Impact of green tea intake on the pharmacokinetics of celiprolol in healthy subjects.

OBJECTIVE: To assess the effect of green tea intake on the pharmacokinetics of the ß-blocker celiprolol. MATERIALS AND METHOD: In an open-label crossover study, 3 healthy subjects were given water or a green tea beverage daily for 3 days. On day 4, each subject received a single oral dose of 200 mg celiprolol with water or green tea. Serum and urinary concentrations of celiprolol were measured for up to 24 hours. RESULTS: Green tea intake decreased the area under the serum concentration-time curve and urinary excretion of celiprolol by 98.6 and 98.0%, respectively. CONCLUSION: Green tea intake might have a negative impact on the clinical effectiveness of celiprolol.

Antibody-mediated activation of the FGFR1/Klothoß complex corrects metabolic dysfunction and alters food preference in obese humans.

Fibroblast growth factor 21 (FGF21) controls metabolic organ homeostasis and eating/drinking behavior via FGF receptor 1/Klothoß (FGFR1/KLB) complexes expressed in adipocytes, pancreatic acinar cells, and the nervous system in mice. Chronic administration of recombinant FGF21 or engineered variants improves metabolic health in rodents, nonhuman primates, and humans; however, the rapid turnover of these molecules limits therapeutic utility. Here we show that the bispecific anti-FGFR1/KLB agonist antibody BFKB8488A induced marked weight loss in obese cynomolgus monkeys while elevating serum adiponectin and the adipose expression of FGFR1 target genes, demonstrating its action as an FGF21 mimetic. In a randomized, placebo-controlled, single ascending-dose study in overweight/obese human participants, subcutaneous BFKB8488A injection caused transient body weight reduction, sustained improvement in cardiometabolic parameters, and a trend toward reduction in preference for sweet taste and carbohydrate intake. These data suggest that specific activation of the FGFR1/KLB complex in humans can be used as therapy for obesity-related metabolic defects.

Adaptive adipose tissue stromal plasticity in response to cold stress and antibody-based metabolic therapy.

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.

Drug-drug interactions among drugs prescribed for nontuberculous mycobacterial infection and epilepsy: A case report.

WHAT IS KNOWN AND OBJECTIVE: Multi-drug combinations often make chemotherapy difficult owing to drug-drug interactions (DDIs). We report a rare and difficult-to-treat case due to DDIs between drugs for Mycobacterium avium complex (MAC) infection and antiepileptic drugs. CASE DESCRIPTION: A 70-year-old Japanese woman was diagnosed as having pulmonary MAC disease. She had a history of symptomatic epilepsy, which was successfully treated with phenytoin and phenobarbital. Serum phenytoin concentrations increased upon the initiation of MAC infection treatment. WHAT IS NEW AND CONCLUSION: We evaluated DDIs and adjusted the dosage of drugs by monitoring the serum drug level.

A pharmacology guided approach for setting limits on product-related impurities for bispecific antibody manufacturing.

INTRODUCTION: bFKB1 is a humanized bispecific IgG1 antibody, created by conjoining an anti-Fibroblast Growth Factor Receptor 1 (FGFR1) half-antibody to an anti-Klothoß (KLB) half-antibody, using the knobs-into-holes strategy. bFKB1 acts as a highly selective agonist for the FGFR1/KLB receptor complex and is intended to ameliorate obesity-associated metabolic defects by mimicking the activity of the hormone FGF21. An important aspect of the biologics product manufacturing process is to establish meaningful product specifications regarding the tolerable levels of impurities that copurify with the drug product. The aim of the current study was to determine acceptable levels of product-related impurities for bFKB1. METHODS: To determine the tolerable levels of these impurities, we dosed obese mice with bFKB1 enriched with various levels of either HMW impurities or anti-FGFR1-related impurities, and measured biomarkers for KLB-independent FGFR1 signaling. RESULTS: Here, we show that product-related impurities of bFKB1, in particular, high molecular weight (HMW) impurities and anti-FGFR1-related impurities, when purposefully enriched, stimulate FGFR1 in a KLB-independent manner. By taking this approach, the tolerable levels of product-related impurities were successfully determined. DISCUSSION: Our study demonstrates a general pharmacology-guided approach to setting a product specification for a bispecific antibody whose homomultimer-related impurities could lead to undesired biological effects.

