Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of [11C]acetylcoenzyme A

The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (baker's yeast) is used for the synthesis of 1 mumol [11C]acetylcoenzyme A. (CoA-[11C]Ac). A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125-180 micron particle size) was performed. Several silanes, spacer arms and terminal reactive groups were tested. The immobilized enzyme was subjected to storage stability tests. From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass. The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads. The latter were packed in a glass column. The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 mumol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes. This is realized with an enzyme reactor (13.0 x 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.     

Purification and physical-chemical properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver

Acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver was purified by protamine sulfate precipitation, ion exchange chromatography on DEAE-A-25 Sephadex, gel filtration on Sephadex G-75, amethopterin-AH-Sepharose 4B affinity chromatography, and finally, gel filtration on Sephadex G-100. The enzyme preparation was homogeneous as judged by ultracentrifugation studies, SDS-polyacrylamide gel electrophoresis and gel filtration. The N-terminal amino acid was detected to be histidine and the complete amino acid composition is reported. The enzyme contains one disulfide bridge and two cysteine residues/mol monomer. The isoelectric point was estimated to be 4.8. The molecular weight was determined to be 32900 by high-speed sedimentation equilibrium analysis, 33000 by Sephadex G-100 gel filtration and 31600 by SDS-disc gel electrophoresis. The sedimentation coefficient from conventional sedimentation velocity runs was 3.1 S observed by ultraviolet optics. 'Active enzyme centrifugation' showed a sedimentation constant of 5.0 and 4.8 S for the purified enzyme and crude extract from pigeon liver, respectively, indicating that the enzyme forms a dimer under conditions of catalysis. It could be demonstrated that the inhibitor amethopterin was noncompetitive with respect to the acetyl donor and the acetyl acceptor. Acetyl-CoA:arylamine N-acetyltransferase was examined in different organs of pigeon. The enzyme was not inducible by 1,3-phenylenediamine and hexobarbital in vivo.     

New spectrophotometric and radiochemical assays for acetyl-CoA: arylamine N-acetyltransferase applicable to a variety of arylamines

Simple and sensitive spectrophotometric and radiochemical procedures are described for the assay of acetyl-CoA:arylamine N-acetyltransferase (NAT; EC 2.3.1.5), which catalyzes the reaction acetyl-CoA + arylamine----N-acetylated arylamine + CoASH. The methods are applicable to crude tissue homogenates and blood lysates. The spectrophotometric assay is characterized by two features: (i) NAT activity is measured by quantifying the disappearance of the arylamine substrate as reflected by decreasing Schiff's base formation with dimethylaminobenzaldehyde. (ii) During the enzymatic reaction, the inhibitory product CoASH is recycled by the system acetyl phosphate/phosphotransacetylase to the substrate acetyl-CoA. The radiochemical procedure depends on enzymatic synthesis of [3H]acetyl-CoA in the assay using [3H]acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-[3H]acetylarylamine after separation from [3H]acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase.     

A comparison of the brain uptake of N-(cyclopropyl[11C]methyl)norbuprenorphine ([11C]buprenorphine) and N-(cyclopropyl[11C]methyl)nordiprenorphme ([11C]diprenorphine) in baboon using PET

Buprenorphine and diprenorphine were radiolabeled with ¹¹C and their distributions in the baboon brain were studied using positron emission tomography (PET). Specific binding was demonstrated in the striatum (but not in the cerebellum) by pretreating the baboon with (−)naloxone. The absolute striatal uptakes and time courses were similar for these two radioligands but the ratio of radioactivity in the striatum to cerebellum in the baboon was higher for [¹¹C]diprenorphine than for [¹¹C]buprenorphine. Analysis of baboon plasma indicated that both [¹¹C]diprenorphine and [¹¹CJbuprenorphine are rapidly metabolized. Analysis of radioactivity in mouse brain indicated that these two radioligands are stable to metabolic transformation. At 30 min after injection, 86–90% of extracted radioactivity was due to unchanged ¹¹C-labeled radioligands. These results suggest that both [¹¹C]diprenorphine and [¹¹C]buprenorphine may be useful radioligands for studying opioid receptors in humans, although [¹¹C]diprenorphine may be a better radioligand than [¹¹C]buprenorphine for this purpose because of its more rapid clearance from the cerebellum.     

Purification and characterization of an arylamine N-acetyltransferase in the nematode Enterobius vermicularis.

