Shuiyu Lu

D2 dopamine receptor internalization prolongs the decrease of radioligand binding after amphetamine: A PET study in a receptor internalization-deficient mouse model

Dopamine released by amphetamine decreases the in vivo binding of PET radioligands to the dopamine D(2) receptor. Although concentrations of extracellular dopamine largely return to baseline within 1 to 2 h after amphetamine treatment, radioligand binding remains decreased for several hours. The purpose of this study was to determine whether the prolonged decrease of radioligand binding after amphetamine administration is caused by receptor internalization. To distinguish dopamine displacement from receptor internalization, we used wild-type and arrestin3 (arr3) knockout mice, which are incapable of internalizing D(2) receptors. In addition, we used both the D(2) selective agonist [(11)C]MNPA (which is thought to bind to the high affinity state of the receptor) and the D(2) selective antagonist [(18)F]fallypride (which does not differentiate between high and low affinity state). After an initial baseline scan, animals were divided in three groups for a second scan: either 30 min or 4 h after amphetamine administration (3 mg/kg, i.p.) or as retest. At 30 min, [(11)C]MNPA showed greater displacement than [(18)F]fallypride, but each radioligand gave similar displacement in knockout and wild-type mice. At 4 h, the binding of both radioligands returned to baseline in arr3 knockout mice, but remained decreased in wild-type mice. Radioligand binding was unaltered on retest scanning. Our results suggest that the prolonged decrease of radioligand binding after amphetamine is mainly due to internalization of the D(2) receptor rather than dopamine displacement. In addition, this study demonstrates the utility of small animal PET to study receptor trafficking in vivo in genetically modified mice.

CuI-Catalyzed 11C Carboxylation of Boronic Acid Esters: A Rapid and Convenient Entry to 11C-Labeled Carboxylic Acids, Esters, and Amides†

Rapid and direct: the carboxylation of boronic acid esters with (11)CO(2) provides [(11)C]carboxylic acids as a convenient entry into [(11)C]esters and [(11)C]amides. This conversion of boronates is tolerant to diverse functional groups (e.g., halo, nitro, or carbonyl).

Single-Step High-Yield Radiosynthesis and Evaluation of a Sensitive 18F-Labeled Ligand for Imaging Brain Peripheral Benzodiazepine Receptors with PET

Elevated levels of peripheral benzodiazepine receptors (PBR) are associated with activated microglia in their response to inflammation. Hence, PBR imaging in vivo is valuable for investigating brain inflammatory conditions. Sensitive, easily prepared, and readily available radioligands for imaging with positron emission tomography (PET) are desirable for this purpose. We describe a new 18F-labeled PBR radioligand, namely [18F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([18F]9). [18F]9 was produced easily through a single and highly efficient step, the reaction of [18F]fluoride ion with the corresponding bromo precursor, 8. Ligand 9 exhibited high affinity for PBR in vitro. PET showed that [18F]9 was avidly taken into monkey brain and gave a high ratio of PBR-specific to nonspecific binding. [18F]9 was devoid of defluorination in rat and monkey and gave predominantly polar radiometabolite(s). In rat, a low level radiometabolite of intermediate lipophilicity was identified as [18F]2-fluoro-N-(2-phenoxyphenyl)acetamide ([18F]11). [18F]9 is a promising radioligand for future imaging of PBR in living human brain.

Synthesis of [18F]fallypride in a micro-reactor: rapid optimization and multiple-production in small doses for micro-PET studies

A commercial coiled-tube micro-reactor (NanoTek; Advion) was used as a convenient platform for the synthesis of [(18)F]fallypride in small doses (0.5-1.5 mCi) for micro-PET studies of brain dopamine subtype-2 receptors in rodents. Each radiosynthesis used low amounts (20-40 mug; 39-77 nmol) of tosylate precursor and [(18)F]fluoride ion (0.5-2.5 mCi). Optimization of the labeling reaction in the apparatus, with respect to the effects of precursor amount, reaction temperature, flow rate and [(18)F]fluoride ion to precursor ratio, was achieved rapidly and the decay-corrected radiochemical yield of [(18)F]fallypride (up to 88%) was reproducible. The low amounts of material used in each radiosynthesis allowed crude [(18)F]fallypride to be purified rapidly on an analytical-size reverse phase HPLC column, preceding formulation for intravenous injection. Scale-up of the reaction was easily achieved by continuously infusing reagent precursor solutions to obtain [(18)F]fallypride in much greater quantity.

