Biodistribution of [11C]methylaminoisobutyric acid, a tracer for PET studies on system A amino acid transport in vivo

[N-methyl-¹¹C]!-Methylaminoisobutyric acid (¹¹C-MeAIB) is a potentially useful tracer for positron emission tomography (PET) studies on hormonally regulated system A amino acid transport. ¹¹C-MeAIB is a metabolically stable amino acid analogue specific for system A amino acid transport. We evaluated the biodistribution of ¹¹C-MeAIB in rats and humans to estimate the usefulness of the tracer for in vivo human PET studies, for example, on regulation of system A amino acid transport and on tumour imaging. Healthy Sprague-Dawley rats (n=14) were killed 5, 20, 40 or 60 min after the injection of ¹¹C-MeAIB, and the tissue samples were weighed and counted for ¹¹C radioactivity. Ten lymphoma patients with relatively limited tumour burden underwent whole-body (WB) PET imaging with ¹¹C-MeAIB. In addition, three other patients had dynamic PET scanning of the head and neck area, and the tracer uptake was quantitated by calculating the kinetic influx constants (K_i values) for the tracer. In animal studies, the highest activity was detected in the kidney, pancreas, adrenal gland and intestines. In humans, the highest activity was found in the salivary glands, and after that in the kidney and pancreas, similar to the results in animal studies. Rapid uptake was also detected in the skeletal muscle. In the graphical analysis, linear plots were obtained, and the mean fractional tracer uptake values (K_i) of the parotid glands (n=3) and cervical muscles (n=3) were 0.039ǂ.008 min^-1 and 0.013ǂ.006 min^-1, respectively. The K_i value of the tumour (n=1) was 0.064 min^-1. Higher uptake of ¹¹C-MeAIB into the tumour tissue was encountered. These results encourage further ¹¹C-MeAIB PET studies in humans on the physiology and pathology of system A amino acid transport and on tumour detection.     

Human radiation dosimetry of [11C]MeAIB, a new tracer for imaging of system A amino acid transport

Purpose [N-methyl-¹¹C]α-methylaminoisobutyric acid ([¹¹C]MeAIB) is a promising positron emission tomography (PET) tracer for imaging hormonally regulated system A amino acid transport. Uptake of [¹¹C]MeAIB is totally specific for amino acid transport since [¹¹C]MeAIB is metabolically stable both extra- and intracellularly. The aim of this study was to measure cumulated radioactivity in different organs and estimate the absorbed radiation doses to humans with the Medical Internal Radiation Dosimetry (MIRD) method.Methods Radiation absorbed doses were calculated from PET images for 25 volunteers. Dynamic acquisition data were obtained for the thoracic, abdominal, femoral and head and neck regions. The median dose of intravenously injected [¹¹C]MeAIB was 422±35 MBq, with a range of 295–493 MBq. After PET imaging the radioactivity in voided urine was measured. Experimental human data were used for residence time estimates. Radiation doses were calculated with commonly used software.Results The effective dose for a 70-kg adult was 0.004 mSv/MBq, corresponding to a 1.72 mSv effective dose from the PET study with injection of 430 MBq [¹¹C]MeAIB. The highest absorbed doses were in the pancreas (0.018 mGy/MBq), kidneys (0.017 mGy/MBq), intestine (0.014 mGy/MBq), liver (0.008 mGy/MBq) and stomach (0.005 mGy/MBq). Only 0.57% of injected activity was excreted to urine within 1 h after injection.Conclusion Biodistribution of [¹¹C]MeAIB in the abdominal region reflected the high activity of the transportation of amino acids via system A and these organs also had the highest radiation doses. An effective dose of 0.004 mSv/MBq is fully justified when [¹¹C]MeAIB PET is performed to study system A activity in vivo.     

The pharmacokinetics of [11C]methoxy-norchloroprogabidic acid, a potential PET tracer for GABA receptors in the brain.

