Analysis of residual solvents in 2-[(18)F]FDG by GC.

A gas chromatography method has been developed for the measurement of the residual acetone, ethanol and acetonitrile in 2-deoxy-2-[(18)F] fluoro-D-glucose (2-[(18)F]FDG), in accordance with the pending FDA revision on the drug. The detections limits were 0.1 ppm for all three solvents. Good precision and linearity were obtained over ranges spanning the allowable concentration levels proposed by FDA. The amounts of the three solvents in our routine 2-[(18)F]FDG products have been found well below the maximum permissible levels. The method is very amenable to quality control testing for the radiopharmaceutical.

Noninvasive estimation of the aorta input function for measurement of tumor blood flow with.

Quantitative measurement of tumor blood flow with [15O]water can be used to evaluate the effects of tumor treatment over time. Since quantitative flow measurements require an input function, we developed the profile fitting method (PFM) to measure the input function from positron emission tomography images of the aorta. First, a [11C]CO scan was acquired and the aorta region was analyzed. The aorta diameter was determined by fitting the image data with a model that includes scanner resolution, the measured venous blood radioactivity concentration, and the spillover of counts from the background. The diameter was used in subsequent fitting of [15O]water dynamic images to estimate the aorta and background radioactivity concentrations. Phantom experiments were performed to test the model. Image quantification biases (up to 15%) were found for small objects, particularly for those in a large elliptical phantom. However, the bias in the PFM concentration estimates was much smaller (2%-6%). A simulation study showed that PFM had less bias and/or variability in flow parameter estimates than an ROI method. PFM was applied to human [11C]CO and [15O]water dynamic studies with left ventricle input functions used as the gold standard. PFM parameter estimates had higher variability than found in the simulation but with minimal bias. These studies suggest that PFM is a promising technique for the noninvasive measurement of the aorta [15O]water input function.

Kinetic analysis of the 5-HT2A ligand [11C]MDL 100,907.

The goal of this study was to develop a suitable kinetic analysis method for quantification of 5-HT2A receptor parameters with [11C]MDL 100,907. Twelve control studies and four preblocking studies (400 nmol/kg unlabeled MDL 100,907) were performed in isoflurane-anesthetized rhesus monkeys. The plasma input function was determined from arterial blood samples with metabolite measurements by extraction in ethyl acetate. The preblocking studies showed that a two-tissue compartment model was necessary to fit the time activity curves of all brain regions including the cerebellum--in other words, the need for two compartments is not proof of specific binding. Therefore, a three-tissue compartment model was used to analyze the control studies, with three parameters fixed based on the preblocking data. Reliable fits of control data could be obtained only if no more than three parameters were allowed to vary. For routine use of [11C]MDL 100,907, several simplified methods were evaluated. A two-tissue (2T') compartment with one fixed parameter was the most reliable compartmental approach; a one-compartment model failed to fit the data adequately. The Logan graphical approach was also tested and produced comparable results to the 2T' model. However, a simulation study showed that Logan analysis produced a larger bias at higher noise levels. Thus, the 2T' model is the best choice for analysis of [11C]MDL 100,907 studies.

Biologically stable [(18)F]-labeled benzylfluoride derivatives.

