Evidence that formulations of the selective MAO-B inhibitor, selegiline, which bypass first-pass metabolism, also inhibit MAO-A in the human brain.

Selegiline (L-deprenyl) is a selective, irreversible inhibitor of monoamine oxidase B (MAO-B) at the conventional dose (10 mg/day oral) that is used in the treatment of Parkinson's disease. However, controlled studies have demonstrated antidepressant activity for high doses of oral selegiline and for transdermal selegiline suggesting that when plasma levels of selegiline are elevated, brain MAO-A might also be inhibited. Zydis selegiline (Zelapar) is an orally disintegrating formulation of selegiline, which is absorbed through the buccal mucosa producing higher plasma levels of selegiline and reduced amphetamine metabolites compared with equal doses of conventional selegiline. Although there is indirect evidence that Zydis selegiline at high doses loses its selectivity for MAO-B, there is no direct evidence that it also inhibits brain MAO-A in humans. We measured brain MAO-A in 18 healthy men after a 28-day treatment with Zydis selegiline (2.5, 5.0, or 10 mg/day) and in 3 subjects receiving the selegiline transdermal system (Emsam patch, 6 mg/day) using positron emission tomography and the MAO-A radiotracer [(11)C]clorgyline. We also measured dopamine transporter (DAT) availability in three subjects from the 10 mg group. The 10 mg Zydis selegiline dose significantly inhibited MAO-A (36.9±19.7%, range 11-70%, p<0.007)) but not DAT; and while Emsam also inhibited MAO-A (33.2±28.9 (range 9-68%) the difference did not reach significance (p=0.10)) presumably because of the small sample size. Our results provide the first direct evidence of brain MAO-A inhibition in humans by formulations of selegiline, which are currently postulated but not verified to target brain MAO-A in addition to MAO-B.

Image-guided synthesis reveals potent blood-brain barrier permeable histone deacetylase inhibitors.

Recent studies have revealed that several histone deacetylase (HDAC) inhibitors, which are used to study/treat brain diseases, show low blood-brain barrier (BBB) penetration. In addition to low HDAC potency and selectivity observed, poor brain penetrance may account for the high doses needed to achieve therapeutic efficacy. Here we report the development and evaluation of highly potent and blood-brain barrier permeable HDAC inhibitors for CNS applications based on an image-guided approach involving the parallel synthesis and radiolabeling of a series of compounds based on the benzamide HDAC inhibitor, MS-275 as a template. BBB penetration was optimized by rapid carbon-11 labeling and PET imaging in the baboon model and using the imaging derived data on BBB penetration from each compound to feed back into the design process. A total of 17 compounds were evaluated, revealing molecules with both high binding affinity and BBB permeability. A key element conferring BBB penetration in this benzamide series was a basic benzylic amine. These derivatives exhibited 1-100 nM inhibitory activity against recombinant human HDAC1 and HDAC2. Three of the carbon-11 labeled aminomethyl benzamide derivatives showed high BBB penetration (∼0.015%ID/cc) and regional binding heterogeneity in the brain (high in thalamus and cerebellum). Taken together this approach has afforded a strategy and a predictive model for developing highly potent and BBB permeable HDAC inhibitors for CNS applications and for the discovery of novel candidate molecules for small molecule probes and drugs.

Therapeutic doses of buspirone block D3 receptors in the living primate brain.

Dopamine D3 receptor (D3R) antagonists may be effective medications for multiple substance use disorders (SUDs). However, no selective D3R antagonists are currently available for clinical testing. Buspirone, originally characterized as a 5-HT1A partial agonist and used as an anxiolytic, also binds to D3R and D4R with high affinity, with lower affinity to D2R, and interferes with cocaine reward. Here we used PET with [11C]PHNO (D3R-preferring radioligand), [11C]raclopride (D2R/D3R radioligand) and [11C]NNC-112 (D1R radioligand) to measure occupancy of oral and parenteral buspirone in the primate brain. Intramuscular buspirone (0.19 and 0.5 mg/kg) blocked both [11C]PHNO and [11C]raclopride binding to striatum, exhibiting high occupancy (50-85%) at 15 min and rapid wash-out over 2-6 h. In contrast, oral buspirone (3 mg/kg) significantly blocked [11C]PHNO binding in D3-rich regions (globus pallidum and midbrain) at 3 h, but had minimal effects on [11C]raclopride binding (28-37% at 1 h and 10% at 3 h). Buspirone did not block [11C]NNC-112. Our findings provide evidence that i.m. buspirone blocks D3R and D2R, whereas oral buspirone is more selective towards D3R blockade in vivo, consistent with extensive first pass metabolism and supporting the hypothesis that its metabolites (5- and 6'-hydroxybuspirone) merit evaluation for treating SUDs. They also indicate that for oral buspirone to achieve greater than 80% sustained D3R occupancy, as might be needed to treat addiction, higher doses (at least three-fold) than those used to treat anxiety (maximal 60 mg) will be required. Nonetheless, based on previous clinical studies, these doses would be safe and well tolerated.

