One-shot reflectance direction field imaging for measuring the surface slope distribution of a capillary wave.

A method for measuring a surface slope distribution of a capillary wave is proposed. The method uses an optical imaging system that can capture a one-shot image of a light-reflectance direction field in a two-dimensional image plane. A dispersion relation between the wavelength and frequency of the capillary wave is shown to be obtainable by the imaging system, which agrees well with the theoretical prediction.

Points-connecting neural network ray tracing.

Unsupervised neural network ray tracing (NNRT) to calculate a light ray path connecting given points in a gradient-index medium is proposed here. If two points are given, the NNRT can provide a light ray path passing through these points without knowledge of the light ray direction. Maxwell's fisheye lens having a spherical gradient-index is used to demonstrate how well the NNRT works. Light rays calculated using the NNRT are shown to trace an ideal path passing through given points.

Gradient-index dark hole based on conformal mapping with etendue conservation.

An omnidirectional light collector consisting of an axisymmetric spatially gradient refractive index medium can almost perfectly absorb light rays, regardless of where they come from. Based on the conformal mapping with complex gradient-index medium, the omnidirectional light collector, which is here called a dark hole, is able to be designed with an exponential function. The dark hole, however, has a reflective boundary where the Fresnel reflection occurs, which might lessen the absorption efficiency. To design a dark hole with consideration of the Fresnel reflection loss, a method to estimate its absorptance is necessary. Therefore, a formula to calculate the absorptance of the dark hole is derived based on the Lagrangian optics with the etendue conservation. Absorptances calculated using the formula agree well with those calculated using the Mie scattering theory in refractive index small-difference limit, which validates the formula. Absorptance of a dark hole with a silicon core and another dark hole with a complex gradient-index intermediate medium are calculated using the formula to be more than 98.8%. A micro-size dark hole is also shown to efficiently collect light rays with an absorptance of more than 95% using FDTD (finite-difference time-domain) simulation.

GM-CSF but not IL-17 is critical for the development of severe interstitial lung disease in SKG mice.

Interstitial lung disease (ILD) is a common complication and sometimes a prognostic factor of connective tissue diseases (CTDs) in humans. However, suitable animal model of severe CTD-associated ILD (CTD-ILD) has been limited. In this study, we showed that zymosan-treated SKG mice developed not only arthritis but also chronic-progressive ILD with high mortality over several months. The pathological and clinical features of ILD in zymosan-treated SKG mice were similar to that of human severe CTD-ILD. ILD in this mouse was characterized by massive infiltration of Th17 cells, GM-CSF-producing CD4(+) T cells, and CD11b(+) Gr1(+) neutrophils with fibrosis. Naive SKG T cells were skewed to differentiate into GM-CSF-producing cells, and GM-CSF secreted by T cells enhanced IL-6 and IL-1ß production by macrophages, which in turn enhanced differentiation of IL-17A- and/or GM-CSF-producing T cells and infiltration of neutrophils into lung. Neutralization of GM-CSF completely blocked the development of this ILD, and the blocking of IL-6 signaling resulted in partial prevention of it, whereas neutralization of IL-17A did not. In contrast, the progression of arthritis was inhibited by the neutralization of GM-CSF and slightly by the neutralization of IL-17A, but not by the blocking of IL-6 signaling. These data suggested zymosan-treated SKG mice could be a useful mouse model of severe CTD-ILD, and GM-CSF, rather than IL-17A or IL-6, contributed to the development of ILD in zymosan-treated SKG mice, indicating that neutralization of GM-CSF would be a useful therapeutic strategy for severe CTD-ILD.

Murine Cyp3a knockout chimeric mice with humanized liver: prediction of the metabolic profile of nefazodone in humans.

Chimeric mice with humanized livers (PXB mice) are used to investigate the metabolism and pharmacokinetics of drugs in humans. However, residual murine enzymatic activities derived from the liver and the presence of mouse small intestinal metabolism can hamper the prediction of human drug metabolism. Recently murine Cytochrome P450 3a gene knockout chimeric mice with humanized livers (Cyp3a KO CM) were developed. To evaluate the prediction of drug metabolism, nefazodone (NEF) was administered orally at 10 mg/kg to the following mouse strains: Cyp3a KO CM, murine Cyp3a gene knockout (Cyp3a KO), PXB and severe combined immunodeficiency (SCID) mice. Liquid chromatography-mass spectrometry was used for metabolic profiling of plasma, urine and bile. The prediction of human metabolite levels such as hydroxy nefazodone (OH-NEF), triazoledione form (TD), m-chlorophenylpiperazine and dealkyl metabolites in Cyp3a KO CM was superior to that in Cyp3a KO, PXB or SCID mice. Further, clinical exposure levels of NEF, OH-NEF and TD were reproduced in Cyp3a KO CM. In contrast, NEF was rapidly metabolized to TD in both PXB and SCID mice but not in Cyp3a KO mice, suggesting that murine CYP3A is involved in the elimination of NEF in these mice. These findings demonstrate that the metabolic profile of NEF in Cyp3a KO CM differs qualitatively and quantitatively from that in PXB mice due to the higher metabolic rate of NEF and its metabolites via murine CYP3A. Therefore Cyp3a KO CM might be useful in predicting the metabolic profiles of drug candidates in humans.

Functional engraftment of human peripheral T and B cells and sustained production of autoantibodies in NOD/LtSzscid/IL-2Rγ(-/-) mice.

