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Dopamine released by amphetamine decreases the in vivo binding of PET radioligands to the dopamine D(2) receptor. Although concentrations of extracellular dopamine largely return to baseline within 1 to 2 h after amphetamine treatment, radioligand binding remains decreased for several hours. The purpose of this study was to determine whether the prolonged decrease of radioligand binding after amphetamine administration is caused by receptor internalization. To distinguish dopamine displacement from receptor internalization, we used wild-type and arrestin3 (arr3) knockout mice, which are incapable of internalizing D(2) receptors. In addition, we used both the D(2) selective agonist [(11)C]MNPA (which is thought to bind to the high affinity state of the receptor) and the D(2) selective antagonist [(18)F]fallypride (which does not differentiate between high and low affinity state). After an initial baseline scan, animals were divided in three groups for a second scan: either 30 min or 4 h after amphetamine administration (3 mg/kg, i.p.) or as retest. At 30 min, [(11)C]MNPA showed greater displacement than [(18)F]fallypride, but each radioligand gave similar displacement in knockout and wild-type mice. At 4 h, the binding of both radioligands returned to baseline in arr3 knockout mice, but remained decreased in wild-type mice. Radioligand binding was unaltered on retest scanning. Our results suggest that the prolonged decrease of radioligand binding after amphetamine is mainly due to internalization of the D(2) receptor rather than dopamine displacement. In addition, this study demonstrates the utility of small animal PET to study receptor trafficking in vivo in genetically modified mice., (Published by Elsevier Inc.)

Unlabelled: Permeability-glycoprotein (P-gp), an efflux transporter in several organs, acts at the blood-brain barrier to protect the brain from exogenous toxins. P-gp almost completely blocks brain entry of the PET radiotracer (11)C-N-desmethyl-loperamide ((11)C-dLop). We examined the ability of (11)C-dLop to quantify P-gp function in humans after increasing doses of tariquidar, an inhibitor of P-gp., Methods: Seventeen healthy volunteers had a total of 23 PET scans with (11)C-dLop at baseline and after increasing doses of tariquidar (2, 4, and 6 mg/kg intravenously). A subset of subjects received PET with (15)O-H(2)O to measure cerebral blood flow. Brain uptake of (11)C-dLop was quantified in 2 ways. Without blood data, uptake was measured as area under the time-activity curve in the brain from 10 to 30 min (AUC(10-30)). With arterial blood data, brain uptake was quantified with compartmental modeling to estimate the rates of entry into (K(1)) and efflux from (k(2)) the brain., Results: Brain uptake of radioactivity was negligible at baseline and increased only slightly (approximately 30%) after 2 mg of tariquidar per kilogram. In contrast, 4 and 6 mg of tariquidar per kilogram increased brain uptake 2- and 4-fold, respectively. Greater brain uptake reflected greater brain entry (K(1)), because efflux (k(2)) and cerebral blood flow did not differ between tariquidar-treated and untreated subjects. In the subjects who received the highest dose of tariquidar (and had the highest brain uptake), regional values of K(1) correlated linearly with absolute cerebral blood flow, consistent with high single-pass extraction of (11)C-dLop. AUC(10-30) correlated linearly with K(1)., Conclusion: P-gp function at the blood-brain barrier in humans can be quantified using PET and (11)C-dLop. A simple measure of brain uptake (AUC(10-30)) may be used as a surrogate of the fully quantified rate constant for brain entry (K(1)) and thereby avoid arterial sampling. However, to dissect the function of P-gp itself, both brain uptake and the influx rate constant must be corrected for radiotracer delivery (blood flow).

Introduction: [(11)C]Loperamide and [(11)C]N-desmethyl-loperamide ([(11)C]dLop) have been proposed as radiotracers for imaging brain P-glycoprotein (P-gp) function. A major route of [(11)C]loperamide metabolism is N-demethylation to [(11)C]dLop. We aimed to test whether inhibition of CYP3A4 with ketoconazole might reduce the metabolism of [(11)C]loperamide and [(11)C]dLop in mice, and thereby improve the quality of these radiotracers., Methods: Studies were performed in wild-type and P-gp knockout (mdr-1a/b -/-) mice. During each of seven study sessions, one pair of mice, comprising one wild-type and one knockout mouse, was pretreated with ketoconazole (50 mg/kg, ip), while another such pair was left untreated. Mice were sacrificed at 30 min after injection of [(11)C]loperamide or [(11)C]dLop. Whole brain and plasma samples were measured for radioactivity and analyzed with radio-high-performance liquid chromatography., Results: Ketoconazole increased the plasma concentrations of [(11)C]loperamide and its main radiometabolite, [(11)C]dLop, by about twofold in both wild-type and knockout mice, whereas the most polar radiometabolite was decreased threefold. Furthermore, ketoconazole increased the brain concentrations of [(11)C]loperamide and the radiometabolite [(11)C]dLop by about twofold in knockout mice, and decreased the brain concentrations of the major and most polar radiometabolite in wild-type and knockout mice by 82% and 49%, respectively. In contrast, ketoconazole had no effect on plasma and brain distribution of administered [(11)C]dLop and its radiometabolites in either wild-type or knockout mice, except to increase the low plasma [(11)C]dLop concentration. The least polar radiometabolite of [(11)C]dLop was identified with LC-MS(n) as the N-hydroxymethyl analog of [(11)C]dLop and this also behaved as a P-gp substrate., Conclusion: In this study, ketoconazole (50 mg/kg, ip) proved partially effective for inhibiting the N-demethylation of [(11)C]loperamide in mouse in vivo but had relatively smaller or no effect on [(11)C]dLop., (Published by Elsevier Inc.)

Unlabelled: Ten percent of humans lack specific binding of [(11)C]PBR28 to 18 kDa translocator protein (TSPO), a biomarker for inflammation. "Non-binders" have not been reported using another TSPO radioligand, [(11)C]-(R)-PK 11195, despite its use for more than two decades. This study asked two questions: (1) What is the cause of non-binding to PBR28? and (2) Why has this phenomenon not been reported using [(11)C]-(R)-PK 11195?, Methods: Five binders and five non-binders received whole-body imaging with both [(11)C]-(R)-PK 11195 and [(11)C]PBR28. In vitro binding was performed using leukocyte membranes from binders and non-binders and the tritiated versions of the ligand. Rhesus monkeys were imaged with [(11)C]-(R)-PK 11195 at baseline and after blockade of TSPOs., Results: Using [(11)C]PBR28, uptake in all five organs with high densities of TSPO (lung, heart, brain, kidney, and spleen) was 50% to 75% lower in non-binders than in binders. In contrast, [(11)C]-(R)-PK 11195 distinguished binders and non-binders in only heart and lung. For the in vitro assay, [(3)H]PBR28 had more than 10-fold lower affinity to TSPO in non-binders than in binders. The in vivo specific binding of [(11)C]-(R)-PK 11195 in monkey brain was approximately 80-fold lower than that reported for [(11)C]PBR28., Conclusions: Based on binding of [(3)H]PK 11195 to leukocyte membranes, both binders and non-binders express TSPO. Non-binding to PBR28 is caused by its low affinity for TSPO in non-binders. Non-binding may be differentially expressed in organs of the body. The relatively low in vivo specific binding of [(11)C]-(R)-PK 11195 may have obscured its detection of non-binding in peripheral organs., (Copyright 2009. Published by Elsevier Inc.)

Rolipram is a selective inhibitor of phosphodiesterase-4 (PDE4), and positron emission tomography (PET) using [(11)C]rolipram can monitor the in vivo activity of this enzyme that is part of the cAMP second messenger cascade. cAMP-dependent protein kinase (PKA) phosphorylates PDE4 and increases both enzyme activity and affinity for rolipram. In the present PET study, we examined effects of PKA modulators in conscious rats on the binding of [(11)C](R)-rolipram in comparison to the much less active enantiomer [(11)C](S)-rolipram. Unilateral injection of a PKA activator (dibutyryl-cAMP) and a PKA inhibitor (Rp-adenosine-3',5'-cyclic monophosphorothioate) into the striatum significantly increased and decreased, respectively, the binding of [(11)C](R)-rolipram. These effects were not caused by changes in blood flow or delivery of radioligand to brain, since these agents had no effect on the binding of [(11)C](S)-rolipram binding. These results support the value of measuring in vivo [(11)C](R)-rolipram binding in brain to assess responses to physiological or pharmacological challenges to the cAMP second messenger system.