Use of the [(18)F]-fluoromethyl phenyl group is an attractive alternative to direct fluorination of phenyl groups because the fluorination of the methyl group takes place under milder reaction conditions. However, we have found that 4-FMeBWAY showed femur uptake equal to that of fluoride up to 30 min in rat whereas 4-FMeQNB had a significantly lower percent injected dose per gram in femur up to 120 min. For these and other benzylfluoride derivatives, there was no clear in vivo structure-defluorination relationship. Because benzylchlorides (BzCls) are known alkylating agents, benzylfluorides may be alkylating agents as well, which may be the mechanism of defluorination. On this basis, the effects of substitution on chemical stability were evaluated by the 4-(4-nitro-benzyl)-pyridine (NBP) test, which is used to estimate alkylating activity with NBP. The effect of substitution on the alkylating activity was evaluated for nine BzCl derivatives: BzCl; 3- or 4-methoxy (electron donation) substituted BzCl; 2-, 3-, or 4-nitro (electron withdrawing) substituted BzCl; and 2-, 3-, or 4-chloro (electron withdrawing) substituted BzCl. Taken together, the alkylating reactivity of 3-chloro-BzCl was the weakest. This result was then applied to [(18)F]-benzylfluoride derivatives and in vivo and in vitro stability were evaluated. Consequently, 3-chloro-[(18)F]-benzylfluoride showed a 70-80% decrease of defluorination in both experiments in comparison with [(18)F]-benzylfluoride, as expected. Moreover, a good linear relationship between in vivo femur uptake and in vitro hepatocyte metabolism was observed with seven (18)F-labeled radiopharmaceuticals, which were benzylfluorides, alkylfluorides, and arylfluorides. Apparently, the [(18)F]-fluoride ion is released by metabolism in the liver in vivo. In conclusion, 3-chloro substituted BzCls are the most stable, which suggests that 3-chloro benzylfluorides will be the most chemically stable compound. This result should be important in future design of radioligands labeled with a benzylfluoride moiety.

Brain incorporation of [1-11C]arachidonate in normocapnic and hypercapnic monkeys, measured with positron emission tomography.

Positron emission tomography (PET) was used to determine brain incorporation coefficients k* of [1-11C]arachidonate in isoflurane-anesthetized rhesus monkeys, as well as cerebral blood flow (CBF) using [15O]water. Intravenously injected [1-11C]arachidonate disappeared from plasma with a half-life of 1.1 min, whereas brain radioactivity reached a steady-state by 10 min. Mean values of k* were the same whether calculated by a single-time point method at 20 min after injection began, or by least-squares fitting of an equation for total brain radioactivity to data at all time points. k* equalled 1.1-1.2 x 10(-4) ml x s(-1) x g(-1) in gray matter and was unaffected by a 2.6-fold increase in CBF caused by hypercapnia. These results indicate that brain incorporation of [1-11C]arachidonate can be quantified in the primate using PET, and that incorporation is flow-independent.

Synthesis and evaluation of radioiodinated 6-iodo-L-DOPA as a cerebral L-amino acid transport marker.

Regioselective radioiodination of N-trifluoroacetyl 3,4-dimethoxy-6-trifluoroacetoxymercurio-L-phenylalanine ethyl ester 1 under no-carrier-added condition gave 6-[125I]iodo protected L-DOPA with a labeling efficiency of more than 85%, and no-carrier-added 6-[125I]I-L-DOPA was obtained with a radio-chemical purity of over 95% after hydrolysis and chromatography. A nonradioactive standard of 6-iodo protected L-DOPA was synthesized by the iododemercuration of 6-mercuric trifluoroacetate protected L-DOPA 1 using I2 in chloroform. 6-[125I]I-L-DOPA showed high brain accumulation and rapid blood clearance in mice. The rat brain slice studies indicated high affinity of 6-[125I]I-L-DOPA for carrier-mediated and stereoselective active transport systems. The tissue homogenate analysis revealed that most of the accumulated radioactivity was intact 6-[125I]I-L-DOPA. Thus, 6-[123I]I-L-DOPA appears to be a suitable single photon emission computed tomography (SPECT) tracer for the selective measurement of cerebral L-amino acid transport, having no affinity for dopamine metabolism.

Synthesis of polymer-bound 6-mercuric carboxylate DOPA precursors and solid phase labelling method of 6-radioiodinated L-DOPA.

In order to avoid separating unreacted mercury precursor and other mercury-containing compounds after the halodemercuration of a 6-mercury DOPA precursor, we developed a polymer-bound mercury precursor for the preparation of 6-halogenated DOPA. In this study, polymer-bound 6-mercuric carboxylate DOPA derivatives were synthesized from ion-exchange resin and Merrifield-type resin. Iododemercuration of polymer-bound 6-mercuric carboxylate DOPA derivatives gave higher yields (49-54%) compared with monomeric 6-mercuric trifluoroacetate protected DOPA. The radioiodination of the resin with no-carrier added iodine-125 afforded protected 6-[125I]I-L-DOPA with labelling efficiency of 92-97% with both polymer-bound 6-mercuric carboxylate DOPA derivatives.

