Efficient 11C-carbonylation of isolated aryl palladium complexes for PET: application to challenging radiopharmaceutical synthesis.

We describe the successful implementation of palladium-aryl oxidative addition complexes as stoichiometric reagents in carbonylation reactions with (11)CO to produce structurally challenging, pharmaceutically relevant compounds. This method enables the first (11)C-carbonyl labeling of an approved PET tracer, [(11)C]raclopride, for the dopamine D2/D3 receptor by carbonylation with excellent radiochemical purity and yield. Two other molecules, [(11)C]olaparib and [(11)C]JNJ 31020028, were efficiently labeled in this manner. The technique distinguishes itself from existing methods by the markedly improved purity profiles of the tracer molecules produced and provides access to complex structures in synthetically useful yields, hereby offering a viable alternative to other (11)C-labeling strategies.

Application of Microreactor to the Preparation of C-11-Labeled Compounds via O-[11C]Methylation with [11C]CH3I: Rapid Synthesis of [11C]Raclopride.

A new radiolabeling method using a microreactor was developed for the rapid synthesis of [(11)C]raclopride. A chip bearing a Y-shaped mixing junction with a 200 µm (width)×20 µm (depth)×250 mm (length) flow channel was designed, and the efficiency of O-[11C]methylation was evaluated. Dimethyl sulfoxide solutions containing the O-desmethyl precursor or [11C]CH3I were introduced into separate injection ports by infusion syringes, and the radiochemical yields were measured under various conditions. The decay-corrected radiochemical yield of microreactor-derived [11C]raclopride reached 12% in 20 s at 25 °C, which was observed to increase with increasing temperature. In contrast, batch synthesis at 25 °C produced a yield of 5%: this indicates that this device could effectively achieve O-[11C]methylation in a shorter period of time. The microreactor technique may facilitate simple and efficient routine production of 11C-labeled compounds via O-[11C]methylation with [11C]CH3I.

A highly sensitive LC-MS/MS method for the determination of S-raclopride in rat plasma: application to a pharmacokinetic study in rats.

A highly sensitive and rapid assay method has been developed and validated for the estimation of S-(-)-raclopride (S-RCP) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive ion mode. The assay procedure involves a simple liquid-liquid extraction technique for extraction of S-RCP and phenacetin (internal standard, IS) from rat plasma. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (80:20, v/v) at a flow rate of 0.30 mL/min on a Phenomenex Prodigy C(18) column with a total run time of 4.5 min. The MS/MS ion transitions monitored were 347.2 → 112.1 for S-RCP and 180.1 → 110.1 for IS. Method validation and pre-clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range was extended from 0.05 to 152 ng/mL in rat plasma. The intra-day and inter-day precisions were 0.23-10.5 and 3.74-7.29%, respectively. This novel method was applied to a pharmacokinetic study of S-RCP in rats.

A method based on Monte Carlo simulations and voxelized anatomical atlases to evaluate and correct uncertainties on radiotracer accumulation quantitation in beta microprobe studies in the rat brain.

The beta-microprobe is a simple and versatile technique complementary to small animal positron emission tomography (PET). It relies on local measurements of the concentration of positron-labeled molecules. So far, it has been successfully used in anesthetized rats for pharmacokinetics experiments and for the study of brain energetic metabolism. However, the ability of the technique to provide accurate quantitative measurements using (18)F, (11)C and (15)O tracers is likely to suffer from the contribution of 511 keV gamma rays background to the signal and from the contribution of positrons from brain loci surrounding the locus of interest. The aim of the present paper is to provide a method of evaluating several parameters, which are supposed to affect the quantification of recordings performed in vivo with this methodology. We have developed realistic voxelized phantoms of the rat whole body and brain, and used them as input geometries for Monte Carlo simulations of previous beta-microprobe reports. In the context of realistic experiments (binding of (11)C-Raclopride to D2 dopaminergic receptors in the striatum; local glucose metabolic rate measurement with (18)F-FDG and H(2)O(15) blood flow measurements in the somatosensory cortex), we have calculated the detection efficiencies and corresponding contribution of 511 keV gammas from peripheral organs accumulation. We confirmed that the 511 keV gammas background does not impair quantification. To evaluate the contribution of positrons from adjacent structures, we have developed beta-Assistant, a program based on a rat brain voxelized atlas and matrices of local detection efficiencies calculated by Monte Carlo simulations for several probe geometries. This program was used to calculate the 'apparent sensitivity' of the probe for each brain structure included in the detection volume. For a given localization of a probe within the brain, this allows us to quantify the different sources of beta signal. Finally, since stereotaxic accuracy is crucial for quantification in most microprobe studies, the influence of stereotaxic positioning error was studied for several realistic experiments in favorable and unfavorable experimental situations (binding of (11)C-Raclopride to D2 dopaminergic receptors in the striatum; binding of (18)F-MPPF to 5HT1A receptors in the dorsal raphe nucleus).

Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D1 and D2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D1 and D2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D1 and D2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays.

Cocaine Cue-Induced Dopamine Release in Recreational Cocaine Users.

It has been proposed that the acquisition of drug seeking is related to the development of conditioned dopamine responses in the ventral striatum. As drug use continues and becomes habit-like, conditioned responses have been shown to shift to the dorsal striatum. Here, using the PET [11C]raclopride method and highly personalized cocaine cues, we report the first evidence in humans of the dorsal dopamine response prior to the onset of addiction.

ORGAN DOSES AND EFFECTIVE DOSE FOR FIVE PET RADIOPHARMACEUTICALS.

Diagnostic investigations with positron-emitting radiopharmaceuticals are dominated by (18)F-fluorodeoxyglucose ((18)F-FDG), but other radiopharmaceuticals are also commercially available or under development. Five of them, which are all clinically important, are (18)F-fluoride, (18)F-fluoroethyltyrosine ((18)F-FET), (18)F-deoxyfluorothymidine ((18)F-FLT), (18)F-fluorocholine ((18)F-choline) and (11)C-raclopride. To estimate the potential risk of stochastic effects (mainly lethal cancer) to a population, organ doses and effective dose values were updated for all five radiopharmaceuticals. Dose calculations were performed using the computer program IDAC2.0, which bases its calculations on the ICRP/ICRU adult reference voxel phantoms and the tissue weighting factors from ICRP publication 103. The biokinetic models were taken from ICRP publication 128. For organ doses, there are substantial changes. The only significant change in effective dose compared with previous estimations was a 46 % reduction for (18)F-fluoride. The estimated effective dose in mSv MBq(-1) was 1.5E-02 for (18)F-FET, 1.5E-02 for (18)F-FLT, 2.0E-02 for (18)F-choline, 9.0E-03 for (18)F-fluoride and 4.4E-03 for (11)C-raclopride.

Synthesis of ([(11)C]carbonyl)raclopride and a comparison with ([(11)C]methyl)raclopride in a monkey PET study.