Unlabelled: As a PET biomarker for inflammation, translocator protein (18 kDa) (TSPO) can be measured with an (18)F-labeled aryloxyanilide, (18)F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ((18)F-PBR06), in the human brain. The objective of this study was to estimate the radiation absorbed doses of (18)F-PBR06 based on biodistribution data in humans., Methods: After the injection of (18)F-PBR06, images were acquired from head to thigh in 7 healthy humans. Urine was collected at various time points. Radiation absorbed doses were estimated by the MIRD scheme., Results: Moderate to high levels of radioactivity were observed in organs with high densities of TSPO and in organs of metabolism and excretion. Bone had low levels of radioactivity. The effective dose was 18.5 muSv/MBq., Conclusion: The effective dose of (18)F-PBR06, compared with other (18)F radioligands, was moderate. This radioligand had negligible defluorination, as indirectly assessed by bone radioactivity. Doses to the gallbladder wall and spleen may limit the amount of permissible injected radioactivity.

In vitro and ex vivo measurements have shown that the binding of the selective high-affinity agonist, S14506 (1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxy-naphthyl)piperazine), to 5-HT(1A) receptors, is similar in affinity (K(d) = 0.79 nM) and extent (B(max)) to that of the antagonist, WAY 100635. We aimed to test whether S14506, labeled with a positron-emitter, might serve as a radioligand for imaging brain 5-HT(1A) receptors in vivo with positron emission tomography (PET). Here we evaluated [(11)C]S14506 and [(18)F]S14506 in rat and rhesus monkey in vivo. After intravenous administration of [(11)C]S14506 into rat, radioactivity entered brain, reaching 210% SUV at 2 min. Radioactivity uptake into brain was higher (~ 350% SUV) in rats pre-treated with the P-glycoprotein (P-gp) inhibitor, cyclosporin A. In rhesus monkey, peak brain uptake of radioactivity after administration of [(11)C]S14506 or [(18)F]S14506 was also moderate and for [(11)C]S14506 increased from ~ 170% SUV after 7 min, to 240% SUV in a monkey pre-treated with the P-gp inhibitor, tariquidar. The ratios of radioactivity in 5-HT(1A) receptor-rich regions, such as cingulate or hippocampus to that in receptor-poor cerebellum reached between 1.35 and 1.5 at 60 min for both [(11)C]S14506 and [(18)F]S14506. [(11)C]S14506 gave one major polar radiometabolite in monkey plasma, and [(18)F]S14506 gave three and two more polar radiometabolites in rat and monkey plasma, respectively. The rat radiometabolites of [(18)F]S14506 did not accumulate in brain. [(18)F]S14506 was not radiodefluorinated in monkey. Thus, despite high-affinity and lack of troublesome brain radiometabolites, both [(11)C]S14506 and [(18)F]S14506 were ineffective for imaging rat or monkey brain 5-HT(1A) receptors in vivo, even under P-gp inhibited conditions. Explanations for the failure of these radioligands are offered.

Unlabelled: We recently demonstrated that (11)C-MePPEP, a PET ligand for CB(1) receptors, has such high uptake in the human brain that it can be imaged for 210 min and that receptor density can be quantified as distribution volume (V(T)) using the gold standard of compartmental modeling. However, (11)C-MePPEP had relatively poor retest and intersubject variabilities, which were likely caused by errors in the measurements of radioligand in plasma at low concentrations by 120 min. We sought to find an analog of (11)C-MePPEP that would provide more accurate plasma measurements. We evaluated several promising analogs in the monkey brain and chose the (18)F-di-deutero fluoromethoxy analog ((18)F-FMPEP-d(2)) to evaluate further in the human brain., Methods: (11)C-FMePPEP, (18)F-FEPEP, (18)F-FMPEP, and (18)F-FMPEP-d(2) were studied in 5 monkeys with 10 PET scans. We calculated V(T) using compartmental modeling with serial measurements of unchanged parent radioligand in arterial plasma and radioactivity in the brain. Nonspecific binding was determined by administering a receptor-saturating dose of rimonabant, an inverse agonist at the CB(1) receptor. Nine healthy human subjects participated in 17 PET scans using (18)F-FMPEP-d(2), with 8 subjects having 2 PET scans to assess retest variability. To identify sources of error, we compared intersubject and retest variability of brain uptake, arterial plasma measurements, and V(T)., Results: (18)F-FMPEP-d(2) had high uptake in the monkey brain, with greater than 80% specific binding, and yielded less radioactivity uptake in bone than did (18)F-FMPEP. High brain uptake with (18)F-FMPEP-d(2) was also observed in humans, in whom V(T) was well identified within approximately 60 min. Retest variability of plasma measurements was good (16%); consequently, V(T) had a good retest variability (14%), intersubject variability (26%), and intraclass correlation coefficient (0.89). V(T) increased after 120 min, suggesting an accumulation of radiometabolites in the brain. Radioactivity accumulated in the skull throughout the entire scan but was thought to be an insignificant source of data contamination., Conclusion: Studies in monkeys facilitated our development and selection of (18)F-FMPEP-d(2), compared with (18)F-FMPEP, as a radioligand demonstrating high brain uptake, high percentage of specific binding, and reduced uptake in bone. Retest analysis in human subjects showed that (18)F-FMPEP-d(2) has greater precision and accuracy than (11)C-MePPEP, allowing smaller sample sizes to detect a significant difference between groups.

[11C]MePPEP is a high affinity, CB1 receptor-selective, inverse agonist that has been studied in rodents and monkeys. We examined the ability of [11C]MePPEP to quantify CB1 receptors in human brain as distribution volume calculated with the "gold standard" method of compartmental modeling and compared results with the simple measure of brain uptake. A total of 17 healthy subjects participated in 26 positron emission tomography (PET) scans, with 8 having two PET scans to assess retest variability. After injection of [11C]MePPEP, brain uptake of radioactivity was high (e.g., 3.6 SUV in putamen at approximately 60 min) and washed out very slowly. A two-tissue compartment model yielded values of distribution volume (which is proportional to receptor density) that were both well identified (SE 5%) and stable between 60 and 210 min. The simple measure of brain uptake (average concentration of radioactivity between 40 and 80 min) had good retest variability ( approximately 8%) and moderate intersubject variability (16%, coefficient of variation). In contrast, distribution volume had two-fold greater retest variability ( approximately 15%) and, thus, less precision. In addition, distribution volume had three-fold greater intersubject variability ( approximately 52%). The decreased precision of distribution volume compared to brain uptake was likely due to the slow washout of radioactivity from brain and to noise in measurements of the low concentrations of [11C]MePPEP in plasma. These results suggest that brain uptake can be used for within subject studies (e.g., to measure receptor occupancy by medications) but that distribution volume remains the gold standard for accurate measurements between groups.

P-glycoprotein (P-gp), an efflux transporter, controls the pharmacokinetics of various compounds under physiological conditions. P-gp-mediated drug efflux has been suggested as playing a role in various disorders, including multidrug-resistant cancer and medication-refractory epilepsy. However, P-gp inhibition has had, to date, little or no clinically significant effect in multidrug-resistant cancer. To enhance our understanding of its in vivo function under pathophysiological conditions, substrates of P-gp have been radiolabeled and imaged using single-photon emission computed tomography (SPECT) and positron emission tomography (PET). To accurately quantify P-gp function, a radiolabeled P-gp substrate should be selective for P-gp, produce a large signal after P-gp blockade, and generate few radiometabolites that enter the target tissue. Furthermore, quantification of P-gp function via imaging requires pharmacological inhibition of P-gp, which requires knowledge of P-gp density at the target site. By meeting these criteria, imaging can elucidate the function of P-gp in various disorders and improve the efficacy of treatments.

Background: Positron emission tomography (PET) can localize and quantify neurokinin-1 (NK(1)) receptors in brain using the nonpeptide antagonist radioligand, [(18)F]SPA-RQ. We sought to determine if patients with panic disorder have altered density of NK(1) receptors in brain because of their history of recurrent panic attacks. We also sought to determine if a drug-induced panic attack releases substance P in brain, as measured by decreased binding of [(18)F]SPA-RQ., Methods: Positron emission tomography scans with [(18)F]SPA-RQ were performed in 14 patients with panic disorder and 14 healthy subjects. Of these two groups, 7 patients and 10 healthy subjects were scanned twice, once at baseline and once after injection of doxapram, a drug that induces panic attacks., Results: NK(1) receptor binding in patients (n = 14) compared with that in healthy subjects (n = 14) was significantly decreased by 12% to 21% in all brain regions. Doxapram effectively produced panic attacks in 6 of 7 patients with panic disorder but only 2 of 10 healthy subjects. Doxapram caused no significant change of [(18)F]SPA-RQ binding in either patients or healthy subjects., Conclusions: Although induction of a panic attack has no significant effect on [(18)F]SPA-RQ binding to NK(1) receptors, patients with panic disorder have widespread reduction of NK(1) receptor binding in brain.

