Radiation dosimetry and pharmacokinetics of the tau PET tracer florzolotau (18F) in healthy Japanese subjects.

OBJECTIVE: Abnormal aggregation of tau in the brain is a major contributing factor in various neurodegenerative diseases. Florzolotau (18F) (florzolotau, APN-1607, PM-PBB3) has been shown to be a probe for tau fibrils in an animal model and patients with Alzheimer's disease and those with non-Alzheimer's disease tauopathies. The objective of this study is to evaluate the safety, pharmacokinetics, and radiation dose following a single intravenous administration of florzolotau in healthy Japanese subjects. METHODS: Three healthy male Japanese subjects aged between 20 and 64 were enrolled in this study. Subjects were determined to be eligible based on the screening assessments at the study site. Subjects received a single intravenous dose of 195.0 ± 0.5 MBq of florzolotau and underwent the whole-body PET scan 10 times in total to calculate absorbed doses to major organs/tissues and effective dose. Radioactivities in whole blood and urine were also measured for pharmacokinetic evaluation. Absorbed doses to major organs/tissues and effective dose were estimated using the medical internal radiation dose (MIRD) method. Vital signs, electrocardiography (ECG), and blood tests were done for safety evaluation. RESULTS: The intravenous injection of florzolotau was well tolerated. There were no adverse events or clinically detectable pharmacologic effects related to the tracer in any subjects. No significant changes in vital signs and ECG were observed. The highest mean initial uptake at 15 min after injection was in the liver (29.0 ± 4.0%ID), intestine (4.69 ± 1.65%ID), and brain (2.13 ± 0.18%ID). The highest absorbed dose was 508 µGy/MBq of the gallbladder wall, followed by the liver of 79.4 µGy/MBq, the pancreas of 42.5 µGy/MBq, and the upper large intestine of 34.2 µGy/MBq. The effective dose was calculated as 19.7 µSv/MBq according to the tissue weighting factor reported by ICRP-103. CONCLUSION: Florzolotau intravenous injection was well tolerated in healthy male Japanese subjects. The effective dose was determined as 3.61 mSv when 185 MBq florzolotau was given.

Pharmacological characterization of M-II, the major human metabolite of ramelteon.

The duration of action of melatonin may be important for improvements in sleep efficiency in insomniacs. Ramelteon, a selective melatonin agonist, is primarily metabolized to the active metabolite M-II, which has a longer half-life and greater systemic exposure than ramelteon. Hence, M-II may contribute significantly to the hypnotic benefits of ramelteon. We assessed the ramelteon-like activity of M-II in vitro and in vivo using cats. Binding and functional studies in Chinese hamster ovary cells expressing human melatonin receptors (MT1 or MT2) revealed that M-II binds melatonin receptors with lower affinity (Ki: 114 and 566 pmol/l for MT1 and MT2, respectively) and has lower potency (IC50: 208 and 1,470 pmol/l for MT1 and MT2, respectively) compared with ramelteon. However, higher M-II doses significantly improved sleep in cats. Thus, M-II may contribute to the clinical efficacy of ramelteon.

Ameliorative effects of a non-competitive BACE1 inhibitor TAK-070 on Aß peptide levels and impaired learning behavior in aged rats.

We examined the effects of TAK-070, a novel non-competitive ß-secretase (BACE1) inhibitor, on the levels of Aß peptides and behavioral deficits in rats. TAK-070 reduced soluble Aß40 and Aß42 levels of the cerebral cortex in a time- and dose-dependent manner in young rats. We found that the insoluble Aß42 content increased significantly with aging from 22 months old without changing Aß40 content. TAK-070 normalized the Aß42 levels to those in young rats when they were fed chow containing TAK-070 starting at 19 months old for 6.5 months. Repeated administration of TAK-070 to aged rats for 2 weeks ameliorated the impaired spatial learning in the Morris water maze task and reduced the levels of soluble and insoluble Aß peptides at doses of 0.3-1mg/kg, (p.o.). Interestingly, TAK-070 significantly recovered the reduced brain synaptophysin levels in aged rats to those in young rats. Our findings support the idea that partial inhibition of BACE1 by TAK-070 exerts symptomatic as well as disease-modifying effects for the treatment of Alzheimer's disease.

A noncompetitive BACE1 inhibitor TAK-070 ameliorates Abeta pathology and behavioral deficits in a mouse model of Alzheimer's disease.