Whole-body pharmacokinetics of HDAC inhibitor drugs, butyric acid, valproic acid and 4-phenylbutyric acid measured with carbon-11 labeled analogs by PET.

The fatty acids, n-butyric acid (BA), 4-phenylbutyric acid (PBA) and valproic acid (VPA, 2-propylpentanoic acid) have been used for many years in the treatment of a variety of CNS and peripheral organ diseases including cancer. New information that these drugs alter epigenetic processes through their inhibition of histone deacetylases (HDACs) has renewed interest in their biodistribution and pharmacokinetics and the relationship of these properties to their therapeutic and side effect profiles. In order to determine the pharmacokinetics and biodistribution of these drugs in primates, we synthesized their carbon-11 labeled analogues and performed dynamic positron emission tomography (PET) in six female baboons over 90 min. The carbon-11 labeled carboxylic acids were prepared by using (11)CO2 and the appropriate Grignard reagents. [(11)C]BA was metabolized rapidly (only 20% of the total carbon-11 in plasma was parent compound at 5 min post injection) whereas for VPA and PBA 98% and 85% of the radioactivity were the unmetabolized compound at 30 min after their administration respectively. The brain uptake of all three carboxylic acids was very low (<0.006%ID/cc, BA>VPA>PBA), which is consistent with the need for very high doses for therapeutic efficacy. Most of the radioactivity was excreted through the kidneys and accumulated in the bladder. However, the organ biodistribution between the drugs differed. [(11)C]BA showed relatively high uptake in spleen and pancreas whereas [(11)C]PBA showed high uptake in liver and heart. Notably, [(11)C]VPA showed exceptionally high heart uptake possibly due to its involvement in lipid metabolism. The unique biodistribution of each of these drugs may be of relevance in understanding their therapeutic and side effect profile including their teratogenic effects.

Blockade of the brain histamine H3 receptor by JNJ-39220675: preclinical PET studies with [¹¹C]GSK189254 in anesthetized baboon.

RATIONALE: The preclinical characterization of a series of aryloxypyridine amides has identified JNJ-39220675 ((4-cyclobutyl-1,4-diazepan-1-yl)(6-(4-fluorophenoxy)pyridin-3-yl)methanone) as a high-affinity histamine H(3) receptor antagonist and a candidate for further drug development particularly in the treatment of alcohol-related behaviors. OBJECTIVE: This study measured brain histamine H(3) receptor blockade by JNJ-39220675 (1 mg/kg) in the female baboon. METHODS: Positron emission tomography imaging and [(11)C]GSK189254, a reversible high-affinity radiotracer with specificity for the histamine H(3) receptor, was used to measure histamine H(3) receptor availability at baseline and after i.v. and oral administration of JNJ-39220675 (1 mg/kg) in the anesthetized baboon. Histamine H(3) receptor availability was estimated as the total distribution volume (V (T)) in brain regions. The sensitivity of [(11)C]GSK189254 binding to injected mass and carryover effects was determined. RESULTS: JNJ-39220675 produces robust (ca. 90 %) blockade of [(11)C]GSK189254 binding after i.v. and oral administration. After oral administration of JNJ-39220675 (1 mg/kg), the fractional receptor occupancy was >0.9 at 90 min with a slight increase from 90 to 240 min. Similar to prior studies in humans, V (T) was highly sensitive to the mass of GSK189254 with ED(50) estimated to be 0.16 µg/kg. CONCLUSIONS: The robust blockade of binding of [(11)C]GSK189254 by JNJ-39220675 demonstrates that this compound readily penetrates the blood-brain barrier and occupies the histamine H(3) receptor after oral administration at low plasma concentrations (∼1 ng/cc) supporting further drug development for alcohol addiction and other disorders. This study corroborates prior reports of the high sensitivity of [(11)C]GSK189254 to injected mass at doses >0.1 µg/kg.

Unique distribution of aromatase in the human brain: in vivo studies with PET and [N-methyl-11C]vorozole.

