Hirotaka Kushida

The blood-brain barrier permeability of geissoschizine methyl ether in Uncaria hook, a galenical constituent of the traditional Japanese medicine yokukansan.

Geissoschizine methyl ether (GM) in Uncaria hook, a galenical constituent of yokukansan is thought to be one of active components in the psychotropic effect of yokukansan, a traditional Japanese medicine (kampo medicine). However, there is no data on the blood–brain barrier (BBB) permeability of Uncaria hook-derived alkaloids containing GM. In this study, we investigated the BBB permeability of seven Uncaria hook alkaloids (GM, isocorynoxeine, isorhynchophylline, hirsuteine, hirsutine, rhynchophylline, and corynoxeine) using in vivo and in vitro methods. In the in vivo experiment, seven alkaloids in the plasma and brain of rats orally administered with yokukansan were measured by liquid chromatography–mass spectroscopy/mass spectrometric multiple reaction monitoring assay. In the in vitro experiment, the BBB permeability of seven alkaloids were examined using the BBB model composed of co-culture of endothelial cells, pericytes, and astrocytes. In the in vivo study, six components containing GM but not isocorynoxeine were detected in the plasma, and three (GM, hirsuteine, and corynoxeine) of components were detected in the brain. The in vitro BBB permeability data indicated that seven alkaloids were able to cross brain endothelial cells in culture conditions and that the BBB permeability of GM was higher than those of the other six alkaloids. These results suggest that target ingredient GM in yokukansan administered orally is absorbed into the blood and then reaches the brain through the BBB. This evidence further supports the possibility that GM is an active component in the psychotropic effect of yokukansan.

Simultaneous quantitative analyses of indole and oxindole alkaloids of Uncaria Hook in rat plasma and brain after oral administration of the traditional Japanese medicine Yokukansan using high-performance liquid chromatography with tandem mass spectrometry

Uncaria Hook (UH) alkaloids are involved in the beneficial effects of Yokukansan. However, the pharmacokinetics of UH alkaloids after oral administration of Yokukansan has not yet been sufficiently investigated. Therefore, we developed and validated a sensitive and specific high-performance liquid chromatography with tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of seven UH alkaloids (corynoxeine, isocorynoxeine, rhynchophylline, isorhynchophylline, hirsutine, hirsuteine and geissoschizine methyl ether) in rat plasma and brain. After protein precipitation with acetonitrile, chromatographic separation was performed using an Ascentis Express RP-amide column, with gradient elution with 0.2% formic acid and acetonitrile at 0.3 mL/min. All analytes in the plasma and brain showed good linearity over a wide concentration range (r > 0.995). Intra-day and inter-day variations of each constituent were 8.6 and 8.0% or less in the plasma, and 14.9 and 15.0% or less in the brain, respectively. The validated LC/MS/MS method was applied in the pharmacokinetic studies of UH alkaloids after oral administration of Yokukansan to rats. In the plasma, rhynchophylline, hirsutine, hirsuteine and geissoschizine methyl ether were detected, but only geissoschizine methyl ether was detected in the brain. These results suggest that geissoschizine methyl ether is an important constituent of the pharmacological effects of Yokukansan.

Specific binding and characteristics of geissoschizine methyl ether, an indole alkaloid of Uncaria Hook, in the rat brain.

