Evaluation of Radiation Dose Resulting from the Ingestion of [3H]- and [14C]thymidine in the Rat

Summary

Average doses to rat tissues from the ingestion of 2-[¹⁴C]thymidine were compared with those from methyl-[³H]thymidine or 6-[³H]thymidine. Among the three precursors, [¹⁴C]thymidine gave the highest dose to spleen and small intestine. The doses to other tissues from [¹⁴C]thymidine were almost the same or lower as compared with those from [³H]thymidine, irrespective of the 9 times higher β-ray energy of ¹⁴C than that of ³H. In the case of [¹⁴C]thymidine, most of the dose was given by radioactivity incorporated into the organic tissue constituents (non-volatile radioactivity). In the case of [³H]thymidine, however, the dose contributions by non-volatile radioactivity were very small and the major contributions were rather from volatile radioactivity (³HHO), formed by degradation of [³H]thymidine. No significant difference in their total doses was found between the two [³H]precursors, but the dose from non-volatile radioactivity alone was 2–3 times higher with methyl-[³H]thymidine than with 6-[³H]thymidine. Estimates of the dose to cell nuclei in various tissues after the ingestion of [³H]thymidine were also made in order to predict more precisely possible radiation hazards.     

Role of NMDA receptor upon [14C]acetate uptake into intact rat brain

Objective  To clarify the role of N-methyl-d-aspartate (NMDA) receptors upon [¹⁴C]acetate uptake in the rodent central nervous system (CNS), ibotenic acid (IBO) was infused into the right striatum of the rat brain. Methods  Autoradiograms of [¹⁴C]acetate uptake in the brain for 2 h following the infusion of IBO (10 μg/μl) were obtained in both non-treated and MK-801 (1 mg/kg, i.v.) pretreated rats. The effect of MK-801 on [¹⁴C]acetate uptake in the normal rat brain was also studied. Results  Infusion of IBO significantly decreased [¹⁴C]acetate uptake in the infused side of the striatum. The expression of monocarboxylate transporter-1 was not altered, suggesting that the activity of tricarboxylic acid (TCA) cycle in glial cells might be depressed. Pretreatment with MK-801 completely blocked the decreasing effect of IBO on [¹⁴C]acetate uptake. MK-801 also increased [¹⁴C]acetate uptake in the whole brain of normal rats. Conclusions  These results indicate the important roles of NMDA receptors on [¹⁴C]acetate uptake in the intact rat brain.     

Analysis of the radiorespirometric pattern after administration of [U-14C] glucose to Ehrlich ascites tumor-bearing mice

Ehrlich ascites tumor-bearing mice were subjected to ¹⁴CO₂ radiorespirometric analysis after administration of [U-¹⁴C]glucose, and the results were compared with the levels of host liver glycolytic enzyme activities and the uptake of the radioactivity into the liver. After IP administration of [U-¹⁴C]glucose, there was a progressive decline in respiratory ¹⁴CO₂ after the transplantation of the Ehrlich ascites tumor cells. The peak time (PT) was about 10 min on day 1, but thereafter was increasingly delayed, and could not be determined on day 13. Peak height (PH) and yield value (YV) were both considerably decreased, and again the magnitudes of the changes increased with the time after transplantation of the tumor cells. Glycolytic enzyme activities in the host liver were at normal levels 13 days after transplantation of the tumor cells. The uptake of the radioactivity into the liver after IP administration of [U-¹⁴C]glucose began to decline from day 5 and was 50% the value in normal mice 13 days after transplantation of the tumor cells. These results indicate that the radiorespirometric patterns with [U-¹⁴C]glucose reflects hepatic biochemical changes rather well.     