Aromatase catalyzes the last step in estrogen biosynthesis. Brain aromatase is involved in diverse neurophysiological and behavioral functions including sexual behavior, aggression, cognition, and neuroprotection. Using positron emission tomography (PET) with the radiolabeled aromatase inhibitor [N-methyl-(11)C]vorozole, we characterized the tracer distribution and kinetics in the living human brain. Six young, healthy subjects, three men and three women, were administered the radiotracer alone on two separate occasions. Women were scanned in distinct phases of the menstrual cycle. Specificity was confirmed by pretreatment with a pharmacological (2.5 mg) dose of the aromatase inhibitor letrozole. PET data were acquired over a 90-min period and regions of interest placed over selected brain regions. Brain and plasma time activity curves, corrected for metabolites, were used to derive kinetic parameters. Distribution volume (V(T)) values in both men and women followed the following rank order: thalamus > amygdala = preoptic area > medulla (inferior olive) > accumbens, pons, occipital and temporal cortex, putamen, cerebellum, and white matter. Pretreatment with letrozole reduced V(T) in all regions, though the size of the reduction was region-dependent, ranging from ∼70% blocking in thalamus andpreoptic area to ∼10% in cerebellum. The high levels of aromatase in thalamus and medulla (inferior olive) appear to be unique to humans. These studies set the stage for the noninvasive assessment of aromatase involvement in various physiological and pathological processes affecting the human brain.

Reversible inhibitors of monoamine oxidase-A (RIMAs): robust, reversible inhibition of human brain MAO-A by CX157.

Reversible inhibitors of monoamine oxidase-A (RIMA) inhibit the breakdown of three major neurotransmitters, serotonin, norepinephrine and dopamine, offering a multi-neurotransmitter strategy for the treatment of depression. CX157 (3-fluoro-7-(2,2,2-trifluoroethoxy)phenoxathiin-10,10-dioxide) is a RIMA, which is currently in development for the treatment of major depressive disorder. We examined the degree and reversibility of the inhibition of brain monoamine oxidase-A (MAO-A) and plasma CX157 levels at different times after oral dosing to establish a dosing paradigm for future clinical efficacy studies, and to determine whether plasma CX157 levels reflect the degree of brain MAO-A inhibition. Brain MAO-A levels were measured with positron emission tomography (PET) imaging and [(11)C]clorgyline in 15 normal men after oral dosing of CX157 (20-80 mg). PET imaging was conducted after single and repeated doses of CX157 over a 24-h time course. We found that 60 and 80 mg doses of CX157 produced a robust dose-related inhibition (47-72%) of [(11)C]clorgyline binding to brain MAO-A at 2 h after administration and that brain MAO-A recovered completely by 24 h post drug. Plasma CX157 concentration was highly correlated with the inhibition of brain MAO-A (EC(50): 19.3 ng/ml). Thus, CX157 is the first agent in the RIMA class with documented reversible inhibition of human brain MAO-A, supporting its classification as a RIMA, and the first RIMA with observed plasma levels that can serve as a biomarker for the degree of brain MAO-A inhibition. These data were used to establish the dosing regimen for a current clinical efficacy trial with CX157.

Effects of modafinil on dopamine and dopamine transporters in the male human brain: clinical implications.

CONTEXT: Modafinil, a wake-promoting drug used to treat narcolepsy, is increasingly being used as a cognitive enhancer. Although initially launched as distinct from stimulants that increase extracellular dopamine by targeting dopamine transporters, recent preclinical studies suggest otherwise. OBJECTIVE: To measure the acute effects of modafinil at doses used therapeutically (200 mg and 400 mg given orally) on extracellular dopamine and on dopamine transporters in the male human brain. DESIGN, SETTING, AND PARTICIPANTS: Positron emission tomography with [(11)C]raclopride (D(2)/D(3) radioligand sensitive to changes in endogenous dopamine) and [(11)C]cocaine (dopamine transporter radioligand) was used to measure the effects of modafinil on extracellular dopamine and on dopamine transporters in 10 healthy male participants. The study took place over an 8-month period (2007-2008) at Brookhaven National Laboratory. MAIN OUTCOME MEASURES: Primary outcomes were changes in dopamine D(2)/D(3) receptor and dopamine transporter availability (measured by changes in binding potential) after modafinil when compared with after placebo. RESULTS: Modafinil decreased mean (SD) [(11)C]raclopride binding potential in caudate (6.1% [6.5%]; 95% confidence interval [CI], 1.5% to 10.8%; P = .02), putamen (6.7% [4.9%]; 95% CI, 3.2% to 10.3%; P = .002), and nucleus accumbens (19.4% [20%]; 95% CI, 5% to 35%; P = .02), reflecting increases in extracellular dopamine. Modafinil also decreased [(11)C]cocaine binding potential in caudate (53.8% [13.8%]; 95% CI, 43.9% to 63.6%; P < .001), putamen (47.2% [11.4%]; 95% CI, 39.1% to 55.4%; P < .001), and nucleus accumbens (39.3% [10%]; 95% CI, 30% to 49%; P = .001), reflecting occupancy of dopamine transporters. CONCLUSIONS: In this pilot study, modafinil blocked dopamine transporters and increased dopamine in the human brain (including the nucleus accumbens). Because drugs that increase dopamine in the nucleus accumbens have the potential for abuse, and considering the increasing use of modafinil, these results highlight the need for heightened awareness for potential abuse of and dependence on modafinil in vulnerable populations.

