Enhancement of Haloperidol-Induced Catalepsy by GPR143, an L-Dopa Receptor, in Striatal Cholinergic Interneurons.

Dopamine neurons play crucial roles in pleasure, reward, memory, learning, and fine motor skills and their dysfunction is associated with various neuropsychiatric diseases. Dopamine receptors are the main target of treatment for neurologic and psychiatric disorders. Antipsychotics that antagonize the dopamine D2 receptor (DRD2) are used to alleviate the symptoms of these disorders but may also sometimes cause disabling side effects such as parkinsonism (catalepsy in rodents). Here we show that GPR143, a G-protein-coupled receptor for L-3,4-dihydroxyphenylalanine (L-DOPA), expressed in striatal cholinergic interneurons enhances the DRD2-mediated side effects of haloperidol, an antipsychotic agent. Haloperidol-induced catalepsy was attenuated in male Gpr143 gene-deficient (Gpr143-/y ) mice compared with wild-type (Wt) mice. Reducing the endogenous release of L-DOPA and preventing interactions between GPR143 and DRD2 suppressed the haloperidol-induced catalepsy in Wt mice but not Gpr143-/y mice. The phenotypic defect in Gpr143-/y mice was mimicked in cholinergic interneuron-specific Gpr143-/y (Chat-cre;Gpr143flox/y ) mice. Administration of haloperidol increased the phosphorylation of ribosomal protein S6 at Ser240/244 in the dorsolateral striatum of Wt mice but not Chat-cre;Gpr143flox/y mice. In Chinese hamster ovary cells stably expressing DRD2, co-expression of GPR143 increased cell surface expression level of DRD2, and L-DOPA application further enhanced the DRD2 surface expression. Shorter pauses in cholinergic interneuron firing activity were observed after intrastriatal stimulation in striatal slice preparations from Chat-cre;Gpr143flox/y mice compared with those from Wt mice. Together, these findings provide evidence that GPR143 regulates DRD2 function in cholinergic interneurons and may be involved in parkinsonism induced by antipsychotic drugs.

Dynamics of AMPA receptors regulate epileptogenesis in patients with epilepsy.

The excitatory glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) contribute to epileptogenesis. Thirty patients with epilepsy and 31 healthy controls are scanned using positron emission tomography with our recently developed radiotracer for AMPARs, [11C]K-2, which measures the density of cell-surface AMPARs. In patients with focal-onset seizures, an increase in AMPAR trafficking augments the amplitude of abnormal gamma activity detected by electroencephalography. In contrast, patients with generalized-onset seizures exhibit a decrease in AMPARs coupled with increased amplitude of abnormal gamma activity. Patients with epilepsy had reduced AMPAR levels compared with healthy controls, and AMPARs are reduced in larger areas of the cortex in patients with generalized-onset seizures compared with those with focal-onset seizures. Thus, epileptic brain function can be regulated by the enhanced trafficking of AMPAR due to Hebbian plasticity with increased simultaneous neuronal firing and compensational downregulation of cell-surface AMPARs by the synaptic scaling.

Coupling between GPR143 and dopamine D2 receptor is required for selective potentiation of dopamine D2 receptor function by L-3,4-dihydroxyphenylalanine in the dorsal striatum.

Dopamine (DA) is involved in neurological and physiological functions such as motor control. L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of DA, is conventionally believed to be an inert amino acid precursor of DA, and its major therapeutic effects in Parkinson's disease (PD) are mediated through its conversion to DA. On the contrary, accumulating evidence suggests that L-DOPA itself is a neurotransmitter. We here show that L-DOPA potentiates DA D2 receptor (DRD2) signaling through GPR143, the gene product of X-linked ocular albinism 1, a G-protein-coupled receptor for L-DOPA. In Gpr143-gene-deficient (Gpr143-/y ) mice, quinpirole, a DRD2/DRD3 agonist, -induced hypolocomotion was attenuated compared to wild-type (WT) mice. Administration of non-effective dose of L-DOPA methyl ester augmented the quinpirole-induced hypolocomotion in WT mice but not in Gpr143-/y mice. In cells co-expressing GPR143 and DRD2, L-DOPA enhanced the interaction between GPR143 and DRD2 and augmented quinpirole-induced decrease in cAMP levels. This augmentation by L-DOPA was not observed in cells co-expressing GPR143 and DRD1 or DRD3. Chimeric analysis in which the domain of GPR143 was replaced with GPR37 revealed that GPR143 interacted with DRD2 at the fifth transmembrane domain. Intracerebroventricular administration of a peptide that disrupted the interaction mitigated quinpirole-induced behavioral changes in WT mice but not in Gpr143-/y mice. These findings provide evidence that coupling between GPR143 and DRD2 is required for selective DRD2 modulation by L-DOPA in the dorsal striatum.

