A Kinetic Analysis of 6-[18F]Fluoro-l-Dihydroxyphenylalanine Metabolism in the Rat

Abstract: A previous study of the metabolism of 6-[¹⁸F]-fluoro-l -3,4-dihydroxyphenylalanine (FDOPA) in rats pretreated with carbidopa contained information amenable to kinetic analysis. Using these data, tracer transfer coefficients and metabolic rate constants were estimated. After intravenous injection, FDOPA in circulation was O-methylated (k^D₀ = 0.055 min^?1), and the metabolite (O-methyl-FDOPA) escaped from plasma with a rate constant (k^M_?1) of 0.01 min^?1. The initial clearance of FDOPA to striatum (K^D₁) was 0.07 ml g^?1 min^?1, and the equilibrium distribution volume (V^D_e) was 0.67 ml g^?1. The initial clearance of O-methyl-FDOPA to striatum (K^M₁) was 0.08 ml g^?1 min^?1, and the equilibrium distribution volume (V^M_e) was 0.75 ml g^?1. The rate constant of FDOPA decarboxylation (k^D₃) was 0.17 min^?1 in striatum. The elimination of 6-[¹⁸F]fluorodopamine (FDA) from striatum suggested an apparent rate constant for monoamine oxidase activity (k′₇) of 0.055 min^?1. 6-[¹⁸F]Fluorohomovanillic acid (FHVA) was formed from 6-[¹⁸F]fluoro-l -3,4-dihydroxyphenylacetic acid with a rate constant (k₁₁) of 0.083 min^?1, and FHVA was eliminated from striatum (k₉) with a rate constant of 0.12 min^?1. The steady-state concentration ratios of FDA and its metabolites were shown to be functions of these rate constants.     

Synthesis and evaluation of 6-[1-(2-[(18)F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline for positron emission tomography imaging of the metabotropic glutamate receptor type 1 in brain

The purpose of this study was to synthesize 6-[1-(2-[¹⁸F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline ([¹⁸F]FPTQ, [¹⁸F]7a) and to evaluate its potential as a positron emission tomography ligand for imaging metabotropic glutamate receptor type 1 (mGluR1) in the rat brain. Compound [¹⁸F]7a was synthesized by [¹⁸F]fluorination of 6-[1-(2-bromo-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline (7b) with potassium [¹⁸F]fluoride. At the end of synthesis, 1280-1830 MBq (n = 8) of [¹⁸F]7a was obtained with >98% radiochemical purity and 118-237 GBq/??mol specific activity using 3300-4000 MBq of [¹⁸F]F⁻. In vitro autoradiography showed that [¹⁸F]7a had high specific binding with mGluR1 in the rat brain. Biodistribution study using a dissection method and small-animal PET showed that [¹⁸F]7a had high uptake in the rat brain. The uptake of radioactivity in the cerebellum was reduced by unlabeled 7a and mGluR1-selective ligand JNJ-16259685 (2), indicating that [¹⁸F]7a had in vivo specific binding with mGluR1. Because of a low amount of radiolabeled metabolite present in the brain, [¹⁸F]7a may have a limiting potential for the in vivo imaging of mGluR1 by PET.     

Imaging the Dopamine Uptake Site with Ex Vivo [18F]GBR 13119 Binding Autoradiography in Rat Brain

We studied the binding of [18F]GBR 13119 (1-[[(4-[18F]fluorophenyl) (phenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine) to rat brain with autoradiography after intravenous injection. The rank order of binding was dorsal striatum greater than nucleus accumbens = olfactory tubercle greater than substantia nigra = ventral tegmental area greater than other areas. Binding was blocked by prior injection of dopamine uptake blockers but not by injection of dopamine receptor antagonists or drugs that bind to the dialkylpiperazine site. Unilateral 6-hydroxy-dopamine lesions of dopamine neurons caused a marked decrease in striatal and nigral binding on the side of the lesion. We conclude that intravenous injection of [18F]GBR 13119 provides a useful marker of presynaptic dopamine uptake sites.     