Reinvestigation of the synthesis and evaluation of [N-methyl-(11)C]vorozole, a radiotracer targeting cytochrome P450 aromatase.

INTRODUCTION: We reinvestigated the synthesis of [N-methyl-(11)C]vorozole, a radiotracer for aromatase, and discovered the presence of an N-methyl isomer which was not removed in the original purification method. Herein we report the preparation and positron emission tomography (PET) studies of pure [N-methyl-(11)C]vorozole. METHODS: Norvorozole was alkylated with [(11)C]methyl iodide as previously described and also with unlabeled methyl iodide. A high-performance liquid chromatography (HPLC) method was developed to separate the regioisomers. Nuclear magnetic resonance (NMR) spectroscopy ((13)C and 2D-nuclear Overhauser effect spectroscopy NMR) was used to identify and assign structures to the N-methylated products. Pure [N-methyl-(11)C]vorozole and the contaminating isomer were compared by PET imaging in the baboon. RESULTS: Methylation of norvorozole resulted in a mixture of isomers (1:1:1 ratio) based on new HPLC analysis using a pentafluorophenylpropyl bonded silica column, in which vorozole coeluted one of its isomers under the original HPLC conditions. Baseline separation of the three labeled isomers was achieved. The N-3 isomer was the contaminant of vorozole, thus correcting the original assignment of isomers. PET studies of pure [N-methyl-(11)C]vorozole with and without the contaminating N-3 isomer revealed that only [N-methyl-(11)C]vorozole binds to aromatase. [N-methyl-(11)C]Vorozole accumulated in all brain regions with highest accumulation in the aromatase-rich amygdala and preoptic area. Accumulation was blocked with vorozole and letrozole consistent with reports of some level of aromatase in many brain regions. CONCLUSIONS: The discovery of a contaminating labeled isomer and the development of a method for isolating pure [N-methyl-(11)C]vorozole combine to provide a new scientific tool for PET studies of the biology of aromatase and for drug research and development.

Fast uptake and long-lasting binding of methamphetamine in the human brain: comparison with cocaine.

Methamphetamine is one of the most addictive and neurotoxic drugs of abuse. It produces large elevations in extracellular dopamine in the striatum through vesicular release and inhibition of the dopamine transporter. In the U.S. abuse prevalence varies by ethnicity with very low abuse among African Americans relative to Caucasians, differentiating it from cocaine where abuse rates are similar for the two groups. Here we report the first comparison of methamphetamine and cocaine pharmacokinetics in brain between Caucasians and African Americans along with the measurement of dopamine transporter availability in striatum. Methamphetamine's uptake in brain was fast (peak uptake at 9 min) with accumulation in cortical and subcortical brain regions and in white matter. Its clearance from brain was slow (except for white matter which did not clear over the 90 min) and there was no difference in pharmacokinetics between Caucasians and African Americans. In contrast cocaine's brain uptake and clearance were both fast, distribution was predominantly in striatum and uptake was higher in African Americans. Among individuals, those with the highest striatal (but not cerebellar) methamphetamine accumulation also had the highest dopamine transporter availability suggesting a relationship between METH exposure and DAT availability. Methamphetamine's fast brain uptake is consistent with its highly reinforcing effects, its slow clearance with its long-lasting behavioral effects and its widespread distribution with its neurotoxic effects that affect not only striatal but also cortical and white matter regions. The absence of significant differences between Caucasians and African Americans suggests that variables other than methamphetamine pharmacokinetics and bioavailability account for the lower abuse prevalence in African Americans.

PET studies of d-methamphetamine pharmacokinetics in primates: comparison with l-methamphetamine and ( --)-cocaine.