ETHNOPHARMACOLOGICAL RELEVANCE Geissoschizine methyl ether (GM) is an indole alkaloid that is a component of Uncaria Hook, and has been identified as the active component responsible for the anti-aggressive effects of the Uncaria Hook-containing traditional Japanese medicine, yokukansan. Recently, GM was shown to reach the brain by crossing the blood-brain barrier in rats following the oral administration of yokukansan. This finding suggested that there may be specific binding sites for GM in the brain. Here we show evidence that tritium-labeled GM ([(3)H]GM) binds specifically to several brain areas of rats. MATERIALS AND METHODS Male rats were used. [(3)H]GM was synthesized from a demethylated derivative of GM. Specific binding sites of [(3)H]GM on brain sections were determined by quantitative autoradiography, and maximum binding densities (Bmax) and dissociation constants (Kd) were calculated. Several chemical compounds were used to clarify the molecules that recognize [(3)H]GM in the completion-binding assay. Emulsion microautoradiography was also performed to identify the cells that bind [(3)H]GM. RESULTS Specific binding of [(3)H]GM was observed in the frontal cortex, including the prefrontal cortical region (e.g., prelimbic cortex (PrL)), hippocampus, caudate putamen, amygdala, central medial thalamic nucleus, dorsal raphe nucleus (DR), and cerebellum. Bmax ranged between 0.65 and 8.79pmol/mg tissue, and Kd was between 35.0 and 232.6nM. Specific binding with relatively high affinity (Kd less than 62nM) was dense in the frontal cortical region, moderate in the DR, and sparse in the cerebellum. The specific binding of [(3)H]GM in the PrL was significantly replaced by the serotonin 1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), 5-HT2A receptor antagonist ketanserin, 5-HT2B receptor agonist BW 723C86, 5-HT2C receptor agonist RO60-0175, adrenergic α2A receptor antagonist yohimbine, L-type Ca(2+) channel blocker verapamil, and μ-opioid receptor antagonist naloxone. Similar results were obtained in the frontal cortex and DR, but not in the cerebellum. Microautoradiography revealed that [(3)H]GM signals were distributed throughout the frontal cortex, which included neuron-like large cells. CONCLUSION These results demonstrate that specific binding sites for GM exist in rat brain tissue, and suggest that the pharmacological actions of GM are mainly associated with 5-HT receptors in the frontal cortex and DR. These results provide an insight into the neuropharmacology of GM and GM-containing herbal medicines.

Intestinal, portal, and peripheral profiles of daikenchuto (TU-100)'s active ingredients after oral administration

A pharmaceutical grade Japanese traditional medicine, daikenchuto (TU‐100), consisting of Japanese pepper, processed ginger, and ginseng, has been widely used for various intestinal disorders in Japan and now under development as a new therapeutic drug in the US. It is suggested that TU‐100 ingredients exert pharmacological effects on intestines via two routes, from the luminal side before absorption and the peripheral blood stream after absorption. Therefore, in order to fully understand the pharmacological actions of TU‐100, it is critically important to know the intraluminal amounts and forms of ingested TU‐100 ingredients. In the present study, after administrating TU‐100 to rats, the concentrations of TU‐100 ingredients and their conjugates in the peripheral and portal blood and ileal contents were determined by LC‐MS/MS. Next, TU‐100 was administered to patients with ileostomy bags, but whose small intestines are diagnosed as healthy, and the ingredients/conjugates in the ileal effluent were analyzed. The results suggest that: (1) Pepper ingredients hydroxysanshools are rapidly absorbed and enter systemic circulation, (2) Ginseng ingredients ginsenosides are transported to the colon with the least absorption, (3) Ginger ingredients gingerols are absorbed and some conjugated in the small intestine and transported via the portal vein. While only a small amount of gingerols/gingerol conjugates enter systemic circulation, considerable amounts reappear in the small intestine. Thus, the effect of TU‐100 on the intestines is believed to be a composite of multiple actions by multiple compounds supplied via multiple routes.

Effects of shakuyakukanzoto and its absorbed components on twitch contractions induced by physiological Ca2+ release in rat skeletal muscle

Shakuyakukanzoto (SKT) is a kampo medicine composed of equal proportions of Glycyrrhizae radix (G. radix) and Paeoniae radix (P. radix). A double-blind study reported that SKT significantly ameliorated painful muscle cramp in cirrhosis patients without the typical severe side effects of muscle weakness and central nervous system (CNS) depression. Previous basic studies reported that SKT and its active components induced relaxation by a direct action on skeletal muscle and that SKT did not depress CNS functions; however, why SKT has a lower incidence of muscle weakness remains unknown. In the present study, we investigated which components are absorbed into the blood of rats after a single oral administration of SKT to identify the active components of SKT. We also investigated the effects of SKT and its components on the twitch contraction induced by physiological Ca2+ release. Our study demonstrated that SKT and five G. radix isolates, which are responsible for the antispasmodic effect of SKT, did not inhibit the twitch contraction in contrast to dantrolene sodium, a direct-acting peripheral muscle relaxant, indicating that the mechanisms of muscle contraction of SKT and dantrolene in skeletal muscle differ. These findings suggest that SKT does not reduce the contractile force in skeletal muscle under physiological conditions, i.e., SKT may have a low risk of causing muscle weakness in clinical use. Considering that most muscle relaxants and anticonvulsants cause various harmful side effects such as weakness and CNS depression, SKT appears to have a benign safety profile.