INTRODUCTION: The selective dopamine D2 receptor antagonist raclopride is usually labeled with carbon-11 using [(11)C]methyl iodide or [(11)C]methyl triflate for use in the quantification of dopamine D2 receptors in human brain. The aim of this work was to label raclopride at the carbonyl position using [(11)C]carbon monoxide chemistry and to compare ([(11)C]carbonyl)raclopride with ([(11)C]methyl)raclopride in non-human primate (NHP) using PET with regard to quantitative outcome measurement, metabolism of the labeled tracers and protein binding. METHODS: Palladium-mediated carbonylation using [(11)C]carbon monoxide, 4,6-dichloro-2-iodo-3-methoxyphenol and (S)-(-)-2-aminomethyl-1-ethylpyrrolidine was applied in the synthesis of ([(11)C]carbonyl)raclopride. The reaction was performed at atmospheric pressure using xantphos as supporting phosphine ligand and palladium (π-cinnamyl) chloride dimer as the palladium source. ([(11)C]Methyl)raclopride was prepared by a previously published method. In the PET study, two female cynomolgus monkeys were used under gas anesthesia of sevoflurane. A dynamic PET measurement was performed for 63 min with an HRRT PET camera after intravenous injection of ([(11)C]carbonyl)raclopride and ([(11)C]methyl)raclopride, respectively, during the same day. The order of injection of the two PET radioligands was changed between the two monkeys. The venous blood sample for measurement of protein binding was taken 3 min prior to the PET scan. Binding potential (BPND) of the putamen and caudate was calculated with SRTM using the cerebellum as a reference region. RESULTS: The target compound ([(11)C]carbonyl)raclopride was obtained with 50 ± 5% decay corrected radiochemical yield and 95% radiochemical purity. The trapping efficiency (TE) of [(11)C]carbon monoxide was 65 ± 5% and the specific radioactivity of the final product was 34 ± 1 GBq/µmol after a 50 min of synthesis time. The radiochemical yield of ([(11)C]methyl)raclopride was in the same range as published previously i. e. 50-60% and specific radioactivity of those two batches which were used in the present PET study were 192 GBq/µmol and 638 GBq/µmol respectively after a synthesis time of 32 min. In monkey PET studies, the percentage difference of BPND in putamen was <3% and that in caudate was <9% for the two radioligands. The plasma protein binding was 86.2 ± 0.3% and 85.7 ± 0.6% for ([(11)C]carbonyl)raclopride and ([(11)C]methyl)raclopride, respectively. The radiometabolite pattern was similar for both radioligands. CONCLUSION: Raclopride was (11)C-labeled at the carbonyl position using a palladium-mediated [(11)C]carbonylation reaction. A comparison between ([(11)C]carbonyl)raclopride and ([(11)C]methyl)raclopride with regard to quantitative PET outcome measurements, metabolism of radioligands and protein binding in monkey was performed. The monkey PET study with ([(11)C]carbonyl)raclopride showed similar results as for ([(11)C]methyl)raclopride. The PET studies were performed on 2 subjects.

Amphiphilic drug interactions with model cellular membranes are influenced by lipid chain-melting temperature.

Small-molecule amphiphilic species such as many drug molecules frequently exhibit low-to-negligible aqueous solubility, and generally have no identified transport proteins assisting their distribution, yet are able to rapidly penetrate significant distances into patient tissue and even cross the blood-brain barrier. Previous work has identified a mechanism of translocation driven by acid-catalysed lipid hydrolysis of biological membranes, a process which is catalysed by the presence of cationic amphiphilic drug molecules. In this study, the interactions of raclopride, a model amphiphilic drug, were investigated with mixtures of biologically relevant lipids across a range of compositions, revealing the influence of the chain-melting temperature of the lipids upon the rate of acyl hydrolysis.

Enhanced radiosyntheses of [¹¹C]raclopride and [¹¹C]DASB using ethanolic loop chemistry.

INTRODUCTION: To improve the synthesis and quality control of carbon-11 labeled radiopharmaceuticals, we report the fully automated loop syntheses of [¹¹C]raclopride and [¹¹C]DASB using ethanol as the only organic solvent for synthesis module cleaning, carbon-11 methylation, HPLC purification, and reformulation. METHODS: Ethanolic loop chemistry is fully automated using a GE TRACERLab FX(C-Pro) synthesis module, and is readily adaptable to any other carbon-11 synthesis apparatus. Precursors (1 mg) were dissolved in ethanol (100 µL) and loaded into the HPLC loop. [¹¹C]MeOTf was passed through the HPLC loop and then the labeled products were purified by semi-preparative HPLC and reformulated into ethanolic saline. RESULTS: Both [¹¹C]raclopride (3.7% RCY; >95% RCP; SA=20831 Ci/mmol; n=64) and [¹¹C]DASB, both with (3.0% RCY; >95% RCP; SA=15152Ci/mmol; n=9) and without (3.0% RCY; >95% RCP; SA=10931 Ci/mmol; n=3) sodium ascorbate, have been successfully prepared using the described methodology. Doses are suitable for human use and the described methods are now employed for routine clinical production of both radiopharmaceuticals at the University of Michigan. CONCLUSIONS: Ethanolic loop chemistry is a powerful technique for preparing [¹¹C]raclopride and [¹¹C]DASB, and we are in the process of adapting it for other carbon-11 radiopharmaceuticals prepared in our laboratories ([¹¹C]PMP, [¹¹C]PBR28 etc.).

Fully automated and reproducible radiosynthesis of high specific activity [¹¹C]raclopride and [¹¹C]Pittsburgh compound-B using the combination of two commercial synthesizers.