Transport mechanisms of trans-1-amino-3-fluoro[1-(14)C]cyclobutanecarboxylic acid in prostate cancer cells

IntroductionWe investigated the mechanisms of trans-1-amino-3-fluoro[1-¹⁴C]cyclobutanecarboxylic acid (anti-[¹⁴C]FACBC) transport by human-derived prostate cancer (PCa) cells and normal human prostatic epithelial cells (PrECs).MethodsUsing PCa cells (DU145, PC-3, LNCaP) and PrECs, we performed the following in vitro experiments: time-course, kinetics, competitive inhibition by synthetic/naturally occurring amino acids (AAs), exchange transport with synthetic/naturally occurring AAs and pH-dependency of anti-[¹⁴C]FACBC uptake. We also examined the amino acid transporter (AAT) expression using flow cytometry.ResultsThe uptake of anti-[¹⁴C]FACBC by LNCaP and DU145 cells was higher than that by PC-3 and PrECs. The K_m values for anti-[¹⁴C]FACBC were 64.4 and 191.7 μmol/L in the DU145 cells and PrECs, respectively. Total levels of anti-[¹⁴C]FACBC uptake were positively correlated with the expression level of system ASC in PCa cells. The contributions of Na⁺-dependent AATs to anti-[¹⁴C]FACBC uptake were greater than those of Na⁺-independent AATs, especially in PCa cells. In the presence of Na⁺, glutamine and serine showed the strongest inhibitory effect against anti-[¹⁴C]FACBC uptake, suggesting that system ASC, especially ASCT2, is an important AAT for anti-[¹⁴C]FACBC. In contrast, phenylalanine and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid, but not N-ethylmaleimide, almost completely inhibited the anti-[¹⁴C]FACBC uptake in the absence of Na⁺, indicating the contribution of LAT1. In the exchange transport experiments, glutamine showed the strongest transstimulation of intracellular anti-[¹⁴C]FACBC efflux in DU145 cells. Furthermore, the contributions of Na⁺-independent AATs to the uptake of anti-[¹⁴C]FACBC in DU145 and PrECs were greater under acidic pH conditions than under neutral or alkaline pH conditions.ConclusionsTotal uptake of anti-[¹⁴C]FACBC by PCa cells correlates with the expression level of system ASC in PCa cells. Furthermore, LAT1 is an important transport system for anti-[¹⁴C]FACBC uptake, especially in an acidic environment, such as the intra-tumoural environment.     

Preparation of [11C]carbon dioxide from [11C]cyanide

[¹¹C]carbon dioxide was prepared from [¹¹C]cyanide. First, [¹¹C]CH₄ was produced by the (p, α) reaction on a N₂/H₂ mixture and was converted to [¹¹C]CN⁻ by reaction with ammonia over platinum at 1273 K. The [¹¹C]CN⁻ was adsorbed on a cobalt(II, III) oxide/ceramic furnace material at room temperature and was subsequently converted to [¹¹C]CO₂ by heating the cobalt(II, III) oxide to 948 K over a 10 min period and collecting the [¹¹C]CO₂ in a trap at 77 K. Specific activities as high as 600 mCi/μmol at end of bombardment (EOB) were obtained in a 15 μA irradiation for 10 min.     

A simple,versatile, low-cost and remotely operated apparatus for [11C]acetate, [11C]choline, [11C]methionine and [11C]PIB synthesis

A simple, efficient and remotely operated synthesis apparatus for carrying out routine [¹¹C]carboxylation, on-column and bubbling [¹¹C]methylation was essential for reliable, day-to-day production of [¹¹C]-labelled PET radiopharmaceuticals. We developed an in-house apparatus specifically applied to the synthesis of [¹¹C]acetate, [¹¹C]choline, [¹¹C]methionine and 2-(4′-N-[¹¹C]methylaminophenyl)-6-hydroxybenzothiazole ([¹¹C]PIB), where high radiochemical purity (⩾97%) and moderate radiochemical yields (18% for [¹¹C]PIB, 41–55% for the others) could be achieved. These findings provided evidence that this was a fast, versatile and reliable apparatus suitable for a PET/CT centre with limited financial budget and hot cell space for synthesis of [¹¹C]-labelled radiopharmaceuticals.     