We have described the kinetic properties of [¹¹C]methoxy-norchloroprogabidic acid, a potential radioligand for the gamma-aminobutyric acid (GABA) receptor. Early metabolism in mice was negligible. Protein binding in human plasma was 89 ± 7%. Distribution volumes were 89.6 ± 29.4 l whereas the elimination half-life was 41.6 ± 14.3 min. Animal and human positron emission tomography (PET) data demonstrate limited uptake of the activity in brain tissue. Displacement studies in mice suggest a nonsignificant fraction of specific receptor binding. [¹¹C] Methoxy-norchloroprogabidic acid is therefore unsuitable for brain PET.     

Preclinical evaluation of [11C]NE40, a type 2 cannabinoid receptor PET tracer

IntroductionUp-regulation of the type 2 cannabinoid receptor (CB₂R) has been reported in (neuro)inflammatory diseases. In this study, we report the preclinical evaluation of [¹¹C]NE40 as positron emission tomography (PET) radioligand for visualization of the CB₂R.MethodsThe selectivity of NE40 for CB₂R and its toxicity and mutagenicity were determined. [¹¹C]NE40 was evaluated by biodistribution and autoradiography studies in normal rats and a microPET study in normal mice, rats and a rhesus monkey. Specific in vivo binding of [¹¹C]NE40 to human CB₂R (hCB₂R) was studied in a rat model with hCB₂R overexpression.Results[¹¹C]NE40 shows specific CB₂R binding in the spleen and blood of normal rats and high brain uptake in rhesus monkey. [¹¹C]NE40 showed specific and reversible binding to hCB₂R in vivo in a rat model with local hCB₂R overexpression.Conclusions[¹¹C]NE40 shows favorable characteristics as radioligand for in vivo visualization of the CB₂R and is a promising candidate for hCB₂R PET imaging.     

Uptake of radioactive octanoate in astrocytoma cells: Basic studies for application of [11C]octanoate as a PET tracer

Fatty acids are taken up and metabolized in the brain.In vitro uptake experiments on astrocytoma cells were carried out to assess the potential use of [1-¹¹C]octanoate as a positron emission tomography (PET) tracer for astroglial functions. Uptake of [1-¹⁴C]octanoate increased in a time-dependent fashion until 60 min after application. The uptake of [1-¹¹C]octanoate showed similar results to that of [1-¹⁴C]octanoate until 10 min. As for medium pH, [1-¹⁴C]octanoate uptake increased gradually with the decrease in pH. We also examined the effects of glutamate, glucose deprivation and hypoxia on the uptake of octanoate and found that these conditions did not bring about any change in the extent of [1-¹⁴C]octanoate uptake. These results show that the octanoate uptake was not influenced by any of several pathological conditions. When the number of astrocytes increases in the area of hypoglycemia or hypoxia near a brain lesion, the amount of octanoate uptake also increases, so this indicates the possibility that¹¹C-octanoate will detect a brain lesion.     

Pharmacological evaluation of [11C]donepezil as a tracer for visualization of acetylcholinesterase by PET

Donepezil is a highly potent and selective reversible achetylcholinesterase inhibitor. [(11)C]Donepezil is prepared by methylation with [(11)C]CH(3)I of the corresponding 6'-O-desmethylprecursor. Tissue distribution in mice revealed a high uptake in brain and rapid clearance from the blood. Metabolization studies in mice indicated the formation of one (11)C-labeled polar metabolite that didn't penetrate the blood-brain barrier. Regional brain distribution in rabbits didn't reflect the measured achetylcholinesterase distribution in rabbit brain.     

Biodistribution of 3,4-dihydro-5-[11C]methoxy-1(2H)-isoquinolinone, a potential PET tracer for poly(ADP-ribose) synthetase