UNLABELLED: The methamphetamine molecule has a chiral center and exists as 2 enantiomers, d-methamphetamine (the more active enantiomer) and l-methamphetamine (the less active enantiomer). d-Methamphetamine is associated with more intense stimulant effects and higher abuse liability. The objective of this study was to measure the pharmacokinetics of d-methamphetamine for comparison with both l-methamphetamine and (-)-cocaine in the baboon brain and peripheral organs and to assess the saturability and pharmacologic specificity of binding. METHODS: d- and l-methamphetamine and (-)-cocaine were labeled with (11)C via alkylation of the norprecursors with (11)C-methyl iodide using literature methods. Six different baboons were studied in 11 PET sessions at which 2 radiotracer injections were administered 2-3 h apart to determine the distribution and kinetics of (11)C-d-methamphetamine in brain and peripheral organs. Saturability and pharmacologic specificity were assessed using pretreatment with d-methamphetamine, methylphenidate, and tetrabenazine. (11)C-d-Methamphetamine pharmacokinetics were compared with (11)C-l-methamphetamine and (11)C-(-)-cocaine in both brain and peripheral organs in the same animal. RESULTS: (11)C-d- and l-methamphetamine both showed high uptake and widespread distribution in the brain. Pharmacokinetics did not differ between enantiomers, and the cerebellum peaked earlier and cleared more quickly than the striatum for both. (11)C-d-Methamphetamine distribution volume ratio was not substantially affected by pretreatment with methamphetamine, methylphenidate, or tetrabenazine. Both enantiomers showed rapid, high uptake and clearance in the heart and lungs and slower uptake and clearance in the liver and kidneys. A comparison of (11)C-d-methamphetamine and (11)C-(-)-cocaine showed that (11)C-d-methamphetamine peaked later in the brain than did (11)C-(-)-cocaine and cleared more slowly. The 2 drugs showed similar behavior in all peripheral organs examined except the kidneys and pancreas, which showed higher uptake for (11)C-d-methamphetamine. CONCLUSION: Brain pharmacokinetics did not differ between d-and l-methamphetamine and thus cannot account for the more intense stimulant effects of d-methamphetamine. Lack of pharmacologic blockade by methamphetamine indicates that the PET image represents nonspecific binding, though the fact that methamphetamine is both a transporter substrate and an inhibitor may also play a role. A comparison of (11)C-d-methamphetamine and (11)C-(-)-cocaine in the same animal showed that the slower clearance of methamphetamine is likely to contribute to its previously reported longer-lasting stimulant effects relative to those of (-)-cocaine. High kidney uptake of d-methamphetamine or its labeled metabolites may account for the reported renal toxicity of d-methamphetamine in humans.

Imaging the norepinephrine transporter in humans with (S,S)-[11C]O-methyl reboxetine and PET: problems and progress.

UNLABELLED: Results from human studies with the PET radiotracer (S,S)-[(11)C]O-methyl reboxetine ([(11)C](S,S)-MRB), a ligand targeting the norepinephrine transporter (NET), are reported. Quantification methods were determined from test/retest studies, and sensitivity to pharmacological blockade was tested with different doses of atomoxetine (ATX), a drug that binds to the NET with high affinity (K(i)=2-5 nM). METHODS: Twenty-four male subjects were divided into different groups for serial 90-min PET studies with [(11)C](S,S)-MRB to assess reproducibility and the effect of blocking with different doses of ATX (25, 50 and 100 mg, po). Region-of-interest uptake data and arterial plasma input were analyzed for the distribution volume (DV). Images were normalized to a template, and average parametric images for each group were formed. RESULTS: [(11)C](S,S)-MRB uptake was highest in the thalamus (THL) and the midbrain (MBR) [containing the locus coeruleus (LC)] and lowest for the caudate nucleus (CDT). The CDT, a region with low NET, showed the smallest change on ATX treatment and was used as a reference region for the DV ratio (DVR). The baseline average DVR was 1.48 for both the THL and MBR with lower values for other regions [cerebellum (CB), 1.09; cingulate gyrus (CNG) 1.07]. However, more accurate information about relative densities came from the blocking studies. MBR exhibited greater blocking than THL, indicating a transporter density approximately 40% greater than THL. No relationship was found between DVR change and plasma ATX level. Although the higher dose tended to induce a greater decrease than the lower dose for MBR (average decrease for 25 mg=24+/-7%; 100 mg=31+/-11%), these differences were not significant. The different blocking between MBR (average decrease=28+/-10%) and THL (average decrease=17+/-10%) given the same baseline DVR indicates that the CDT is not a good measure for non-NET binding in both regions. Threshold analysis of the difference between the average baseline DV image and the average blocked image showed the expected NET distribution with the MBR (LC) and hypothalamus>THL>CNG and CB, as well as a significant change in the supplementary motor area. DVR reproducibility for the different brain regions was approximately 10%, but intersubject variability was large. CONCLUSIONS: The highest density of NETs was found in the MBR where the LC is located, followed by THL, whereas the lowest density was found in basal ganglia (lowest in CDT), consistent with the regional localization of NETs in the nonhuman primate brain. While all three doses of ATX were found to block most regions, no significant differences between doses were found for any region, although the average percent change across subjects of the MBR did correlate with ATX dose. The lack of a dose effect could reflect a low signal-to-noise ratio coupled with the possibility that a sufficient number of transporters were blocked at the lowest dose and further differences could not be detected. However, since the lowest (25 mg) dose is less than the therapeutic doses used in children for the treatment of attention-deficit/hyperactivity disorder ( approximately 1.0 mg/kg/day), this would suggest that there may be additional targets for ATX's therapeutic actions.

