[18F]Fallypride dopamine D2 receptor studies using delayed microPET scans and a modified Logan plot

[¹⁸F]Fallypride PET studies can be used to estimate the nondisplaceable binding potential (BP_ND) in vivo of dopamine D2/D3 receptor-rich regions of the brain. These studies often take considerable time, up to ≥2 h, limiting the throughput. In this work, we investigated whether limited-duration scans performed subsequent to tracer administration yielded stable BP_ND estimates. In particular, we applied a modified version of the Logan plot method on the last 60 min of 120-min data and compared the results to those from analysis of the full data set.MethodsFourteen male Sprague-Dawley rats were injected with [¹⁸F]fallypride intravenously while under isoflurane anesthesia, and dynamic data were acquired on the microPET Focus 220 scanner for 120 min. The distribution volume ratio (DVR=BP_ND+1) was calculated from a Logan plot using 120 min of data and from a modified version using only the last 60 min. Three of these rats were imaged again on a second day to test the reproducibility. A two-tissue compartment model also was used to fit the time–activity curves (TACs) of the 120-min scans to estimate the parameters K₁, k₂, k_on, k₄ and B_max. These parameters were then used to simulate similar TACs while changing k_on to reflect changes in the dopaminergic system. The simulated TACs were used as a means for exploring the differences in DVR estimates between the last 60 min only and the full 120 min of simulated data.ResultsThe average DVR from the full 120-min scans was 13.8±0.9, whereas the average DVR estimated from only the last 60 min of data (DVR′) was 16.3±1.0. The DVR estimates showed good reproducibility in the three rats (mean DVR=13.8±1.5 on Day 1 and DVR=13.8±0.9 on Day 2). The simulations showed that the relationship between DVR′ and DVR estimates follows a semilinear form with varying k_on.ConclusionAlthough the BP_ND estimates are slightly overestimated in a delayed scan mode (i.e., no initial radiotracer uptake measurements) compared to a full scan, this overestimation depends primarily on k₃ (≈k_on×B_max) and has been evaluated in this work for a wide range of k_on values using simulated TACs. In particular, the sensitivity of DVR′ to changes in k_on is similar to that of DVR. This method of delayed scans eliminates the necessity of imaging during the initial uptake of the radiotracer and, thus, can be used to increase the throughput of studies.     

A simplified method for the measurement of nonmetabolized 2-[18F]F-A-85380 in blood plasma using solid-phase extraction

Quantification of alpha(4)beta(2)* nicotinic acetylcholine receptors using 2-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[(18)F]FA) and positron emission tomography (PET) imaging requires measurement of nonmetabolized radioligand in blood plasma, which was previously accomplished using high-performance liquid chromatography (HPLC). Here, we introduce a one-step solid-phase extraction (SPE) method for measuring the concentration of nonmetabolized 2-[(18)F]FA. This method allows many samples to be processed in a short period of time. SPE effectively separated 2-[(18)F]FA from radioactive metabolites typically observed in blood plasma after administration of radioligand in humans. Measurements of the 2-[(18)F]FA parent fraction in healthy human volunteers obtained using the SPE method were nearly identical to those obtained using HPLC (1.3+/-5% average underestimation of SPE), and reproducibility was good within and between runs (2% and 6% coefficient of variation, respectively). SPE recovery of 2-[(18)F]FA from blood plasma was not appreciably diminished (3+/-0.6%) by a larger volume of blood plasma loaded onto the cartridge, suggesting the possibility of increasing the plasma sample volume at later times in a PET study to improve measurement sensitivity. 2-[(18)F]FA was stable in blood stored on ice over 8 h and in saline at low concentrations (<2 MBq/ml) but not at high concentrations (ca. 130 MBq/ml). Using SPE, the elimination half-life and full body distribution volume of 2-[(18)F]FA in healthy human volunteers were estimated as 4.2+/-0.8 h and 220+/-70 L, respectively. These results suggest that SPE is the method of choice for the determination of the plasma 2-[(18)F]FA concentration when measurement of individual metabolites is not required.     