Poly(adenosine diphosphate-ribose) synthetase (PARS) is a nuclear enzyme that is activated by deoxyribonucleic acid (DNA) strand breaks and participates in DNA repair. Excessive PARS activation, however, leads to cell death due to depletion of adenosine triphosphate (ATP). To evaluate whether it is possible to detect excessive activation of PARS with positron emission tomography (PET), we examined the pharmacokinetics of 3,4-dihydro-5-[(11)C]methoxy-1(2H)-isoquinolinone ([(11)C]MIQO), a potent poly(ADP-ribose) synthetase inhibitor, in the brain of rats and monkeys. Although the uptake of [(11)C]MIQO in the brain of normal rats was low, [(11)C]MIQO was rapidly incorporated into and then quickly washed out from the brain. The uptake of the radiotracer in the brain of normal monkeys was also low; however, [(11)C]MIQO gave a distribution image that differed from that of cerebral blood flow obtained by [(15)O]water-PET. No localization of [(11)C]MIQO in the brain of normal monkeys was observed. Low accumulation of some radioactivity was also observed in muscles surrounding the brain of monkeys, but did not seem to interfere with measurement of [(11)C]MIQO uptake in the brain with PET. Thus, detection of [(11)C]MIQO uptake with PET may be useful for detecting PARS activity in ischemic injury.     

Use of 11C as a tracer for studying the synthesis of [11C]urea from [11C]cyanide

The use of reversed-phase liquid chromatography and radiochemical detection with carbon-11 (t1/2 = 20.4 min) as a tracer allowed the study of the preparation of [11C]urea from [11C]cyanide at no-carrier-added concentrations. [11C]cyanate was readily prepared by permanganate oxidation of [11C]cyanide at 75 degrees C. The conversion of NH4O11CN (approximately 0.03 mM) to [11C]urea in the presence of excess ammonium ions (0.28 M) was found to best fit pseudo first order reaction kinetics with a rate constant of 0.065 +/- 0.008 min-1 at 75 degrees C. Heating at higher temperatures (180-200 degrees C) revealed that the conversion of NH4O11CN to [11C]urea occurred in high yield in less than 3 min. The hydrolysis of [11C]cyanate to [11C]carbonate, a possible side reaction, was found to proceed at a rate of 0.010 +/- 0.001 min-1 at 113 degrees C.     

Preparation of [11C] radioligands with high specific radioactivity on a commercial PET tracer synthesizer

High specific radioactivity is required for receptor studies with PET. Hereby we wish to report our experience using Nuclear Interface PET Tracer Synthesizer for preparation of Carbon-11 radioligands and module's modifications, which allowed to achieve high specific radioactivity.     

Radiosynthesis of 1-[F]fluoroethyl-L-tryptophan as a novel potential amino acid PET tracer

¹⁸F labeled natural amino acids have been introduced as promising tumor imaging agents. A novel [¹⁸F]fluoro amino acid analog 1-[¹⁸F]fluoroethyl-L-tryptophan (1-[¹⁸F]FETrp) was designed and synthesized by a two-pot three-step procedure, including the synthesis of 1-[¹⁸F]fluoro-2- (tosyloxy)ethane, the [¹⁸F]fluoroethylation of the precursor N-Boc-L-tryptophan ethyl ester and following the deprotection of the tert-butoxycarbonyl and ethyl ester protecting groups. 1-[¹⁸F]FETrp was resulted in 0.9±0.2% (n=5) radiochemical yields (no decay corrected) by HPLC purification, within a total synthesis time of 65 min. The radiochemical purity of 1-[¹⁸F]FETrp was 95-97%. The radiosynthetic method needs to be further optimized to get a satisfying radiochemical yield.     

Synthesis and preliminary evaluation of [1-11C]hexanoate as a PET tracer of fatty acid metabolism

The potential of [1-¹¹C]hexanoate (¹¹C-HA) as a radiopharmaceutical assessing fatty acid metabolism of the myocardium and brain tissues by PET studies was evaluated.¹¹C-HA was synthesized by the Grignard reaction of pentylmagnesium bromide and¹¹CO₂.¹¹C-HA, [1-¹⁴C]acetate and [³H]deoxyglucose were simultaneously injected i.v. into mice, and the tissue distribution of the three radionuclides was measured. In the heart, high uptake and rapid clearance of¹¹C and¹⁴C was found. The brain uptake of¹¹C was twice as high as that of¹⁴C, and both¹¹C and¹⁴C decreased slowly compared to the heart. The level of³H increased with time in both the heart and brain. In fasting conditions, the uptake of¹¹C by the heart was enhanced and the level of³H decreased with time. The brain uptake of¹¹C and³H was also enhanced. The fasting conditions did not affect the distribution of¹⁴C. The radiation absorbed dose of¹¹C-HA was also estimated.     