Metabolic Profiling of the Uncaria Hook Alkaloid Geissoschizine Methyl Ether in Rat and Human Liver Microsomes Using High-Performance Liquid Chromatography with Tandem Mass Spectrometry

Geissoschizine methyl ether (GM) is an indole alkaloid found in Uncaria hook, which is a galenical constituent of yokukansan, a traditional Japanese medicine. GM has been identified as the active component responsible for anti-aggressive effects. In this study, the metabolic profiling of GM in rat and human liver microsomes was investigated. Thirteen metabolites of GM were elucidated and identified using a high-performance liquid chromatography with tandem mass spectrometry method, and their molecular structures were proposed on the basis of the characteristics of their precursor ions, product ions, and chromatographic retention times. There were no differences in the metabolites between the rat and human liver microsomes. Among the 13 identified metabolites, there were two demethylation metabolites, one dehydrogenation metabolite, three methylation metabolites, three oxidation metabolites, two water-adduct metabolites, one di-demethylation metabolite, and one water-adduct metabolite followed by oxidation. The metabolic pathways of GM were proposed on the basis of this study. This study will be helpful in understanding the metabolic routes of GM and related Uncaria hook alkaloids, and provide useful information on the pharmacokinetics and pharmacodynamics. This is the first report that describes the separation and identification of GM metabolites in rat and human liver microsomes.

Distribution Analysis via Mass Spectrometry Imaging of Ephedrine in the Lungs of Rats Orally Administered the Japanese Kampo Medicine Maoto

Maoto, a traditional Japanese Kampo medicine, has been used to treat various respiratory diseases, including respiratory infections and influenza. Ephedrine (EPD), the main ingredient in maoto, is also clinically used to treat respiratory diseases. However, the pharmacokinetics and distribution of EPD in the lungs after the administration of maoto have not been demonstrated. This study aimed to determine the concentrations, distribution, and pharmacokinetics of EPD and its precursor methylephedrine (MEPD) in the lungs of rats orally administered maoto (1 and 4 g/kg). We used liquid chromatography–electrospray ionization-tandem mass spectrometry to measure the ingredient concentrations. Both ingredients were detected in maoto-treated lung homogenates. Next, we examined the distribution of both ingredients in lung sections by using matrix-assisted laser desorption/ionization-mass spectrometry imaging, a powerful tool for the visualization of the distribution of biological molecules. The mass spectrometry imaging analysis detected only EPD and provided the first visual demonstration that EPD is distributed in the alveoli, bronchi, and bronchioles in the lungs of rats orally administered maoto (4 g/kg, three times at 2-h intervals). These results suggest that the pharmacological efficacy of maoto for the amelioration of respiratory symptoms is related to the distribution of EPD in the lung.

Deconstructing the traditional Japanese medicine “Kampo”: compounds, metabolites and pharmacological profile of maoto, a remedy for flu-like symptoms

