-B) coefficient, indicative of net mucosal secretion. A concentration dependent decrease of this secretion was obtained by the P-gp substrate verapamil, while no effect was evident by the MRP2 inhibitor MK-571 and the BCRP inhibitor FTC. [3H]-TPP transfer across rat jejunum wall was directional and concentration-dependent. 2,4-Dinitrophenol, cyclosporin A (CsA), verapamil and PSC-833 enhanced A-B transport of TPP 3.6-fold, 4-fold, 4.6-fold and 5.3-fold respectively. Likewise, PappA-B of [3H]-TPP was 5-fold greater in P-gp knockout mice than in controls. In vivo, PSC-833, P-gp inhibitor, significantly increased the uptake of [3H]-TPP in the liver, heart, small intestine and the lungs but not the brain. Similar results were obtained in P-gp knockout mice. Our study demonstrates that P-gp mediates TPP efflux in vitro and in vivo; however, the consistently poor BBB permeation of TPP in all in vivo studies including P-gp knockout animals indicates that it is most likely mediated by other mechanisms. These findings are important for optimized clinical application of TPP as an imaging agent in cancer.

Detection and quantification of the evolution dynamics of apoptosis using the PET voltage sensor 18F-fluorobenzyl triphenyl phosphonium.

UNLABELLED: Apoptosis is a key mechanism in numerous pathologies. However, there are no effective noninvasive means available for an early detection and quantitative assessment of evolution dynamics of the apoptotic process. Here, we have characterized the ability of the novel PET voltage sensor (18)F-fluorobenzyl triphenyl phosphonium ((18)F-FBnTP) to quantify the time-dependent apoptotic action of the taxanes paclitaxel and docetaxel in vitro and in vivo. METHODS: The duration-dependent treatment effect of paclitaxel on (18)F-FBnTP uptake was assayed in human MDA-MB-231 breast carcinoma cells. The expression of the proapoptotic Bax and antiapoptotic Bcl-2 mitochondrial proteins, release of the apoptogen cytochrome c, and activation of executioner caspase-3 were determined by Western blotting. The fraction of viable cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The effect of docetaxel on (18)F-FBnTP and (18)F-FDG uptake in orthotopic prostate tumors in mice was compared. RESULTS: (18)F-FBnTP cellular uptake in viable cells declined linearly with the increasing duration of paclitaxel treatment, from 3 to 24 h, and plateaued at 48 h. The extent of decrease of (18)F-FBnTP correlated strongly with the Bax-to-Bcl-2 ratio (R(2) = 0.83) and release of cytochrome c (R(2) = 0.92), but preceded in time the caspase-3 cleavage. The P-glycoprotein blocker verapamil did not interfere with (18)F-FBnTP cellular uptake. (18)F-FBnTP prostate tumor contrast was greater than (18)F-FDG prostate tumor contrast. Docetaxel caused a marked decrease (52.4%) of (18)F-FBnTP tumor uptake, within 48 h, whereas (18)F-FDG was much less affected (12%). CONCLUSION: The voltage sensor (18)F-FBnTP is a viable means for quantification of paclitaxel pharmacodynamics. (18)F-FBnTP permits the detection of paclitaxel apoptotic action in vivo earlier than does (18)F-FDG. (18)F-FBnTP may afford a novel approach for early detection and quantitative assessment of the cumulative-effect kinetics of proapoptotic drugs and conditions using PET.

Characterization of membrane potential-dependent uptake of the novel PET tracer 18F-fluorobenzyl triphenylphosphonium cation.