Incorporation of [1-carbon-11]palmitate in monkey brain using PET.

UNLABELLED: We determined regional incorporation coefficients (k*) of plasma [1-11C]palmitate into stable brain lipids of anesthetized monkeys with PET. METHODS: Carbon-11-palmitate was injected intravenously in untreated animals and in animals pretreated with methyl palmoxirate (MEP), an inhibitor of beta-oxidation of palmitate in the brain and periphery. Plasma radioactivity was followed, and brain radioactivity was determined at various times using PET. A least-squares method was used to fit the data to an operational equation to obtain regional values of k* and of cerebral blood volume (Vb) in individual experiments. RESULTS: MEP significantly decreased the integral of plasma [11C]CO2 following 11C-palmitate infusion. Mean values of k* in monkeys not given MEP were 4.9, 4.2, 4.9, 4.0 and 2.9 x 10(-5) ml/sec.g for the temporal, frontal, parietal and occipital cortices and white matter, respectively. With the exception of k* in white matter, which was increased by MEP, k* in the other brain regions was not significantly changed by MEP. The Vb ranged from 0.035 ml/g to 0.048 ml/g in gray matter regions and equaled 0.022 ml/g in white matter. CONCLUSION: PET can be used to determine regional incorporation coefficients of 11C-palmitate into the primate brain in vivo. Combined with MEP, 11C-palmitate could be used with PET to examine regional brain phospholipid metabolism in humans in normal and pathological conditions.

Synthesis of polymer-bound 6-thiolatomercury and 6-mercuric sulfonate DOPA precursors and their halodemercuration reactivity.

Fluorodemercuration has the greatest utility for the preparation of 6-[18F]DOPA, but requires separation from unreacted mercury precursor and other mercury-containing compounds. One approach is the development of a polymer-bound mercury precursor. In this study, polymer-bound 6-thiolatomercury and 6-mercuric sulfonate DOPA derivatives, and its monomeric analogs were synthesized. Fluorodemercuration of monomeric analog of mercuric sulfonate gave half the yield (14-15%) while iododemercuration gave the same yield (38%) compared with a 6-mercuric trifluoroacetate protected DOPA. The mercuric sulfonate undergoes halodemercuration, so polymer-bound halodemercuration precursors may be useful as precursors of 6-[18F]DOPA.

Comparison of bolus and infusion methods for receptor quantitation: application to [18F]cyclofoxy and positron emission tomography.

Positron emission tomography studies with the opiate antagonist [18F]cyclofoxy ([18F]CF) were performed in baboons. Bolus injection studies demonstrated initial uptake dependent on blood flow. The late uptake showed highest binding in caudate nuclei, amygdala, thalamus, and brainstem and the least accumulation in cerebellum. By 60 min postinjection, regional brain radioactivity cleared at the same rate as metabolite-corrected plasma, i.e., transient equilibrium was achieved. Compartmental modeling methods were applied to time-activity curves from brain and metabolite-corrected plasma. Individual rate constants were estimated with poor precision. The model estimate of the total volume of distribution (VT), representing the ratio of tissue radioactivity to metabolite-corrected plasma at equilibrium, was reliably determined. The apparent volume of distribution (Va), the concentration ratio of tissue to metabolite-corrected plasma during transient equilibrium, was compared with the fitted VT values to determine if single-scan methods could provide accurate receptor measurements. Va significantly overestimated VT and produced artificially high image contrast. These differences were predicted by compartment model theory and were caused by a plasma clearance rate that was close to the slowest tissue clearance rate. To develop a simple method to measure VT, an infusion protocol consisting of bolus plus continuous infusion (B/I) of CF was designed and applied in a separate set of studies. The Va values from the B/I studies agreed with the VT values from both B/I and bolus studies. This infusion approach can produce accurate receptor measurements and has the potential to shorten scan time and simplify the acquisition and processing of scan and blood data.

In vivo imaging of insulin receptors in monkeys using 18F-labeled insulin and positron emission tomography.