N-acetyltransferase (NAT) activities were determined by incubation of Enterobius vermicularis cytosols with 2-aminofluorene (2-AF) as the substrate followed by high pressure liquid chromatography assays. The NAT activity from E. vermicularis was found to be 0.41 +/- 0.08 nmol/min/mg protein for 2-AF. The apparent K(m) and Vmax values obtained were 0.81 +/- 0.11 mM and 2.25 +/- 0.22 nmol/min/mg protein respectively, for 2-AF. The optimal pH value for the enzyme activity was 7.5 for 2-AF. The optimal temperature for enzyme activity was 37 degrees C for the 2-AF substrate. The molecular weight of NAT from E. vermicularis was 44.9 kD. Among a series of divalent cations and salts, Zn2+, Ca2+, and Fe2+ were the most potent inhibitors. Of the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected the NAT. Iodoacetate, in contrast to other agents, markedly inhibited NAT activity. This report is the first demonstration of acetyl coenzyme A-dependent arylamine NAT activity in E. vermicularis and extends the number of phyla in which this activity has been found.     

Studies with Differentially Labeled [11C]Cocaine, [11C]Norcocaine, [11C]Benzoylecgonine,and [11C]-and 4′-[18F]Fluorococaine to Probe the Extent to Which [11C]Cocaine Metabolites Contribute to PET Images of the Baboon Brain

Abstract: The psychostimulant drug of abuse, cocaine (benzoylecgonine methyl ester), is rapidly metabolized by cleavage of its two ester groups, to give benzoylecgonine (BE) and ecgonine methyl ester, and by N-demethylation, to give N-norcocaine (NC). The recent use of [N-methyl-¹¹CH₃]cocaine to image brain cocaine binding sites with positron emission tomography (PET) raises the question of whether PET images partially reflect the distribution and kinetics of labeled cocaine metabolites. We prepared [O-metty/-¹¹CH₃]cocaine by methylation of the sodium salt of BE with [¹¹C]CH₃l, and showed that PET baboon brain scans, as well as regional brain kinetics and plasma time-activity curves corrected for the presence of labeled metabolites, are nearly identical to those seen with [N-methyl-¹¹CH₃]cocaine. This strongly suggests that ¹¹C metabolites do not significantly affect PET images, because the metabolite pattern is different for the two labeled forms of cocaine. In particular, nearly half the ¹¹C in blood plasma at 30 min was [¹¹C]CO₂ when [N-methy/-¹¹CH₃]cocaine was administered, whereas [¹¹C]CO₂ was not formed from [O-methy/-¹¹CH₃]cocaine. Only a trace of [¹¹C]NC was detected in plasma after [O-methyl-¹¹CH₃]cocaine administration. Nearly identical brain PET data were also obtained when 4′-[N-methy/-¹¹CH₃]fluorococaine and 4′-[¹⁸F]fluoro-cocaine (prepared by nucleophilic aromatic substitution from [¹⁸F]fluoride-and 4′-nitrococaine) were compared with [N-methy/-¹¹CH₃]cocaine. In vitro assays with rat brain membranes showed that cocaine and 4′-fluoroco-caine were equipotent at the dopamine reuptake site, but that 4′-fluorococaine was about 100 times more potent at the 5-hydroxytryptamine reuptake site. The studies with positron-emitting 4′-fluorococaines thus support the lack of significance of labeled metabolites or of binding to 5-hydroxytryptamine reuptake sites to PET images taken with [N-methy/-¹¹CH₃]cocaine. [¹¹C]NC prepared by O-methylation of norbenzoylecgonine gave PET images with preferential uptake in striatum, but slower clearance from all brain regions than [O-methy/-¹¹CH₃]cocaine. [¹¹C]BE prepared by N-methylation of norbenzoylecgonine did not show brain uptake.     

Rapid C-carboxylation of nitro[(11)C]methane for the synthesis of ethyl nitro[2-(11)C]acetate

The labelling synthesis of ethyl nitro[2-(11)C]acetate, a synthetic intermediate feasible for (11)C-labelled PET tracers, by C-carboxylation of [(11)C]MeNO(2) with 1-ethoxycarbonylbenzotriazole, and its simple application are presented.     