Role of acetyl-L-carnitine in the brain: revealed by Bioradiography

To elucidate the role of acetyl-l-carnitine in the brain, we used a novel method, ‘Bioradiography,’ in which the dynamic process could be followed in living slices by use of positron-emitter labeled compounds and imaging plates. We studied the incorporation of 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) into rat brain slices incubated in oxygenated Krebs-Ringer solution. Under the glucose-free condition, [¹⁸F]FDG uptake rate decreased with time and plateaued within 350 min in the cerebral cortex and cerebellum, and the addition of 1 or 5 mM acetyl-l-carnitine did not alter the [¹⁸F]FDG uptake rate. When a glutaminase inhibitor, 0.5 mM 6-diazo-5-oxo-l-norleucine (DON), was added under the normal glucose condition, [¹⁸F]FDG uptake rate decreased. Acetyl-l-carnitine (1 mM), which decreased [¹⁸F]FDG uptake rate, reversed this DON-induced decrease in [¹⁸F]FDG uptake rate in the cerebral cortex. These results suggest that acetyl-l-carnitine can be used for the production of releasable glutamate rather than as an energy source in the brain.     

Cerebral Metabolism of 6–[18F]Fluoro-l-3,4-Dihydroxyphenylalanine in the Primate

The tracers 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (6-[18F]fluoro-L-DOPA) and L-[14C]DOPA were injected simultaneously into rhesus monkeys, and the time course of their metabolites was measured in the striatum and in the occipital and frontal cortices. In the striatum, 6-[18F]fluoro-L-DOPA was metabolized to 6-[18F]fluorodopamine, 3,4-dihydroxy-6-[18F]fluorophenylacetic acid, and 6-[18F]fluorohomovanillic acid. The metabolite pattern was qualitatively similar to that of L-[14C]DOPA. 6-[18F]Fluorodopamine was synthesized faster than [14C]dopamine. In the frontal cortex, the major metabolite was also 6-[18F]fluorodopamine or [14C]dopamine. In the occipital cortex, the major metabolite was 3-O-methyl-6-[18F]fluoro-L-DOPA. On the basis of these data, the images obtained with 6-[18F]fluoro-L-DOPA and positron emission tomography in humans can now be interpreted in neurochemical terms.     

An In Vivo Microdialysis Study of Striatal 6-[18F]Fluoro-L-m-Tyrosine Metabolism

In vivo brain microdialysis was used to monitor 6-[¹⁸F]fluoro-L-m-tyrosine (FMT) uptake and metabolism in the striatum of conscious freely moving rats for 3 hours after FMT injection (25 mg/kg, iv). Microdialysate collected 20 to 120 min post-dose, contained FMT at a concentration (0.2 to 0.3 nM) approximately ten-fold below that of its metabolite [¹⁸F]fluoro-3-hydroxyphenylacetic acid (FPAC; 3.2 to 3.3 nM). D-amphetamine (2.5 mg/kg, i.p.) injected 120 min after significantly increased microdialysate FPAC (3.27 ± 0.31 nM to 4.51 ± 0.45 nM) in control but not reserpinized rats. Taken together these data demonstrate FMT is heavily metabolized following its entry into the striatum yielding FPAC which appears to be stored, at least in part, in reserpine sensitive cytoplasmic vesicles. Presynaptic retention of FPAC may contribute to the preferential accumulation of FMT positron emission tomography (PET) signaling in dopaminergic brain areas.     

Comparison of Three 18F-Labeled Butyrophenone Neuroleptic Drugs in the Baboon Using Positron Emission Tomography

The butyrophenone neuroleptics spiroperidol, benperidol, and haloperidol were radiolabeled with fluorine-18 and studied in baboon brain using positron emission transaxial tomography (PETT). Pretreatment of the baboon with a high pharmacological dose of (+)-butaclamol reduced the specifically bound component of radioactivity distribution in the striatum to approximately the radioactivity distribution found in the cerebellum. Comparative studies of brain distribution kinetics over a 4-h period indicated that either [18F]spiroperidol or [18F]benperidol may be suitable for specific labeling of neuroleptic receptors. In an 8-h study with [18F]spiroperidol, striatal radioactivity did not decline, suggesting that spiroperidol either has a very slow dissociation rate or that it binds irreversibly to these receptors in vivo. [18F]Haloperidol may not be suitable for in vivo PETT studies, because of a relatively high component of nonspecific distribution and a faster dissociation from the receptor. Analysis of 18F in plasma after injection of [18F]spiroperidol indicated rapid metabolism to polar and acidic metabolites, with only 40% of the total radioactivity being present as unchanged drug after 30 min. Analysis of the metabolic stability of the radioactively labeled compound in rat striatum indicated that greater than 95% of [18F]spiroperidol remains unchanged after 4 h.     