Synthesis and positron emission tomography studies of C-11-labeled isotopomers and metabolites of GTS-21, a partial alpha7 nicotinic cholinergic agonist drug.

INTRODUCTION: (3E)-3-[(2,4-dimethoxyphenyl)methylene]-3,4,5,6-tetrahydro-2,3'-bipyridine (GTS-21), a partial alpha7 nicotinic acetylcholine receptor agonist drug, has recently been shown to improve cognition in schizophrenia and Alzheimer's disease. One of its two major demethylated metabolites, 4-OH-GTS-21, has been suggested to contribute to its therapeutic effects. METHODS: We labeled GTS-21 in two different positions with carbon-11 ([2-methoxy-(11)C]GTS-21 and [4-(11)C]GTS-21) along with two corresponding demethylated metabolites ([2-methoxy-(11)C]4-OH-GTS-21 and [4-methoxy-(11)C]2-OH-GTS-21) for pharmacokinetic studies in baboons and mice with positron emission tomography (PET). RESULTS: Both [2-(11)C]GTS-21 and [4-methoxy-(11)C]GTS-21 showed similar initial high rapid uptake in baboon brain, peaking from 1 to 3.5 min (0.027-0.038%ID/cc) followed by rapid clearance (t(1/2)<15 min), resulting in low brain retention by 30 min. However, after 30 min, [2-methoxy-(11)C]GTS-21 continued to clear while [4-methoxy-(11)C]GTS-21 plateaued, suggesting the entry of a labeled metabolite into the brain. Comparison of the pharmacokinetics of the two labeled metabolites confirmed expected higher brain uptake and retention of [4-methoxy-(11)C]2-OH-GTS-21 (the labeled metabolite of [4-methoxy-(11)C]GTS-21) relative to [2-methoxy-(11)C]4-OH-GTS-21 (the labeled metabolite of [2-methoxy-(11)C]GTS-21), which had negligible brain uptake. Ex vivo studies in mice showed that GTS-21 is the major chemical form in the mouse brain. Whole-body dynamic PET imaging in baboon and mouse showed that the major route of excretion of C-11 is through the gallbladder. CONCLUSIONS: The major findings are as follows: (a) extremely rapid uptake and clearance of [2-methoxy-(11)C]GTS-21 from the brain, which may need to be considered in developing optimal dosing of GTS-21 for patients, and (b) significant brain uptake of 2-OH-GTS-21, suggesting that it might contribute to the therapeutic effects of GTS-21. This study illustrates the value of comparing different label positions and labeled metabolites to gain insight on the behavior of a central nervous system drug and its metabolites in the brain, providing an important perspective on drug pharmacokinetics.

Comparison of monoamine oxidase a in peripheral organs in nonsmokers and smokers.

UNLABELLED: Smokers have reduced levels of brain monoamine oxidase A (MAO A) leading to speculation that MAO A inhibition by tobacco smoke may underlie some of the neurophysiologic effects of smoking. Because smoking exposes peripheral organs as well as the brain to MAO A-inhibitory compounds, we determined whether smokers would also have reduced MAO A in peripheral organs. METHODS: We measured MAO A in peripheral organs in a group of 9 smokers and compared it with a group of nonsmokers studied previously. MAO A was measured using PET and serial scans with the MAO A-specific radiotracers (11)C-clorgyline and deuterium-substituted (11)C-clorgyline ((11)C-clorgyline-D2) using the deuterium isotope effect to assess binding specificity. The time course of radiotracer in the arterial plasma was also measured and data from the tissue time-activity curves and the arterial input function were analyzed using a 3-compartment model to estimate k(3), which represents the rate-limiting step for the irreversible binding of labeled clorgyline to MAO A. RESULTS: Tracer uptake at plateau was reduced with deuterium substitution for the heart, lungs, and kidneys, indicating specificity for MAO. There was no difference in organ uptake at plateau between nonsmokers and smokers though, for the smokers, the efflux of tracer from peak uptake to plateau was slower for the lungs. The area under the time-activity curve for the arterial plasma was also significantly reduced for smokers versus nonsmokers and the reduction occurred in the first few minutes after radiotracer injection. Smokers had an approximately 50% reduction in k(3) when compared with nonsmokers; however, k(3) did not differ for nonsmokers and smokers for the heart and the kidneys. CONCLUSION: Because MAO A breaks down serotonin, norepinephrine, dopamine, and tyramine, and because the lung is a major metabolic organ in degrading some of these substances, reduced lung MAO A may contribute to some of the physiologic effects of smoking. This study also revealed that the concentration of the radiotracers in the arterial plasma is significantly lower for the smoker versus the nonsmoker and that this appears to be caused in part by retention of the radiotracer in lungs. If this is generally true for other substances that are administered intravenously, then this needs to be considered as a variable that may contribute to different short-term behavioral responses to intravenously administered drugs for nonsmokers versus smokers.