A facile no-carrier-added one pot micro-molar scale radiosynthesis of 2-[14C]–uracil

A facile no-carrier-added one pot micro-molar scale radiolabelled synthesis of 2-[¹⁴C]–uracil from [¹⁴C]–urea and propiolic acid in the presence of polyphosphoric acid (PPA) with an yield of 48.33% and with radiochemical purity of 98% is reported in this paper.     

[11C]octopamine synthesis using [11C]cyanide: Chemical and enzymatic approaches for the [11C]cyanohydrin synthesis

[¹¹C]-p- and m-octopamine hydrochloride were synthesized from [¹¹C]HCN in a two-step sequence. Chemical and enzymatic approaches were used for the formation of the [¹¹C]cyanohydrin intermediates as the key step. Isolated radiochemical yields of 0.7–2.3% at the end-of-synthesis were obtained with an overall preparation time of 40–60 min. The enantiomeric purity of the [¹¹C]-p-octopamine obtained through the enzymatic process was 92% e.e. in the (S)-enantiomer, whereas that of the [¹¹C]-m-octopamine was 42% e.e. in the (R)-enantiomer, as determined by HPLC without any derivatization.     

Synthesis of racemic [3-11C]phenylalanine and [3-11C]DOPA

The synthesis of racemic [3-¹¹C]phenylalanine and [3-¹¹C]DOPA is reported. The [¹¹C]benzaldehyde and [¹¹C]veratraldehyde prepared in a two-step reaction from the corresponding [¹¹C]acid salt and [¹¹C]alcohol, by means of selective oxidation with tetrabutylammonium hydrogen chromate, were reacted with 2-phenyl-5-oxazolone or 2-(4-chloro)phenyl-5-oxazolone in the presence of a tertiary amine to give the corresponding [α-¹¹C]-4-arylene-2-aryl-5-oxazolones. Ring opening of these olefins, hydrogenation, and removal of protecting groups was carried out in one step using hydroiodic acid/phosphorus, with the production of the racemic [3-¹¹C]amino acids in 8–30% radiochemical yield (starting with ¹¹CO₂) within 52–60 min (including LC separation).     

An alternative synthesis of DL-[1-11C]Alanine from [11C]HCN

The synthesis of dl-[1-¹¹C]alanine from [1-¹¹C]HCN via dl-[1-¹¹C]2-aminopropanenitrile and its use in an enzymatic synthesis of [1-¹¹C]pyruvic acid is reported. The purified dl-[1-¹¹C]alanine was obtained in a radiochemical yield of 75% with a radiochemical purity higher than 98% within 40 min after end of bombardment (EOB). [1-¹¹C]Pyruvic acid was prepared by enzymatic synthesis from crude dl-[1-¹¹C]alanine using a previously reported procedure for the synthesis of [3-¹¹C]pyruvic acid. dl-[1-¹¹C]Alanine and [1-¹¹C]pyruvic acid prepared by these methods have been found to be sterile and free of pyrogens, and can thus be used in studies of the distribution of these radiopharmaceuticals in man using positron emission tomography (PET).     

A semi-automated synthesis system for routine preparation of [11C]YM-09151-2 and [11C]pyrilamine from [11C]methyl iodide

A synthesis system has been developed for routine preparation of the ¹¹C-labeled receptor ligands, [¹¹C]YM-09151-2 and [¹¹C]pyrilamine, from [¹¹C]methyl iodide produced automatically. The system features semi-automated operation, from the reaction of the desmethyl derivative with [¹¹C]methyl iodide to filtration with a specifically developed syringe pump of the final product in saline into a sterile vial. The preparations were completed within 45 min after irradiation and approx. 1 GBq (27 mCi) of [¹¹C]YM-09151-2 or [¹¹C]pyrilamine was obtained with a radiochemical and chemical purity of >99% and an average specific activity of 44 GBq/μmol (1.2 Ci/μmol) at the end of synthesis. Sterile and pyrogen-free ¹¹C-labeled receptor ligands suitable for human injection are routinely prepared using the present synthesis system.     