Fluorine-18 labeled galactosyl-neoglycoalbumin for imaging the hepatic asialoglycoprotein receptor

Asialoglycoprotein receptors (ASGP-R) are well known to exist on the mammalian liver, situate on the surface of hepatocyte membrane. Quantitative imaging of asialoglycoprotein receptors could estimate the function of the liver. ^99mTc labeled galactosyl-neoglycoalbumin (NGA) and diethylenetriaminepentaacetic acid galactosyl human serum albumin (GSA) have been developed for SPECT imaging and clinical used in Japan. In this study, we labeled the NGA with ¹⁸F to get a novel PET tracer [¹⁸F]FNGA and evaluated its hepatic-targeting efficacy and pharmacokinetics. Methods: NGA was labeled with ¹⁸F by conjugation with N-succinimidyl-4-¹⁸F-fluorobenzoate ([¹⁸F]SFB) under a slightly basic condition. The in vivo metabolic stability of [¹⁸F]FNGA was determined. Ex vivo biodistribution of [¹⁸F]FNGA and blocking experiment was investigated in normal mice. MicroPET images were acquired in rat with and without block at 5 min and 15 min after injection of the radiotracer (3.7 MBq/rat), respectively. Results: Starting with ¹⁸F⁻ Kryptofix 2.2.2./K₂CO₃ solution, the total reaction time for [¹⁸F]FNGA is about 150 min. Typical decay-corrected radiochemical yield is about 8–10%. After rapid purified with HiTrap desalting column, the radiochemical purity of [¹⁸F]FNGA was more than 99% determined by radio-HPLC. [¹⁸F]FNGA was metabolized to produce [¹⁸F]FB-Lys in urine at 30 min. Ex vivo biodistribution in mice showed that the liver accumulated 79.18 ± 7.17% and 13.85 ± 3.10% of the injected dose per gram at 5 and 30 min after injection, respectively. In addition, the hepatic uptake of [¹⁸F]FNGA was blocked by pre-injecting free NGA as blocking agent (18.55 ± 2.63%ID/g at 5 min pi), indicating the specific binding to ASGP receptor. MicroPET study obtained quality images of rat at 5 and 15 min post-injection. Conclusion: The novel ASGP receptor tracer [¹⁸F]FNGA was synthesized with high radiochemical yield. The promising biological properties of [¹⁸F]FNGA afford potential applications for assessment of hepatocyte function in the future. It may provide quantitative information and better resolution which particularly help to the liver surgery.     

<Superscript>18</Superscript>F-labeled tyrosine derivatives: Synthesis and experimental studies on accumulation in tumors and abscesses

Tyrosine derivatives labeled with a short-lived fluorine-18 isotope (T _1/2 110 min), namely 2-[¹⁸F]fluoro-L-tyrosine (FTYR) and O-(2′-[¹⁸F]fluoroethyl)-L-tyrosine (FET), promising radiopharmaceuticals (RPs) for positron emission tomography (PET), were obtained by asymmetric syntheses. Accumulation of FTYR and FET in the rat tumor “Glioma 35 rats tumor” and in abscesses induced in Wistar rats muscles was studied and compared with that of a well-known glycolysis radiotracer 2-[¹⁸F]fluoro-2-deoxy-D-glucose (FDG). It was shown that the relative accumulation indices of amino acid RPs were considerably lower than those of FDG. At the same time, tumor/muscle ratios were high enough (2.9 for FET and 3.9 for FTYR 120 min after injection) for reliable tumor visualization. The data obtained indicated a possibility in principle to use FTYR and FET for differentiated PET diagnostics of brain tumors and inflammation lesions. Of the tyrosine derivatives studied, FET seems to be the most promising agent due to a simple and easily automated method of preparation based on direct nucleophilic substitution of the leaving tosyloxy group of an enantiomerically pure Ni-(S)-BPS-(S)-Tyr(CH₂CH₂OTs) precursor by an activated [¹⁸F]fluoride.     

