Igal Madar

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

PurposeMitochondrial 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 (Δψm). This suggests that Δψm measurement can be highly useful for diagnostic purposes. In the current study, we characterized the capability of the novel PET agent 18F-fluorobenzyl triphenylphosphonium (18F-FBnTP) to assess Δψm, compared with the well-established voltage sensor 3H-tetraphenylphosphonium (3H-TPP).Methods18F-FBnTP and 3H-TPP uptake under conditions known to alter Δψm and plasma membrane potential (Δψp) was assayed in the H345 lung carcinoma cell line. 18F-FBnTP biodistribution was assessed in CD1 mice using dynamic PET and ex vivo gamma well counting.Results18F-FBnTP and 3H-TPP demonstrated similar uptake kinetics and plateau concentrations in H345 cells. Stepwise membrane depolarization resulted in a linear decrease in 18F-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 3H-TPP (−0.63±0.06 and 0.96±0.05, respectively). Selective collapse of Δψm caused a substantial decrease in cellular uptake for 18F-FBnTP (81.6±8.1%) and 3H-TPP (85.4±6.7%), compared with control. Exposure to the proapoptotic staurosporine, known to collapse Δψm, resulted in a decrease of 68.7±10.1% and 71.5±8.4% in 18F-FBnTP and 3H-TPP cellular uptake, respectively. 18F-FBnTP accumulated mainly in kidney, heart and liver.Conclusion18F-FBnTP is a mitochondria-targeting PET radiopharmaceutical responsive to alterations in membrane potential with voltage-dependent performance similar to that of 3H-TPP. 18F-FBnTP is a promising new voltage sensor for detection of physiological and pathological processes associated with mitochondrial dysfunction, such as apoptosis, using PET.

Quantification of brain μ-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.

Imaging of δ opioid receptors in human brain by N1′‐ ([11C]methyl)naltrindole and PET

Recently, we have developed the positron emitting radiotracer N1′‐([11C]methyl)naltrindole ([11C]MeNTI) and demonstrated its high selectivity for δ opioid receptors in the mouse brain [Lever et al. (1992) Eur. J. Pharmacol., 216:449‐450]. In the present study, we examined the selectivity of [11C]MeNTI for the δ opioid receptor in the human brain, using positron emission tomography (PET). The regional kinetics and distribution as well as the pharmacology confirmed the selectivity of [11C]MeNTI for δ opioid receptor in the human brain. First, the regional kinetics of [11C]MeNTI are in accordance with the density of the δ opioid receptor. Rapid washout in receptor‐poor areas and prolonged retention in receptor‐rich areas were observed. Second, the regional distribution of [11]MeNTI correlated well (r = 0.91) with the in vitro distribution of δ opioid sites but not with μ or κ site densities (r ≤ 0.008 or r ≤ 0.014, respectively). [11C]MeNTI binding was highest in regions of the neocortex (insular, parietal, frontal, cingulate, and occipital), caudate nucleus, and putamen. Binding was intermediate in the amygdala and lowest in the cerebellum and thalamus. Third, studies using the competitive antagonist naltrexone demonstrated the inhibition of [11C]MeNTI binding. Naltrexone inhibition of [11C]MeNTI binding was most effective in δ receptor‐rich regions, and its inhibitory potency correlated well (r = 0.88) with the regional distribution of δ opioid sites. [11C]MeNTI is the first radioligand which selectively labels δ opioid receptors in vivo in the human brain following systemic administration. The availability of [11C]MeNTI will enable a receptor specific analysis of the role of [11C]MeNTI receptors in normal and abnormal human brain.

Stable Delineation of the Ischemic Area by the PET Perfusion Tracer 18F-Fluorobenzyl Triphenyl Phosphonium After Transient Coronary Occlusion

18F-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, 18F-FBnTP (92.5 MBq) and 201Tl-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 18F-FBnTP PET was performed on a subgroup of animals (n = 4). Results: 18F-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, R2 = 0.94, y = blue-dye defect, x = 18F-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, 201Tl 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 18F-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 18F-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.

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

BackgroundPartial 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 ResultsThe 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.ResultsResults 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.ConclusionsPVC 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.

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.