Dose-responsive effect of radiotherapy on the tumor uptake of l-[methyl-11C]methionine; feasibility for monitoring recurrence of tumor

The l-[methyl-¹¹C]methionine ([¹¹C]Met) uptake by rat AH109A tumor was decreased irradiation-dose dependently from the control to 5, 10 and 20 Gy. After 10 Gy irradiation, the [¹¹C]Met uptake decreased earlier than the tumor volume reduction, and later, it significantly increased earlier than the recurrent growth. Double tracer autoradiography with [¹⁴C]Met and 4-[¹⁸F]fluoroantipyrine showed a decrease in the [¹⁴C]Met tumor uptake without change of blood flow after irradiation. The [¹¹C]Met uptake representing amino acid metabolism is a sensitive indicator for monitoring radiotherapeutic effect on tumor.     

Tumor uptake studies of d,l-[5-14C]ornithine and d,l-2-difluoromethyl[5-14C]ornithine

The feasibility of d,l-[5-¹⁴C]ornithine ([¹⁴C]ornithine), a precursor for polyamine synthesis, and d,l-2-difluoromethyl[5-¹⁴C]ornithine ([¹⁴C]DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) were investigated for tumor localization. As an animal model, mice bearing mammary carcinoma, FM3A, were used. After i.v. injection of [¹⁴C]ornithine accumulation of radioactivity was observed in the FM3A, in which 43% of the ¹⁴C radioactivity was measured in the polyamine pool and 41% in the amino acid pool at 60 min after injection. Tumor uptake of [¹⁴C]DFMO was relatively low but constant during 60 min after injection. At 60 min after injection, 11% of the ¹⁴C was present in the acid-precipitable fraction of the FM3A, which suggests the formation of an irreversible complex of [¹⁴C]DFMO with ODC. For both compounds rapid blood clearance and high tumor-to-organ ratios were observed. Our results indicate that in connection with an enhanced polyamine synthesis in the tumors, the compounds investigated have potential as tracers for tumor detection.     

A comparative PET study using different 11C-labelled amino acids in Walker 256 carcinosarcoma-bearing rats

In Walker 256 carcinosarcoma-bearing rats, the dynamic distribution of l-[1-^11C]tyrosine, l-[methyl-¹¹C]methionine, l-[1-¹¹C]methionine and d-[1-¹¹C]methionine has been measured by PET. An equivalent tumor-imaging potential was observed for each of the three l-amino acids. Thirty minutes after injection, the tumors accumulated 57% (P < 0.01) more ¹¹C-activity from l-[1-¹¹C]methionine than from l-[methyl-¹¹C]methionine. At the same point of time, the livers showed a 33% (P < 0.001) higher ¹¹C-uptake with l-[methyl-¹¹C]methionine than with l-[1-¹¹C]methionine. The dynamic tissue data are in agreement with the findings in experiments with ¹⁴C-analogs.     

Visualizing real time [11C]methionine translocation in Fe-sufficient and Fe-deficient barley using a Positron Emitting Tracer Imaging System (PETIS)

[¹¹C]methionine was supplied to Fe-deficient and Fe-sufficient barley plants through a single leaf, and real time ¹¹C movement was monitored using a Positron Emitting Tracer Imaging System (PETIS). In Fe-deficient plants, [¹¹C]methionine was translocated from the tip of the absorbing leaf to the 'discrimination centre' located at the base of the shoot, and then retranslocated to all the chlorotic leaves within 60 min, while a negligible amount was retranslocated to the roots. In Fe-sufficient plants, methionine was translocated to the discrimination centre and then only to the newest leaf on the main shoot within 60 min. A negligible amount was also retranslocated to the roots. In conclusion, methionine from the above-ground parts of a plant is not a precursor of mugineic acid under Fe-deficiency. The discrimination centre is suggested to play a vital role in the distribution of mineral elements and metabolites in graminaceous monocots.Keywords: [¹¹C]methionine, discrimination centre, Fe deficiency, mugineic acid, PETIS.      

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 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.     

Real-time [11C]methionine translocation in barley in relation to mugineic acid phytosiderophore biosynthesis

[11C]Methionine was supplied through barley roots and the 11C signal was followed for 90 min using a real-time imaging system (PETIS), with subsequent development of autoradiographic images of the whole plant. In all cases, [11C]methionine was first translocated to the 'discrimination center', the basal part of the shoot, and this part was most strongly labeled. Methionine absorbed by the roots of the plants was subsequently translocated to other parts of the plant. In Fe-deficient barley plants, a drastic reduction in [11C]methionine translocation from the roots to the shoot was observed, while a greater amount of 11C was found in the leaves of Fe-sufficient or methionine-pretreated Fe-deficient plants. Treatment of Fe-deficient plants with aminooxyacetic acid, an inhibitor of nicotianamine aminotransferase, increased the translocation of [11C]methionine to the shoot. The retention of exogenously supplied [11C]methionine in the roots of Fe-deficient barley indicates that the methionine is used in the biosynthesis of mugineic acid phytosiderophores in barley roots. This and the absence of methionine movement from shoots to the roots suggest that the mugineic acid precursor methionine originates in the roots of plants.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Synthesis and biodistribution of [11C]fludiazepam for imaging benzodiazepine receptors