Kinetic modeling of [18F]VAT,a novel radioligand for positron emission tomography imaging vesicular acetylcholine transporter in non‐human primate brain

Molecular imaging of vesicular acetylcholine transporter (VACh T) in the brain provides an important cholinergic biomarker for the pathophysiology and treatment of dementias including Alzheimer's disease. In this study, kinetics modeling methods were applied and compared for quantifying regional brain uptake of the VACh T‐specific positron emission tomography radiotracer, ((?)‐(1‐(‐8‐(2‐fluoroethoxy)‐3‐hydroxy‐1,2,3,4‐tetrahydronaphthalen‐2‐yl)piperidin‐4‐yl)(4‐fluorophenyl)‐methanone) ([¹⁸F]VAT ) in macaques. Total volume distribution (V _T ) estimates were compared for one‐tissue compartment model (1TCM ), two‐tissue compartment model (2TCM ), Logan graphic analysis (LoganAIF ) and multiple linear analysis (MA 1) with arterial blood input function using data from three macaques. Using the cerebellum‐hemispheres as the reference region with data from seven macaques, three additional models were compared: reference tissue model (RTM ), simplified RTM (SRTM ), and Logan graphic analysis (LoganREF ). Model selection criterion indicated that a) 2TCM and SRTM were the most appropriate kinetics models for [¹⁸F]VAT ; and b) SRTM was strongly correlated with 2TCM (Pearson's coefficients r  > 0.93, p  < 0.05). Test–retest studies demonstrated that [¹⁸F]VAT has good reproducibility and reliability (TRV < 10%, ICC > 0.72). These studies demonstrate [¹⁸F]VAT is a promising VACh T positron emission tomography tracer for quantitative assessment of VACh T levels in the brain of living subjects.

     

Direct labelling of peptides with 2-[F]fluoro-2-deoxy-d-glucose ([F]FDG)

The study describes the use of [¹⁸F]FDG as ¹⁸F building block for the direct labelling of various aminooxy-functionalised peptides via chemoselective oxime formation.     

Synthesis and biological evaluation of novel F-18 labeled pyrazolo[1,5-a]pyrimidine derivatives: potential PET imaging agents for tumor detection

Two novel pyrazolo[1,5-a]pyrimidine derivatives, 7-(2-[¹⁸F]fluoroethylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile ([¹⁸F]FEMPPC, [¹⁸F]1) and N-(2-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)ethyl)-2-[¹⁸F]fluoro-4-nitrobenzamide ([¹⁸F]FCMPPN, [¹⁸F]2), have been designed and successively labeled with ¹⁸F by the nucleophilic substitution employing tosylate and nitryl as leaving groups, respectively. The radiochemical synthesis of both compounds was completed within 60 min with final high-performance liquid chromatography purification included. The corresponding radiochemical yields (without decay correction) were approximately 35% and 30%, respectively. Meanwhile, we compared the uptake characteristics of [¹⁸F]1 and [¹⁸F]2 with those of [¹⁸F]FDG and L-[¹⁸F]FET in S180 tumor cells. Furthermore, the tumor uptake of [¹⁸F]1 and [¹⁸F]2 was assessed in mice bearing S180 tumor and compared with [¹⁸F]FDG and L-[¹⁸F]FET in the same animal model. In vitro cell uptake studies showed [¹⁸F]1 had higher uptake than [¹⁸F]FDG, [¹⁸F]2 and L-[¹⁸F]FET over the 2 h period. In ex vivo biodistribution showed tumor/brain uptake ratios of [¹⁸F]2 were 12.35, 10.44, 8.69 and 5.13 at 15 min, 30 min, 60 min and 120 min post-injection, much higher than those of L-[¹⁸F]FET (2.43, 2.54, 2.93 and 2.95) and [¹⁸F]FDG (0.59, 0.61, 1.02 and 1.33) at the same time point. What’s more, the uptake of [¹⁸F]1 in tumor was 1.88, 4.37, 5.51, 2.95 and 2.88 at 5 min, 15 min, 30 min, 60 min and 120 min post-injection, respectively. There was a remarkable increasing trend before 30 min. The same trend was present for L-[¹⁸F]FET before 30 min and [¹⁸F]FDG before 60 min. Additionally, the tumor/brain uptake ratios of [¹⁸F]1 were superior to those of [¹⁸F]FDG at all the selected time points, the tumor/muscle and tumor/blood uptake ratios of [¹⁸F]1 at 30 min were higher than those of L-[¹⁸F]FET at the same time point. MicroPET image of [¹⁸F]1 administered into S180 tumor-bearing mouse acquired at 30 min post-injection illustrated that the uptake in S180 tumor was obvious. These results suggest that compound [¹⁸F]1 could be a new probe for PET tumor imaging.     