PURPOSE: Mitochondrial dysfunction has been attributed a critical role in the etiology and pathogenesis of numerous diseases, and is manifested by alterations of the organelle's membrane potential (Deltapsi(m)). This suggests that Deltapsi(m) measurement can be highly useful for diagnostic purposes. In the current study, we characterized the capability of the novel PET agent (18)F-fluorobenzyl triphenylphosphonium ((18)F-FBnTP) to assess Deltapsi(m), compared with the well-established voltage sensor (3)H-tetraphenylphosphonium ((3)H-TPP). METHODS: (18)F-FBnTP and (3)H-TPP uptake under conditions known to alter Deltapsi(m) and plasma membrane potential (Deltapsi(p)) was assayed in the H345 lung carcinoma cell line. (18)F-FBnTP biodistribution was assessed in CD1 mice using dynamic PET and ex vivo gamma well counting. RESULTS: (18)F-FBnTP and (3)H-TPP demonstrated similar uptake kinetics and plateau concentrations in H345 cells. Stepwise membrane depolarization resulted in a linear decrease in (18)F-FBnTP cellular uptake, with a slope (-0.58+/-0.06) and correlation coefficient (0.94+/-0.07) similar (p>0.17) to those measured for (3)H-TPP (-0.63+/-0.06 and 0.96+/-0.05, respectively). Selective collapse of Deltapsi(m) caused a substantial decrease in cellular uptake for (18)F-FBnTP (81.6+/-8.1%) and (3)H-TPP (85.4+/-6.7%), compared with control. Exposure to the proapoptotic staurosporine, known to collapse Deltapsi(m), resulted in a decrease of 68.7+/-10.1% and 71.5+/-8.4% in (18)F-FBnTP and (3)H-TPP cellular uptake, respectively. (18)F-FBnTP accumulated mainly in kidney, heart and liver. CONCLUSION: (18)F-FBnTP is a mitochondria-targeting PET radiopharmaceutical responsive to alterations in membrane potential with voltage-dependent performance similar to that of (3)H-TPP. (18)F-FBnTP is a promising new voltage sensor for detection of physiological and pathological processes associated with mitochondrial dysfunction, such as apoptosis, using PET.

Assessment of severity of coronary artery stenosis in a canine model using the PET agent 18F-fluorobenzyl triphenyl phosphonium: comparison with 99mTc-tetrofosmin.

UNLABELLED: Myocardial perfusion imaging plays an important role in clinical management of coronary artery disease, but the most commonly used radionuclides significantly underestimate the severity of coronary artery stenosis. The objective of this study was to evaluate the potential clinical utility of the PET compound (18)F-fluorobenzyl triphenyl phosphonium ((18)F-FBnTP) and characterize its capacity to assess the severity of coronary artery stenosis in a canine model in vivo and ex vivo. METHODS: (18)F-FBnTP myocardial uptake was measured in 17 dogs with various degrees of stenosis of the left anterior descending (LAD) or circumflex (LCx) coronary arteries during adenosine vasodilation, using dynamic PET and gamma-well counting. True myocardial blood flow in ischemic (IS) and nonischemic (NIS) beds of the left ventricle was determined with radioactive microspheres. (18)F-FBnTP and (99m)Tc-tetrofosmin activities were compared in 8 dogs ex vivo. RESULTS: The quantitative assessment of the perfusion defect was significantly (P < 0.03) more accurate with (18)F-FBnTP than with (99m)Tc-tetrofosmin, in mild (IS/NIS; 0.72 +/- 0.08, 0.93 +/- 0.07, respectively, mean +/- SE) and severe stenosis (0.42 +/- 0.05, 0.64 +/- 0.08, respectively), compared with microsphere flow (mild, 0.43 +/- 0.06; severe, 0.22 +/- 0.04). The IS/NIS ratio of both radionuclides correlated linearly with microsphere flow disparity with a similar slope. Flow defect contrast was 2.7 times greater for (18)F-FBnTP than for (99m)Tc-tetrofosmin, as inferred from the regression line intercept (0.14 vs. 0.38, respectively). The (18)F-FBnTP PET IS/NIS ratio (mild, 0.70 +/- 0.04; severe, 0.46 +/- 0.02), did not differ statistically (P >or= 0.330) from that measured ex vivo. A nearly identical qualitative and quantitative estimate of stenosis severity was obtained by early, short (5-15-min) and delayed, prolonged (30-60-min) (18)F-FBnTP PET scans. The stenotic area measured by PET was 16% smaller than that defined by tissue staining. CONCLUSION: (18)F-FBnTP PET is a promising new technology for rapid noninvasive detection and assessment of perfusion defect severity in the myocardium.