We discovered a nonpeptidic compound, TAK-070, that inhibited BACE1, a rate-limiting protease for the generation of Abeta peptides that are considered causative for Alzheimer's disease (AD), in a noncompetitive manner. TAK-070 bound to full-length BACE1, but not to truncated BACE1 lacking the transmembrane domain. Short-term oral administration of TAK-070 decreased the brain levels of soluble Abeta, increased that of neurotrophic sAPPalpha by approximately 20%, and normalized the behavioral impairments in cognitive tests in Tg2576 mice, an APP transgenic mouse model of AD. Six-month chronic treatment decreased cerebral Abeta deposition by approximately 60%, preserving the pharmacological efficacy on soluble Abeta and sAPPalpha levels. These results support the feasibility of BACE1 inhibition with a noncompetitive inhibitor as disease-modifying as well as symptomatic therapy for AD.

[A novel therapeutic drug: ramelteon].

Current treatment of insomnia with hypnotics, GABA(A) receptor modulators, induces various side effects, including cognitive impairment, motor disturbance, dependence, tolerance, hang-over, and rebound insomnia. Ramelteon (Rozerem) is an orally active, highly selective melatonin MT1/MT2 receptor agonist. Unlike the sedative hypnotics that target GABA(A) receptor complexes, ramelteon is a chronohypnotic that acts on the melatonin MT1 and MT2 receptors, which are primarily located in the suprachiasmatic nucleus. Ramelteon has demonstrated sleep-promoting effects in clinical trials, and coupled with its favorable safety profile and lack of abuse potential or dependence, this chronohypnotic provides an important treatment option for insomnia.

Pharmacology of ramelteon, a selective MT1/MT2 receptor agonist: a novel therapeutic drug for sleep disorders.

An estimated one-third of the general population is affected by insomnia, and this number is increasing due to more stressful working conditions and the progressive aging of society. However, current treatment of insomnia with hypnotics, gamma-aminobutyric acid A (GABA(A)) receptor modulators, induces various side effects, including cognitive impairment, motor disturbance, dependence, tolerance, hangover, and rebound insomnia. Ramelteon (Rozerem; Takeda Pharmaceutical Company Limited, Osaka, Japan) is an orally active, highly selective melatonin MT(1)/MT(2) receptor agonist. Unlike the sedative hypnotics that target GABA(A) receptor complexes, ramelteon is a chronohypnotic that acts on the melatonin MT(1) and MT(2) receptors, which are primarily located in the suprachiasmatic nucleus, the body's "master clock." As such, ramelteon possesses the first new therapeutic mechanism of action for a prescription insomnia medication in over three decades. Ramelteon has demonstrated sleep-promoting effects in clinical trials, and coupled with its favorable safety profile and lack of abuse potential or dependence, this chronohypnotic provides an important treatment option for insomnia.

Effect of ramelteon (TAK-375), a selective MT1/MT2 receptor agonist, on motor performance in mice.

Effect of (S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl]propionamide (ramelteon, TAK-375), a selective MT1/MT2 receptor agonist, on motor coordination was studied using rota-rod performance in mice. Ramelteon did not impair rota-rod performance at doses of 3, 10 and 30 mg/kg, p.o. Melatonin and N-acetyl-5-hydroxytryptamine (N-acetyl-5-HT), a ligand of MT3 biding site, also had no impairment on the performance, per se. However, in combination with a low dose of diazepam (3mg/kg, p.o.), treatment with melatonin and N-acetyl-5-HT exacerbated the impairment by diazepam. Ramelteon had no significant effect on the diazepam-induced impairment of motor coordination.

Neurochemical properties of ramelteon (TAK-375), a selective MT1/MT2 receptor agonist.

Ramelteon (TAK-375) is a novel melatonin receptor agonist currently under investigation for the treatment of insomnia. This study describes the neurochemical and receptor binding characteristics of ramelteon in vitro. Ramelteon showed very high affinity for human MT1 (Mel1a) and MT2 (Mel1b) receptors (expressed in Chinese hamster ovary [CHO] cells), and chick forebrain melatonin receptors (consisting of Mel1a and Mel1c receptors) with Ki values of 14.0, 112, and 23.1 pM, respectively, making the affinities of ramelteon for these receptors 3-16 times higher than those of melatonin. The affinity of ramelteon for hamster brain MT3 binding sites was extremely weak (Ki: 2.65 microM) compared to melatonin's affinity for the MT3 binding site (Ki: 24.1 nM). In addition, ramelteon showed no measurable affinity for a large number of ligand binding sites (including benzodiazepine receptors, dopamine receptors, opiate receptors, ion channels, and transporters) and no effect on the activity of various enzymes. Ramelteon inhibited forskolin-stimulated cAMP production in the CHO cells that express the human MT1 or MT2 receptors. Taken together, these results indicate that ramelteon is a potent and highly selective agonist of MT1/MT2 melatonin receptors.

Ramelteon (TAK-375) accelerates reentrainment of circadian rhythm after a phase advance of the light-dark cycle in rats.