Synthesis of [18F] fluorine-labeled K-2 derivatives as radiotracers for AMPA receptors.

INTRODUCTION: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) receptors play a central role in neurotransmission and neuronal function. A positron emission tomography (PET) tracer for AMPA receptors, [11C]K-2, was recently developed by us to visualize AMPA receptors in the living human brain. [11C]K-2 is a derivative of 4-[2-(phenylsulphonylamino)ethylthio]-2,6-difuluoro-phenoxyacetamide (PEPA), and is labeled with the radioactive isotope 11C, which has a short half-life. PET drugs are usually labeled with 18F because of its long half-life. Therefore, we screened and identified potential 18F-labeled PET drugs for AMPA receptors (AMPA-PET drugs), which could provide an image equivalent to that of [11C]K-2. METHODS: Derivatives of K-2 labeled with 18F were synthesized and administered to rats and PET imaging was performed. The transferability of each compound to the brain and its correlation with the PET image of [11C]K-2 were evaluated from the obtained PET images. Furthermore, the specific binding ability of promising compounds to the AMPA receptor was evaluated by the PET imaging of rats, which we specifically knocked down the expression of AMPA by the lentivirus-mediated introduction of short hairpin RNA (shRNA) targeted to subunits of the AMPA receptor (GluA1-A3). The specific binding ability was also evaluated through electrophysiological experiments with acute brain slices. RESULTS: Some of the synthesized 18F-labeled candidate compounds showed a distribution similar to that of K-2, with reasonable transferability to the brain. In addition, from the evaluation of the specific binding ability to the AMPA receptor, a promising structure of an 18F-labeled AMPA PET drug was identified. This study also revealed that the alkylation of the sulfonamide group of PEPA enhances brain transferability.

[11C]K-2 image with positron emission tomography represents cell surface AMPA receptors.

The glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) is an important molecule in neurotransmission. We have recently developed the first positron emission tomography (PET) tracer [11C]K-2 to visualize and quantify AMPARs in the living human brain. After injection, [11C]K-2 is hydrolyzed at the terminal amide (and is thus metabolized to a major metabolite, [11C]K-2OH) within 10 min, representing the PET image in rodents and humans. Here, we found that K-2OH did not penetrate the cell membrane but slowly passed through the blood brain barrier (BBB) with paracellular transport. Furthermore, major efflux transporters in the BBB did not carry K-2OH. Logan graphical analysis exhibited reversible binding kinetics of this radiotracer in healthy individuals; these results demonstrated that the PET image of this tracer represents cell surface AMPARs with passive penetration of [11C]K-2OH through the BBB, resulting in reversible binding kinetics. Thus, PET images with this tracer depict the physiologically crucial fraction of AMPARs.

Biodistribution and radiation dosimetry of the positron emission tomography probe for AMPA receptor, [11C]K-2, in healthy human subjects.

[11C]K-2, a radiotracer exhibiting high affinity and selectivity for α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), is suitable for the quantification of AMPARs in living human brains and potentially useful in the identification of epileptogenic foci in patients. This study aimed to estimate the radiation doses of [11C]K-2 in various organs and calculate the effective dose after injection of [11C]K-2 in healthy human subjects. Twelve healthy male subjects were registered and divided into two groups (370 or 555 MBq of [11C]K-2), followed by 2 h whole-body scans. We estimated the radiation dose of each organ and then calculated the effective dose for each subject. The highest uptake of [11C]K-2 was observed in the liver, while the brain also showed relatively high uptake. The urinary bladder exhibited the highest radiation dose. The kidneys and liver also showed high radiation doses after [11C]K-2 injections. The effective dose of [11C]K-2 ranged from 5.0 to 5.2 µSv/MBq. Our findings suggest that [11C]K-2 is safe in terms of the radiation dose and adverse effects. The injection of 370-555 MBq (10 to 15 mCi) for PET studies using this radiotracer is applicable in healthy human subjects and enables serial PET scans in a single subject.

Visualization of AMPA receptors in living human brain with positron emission tomography.

Although aberrations in the number and function of glutamate AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors are thought to underlie neuropsychiatric disorders, no methods are currently available for visualizing AMPA receptors in the living human brain. Here we developed a positron emission tomography (PET) tracer for AMPA receptors. A derivative of 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide radiolabeled with 11C ([11C]K-2) showed specific binding to AMPA receptors. Our clinical trial with healthy human participants confirmed reversible binding of [11C]K-2 in the brain according to Logan graphical analysis (UMIN000020975; study design: non-randomized, single arm; primary outcome: dynamics and distribution volumes of [11C]K-2 in the brain; secondary outcome: adverse events of [11C]K-2 during the 4-10 d following dosing; this trial met prespecified endpoints). In an exploratory clinical study including patients with epilepsy, we detected increased [11C]K-2 uptake in the epileptogenic focus of patients with mesial temporal lobe epilepsy, which was closely correlated with the local AMPA receptor protein distribution in surgical specimens from the same individuals (UMIN000025090; study design: non-randomized, single arm; primary outcome: correlation between [11C]K-2 uptake measured with PET before surgery and AMPA receptor protein density examined by biochemical study after surgery; secondary outcome: adverse events during the 7 d following PET scan; this trial met prespecified endpoints). Thus, [11C]K-2 is a potent PET tracer for AMPA receptors, potentially providing a tool to examine the involvement of AMPA receptors in neuropsychiatric disorders.