The inhibitor Ko143 is not specific for ABCG2

Imaging ATP-binding cassette (ABC) transporter activity in vivo with positron emission tomography requires both a substrate and a transporter inhibitor. However, for ABCG2, there is no inhibitor proven to be specific to that transporter alone at the blood-brain barrier. Ko143 [[(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4- b]indole-3-propanoic acid 1,1-dimethylethyl ester], a nontoxic analog of fungal toxin fumitremorgin C, is a potent inhibitor of ABCG2, although its specificity in mouse and human systems is unclear. This study examined the selectivity of Ko143 using human embryonic kidney cell lines transfected with ABCG2, ABCB1, or ABCC1 in several in vitro assays. The stability of Ko143 in rat plasma was measured using high performance liquid chromatography. Our results show that, in addition to being a potent inhibitor of ABCG2, at higher concentrations (≥1 μM) Ko143 also has an effect on the transport activity of both ABCB1 and ABCC1. Furthermore, Ko143 was found to be unstable in rat plasma. These findings indicate that Ko143 lacks specificity for ABCG2 and this should be taken into consideration when using Ko143 for both in vitro and in vivo experiments.

Nucleophile assisting leaving groups: a strategy for aliphatic 18F-fluorination.

A series of arylsulfonate nucleophile assisting leaving groups (NALGs) were prepared in which the metal chelating unit is attached to the aryl ring via an ether linker. These NALGs exhibited significant rate enhancements in halogenation reactions using metal halides. Studies with a NALG containing a macrocyclic ether unit suggest that rate enhancements of these nucleophilic halogenation reactions are facilitated by stabilization of charge in the transition state rather than through strong precomplexation with metal cation. In several cases, a primary substrate containing one of the new leaving groups rivaled or surpassed the reactivity of triflates when exposed to nucleophile but was otherwise highly stable and isolable. These and previously disclosed chelating leaving groups were used in (18)F-fluorination reactions using no-carrier-added [18F]fluoride ion (t(1/2) = 109.7 min, beta+ = 97%) in CH3CN. Under microwave irradiation and without the assistance of a cryptand, such as K2.2.2, primary substrates with select NALGs led to a substantial improvement (2-3-fold) in radiofluorination yields over traditional leaving groups.

Radiosynthesis and evaluation of an 18F-labeled positron emission tomography (PET) radioligand for brain histamine subtype-3 receptors based on a nonimidazole 2-aminoethylbenzofuran chemotype

A known chemotype of H(3) receptor ligand was explored for development of a radioligand for imaging brain histamine subtype 3 (H(3)) receptors in vivo with positron emission tomography (PET), namely nonimidazole 2-aminoethylbenzofurans, represented by the compound (R)-(2-(2-(2-methylpyrrolidin-1-yl)ethyl)benzofuran-5-yl)(4-fluorophenyl)methanone (9). Compound 9 was labeled with fluorine-18 (t(1/2) = 109.7 min) in high specific activity by treating the prepared nitro analogue (12) with cyclotron-produced [(18)F]fluoride ion. [(18)F]9 was studied with PET in mouse and in monkey after intravenous injection. [(18)F]9 showed favorable properties as a candidate PET radioligand, including moderately high brain uptake with a high proportion of H(3) receptor-specific signal in the absence of radiodefluorination. The nitro compound 12 was found to have even higher H(3) receptor affinity, indicating the potential of this chemotype for the development of further promising PET radioligands.