Determination of [18F]FCWAY, [18F]FP-TZTP,and their metabolites in plasma using rapid and efficient liquid-liquid and solid phase extractions

Liquid-liquid and solid phase extraction methods were developed for the accurate and rapid quantitation of radioactive components in human plasma following injection of two PET ligands. A solid phase extraction (SPE) method was developed for the determination of the 5HT(1A) receptor ligand [N-[2-[4-(2-methoxyphenyl) piperazino]ethyl]-N-(2-pyridinyl) trans-4-[(18)F]fluorocyclohexanecarboxamide (FCWAY), and its acidic metabolite, 4-[(18)F]fluorocyclohexane carboxylic acid (FC). In both cases, the extraction method was much faster and easier to use, yet provided results comparable to HPLC and TLC methods. In addition, an easy to perform two-step liquid-liquid extraction was developed for quantitation of 3-(3-((3-[(18)F]fluoropropyl)thio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine ([(18)F]FP-TZTP), a selective M2 muscarinic agonist.     

Simplified quantification of small animal [18F]FDG PET studies using a standard arterial input function

Purpose Arterial input function (AIF) measurement for quantification of small animal PET studies is technically challenging and limited by the small blood volume of small laboratory animals. The present study investigated the use of a standard arterial input function (SAIF) to simplify the experimental procedure.Methods Twelve [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) PET studies accompanied by serial arterial blood sampling were acquired in seven male Sprague-Dawley rats under isoflurane anaesthesia without (every rat) and with additional (five rats) vibrissae stimulation. A leave-one-out procedure was employed to validate the use of a SAIF with individual scaling by one (1S) or two (2S) arterial blood samples.Results Automatic slow bolus infusion of [¹⁸F]FDG resulted in highly similar AIF in all rats. The average differences of the area under the curve of the measured AIF and the individually scaled SAIF were 0.11±4.26% and 0.04±2.61% for the 1S (6-min sample) and the 2S (4-min/43-min samples) approach, respectively. The average differences between the cerebral metabolic rates of glucose (CMR_glc) calculated using the measured AIF and the scaled SAIF were 1.31±5.45% and 1.30±3.84% for the 1S and the 2S approach, respectively.Conclusion The use of a SAIF scaled by one or (preferably) two arterial blood samples can serve as a valid substitute for individual AIF measurements to quantify [¹⁸F]FDG PET studies in rats. The SAIF approach minimises the loss of blood and should be ideally suited for longitudinal quantitative small animal [¹⁸F]FDG PET studies.     

20-[18F]fluoromibolerone, a positron-emitting radiotracer for androgen receptors: synthesis and tissue distribution studies

To develop an androgen receptor-based, positron-emitting imaging agent for prostate tumors, we have prepared 20-fluoromibolerone (F-Mib) and evaluated its tissue distribution. This compound was synthesized in eight steps from 7 alpha-methyl-19-nortestosterone, with fluorine introduced in the penultimate step by fluoride ion displacement on a spirocyclic sulfate. Fluoromibolerone was obtained in 9%-19% radiochemical yield (decay corrected), at 1.5 hr after bombardment, with an effective specific activity of 217-283 Ci/mmol. The relative binding affinity of F-Mib is 53 (versus R1881 = 100 or mibolerone = 118). In tissue distribution studies in diethylstilbestrol-treated male rats, 18F-Mib demonstrates high target/tissue uptake efficiency and selectivity: the prostate uptake at 0.5 hr and 4 hr is 1.0%-1.3% injected dose/gram tissue (ID/g) and 0.5%-0.6% ID/g, respectively; the prostate-to-blood and the prostate-to-muscle (non-target) ratios are both ca. 4 at 0.5 hr, and increase to ca. 12 by 4 hr after injection. The observed distribution of 18F-Mib suggests that it may be useful for in vivo imaging of prostatic tumors in man by positron emission tomography.     