Preparation of 1-[125I]iodo-[114C]dodecane

1-[¹²⁵I]Iodo-[1¹⁴C]dodecane was prepared using the phosphorus halide/alcohol-reaction. Since the conventional synthesis of iodoalkanes requires apparatus and procedures which do not conform to radioprotection standards, a new apparatus was also constructed. Apparatus and method can also be used for the synthesis of other halogen-alkanes.     

Two routes to [C-carbonyl]organo-isocyanates utilizing [C]phosgene ([C]organo-isocyanates from [C]phosgene)

Two generic radiosynthetic routes for the preparation of [¹¹C-carbonyl]isocyanates have been developed. Reaction of N-organo-sulfinylamines; RNSO, (R = Me, Et, allyl, cyclohexyl and phenyl) with [¹¹C]phosgene gave the corresponding [¹¹C-carbonyl]isocyanates in good radiochemical yield (53–68%) from [¹¹C]phosgene (decay corrected) in ca 16 min from EOB. Alternatively, the reaction of [¹¹C]phosgene with N,N′-organo-ureas; (RNH)₂CO, (R = Me, Et, Pr and phenyl) also gave the corresponding [¹¹C-carbonyl]isocyanates in moderate radiochemical yield (9–37%) from [¹¹C]phosgene (decay corrected) in ca 16 min from EOB. For identification, the [¹¹C-carbonyl]organo-isocyanates were derivatized with 1-(2-methoxyphenyl)piperazine in situ to [¹¹C-carbonyl]carboxamides and the position of radiolabelling in the carbonyl group confirmed by [^11/13C]co-labeling and subsequent carbon-13 NMR spectroscopy.     

DNA Strand Breakage in Chinese Hamster V79 Cells Caused by Low Levels of Incorporated [3H] and [14C]thymidine

Summary

A sensitive alkali-unwinding assay was used to measure DNA strand breakage in Chinese hamster V79 cells caused by low-level incorporation of methyl-labelled [³H] and [¹⁴C] thymidine, and to estimate the effective dose per disintegration relative to low doses of gamma-irradiation. Damage equivalent to 0·0035 ± 0·0006 and 0·0014 ± 0·0005 Gy was observed for each ³H and ¹⁴C disintegration respectively. These values agree well with those expected from the estimated nuclear radiation dose delivered by the beta particles if a relative biological effect (r.b.e.) of 1·0 is assumed, and suggest that strand-breakage produced by these isotopes is determined by the nuclear radiation dose delivered by the beta particles.     

PET imaging of focal demyelination and remyelination in a rat model of multiple sclerosis: comparison of [11C]MeDAS, [11C]CIC and [11C]PIB

Purpose

In this study, we compared the ability of [¹¹C]CIC, [¹¹C]MeDAS and [¹¹C]PIB to reveal temporal changes in myelin content in focal lesions in the lysolecithin rat model of multiple sclerosis. Pharmacokinetic modelling was performed to determine the best method to quantify tracer uptake.

Methods

Sprague-Dawley rats were stereotactically injected with either 1 % lysolecithin or saline into the corpus callosum and striatum of the right brain hemisphere. Dynamic PET imaging with simultaneous arterial blood sampling was performed 7 days after saline injection (control group), 7 days after lysolecithin injection (demyelination group) and 4 weeks after lysolecithin injection (remyelination group).

Results

The kinetics of [¹¹C]CIC, [¹¹C]MeDAS and [¹¹C]PIB was best fitted by Logan graphical analysis, suggesting that tracer binding is reversible. Compartment modelling revealed that all tracers were fitted best with the reversible two-tissue compartment model. Tracer uptake and distribution volume in lesions were in agreement with myelin status. However, the slow kinetics and homogeneous brain uptake of [¹¹C]CIC make this tracer less suitable for in vivo PET imaging. [¹¹C]PIB showed good uptake in the white matter in the cerebrum, but [¹¹C]PIB uptake in the cerebellum was low, despite high myelin density in this region. [¹¹C]MeDAS distribution correlated well with myelin density in different brain regions.