INTRODUCTION: The use of ¹¹C-labeled radiotracers in routine positron emission tomography studies is dependent on the production capability of radiochemistry laboratories. Therefore, considerable efforts are being focused on the development of fast, efficient, and robust methods for the preparation of such radiotracers. METHODS: The fully automated syntheses of [¹¹C]raclopride and [¹¹C]Pittsburgh compound-B (PIB) starting from cyclotron-produced [¹¹C]CH4 are reported. [¹¹C]methyl iodide and [¹¹C]methyl triflate were produced in the TRACERlab FXC Pro synthesis box. Methylation reactions and the final formulation were performed using the AutoLoop (captive solvent method) and the ReFORM-plus systems, respectively. RESULTS: [¹¹C]raclopride (n=30) and [¹¹C]PIB (n=24) were synthesized by O-[¹¹C]-methylation and N-[¹¹C]-methylation of (S)-O-desmethylraclopride and 6-OH-BTA-0 using [¹¹C]methyl iodide and [¹¹C]methyl triflate, respectively. Good radiochemical yields (51.3 ± 11.2 and 32.9 ± 6.6%, referred to as [¹¹C]methyl iodide, decay corrected) and specific activities (109 ± 20 and 143 ± 26 GBq/µmol) were obtained for [¹¹C]raclopride and [¹¹C]PIB, respectively, in a fully automated process. Radiochemical purity was higher than 99% in all cases. CONCLUSION: The fast, robust and fully automated processes reported here allow [¹¹C]raclopride and [¹¹C]PIB synthesis with good radiochemical yields and high specific activities. Consecutive productions can be performed with minimal intervention on the synthesis modules and minimal exposure to radiation.

A simple modification of GE tracerlab FX C Pro for rapid sequential preparation of [11C]carfentanil and [11C]raclopride.

A simple modification of the GE Tracerlab FX C Pro system which enabled performance of both solvent capture (loop method) and conventional solution phase [(11)C]methylation in the same module is described. By the quick setup and automated method, [(11)C]carfentanil and [(11)C]raclopride could be prepared in rapid succession without opening the hot cell. The radiochemical yields were over 40% and 30% (decay-corrected and based on [(11)C]methyl iodide) for [(11)C]carfentanil and [(11)C]raclopride, respectively. The radiochemical purities were greater than 95% and specific activities over 5 Ci/mmol for both tracers. The modification is extremely easy and can be utilized for multiple syntheses of other (11)C-labeled radiopharmaceuticals in a fast and reliable manner.

Antagonist-radioligand binding to D2L-receptors in intact cells.

D(2)-dopamine receptors mediate most of the physiological actions of dopamine and are important recognition sites for antipsychotic drugs. Earlier binding studies were predominantly done with broken cell preparations with the tritiated D(2)-receptor antagonists [(3)H]-raclopride, a hydrophilic benzamide, and [(3)H]-spiperone, a highly hydrophobic butyrophenone. Here we compared [(3)H]-raclopride and [(3)H]-spiperone binding properties in intact Chinese Hamster Ovary cells stably expressing recombinant human D(2L)-receptors. Specific binding of both radioligands occurred to a comparable number of sites. In contrast to the rapid dissociation of [(3)H]-raclopride in both medium only and in the presence of an excess of unlabelled ligand [(3)H]-spiperone dissociation was only observed in the latter condition, and it was still slower than in broken cell preparations. However, this could not explain the pronounced difference in the potency of some unlabelled ligands to compete with both radioligands. To integrate these new findings, a model is proposed in which raclopride approaches the receptor from the aqueous phase, while spiperone approaches the receptor by lateral diffusion within the membrane.

Purification of carbon-11 PET radiotracers from unlabeled precursors by preparative HPLC and SPE.

A general methodology for the rapid purification of carbon-11 positron emission tomography (PET) radiotracers from radiolabeling reaction mixtures has been developed. Preparative HPLC and solid-phase extraction (SPE) techniques are described which can separate some commonly used radiopharmaceuticals such as [(11)C]raclopride, [(11)C]beta-CFT and [(11)C]choline from their unlabeled precursors.