A simple method for the quality control of [18F]FDG

Most automated synthesis modules produce [¹⁸F]FDG within half an hour, but the quality control involving up to three separate methods and three different analytical systems is time consuming. The use of HPLC, TLC, and GC for the quality control of [¹⁸F]FDG is both time consuming and expensive (high purchase costs). Presented here is a method using a single HPLC system for all three analyses.     

Rapid solid-phase extraction method to quantify [C]-verapamil, and its [C]-metabolites, in human and macaque plasma

IntroductionP-glycoprotein (P-gp), an efflux transporter, is a significant barrier to drug entry into the brain and the fetus. The positron emission tomography (PET) ligand, [¹¹C]-verapamil, has been used to measure in vivo P-gp activity at various tissue–blood barriers of humans and animals. Since verapamil is extensively metabolized in vivo, it is important to quantify the extent of verapamil metabolism in order to interpret such P-gp activity. Therefore, we developed a rapid solid-phase extraction (SPE) method to separate, and then quantify, verapamil and its radiolabeled metabolites in plasma.MethodsUsing high-performance liquid chromatography (HPLC), we established that the major identifiable circulating radioactive metabolite of [¹¹C]-verapamil in plasma of humans and the nonhuman primate, Macaca nemestrina, was [¹¹C]-D-617/717. Using sequential and differential pH elution on C₈ SPE cartridges, we developed a rapid method to separate [¹¹C]-verapamil and [¹¹C]-D-617/717. Recovery was measured by spiking the samples with the corresponding nonradioactive compounds and assaying these compounds by HPLC.ResultsVerapamil and D-617/717 recovery with the SPE method was >85%. When the method was applied to PET studies in humans and nonhuman primates, significant plasma concentration of D-617/717 and unknown polar metabolite(s) were observed. The SPE and the HPLC methods were not significantly different in the quantification of verapamil and D-617/717.ConclusionsThe SPE method simultaneously processes multiple samples in less than 5 min. Given the short half-life of [¹¹C], this method provides a valuable tool to rapidly determine the concentration of [¹¹C]-verapamil and its [¹¹C]-metabolites in human and nonhuman primate plasma.     

Advances in processes for PET radiotracer synthesis: Separation of [18F]fluoride from enriched [18O]water

Positron emission tomography (PET) is a powerful scientific and clinical tool for the study and visualization of human physiology that can provide important information about metabolism and diseases such as cancer. At present, [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) is the most frequently used radiotracer for the routine clinical evaluation of malignant tumors in a range of body tissues. FDG synthesis is continuously being developed to improve and simplify the synthetic procedure including the isolation of [¹⁸F]fluoride from [¹⁸O]water. There are many methods reported in literature for the isolation of [¹⁸F]fluoride, including evaporation, coat-capture–elution, the use of cation-exchange resin and electrode trapping. This review article gives an overview of some of the most common methods for the separation of [¹⁸F]fluoride ions from [¹⁸O]water, highlighting the potential strength of the methods and also problems and weaknesses for synthesis of ¹⁸F PET tracers.     

GMP-compliant radiosynthesis of [18F]altanserin and human plasma metabolite studies

[¹⁸F]altanserin is the preferred radiotracer for in-vivo labeling of serotonin 2A receptors by positron emission tomography (PET). We report a modified synthesis procedure suited for reliable production of multi-GBq amounts of [¹⁸F]altanserin useful for application in humans. We introduced thermal heating for drying of [¹⁸F]fluoride as well as for the reaction instead of microwave heating. We furthermore describe solid phase extraction and HPLC procedures for quantitative determination of [¹⁸F]altanserin and metabolites in plasma. The time course of arterial plasma activity with and without metabolite correction was determined. 90 min after bolus injection, 38.4% of total plasma activity derived from unchanged [¹⁸F]altanserin. Statistical comparison of kinetic profiles of [¹⁸F]altanserin metabolism in plasma samples collected in the course of two ongoing studies employing placebo, the serotonin releaser dexfenfluramine and the hallucinogen psilocybin, revealed the same tracer metabolism. We conclude that metabolite analysis for correction of individual plasma input functions used in tracer modeling is not necessary for [¹⁸F]altanserin studies involving psilocybin or dexfenfluramine treatment.     

