Population pharmacokinetic analysis of daikenchuto, a traditional Japanese medicine (Kampo) in Japanese and US health volunteers.

We constructed population pharmacokinetic (PK) models for the five constituents of daikenchuto (DKT), a traditional Japanese herbal medicine. Data were collected from two randomized PK studies conducted in Japan and the United States. Participants received single oral doses of 2.5 g, 5 g, and 10 g of DKT. The plasma concentrations of five DKT constituents--hydroxy-α-sanshool (HAS), hydroxyl-ß-sanshool (HBS), 6-shogaol (6S), 10-shogaol (10S), and ginsenoside Rb1 (GRB1)--were determined by liquid chromatography-tandem mass spectrometry. A total of 1859 samples from 55 participants (US, n = 36; Japanese, n = 19) were included in the analysis. Population PK models of HAS, HBS, 6S, and 10S were best described by a one or two-compartment model with a bolus input. On the other hand, the model of GRB1 was best described by a one-compartment model with nonlinear extravascular input. Among the covariates evaluated, body mass index (BMI) and age were found to influence oral clearance (CL/F) and volume of distribution (Vd/F) for HAS and HBS, respectively. The influence of body weight on CL/F and Vd/F for 6S was demonstrated. Marked differences were observed in mean plasma concentrations of HAS and HBS between Japanese and US participants. However, the simulation results indicated that the difference in plasma concentrations may be attributed to the difference in demographic factors such as BMI, body weight, and age, whereas ethnic difference between the Japanese and US participants was considered minimal.

Evaluation of a bioreactor with stacked sheet shaped organoids of primary hepatocytes.

Hepatocyte organoids have an in vivo-like cell morphology and maintain cell viability and function in vitro. On the other hand, the oxygen supply to hepatocytes is sometimes limited in the core of organoids that are more than 100 mum in thickness. In this study, we designed and examined a new bioreactor using sheet-shaped organoids (organoid-sheets) in which the thickness was controlled to prevent hepatocyte death in the core of organoid due to limitation of oxygen supply. The cell culture space consisted of stacked organoid formation spaces and medium flow channels. Each space was separated by flat porous polycarbonate membranes, and the organoid thickness was controlled at 100 microm with a stainless steel spacer. Freshly isolated hepatocytes (7.0 x 10(7)) were immobilized in the bioreactor, yielding a cell density of 4.5 x 10(7) cells/cm(3)-bioreactor. Of the five flow rates tested (1.0, 5.0, 10, 20, and 50 mL/min), the bioreactor with the 10 mL/min had the highest ammonia removal and albumin secretion activities for at least 14 days. In conclusion, a new bioreactor controlling organoid thickness is useful for achieving high cell density culture and the maintenance of hepatocyte function to avoid cell death in the core of the organoids due to limitation of oxygen supply. The bioreactor may be useful for the development of various applications using cultured hepatocytes.

A new culture technique for hepatocyte organoid formation and long-term maintenance of liver-specific functions.

To develop a useful hybrid artificial liver, it is important to use cultured hepatocytes that maintain liver-specific functions for a long time. These requirements were achieved recently by the use of a hepatocyte multicellular aggregate (organoid) with a tissue-like structure. In this study, we developed a three-dimensional culture of hepatocytes that formed an organoid. Primary rat hepatocytes were immobilized inside hollow fibers (for plasma separation) by centrifugation. Hepatocytes formed a cylindrical organoid (cylindroid) of 200 mum in diameter by day 2 of culture. We used two types of culture media, medium A (Williams' medium E containing insulin and epidermal growth factor) and medium B (Dulbecco's modified Eagle's medium containing insulin, epidermal growth factor, and hydrocortisone). In medium A, the hepatocyte cylindroid diminished after 14 days of culture and liver-specific functions of the hepatocyte cylindroid nearly disappeared after 1 month of culture. In contrast, hepatocyte cylindroid cultured in medium B maintained its morphology and liver-specific functions for 2-5 months. These results indicate that a combination of the new culture technique and suitable culture medium is effective for expression and maintenance of liver-specific functions of hepatocytes. This culture technique will be helpful in the development of a hybrid artificial liver.

The influence of the sennosides on absorption of glycyrrhetic acid in rats.

In the course of our clinical studies of Kampo medicine (traditional Japanese medicines), we observed the pharmacokinetic interactions between two herbs. When Onpito (TJ-8117, Kampo medicine) containing licorice and rhubarb was administered orally to human subjects, we observed that the AUC(0-lim) and Cmax of glycyrrhetic acid (GA) in plasma were lower than those treated with other Kampo medicines containing licorice. In this study, we demonstrate the pharmacokinetic interactions of GA derived from glycyrrhizinic acid (GL) in licorice and anthraquinones derived from rhubarb. To our knowledge, this is the first report to investigate the pharmacokinetic interactions between two herbs. When GL was orally co-administrated to rats with a non-effective dose of sennoside A having purgative activity, the AUC(0-lim) and Cmax of GA decreased. In addition, sennoside A did not affect the metabolism of GL by the intestinal bacteria in vitro. In the examination using an in situ loop of rat colon, the remaining ratio of GA rose drastically by the co-administration of sennoside A, sennidin A and rhein. Observed inhibition activity of these anthraquinones on GA absorption depended on the concentration of the components added. The maximum inhibition ratio was approximately 75% by rhein, 60% by sennoside A and 25% by sennidin A. We conclude that the decrease of the pharmacokinetic parameters of GA in human plasma observed in the clinical study of TJ-8117 is attributable to an interactive action of absorption from the intestinal tract by anthraquinones contained in or derived from rhubarb.

