Eugene Malveaux

18F-labeled FECNT: a selective radioligand for PET imaging of brain dopamine transporters

Fluorine-18 labeled 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-fluoroethyl)nort ropane (FECNT) was synthesized in the development of a dopamine transporter (DAT) imaging ligand for positron emission tomography (PET). The methods of radiolabeling and ligand synthesis of FECNT, and the results of the in vitro characterization and in vivo tissue distribution in rats and in vivo PET imaging in rhesus monkeys of [18F]FECNT are described. Fluorine-18 was introduced into 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-fluoroethyl)nort ropane (4) by preparation of 1-[18F]fluoro-2-tosyloxyethane (2) followed by alkylation of 2beta-carbomethoxy-3beta-(4-chlorophenyl)nortropane (3) in 21% radiochemical yield (decay corrected to end of bombardment [EOB]). Competition binding in cells stably expressing the transfected human DAT serotonin transporter (SERT) and norepinephrine transporter (NET) labeled by [3H]WIN 35428, [3H]citalopram, and [3H]nisoxetine, respectively, indicated the following order of DAT affinity: GBR 12909 > CIT >> 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(3-fluoropropyl) nortropane (FPCT) > FECNT. The affinity of FECNT for SERT and NET was 25- and 156-fold lower, respectively, than for DAT. Blocking studies were performed in rats with a series of transporter-specific agents and demonstrated that the brain uptake of [18F]FECNT was selective and specific for DAT-rich regions. PET brain imaging studies in monkeys demonstrated high [18F]FECNT uptake in the caudate and putamen that resulted in caudate-to-cerebellum and putamen-to-cerebellum ratios of 10.5 at 60 min. [18F]FECNT uptake in the caudate/putamen peaked in less than 75 min and exhibited higher caudate- and putamen-to-cerebellum ratios at transient equilibrium than reported for 11C-WIN 35,428, [11C]CIT/RTI-55, or [18F]beta-CIT-FP. Analysis of monkey arterial plasma samples using high performance liquid chromatography determined that there was no detectable formation of lipophilic radiolabeled metabolites capable of entering the brain. In equilibrium displacement experiments with CIT in rhesus monkeys, radioactivity in the putamen was displaced with an average half-time of 10.2 min. These results indicate that [18F]FECNT is a radioligand that is superior to 11C-WIN 35,428, [11C]CIT/RTI-55, [18F]beta-CIT-FP, and [18F]FPCT for mapping brain DAT in humans using PET.

Stereoselective synthesis and biological evaluation of syn-1-amino-3-[18F]fluorocyclobutyl-1-carboxylic acid as a potential positron emission tomography brain tumor imaging agent

Amino acid syn-1-amino-3-fluoro-cyclobutyl-1-carboxylic acid (syn-FACBC) 12, the isomer of anti-FACBC, has been selectively synthesized and [(18)F] radiofluorinated in 52% decay-corrected yield using no-carrier-added [(18)F]fluoride. The key step in the synthesis of the desired isomer involved stereoselective reduction using lithium alkylborohydride/zinc chloride, which improved the ratio of anti-alcohol to syn-alcohol from 17:83 to 97:3. syn-FACBC 12 entered rat 9L gliosarcoma cells primarily via L-type amino acid transport in vitro with high uptake of 16% injected dose per 5 x 10(5) cells. Biodistribution studies in rats with 9L gliosarcoma brain tumors demonstrated high tumor to brain ratio of 12:1 at 30 min post injection. In this model, amino acid syn-[(18)F]FACBC 12 is a promising metabolically based radiotracer for positron emission tomography brain tumor imaging.