Cardiovascular Effects of Renal Distal Tubule Deletion of the FGF Receptor 1 Gene.

The bone-derived hormone fibroblast growth factor-23 (FGF-23) activates complexes composed of FGF receptors (FGFRs), including FGFR1, and α-Klotho in the kidney distal tubule (DT), leading to increased sodium retention and hypertension. However, the role of FGFR1 in regulating renal processes linked to hypertension is unclear. Here, we investigated the effects of selective FGFR1 loss in the DT. Conditional knockout (cKO) of FGFR1 in the DT (FGFR1DT-cKO mice) resulted in left ventricular hypertrophy (LVH) and decreased kidney expression of α-Klotho in association with enhanced BP, decreased expression of angiotensin converting enzyme 2, and increased expression of the Na+-K+-2Cl- cotransporter. Notably, recombinant FGF-23 administration similarly decreased the kidney expression of α-Klotho and induced LVH in mice. Pharmacologic activation of FGFR1 with a monoclonal anti-FGFR1 antibody (R1MAb1) normalized BP and significantly attenuated LVH in the Hyp mouse model of excess FGF-23, but did not induce a response in FGFR1DT-cKO mice. The hearts of FGFR1DT-cKO mice showed increased expression of the transient receptor potential cation channel, subfamily C, member 6 (TRPC6), consistent with cardiac effects of soluble Klotho deficiency. Moreover, administration of recombinant soluble Klotho lowered BP in the Hyp mice. Thus, FGFR1 in the DT regulates systemic hemodynamic responses opposite to those predicted by the actions of FGF-23. These cardiovascular effects appear to be mediated by paracrine FGF control of kidney FGFR1 and subsequent regulation of soluble Klotho and TRPC6. FGFR1 in the kidney may provide a new molecular target for treating hypertension.

FGF21 mimetic antibody stimulates UCP1-independent brown fat thermogenesis via FGFR1/ßKlotho complex in non-adipocytes.

OBJECTIVE: Fibroblast Growth Factor 21 (FGF21) is a potent stimulator of brown fat thermogenesis that improves insulin sensitivity, ameliorates hepatosteatosis, and induces weight loss by engaging the receptor complex comprised of Fibroblast Growth Factor Receptor 1 (FGFR1) and the requisite coreceptor ßKlotho. Previously, recombinant antibody proteins that activate the FGFR1/ßKlotho complex were proposed to act as an FGF21-mimetic; however, in vivo action of these engineered proteins has not been well studied. METHODS: We investigated the mechanism by which anti-FGFR1/ßKlotho bispecific antibody (bFKB1) stimulates thermogenesis in UCP1-expressing brown adipocytes using genetically engineered mice. Anti-FGFR1 agonist antibody was also used to achieve brown adipose tissue restricted activation in transgenic mice. RESULTS: Studies with global Ucp1-deficient mice and adipose-specific Fgfr1 deficient mice demonstrated that bFKB1 acts on targets distal to adipocytes and indirectly stimulates brown adipose thermogenesis in a UCP1-independent manner. Using a newly developed transgenic system, we also show that brown adipose tissue restricted activation of a transgenic FGFR1 expressed under the control of Ucp1 promoter does not stimulate energy expenditure. Finally, consistent with its action as a FGF21 mimetic, bFBK1 suppresses intake of saccharin-containing food and alcohol containing water in mice. CONCLUSIONS: Collectively, we propose that FGFR1/ßKlotho targeted therapy indeed mimics the action of FGF21 in vivo and stimulates UCP1-independent brown fat thermogenesis through receptors outside of adipocytes and likely in the nervous system.