11C-CUMI-101, a PET Radioligand, Behaves as a Serotonin 1A Receptor Antagonist and Also Binds to α1 Adrenoceptors in Brain

The PET radioligand 11C-CUMI-101 was previously suggested as a putative agonist radioligand for the serotonin 1A (5-hydroxytryptamine 1A [5-HT1A]) receptor in recombinant cells expressing human 5-HT1A receptor. However, a recent study showed that CUMI-101 behaved as a potent 5-HT1A receptor antagonist in rat brain. CUMI-101 also has moderate affinity (Ki = 6.75 nM) for α1 adrenoceptors measured in vitro. The current study examined the functional properties and selectivity of CUMI-101, both in vitro and in vivo. Methods: The functional assay was performed using 35S-GTPγS (GTP is guanosine triphosphate) in primate brains. The cross-reactivity of CUMI-101 with α1 adrenoceptors was performed using in vitro radioligand binding studies in rat, monkey, and human brains as well as in vivo PET imaging in mouse, rat, and monkey brains. Results: CUMI-101 did not stimulate 35S-GTPγS binding in primate brain, in contrast to 8-OH-DPAT, a potent 5-HT1A receptor agonist. Instead, CUMI-101 behaved as a potent 5-HT1A receptor antagonist by dose-dependently inhibiting 8-OH-DPAT–stimulated 35S-GTPγS binding. Both in vitro and in vivo studies showed that CUMI-101 had significant α1 adrenoceptor cross-reactivity. On average, across all 3 species examined, cross-reactivity was highest in the thalamus (>45%) and lowest in the neocortex and cerebellum (<10%). PET imaging further confirmed that only preblocking with WAY-100635 plus prazosin decreased 11C-CUMI-101 brain uptake to that of self-block. Conclusion: CUMI-101 behaves as a 5-HT1A receptor antagonist in primate brain, with significant, regional-dependent α1 adrenoceptor cross-reactivity, limiting its potential use as a PET radioligand in humans.

[11 C]Rhodamine-123: Synthesis and biodistribution in rodents

INTRODUCTION Rhodamine-123 is a known substrate for the efflux transporter, P-glycoprotein (P-gp). We wished to assess whether rhodamine-123 might serve as a useful substrate for developing probes for imaging efflux transporters in vivo with positron emission tomography (PET). For this purpose, we aimed to label rhodamine-123 with carbon-11 (t(1/2)=20.4min) and to study its biodistribution in rodents. METHODS [¹¹C]Rhodamine-123 was prepared by treating rhodamine-110 (desmethyl-rhodamine-123) with [¹¹C]methyl iodide. The biodistribution of this radiotracer was studied with PET in wild-type mice and rats, in efflux transporter knockout mice, in wild-type rats pretreated with DCPQ (an inhibitor of P-gp) or with cimetidine (an inhibitor of organic cation transporters; OCT), and in P-gp knockout mice pretreated with cimetidine. Unchanged radiotracer in forebrain, plasma and peripheral tissues was also measured ex vivo at 30min after radiotracer administration to wild-type and efflux transporter knockout rodents. RESULTS [(¹¹C]Rhodamine-123 was obtained in 4.4% decay-corrected radiochemical yield from cyclotron-produced [¹¹C]carbon dioxide. After intravenous administration of [¹¹C]rhodamine-123 to wild-type rodents, PET and ex vivo measurements showed radioactivity uptake was very low in brain, but relatively high in some other organs such as heart, and especially liver and kidney. Inhibition of P-gp increased uptake in brain, heart, kidney and liver, but only by up to twofold. Secretion of radioactivity from kidney was markedly reduced by OCT knockout or pretreatment with cimetidine. CONCLUSIONS [¹¹C]Rhodamine-123 was unpromising as a PET probe for P-gp function and appears to be a strong substrate of OCT in kidney. Cimetidine appears effective for blocking OCT in kidney in vivo.

An Investigation of (Diacetoxyiodo)arenes as Precursors for Preparing No-Carrier-Added [18F]Fluoroarenes from Cyclotron-Produced [18F]Fluoride Ion

Treatment of (diacetoxyiodo)arenes (1a-1u) with cyclotron-produced [(18)F]fluoride ion rapidly affords no-carrier-added [(18)F]fluoroarenes (2a-2u) in useful yields and constitutes a new method for converting substituted iodoarenes into substituted [(18)F]fluoroarenes in just two steps.