As a tracer for in vivo studies on benzodiazepine receptors, 7-chloro-1,3-dihydro-5-(2-fluorophenyl)-1-[¹¹C]methyl-2H-1, 4-benzodiazepin-2-one, [¹¹C]fludiazepam, was synthesized by the methylation of norderivative with [¹¹C]CH₃I, and purified by high-performance liquid chromatography. Within 60 min [¹¹C]fludiazepam was obtained for injection in high radiochemical yields and in high radiochemical purity with a specific activity of up to 230mCi/μmol.After i.v. injection of [¹¹C]fludiazepam in rats the radioactivity was rapidly incorporated into many tissues and the blood clearance of the radioactivity was very rapid. The brain uptake was high and decreased gradually. The adrenal uptake was the highest and decreased with high loading doses. The effect of the loading dose on the uptake was also found in the heart and lungs. By autoradiography using [¹¹C]fludiazepam, a higher accumulation was visualized in the cortex and thalamus than in other regions.     

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.     

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.     

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.     

Changes in the Contents of S-Adenosylmethionine and 1-Methylhistidine in Hiproly Barley Callus after Auxin Withdrawal

When Hiproly barley callus was placed on an auxin-free Murashige and Skoog’s medium, adventitious roots were formed. The contents of S-adenosylmethionine (SAM) and 1-methylhistidine increased gradually in the callus throughout the root differentiation, along with increases in the levels of their precursors, methionine, histidine, and adenosine 5′-triphosphate. In the callus after auxin withdrawal, the addition of methionine resulted in rapid increases in the content of SAM and the incorporation of [methyl-¹⁴C]methionine into SAM. The accumulation of SAM was closely related to the elevation in methionine level in the callus. 1-Methylhistidine also increased greatly in the callus treated with methionine, but the addition of histidine had little effect on the change in 1-methylhistidine in the callus.     

Radiosynthesis and evaluation of [11C]CMP,a high affinity GSK3 ligand

Dysfunction of GSK3 is implicated in the etiology of many brain, inflammatory, cardiac diseases, and cancer. PET imaging would enable in vivo detection and quantification of GSK3 and can impact the choice of therapy, allow non-invasive monitoring of disease progression and treatment effects. In this report, the synthesis and evaluation of a high affinity GSK3 ligand, [¹¹C]2-(cyclopropanecarboxamido)-N-(4-methoxypyridin-3-yl)isonicotinamide, ([¹¹C]CMP, (3), (IC₅₀?=?3.4?nM, LogP?=?1.1) is described. [¹¹C]CMP was synthesized in 25?±?5% yield by radiomethylating the corresponding phenolate using [¹¹C]CH₃I. The radioligand exhibited modest uptake in U251 human glioblastoma cell lines with ～50% specific binding. MicroPET studies in rats indicated negligible blood–brain barrier (BBB) penetration of [¹¹C]CMP, despite its high affinity and suitable logP value for BBB penetration. However, administration of cyclosporine prior to [¹¹C]CMP injection showed significant improvement in brain radioactivity uptake and the tracer binding. This finding indicates that [¹¹C]CMP might be a P-gp efflux substrate and therefore has some limitations for routine in vivo PET evaluations in brain.     

Effects of dietary ethionine on methionine metabolism in a dipterous parasitoid Agria housei schwel

Metabolism of injected l-[methyl-¹⁴C]-methionine and l-[ethyl-1-¹⁴C]-ethionine was studied in female adults of Agria housei fed chemically-defined diet (control) or ethionine-supplemented diet (0.07% w/v). Flies fed ethionine-supplemented diet showed a 50% reduction in the amount of radioactivity incorporated into proteins from injected l-[methyl-¹⁴C]-methionine compared to flies fed the control diet. Small amounts of ethionine were also incorporated into proteins possibly giving rise to functionally abnormal proteins. These observations appear to explain, at least in part, the ability of ethionine to inhibit ovarian growth in A. housei. Both methionine and ethionine are oxidized to their corresponding sulphoxides; in addition to being incorporated into proteins and lipids, radioactivity was also detected in several unidentified products.     

Methionine metabolism in apple tissue: implication of s-adenosylmethionine as an intermediate in the conversion of methionine to ethylene

If S-adenosylmethionine (SAM) is the direct precursor of ethylene as previously proposed, it is expected that 5′-S-methyl-5′-thioadenosine (MTA) would be the fragment nucleoside. When [Me-¹⁴C] or [³⁵S]methionine was fed to climacteric apple (Malus sylvestris Mill) tissue, radioactive 5-S-methyl-5-thioribose (MTR) was identified as the predominant product and MTA as a minor one. When the conversion of methionine into ethylene was inhibited by _l-2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid, the conversion of [³⁵S] or [Me¹⁴C]methionine into MTR was similarly inhibited. Furthermore, the formation of MTA and MTR from [³⁵S]methionine was observed only in climacteric tissue which produced ethylene and actively converted methionine to ethylene but not in preclimacteric tissue which did not produce ethylene or convert methionine to ethylene. These observations suggest that the conversion of methionine into MTA and MTR is closely related to ethylene biosynthesis and provide indirect evidence that SAM may be an intermediate in the conversion of methionine to ethylene.     