Imaging delta- and mu-opioid receptors by PET in lung carcinoma patients.

UNLABELLED: In the present study, we measured the kinetics and distribution in vivo of the selective delta-opioid antagonist 11C-methylnaltrindole (11C-MeNTI) and the mu-opioid agonist 11C-carfentanil (11C-CFN) in patients with lung carcinoma using PET. METHODS: Paired measurements of 11C-MeNTI and 11C-CFN binding were performed in biopsy-proven small-cell (n = 2), squamous (n = 2), and adenocarcinoma (n = 3) lung cancer patients. Dynamic PET scans of increasing duration (0.5-8 min) were acquired over 90 min after an intravenous bolus injection of 370 MBq of tracer. Time-activity curves for tumor and normal lung parenchyma binding were generated using the region-of-interest (ROI) method. The mean activity at equilibrium was measured, and the specific-to-nonspecific binding ratio (tumor - lung)/lung was calculated. Four of 7 patients underwent an additional static 18F-FDG PET scan for clinical indications. Three of 7 patients underwent surgery, and stained sections of tumor were inspected for inflammation, necrosis, and scar tissue. RESULTS: Increased binding of 11C-MeNTI and 11C-CFN was detected in all tumor types studied. 11C-MeNTI binding in tumor and healthy lung tissue was significantly more intense than that of 11C-CFN. The average specific-to-nonspecific binding ratio across cell types for 11C-MeNTI (4.32 +/- 1.31; mean +/- SEM) was greater than that of 11C-CFN (2.42 +/- 1.17) but lower than that of 18F-FDG (7.74 +/- 0.53). Intravenous naloxone produced 50% and 44% decreases in the specific-to-nonspecific binding ratios of 11C-MeNTI and 11C-CFN, respectively. CONCLUSION: These data provide in vivo evidence for the presence of delta- and mu-opioid receptor types in the 3 major human lung carcinomas and suggest the suitability of 11C-MeNTI and 11C-CFN as investigational probes of lung carcinoma biology.

Characterization of uptake of the new PET imaging compound 18F-fluorobenzyl triphenyl phosphonium in dog myocardium.

UNLABELLED: 18F-Labeled p-fluorobenzyl triphenyl phosphonium cation (18F-FBnTP) is a member of a new class of positron-emitting lipophilic cations that may act as myocardial perfusion PET tracers. Here, we characterize the 18F-FBnTP uptake and retention kinetics, in vitro and in vivo, as well as the myocardial and whole-body biodistribution in healthy dogs, using PET. METHODS: Time-dependent accumulation and retention of 18F-FBnTP in myocytes in vitro was studied. Seven anesthetized, mongrel dogs underwent dynamic PET scans of the heart after intravenous administration of 126-240 MBq 18F-FBnTP. In 4 of the 7 dogs, at the completion of a 60-min dynamic scan, whole-body scans (4 bed positions, 5-min emission and 3-min transmission per bed) were acquired. Arterial blood samples were collected at 0, 5, 10, 20, 30, and 60 min after administration, plasma activity was counted, and high-performance liquid chromatographic analyses for metabolites were performed. The extent of defluorination was assessed by measuring 18F-FBnTP bone uptake in mice, compared with 18F-fluoride. RESULTS: The metabolite fraction comprised <5% of total activity in blood at 5 min and gradually increased to 25% at 30 min after injection. In vivo, 18F-FBnTP myocardial concentration reached a plateau level within a few minutes, which was retained throughout the scanning time. In contrast, activity in the blood pool and lungs cleared rapidly (half-life = 19.5 +/- 4.4 and 30.7 +/- 11.6 s, respectively). Liver uptake did not exceed the activity measured in the myocardium. At 60 min, the uptake ratios of left ventricular wall to blood, lung, and liver (mean of 7 dogs) were 16.6, 12.2, and 1.2, respectively. Summation of activity from 5 to 15 min and from 30 to 60 min after injection produced high-quality cardiac images of similar contrast. Circumferential sampling and a polar plot revealed a uniform distribution, near unitary value, throughout the entire myocardium. The mean coefficient of variance, on 30- to 60-min images along the septum-to-anterior wall and the apex-to-base axes was 7.58% +/- 1.04% and 6.11% +/- 0.89% (mean +/- SD; n = 7), respectively, and on 5- to 15-min images was 7.25% +/- 1.43% and 6.12% +/- 1.88%, respectively. 18F-FBnTP whole-body distribution was highly organ specific with the kidney cortex being the major target organ, followed by the heart and the liver. CONCLUSION: 18F-FBnTP is a promising new radionuclide for cardiac imaging using PET with rapid kinetics, uniform myocardial distribution, and favorable organ biodistribution.