Conclusion

This study showed that PET imaging of demyelination and remyelination processes in focal lesions is feasible. Our comparison of three myelin tracers showed that [¹¹C]MeDAS has more favourable properties for quantitative PET imaging of demyelinated and remyelinated lesions throughout the CNS than [¹¹C]CIC and [¹¹C]PIB.     

In-target produced [11C]methane: Increased specific radioactivity

The ¹⁴N(ρ, α)¹¹C reaction on N₂–O₂ or N₂–H₂ gaseous systems as targets in proton bombardment allows for the production of [¹¹C]CO₂ and [¹¹C]CH_4. We report the target production of [¹¹C]CH₄ and the gas phase iodination to produce [¹¹C]CH₃I with high specific radioactivity (SA). SA was calculated for four different radiopharmaceuticals produced in-house from both target produced [¹¹C]CO₂ and [¹¹C]CH_4. For [¹¹C]raclopride we obtained an average SA of 3908 GBq/μmol (106 000 Ci/mmol) at the end of bombardment for the last 52 productions, which is a 32-fold increase compared to when using the in-house [¹¹C]CO₂ target.     

A comparative small-animal PET evaluation of [11C]tariquidar, [11C]elacridar and (R)-[11C]verapamil for detection of P-glycoprotein-expressing murine breast cancer

Purpose

One important mechanism for chemoresistance of tumours is overexpression of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp). Pgp reduces intracellular concentrations of chemotherapeutic drugs. The aim of this study was to compare the suitability of the radiolabelled Pgp inhibitors [¹¹C]tariquidar and [¹¹C]elacridar with the Pgp substrate radiotracer (R)-[¹¹C]verapamil for discriminating tumours expressing low and high levels of Pgp using small-animal PET imaging in a murine breast cancer model.

Methods

Murine mammary carcinoma cells (EMT6) were continuously exposed to doxorubicin to generate a Pgp-overexpressing, doxorubicin-resistant cell line (EMT6AR1.0 cells). Both cell lines were subcutaneously injected into female athymic nude mice. One week after implantation, animals underwent PET scans with [¹¹C]tariquidar (n?=?7), [¹¹C]elacridar (n?=?6) and (R)-[¹¹C]verapamil (n?=?7), before and after administration of unlabelled tariquidar (15?mg/kg). Pgp expression in tumour grafts was evaluated by Western blotting.

Results

[¹¹C]Tariquidar showed significantly higher retention in Pgp-overexpressing EMT6AR1.0 compared with EMT6 tumours: the mean?±?SD areas under the time?Cactivity curves in scan 1 from time 0 to 60?min (AUC_0?C60) were 38.8?±?2.2?min and 25.0?±?5.3?min (p?=?0.016, Wilcoxon matched pairs test). [¹¹C]Elacridar and (R)-[¹¹C]verapamil were not able to discriminate Pgp expression in tumour models. Following administration of unlabelled tariquidar, both EMT6Ar1.0 and EMT6 tumours showed increases in uptake of [¹¹C]tariquidar, [¹¹C]elacridar and (R)-[¹¹C]verapamil.

Conclusion

Among the tested radiotracers, [¹¹C]tariquidar performed best in discriminating tumours expressing high and low levels of Pgp. Therefore [¹¹C]tariquidar merits further investigation as a PET tracer to assess Pgp expression levels in solid tumours.     

Equivalence of arterial and venous blood for [11C]CO2-metabolite analysis following intravenous administration of 1-[11C]acetate and 1-[11C]palmitate