Utilization of [11C]phosgene for radiosynthesis of N-(2-{3-[3,5-bis(trifluoromethyl)]phenyl[11C]ureido}ethyl)glycyrrhetinamide, an inhibitory agent for proteasome and kinase in tumors

N-(2-{3-[3,5-Bis(trifluoromethyl)]phenylureido}ethyl)glycyrrhetinamide (2), an ureido-substituted derivative of glycyrrhetinic acid (1), has been reported to display potent inhibitory activity for proteasome and kinase, which are overexpressed in tumors. In this study, we labeled this unsymmetrical urea 2 using [(11)C]phosgene ([(11)C]COCl(2)) as a labeling agent with the expectation that [(11)C]2 could become a positron emission tomography ligand for the imaging of proteasome and kinase in tumors. The strategy for the radiosynthesis of [(11)C]2 was to react hydrochloride of 3,5-bis(trifluoromethyl)aniline (4·HCl) with [(11)C]COCl(2) to possibly give isocyanate [(11)C]6, followed by the reaction of [(11)C]6 with N-(2-aminoethyl)glycyrrhetinamide (3).     

Radiosynthesis of [11C]BBAC and [11C]BBPC as potential PET tracers for orexin2 receptors

Radiosynthesis of [N-methyl-(11)C](S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide ([(11)C]BBAC or [(11)C]3) and [N-methyl-(11)C] (S)-N-([1,1'-biphenyl]-2-yl)-1-(3-(1-methyl-1H-benzo[d]imidazol-2-yl)propanoyl)pyrrolidine-2-carboxamide ([(11)C]BBPC or [(11)C]-4), two potential PET tracers for orexin2 receptors are described. Syntheses of non-radioactive standards 3, 4 and corresponding desmethyl precursors 1, 2 were achieved from common intermediate (S)-2-([1,1'-biphenyl]-2-yl)-1-(pyrrolidin-2-yl)ethanone. Methylation using [(11)C]CH(3)OTf in the presence of base in acetone afforded [(11)C]3 and [(11)C]4 in 30±5% yield (EOS) with >99 % radiochemical purities with a specific activity ranged from 2.5±0.5 Ci/μmol (EOB). The logP of [(11)C]3 and [(11)C]4 were determined as 3.4 and 2.8, respectively. The total synthesis time was 30 min from EOB. However, PET scans performed in a rhesus monkey did not show tracer retention or appropriate brain uptake. Hence [(11)C]3 and [(11)C]4 cannot be used as PET tracers for imaging orexin2 receptors.     

Synthesis of 11C-radiopharmaceuticals using direct fixation of [11C]carbon dioxide and [11C]carbon monoxide

Synthesis of ¹¹C-labeled radiopharmaceuticals via direct fixation of [¹¹C]carbon dioxide and [¹¹C]carbon monoxide are described.     

Tumor uptake studies of S-adenosyl-l-[methyl-11C]methionine and l-[methyl-11C]methionine

Tumor accumulation of S-adenosyl-l-[methyl-¹¹C]methionine ([¹¹C]SAM) was investigated in mice bearing mammary carcinoma (FM3A) and in rats bearing ascitic hepatoma (AH109A). After injection of [¹¹C]SAM the blood clearance of ¹¹C radioactivity was rapid. The ¹¹C level was relatively high in both tumors. The uptake ratios of tumor to organ increased with time in several organs, especially in brain and muscle. In FM3A tumor tissue the ¹¹C was incorporated with time into the acid-precipitable fraction and 38% of the ¹¹C was detected in this fraction at 60 min after injection. This fraction reflects the amount of ¹¹C-methyl group transferred into macromolecules in tumor tissue. In AH109A-bearing rats the metabolisms of [¹¹C]SAM and l-[methyl-¹¹C]methionine ([¹¹C]Met), in vivo precursor of SAM, were compared. Tumor uptake of [¹¹C]SAM was about two thirds of that of [¹¹C]Met at 20 min after injection. At this time, for the [¹¹C]SAM 27 and 8% of the ¹¹C in the AH109A tissue were detected in the acid-precipitable and the lipid fractions, respectively. The corresponding figures for [¹¹C]Met were 61% and 2%. In the liver considerable amounts of ¹¹C were observed in the lipid fraction for both tracers.These results show that [¹¹C]SAM has potential as a tracer for tumor localization with positron emission tomography (PET) and suggest that in tumor studies combining [¹¹C]Met and PET, it should be taken into account that the ¹¹C-labeled methyl group of [¹¹C]Met is not only incorporated into protein but also other macromolecules and lipids via [¹¹C]SAM.     

Fully automated synthesis and initial PET evaluation of [11C]PBR28

Fully automated synthesis and initial PET evaluation of a TSPO radioligand, [¹¹C]PBR28 (N-(2-[¹¹C]methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide), are reported. These results facilitate the potential preclinical and clinical PET studies of [¹¹C]PBR28 in animals and humans.     