Evaluation of [1-11C]-alpha-aminoisobutyric acid for tumor detection and amino acid transport measurement: spontaneous canine tumor studies

Alpha-aminoisobutyric acid (AIB), or alpha-methyl alanine, is a nonmetabolized amino acid transported into cells, particularly malignant cells, predominantly by the 'A' amino acid transport system. Since it is not metabolized, [1-11C]-AIB can be used to quantify A-type amino acid transport into cells using a relatively simple compartmental model and quantitative imaging procedures (e.g. positron tomography). The tissue distribution of [1-11C]-AIB was determined in six dogs bearing spontaneous tumors, including lymphosarcoma, osteogenic sarcoma, mammary carcinoma, and adenocarcinoma. Quantitative imaging with tissue radioassay confirmation at necropsy showed poor to excellent tumor localization. However, in all cases the concentrations achieved appear adequate for amino acid transport measurement at known tumor locations. The observed low normal brain (due to blood-brain barrier exclusion) and high (relative to brain) tumor concentrations of [1-11C]-AIB suggest that this agent may prove effective for the early detection of human brain tumors.     

A fast chemoenzymatic synthesis of [11C]-N5,N10-methylenetetrahydrofolate as a potential PET tracer for proliferating cells

IntroductionThymidylate synthase and folate receptors are well-developed targets of cancer therapy. Discovery of a simple and fast method for the conversion of ¹¹CH₃Ito[¹¹C]-formaldehyde (¹¹CH₂O) encouraged us to label the co-factor of this enzyme. Preliminary studies conducted on cell lines have demonstrated a preferential uptake of [11-¹⁴C]-(R)-N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (¹⁴CH₂H₄folate) by cancerous cell vs. normal cells from the same organ (Saeed M., Sheff D. and Kohen A. Novel positron emission tomography tracer distinguishes normal from cancerous cells. J Biol Chem 2011;286:33872–33878), pointing out ¹¹CH₂H₄folate as a positron emission tomography (PET) tracer for cancer imaging. Herein we report the synthesis of ¹¹CH₂H₄folate, which may serve as a potential PET tracer.MethodsIn a remotely controlled module, methyl iodide (¹¹CH₃I) was bubbled into a reaction vial containing trimethylamine N-oxide in N,N-Dimethylformamide (DMF) and heated to 70°C for 2 min. Formaldehyde (¹¹CH₂O) formed after the completion of reaction was then mixed with a solution of freshly prepared tetrahydrofolate (H₄folate) by using a fast chemoenzymatic approach to accomplish synthesis of ¹¹CH₂H₄folate. Purification of the product was carried out by loading the crude reaction mixture on a SAX cartridge, washing with water to remove unbound impurities and finally eluting with a saline solution.ResultsThe synthesis and purification of ¹¹CH₂H₄folate were completed within 5 min. High-performance liquid chromatography analysis of the product after SAX purification indicates that more than 90% of the radioactivity that was retained on the SAX cartridge was in ¹¹CH₂H₄folate, with minor (<10%) radioactivity due to unreacted ¹¹CH₂O.ConclusionWe present a fast (～5 min) synthesis and purification of ¹¹CH₂H₄folate as a potential PET tracer. The final product is received in physiologically compatible buffer (100 mM sodium phosphate, pH 7.0 containing 500 mM NaCl) and ready for use in vivo.     