Unlabelled: Translocator protein (TSPO) (18 kDa), formerly called the peripheral benzodiazepine receptor, is upregulated on activated microglia and macrophages and is, thus, a biomarker of inflammation. We previously reported that an (11)C-labeled aryloxyanilide (half-life, 20 min) was able to quantify TSPOs in the healthy human brain. Because many PET centers would benefit from a longer-lived (18)F-labeled radioligand (half-life, 110 min), the objective of this study was to evaluate the ability of a closely related aryloxyanilide ((18)F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline [(18)F-PBR06]) to quantify TSPOs in the healthy human brain., Methods: A total of 9 human subjects were injected with (18)F-PBR06 (approximately 185 MBq) and scanned for 5 h, with rest periods outside the camera. The concentrations of (18)F-PBR06, separated from radiometabolites, were measured in arterial plasma., Results: Modeling of regional brain and plasma data showed that a 2-tissue-compartment model was superior to a 1-tissue-compartment model. Even if data for all time points were used for the fitting, concentrations of brain activity measured with PET were consistently greater than the modeled values at late (280-300 min) but not at early time points. The greater values may have been caused by the slow accumulation of radiometabolites in the brain. To determine an adequate time for more accurate measurement of distribution volume (V(T)), which is the summation of receptor binding and nondisplaceable activity, we investigated which scan duration would be associated with maximal or near-maximal identifiability. We found that a scan of 120 min provided the best identifiability of V(T) (approximately 2%). The images showed no significant defluorination., Conclusion: (18)F-PBR06 can quantify TSPOs in the healthy human brain using 120 min of image acquisition and concurrent measurements of radioligand in plasma. Although brain activity is likely contaminated with radiometabolites, the percentage contamination is thought to be small (<10%), because values of distribution volume are stable during 60-120 min and vary by less than 10%. (18)F-PBR06 is a longer-lived and promising alternative to (11)C-labeled radioligands to measure TSPOs as a biomarker of inflammation in the brain.

Dopaminergic signaling pathways have been extensively investigated using PET imaging, primarily with antagonist radioligands of D(2) and D(3) dopamine receptors (DARs). Recently, agonist radioligands of D(2)/D(3) DARs have begun to be developed and employed. One such agonist is (R)-2-(11)CH(3)O-N-n-propylnorapomorphine (MNPA). Here, we perform a pharmacological characterization of MNPA using recombinant D(2) and D(3) DARs expressed in HEK293 cells. MNPA was found to robustly inhibit forskolin-stimulated cAMP accumulation to the same extent as dopamine in D(2) or D(3) DAR-transfected cells, indicating that it is a full agonist at both receptors. MNPA is approximately 50-fold more potent than dopamine at the D(2) DAR, but equally potent as dopamine at the D(3) DAR. MNPA competition binding curves in membrane preparations expressing D(2) DARs revealed two binding states of high and low-affinity. In the presence of GTP, only one binding state of low affinity was observed. Direct saturation binding assays using [(3)H]MNPA revealed similar results as with the competition experiments leading to the conclusion that MNPA binds to the D(2) DAR in an agonist-specific fashion. In contrast to membrane preparations, using intact cell binding assays, only one site of low affinity was observed for MNPA and other agonists binding to the D(2) DAR. MNPA was also found to induce D(2) DAR internalization to an even greater extent than dopamine as determined using both cell surface receptor binding assays and confocal fluorescence microscopy. Taken together, our data indicate that the PET tracer, MNPA, is a full and potent agonist at both D(2) and D(3) receptors., (Copyright 2009 Wiley-Liss, Inc.)

Unlabelled: A variety of phosphodiesterases hydrolyze and terminate the effects of the intracellular second messenger 3',5'-cyclic adenosine monophosphate (cAMP). Phosphodiesterase subtype 4 (PDE4) is particularly abundant in the brain and has been imaged with (11)C-(R)-rolipram, a selective inhibitor of PDE4. We sought to measure in vivo both the binding site density (B(max)) and the radioligand affinity (1/K(D)) of (11)C-(R)-rolipram in the rat brain. We also studied 2 critical factors in small-animal PET scans: the influence of anesthesia and the difference in binding under in vivo and in vitro conditions., Methods: In vivo, B(max) and K(D) were measured in PET saturation experiments by the administration of (11)C-(R)-rolipram and various doses of carrier (R)-rolipram in conscious and isoflurane-anesthetized rats. The metabolite-corrected arterial input function was measured in each scan. To image conscious rats, the head of the rat was fixed in a holder and the animals were trained to comply with this apparatus. Bound and free (R)-rolipram levels were calculated under transient equilibrium conditions (i.e., at the time of peak specific binding)., Results: The B(max) and K(D) of conscious rats were significantly greater than those of anesthetized rats, by 29% and 59%, respectively. In addition, the in vitro K(D) was 3-7 times greater than was the in vivo K(D), although the B(max) was similar in both conditions., Conclusion: The in vivo B(max) and K(D) of (R)-rolipram were successfully measured in both conscious and anesthetized rats. K(D) was affected to a greater extent than was B(max) by the 2 conditions. That is, K(D) was increased in the conscious rat, compared with in the anesthetized rat, and K(D) was increased in vitro, compared with in vivo. The current study shows that the rat, a readily available species for research, can be used to measure in vivo both affinity and density of radioligand targets, which can later be directly assessed with standard in vitro techniques.

Unlabelled: P-glycoprotein (P-gp) is a membrane-bound efflux pump that limits the distribution of drugs to several organs of the body. At the blood-brain barrier, P-gp blocks the entry of both loperamide and its metabolite, N-desmethyl-loperamide (N-dLop), and thereby prevents central opiate effects. Animal studies have shown that (11)C-dLop, compared with (11)C-loperamide, is an especially promising radiotracer because it generates negligible radiometabolites that enter the brain. The purposes of this study were to determine whether (11)C-dLop is a substrate for P-gp at the blood-brain barrier in humans and to measure the distribution of radioactivity in the entire body to estimate radiation exposure., Methods: Brain PET scans were acquired in 4 healthy subjects for 90 min and included concurrent measurements of the plasma concentration of unchanged radiotracer. Time-activity data from the whole brain were quantified using a 1-tissue-compartment model to estimate the rate of entry (K(1)) of radiotracer into the brain. Whole-body PET scans were acquired in 8 healthy subjects for 120 min., Results: For brain imaging, after the injection of (11)C-dLop the concentration of radioactivity in the brain was low (standardized uptake value, approximately 15%) and stable after approximately 20 min. In contrast, uptake of radioactivity in the pituitary was about 50-fold higher than that in the brain. The plasma concentration of (11)C-dLop declined rapidly, but the percentage composition of plasma was unusually stable, with the parent radiotracer constituting 85% of total radioactivity after approximately 5 min. The rate of brain entry was low (K(1) = 0.009 +/- 0.002 mL.cm(-3).min(-1); n = 4). For whole-body imaging, as a measure of radiation exposure to the entire body the effective dose of (11)C-dLop was 7.8 +/- 0.6 muSv/MBq (n = 8)., Conclusion: The low brain uptake of radioactivity is consistent with (11)C-dLop being a substrate for P-gp in humans and confirms that this radiotracer generates negligible quantities of brain-penetrant radiometabolites. In addition, the low rate of K(1) is consistent with P-gp rapidly effluxing substrates while they transit through the lipid bilayer. The radiation exposure of (11)C-dLop is similar to that of many other (11)C-radiotracers. Thus, (11)C-dLop is a promising radiotracer to study the function of P-gp at the blood-brain barrier, at which impaired function would allow increased uptake into the brain.

Elevated levels of peripheral benzodiazepine receptors (PBR) are associated with activated microglia in their response to inflammation. Hence, PBR imaging in vivo is valuable for investigating brain inflammatory conditions. Sensitive, easily prepared, and readily available radioligands for imaging with positron emission tomography (PET) are desirable for this purpose. We describe a new 18F-labeled PBR radioligand, namely [18F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([18F]9). [18F]9 was produced easily through a single and highly efficient step, the reaction of [18F]fluoride ion with the corresponding bromo precursor, 8. Ligand 9 exhibited high affinity for PBR in vitro. PET showed that [18F]9 was avidly taken into monkey brain and gave a high ratio of PBR-specific to nonspecific binding. [18F]9 was devoid of defluorination in rat and monkey and gave predominantly polar radiometabolite(s). In rat, a low level radiometabolite of intermediate lipophilicity was identified as [18F]2-fluoro-N-(2-phenoxyphenyl)acetamide ([18F]11). [18F]9 is a promising radioligand for future imaging of PBR in living human brain.