Permeability of the flavonoids liquiritigenin and its glycosides in licorice roots and davidigenin, a hydrogenated metabolite of liquiritigenin, using human intestinal cell line Caco-2.

The present investigation focused on the transepithelial flux of liquiritigenin (LG), davidigenin (DG), liquiritin (LQ), and liquiritin apioside (LA) using the human colonic cell line Caco-2 as a model of human intestinal absorption. Apparent permeability coefficients (Papp) for the apical to basolateral flux of LG and DG were (16.0 +/- 0.727) x 10(-6) cm/s and (18.2 +/- 1.67) x 10(-6) cm/s, respectively. These Papp were higher than that of the transcellular transport marker propranolol (13.5 +/- 0.34) x 10(-6) cm/s (P < 0.01). Papp for the apical to basolateral flux of LQ and LA were (0.26 +/- 0.12) x 10(-6) cm/s and (0.075 +/- 0.005) x 10(-6) cm/s, respectively. These Papp were lower than that of the paracellular transport marker mannitol (0.64 +/- 0.04) x 10(-6) cm/s (LG, P < 0.01; LA, P < 0.001). These data suggested excellent absorption of LG and DG through the human intestinal epithelial cell line. On the contrary, poor absorption of LQ and LA was expected due to the little transepithelial flux of these compounds in the human colonic cell line Caco-2.

Toxicological aspects of Kampo medicines in clinical use.

Among 210 medicinal prescriptions used in present-day Japan, the clinical uses and the acute, chronic and mutagenic toxicity study of 16 Kampo (Japanese herbal) medicines are summarized. These Kampo medicines are classified into two categories; eight prescriptions containing Bupleurum root (Bupleurum falcatum L.) such as Sho-saiko-to and Saiko-keishi-to, and eight prescriptions not containing Bupleurum root such as Juzen-taiho-to and Ninjin-yoei-to. Studies of some potential interaction between herbal medicine and western drugs are also described.

Absorption, distribution and excretion of 3H-labeled cephaeline- and emetine-spiked ipecac syrup in rats.

The maximum plasma radioactivity levels of tritium (3H)-labeled cephaeline, (24.3, 28.7 and 40.6 ng eq./mL) were reached at 2.00-3.33 hours following oral dosing of ipecac syrup. The maximum plasma radioactivity levels of 3H-emetine (2.71, 6.47 and 9.62 ng eq./mL) were reached at 1.08-2.33 hours following ipecac syrup administration. The Cmax values of 3H-cephaeline were followed by a biexponential decrease with half-lives t 1/2(lambda z) of 3.45-9.40 hours. On the other hand, the t 1/2 (lambda z)of 3H-emetine were 65.4-163 hours, which revealed a biexponential decrease. The radioactivity of both tritium-labeled compounds was distrbuted maximally in most tissues at 24 hours. For 3H-cephaeline, the maximum radioactivity levels in tissues were approximately 100-150 times greater than in plasma. For 3H-emetine, the radioactivity levels in tissues were approximately 1000-3000 times greater than in plasma. Tissue radioactivity levels decreased at a substantially slower rate than that observed in plasma. Tissue radioactivity of 3H-emetine decreased more slowly than that of 3H-cephaeline. For 3H-cephaeline, the cumulative biliary excretion of radioactivity was 57.5% at 48 hours. The cumulative urinary and fecal excretion of radioactivity in these rats was 16.5% and 29.1%, respectively, of the dose at 48 hours following dosing. For 3H-emetine, the cumulative biliary excretion of radioactivity was 12.5% at 48 hours. The cumulative urinary and fecal excretion of radioactivity was 9.4% and 34.1%, respectively, of the administered dose at 48 hours. The radioactivity level of 3H-emetine remaining in the carcasses at 48 hours was equivalent to approximately 50% of the dose. A portion of each tritium-labeled compound was subjected to entero-hepatic circulation. Thus, the absorption rate of 3H-cephaeline and 3H-emetine was estimated to be approximately 70% on the basis of the data obtained from excretion studies. There was no difference in the absorption process between these two compounds. However, the difference was admitted in the biliary clearance, which is the main excretion route of both compounds. Delayed excretion of 3H-emetine may be primarily due to its resorption as related to entero-hepatic circulation and tissue retention. This study has determined the absorption, distribution and excretion of 3H-cephaeline and 3H-emetine in rats.

Biotransformation of the ipecac alkaloids cephaeline and emetine from ipecac syrup in rats.

The metabolism of cephaeline and emetine, which are the primary active components of ipecac syrup, were investigated in rats. Cephaeline-6'-O-glucuronide was found to be a biliary metabolite of cephaeline. Cephaeline (6'-O-demethylemetine) and 9-O-demethylemetine were observed to be enzyme-hydrolyzed biliary metabolites of emetine. Cephaeline was conjugated to glucuronide, while emetine was demethylated to cephaeline and 9-0-demethylemetine, and may be conjugated to glucuronides afterwards. Urine, feces and bile were collected from rats within 48 hours following the administration of ipecac syrup containing tritium (3H)--labeled cephaeline or emetine. Metabolites were separated and quantified by thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Biliary and urinary excretion rates of 3H-cephaeline were 57.5% and 16.5% of the dose, respectively. Cephaeline-6'-O-glucuronide was comprised 79.5% of biliary radioactivity and 84.3% of urinary radioactivity. Unchanged cephaeline was detected in 42.4% of the dose in feces. Biliary excretion rate of 3H-emetine was 6.9% of the dose. Emetine, cephaeline and 9-0-demethylemetine comprised 5.8%, 43.2% and 13.6% in hydrolyzed bile, respectively. There were no emetine-derived metabolites in urine or feces. The occurrence of unchanged emetine was 6.8% and 19.7% of the dose in urine and feces, respectively.