Clinical Usefulness of Somatostatin Receptor Scintigraphy in Japanese Patients with Gastroenteropancreatic Neuroendocrine Tumors.

BACKGROUND/AIMS: Somatostatin receptor (SSTR) scintigraphy (SRS) is the standard imaging modality for evaluation of gastroenteropancreatic neuroendocrine tumor (GEP-NET) in Western countries. However, this modality was not approved in Japan until recently. The purpose of this study was to evaluate the clinical efficacy of SRS for detecting GEP-NET in Japanese patients. METHODS: Japanese patients with advanced GEP-NET were enrolled and evaluated by the SRS and CT. We also compared SRS and immunohistochemical expression of SSTR type 2a (SSTR2a). RESULTS: We enrolled 16 patients and the primary sites were the pancreas in 9, the stomach in 1, the small intestine in 2, the colon in 3, and unknown in 1. SRS showed positive findings in 3 (100%) of grade 1 (G1) and in 12 (92.3%) of grade 2 (G2) lesions. In the liver, SRS and CT detected lesions in 13 and 14 cases, respectively. The concordance rate of SSTR2a expression with SRS findings was 93.8% in the whole body and 92.9% in the liver. CONCLUSIONS: SRS could detect almost all of G1 and G2. SRS could be useful to detect lesions, with a high concordance rate with CT and pathological findings. We confirmed that SRS is a useful and reliable modality for Japanese patients.

Carbon-11 radiolabeling of an oligopeptide containing tryptophan hydrochloride via a Pictet-Spengler reaction using carbon-11 formaldehyde.

A procedure for the synthesis of a(11)C-labeled oligopeptide containing [1-(11)C]1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid ([1-(11)C]Tpi) from the corresponding Trp•HCl-containing peptides has been developed involving a Pictet-Spengler reaction with [(11) C]formaldehyde. The synthesis of [1-(11)C]Tpi from Trp and [(11)C]formaldehyde was examined as a model reaction with the aim of developing a facile and effective method for the labeling of peptides with carbon-11. The Pictet-Spengler reaction of Trp and [(11)C]formaldehyde in acidic media (TsOH or HCl) afforded the desired [1-(11)C]Tpi in a moderate radiochemical yield. Herein, the application of a Pictet-Spengler reaction to an aqueous solution of Trp•HCl gave the desired product with a radiochemical yield of 45.2%. The RGD peptide cyclo[Arg-Gly-Asp-D-Tyr-Lys] was then selected as a substrate for the labeling reaction with [(11)C]formaldehyde. The radiolabeling of a Trp•HCl-containing RGD peptide using the Pictet-Spengler reaction was successful. Furthermore, the remote-controlled synthesis of a [1-(11)C]Tpi-containing RGD peptide was attempted by using an automatic production system to generate [(11)C]CH3 I. The radiochemical yield of the [1-(11) C]Tpi-containing RGD at the end of synthesis (EOS) was 5.9 ± 1.9% (n = 4), for a total synthesis time of about 35 min. The specific activity was 85.7 ± 9.4 GBq/µmol at the EOS.

Rational approach to the synthesis, evaluation, and (68)ga labeling of a novel 4-anilinoquinoline epidermal growth factor receptor inhibitor as a new imaging agent that selectively targets the epidermal growth factor receptor tyrosine kinase.

Certain small-molecule inhibitors that target epidermal growth factor receptor (EGFR), such as Gefitinib, Erlotinib, and Lapatinib, provide a new approach for cancer treatment. In accordance with the pharmacophore model for inhibitor competition at EGFR-binding site, this study proposes a rationalized design for a novel 4-anilinoquinoline EGFR tyrosine kinase inhibitor, [6,7-dimethoxyethoxy]-quinolin-4-yl]-(3-ethynylphenyl)-amine (YCU07). This is the first study to apply ring-closing metathesis toward synthesis of the quinoline nucleus for this 4-anilinoquinoline EGFR inhibitor. YCU07 expressed significant inhibitory activity for EGFR tyrosine kinase in A431 cells, as confirmed by an ABTS microwell peroxidase substrate system read colorimetrically at 405 nm. Injection of (68)Ga-labeled glutamic acid polypeptide (GAP)-YCU07 conjugate in nude mice implanted with A431 was imaged by animal PET camera (LabPET8; Gamma Medica-Ideas) and computed tomography (eXplore Locus; GE Healthcare), to evaluate its biodistribution. (68)Ga-GAP-YCU accumulated in the receptor-positive tumors, with uptake values of 1.50% +/- 0.09% and 2.36% +/- 0.36% of injected activity per gram tissue at 30 and 90 minutes, respectively.