Human whole-body biodistribution and dosimetry of a new PET tracer, [11C]ketoprofen methyl ester,for imagings of neuroinflammation

IntroductionNeuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease and other brain disorders, and nonsteroidal anti-inflammatory drugs (NSAIDs) are considered therapeutic candidates. As a biomarker of neuroinflammatory processes, ¹¹C-labeled ketoprofen methyl ester ([¹¹C]KTP-Me) was designed to allow cerebral penetration of ketoprofen (KTP), an active form of a selective cyclooxygenase-1 inhibitor that acts as an NSAID. Rat neuroinflammation models indicate that [¹¹C]KTP-Me enters the brain and is retained in inflammatory lesions, accumulating in activated microglia. [¹¹C]KTP-Me is washed out from normal tissues, leading to the present first-in-human exploratory study.Methods[¹¹C]KTP-Me was synthesized by rapid C-[¹¹C]methylation of [¹¹C]CH₃I and the corresponding arylacetate precursor, purified with high-performance liquid chromatography, and prepared as an injectable solution including PEG400, providing radiochemical purity of > 99% and specific activity of > 25 GBq/μmol at injection. Six young healthy male humans were injected with [¹¹C]KTP-Me and scanned with PET camera to determine the early-phase brain time course followed by three whole-body scans starting 8, 20, and 40 min post-injection, together with sequential blood sampling and labeled metabolite analysis.ResultsNo adverse effects were observed during PET scanning after [¹¹C]KTP-Me injection. [¹¹C]KTP-Me was rapidly metabolized to ¹¹C-labeled ketoprofen ([¹¹C]KTP) within 2–3 min and was gradually cleared from blood. The radioactivity entered the brain with an average peak cortical SUV of 1.5 at 2 min. The cortical activity was gradually washed out. Whole-body images indicated that the urinary bladder was the major excretory pathway. The organ with the highest radiation dose was the urinary bladder (average dose of 41μGy/MBq, respectively). The mean effective dose was 4.7 μSv/MBq, which was comparable to other ¹¹C-labeled radiopharmaceuticals.Conclusion[¹¹C]KTP-Me demonstrated a favorable dosimetry, biodistribution, and safety profile. [¹¹C]KTP-Me entered the human brain, and the radioactivity was washed out from cerebral tissue. These data warrant further exploratory studies on patients with neuroinflammation.     

Synthesis and evaluation of [11C]FR194921 as a nonxanthine-type PET tracer for adenosine A1 receptors in the brain

This report describes the synthesis of [¹¹C]2-(1-methyl-4-piperidinyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone ([¹¹C]FR194921), a highly selective, nonxanthine-type adenosine A₁ receptor antagonist, used in brain imaging in rats and conscious monkeys as a potential novel PET tracer. [¹¹C]FR194921 was successfully synthesized in 19 min after [¹¹C]CH₃I formation. The radiochemical yield was 38±3%; and radioactivity was 4.1±0.4 GBq, calculated from end of synthesis; radiochemical purity was higher than 99%; and the specific radioactivity was 25.0±8.1 GBq μmol⁻¹ (n=5). In a rat experiment, the distribution of [¹¹C]FR194921 was higher in the hippocampus, striatum and cerebellum regions. This accumulation was significantly decreased by approximately 50% by pretreatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A₁ receptor antagonist, which indicated specific binding of the radioligand to adenosine A₁ receptors. In conscious monkey PET experiments, [¹¹C]FR194921 accumulated in several regions of the brain, especially in the occipital cortex, thalamus and striatum. These results suggest that [¹¹C]FR194921 can be used as an agent for imaging adenosine A₁ receptors in vivo by positron emission tomography (PET).     

Comparison of L-[1-11C]methionine and L-methyl-[11C]methionine for measuring in vivo protein synthesis rates with PET

To evaluate the feasibility of using either L-[1-11C]-methionine or L-[methyl-11C]methionine for measuring protein synthesis rates by positron emission tomography (PET) in normal and neoplastic tissues, distribution and metabolic studies with 14C- and 11C-labeled methionines were carried out in rats bearing Walker 256 carcinosarcoma. The tissue distributions of the two 14C-labeled methionines were similar except for liver tissue. Similar distribution patterns were observed in vivo by PET using 11C-labeled methionines. The highest 14C incorporation rate into the protein-bound fraction was found in the liver followed by tumor, brain, and pancreas. The incorporation rates in liver and pancreas were different for the two methionines. By chloroform-methanol fractionation of these four tissues, in liver significantly different amounts of 14C were observed in macromolecules. Also in brain tissue slight differences were found. By HPLC analyses of the protein-free fractions of plasma, tumor, and brain tissue at 60 min after injection, for both methionines several 14C-labeled metabolites in different amounts, were detected. About half of the 14C-labeled material in the protein-free fraction was found to be methionine. In these three tissues the amount of nonprotein metabolites and [14C]bicarbonate amount ranged from 10% to 17% and 12% to 15% for L-[1-14C]methionine and L-[methyl-14C]methionine, respectively. From these results it can be concluded that the minor metabolic pathways have to be investigated in order to quantitatively model the protein synthesis by PET.     