Tethered-variable CL bispecific IgG: an antibody platform for rapid bispecific antibody screening.

Bispecific antibodies offer a clinically validated platform for drug discovery. In generating functionally active bispecific antibodies, it is necessary to identify a unique parental antibody pair to merge into a single molecule. However, technologies that allow high-throughput production of bispecific immunoglobulin Gs (BsIgGs) for screening purposes are limited. Here, we describe a novel bispecific antibody format termed tethered-variable CLBsIgG (tcBsIgG) that allows robust production of intact BsIgG in a single cell line, concurrently ensuring cognate light chain pairing and preserving key antibody structural and functional properties. This technology is broadly applicable in the generation of BsIgG from a variety of antibody isotypes, including human BsIgG1, BsIgG2 and BsIgG4. The practicality of the tcBsIgG platform is demonstrated by screening BsIgGs generated from FGF21-mimetic anti-Klotho-ß agonistic antibodies in a combinatorial manner. This screen identified multiple biepitopic combinations with enhanced agonistic activity relative to the parental monoclonal antibodies, thereby demonstrating that biepitopic antibodies can acquire enhanced functionality compared to monospecific parental antibodies. By design, the tcBsIgG format is amenable to high-throughput production of large panels of bispecific antibodies and thus can facilitate the identification of rare BsIgG combinations to enable the discovery of molecules with improved biological function.

FGF19, FGF21, and an FGFR1/ß-Klotho-Activating Antibody Act on the Nervous System to Regulate Body Weight and Glycemia.

Despite the different physiologic functions of FGF19 and FGF21 as hormonal regulators of fed and fasted metabolism, their pharmacologic administration causes similar increases in energy expenditure, weight loss, and enhanced insulin sensitivity in obese animals. Here, in genetic loss-of-function studies of the shared co-receptor ß-Klotho, we show that these pharmacologic effects are mediated through a common, tissue-specific pathway. Surprisingly, FGF19 and FGF21 actions in liver and adipose tissue are not required for their longer-term weight loss and glycemic effects. In contrast, ß-Klotho in neurons is essential for both FGF19 and FGF21 to cause weight loss and lower glucose and insulin levels. We further show an FGF21 mimetic antibody that activates the FGF receptor 1/ß-Klotho complex also requires neuronal ß-Klotho for its metabolic effects. These studies highlight the importance of the nervous system in mediating the beneficial weight loss and glycemic effects of endocrine FGF drugs.

Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP).

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

Preclinical pharmacokinetic characterization of an adipose tissue-targeting monoclonal antibody in obese and non-obese animals.

Target receptor levels can influence pharmacokinetics (PK) or pharmacodynamics (PD) of monoclonal antibodies (mAbs), and can affect drug development of this class of molecules. We generated an effector-less humanized bispecific antibody that selectively activates fibroblast growth factor receptor (FGFR)1 and ßKlotho receptor, a FGF21 receptor complex highly expressed in both white and brown adipocytes. The molecule shows cross-species binding with comparable equilibrium binding affinity (Kd) for human, cynomolgus monkey, and mouse FGFR1/ßKlotho. To understand the PK/PD relationship in non-obese and obese animals, we evaluated the adipose tissue distribution of the antibody, serum exposures, and an associated PD marker (high-molecular-weight adiponectin), in both non-obese and obese mice and monkeys. Antibody uptake into fat tissue was found to be higher on a per gram basis in non-obese animals compared to obese animals. Since obesity has been reported to be associated with reduced expression of FGFR1 and ßKlotho receptor in white adipose tissues in mice, our results suggest that the distribution in adipose tissues was influenced by target expression levels. Even so, the overall dose-normalized serum exposures were comparable between non-obese and obese mice and monkeys, suggesting that adipose tissue uptake plays a limited role in overall systemic PK determination. It remains to be determined if and how obesity and receptor expression in humans influence the PK and PD profile of this novel therapeutic candidate.