In vivo pharmacological effects of ramelteon (TAK-375), a novel, highly MT1/MT2-selective receptor agonist, were studied in rats to determine ramelteon's ability to reentrain the circadian rhythm after an abrupt phase advance. Experiments were also conducted to assess the potential cognitive side effects of ramelteon and its potential to become a drug of abuse. After an abrupt 8-h phase shift, ramelteon (0.1 and 1 mg/kg, p.o.) and melatonin (10 mg/kg, p.o.) accelerated reentrainment of running wheel activity rhythm to the new lightdark cycle. Ramelteon (3-30 mg/kg, p.o.) and melatonin (10-100 mg/kg, p.o.) did not affect learning or memory in rats tested by the water maze task and the delayed match to position task, although diazepam and triazolam impaired both of the tasks. Neither ramelteon (3-30 mg/kg, p.o.) nor melatonin (10-100 mg/kg, p.o.) demonstrated a rewarding property in the conditioned place-preference test, implying that MT1/MT2 receptor agonists have no abuse potential. In contrast, benzodiazepines and morphine showed rewarding properties in this test. The authors' results suggest that ramelteon may be useful for treatment of circadian rhythm sleep disorders without adverse effects typically associated with benzodiazepine use, such as learning and memory impairment, and drug dependence.

The sleep-promoting action of ramelteon (TAK-375) in freely moving cats.

INTRODUCTION: Ramelteon (TAK-375) is an MT1/MT2 receptor agonist being studied for the treatment of insomnia and circadian rhythm sleep disorders. We compared the behavioral effects of ramelteon and exogenous melatonin in freely moving cats. METHODS: Ramelteon and melatonin were each suspended in a 0.5% (weight per volume) methylcellulose solution and administered orally to freely moving cats. In the control trial, each cat was given vehicle. Each dose of ramelteon or melatonin was compared with the vehicle control in a crossover design. Electroencephalogram, electromyogram, and electrooculogram recordings were assessed. RESULTS: Ramelteon significantly decreased wakefulness at doses of 0.001,0.01, and 0.1 mg/kg, increased slow-wave sleep at doses of 0.001, 0.01, and 0.1 mg/kg, and increased rapid eye movement sleep at a dose of 0.1 mg/kg, compared with the vehicle controls, as assessed by analysis of variance. The effects of ramelteon lasted for up to 6 hours when evaluated by reduction of wakefulness. Exogenous melatonin (0.01-1 mg/kg) significantly increased slow-wave sleep, but the effect was weaker than that of ramelteon and lasted for only 2 hours. The lowest doses of ramelteon (0.0001 mg/kg) and melatonin (0.001 mg/kg) had no significant effect on sleep-wakefulness stage. CONCLUSIONS: Ramelteon was more effective than exogenous melatonin in promoting and maintaining sleep in freely moving cats. Based on its unique mechanism of action, ramelteon should be studied further to evaluate its potential for the treatment of sleep disorders.

Effects of ramelteon (TAK-375) on nocturnal sleep in freely moving monkeys.

We investigated the effects of (S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4]furan-8-yl)ethyl]propionamide (ramelteon, TAK-375), a novel MT1/MT2 receptor agonist, on nocturnal sleep in freely moving monkeys and compared these results with those of melatonin and zolpidem. Treatment with ramelteon (0.03 and 0.3 mg/kg, p.o.) significantly shortened latency to sleep onset and significantly increased total duration of sleep. Treatment with melatonin (0.3, 1, and 3 mg/kg, p.o.) also decreased sleep latency, but the effect was weak; the only significant reduction was seen with the 0.3 mg/kg dose on latency to light sleep. Melatonin had no significant effects on the duration of sleep. Zolpidem had no significant effects on latency to sleep onset in this study at any dose (1, 3, 10, and 30 mg/kg, p.o.). The highest dose (30 mg/kg) of zolpidem had a tendency to increase slow wave sleep; however, it also induced apparent sedation and myorelaxation. Treatment with ramelteon and melatonin had no evident effect on the general behavior of the monkeys. Spectral analysis (fast Fourier transform, FFT) of both ramelteon and melatonin revealed sleep patterns that were indistinguishable from those of naturally occurring sleep. The EEG power spectra of zolpidem were qualitatively different from that of naturally occurring physiological sleep. Results of the present study support the investigation of ramelteon as a sleep-promoting agent in humans.

Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome.