Measurement of [<Superscript>18</Superscript>F]-fluorodeoxyglucose incorporation into human osteoblast–An experimental method

An evaluation of human osteoblast metabolism usually involves measurements of the by-products of bone matrix elaboration. The assessment of glycolytic activity of osteoblasts is not a standard tool in most of the reports, but might be of value by providing a direct indicator of cellular metabolism. Measurement of the incorporation of [¹⁸F]-fluorodeoxyglucose, which is not further degradable following its conversion into glycose-6-phosphate during glycolysis and is trapped in this form within the cells, can be used as an effective research tool for estimation of osteoblast metabolism. In order to estimate the [¹⁸F]-fluorodeoxyglucose incorporation we used cultured human osteoblast-like cells. Following incubation of the culture samples in a glucose free medium with 5 μ Ci [¹⁸F]-fluorodeoxyglucose we measured the radioactivity of the cell fraction, as a percent from the initial dose, and compared to the incorporation values in cells treated by protoporphyrine IX (10⁻⁵ M), an endogenous pro-apoptotic agent. To compare the response of [¹⁸F]-fluorodeoxyglucose incorporation studies, following treatment of cells with the protoporphyrine IX, to other experimental cell metabolism evaluation methods, we performed a parallel comparison of alkaline phospatase activity, which is a standard measurement tool of osteoblast metabolism, in the control and treatment groups. A narrow range of 0.22–1.36% of [¹⁸F]-fluorodeoxyglucose incorporation per million cells was found. Additionally in the protoporphyrine IX treated cells a significant 62% decrease of [¹⁸F]-fluorodeoxyglucose incorporation was observed (p < .05). A parallel significant decrease in alkaline phosphatase activity (p < .001) was found in the cells treated by the protoporphyrine IX. Therefore we suggest that the presented method of [¹⁸F]-fluorodeoxyglucose incorporation measurement can be utilized as an effective research tool for estimation of the cellular glycolitic activity in human osteoblast-like cells in vitro.     

Synthesis and preclinical evaluation of [18F]FSL25.1188, a reversible PET radioligand for monoamine oxidase-B

Carbon-11 labeled SL25.1188 is a promising reversible monoamine oxidase-B (MAO-B) radioligand that was recently translated for human positron emission tomography (PET) imaging. Herein, we report the development of a novel fluorinated derivative, namely, [¹⁸F](S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one ([¹⁸F]FSL25.1188; [¹⁸F]6), as a candidate ¹⁸F-labeled MAO-B radioligand, and, its subsequent preclinical evaluation in non-human primates (NHP). [¹⁸F]6 was produced and isolated (>6 GBq) with high radiochemical purity (>99%), and molar activity (>100 GBq/µmol at time of injection). Autoradiography studies conducted in post-mortem human brain sections revealed [¹⁸F]6 binding in MAO-B rich regions. PET imaging study of [¹⁸F]6 in NHP showed high brain uptake (SUV > 2.5) as well as a regional brain radioactivity distribution in accordance with MAO-B expression. [¹⁸F]6 displayed favorable in vivo kinetics, with an early peak in the time-activity curve followed by progressive wash-out from the NHP brain. Specificity of [¹⁸F]6 was investigated in a pre-treatment study with l-deprenyl (1.0 mg/kg) wherein reduced radioligand uptake was observed in all MAO-B rich regions. Results from the current preclinical investigation suggests [¹⁸F]6 is a promising MAO-B PET radioligand. Further evaluation of [¹⁸F]6 and structurally related ¹⁸F-analogs are underway to identify an optimized candidate for clinical research studies.     