Synthesis and evaluation of 2-amino-4-[18F]fluoro-2-methylbutanoic acid (FAMB): relationship of amino acid transport to tumor imaging properties of branched fluorinated amino acids

Radiolabeled amino acids represent a promising class of tumor imaging agents, and the determination of the optimal characteristics of these tracers remains an area of active investigation. A new (18)F-labeled branched amino acid, 2-amino-4-[(18)F]fluoro-2-methylbutanoic acid (FAMB), has been prepared in 36% decay-corrected yield using no-carrier-added [(18)F]fluoride. In vitro uptake assays with rat 9L gliosarcoma cells suggest that [(18)F]FAMB was transported primarily via the L type amino acid transport system. In vivo studies with [(18)F]FAMB demonstrated tumor to normal brain ratios of 14:1 in rats with intracranial 9L gliosarcoma tumors at 60 minutes after injection. Comparison of [(18)F]FAMB with structurally related (18)F-labeled branched amino acids demonstrated that A type transport in vitro was positively correlated with the tumor to brain ratios observed in vivo.

Synthesis, Radiolabeling and Biological Evaluation of (R)- and (S)-2-Amino-3-[18F]Fluoro-2-Methylpropanoic Acid (FAMP) and (R)- and (S)-3-[18F]Fluoro-2-Methyl-2-N-(Methylamino)propanoic Acid (NMeFAMP) as Potential PET Radioligands for Imaging Brain Tumors

The non-natural amino acids (R)- and (S)-2-amino-3-fluoro-2-methylpropanoic acid 5 and (R)- and (S)-3-fluoro-2-methyl-2-N-(methylamino)propanoic acid 8 were synthesized in shorter reaction sequences than in the original report starting from enantiomerically pure (S)- and (R)-alpha-methyl-serine, respectively. The reaction sequence provided the cyclic sulfamidate precursors for radiosynthesis of (R)- and (S)-[(18)F]5 and (R)- and (S)-[(18)F]8 in fewer steps than in the original report. (R)- and (S)-[(18)F]5 and(R)- and (S)-[(18)F]8 were synthesized by no-carrier-added nucleophilic [(18)F]fluorination in 52-66% decay-corrected yields with radiochemical purity over 99%. The cell assays showed that all four compounds were substrates for amino acid transport and enter 9L rat gliosarcoma cells in vitro at least in part by system A amino acid transport. The biodistribution studies demonstrated that in vivo tumor to normal brain ratios for all compounds were high with ratios of 20:1 to115:1 in rats with intracranial 9L tumors. The (R)-enantiomers of [(18)F]5 and [(18)F]8 demonstrated higher tumor uptake in vivo compared to the (S)-enantiomers.

Synthesis, Radiosynthesis, and Biological Evaluation of Fluorine-18 Labeled 2β-Carbo(fluoroalkoxy)-3β-(3′-((Z)-2-haloethenyl)phenyl)nortropanes: Candidate Radioligands for In Vivo Imaging of the Serotonin Transporter with Positron Emission Tomography

The meta-vinylhalide fluoroalkyl ester nortropanes 1-4 were synthesized as ligands of the serotonin transporter (SERT) for use as positron emission tomography (PET) imaging agents. In vitro competition binding assays demonstrated that 1-4 have a high affinity for the SERT (K(i) values = 0.3-0.4 nM) and are selective for the SERT over the dopamine and norepinephrine transporters (DAT and NET). MicroPET imaging in anesthetized cynomolgus monkeys with [(18)F]1-[(18)F]4 demonstrated that all four tracers behave similarly with peak uptake in the SERT-rich brain regions achieved after 45-55 min, followed by a steady washout. An awake monkey study was performed with [(18)F]1, which demonstrated that the uptake of [(18)F]1 was not influenced by anesthesia. Chase studies with the SERT ligand 15 displaced [(18)F]1-[(18)F]4, but chase studies with the DAT ligand 16 did not displace [(18)F]1-[(18)F]4 thus indicating that the tracers were binding specifically to the SERT.

Cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and phosphodieterases in arteries and veins of dogs Distribution and effects of arteriovenous fistula and arterial occlusion

Possible involvement of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and cyclic nucleotide phosphodiesterases in functions of vascular tissues were investigated in the dog. All of the above activities, localized in the smooth muscle-rich inner layer of the blood vessels, were found to be higher in the arteries than in the veins. The peripheral arteries were disproportionately richer in cyclic GMP-dependent protein kinase (as indicated by high ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase) than were the veins, with the exception of the pulmonary artery, an atypical arterial tissue exposed to low blood pressure. Interestingly, the protein kinase ratio for the aorta, an artery with no significant role in blood pressure regulation, was not higher than that for the vena cava. Creation of femoral arteriovenous fistulae in the dogs led to preferential reductions in the cyclic GMP-dependent enzyme activity both in the proximal and distal arteries, whereas it was elevated in the stressed vein distal to the anastomotic site. The cyclic GMP-dependent enzyme was preferentially reduced in the saphenous artery distal to occlusion. Changes in the cyclic GMP-dependent enzyme activity appeared to precede gross atrophy or hypertrophy of the vessels. It is suggested that the vascular cyclic GMP-dependent protein kinase may be closely related to peripheral resistance and its regulation.

Impaired monoamine oxidase activity in dogs with portacaval shunt

Abstract In dogs with portacaval shunt, hypertyraminemia could result from either impaired degradation by monoamine oxidase (MAO) and/or from failure of tyramine to reach this enzyme. MAO activity was evaluated in liver obtained from dogs before and after the construction of an end-to-side portacaval shunt. Diversion of portal blood from the liver by a portacaval shunt resulted in a significant (P −1 · hr −1 ] as compared to controls [28.7 ± 6.3 nmoles PHA formed · (mg protein) −1 · hr −1 ]. Activity was maximally reduced in shunted dogs with stages II and III hepatic encephalopathy. In addition, more than a 50 per cent reduction in both the V max and the K m of hepatic MAO for tyramine was noted in shunted dogs as compared to controls. Similar kinetic abnormalities [post-shunt, K m 52.63 ± 14.3, V max 3.8 ± 1.2 vs sham group, K m 120.1 ± 22.3 μM tyramine, V max 14.3 ± 4.5 nmoles PHA · (mg protein) −1 · hr −1 ], as well as decreased MAO activity [post-shunt, 1.85 ± 0.83 vs sham group, 6.7 ± 2.1 nmoles PHA formed · (mg protein) −1 · hr −1 ], were found in cerebral cortex from encephalopathic dogs with portacaval shunt. In summary, defective MAO activity may contribute to many of the pathophysiologic events observed in dogs with portacaval anastomosis. Such abnormalities could explain the hypertyraminemia and encephalopathy that have been reported in patients and experimental animals with liver disease.

Tyramine-Induced Brain Injury in Phenelzine-Treated Dogs: An Animal Model for Cerebral Edema

We have recently demonstrated that p-tyramine (p-TA) induced behavioral side-effects and precipitated coma in chronically treated dogs with the monoamine oxidase inhibitor (MAOI) phenelzine (PEH) (Faraj et al, 1983). The objective of the present investigation was to determine whether p-TA-induced neurotoxicity in PEH-treated dogs is a reflection of the development of brain edema and whether the edema is associated with increased permeability into the brain from peripheral circulation of diffusion-limited substances. We chose to examine the influence of p-TA in dogs before and after treatment with PEH on the first-pass brain image of technetium-99m-diethylenetriamine pentaacetic acid (Tc-99m-DTPA). Furthermore, kinetics of brain Tc-99m-DTPA were correlated with cerebrospinal fluid (CSF) pressure measurements and determination of CSF concentrations of catecholamines.

A Quantitative Radionuclide Imaging Technique to Study Cerebral Edema in Dogs with Impaired Monoamine Oxidase

Under normal circumstances, the brain vasculature and astrocyte cells effectively exclude most substances that are not highly lipid soluble or are not used directly in brain cellular metabolism. However, in the presence of cerebral pathology such as neoplasm, infarction, Reye’s syndrome and hydrocephalus, sufficient alterations in the blood-brain barrier occur to allow passage of diffusion-limited radioactive tracers into the region of abnormality. The most commonly employed diffusion-limited tracer for cerebral imaging is Tc-99m-DTPA (Russell et al, 1980). The tracer Tc-99m-diethylenetriamine pentaacetic acid (Tc-99m-DTPA) is a low molecular weight, negatively charged radiopharmaceutical.