Radiosynthesis and mouse brain distribution studies of [11C] CP-126,998: a PET ligand for in vivo study of acetylcholinesterase

The selective, reversible acetylcholinesterase inhibitor 5,7-Dihydro-7-methyl-3- [2-[1-(phenylmethyl]-4-piperidinyl]ethyl]-6H-pyrrolo[3,2-f]-1,2-benzisoxazol3-6-one (CP-126,998) was labeled with C-11 iodomethane via base-promoted alkylation of the lactam nitrogen. [11C] CP-126,998 was synthesized in good radiochemical yield (13-29% non-decay corrected) and high specific radioactivity (177-418 GBq/micromol). In vivo mouse biodistribution studies reveal [11C] CP-126,998 to localize preferentially in striatal tissue, a region known to be rich in acetylcholinesterase. Competitive blocking studies using a variety of acetylcholinesterase inhibitors (diisopropylfluorophosphate, tacrine, CP-118,954) verified the specificity of the PET radiotracer for brain acetylcholinesterase.     

Synthesis and quality assurance of [11C]alpha-aminoisobutyric acid (AIB), a potential radiotracer for imaging and amino acid transport studies in normal and malignant tissues

Carbon-11 labeled alpha-aminoisobutyric acid (AIB), a synthetic amino acid, was prepared by the modified Bucherer-Strecker amino acid synthesis from acetone, ammonium carbonate and [11C]KCN in the presence of carrier KCN. This method results in the labeling of AIB in the carboxyl group. The label is stable in this position because AIB is not a metabolized after cellular uptake. AIB is rapidly accumulated in viable cells including malignant cells. Since it is a non-metabolized amino acid, AIB offers the possibility of studying amino acid transport in vivo without interference by radiolabeled metabolic products. Radiochemical yields of [11C]AIB of 35-60% have been obtained in 70-80 min with radiopurities greater than 99%. Carrier added syntheses gave 15-25 mCi of [11C]AIB with specific activities of 0.3 Ci/mmol. Our quality control program which insures that [11C]AIB is suitable for imaging studies in patients with cancer includes HPLC analyses of product identity and purity, apyrogenecity and isotonicity assays, and a sensitive test for cyanide.     

Comparison of three PET dopamine D2-like receptor ligands, [11C]raclopride, [11C]nemonapride and [11C]N-methylspiperone, in rats

We studied the tracer kinetics of three dopamine D2-like receptor ligands, [11C]raclopride ([11C]RAC), [11C]nemonapride ([11C]NEM) and [11C]N-methylspiperone ([11C]MSP), in anesthetized rats by tissue dissection, ex vivo ARG and PET in order to clarify their characteristics for PET imaging. The in vivo affinity of the three ligands for the striatum ([11C]MSP > [11C]NEM > [11C]RAC) obeyed the in vitro affinity for dopamine D2 receptors. The affinity of [11C]RAC and [11C]MSP for the cerebellum was very low, but the affinity of [11C]NEM for the cerebellum was compatible to that for the cortex and was not to be ignored. Also the affinity of [11C]MSP for the cortex was relatively high. [11C]RAC showed the highest selectivity. The striatal PET image with [11C]RAC was clearer than that with [11C]NEM or [11C]MSP, but the activity decreased much faster than that measured by tissue dissection because of the partial volume effect. The striatal activity with [11C]NEM remained high and that with [11C]MSP gradually increased. [11C]RAC and [11C]MSP, but not [11C]NEM, showed a high accumulation in the periorbital region.