In Vivo Binding to Peripheral Benzodiazepine Binding Sites in Lesioned Rat Brain: Comparison Between [3H]PK11195 and [18F]PK14105 as Markers for Neuronal Damage

Peripheral-type benzodiazepine binding sites are not normally present in most cerebral tissues, but following neuronal damage, the cells involved in the ensuing gliosis show a marked expression of these sites. In a unilateral excitotoxic striatal lesion in the rat, we sought to determine whether the isoquinoline derivatives PK11195 and PK14105 bind to these sites in vivo and whether demonstration of these sites offers the potential of indirectly localising areas of neuronal damage. Binding was studied at several intervals after coinjection of [3H]PK11195 and [18F]PK14105 to determine the time courses of specific binding. Both compounds were rapidly extracted into all cerebral tissues, but in the absence of binding sites in nonlesioned tissues, this was followed by a rapid clearance of radioactivity. In lesioned areas, both [3H]PK11195 and [18F]PK14105 accumulated over the first 5 min followed by a much slower clearance of radioactivity, resulting in a "specific signal." [3H]PK11195 binding peaked at 20-30 min postinjection, with radioactivity in the lesioned striatum being three times greater than in its contralateral homologue. The specific signal was present for at least 60 min. The maximal [18 F]PK14105-specific signal was of similar magnitude but peaked earlier and was retained for only 45 min. Specific signals with both ligands were also detected in regions remote from the primary lesion site, e.g., in the hippocampus and substantia nigra. Predosing animals with a large dose of PK11195 (3 mg/kg), sufficient to saturate peripheral-type benzodiazepine binding sites, abolished in vivo binding of both [3H]PK11195 and [18F]PK14105 to both primary- and remote-lesioned tissues. The specific signal with both ligands could be of sufficient magnitude and duration to make tomographic studies in humans feasible.     

Labeling a TCO-functionalized single domain antibody fragment with 18F via inverse electron demand Diels Alder cycloaddition using a fluoronicotinyl moiety-bearing tetrazine derivative

Single domain antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with ¹⁸F (t_½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for ¹⁸F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel’s Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [¹⁸F]AlF-NOTA moiety with [¹⁸F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[¹⁸F]fluoronicotinyl-PEG₄-methyltetrazine to provide [¹⁸F]FN-PEG₄-Tz-TCO-GK-PEG₄-5F7 ([¹⁸F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[¹²⁵I]iodobenzoate (iso-[¹²⁵I]SGMIB) also were synthesized. Radiochemical purity, affinity (K_D) and immunoreactive fraction of [¹⁸F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [¹⁸F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[¹²⁵I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [¹⁸F]FN-GK-5F7 were 3–4 times higher than those for co-injected iso-[¹²⁵I]SGMIB-5F7 as well as those observed for the ¹⁸F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [¹⁸F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [¹⁸F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.     

Synthesis of [¹¹C]PBR06 and [¹⁸F]PBR06 as agents for positron emission tomographic (PET) imaging of the translocator protein (TSPO)