Fully automated synthesis of [18F]fluoromisonidazole using a conventional [18F]FDG module

We developed a new fully automated method for the synthesis of [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO) by modifying a commercial FDG synthesizer and its disposable fluid pathway. A three-step procedure was used to prepare the tosylate precursor, 1-(2′-nitro-1′-imidazolyl)-2-O-tetrahydrofuranyl-3-O-toluenesulfonylpropanediol. Using glycerol as the starting material, the precursor was synthesized with a yield of 21%. The optimal labeling conditions for the automated synthesis of [¹⁸F]FMISO was 10 mg of precursor in acetonitrile (2 ml heated at 105°C for 360 s, followed by heating at 75°C for 280 s and hydrolysis with 1 N HCl at 105°C for 300 s. Using 3.7 GBq of [¹⁸F]F⁻ as a starting activity, [¹⁸F]FMISO was obtained with high end-of-synthesis (EOS) radiochemical yields of 58.5±3.5% for 60.0±5.2 min with high-performance liquid chromatography (HPLC) purification. When solid-phase purification steps were added, the EOS radiochemical yields were 54.5±2.8% (337±25 GBq/μmol) for 70.0±3.8 min (n=10 for each group, decay-corrected). With a high starting radioactivity of 37.0 GBq, we obtained radiochemical yields of 54.4±2.9% and 52.8±4.2%, respectively (n=3). The solid-phase purification removed unreacted [¹⁸F]fluoride and polar impurities before the HPLC procedure. Long-term tests showed a good stability of 98.2±1.5%. This new automated synthesis procedure combines high and reproducible yields with the advantage of using a disposable cassette system.     

Difficulties in dopamine transporter radioligand PET analysis: the example of LBT-999 using [18F] and [11C] labelling: part II: Metabolism studies

IntroductionLBT-999, (E)-N-(4-fluorobut-2-enyl)-2β-carbomethoxy-3β-(4'-tolyl)nortropane, has been developed for PET imaging of the dopamine transporter. [¹⁸F]LBT-999 PET studies in baboons showed a lower brain uptake than [¹¹C]LBT-999 and a high bone uptake, suggesting the presence of interfering metabolites. Therefore, in vitro and in vivo metabolism of these radiotracers was investigated.MethodsRat and human liver microsomal incubations, baboon plasma and rat brain extracts were analyzed by radio-HPLC and LC-MS-MS.ResultsIn vitro experiments demonstrated the formation by P450s of five polar metabolites. The main routes of LBT-999 metabolism proposed were N-dealkylation, tolyl-hydroxylation and dealkylation plus tolyl-hydroxylation. In vivo in baboons, [¹⁸F]LBT-999 was rapidly converted into a [¹⁸F]hydroxylated metabolite likely oxidized in plasma into a [¹⁸F]carboxylic acid and into unlabeled N-dealkyl-LBT-999. The latter was detected in baboon plasma and in rat brain by LC-MS-MS. The time course of unchanged [¹⁸F]LBT-999 decreased rapidly in plasma and was higher than that of [¹¹C]LBT-999 due to the formation of unlabeled N-dealkyl-LBT-999. In rats, striatum-to-cerebellum ratios of [¹⁸F]LBT-999, [¹⁸F]hydroxylated and [¹⁸F]acidic metabolite were 20, 4.2 and 1.65, respectively, suggesting a possible accumulation of the hydroxylated compound in the striatum.ConclusionP450s catalyzed the formation of dealkylated and hydroxylated metabolites of LBT-999. In baboons, an extensive metabolism of [¹⁸F]LBT-999, with formation of unlabeled N-dealkyl-LBT-999, [¹⁸F]fluorobutenaldehyde (or its oxidation product) and [¹⁸F]hydroxy-LBT-999 able to penetrate the brain, prevented an easy and accurate estimation of the input function of the radiotracer. CYP3A4 being the main P450 involved in the metabolism of LBT-999, a similar pathway may occur in humans and confound PET quantification.     