Brain kinetics of methylphenidate (Ritalin) enantiomers after oral administration.

Methylphenidate (MP) (Ritalin) is widely used for the treatment of attention deficit hyperactivity disorder (ADHD). It is a chiral drug, marketed as the racemic mixture of d- and l-threo enantiomers. Our previous studies (PET and microdialysis) in humans, baboons, and rats confirm the notion that pharmacological specificity of MP resides predominantly in the d-isomer. A recent report that intraperitoneally (i.p.) administered l-threo-MP displayed potent, dose-dependent inhibition of cocaine- or apomorphine-induced locomotion in rats, raises the question of whether l-threo-MP has a similar effect when given orally. It has been speculated that l-threo-MP is poorly absorbed in humans when it is given orally because of rapid presystemic metabolism. To investigate whether l-threo-MP or its metabolites can be delivered to the brain when it is given orally, and whether l-threo-MP is pharmacologically active. PET and MicroPET studies were carried out in baboons and rats using orally delivered C-11-labeled d- and l-threo-MP ([methyl-(11)C]d-threo-MP and [methyl-(11)C]l-threo-MP). In addition, we assessed the effects of i.p. l-threo-MP on spontaneous and cocaine-stimulated locomotor activity in mice. There was a higher global uptake of carbon-11 in both baboon and rat brain for oral [(11)C]l-threo-MP than for oral [(11)C]d-threo-MP. Analysis of the chemical form of radioactivity in rat brain after [(11)C]d-threo-MP indicated mainly unchanged tracer, whereas with [(11)C]l-threo-MP, it was mainly a labeled metabolite. The possibility that this labeled metabolite might be [(11)C]methanol or [(11)C]CO(2), derived from demethylation, was excluded by ex vivo studies in rats. When l-threo-MP was given i.p. to mice at a dose of 3 mg/kg, it neither stimulated locomotor activity nor inhibited the increased locomotor activity due to cocaine administration. These results suggest that, in animal models, l-threo-MP or its metabolite(s) is (are) absorbed from the gastrointestinal tract and enters the brain after oral administration, but that l-threo-MP may not be pharmacologically active. These results are pertinent to the question of whether l-threo-MP contributes to the behavioral and side effect profile of MP during treatment of ADHD.

Evaluation of a new norepinephrine transporter PET ligand in baboons, both in brain and peripheral organs.

Reboxetine is a specific norepinephrine transporter (NET) inhibitor and has been marketed in several countries as a racemic mixture of the (R,R) and (S,S) enantiomers for the treatment of depression. Its methyl analog (methylreboxetine, MRB) has been shown to be more potent than reboxetine itself. We developed a nine-step synthetic procedure to prepare the normethyl precursor, which was used to synthesize [11C]O-methylreboxetine ([11C]MRB). We also developed a convenient resolution method using a chiral HPLC column to resolve the racemic precursor to obtain enantiomerically pure individual precursors that lead to the individual enantiomers (R,R)-[11C]MRB and (S,S)-[11C]MRB. Here we report an evaluation of the racemate and individual enantiomers of [11C]MRB as radioligands for PET imaging studies of NET systems in baboons both in brain and in peripheral organs. The relative regional distribution of the radioactivity after injection of [11C]MRB in baboon brain is consistent with the known distribution of NET. For a NET-poor region such as striatum, there were no significant changes in the striatal uptakes with and without the nisoxetine pretreatment. In contrast, a significant blocking effect was observed in NET-rich regions such as thalamus and cerebellum after injection of racemic [11C]MRB, with an even more dramatic effect after injection of (S,S)-[11C]MRB. These results, along with the fact that there was no regional specificity and no blocking effect by nisoxetine for (R,R)-[11C]MRB, suggest the enantioselectivity of MRB in vivo, consistent with previous in vitro and in vivo studies in rodents. PET studies of baboon torso revealed a blocking effect by desipramine only in the heart, a NET-rich organ, after injection of (S,S)-[11C]MRB, but not the (R,R)-isomer. These studies demonstrate that the use of (S,S)-[11C]MRB would allow a better understanding of the role that NET plays in living systems.

Maternal-fetal in vivo imaging: a combined PET and MRI study.

