Immobilization of acetyl-CoA:arylamine N-acetyltransferase and the preparation of an enzyme reactor for the synthesis of N-[11C]acetylserotonin

The enzyme arylamine acetyltransferase (acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) from pigeon liver is immobilized onto differently derivatized controlled pore glass beads. Different silanes, spacer arms and reactive end-groups were tested, and immobilized enzyme stability tests were performed. From these experiments, the method of choice was selected: immobilization on controlled pore glass beads (24 nm pore size, 75-125 microns particle size) derivatized with gamma-aminopropyl and glutaraldehyde as the reactive end group. The kinetic properties of an enzyme reactor were investigated and optimized. The goal was to obtain a rapid high-yield conversion of 0.5-1 mumol acetyl-CoA to N-acetylserotonin, so that the reactor is useful for the 11C-labelling of N-acetylserotonin. Using an enzyme reactor (9.8 x 0.5 cm i.d.) containing 4.6 U active arylamine acetyltransferase immobilized onto 930 mg carrier, a 70% conversion of acetyl-CoA was obtained within 4 min.     

A simple preparation of an enzyme reactor producing nicotinamidemononucleotide

Summary An enzyme reactor which produces nicotinamidemononucleotide is easily prepared by adsorption of NAD pyrophosphatase to phosphocellulose. The separation and purification of the mononucleotide is achieved in a single chromatographic step. The spectrophotometric data of purified NMN and its cyanide adduct were redetermined.     

A method for the synthesis of [14C]-kynurenine

A new method is described for the synthesis of [14C]-labelled L-kynurenine from [14C]-L-tryptophan, using extracts of tryptophan-adapted cells of Pseudomonas marginalis. It is based on the selective, rapid inactivation of kynureninase by a newly discovered inhibitor of this enzyme, 3-chloro-L-alanine. The yield of [14C]-kynurenine produced in this manner is 76% theoretical.     

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.     

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.     

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.     

A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate

A simple and sensitive radiochemical procedure to assay argininosuccinate synthetase activity in crude tissue homogenates and lysates of cultured cells is described. The new method depends on the location of 14C, uniformly, in the four carbons of aspartate. On incubation in the presence of excess of L-[U-14C]aspartate, L-citrulline, ATP, and an ATP-generating system, argininosuccinase and arginase, the [14C]fumarate formed is measured as the sum of malate and fumarate. After acidification the latter two acids are separated from [14C]aspartate on a small Dowex-50 column by elution with a few milliliters of water; the unutilized amino acid substrates remain on the column. With a specific radioactivity of 9 X 10(4) cpm, 1 to 2 nmol of product can be accurately measured under kinetically optimum conditions.     

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.     

A facile synthesis of [14C] epicholesterol

A facile procedure is described for the preparation of [14C]epicholesterol from [14C]cholesterol. Cholesterol is first converted to cholesteryl mesylate, which is treated with cesium acetate and 18-crown-6 in refluxing toluene to give epicholesteryl acetate. The latter is hydrolyzed, without isolation, with potassium hydroxide in tetrahydrofuran-methanol to give epicholesterol, which is obtained in pure form by preparative thin-layer chromatography.     

A simplified method for the preparation of pure UDP[14C]glucose

A two-step enzymatic synthesis of UDP[¹⁴C]glucose was described which resulted in high yield. Separation of product from labelled intermediates and other contaminants was achieved by a simple ion exchange chromatography method.     

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 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.     

Synthesis of fatty acids from [1-14C]acetylcoenzyme A in subcellular particles of rat epididymal adipose tissue

1. Mitochondrial and microsomal fractions of rat epididymal adipose tissue incorporated [1-(14)C]acetyl-CoA equally well into various fatty acids by a chain-elongation mechanism. C(18) and C(20) fatty acids were the two major products, and comprised about 80% of the total fatty acids synthesized in both particles. 2. When incubated in air, mitochondria synthesized stearic acid, octadecenoic acid and eicosamonoenoic acid in almost equal amounts (about 20% each), whereas in microsomal fractions, the synthesis of octadecenoic acid was more than fivefold the stearic acid formation. In both fractions, major components of synthesized monoenoic fatty acids were the Delta(11:12) isomers. Hexadecenoic acid and octadecenoic acid from whole adipose tissue contained approx. 11 and 14% of the Delta(11:12) isomer respectively. 3. When mitochondria or microsomal fractions were incubated in nitrogen, there was increased synthesis of stearic acid and palmitic acid and less of C(16) and C(18) monoenoic acids; synthesis of C(20) acids remained predominantly of the monoenoic acids. Determination of the position of the double bond in the monoenoic acids supported the view that the synthesis of hexadecenoic acid and octadecenoic acid involves a desaturase activity, whereas eicosamonoenoic acid and eicosadienoic acid are formed only by elongation of endogenous fatty acids. 4. Most of the radioactivity was found in free fatty acids (63%) and the phospholipid (26%) fraction. In phospholipids, phosphatidylcholine and phosphatidylethanolamine were the two major components. 5. Most of the fatty acids synthesized, including those not normally found in particle lipids (arachidic acid, eicosamonoenoic acid and eicosadienoic acid) were distributed fairly evenly in the phospholipid and free fatty acid fractions. However, stearic acid was found predominantly in the phospholipid fraction.     

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.     

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.