The detection of influenza virus at the community pharmacy to improve the management of local residents with influenza or influenza-like disease.

BACKGROUND: As of 2014, community pharmacies in Japan are approved by the Ministry of Health, Labour and Welfare to measure lipid panel, HbA1c, glucose, ALT, AST and γ-GTP, but not to screen for influenza virus. We provided influenza virus screening tests at a community pharmacy to triage people with symptoms suggestive of influenza. Participants were given appropriate advice on how to prevent the spread of and safeguard against influenza. We subsequently evaluated the effects of community pharmacy-based influenza virus screening and prevention measures. METHODS: Local residents with symptoms suggestive of influenza participated in this study. Influenza virus screening tests using nasal samples were provided to the pharmacy, and we assessed samples for the presence of influenza virus. The study consisted of a preliminary interview, informed consent, and screening test on Day 1, and mail-in survey on Day 14. RESULTS: A total 52 local residents participated in the study. The number of participants and influenza virus positive results followed the same trend as the influenza epidemic in the study area. Influenza virus was found in 28.8% of samples. There was no significant difference between the appearance ratios of subjective symptoms among influenza-positive and influenza-negative groups. The percentages of participants who were first screened at the pharmacy, and those who were first screened at a clinic and then tested again at the pharmacy, were 71.2% (37/52) and 28.8% (15/52), respectively. In the latter group, 14 of 15 were negative by screening at the clinic, and one was diagnosed with influenza without testing. Subsequently, 46.8% (7/15) of participants tested positive for influenza by pharmacy-based screening. According to the mail-in survey, all influenza-positive (100%, 7/7) and 35.3% (6/17) of influenza-negative participants visited the clinic after being tested at the community pharmacy; test results between the community pharmacy and clinic were consistent. A total 64.7% (11/17) of symptomatic participants who tested negative recovered spontaneously at home. CONCLUSIONS: Implementation of influenza virus screening followed by provision of appropriate advice for both influenza-positive and influenza-negative participants at the community pharmacy showed a significant effect on improving the health of the local community.

FGF21-receptor agonists: an emerging therapeutic class for obesity-related diseases.

Fibroblast growth factor 21 (FGF21) analogs and FGF21 receptor agonists (FGF21RAs) that mimic FGF21 ligand activity constitute the new "FGF21-class" of anti-obesity and anti-diabetic molecules that improve insulin sensitivity, ameliorate hepatosteatosis and promote weight loss. The metabolic actions of FGF21-class proteins in obese mice are attributed to stimulation of brown fat thermogenesis and increased secretion of adiponectin. The therapeutic utility of this class of molecules is being actively investigated in clinical trials for the treatment of type 2 diabetes and non-alcoholic steatohepatitis (NASH). This review is focused on various FGF21-class molecules, their molecular designs and the preclinical and clinical activities. These molecules include modified FGF21 as well as agonistic antibodies against the receptor for FGF21, namely the complex of FGF receptor 1 (FGFR1) and the obligatory coreceptor ßKlotho (KLB). In addition, a novel approach to increase endogenous FGF21 activity by inhibiting the FGF21-degrading protease fibroblast activation protein (FAP) is discussed.

An efficient route to bispecific antibody production using single-reactor mammalian co-culture.

Bispecific antibodies have shown promise in the clinic as medicines with novel mechanisms of action. Lack of efficient production of bispecific IgGs, however, has limited their rapid advancement. Here, we describe a single-reactor process using mammalian cell co-culture production to efficiently produce a bispecific IgG with 4 distinct polypeptide chains without the need for parallel processing of each half-antibody or additional framework mutations. This method resembles a conventional process, and the quality and yield of the monoclonal antibodies are equal to those produced using parallel processing methods. We demonstrate the application of the approach to diverse bispecific antibodies, and its suitability for production of a tissue specific molecule targeting fibroblast growth factor receptor 1 and klotho ß that is being developed for type 2 diabetes and other obesity-linked disorders.