Aberrant neurotransmissions via glutamate and dopamine receptors have been the focus of biomedical research on the molecular basis of psychiatric disorders, but the mode of their interaction is yet to be uncovered. In this study, we demonstrated the pharmacological reversal of methamphetamine-stimulated dopaminergic overflow by suppression of group I metabotropic glutamate (mGlu) receptor in living primates and rodents. In vivo positron emission tomography (PET) was conducted on cynomolgus monkeys and rats using a full agonistic tracer for dopamine D(2/3) receptor, [(11)C]MNPA [(R)-2-(11)CH(3)O-N-n-propylnorapomorphine], and fluctuation of kinetic data resulting from anesthesia was avoided by scanning awake subjects. Excessive release of dopamine induced by methamphetamine and abolishment of this alteration by treatment with an antagonist of group I mGlu receptors, 2-methyl-6-(phenylethynyl)pyridine (MPEP), were measured in both species as decreased binding potential because of increased dopamine and its recovery to baseline levels, respectively. Counteraction of MPEP to the methamphetamine-induced dopamine spillover was also supported neurochemically by microdialysis of unanesthetized rat striatum. Moreover, patch-clamp electrophysiological assays using acute brain slices prepared from rats indicated that direct targets of MPEP mechanistically involved in the effects of methamphetamine are present locally within the striatum. Because MPEP alone did not markedly alter the baseline dopaminergic neurotransmission according to our PET and electrophysiological data, the present findings collectively extend the insights on dopamine-glutamate cross talk from extrastriatal localization of responsible mGlu receptors to intrastriatal synergy and support therapeutic interventions in case of disordered striatal dopaminergic status using group I mGlu receptor antagonists assessable by in vivo imaging techniques.

Unlabelled: 11C-Loperamide is an avid substrate for P-glycoprotein (P-gp), but it is rapidly metabolized to 11C-N-desmethyl-loperamide (11C-dLop), which is also a substrate for P-gp and thereby contaminates the radioactive signal in the brain. Should further demethylation of 11C-dLop occur, radiometabolites with low entry into the brain are generated. Therefore, we evaluated the ability of 11C-dLop to quantify the function of P-gp at the blood-brain barrier in monkeys., Methods: Six monkeys underwent 12 PET scans of the brain, 5 at baseline and 7 after pharmacologic blockade of P-gp. A subset of monkeys also underwent PET scans with 15O-water to measure cerebral blood flow. To determine whether P-gp blockade affected peripheral distribution of 11C-dLop, we measured whole-body biodistribution in 4 monkeys at baseline and after P-gp blockade., Results: The concentration of 11C-dLop in the brain was low under baseline conditions and increased 5-fold after P-gp blockade. This increase was primarily caused by an increased rate of entry into the brain rather than a decreased rate of removal from the brain. With P-gp blockade, uptake of radioactivity among brain regions correlated linearly with blood flow, suggesting a high single-pass extraction. After correction for cerebral blood flow, the uptake of 11C-dLop was fairly uniform among brain regions, suggesting that the function of P-gp is fairly uniformly distributed in the brain. On whole-body imaging, P-gp blockade significantly affected distribution of radioactivity only to the brain and not to other visually identified source organs. The effective dose estimated for humans was approximately 9 microSv/MBq., Conclusion: PET with 11C-dLop can quantify P-gp function at the blood-brain barrier in monkeys. The single-pass extraction of 11C-dLop is high and requires correction for blood flow to accurately measure the function of this efflux transporter. The low uptake at baseline and markedly increased uptake after P-gp blockade suggest that 11C-dLop will be useful to measure a wide range of P-gp functions at the blood-brain barrier in humans.

N-(4-Fluorobenzyl)-4-[3-(piperidin-1-yl)indole-1-sulfonyl]benzamide] (PipISB, 3) is a selective and high-potency cannabinoid subtype-1 (CB1) receptor inverse agonist. We have previously reported radiosyntheses of [11C]3 and [18F]3. Here, we aimed to evaluate the uptake and CB(1) receptor-specific binding of each radioligand in monkey brain in vivo with positron emission tomography (PET). [11C]3 or [18F]3 was injected intravenously into rhesus or cynomolgus monkey, respectively, and examined with PET at baseline or after pretreatment with a receptor-saturating dose of CB1 receptor-selective ligand (3 for [11C]3 or 8 for [18F]3). In one PET experiment, the dose of 3 was administered at 100 min after [11C]3. Relative plasma concentrations of radioligand and radiometabolites were concurrently measured in baseline experiments with high-performance liquid chromatography. Brain radioactivity uptake was highest in striatum and cerebellum, and it reached 170-270% standardized uptake value (SUV) at 120 min after injection of [11C]3 and 180% SUV at 240 min after injection of [18F]3. Radioactivity was well retained in all CB1 receptor-rich regions. No reference region could be identified for nonspecifically bound radioligand. Under CB1 receptor pretreatment and displacement conditions, initial brain uptakes of radioactivity were similar to those at baseline. Regional brain radioactivity concentrations then became homogeneous and diminished to between 70 and 80% SUV at 120 min after injection of [11C]3 and to 25% SUV at 240 min after injection of [18F]3. [18F]3 was not defluorinated but was metabolized to less lipophilic radiometabolites, as was [11C]3. Hence, [11C]3 and [18F]3 showed high CB1 receptor-specific binding in monkey brain in vivo and merit further investigation as prospective PET radioligands in humans.

Introduction: (S,S)-[(11)C]MeNER ((S,S)-2-(alpha-(2-[(11)C]methoxyphenoxy)benzyl)morpholine) is a positron emission tomography (PET) radioligand recently applied in clinical studies of norepinephrine transporters (NETs) in the human brain in vivo. In view of further assessment of the suitability of (S,S)-[(11)C]MeNER as a NET radioligand, its metabolism and the identity of the in vivo radiometabolites of (S,S)-[(11)C]MeNER are of great interest., Materials and Methods: Thus, PET studies were used to measure brain dynamics of (S,S)-[(11)C]MeNER, and plasma reverse-phase radiochromatographic analysis was performed to monitor and quantify its rate of metabolism. Eighteen healthy human volunteers, five cynomolgus monkeys, and five rats were studied., Results and Discussion: In human subjects, the plasma radioactivity representing (S,S)-[(11)C]MeNER decreased from 88 +/- 5% at 4 min after injection to 82 +/- 7% at 40 min, while a polar radiometabolite increased from 3 +/- 3% to 16 +/- 7% at the same time-points, respectively. A more lipophilic radiometabolite than (S,S)-[(11)C]MeNER decreased from 9 +/- 5% at 4 min to 1 +/- 2% at 40 min. In monkeys, plasma radioactivity representing (S,S)-[(11)C]MeNER decreased from 97 +/- 2% at 4 min to 74 +/- 7% at 45 min, with a polar fraction as the major radiometabolite. A more lipophilic radiometabolite than (S,S)-[(11)C]MeNER, constituted 3 +/- 2% of radioactivity at 4 min and was not detectable later on. In rats, 17 +/- 4% of plasma radioactivity was parent radioligand at 30 min with the remainder comprising mainly a polar radiometabolite. (S,S)-[(11)C]MeNER in rat brain and urine at 30 min after injection were 90% and 4%, respectively. On a brain regional level, parent radioligand ranged from 87.5 +/- 3.9% (57.2 +/- 14.2% SUV [standard uptake values, %injected radioactivity per mL multiplied with animal weight (in g)]; cerebellum) to 92.9 +/- 1.8% (36.1 +/- 4.7% SUV; striatum), with differential distribution of the radiometabolite in the cerebellum (6.7 +/- 0.3% SUV) and the striatum (2.5 +/- 0.3% SUV). Liquid chromatography-mass spectrometry analysis of rat urine identified a hydroxylation product of the methoxyphenoxy ring of (S,S)-MeNER as the main metabolite. In the brain, the corresponding main metabolite was the product from O-de-methylation of (S,S)-MeNER. PET measurements were performed in rats as well as in wild-type and P-gp-knock-out mice. In rats, the brain peak level of radioactivity was found to be very low (65%SUV). In mice, there was only a small difference in peak brain accumulation between P-gp knock-out and wild-type mice (145 vs. 125%SUV) with the following rank order of regional brain radioactivity: cerebellum x thalamus > cortical regions > striatum., Conclusion: It can be concluded that radiometabolites of (S,S)-[(11)C]MeNER are of minor importance in rat and monkey brain imaging. The presence of a transient lipophilic radiometabolite in peripheral human plasma may induce complications with brain imaging, but its kinetics appear favorable in relation to the slow kinetics of (S,S)-[(11)C]MeNER in humans.

Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomography (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 density before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2-[(18)F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([(18)F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was observed in bone, showing release of [(18)F]-fluoride ion from [(18)F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [(18)F]SP203 in rats. To understand the biochemical basis for defluorination, we administered [(18)F]SP203 plus SP203 in rats for ex vivo analysis of metabolites. Radio-high-performance liquid chromatography detected [(18)F]fluoride ion as a major radiometabolite in both brain extract and urine. Incubation of [(18)F]SP203 with brain homogenate also generated this radiometabolite, whereas no metabolism was detected in whole blood in vitro. Liquid chromatography-mass spectrometry analysis of the brain extract detected m/z 548 and 404 ions, assignable to the [M + H](+) of S-glutathione (SP203Glu) and N-acetyl-S-l-cysteine (SP203Nac) conjugates of SP203, respectively. In urine, only the [M + H](+) of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepared by glutathione S-transferase reaction and chemical synthesis, respectively. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [(18)F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand.

Unlabelled: We developed a radioligand, 3-fluoro-5-(2-(2-(18)F-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ((18)F-SP203), for metabotropic glutamate subtype 5 (mGluR5) receptors that showed both promising (high specific binding) and problematic (defluorination) imaging characteristics in animals. The purposes of this initial evaluation in human subjects were to determine whether (18)F-SP203 is defluorinated in vivo (as measured by uptake of radioactivity in the skull) and to determine whether the uptake in the brain can be quantified as distribution volume relative to concentrations of (18)F-SP203 in plasma., Methods: Seven healthy subjects were injected with (18)F-SP203 (323 +/- 87 MBq) and scanned over 5 h, with rest periods outside the camera. The concentrations of (18)F-SP203, separated from radiometabolites, were measured in arterial plasma., Results: The skull was difficult to visualize on PET images in the initial 2 h, because of high radioactivity in the brain. Although radioactivity in the skull and adjacent cortex showed some cross-contamination, the concentration of radioactivity in the skull was less than half of that in the adjacent cortex during the initial 2 h. Modeling of regional brain and plasma data showed that a 2-tissue-compartment model was superior to a 1-tissue-compartment model, consistent with measurable amounts of both receptor-specific and nonspecific binding. The concentrations of activity in the brain measured with PET were consistently greater than the modeled values at late but not early time points and may well have been caused by the slow accumulation of radiometabolites in the brain. To determine an adequate time for more accurate measurement of distribution volume, we selected a scan duration (i.e., 2 h) associated with maximal or near-maximal identifiability. Distribution volume was well identified ( approximately 2%) by only 2 h (and even just 1) of image acquisition., Conclusion: This initial evaluation of (18)F-SP203 in healthy human subjects showed that defluorination is relatively small and that brain uptake can be robustly calculated as distribution volume. The values of distribution volume were well identified and had relatively small variation in this group of 7 subjects. These results suggest that (18)F-SP203 will have good sensitivity to measure mGluR5 receptors for both within-subject studies (e.g., receptor occupancy) and between-subject studies (e.g., patients vs. healthy subjects).

Inflammation in atherosclerotic plaques makes them unstable and can cause thrombosis. Therefore, it is important to detect macrophage activity for clinical management of atherosclerosis. Peripheral benzodiazepine receptor (PBR) is expressed in various tissue and organs including macrophages. In this study, we tested whether inflammation characterized by macrophage infiltration can be detected by PBR binding. Six patients diagnosed as carotid atherosclerosis underwent endarterectomy. Using the fresh frozen sections, presence of PBRs and macrophages was examined by in vitro autoradiography using [(3)H]PK 11195 and immunohistochemical staining of CD68, respectively. All sections showed specific binding of [(3)H]PK 11195, and the staining with CD68 indicating macrophage infiltration. Density and distribution of PBR detected by [(3)H]PK 11195 autoradiography were consistent with those of the immunohistochemical staining. In conclusion, this study demonstrated that macrophage and inflammatory activity in atherosclerotic plaque can be imaged specifically by the binding of PBR indicating future application of PET imaging for PBR.

[(11)C]Loperamide has been proposed for imaging P-glycoprotein (P-gp) function with positron emission tomography (PET), but its metabolism to [N-methyl-(11)C] N-desmethyl-loperamide ([(11)C]dLop; [(11)C]3) precludes quantification. We considered that [(11)C]3 might itself be a superior radiotracer for imaging brain P-gp function and therefore aimed to prepare [(11)C]3 and characterize its efficacy. An amide precursor (2) was synthesized and methylated with [(11)C]iodomethane to give [(11)C]3. After administration of [(11)C]3 to wild-type mice, brain radioactivity uptake was very low. In P-gp (mdr-1a(-/-)) knockout mice, brain uptake of radioactivity at 30 min increased about 3.5-fold by PET measures, and over 7-fold by ex vivo measures. In knockout mice, brain radioactivity was predominantly (90%) unchanged radiotracer. In monkey PET experiments, brain radioactivity uptake was also very low but after P-gp blockade increased more than 7-fold. [(11)C]3 is an effective new radiotracer for imaging brain P-gp function and, in favor of future successful quantification, appears free of extensive brain-penetrant radiometabolites.

Estimates of dopamine D(2/3) receptor occupancy by endogenous dopamine using positron emission tomography (PET) in animals have varied almost threefold. This variability may have been caused by incomplete depletion of dopamine or by the use of antagonist radioligands, which appear less sensitive than agonist radioligands to changes in endogenous dopamine. PET scans were performed in rats with the agonist PET radioligand [(11)C]MNPA ([O-methyl-(11)C]2-methoxy-N-propylnorapomorphine). [(11)C]MNPA was injected as a bolus plus constant infusion to achieve steady-state concentration in the body and equilibrium receptor binding in the brain. Radioligand binding was compared at baseline and after treatment with reserpine plus alpha-methyl-para-tyrosine, which cause approximately 95% depletion of endogenous dopamine. Depletion of dopamine increased radioligand binding in striatum but had little effect in cerebellum. Striatal [(11)C]MNPA binding potential was 0.93 +/- 0.12 at baseline and increased to 1.99 +/- 0.25 after dopamine depletion. Occupancy of D(2/3) receptors by endogenous dopamine at baseline was calculated to be approximately 53%. Striatal binding was displaceable with raclopride, but not with BP 897 (a selective D(3) compound), thus confirming the D(2) receptor specificity of [(11)C]MNPA binding. Radioactivity extracted from rat brain contained only 8-10% radiometabolites and was insignificantly altered by administration of reserpine plus alpha-methyl-para-tyrosine. Hence, dopamine depletion did not increase the PET measurements via an effect on radiotracer metabolism. Our in vivo estimate of dopamine's occupancy of D(2/3) receptors at baseline is higher than that previously reported using antagonist radioligands and PET, but is similar to that reported using agonist radioligands and ex vivo measurements., (Published 2008 Wiley-Liss, Inc.)

With a view to future extension of the use of the agonist radioligand [(11)C]MNPA ([O-methyl-(11)C]2-methoxy-N-propylnorapomorphine) from animals to humans, we performed two positron emission tomography (PET) studies in monkeys. First, we assessed the ability to quantify the brain uptake of [(11)C]MNPA with compartmental modeling. Second, we estimated the radiation exposure of [(11)C]MNPA to human subjects based on whole-body imaging in monkeys. Brain PET scans were acquired for 90 min and included concurrent measurements of the plasma concentration of unchanged radioligand. Time-activity data from striatum and cerebellum were quantified with two methods, a reference tissue model and distribution volume. Whole-body PET scans were acquired for 120 min using four bed positions from head to mid thigh. Regions of interest were drawn on compressed planar whole-body images to identify organs with the highest radiation exposures. After injection of [(11)C]MNPA, the highest concentration of radioactivity in brain was in striatum, with lowest levels in cerebellum. Distribution volume was well identified with a two-tissue compartmental model and was quite stable from 60 to 90 min. Whole-body PET scans showed the organ with the highest radiation burden (muSv/MBq) was the urinary bladder wall (26.0), followed by lungs (22.5), gallbladder wall (21.9), and heart wall (16.1). With a 2.4-h voiding interval, the effective dose was 6.4 muSv/MBq (23.5 mrem/mCi). In conclusion, brain uptake of [(11)C]MNPA reflected the density of D(2/3) receptors, quantified relative to serial arterial measurements, and caused moderate to low radiation exposure., (Published 2008 Wiley-Liss, Inc.)