We previously described a prosthetic group methodology for incorporating 18F into peptides and showed that 18F-labeled insulin (18F-insulin) binds to insulin receptors on human cells (IM-9 lymphoblastoid cells) with affinity equal to that of native insulin (1). We now report studies using 18F-insulin with positron emission tomography to study binding to insulin receptors in vivo. Positron emission tomography scans were performed in six rhesus monkeys injected with 0.3-1.4 mCi of 18F-insulin (approximately 0.1 nmol, SA 4-11 Ci/mumol). Integrity of the tracer in blood, determined by immunoprecipitation, was 94% of control for the first 5 min and decreased to 31% by 30 min. Specific, saturable uptake of 18F was observed in the liver and kidney. Coinjection of unlabeled insulin (200 U, approximately 1 nmol) with the 18F-insulin reduced liver and increased kidney uptake of the labeled insulin. Liver radioactivity was decreased by administration of unlabeled insulin at 3 min, but not 5 min, after administration of the tracer, while some kidney radioactivity could be displaced 5 min after injection. Clearance of 18F was predominantly in bile and urine. 18F-insulin is a suitable analogue for studying insulin receptor-ligand interactions in vivo, especially in the liver and kidney.

A single column, rapid quality control procedure for 6-[18F]fluoro-L-dopa and 6-[18F]fluorodopamine PET imaging agents.

6-[18F]Fluoro-L-dopa and 6-[18F]fluorodopamine are promising PET imaging agents for visualizing cerebral dopaminergic centers and cardiac sympathetic innervation and function. Administration to humans requires a means to determine the purity before injection. We describe such a method using HPLC with u.v. and radioactivity detection and a single high-speed C-18 column with gradient elution. The procedure can resolve within 10 min these fluorinated catechols, their isomers, and dihydroxyphenylalanine. The chemical and radiochemical purity, and specific activity, can be determined before injection.

18F-labeled insulin: a prosthetic group methodology for incorporation of a positron emitter into peptides and proteins.

In the present study we synthesize 18F-labeled insulin of high specific radioactivity. A new prosthetic group methodology, in which [18F]fluoride displaces a bromide group of 4-(bromomethyl)-benzoylamine intermediates, was used. The 4-(fluoromethyl)benzoyl product was chemically stable. 18F-Labeled insulin retains the essential biological properties of native insulin, as measured in vitro by binding to insulin receptors on human cells and stimulation of glucose metabolism in rat adipocytes. The overall process can be carried out speedily to yield a product of sufficient purity to permit in vivo studies. The method appears to be applicable to a wide variety of peptides.

Radiosynthesis of [18F]3-acetylcyclofoxy: a high affinity opiate antagonist.

A convenient method for the preparation of high specific activity [18F]3-acetylcyclofoxy (3-acetyl-6-deoxy-6-beta-18F-fluoronaltrexone) was developed. The method utilizes reactor-produced [18F]-fluoride as its tetraethylammonium (TEA X F) salt in a SN2 displacement on a secondary triflate precursor. Typically, 45% of the 18F activity can be converted to the reactive TEAF in a 70 min preparation. From this, 35% yield (decay corrected) of the [18F]3-acetylcyclofoxy was obtained after HPLC purification with a specific activity of 25 Ci/mmol in a total synthesis time of 60 min.

3-[18F]Acetylcyclofoxy: a useful probe for the visualization of opiate receptors in living animals.

A fluoro-analogue of the potent narcotic antagonist, naltrexone, was synthesized and shown to bind with high affinity to opiate receptors in vitro. 3-[18F]acetylcyclofoxy was prepared via a one-step triflate displacement reaction with the positron emitting 18F ion from tetraethylammonium [18F] fluoride. 3-[18F]acetylcyclofoxy accumulation in opiate receptor rich brain regions of both rat and baboon is shown to be completely displaced by the active enantiomer of naloxone [-)-naloxone) while the identical dose of the pharmacologically inert (+)-naloxone has no detectable effect. Moreover, both rat and baboon brain showed the well documented, typical opiate receptor distribution so that basal ganglia and thalamus are clearly visible in the living baboon brain up to 95 min after intravenous injection of 3-[18F] acetylcyclofoxy. We expect that 3-[18F )acetylcyclofoxy will be a useful probe for visualizing opiate receptors in living humans.