Radiosynthesis of [thiocarbonyl-11C]disulfiram and its first PET study in mice

[Thiocarbonyl-¹¹C]disulfiram ([¹¹C]DSF) was synthesized via iodine oxidation of [¹¹C]diethylcarbamodithioic acid ([¹¹C]DETC), which was prepared from [¹¹C]carbon disulfide and diethylamine. The decay-corrected isolated radiochemical yield (RCY) of [¹¹C]DSF was greatly affected by the addition of unlabeled carbon disulfide. In the presence of carbon disulfide, the RCY was increased up to 22% with low molar activity (A_m, 0.27 GBq/μmol). On the other hand, [¹¹C]DSF was obtained in 0.4% RCY with a high A_m value (95 GBq/μmol) in the absence of carbon disulfide. The radiochemical purity of [¹¹C]DSF was always >98%. The first PET study on [¹¹C]DSF was performed in mice. A high uptake of radioactivity was observed in the liver, kidneys, and gallbladder. The uptake level and distribution pattern in mice were not significantly affected by the A_m value of the [¹¹C]DSF sample used. In vivo metabolite analysis showed the rapid decomposition of [¹¹C]DSF in mouse plasma.     

[11C]WAY 100635: A radioligand for imaging 5-HT1A receptors with positron emission tomography

The potent and selective 5-HT_1A antagonist WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide) was radiolabeled with ¹¹C in high specific activity, and the in vivo properties of this radioligand were assessed in the brains of rats and monkeys. Following i.v. tail vein injection in rats, [¹¹C]WAY 100635 rapidly penetrated into brain tissue and was retained over a 30–90 min time period in a manner consistent with the known distribution of 5-HT_1A receptors. Pretreatment of rats with the selective 5-HT_1A agonist (±)-8-OH-DPAT effectively blocked the retention of radioactivity in brain regions known to contain high densities of 5-HT_1A receptors. The hippocampus-to-cerebellum radioactivity concentration ratio reached a maximum of 16:1 at 60 min post injection. Following i.v. injection of [¹¹C]WAY 100635 in rhesus monkeys, the concentrations of radioactivity in brain regions were consistent with the reported distribution of 5-HT_1A receptors in primates, and the frontal cortex-to-cerebellum ratio reached 5.5:1 at 80 min post injection. Pretreatment of the monkeys with (±)-8-OH-DPAT reduced this ratio to 1.4:1, and injection of (±)-8-OH-DPAT 20 min after the injection of [¹¹C]WAY 100635 significantly displaced frontal cortex binding. The in vivo properties of [¹¹C]WAY 100635 in rats and monkeys strongly support the future utility of this radioligand for imaging 5-HT_1A receptors using positron emission tomography (PET).     

Modulation of the cardiac membrane-bound cyclic nucleotide phosphodiesterase: inhibition due to a contamination of TLC purified S-adenosyl-L-[methyl-3H] methionine by Zn++ ions

Cardiac membranes pretreated with S-Adenosyl-L-[methyl-3H] methionine([3H] SAM) purified on TLC silica gel 60 F254 plates exhibited a marked decrease in cyclic AMP and cyclic GMP phosphodiesterase activity. However, this inhibition did not appear when membranes were incubated with either [14C] SAM or unlabelled SAM. We showed that, during the TLC purification of [3H] SAM, which involved an acidic elution step, minute amounts of the fluorescent indicator F254 (Zn sulfur) were eluted. The contaminating Zn++ ions strongly inhibited cyclic nucleotide phosphodiesterase activity and phospholipid methylation with I50 values in the micromolar range.     

Investigation of tumor metastatic potential with N-[18F]fluoroacetyl-d-glucosamine

In order to investigate the metastatic potential of tumors in vivo by measuring hyaluronic acid metabolism, C57BL/6 mice with B16 melanoma variants and C3H/He mice with FM3A tumor variants were evaluated using N-[¹⁸F]fluoroacetyl-d-glucosamine (¹⁸F-GlcNFAc). The uptake of ¹⁸F-GlcNFAc was slightly higher (P < 0.05) in B16-F10 tumors (high metastatic potential) than in B16-F1 (low metastatic potential). Analysis of metabolites showed that acid-insoluble fraction was the largest one in the liver by 60 min, whereas in the tumors, phosphates fraction was the major metabolite. Slower metabolism in tumors was suggested, and it may be one of the reasons for the difficulty of detecting the characteristics of their hyaluronic acid synthesis. ¹⁸F-GlcNFAc uptake by FM3A variants showed no significant correlation with their metastatic potential. In addition, N-acetyl-d-[l-¹⁴C]glucosamine, 2-deoxy-d-[l-¹⁴C]glucose and [6-³H]thymidine failed to demonstrate any difference between tumors' metastatic variants in vivo.