Comparative evaluation of positron emission tomography radiotracers for imaging the norepinephrine transporter: (S,S) and (R,R) enantiomers of reboxetine analogs ([11C]methylreboxetine, 3-Cl-[11C]methylreboxetine and [18F]fluororeboxetine), (R)-[11C]nisoxetine, [11C]oxaprotiline and [11C]lortalamine

We have synthesized and evaluated several new ligands for imaging the norepinephrine transporter (NET) system in baboons with positron emission tomography (PET). Ligands possessing high brain penetration, high affinity and selectivity, appropriate lipophilicity (log P = 1.0-3.5), high plasma free fraction and reasonable stability in plasma were selected for further studies. Based on our characterization studies in baboons, including 11C-labeled (R)-nisoxetine (Nis), oxaprotiline (Oxap), lortalamine (Lort) and new analogs of methylreboxetine (MRB), in conjunction with our earlier evaluation of 11C and 18F derivatives of reboxetine, MRB and their individual (R,R) and (S,S) enantiomers, we have identified the superiority of (S,S)-[11C]MRB and the suitability of MRB analogs [(S,S)-[11C]MRB > (S,S)-[11C]3-Cl-MRB > (S,S)-[18F]fluororeboxetine] as potential NET ligands for PET. In contrast, Nis, Oxap and Lort displayed high uptake in striatum (higher than in thalamus). The use of these ligands is further limited by high non-specific binding and relatively low specific signal, as is characteristic of many earlier NET ligands. Thus, to our knowledge (S,S)-[11C]MRB remains by far the most promising NET ligand for PET studies.     

Efficient sequential synthesis of PET Probes of the COX-2 inhibitor [11C]celecoxib and its major metabolite [11C]SC-62807 and in vivo PET evaluation

Synthesis of [(11)C]celecoxib, a selective COX-2 inhibitor, and [(11)C]SC-62807, a major metabolite of celecoxib, were achieved and the potential of these PET probes for assessing the function of drug transporter in biliary excretion was evaluated. The synthesis of [(11)C]celecoxib was achieved in one-pot by reacting [(11)C]methyl iodide with an excess of the corresponding pinacol borate precursor using Pd(2)(dba)(3), P(o-tolyl)(3), and K(2)CO(3) (1:4:9) in DMF. The radiochemical yield of [(11)C]celecoxib was 63±23% (decay-corrected, based on [(11)C]CH(3)I) (n=7) with a specific radioactivity of 83±23GBq/μmol (n=7). The average time of synthesis from end of bombardment including formulation was 30min with >99% radiochemical purity. [(11)C]SC-62807 was synthesized from [(11)C]celecoxib by further rapid oxidation in the presence of excess KMnO(4) with microwave irradiation. The radiochemical yield of [(11)C]SC-62807 was 55±9% (n=3) (decay-corrected, based on [(11)C]celecoxib) with a specific radioactivity of 39±4GBq/μmol (n=3). The average time of synthesis from [(11)C]celecoxib including formulation was 20min and the radiochemical purity was >99%. PET studies in rats and the metabolite analyzes of [(11)C]celecoxib and [(11)C]SC-62807 showed largely different excretion processes, and consequently, [(11)C]SC-62807 was rapidly excreted via hepatobiliary excretion without further metabolism. [(11)C]SC-62807 was shown to have a high potential as a PET probe for evaluating drug transporter function in biliary excretion.     

Radiosynthesis of [11C]Vandetanib and [11C]chloro-Vandetanib as new potential PET agents for imaging of VEGFR in cancer

Vandetanib (ZD6474) and its chlorine analogue chloro-Vandetanib are potent and selective vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors with low nanomolar IC₅₀ values. [¹¹C]Vandetanib and [¹¹C]chloro-Vandetanib, new potential PET agents for imaging of VEGFR in cancer, were first designed, synthesized and labeled at nitrogen and oxygen positions from their corresponding N- and O-des-methylated precursors, in 40-50% decay corrected radiochemical yield and 370-555 GBq/μmol specific activity at end of bombardment (EOB).     