PurposeSampling of arterial blood for metabolite correction is often required to define a true radiotracer input function in quantitative modeling of PET data. However, arterial puncture for blood sampling is often undesirable. To establish whether venous blood could substitute for arterial blood in metabolite analysis for quantitative PET studies with 1-[¹¹C]acetate and 1-[¹¹C]palmitate, we compared the results of [¹¹C]CO₂-metabolite analyses performed on simultaneously collected arterial and venous blood samples.MethodsPaired arterial and venous blood samples were drawn from anesthetized pigs at 1, 3, 6, 8, 10, 15, 20, 25 and 30 min after i.v. administration of 1-[¹¹C]acetate and 1-[¹¹C]palmitate. Blood radioactivity present as [¹¹C]CO₂ was determined employing a validated 10-min gas-purge method. Briefly, total blood ¹¹C radioactivity was counted in base-treated [¹¹C]-blood samples, and non-[¹¹C]CO₂ radioactivity was counted after the [¹¹C]-blood was acidified using 6 N HCl and bubbled with air for 10 min to quantitatively remove [¹¹C]CO₂.ResultsAn excellent correlation was found between concurrent arterial and venous [¹¹C]CO₂ levels. For the [¹¹C]acetate study, the regression equation derived to estimate the venous [¹¹C]CO₂ from the arterial values was: y = 0.994x + 0.004 (r² = 0.97), and for the [¹¹C]palmitate: y = 0.964x ? 0.001 (r² = 0.9). Over the 1–30 min period, the fraction of total blood ¹¹C present as [¹¹C]CO₂ rose from 4% to 64% for acetate, and 0% to 24% for palmitate. The rate of [¹¹C]CO₂ appearance in venous blood appears similar for the pig model and humans following i.v. [¹¹C]-acetate administration.ConclusionVenous blood [¹¹C]CO₂ values appear suitable as substitutes for arterial blood samples in [¹¹C]CO₂ metabolite analysis after administration of [¹¹C]acetate or [¹¹C]palmitateAdvances in Knowledge and Implications for Patient CareQuantitative PET studies employing 1-[¹¹C]acetate and 1-[¹¹C]palmitate can employ venous blood samples for metabolite correction of an image-derived tracer arterial input function, thereby avoiding the risks of direct arterial blood sampling.     

[11C]quinidine and [11C]laniquidar PET imaging in a chronic rodent epilepsy model: Impact of epilepsy and drug-responsiveness

IntroductionTo analyse the impact of both epilepsy and pharmacological modulation of P-glycoprotein on brain uptake and kinetics of positron emission tomography (PET) radiotracers [¹¹C]quinidine and [¹¹C]laniquidar.MethodsMetabolism and brain kinetics of both [¹¹C]quinidine and [¹¹C]laniquidar were assessed in naive rats, electrode-implanted control rats, and rats with spontaneous recurrent seizures. The latter group was further classified according to their response to the antiepileptic drug phenobarbital into “responders” and “non-responders”. Additional experiments were performed following pre-treatment with the P-glycoprotein modulator tariquidar.Results[¹¹C]quinidine was metabolized rapidly, whereas [¹¹C]laniquidar was more stable. Brain concentrations of both radiotracers remained at relatively low levels at baseline conditions. Tariquidar pre-treatment resulted in significant increases of [¹¹C]quinidine and [¹¹C]laniquidar brain concentrations. In the epileptic subgroup “non-responders”, brain uptake of [¹¹C]quinidine in selected brain regions reached higher levels than in electrode-implanted control rats. However, the relative response to tariquidar did not differ between groups with full blockade of P-glycoprotein by 15 mg/kg of tariquidar. For [¹¹C]laniquidar differences between epileptic and control animals were only evident at baseline conditions but not after tariquidar pretreatment.ConclusionsWe confirmed that both [¹¹C]quinidine and [¹¹C]laniquidar are P-glycoprotein substrates. At full P-gp blockade, tariquidar pre-treatment only demonstrated slight differences for [¹¹C]quinidine between drug-resistant and drug-sensitive animals.     

On-line preparation of [11C]carbon dioxide from [11C]methane

[¹¹C]Methane, produced from the (p, α) nuclear reaction on a nitrogen-5.6% hydrogen target, has been oxidized in an “on-line” synthetic train to yield [¹¹C]carbon dioxide. [¹¹C]methane is quantitatively adsorbed from the target gas on a Porapak Q column cooled to liquid nitrogen temperature. Following absorptive concentration, the [¹¹C]methane is oxidized by passage over cobalt(II–III) oxide powder heated to 500°C in a stream of nitrogen-2.0% oxygen. The isotopic dilution occurring during this conversion was determined to be 60%.