Robotic production of 2-deoxy-2[18F]fluoro-d-glucose: A routine method of synthesis using tetrabutylammonium [18F]fluoride

Using existing robotic hardware and software programs developed for the synthesis of several positron-emitting radiopharmaceuticals for PET imaging [Brodack et al. (1988) Appl. Radiat. Isot.39, 689], the additional automated synthesis of 2-deoxy-2-[¹⁸F]fluoro-d-glucose (2-[¹⁸F]FDG) has been incorporated into our Zymate Laboratory Automation System. The robotic synthesis of 2-[¹⁸F]FDG took less than one week to implement, including the organization of software subroutines and construction of an additional heating station. The end of synthesis yield (12–17%) and radiochemical purity (96–99%) for the robotic preparation of 2-[¹⁸F]FDG is similar to that of the manual synthesis. This automated method uses anhydrous tetrabutylammonium [¹⁸F]fluoride as the reactive fluoride source in the labeling step. The procedure is a modification of the synthesis reported by Hamacher et al. [Hamacher et al. (1986) J. Nucl. Med.27, 235].     

New approach to fully automated synthesis of sodium [18F]fluoroacetate -- a simple and fast method using a commercial synthesizer

A simple, rapid and fully automated preparation of sodium [(18)F]fluoroacetate has been developed by taking advantage of the similarities between the reaction pathways of [(18)F]fluoroacetate and [(18)F]-2-fluoro-deoxyglucose (FDG). The automated synthesis of sodium [(18)F]fluoroacetate was achieved with a commercial [(18)F]FDG synthesizer, the TRACERlab MX(FDG). The method produced the desired compound in a short synthesis time (32 min) and with a high and reproducible radiochemical yield (50.2 +/- 4.8%, decay corrected). The radiochemical purity of sodium [(18)F]fluoroacetate was greater than 99%.     

Synthesis of [18F]Xeloda as a novel potential PET radiotracer for imaging enzymes in cancers

Xeloda (Capecitabine), a prodrug of antitumor agent 5-fluorouracil, is the first and only oral fluoropyrimidine to be approved for use as second-line therapy in metastatic breast cancer, colorectal cancer, and other solid malignancies. Fluorine-18 labeled Xeloda may serve as a novel radiotracer for positron emission tomography (PET) to image enzymes such as thymidine phosphorylase and uridine phosphorylase in cancers. The precursor 2′,3′-di-O-acetyl-5′-deoxy-5-nitro-N⁴-(pentyloxycarbonyl)cytidine (11) was synthesized from D-ribose and cytosine in 8 steps with approximately 18% overall chemical yield. The reference standard 5′-deoxy-5-fluoro-N⁴-(pentyloxycarbonyl)cytidine (Xeloda; 1) was synthesized from D-ribose and 5-fluorocytosine in eight steps with approximately 28% overall chemical yield. The target radiotracer 5′-deoxy-5-[¹⁸F]fluoro-N⁴-(pentyloxycarbonyl)cytidine ([¹⁸F]Xeloda; [¹⁸F]1) was prepared by nucleophilic substitution of the nitro-precursor with K¹⁸F/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with the HPLC method in 20–30% radiochemical yields.     