The decline in motor performance that accompanies advanced age has unclear neurobiological substrates but may relate, in part, to degeneration of the nigrostriatal dopamine system. This research tested the hypothesis that striatal dopamine transporter (DAT) availability in healthy elderly individuals was related to measures of motor performance. Thirty-six healthy volunteers (18 male, 18 female) who ranged in age from 68 to 88 (75.4+/-4.9 years) received a neuropsychological evaluation that included two primary motor measures (tested with dominant hand): (1) simple reaction time (SRT); and (2) finger tapping (FT). Subjects underwent SPECT scanning with [(123)I]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([(123)I]beta-CIT) for measurement of striatal DAT availability. A ratio of specific to nondisplaceable brain uptake (i.e., radical V3 =[striatal-occipital]/occipital), a measure proportional to the binding potential (B(max)/K(D)), was derived. SRT was significantly correlated with striatal DAT availability with or without controlling for the contribution of age. However, contrary to hypothesis, FT was not correlated with striatal DAT availability. Comparison measures, including episodic memory and general intelligence, were also unrelated to striatal DAT availability. These results demonstrate that a loss of nigrostriatal dopaminergic function likely contributes to slowing of reaction speed with advancing age.

Frontostriatal cognitive dysfunction is common in Parkinson disease (PD), but the explanation for its heterogeneous expressions remains unclear. This study examined the dopamine system within the frontostriatal circuitry with positron emission tomography (PET) to investigate pre- and post-synaptic dopamine function in relation to the executive processes in PD. Fifteen non-demented PD patients and 14 healthy controls underwent [(18)F]FDOPA (for dopamine synthesis) and [(11)C]NNC 112 (for D(1) receptors) PET scans and cognitive testing. Parametric images of [(18)F]FDOPA uptake (K(i)) and [(11)C]NNC 112 binding potential (BP(ND)) were calculated using reference tissue models. Group differences in K(i) and BP(ND) were assessed with both volume of interest and statistical parametric mapping, and were correlated with cognitive tests. Measurement of [(18)F]FDOPA uptake in cerebral cortex was questionable because of higher K(i) values in white than adjacent gray matter. These paradoxical results were likely to be caused by violations of the reference tissue model assumption rendering interpretation of cortical [(18)F]FDOPA uptake in PD difficult. We found no regional differences in D(1) receptor density between controls and PD, and no overall differences in frontostriatal performance. Although D(1) receptor density did not relate to frontostriatal cognition, K(i) decreases in the putamen predicted performance on the Wisconsin Card Sorting Test in PD only. These results suggest that striatal dopamine denervation may contribute to some frontostriatal cognitive impairment in moderate stage PD.

[11C]MePPEP is an inverse agonist and a radioligand developed to image cannabinoid CB1 receptors with positron emission tomography (PET). It provides reversible, high specific signal in monkey brain. We assessed [11C]MePPEP in rodent brain with regard to receptor selectivity, susceptibility to transport by P-glycoprotein (P-gp), sensitivity to displacement by agonists, and accumulation of radiometabolites. We used CB1 receptor knockout mice and P-gp knockout mice to assess receptor selectivity and sensitivity to efflux transport, respectively. Using serial measurements of PET brain activity and plasma concentrations of [11C]MePPEP, we estimated CB1 receptor density in rat brain as distribution volume. CB1 knockout mice showed only nonspecific brain uptake, and [11C]MePPEP was not a substrate for P-gp. Direct acting agonists anandamide (10 mg/kg), methanandamide (10 mg/kg), CP 55,940 (1 mg/kg), and indirect agonist URB597 (0.3 and 0.6 mg/kg) failed to displace [11C]MePPEP, while the inverse agonist rimonabant (3 and 10 mg/kg) displaced >65% of [11C]MePPEP. Radiometabolites represented ~13% of total radioactivity in brain between 30 and 120 min. [11C]MePPEP was selective for the CB1 receptor, was not a substrate for P-gp, and was more potently displaced by inverse agonists than agonists. The low potency of agonists suggests either a large receptor reserve or non-overlapping binding sites for agonists and inverse agonists. Radiometabolites of [11C]MePPEP in brain caused distribution volume to be overestimated by approximately 13%.

Molecular imaging has been used to estimate both drug-induced and tonic dopamine release in the striatum and most recently extrastriatal areas of healthy humans. However, to date, studies of drug-induced and tonic dopamine release have not been performed in the same subjects. This study performed positron emission tomography (PET) with [18F]fallypride in healthy subjects to assess (1) the reproducibility of [18F]fallypride and (2) both D-amphetamine-induced and alpha-methyl-p-tyrosine (AMPT)-induced changes in dopamin release on [(18)F]fallypride binding in striatal and extrastriatal areas. Subjects underwent [18F]fallypride PET studies at baseline and following oral D-amphetamine administration (0.5 mg/kg) and oral AMPT administration (3 g/70 kg/day over 44 h). Binding potential (BP) (BP(ND)) of [18F]fallypride was calculated in striatal and extrastriatal areas using a reference region method. Percent change in regional BP(ND) was computed and correlated with change in cognition and mood. Test-retest variability of [18F]fallypride was low in both striatal and extrastriatal regions. D-Amphetamine significantly decreased BP(ND) by 8-14% in striatal subdivisions, caudate, putamen, substantia nigra, medial orbitofrontal cortex, and medial temporal cortex. Correlation between change in BP(ND) and verbal fluency was seen in the thalamus and substantia nigra. In contrast, depletion of endogenous dopamine with AMPT did not effect [18F]fallypride BP(ND) in both striatum and extrastriatal regions. These findings indicate that [18F]fallypride is useful for measuring amphetamine-induced dopamine release, but may be unreliable for estimating tonic dopamine levels, in striatum and extrastriatal regions of healthy humans.

Introduction: [(11)C](R)-Rolipram is a selective radioligand for positron emission tomography (PET) imaging of phosphodiesterase 4, an enzyme that metabolizes 3',5'-cyclic adenosine monophosphate. The aim of this study was to estimate the human radiation absorbed dose of the radioligand based on its biodistribution in both monkeys and humans., Methods: Whole-body PET images were acquired for 2 h after injecting [(11)C](R)-rolipram in eight healthy humans and three monkeys. The simple method of using a single two-dimensional (2D) planar image was compared to more time-consuming methods that used two (bisected) or four (quadrisected) tomographic images in the anteroposterior direction., Results: Effective dose was 4.8 microGy/MBq based on 2D planar images. The effective dose was only slightly lower by 1% and 5% using the bisected and quadrisected images, respectively. Nevertheless, the two tomographic methods may have more accurately estimated the exposure of some organs (e.g., kidneys) that are asymmetrically located in the body or have radioactivity that appears to overlap on 2D planar images. Monkeys had a different biodistribution pattern compared to humans (e.g., greater urinary excretion) such that their data overestimated the effective dose in humans by 40%., Conclusions: The effective dose of [(11)C](R)-rolipram was modest and comparable to that of other (11)C-labeled radioligands. The simple and far less time-consuming 2D planar method provided accurate and somewhat more conservative estimates of effective dose than the two tomographic methods. Although monkeys are commonly used to estimate human radiation exposures, their data gave a considerable overestimation for this radioligand.

Unlabelled: Loperamide, an opiate receptor agonist, does not cross the blood-brain barrier because it is a substrate for the permeability-glycoprotein (P-gp) efflux pump. We evaluated 11C-loperamide as a PET radiotracer to measure P-gp function in vivo., Methods: Monkeys were injected with 11C-loperamide, and PET brain images were acquired for 120 min. The baseline scans were followed by scans acquired after administration of either of 2 P-gp inhibitors, (2R)-anti-5-{3-[4-(10,11-dichloromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (DCPQ) or tariquidar. Both the PET scans and ex vivo measurements were obtained in P-gp knockout and wild-type mice., Results: Pharmacologic inhibition of P-gp in monkeys dose-dependently increased brain activity, with a 3.7-fold effect at the highest DCPQ dose (8 mg/kg intravenously). This increase of brain activity was not caused peripherally, because DCPQ insignificantly changed the plasma concentration and plasma protein binding of radiotracer. Furthermore, the structurally dissimilar inhibitor, tariquidar, also increased brain uptake with potency equal to that of DCPQ. P-gp knockout mice had 3-fold higher brain activity on PET than did wild-type animals. Four radiometabolites were detected in the plasma and brains of ex vivo mice. The most lipophilic radiometabolite was found to be comobile with reference dLop on high-performance liquid chromatography. The brain concentrations of 11C-loperamide and the putative 11C-dLop were about 16-fold greater in P-gp knockout mice than in wild-type mice., Conclusion: Both 11C-loperamide and its putative radiometabolite 11C-dLop are avid P-gp substrates. 11C-dLop may be superior to 11C-loperamide in measuring P-gp function at the blood-brain barrier, because further demethylation of 11C-dLop will generate radiometabolites that have little entry into the brain.