A high-yield route to synthesize the P-glycoprotein radioligand [11C]N-desmethyl-loperamide and its parent radioligand [11C]loperamide

N-Desmethyl-loperamide and loperamide were synthesized from α,α-diphenyl-γ-butyrolactone and 4-(4-chlorophenyl)-4-hydroxypiperidine in five and four steps with 8% and 16% overall yield, respectively. The amide precursor was synthesized from 4-bromo-2,2-diphenylbutyronitrile and 4-(4-chlorophenyl)-4-hydroxypiperidine in 2 steps with 21–57% overall yield. [¹¹C]N-Desmethyl-loperamide and [¹¹C]loperamide were prepared from their corresponding amide precursor and N-desmethyl-loperamide with [¹¹C]CH₃OTf through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 20–30% and 10–15% radiochemical yields, respectively, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB), with 370–740 GBq/μmol specific activity at EOB.     

Decreased binding of [11C]NNC112 and [11C]SCH23390 in patients with chronic schizophrenia

AimsAbnormality of cognitive function in schizophrenia has been suggested to be related to dopamine D₁ receptor. However, the results of previous positron emission tomography (PET) studies of dopamine D₁ receptor in schizophrenia were not consistent.Main methodsIn this study, six patients with schizophrenia in severe residual phase with chronic antipsychotic treatment and twelve healthy age-matched controls participated. Two different radioligands, [¹¹C]NNC112 and [¹¹C]SCH23390, for dopamine D₁ receptor were used on the same subjects. Binding of the ligands was measured by PET, and statistical analysis was performed using one-way analysis of covariate (ANCOVA) with age as covariate.Key findingsGood correlations between binding potential values (BP_ND) and age were observed in all regions of interest (ROIs) with both ligands. ANCOVA with age as covariate of BP_ND values of all ROIs revealed that the patient group showed significantly lower BP_ND value compared with the control group in both ligands.SignificanceIn patients with chronic schizophrenia in severe residual phase with chronic antipsychotic treatment, the binding potential values of both ligands were significantly lower in the striatum and cortical regions than those of healthy controls.     

Synthesis and evaluation of [11C]cyanoimipramine

[¹¹C]Cyanoimipramine has been prepared by methylation of the desmethyl cyanoimipramine with [¹¹C]methyl iodide. The chemically and radiochemically pure labelled product was obtained with a high specific activity (> 300 mCi/μmol). When ¹¹C (or ³H)-cyanoimipramine was intravenously administered in mice, high accumulations were shown in brain and lung. Thirty minutes after injection of the tracer, differences were found in the radioactivity between the cerebral cortex and the cerebellum. The regional distribution of radioactivity in the rat brain 30 min after i.v. injection of [¹¹C]cyanoimipramine was also examined, and the radioactivity was high in receptor rich areas (striatum, cerebral cortex etc.) but low in receptor poor area (cerebellum). The in vivo stability of [³H]cyanoimipramine was quite stable in the mouse brain for at least 30 min. Thirty minutes after injection, the radioactivity in the cerebral cortex of the carrier-added state was reduced as compared with the carrier-free state. Taken together, the in vivo specific binding of [³H]cyanoimipramine in the cerebral cortex was estimated at about 40–50% of the total radioactivity. Furthermore, the distribution of [³H]cyanoimipramine in the mice forced to swim was examined. Significant changes in the distribution of [³H]cyanoimipramine were observed in the cerebral cortex.     

N-acylation of tyramines: purification and characterization of an arylamine N-acetyltransferase from rat brain and liver.

The N-acylation of tyramine isomers and other biogenic amines has been studied. The liver exhibits the highest activity towards tyramines, while the brain exhibits a low but significant activity. In the brain, tyramine N-acylation activity was heterogenously distributed. The arylamine N-acetyltransferase has been partially purified from both rat liver and brain, the two enzymes being quite similar with respect to their chromatographic properties, optimal pH requirement (pH 7.8), and their kinetic parameters. The product N-acetyltyramine is not oxidized by liver amidohydrolase or monoamine oxidase.     

A general method for the synthesis of aryl [11C]methylsulfones: potential PET probes for imaging cyclooxygenase-2 expression

A general one-pot method has been developed for the conversion of an aryl thiol moiety masked as the butyrate ester to the corresponding 11C-labeled methylsulfone group. The potential of this methodology has been demonstrated by the successful radiosynthesis of carbon-11 analogues of several highly selective cyclooxygenase-2 (COX-2) inhibitors such as Rofecoxib, Etoricoxib, and 3-(4-methylsulfonylphenyl)-4-phenyl-5-trifluoromethyl isoxazole in high yield. The chemical and radiochemical purities obtained for the 11C-labeled COX-2 inhibitors are >99% with a specific activity >1000 Ci/mmol.