Comparative studies of epibatidine derivatives [18F]NFEP and [18F]N-methyl-NFEP: kinetics, nicotine effect, and toxicity

We have previously shown that [18F]norchlorofluoroepibatidine ([18F]NFEP) would be an ideal radiotracer for positron emission tomography (PET) imaging of nicotinic acetylcholine receptors (nAChR); however, its high toxicity is a limiting factor for human studies. We, therefore, synthesized its N-methyl derivative ([18F]N-Me-NFEP) and carried out comparative studies. The distribution volumes for different brain regions were higher for [18F]N-Me-NFEP than those for [18F]NFEP (average: 52.5+/-0.9 vs. 36.4+/-0.7 for thalamus), though the distribution volume (DV) ratios were similar (3.93+/-0.27 vs. 3.65+/-0.19 for thalamus to cerebellum). Treatment with nicotine reduced the binding of both radiotracers. Toxicology studies in awake rats showed that N-methyl-NFEP has a lower mortality (0 vs. 30%) and smaller effect on plasma catecholamines than NFEP at a dose of 1.5 microg/kg. However, marked alterations in cardiorespiratory parameters were observed after injection of N-methyl-NFEP (0.5 microg/kg, IV) to an awake dog. Our results suggest that although the binding characteristics of [18F]NFEP and [18F]N-Me-NFEP appear to be ideally suited for PET imaging studies of the human brain, their relatively small safety margin will limit their use in humans.     

Contamination of 2-deoxy-2-[18F]fluoro-d-mannose in the 2-deoxy-2-[18F]fluoro-d-glucose preparations synthesized from [18F]acetyl hypofluorite and [18F]F2

The presence of 2-deoxy-2-[¹⁸F]fluoro-d-mannose ([¹⁸F]FDM) in 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) prepared by the reaction of 3,4,6-tri-O-acetyl-d-glucal (TAG) with [¹⁸F]acetyl hypofluorite ([¹⁸F]CH₃COOF) or [¹⁸F]F₂ was quantified by radio HPLC analysis of reacetylated [¹⁸F]FDG. The solvent effects on the stereoselectivity of the reaction of TAG with [¹⁸F]CH₃COOF were examined in six kinds of solvent.Reaction of TAG with [¹⁸F]CH₃COOF in Freon-11 results in the least contamination of [¹⁸F]FDM (4–5%). The presence of [¹⁸F]FDM in the [¹⁸F]FDG prepared from TAG with [¹⁸F]F₂ was also indicated, but by careful chromatographic separation of hexopyranosyl difluorides the amount was reduced to nearly that resulting from the [¹⁸F]CH₃COOF method.     

Comparison of [18F]FDOPA, [18F]FMT and [18F]FECNT for imaging dopaminergic neurotransmission in mice

INTRODUCTION: The clinically established positron emission tomography (PET) tracers 6-[(18)F]-fluoro-l-DOPA ([(18)F]FDOPA), 6-[(18)F]-fluoro-l-m-tyrosine ([(18)F]FMT) and 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-[(18)F]-fluoroethyl)-nortropane ([(18)F]FECNT) serve as markers of presynaptic integrity of dopaminergic nerve terminals in humans. This study describes our efforts to adopt the methodology of human Parkinson's disease (PD) PET studies to mice. METHODS: The PET imaging characteristics of [(18)F]FDOPA, [(18)F]FMT and [(18)F]FECNT were analyzed in healthy C57BL/6 mice using the dedicated small-animal PET tomograph quad-HIDAC. Furthermore, [(18)F]FECNT was tested in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. RESULTS: [(18)F]FDOPA and [(18)F]FMT failed to clearly visualize the mouse striatum, whereas PET experiments using [(18)F]FECNT proved that the employed methodology is capable of delineating the striatum in mice with exquisite resolution. Moreover, [(18)F]FECNT PET imaging of healthy and MPTP-lesioned mice demonstrated that the detection and quantification of striatal degeneration in lesioned mice can be accomplished. CONCLUSIONS: This study shows the feasibility of using [(18)F]FECNT PET to analyze noninvasively the striatal degeneration in the MPTP mouse model of PD. This methodology can be therefore considered as a viable complement to established in vivo microdialysis and postmortem techniques.