The peripheral benzodiazepine receptor (PBR) is upregulated on activated microglia and macrophages and thereby is a useful biomarker of inflammation. We developed a novel PET radioligand, [(11)C]PBR28, that was able to image and quantify PBRs in healthy monkeys and in a rat model of stroke. The objective of this study was to evaluate the ability of [(11)C]PBR28 to quantify PBRs in brain of healthy human subjects. Twelve subjects had PET scans of 120 to 180 min duration as well as serial sampling of arterial plasma to measure the concentration of unchanged parent radioligand. One- and two-tissue compartmental analyses were performed. To obtain stable estimates of distribution volume, which is a summation of B(max)/K(D) and nondisplaceable activity, 90 min of brain imaging was required. Distribution volumes in human were only approximately 5% of those in monkey. This comparatively low amount of receptor binding required a two-rather than a one-compartment model, suggesting that nonspecific binding was a sizeable percentage compared to specific binding. The time-activity curves in two of the twelve subjects appeared as if they had no PBR binding-i.e., rapid peak of uptake and fast washout from brain. The cause(s) of these unusual findings are unknown, but both subjects were also found to lack binding to PBRs in peripheral organs such as lung and kidney. In conclusion, with the exception of those subjects who appeared to have no PBR binding, [(11)C]PBR28 is a promising ligand to quantify PBRs and localize inflammation associated with increased densities of PBRs.

Objectives: Peripheral benzodiazepine receptors (PBRs) are upregulated on activated microglia and are thereby biomarkers of neuroinflammation. We developed a PET ligand with an aryloxyanilide structure, [O-methyl-(11)C]N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine ([(11)C]PBR28), to image PBRs. The objectives of the current study were to evaluate kinetics of brain uptake, and the influence of the peripheral binding on the arterial input function in rhesus monkey., Methods: Brain (baseline: n=6, blocking: n=1) and whole-body PET imaging (baseline: n=3, blocking: n=1) of [(11)C]PBR28 were performed with the measurement of radiometabolite-corrected arterial input function in all brain and two whole body scans., Results: Saturating doses of nonradioactive PBR ligands markedly increased [(11)C]PBR28 in plasma (approximately 400% increase) and brain (approximately 200%) at 2 min by displacing radioligand from PBRs in peripheral organs. Brain uptake of radioactivity peaked in baseline scans at approximately 40 min after injection of [(11)C]PBR28 and was high (approximately 300% standardized uptake value). The images showed no receptor-free region that could be used for reference tissue analysis. Thus, quantitation of receptor density required measurement of parent radioligand in arterial plasma. Nondisplaceable uptake was estimated from the blocked scans and was only approximately 5% of total distribution volume measured under baseline conditions. Distribution volume of [(11)C]PBR28 was stably determined within 110 min of scanning., Conclusions: Regional brain uptake of [(11)C]PBR28 in monkey could be quantified as a value proportional to the density of receptors--namely, as equilibrium distribution volume. [(11)C]PBR28 had high levels of specific binding in brain and should provide a sensitive measure of changes in PBRs.

We sought to develop (11)C-labeled ligands for sensitive imaging of brain peripheral benzodiazepine receptors (PBR) in vivo. Two aryloxyanilides with high affinity for PBR were identified and synthesized, namely, N-acetyl- N-(2-methoxycarbonylbenzyl)-2-phenoxyaniline ( 3, PBR01) and N-(2-methoxybenzyl)- N-(4-phenoxypyridin-3-yl)acetamide ( 10, PBR28). 3 was hydrolyzed to 4, which was esterified with [ (11)C]iodomethane to provide [ (11)C] 3. The O-desmethyl analogue of 10 was converted into [ (11)C] 10 with [ (11)C]iodomethane. [ (11)C] 3 and [ (11)C] 10 were each injected into monkey to assess their brain kinetics with positron emission tomography (PET). After administration of either radioligand there was moderately high brain uptake of radioactivity. Receptor blocking and displacement experiments showed that a high proportion of this radioactivity was bound specifically to PBR. In monkey and rat, 3 and 10 were rapidly metabolized by ester hydrolysis and N-debenzylation, respectively, each to a single polar radiometabolite. [ (11)C] 3 and [ (11)C] 10 are effective for imaging PBR in monkey brain. [ (11)C] 10 especially warrants further evaluation in human subjects.

6-Thiolato-substituted 2-(4'- N,N-dimethylamino)phenylimidazo[1,2- a]pyridines ( RS-IMPYs; 1- 4) were synthesized as candidates for labeling with carbon-11 ( t 1/2 = 20.4 min) and imaging of A beta plaques in living human brain using positron emission tomography (PET). K i values for binding of these ligands to Alzheimer's disease brain homogenates were measured in vitro against tritium-labeled 6 (Pittsburgh compound B). MeS-IMPY ( 3, K i = 7.93 nM) was labeled with carbon-11 at its S- or N-methyl position to give [ (11)C] 7 or [ (11)C] 8, respectively. After injection into rats, [ (11)C] 7 or [ (11)C] 8 gave moderately high brain uptakes of radioactivity followed by rapid washout to low levels. The ratio of radioactivity at maximal uptake to that at 60 min reached 18.7 for [ (11)C] 7. [ (11)C] 7 behaved similarly in mouse and monkey. [ (11)C] 7 also bound selectively to A beta plaques in post mortem human Alzheimer's disease brain. Although rapidly metabolized in rat by N-demethylation, [ (11)C] 7 was stable in rat brain homogenates. The ex vivo brain radiometabolites observed in rats have a peripheral origin. Overall, [ (11)C] 7 merits further evaluation in human subjects.

The cannabinoid CB(1) receptor is one of the most abundant G protein-coupled receptors in the brain and is a promising target of therapeutic drug development. Success of drug development for neuropsychiatric indications is significantly enhanced with the ability to directly measure spatial and temporal binding of compounds to receptors in central compartments. We assessed the utility of a new positron emission tomography (PET) radioligand to image CB(1) receptors in monkey brain. [(11)C]MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) has high CB(1) affinity (K(b)=0.574+/-0.207 nM) but also moderately high lipophilicity (measured LogD(7.4)=4.8). After intravenous injection of [(11)C]MePPEP, brain activity reached high levels of almost 600% standardized uptake value (SUV) within 10-20 min. The regional uptake was consistent with the distribution of CB(1) receptors, with high radioactivity in striatum and cerebellum and low in thalamus and pons. Injection of pharmacological doses of CB(1)-selective agents confirmed that the tracer doses of [(11)C]MePPEP reversibly labeled CB(1) receptors. Preblockade or displacement with two CB(1) selective agents (ISPB; (4-(3-cyclopentyl-indole-1-sulfonyl)-N-(tetrahydro-pyran-4-ylmethyl)-benzamide) and rimonabant) showed that the majority (>89%) of brain uptake in regions with high receptor densities was specific and reversibly bound to CB(1) receptors in the high binding regions. [(11)C]MePPEP was rapidly removed from arterial plasma. Regional brain uptake could be quantified as distribution volume relative to the concentration of parent radiotracer in plasma. The P-glycoprotein (P-gp) inhibitor DCPQ ((R)-4-[(1a,6,10b)-1,1-dichloro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-[(5-quinolinyloxy)methyl]-1-piperazineethanol) did not significantly increase brain uptake of [(11)C]MePPEP, suggesting it is not a substrate for this efflux transporter at the blood-brain barrier. [(11)C]MePPEP is a radioligand with high brain uptake, high specific signal to CB(1) receptors, and adequately fast washout from brain that allows quantification with (11)C (half-life=20 min). These promising results in monkey justify studying this radioligand in human subjects.

To develop a potential SPECT probe to evaluate the integrity of the serotoninergic system (5-HTT) whose dysfunction is linked to several disease conditions such as Parkinson's disease, Alzheimer's disease and depression, we report the synthesis, radiolabeling and in vivo baboon imaging of 2beta-carbomethoxy-3beta-(3'-[(123)I]iodophenyl) tropane (YP256, 6). The radiolabeling was performed by iododestannylation using sodium [(123)I]iodide and peracetic acid. Although the ligand displayed high selectivity for 5-HTT over dopamine transporter in vitro, SPECT imaging in baboons did not reveal selective 5-HTT accumulation in brain in vivo.