Pharmacological activities of the traditional Japanese herbal medicine (Kampo) are putatively mediated by complex interactions between multiple herbal compounds and host factors, which are difficult to characterize via the reductive approach of purifying major bioactive compounds and elucidating their mechanisms by conventional pharmacology. Here, we performed comprehensive compound, pharmacological and metabolomic analyses of maoto, a pharmaceutical-grade Kampo prescribed for flu-like symptoms, in normal and polyI:C-injected rats, the latter suffering from acute inflammation via Toll-like receptor 3 activation. In total, 352 chemical composition-determined compounds (CCDs) were detected in maoto extract by mass spectrometric analysis. After maoto treatment, 113 CCDs were newly detected in rat plasma. Of these CCDs, 19 were present in maoto extract, while 94 were presumed to be metabolites generated from maoto compounds or endogenous substances such as phospholipids. At the phenotypic level, maoto ameliorated the polyI:C-induced decrease in locomotor activity and body weight; however, body weight was not affected by individual maoto components in isolation. In accordance with symptom relief, maoto suppressed TNF-α and IL-1β, increased IL-10, and altered endogenous metabolites related to sympathetic activation and energy expenditure. Furthermore, maoto decreased inflammatory prostaglandins and leukotrienes, and increased anti-inflammatory eicosapentaenoic acid and hydroxyl-eicosapentaenoic acids, suggesting that it has differential effects on eicosanoid metabolic pathways involving cyclooxygenases, lipoxygenases and cytochrome P450s. Collectively, these data indicate that extensive profiling of compounds, metabolites and pharmacological phenotypes is essential for elucidating the mechanisms of herbal medicines, whose vast array of constituents induce a wide range of changes in xenobiotic and endogenous metabolism.Systems biology of traditional medicine: comprehensive analysis of a traditional Japanese medicine, maotoPharmacological activities of Kampo, or traditional Japanese herbal medicine, are putatively mediated by complex interactions between the plant-derived compounds and endogenous molecules. To elucidate these properties, we performed comprehensive phytochemical profiling, and pharmacological and metabolomic analyses of maoto, an herbal remedy for flu-like symptoms. In the plasma of maoto-treated rats, we detected maoto-derived compounds, metabolites produced from the chemical transformation of maoto compounds by host metabolism and gut microbes, and endogenous metabolites that were appeared following maoto administration. In an acute inflammatory rat model, maoto ameliorated symptoms of sickness behavior, suppressed inflammatory cytokines, and extensively affected common metabolites and lipid mediators. These data suggest that the diverse chemical composition of Kampo broadly affects the host’s endogenous metabolism and exerts specific pharmacological effects.

In vitro identification of human cytochrome P450 isoforms involved in the metabolism of Geissoschizine methyl ether, an active component of the traditional Japanese medicine Yokukansan

Abstract 1. Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans. 2. Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms. 3. The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible for GM metabolism in human liver microsomes. 4. These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS.

Phenotyping analysis of the Japanese Kampo medicine maoto in healthy human subjects using wide-targeted plasma metabolomics

HighlightsTraditional herbal medicines consist of a vast number of compounds and are assumed to exert their activity by affecting various sites in the body.Plasma metabolomics was used for phenotype analysis of the Japanese kampo medicine maoto in a human clinical study.Maoto influenced plasma amino acids and lipid mediators.Primary compounds in maoto such as ephedrine, prunasin, cinnamic acid, and glyccyrhetinic acid were absorbed in the bloodstream.Alteration of branched‐chain amino acid levels by maoto administration is likely caused by ephedrine and its analogues. Abstract Traditional herbal medicine (THM) consists of a vast number of compounds that exert pharmacological effects throughout the body. Comprehensive phenotyping analysis using omics is essential for understanding the nature of THM in detail. We previously reported that the Japanese Kampo medicine maoto ameliorated flu‐like symptoms in a rat infection model and dynamically changed plasma metabolites as indicated by metabolome analysis. The aim of this study was to apply wide‐targeted plasma metabolomics with quantitative analysis of maoto compounds in a human clinical trial to evaluate the effect of maoto on plasma metabolites. Four healthy human subjects were recruited. Plasma samples were collected before and 0.25, 0.5, 1, 2, 4 and 8 h after maoto treatment. Wide‐targeted metabolomics and quantitative analysis of the main chemical constituents of maoto were then performed. Plasma metabolome analysis revealed that maoto administration decreased essential amino acids including branched‐chain amino acids (BCAAs) and increased various kinds of ω‐3 fatty acids including eicosapentaenoic acid and docosahexaenoic acid, consistent with previous studies in rats. Fifteen of the major compounds in maoto were identified in the systemic circulation. Finally, the correlation between endogenous metabolites and maoto compounds in plasma was analyzed and the results indicated that the decrease in plasma BCAAs might be caused by ephedrines present in maoto. The present study demonstrated that plasma metabolomic studies of endogenous and exogenous metabolites are useful for elucidating the mechanism of action of THM.