Most prostate cancers (PCs) become resistant to combined androgen blockade therapy with surgical or medical castration and antiandrogens after several years. Some of these refractory PCs regress after discontinuation of antiandrogen administration [antiandrogen withdrawal syndrome (AWS)]. Although the molecular mechanisms of the AWS are not fully understood because of the lack of suitable experimental models, one hypothesis of the mechanism is mutation of androgen receptor (AR). However, bicalutamide, which has become the most prevalent pure antiandrogen, does not work as an agonist for any mutant AR detected thus far in PC. To elucidate the mechanisms of the AWS, we established and characterized novel LNCaP cell sublines, LNCaP-cxDs, which were generated in vitro by culturing androgen-dependent LNCaP-FGC human PC cells in androgen-depleted medium with bicalutamide to mimic the combined androgen blockade therapy. LNCaP-FGC cells did not grow at first, but they started to grow after 6-13 weeks of culture. Bicalutamide stimulated LNCaP-cxD cell growth and increased prostate-specific antigen secretion from LNCaP-cxD cells both in vitro and in vivo. Sequencing of AR transcripts revealed that the AR in LNCaP-cxD cells harbors a novel mutation in codon 741, TGG (tryptophan) to TGT (cysteine; W741C), or in codon 741, TGG to TTG (leucine; W741L), in the ligand-binding domain. Transactivation assays showed that bicalutamide worked as an agonist for both W741C and W741L mutant ARs. Importantly, another antiandrogen, hydroxyflutamide, worked as an antagonist for these mutant ARs. In summary, we demonstrate for the first time that within only 6-13 weeks of in vitro exposure to bicalutamide, LNCaP-FGC cells, whose growth had initially been suppressed, came to use bicalutamide as an AR agonist via W741 AR mutation to survive. Our data strongly support the hypothesis that AR mutation is one possible mechanism of the AWS and suggest that flutamide might be effective as a second-line therapy for refractory PC previously treated with bicalutamide.

Synthesis of a novel series of tricyclic indan derivatives as melatonin receptor agonists.

To develop a new therapeutic agent for sleep disorders, we synthesized a novel series of tricyclic indan derivatives and evaluated them for their binding affinity to melatonin receptors. In our previous paper, we proposed a conformation of the methoxy group favorable for the binding of the MT(1) receptor. To fix the methoxy group in an active conformation, we decided to synthesize conformationally restricted tricyclic indan analogues with the oxygen atom in the 6-position incorporated into a furan, 1,3-dioxane, oxazole, pyran, morpholine, or 1,4-dioxane ring system. Among these compounds, indeno[5,4-b]furan analogues were found to be the most potent and selective MT(1) receptor ligands and to have superior metabolic stability. The optimization of substituents led to (S)-(-)-22b, which showed very strong affinity for human MT(1) (K(i) = 0.014 nM), but no significant affinity for hamster MT(3)() (K(i) = 2600 nM) or other neurotransmitter receptors. The pharmacological effects of (S)-(-)-22b were studied in experimental animals, and it was found that a dose of 0.1 mg/kg, po promoted a sleep in freely moving cats, as demonstrated by a decrease in wakefulness and increases in slow wave sleep and rapid eye movement sleep, which lasted for 6 h after administration. Melatonin (1 mg/kg, po) also had a sleep-promoting effect, though it lasted only 2 h. A new chiral method for the synthesis of (S)-(-)-22b starting from 60, which was prepared from 59 employing asymmetric hydrogenation with the (S)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl-Ru complex, was developed. (S)-(-)-22b (TAK-375) is currently under clinical trial for the treatment of insomnia and circadian rhythm disorders.

Synthesis of a novel series of benzocycloalkene derivatives as melatonin receptor agonists.

We synthesized a novel series of benzocycloalkene derivatives and evaluated their binding affinities to melatonin receptors. To control the spatial position of the amide group, one of the important pharmacophores, we incorporated an endo double bond, an exo double bond (E- and Z-configurations), and a chiral center (R- and S-configurations) at position 1. The indan derivatives with the S-configuration at position 1 were the most promising in terms of potency and selectivity for the human melatonin receptor (MT(1) site), while compounds with the R-configuration showed little potential. Our next attempt was to investigate the most favorable conformation of the methoxy group, the other important pharmacophore for binding to the MT(1) receptor. The introduction of a methyl group at position 5 of the indene ring conserved affinity; however, at position 7, it caused a decrease in affinity. These results suggested that the substitution at position 7 forced the methoxy group to adopt an unfavorable orientation. The optimization of the condensed ring size and substituents led to (S)-8d [(S)-N-[2-(2,3-dihydro-6-methoxy-1H-inden-1-yl)ethyl]propionamide], which had high affinity for the human MT(1) receptor (K(i) = 0.041 nM) but no significant affinity for the hamster MT(3)receptor (K(i) = 3570 nM). In addition, a practical synthetic method of chiral N-[2-(2,3-dihydro-1H-inden-1-yl)ethyl]alkanamides employing asymmetric hydrogenation with (S)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl-Ru has been established.

Mice deficient in nervous system-specific carbohydrate epitope HNK-1 exhibit impaired synaptic plasticity and spatial learning.

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P -/- mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.