The translocator protein 18 kDa (TSPO) is an attractive target for molecular imaging of neuroinflammation and tumor progression. [¹⁸F]PBR06, a fluorine-18 labeled form of PBR06, is a promising PET TSPO radioligand originally developed at NIMH. [¹¹C]PBR06, a carbon-11 labeled form of PBR06, was designed and synthesized for the first time. The standard PBR06 was synthesized from 2,5-dimethoxybenzaldehyde in three steps with 71% overall chemical yield. The radiolabeling precursor desmethyl-PBR06 was synthesized from 2-hydroxy-5-methoxybenzaldehyde in five steps with 12% overall chemical yield. The target tracer [¹¹C]PBR06 was prepared by O-[¹¹C]methylation of desmethyl-PBR06 with [¹¹C]CH₃OTf in CH₃CN at 80 °C under basic condition and isolated by HPLC combined with SPE purification with 40–60% decay corrected radiochemical yield and 222–740 GBq/μmol specific activity at EOB. On the similar grounds, [¹⁸F]PBR06 was also designed and synthesized. The previously described Br-PBR06 precursor was synthesized from 2,5-dimethoxybenzaldehyde in two steps with 78% overall chemical yield. A new radiolabeling precursor tosyloxy-PBR06, previously undescribed tosylate congener of PBR06, was designed and synthesized from ethyl 2-hydroxyacetate, 4-methylbenzene-1-sulfonyl chloride, and N-(2,5-dimethoxybenzyl)-2-phenoxyaniline in four steps with 50% overall chemical yield. [¹⁸F]PBR06 was prepared by the nucleophilic substitution of either new tosyloxy-PBR06 precursor or known Br-PBR06 precursor in DMSO at 140 °C with K[¹⁸F]F/Kryptofix 2.2.2 for 15 min and HPLC combined with SPE purification in 20–60% decay corrected radiochemical yield, >99% radiochemical purity, 87–95% chemical purity, and 37–222 GBq/μmol specific activity at EOB. Radiosynthesis of [¹⁸F]PBR06 using new tosylated precursor gave similar radiochemical purity, and higher specific activity, radiochemical yield and chemical purity in comparison with radiosynthesis using bromine precursor.     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

Radiosynthesis and evaluation of a fluorine-18 labeled radioligand targeting vesicular acetylcholine transporter

Vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing the loss of cholinergic neurons in the brain that is associated with cognitive impairment of patients. 5-Hydrotetralin compound (±)-5-OH-VAT is potent (K_i?=?4.64?±?0.32?nM) and selective for VAChT (>1800-fold and 398-fold for σ₁ and σ₂ receptor, respectively) with favorable hydrophilicity (LogD?=?1.78), while (?)-5-OH-VAT originally serves as the radiolabeling precursor of (?)-[¹⁸F]VAT, a promising VAChT radiotracer with a logD value of 2.56. To evaluate (?)-5-OH-[¹⁸F]VAT as a radiotracer for VAChT, we performed in vitro binding assay to determine the potency of the minus enantiomer (?)-5-OH-VAT and plus enantiomer (+)-5-OH-VAT, indicating that (?)-5-OH-VAT is a more potent VAChT enantiomer. Radiosynthesis of (?)-5-OH-[¹⁸F]VAT was explored using three strategies. (?)-5-OH-[¹⁸F]VAT was achieved with a good yield (24?±?6%) and high molar activity (～37?GBq/µmol, at the end of synthesis) using a microwave assisted two-step one-pot procedure that started with di-MOM protected nitro-containing precursor (?)-6. MicroPET studies in the brain of nonhuman primate (NHP) suggest that (?)-5-OH-[¹⁸F]VAT readily penetrated the blood brain barrier and specifically accumulated in the VAChT-enriched striatum with improved washout kinetics from striatum compared to [¹⁸F]VAT. Nevertheless, the lower target to non-target ratio may limit its use for in vivo measurement of the VAChT level in the brain.     

Synthesis and biological evaluation of 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT) for PET imaging of breast cancer

Given the ever-present demand for improved PET radiotracer in oncology imaging, we have synthesized 2-(3,4-dimethoxyphenyl)-6-(2-[¹⁸F]fluoroethoxy)benzothiazole ([¹⁸F]FEDBT), a fluorine-18-containing fluoroethylated benzothiazole to explore its utility as a PET imaging tracer. [¹⁸F]FEDBT was prepared via kryptofix-mediated nucleophilic substitution of the tosyl group precursor. Fractionated ethanol-based solid-phase (SPE cartridge-based) purification afforded [¹⁸F]FEDBT in 60% radiochemical yield (EOB), with radiochemical purity in excess of 98% and the specific activity was 35 GBq/μmol. The radiotracer displayed clearly higher cellular uptake ratio in various breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231. However, both biodistribution and microPET studies have showed an higher abdominal accumulation of [¹⁸F]FEDMBT and the tumor/muscle ratio of 1.8 was observed in the MDA-MB-231 xenograft tumors mice model. Further the lipophilic improvement is needed for the reducement of hepatobilliary accumulation and to promote the tumor uptake for PET imaging of breast cancer.