UNLABELLED: An understanding of how drugs are transferred between mother and fetus during the gestational period is an important medical issue of relevance to both therapeutic drugs and drugs of abuse. Though there are several in vitro and in vivo methods to examine this issue, all have limitations. Furthermore, ethical and safety considerations generally preclude such studies in pregnant humans. PET and appropriately labeled compounds have the ability to provide information on both maternal-fetal drug pharmacokinetics and pharmacodynamics. We present here a nonhuman primate animal model and the methodology for combining PET and MRI to identify fetal organs and to measure maternal and fetal isotope distribution using (18)F-FDG and a whole-body imaging protocol to demonstrate proof-of-principle. METHODS: One nonpregnant nonhuman primate was used for determination of the anesthesia protocol and MRI methods and 3 pregnant nonhuman primates (Macaques radiata) weighing 4.5-7 kg were used for the imaging study and anesthetized with propofol (160-300 micro g/kg/min). Anatomic T2-weighted MR images were acquired on a 4-T MR instrument. Subsequently, whole-body PET images were acquired 35 min after injection of (18)F-FDG, and standardized uptake values (SUVs) were calculated. Image processing and coregistration were performed using commercial software. RESULTS: All animals underwent uneventful general anesthesia for a period of up to 7 h. Coregistration of PET and MR images allowed identification of fetal organs and demonstrated that (18)F-FDG readily crosses the placenta and that (18)F accumulates in both maternal and fetal brain, heart, and bladder. Brain SUVs averaged 1.95 +/- 0.08 (mean +/- SD) and 1.58 +/- 0.11 for mothers and fetuses, respectively. Monkeys delivered healthy babies after a normal gestational term of 170 d following the PET/MRI study. CONCLUSION: The pregnant macaque in combination with PET and MRI technology allows the measurement of radioisotope distribution in maternal and fetal organs. This demonstrates the potential for noninvasively measuring the transfer of drugs across the placenta and for measuring the fetal drug distribution. It also opens up the possibility for studying binding and elimination as well as the effects of a drug on specific cellular elements and physiologic processes during the gestational period in a primate model.

Low monoamine oxidase B in peripheral organs in smokers.

One of the major mechanisms for terminating the actions of catecholamines and vasoactive dietary amines is oxidation by monoamine oxidase (MAO). Smokers have been shown to have reduced levels of brain MAO, leading to speculation that MAO inhibition by tobacco smoke may underlie some of the behavioral and epidemiological features of smoking. Because smoking exposes peripheral organs as well as the brain to MAO-inhibitory compounds, we questioned whether smokers would also have reduced MAO levels in peripheral organs. Here we compared MAO B in peripheral organs in nonsmokers and smokers by using positron emission tomography and serial scans with the MAO B-specific radiotracers,l-[11C]deprenyl and deuterium-substituted l-[11C]deprenyl (l-[11C]deprenyl-D2). Binding specificity was assessed by using the deuterium isotope effect. We found that smokers have significantly reduced MAO B in peripheral organs, particularly in the heart, lungs, and kidneys, when compared with nonsmokers. Reductions ranged from 33% to 46%. Because MAO B breaks down catecholamines and other physiologically active amines, including those released by nicotine, its inhibition may alter sympathetic tone as well as central neurotransmitter activity, which could contribute to the medical consequences of smoking. In addition, although most of the emphases on the carcinogenic properties of smoke have been placed on the lungs and the upper airways, this finding highlights the fact that multiple organs in the body are also exposed to pharmacologically significant quantities of chemical compounds in tobacco smoke.

Monoamine oxidase A imaging in peripheral organs in healthy human subjects.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of many biogenic and dietary amines. Though studies of MAO have focused mainly on its regulatory role in the brain, MAO in peripheral organs also represents a vast mechanism for detoxifying vasoactive compounds as well as for terminating the action of physiologically active amines, which can cross the blood brain barrier. Indeed, robust central and peripheral MAO activity is a major requirement in the safe use of many CNS drugs, particularly antidepressants, and thus an awareness of the MAO inhibitory potential of drugs is essential in therapeutics. In this study, we examined the feasibility of quantifying MAO A in peripheral organs in healthy human subjects using comparative positron emission tomography (PET) imaging with carbon-11 (t(1/2): 20.4 min) labeled clorgyline ([(11)C]clorgyline) a suicide inactivator of MAO A and its deuterium labeled counterpart ([(11)C]clorgyline-D2). Heart, lungs, kidneys, thyroid, and spleen showed a robust deuterium isotope effect characteristic of MAO and the magnitude of the effect was similar to that of trancylcypromine, an irreversible MAO inhibitor used in the treatment of depression. Liver time-activity curves were not affected by deuterium substitution precluding the estimation of liver MAO in vivo. In organs showing an isotope effect, MAO A is greatest in the lungs and kidneys followed by the thyroid and heart. This method, which has been previously applied in the human brain, opens the possibility to also directly assess the effects of different variables including smoking, dietary substances, drugs, disease, and genetics on peripheral MAO A in humans.