PET imaging of opioid receptors in pain: progress and new directions.

The endogenous opioid system plays a central role in pain. Recent advances have permitted imaging of opioid receptors by PET in human subjects while experiencing pain and detection of changes in receptor occupancy. The ability to perform these types of studies is dependent on the development of opioid tracer ligands labeled with positron emitting isotopes. This article follows the development and radiochemistry of opioid tracer molecules through their use in human subjects and subsequent application to the study of pain. The role of mu, delta and kappa opioid receptors in pain is reviewed. Occupancy changes in mu receptors have been observed with PET in human subjects subjected to experimental pain paradigms. The implication of this approach to the study of pain and pain syndromes, and possible clinical applications, is also addressed.

Quantification of brain mu-opioid receptors with [11C]carfentanil: reference-tissue methods.

[(11)C]Carfentanil (CFN) is a mu-opioid agonist used for in vivo positron emission tomography (PET) studies of mu-opioid receptors. Previously, a tissue-ratio method was validated for the quantification of CFN binding. However, since that initial validation, several other blood independent (reference-tissue) methods have become available. To evaluate these methods, CFN PET studies with arterial blood sampling were acquired in six healthy male control subjects. Specific binding estimates obtained from reference-tissue methods were compared to those obtained with a more rigorous blood input modeling technique. It was determined that both a graphical method, and a simplified reference tissue model, were more accurate than the tissue-ratio method for quantification of CFN binding.

Preferential accumulation of (3)H-tetraphenylphosphonium in non-small cell lung carcinoma in mice: comparison with (99m)Tc-MIBI.

UNLABELLED: We have previously shown enhanced accumulation of the delocalized lipophilic cation (11)C-triphenylmethylphosphonium in canine brain glioma, suggesting its potential use for tumor staging in humans using PET. Here, we extend our studies of phosphonium cations to nonbrain tumors and characterize the biodistribution and tumor specificity of (3)H-tetraphenylphosphonium ((3)H-TPP) in non-small cell lung carcinoma in mice. METHODS: (3)H-TPP accumulation in isolated malignant lung nodules of the Lewis lung carcinoma (LLC) cell line, in LLC-bearing lung, and in control lung was measured at various intervals after inoculation. Tumor uptake and biodistribution of (3)H-TPP were compared with those of (99m)Tc-methoxyisobutylisonitrile (MIBI). RESULTS: (3)H-TPP accumulation in LLC nodules at 14 d was significantly greater than that in controls, peaked at 21 d, and declined to lower values at 28 d after injection. At 21 d after injection, (3)H-TPP uptake in LLC nodules was greater than that in control lung tissue and in LLC-bearing lung tissue-by 549% and 230%, respectively-whereas (99m)Tc-MIBI nodule uptake was greater by 90% and 30%, respectively. CONCLUSION: The high tumor accumulation and sensitivity to the phase of tumor development suggest the potential use of radiolabeled phosphonium analogs for in vivo tumor staging and as a tool for investigating tumor evolution.