Unlabelled: (11)C-PBR28 ([methyl-(11)C]N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine) is a recently developed radioligand to image peripheral benzodiazepine receptors (PBRs) in brain. The aim of this study was to estimate the human radiation doses of (11)C-PBR28 based on biodistribution data in monkeys and humans. In addition, we scanned 1 human subject who fortuitously behaved as if he lacked the PBR binding protein., Methods: Whole-body PBR images were acquired after intravenous bolus administration of (11)C-PBR28 in 7 healthy humans (651 +/- 111 MBq) and 2 rhesus monkeys (370 +/- 59.9 MBq). One monkey was scanned after receptor blockade with PK 11195 (10.7 mg/kg intravenously)., Results: For typical subjects (subjects 1-6), the 3 organs with highest exposure were those with the high PBR densities (kidneys, spleen, and lungs), and the effective dose was 6.6 microSv/MBq. The unusual subject (subject 7) had 60%-90% less uptake in these 3 organs, resulting in 28% lower effective dose. The activity in the baseline monkey scans was greater than that in humans for organs with high PBR densities. For this reason, the human effective dose was overestimated by 60% with monkey biodistribution data. The monkey with receptor blockade had an overall distribution qualitatively similar to that of the unusual human subject (subject 7), with decreased exposure to lungs, kidney, and spleen., Conclusion: The effective dose of (11)C-PBR28 was modest and was similar to that of several other (11)C-radioligands. Lack of receptor binding in the unusual human subject and in the monkey with receptor blockade decreased exposure to organs with high PBR densities and enhanced uptake in excretory and metabolic pathways.

Purpose: We aimed to fulfill a need for a radioligand that may be simply labeled with carbon-11 for effective positron emission tomography (PET) imaging of brain 5-HT(1A) receptors., Methods: Racemic RWAY (2,3,4,5,6,7-hexahydro-1-[4-[1-[4-(2-methoxyphenyl)piperazinyl]]-2-phenylbutyryl]-1H-azepine) has high affinity for 5-HT(1A) receptors. The enantiomers of RWAY and O-desmethyl-RWAY, synthesized from commercially available materials, were each labeled with carbon-11 by treating the respective O-desmethyl precursor with [(11)C]iodomethane, and injected into rhesus monkey for measurement of regional brain uptake. The 5-HT(1A) selectivity of (R)-[(11)C]RWAY was checked by administering WAY-100635, before and after radioligand administration. Radiometabolites of (R)-[(11)C]RWAY in blood and urine were analyzed by HPLC with partial elucidation of their structures by LC-MS-MS., Results: (R)-[(11)C]RWAY was a 5-HT(1A) receptor antagonist exhibiting high brain uptake with regional distribution consistent with specific binding to 5-HT(1A) receptors. The similar affinity, (S)-[(11)C]RWAY was a weak partial agonist at 5-HT(1A) receptors exhibiting similar brain peak uptake with much less 5-HT(1A) receptor-specific binding. The maximal ratio in receptor-rich cingulate gyrus to receptor-devoid cerebellum reached 6.4 at 87.5 min after injection of (R)-[(11)C]RWAY. After treatment with WAY-100635 before or after (R)-[(11)C]RWAY administration, radioactivity levels in 5-HT(1A) receptor-rich regions were reduced almost to that in cerebellum. Blood and urine radiometabolites were less lipophilic than parent and were not due to hydrolysis but to ring hydroxylations, oxidation, and dephenylation., Conclusion: (R)-[(11)C]RWAY is simply prepared and an effective antagonist for imaging brain 5-HT(1A) receptors. This radioligand resists hydrolysis in vivo, gives less lipophilic radiometabolites, and warrants further PET studies in human subjects.

A new and extensive set of 4-(6-iodo-H-imidazo[1,2-a]pyridin-2-yl)-N-dimethylbenzeneamine (IMPY) derivatives was synthesized and assayed for affinity toward human Abeta plaques. 6-Ethylthio- (12h), 6-cyano- (12e), 6-nitro- (12f), and 6-p-methoxybenzylthio- (15d) analogues were discovered to have high affinity (KI < 10 nM). However, introduction of a hydrophilic thioether group in the 6-position (15a-c, 15e-g) reduced or abolished affinity. In secondary N-methyl analogues, a bromo substituent in the adjacent ring position (14a) imparted high affinity (KI = 7.4 nM) whereas a methyl substituent did not (14c). The tolerance for nonhydrophilic thioether substituents in the 6-position opens up the possibility of developing new sensitive positron emission tomography radioligands for imaging human Abeta plaques in Alzheimer's disease, especially in view of the amenability of thioethers to be labeled with carbon-11 or fluorine-18 through S-alkylation reactions. The structure-activity relationships revealed in this study extends insight into the topography of the binding site for IMPY-like ligands in human Abeta plaques.

An international group of experts in pharmacokinetic modeling recommends a consensus nomenclature to describe in vivo molecular imaging of reversibly binding radioligands.

[(11)C]MeS-IMPY ([S-methyl-(11)C]N,N-dimethyl-4-(6-(methylthio)imidazo[1,2-a]pyridine-2-yl)aniline) is a potential radioligand for imaging beta-amyloid plaques with positron emission tomography (PET). The aims of this study were to evaluate [(11)C]MeS-IMPY uptake in nonhuman primate brain and to estimate radiation exposure from serial whole-body images. Eight PET studies were performed in rhesus monkeys to measure the brain uptake and washout of [(11)C]MeS-IMPY. Time-activity data were analyzed with one-tissue and two-tissue compartmental models using radiometabolite-corrected plasma input function. In addition, two whole-body PET scans were acquired for 120 min to determine the biodistribution of [(11)C]MeS-IMPY. Tomographic PET images were compressed into a single planar image to identify organs with the highest radiation exposures. Estimates of the absorbed dose of radiation were calculated using OLINDA 1.0. Injection of [(11)C]MeS-IMPY caused little change in pulse rate, blood pressure, respiratory rate and temperature. [(11)C]MeS-IMPY showed high standardized brain uptake values of approximately 500% and 600% between 2 and 3 min in cortical regions and the cerebellum, respectively. The brain uptake of [(11)C]MeS-IMPY was widespread and quite uniform across all cortical regions. Activity rapidly washed out of the brain, with 20% of peak activity remaining at 40 min. Thus, all brain regions showed minimal retention of radioactivity, consistent with these healthy young animals having negligible amyloid plaques. Regional brain activity fitted well into a one-tissue compartment model. The average volume of distribution in all brain regions was 7.66+/-2.14 ml/cm(3) (n=4). The organs with the highest radiation exposure (muSv/MBq) were the gallbladder wall (33.4), urinary bladder (17) and lungs (12.9), with a resulting effective dose of 4.9 microSv/MBq (18 mrem/mCi). The high brain uptake, rapid washout and quantifiable volume of distribution in nonhuman primate brain further support the evaluation of [(11)C]MeS-IMPY. Calculated dosimetry results are comparable with those for other (11)C-labeled brain imaging radioligands.

Peripheral benzodiazepine receptors (PBRs) are upregulated on activated microglia and are, thereby, biomarkers of cellular inflammation in brain. We recently developed two PET ligands with an aryloxyanilide structure to image PBRs and now evaluate the kinetics of these radiotracers in monkey to determine whether they are suitable to explore in human. Baseline and receptor-blocking scans were performed with [(11)C]PBR01 and [(18)F]PBR06 in conjunction with serial measurements of the arterial plasma concentration of parent radiotracer separated from radiometabolite. We used brain and plasma data with compartmental modeling to calculate regional brain distribution volume, which is equal to the ratio at equilibrium of the concentration of radioligand in brain to that of plasma. The distribution volume of [(11)C]PBR01 was inaccurately estimated in the baseline scans, possibly because of the short half-life of (11)C or the presence of radiometabolite in brain. In contrast, the distribution volume of [(18)F]PBR06 was stably determined within 200 min of scanning, and nondisplaceable uptake was only approximately 10% of total brain uptake. [(18)F]PBR06 is promising for use in human because brain activity could be quantified with standard compartmental models and showed higher ratios ( approximately 10:1) of specific to nonspecific uptake. A critical factor for human use will be whether the tracer has adequately fast wash out from brain relative to the half-life of the radionuclide to obtain stable values of distribution volume.

2-fluoromethyl analogs of (3-[(2-methyl-1,3-thiazol-4yl)ethynyl]pyridine) were synthesized as potential ligands for metabotropic glutamate subtype-5 receptors (mGluR5s). One of these, namely, 3-fluoro-5-(2-(2-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile (3), was found to have exceptionally high affinity (IC50 = 36 pM) and potency in a phosphoinositol hydrolysis assay (IC50 = 0.714 pM) for mGluR5. Compound 3 was labeled with fluorine-18 (t1/2 = 109.7 min) in high radiochemical yield (87%) by treatment of its synthesized bromomethyl analog (17) with [18F]fluoride ion and its radioligand behavior was assessed with positron emission tomography (PET). Following intravenous injection of [18F]3 into rhesus monkey, radioactivity was avidly taken up into brain with high uptake in mGluR5 receptor-rich regions such as striata. [18F]3 was stable in monkey plasma and human whole blood in vitro and in monkey and human brain homogenates. In monkey in vivo, a single polar radiometabolite of [18F]3 appeared rapidly in plasma. [18F]3 merits further evaluation as a PET radioligand for mGluR5 in human subjects.