Instructing students to measure their own bone density and prepare a simulated health class during pharmacy school improves their awareness and understanding of osteoporosis prevention.

BACKGROUND: Osteoporosis is estimated to afflict over 200 million people worldwide and healthcare professionals are needed to successfully intervene. The aim of this study was to assess cognitive changes in students pertaining to the primary prevention of osteoporosis after measuring their bone density and having them participate in a simulated health class during pharmacy school. METHODS: Third year pharmacy students participated in the training program, which consisted of measuring their bone density using quantitative ultrasound and preparing educational materials and conducting a simulated health class. The students' knowledge concerning the prevention and education on osteoporosis was surveyed using questionnaires before and after the training. RESULTS: The bone area ratio (BAR) in 24 % of the students was evaluated as category 4 (slightly low) or 5 (low or caution). Regression analysis indicated a significant relationship between the BAR and amount of exercise reported in both males (p = 0.005) and females (p = 0.004). The student-made educational materials were prepared in line with the requirements of the Japanese 2011 guidelines. The student response rates for the importance of food, exercise, and the bone density measurement in youth were significantly increased after the training (p < 0.001 in all). More than 95 % of students reported that the program was useful, improved their understanding, and important, with 94 % satisfied with the experience. CONCLUSIONS: This experience-based educational program combining measuring the bone density and the preparation and presentation of a simulated health class appeared to improve the awareness and understanding of osteoporosis prevention in pharmacy students.

Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21.

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/ßKlotho Complex.

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/ßKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/ßKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/ßKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/ßKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/ßKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

Unliganded fibroblast growth factor receptor 1 forms density-independent dimers.

Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Förster resonance energy transfer combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full-length FGFR1 at the cell-surface with or without the coreceptor ßKlotho. Using this approach we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR) and was further increased by the addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and ßKlotho. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface.

Green tea catechin, epigallocatechin-3-gallate, attenuates the cell viability of human non-small-cell lung cancer A549 cells via reducing Bcl-xL expression.

Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 µM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3-100 µM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 µM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL.

Simultaneous detection of green tea catechins and gallic acid in human serum after ingestion of green tea tablets using ion-pair high-performance liquid chromatography with electrochemical detection.

We developed an analytical method for the simultaneous determination of tea catechins and gallic acid (GA) in human serum using ion-pair high-performance liquid chromatography (HPLC) with electrochemical detection. GA was measured to estimate the amount of gallate moiety produced by degradation of gallated catechins ((-)-epicatechin-3-gallate, ECG; (-)-epigallocatechin-3-gallate, EGCG). Ethyl gallate was adopted as an internal standard to correct for the extraction efficiency. To maximize extraction efficiency, a hydrophobic polytetrafluoroethylene (PTFE) filter was selected for pre-treatment prior to separation. HPLC separation was performed using a C18 reversed-phase column with a gradient mobile phase of phosphate buffer (pH 2.5) containing tetrahexylammonium hydrogensulfate as an ion-pair reagent. Using this method, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), ECG, EGCG, ethyl gallate, and GA were detected as single peaks. The resolution values for target analytes were 4.0-13.0 and the mean values of the absolute recoveries of catechins and GA were 77.3-93.9%. The detection limits for catechins and GA in serum were 0.4-3.1ng/mL. The serum catechin levels of eight healthy volunteers after ingestion of a single dose of green tea tablets were measured using this method. The concentration of total catechins (free+conjugated forms) in serum peaked 60min after ingestion. From these results, this method is thought to enable the simultaneous quantification of GA, the hydrolysis product of gallated catechins, and target catechins, and to be sufficiently sensitive for pharmacokinetic studies of catechins following oral administration of green tea.