Radiosynthesis of [2-(11)C-carbonyl]dantrolene using [(11)C]phosgene for PET.

Automated radiosynthesis of [2-(11)C-carbonyl]dantrolene, the substrate of breast cancer resistance protein (BCRP/ABCG2), was performed for the first time through a multi-step/one-pot labeling sequence that started with ethyl 2-{2-[5-(4-nitrophenyl)furfurylidene]hydrazino}acetate and used [(11)C]phosgene as a labeling agent. After optimization of the automated synthesis conditions and parameters, [2-(11)C-carbonyl]dantrolene was obtained at a radiochemical yield of 34.0+/-8.4% (decay-corrected). The radiochemical purity was greater than 98% and the specific activity was 46.8+/-15.2GBq/micromol at the end of the synthesis.

Simple and effective method for producing [11C]phosgene using an environmental CCl4 gas detection tube.

INTRODUCTION: Carbon-11-labeled phosgene is an important labeling precursor for PET molecular probes. Despite the usefulness of [(11)C]phosgene, some difficulties, especially in the formation of [(11)C]phosgene process from [(11)C]CCl(4), hamper its use. The present article shows a simple preparation method for [(11)C]phosgene. METHOD: [(11)C]CCl(4) was obtained using the conventional method by passing a mixture of [(11)C]CH(4) and Cl(2) through a heated quartz tube. The [(11)C]CCl(4) was transformed to [(11)C]phosgene simply by passing through a pretreatment tube of a Kitagawa gas detection system for the working-environmental CCl(4) concentration measurement at room temperature with a flow rate of 50 ml/min. RESULT: This tube successfully transformed [(11)C]CCl(4) to [(11)C]phosgene at room temperature. [(11)C]Phosgene was obtained at nearly 80% radiochemical yield (EOB) in a short synthesis time with high reproducibility. CONCLUSION: A high yield and reliable [(11)C]phosgene production method using a gas detector tube system for working-environmental CCl(4) concentration measurement was developed.

Ochtodene derivatives from the red alga Carpopeltis crispata.

Red algae are well known as a source of halogenated monoterpenes such as derivatives of ochtodene. From Carpopeltis crispata, we have isolated four new ochtodene derivatives: dibromodichloro-, dibromochloro-, and bromodichlorocyclomonoterpenes. The structures of these monoterpenes were confirmed by NMR and mass spectroscopy and compared with spectral data in the literature.

Kulokekahilide-2, a cytotoxic depsipeptide from a cephalaspidean mollusk Philinopsis speciosa.

A cytotoxic depsipeptide, kulokekahilide-2 (1), was isolated from a cephalaspidean mollusk, Philinopsis speciosa. The structure elucidation of kulokekahilide-2 was carried out by spectroscopic analysis and chemical degradation. Kulokekahilide-2 showed potent cytotoxicity against several cell lines (P388, SK-OV-3, MDA-MB-435, and A-10 with IC50 values ranging from 4.2 to 59.1 nM) indicating cancer cell selectivity.

Novel oxylipin metabolites from the brown alga Eisenia bicyclis.

Nine novel oxylipin metabolites together with several known ones were isolated from the brown alga Eisenia bicyclis. Five (1-5) of them are ecklonialactone derivatives containing a chlorine or an iodine atom, and two (6 and 7) are cymathere type oxylipins with a lactone ring or a chlorine atom. The structures of these oxylipin metabolites were confirmed by NMR and mass spectroscopy and compared with spectral data in the literature. The postulated biosynthetic pathway of these metabolites is discussed.

Kulokekahilide-1, a cytotoxic depsipeptide from the cephalaspidean mollusk Philinopsis speciosa.

The cytotoxic depsipeptide kulokekahilide-1, which contains two unusual amino acids, 4-phenylvaline and 3-amino-2-methylhexanoic acid, was isolated from the cephalaspidean mollusk Philinopsis speciosa. Structure elucidation of kulokekahilide-1 was carried out by spectroscopic analysis and chemical degradation. The absolute stereochemistry was determined by Marfey analysis for amino acids and chiral HPLC analysis for hydroxy acids. All four stereoisomers of 4-phenylvaline and 3-amino-2-methylhexanoic acid, which were necessary for Marfey analysis, were synthesized by use of the Heck reaction and Evans's method, respectively. Kulokekahilide-1 showed cytotoxicity against P388 murine leukemia cells with an IC(50) value of 2.1 microg/mL.