NOD/LtSzscid/IL-2Rγ(-/-) (NSG) mice have advantages in establishing humanized mouse models. However, transferring human PBMCs into these mice often causes lethal GVH disease. In this study, we discovered an improved method for the engraftment of normal or pathological human PBMCs into NSG mice and examined the subsequent induction of specific immune responses. We sequentially transferred human CD4+ memory T (Tm) and B cells obtained from PBMCs of healthy adults or patients with autoimmune diseases into NSG mice. Removing naïve CD4+ T cells from the transferred PBMCs allowed successful engraftment without lethal GVH disease. The transferred Tm cells were found to reside mainly in the spleen and the lymphoid nodules, where they expressed MHC class II molecules and produced cytokines, including IL-21. Surprisingly, the transferred B cells were also well maintained in the lymphoid organs, underwent de novo class-switch recombination, and secreted all isotypes of human Igs at significant levels. Moreover, transferring patient-derived Tm and B cells resulted in sustained production of IgM-rheumatoid factor and antiaminoacyl transfer RNA synthetase Abs in these mice. These results suggest that transfer of Tm and B cells derived from human PBMCs into NSG mice could be a useful method for the study of human autoimmune mechanisms.

A novel splice variant of human L-selectin encodes a soluble molecule that is elevated in serum of patients with rheumatic diseases.

L-selectin, a type I membrane protein, is a leukocyte adhesion molecule that mediates both lymphocyte homing to peripheral lymph nodes and leukocyte accumulation at sites of inflammation. L-selectin is rapidly shed from the cell surface after cellular activation, and the ectodomain thus released is thought to account for high levels of soluble L-selectin in serum. In this study, we report the identification of a novel, naturally occurring isoform of the human L-selectin gene. Sequence analysis revealed that this isoform is generated by an alternative splicing event: the 7th exon of the human L-selectin gene, which encodes the region containing the transmembrane domain, is excluded, predicting a soluble protein product. The mRNA for this splice variant was expressed in lymphoid organs, where conventional L-selectin mRNA was also expressed. Activating T cells increased the variant mRNA and its ratio to the membrane form. Soluble L-selectin translated from the variant mRNA was present in human serum, albeit at a much lower level than that arising from ectodomain shedding, and was markedly elevated in patients with various rheumatic diseases, including rheumatoid arthritis and systemic lupus erythematosus. These observations indicate that some of the soluble L-selectin present in human serum arises through alternative splicing, which may be upregulated during lymphocyte activation in patients with various clinical conditions.

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice.

Pivotal roles of GM-CSF in autoimmunity and inflammation.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor, which stimulates the proliferation of granulocytes and macrophages from bone marrow precursor cells. In autoimmune and inflammatory diseases, Th17 cells have been considered as strong inducers of tissue inflammation. However, recent evidence indicates that GM-CSF has prominent proinflammatory functions and that this growth factor (not IL-17) is critical for the pathogenicity of CD4(+) T cells. Therefore, the mechanism of GM-CSF-producing CD4(+) T cell differentiation and the role of GM-CSF in the development of autoimmune and inflammatory diseases are gaining increasing attention. This review summarizes the latest knowledge of GM-CSF and its relationship with autoimmune and inflammatory diseases. The potential therapies targeting GM-CSF as well as their possible side effects have also been addressed in this review.

Laparoscopic Cholecystectomy for a Patient with Left-sided Gallbladder.

A 47-year-old man who presented with epigastric pain after a meal was diagnosed with biliary sludge present in the gallbladder. Endoscopic retrograde cholangiopancreatography showed normal anatomy of the biliary tree. During the exploratory phase of a laparoscopic cholecystectomy using four ports positioned as usual, surgeons observed a left-sided gallbladder. A review of the preoperative imaging by computed tomography confirmed a round ligament connected to the right portal umbilical portion. It also established that the gallbladder was located to the left of the round ligament, and attached to the left lateral segment of the liver. Laparoscopic cholecystectomy was performed successfully in this patient with the usual port site and careful dissection with a normograde approach. The patient was discharged on the second postoperative day with an uneventful course. Prior identification of a left-sided gallbladder is possible with cautious attention. In particular, it is important for the surgeon to be aware of unusual alterations in the portal and biliary anatomy associated with this anomaly to safely complete a laparoscopic cholecystectomy.

[A case of recurrent gastric cancer successfully treated with S-1 oral administration].

This report describes a case of recurrent gastric cancer successfully treated with S-1 oral administration. A 77-year old female patient underwent distal gastrectomy for gastric cancer, followed by adjuvant chemotherapy with tegafur-uracil (UFT). However, 1 year after surgical resection, recurrence in the lymph node of the hepatic hilum was diagnosed by abdominal computed tomography. The patient was treated with S-1 alone after refusing in travenous infusion chemotherapy. Three months after treatment, the size of the target lesion decreased significantly, and a complete response was seen on imaging examination during the 2 years of chemotherapy treatment. One year and 5 months after the discontinuation of chemotherapy, recurrence was noted again. Although supportive care was eventually provided to the patient, S-1 oral administration was resumed that resulted in tumor growth control for>6 months. In this patient, S-1 treatment was effective in tumor growth suppression without deteriorating the patient's quality of life (QOL). Further studies are needed to identify patients for whom S-1 therapy is optimal treatment.

Significant association between CYP3A5 polymorphism and blood concentration of tacrolimus in patients with connective tissue diseases.

Although the association between CYP3A5 polymorphism and blood concentration of tacrolimus (TAC) in patients with solid organ transplantation was established, whether the association is also true in patients with connective tissue disease (CTD) who usually receive small amount of TAC is uncertain. Here, we performed a quantitative linear regression analysis to address the association between CYP3A5 and blood TAC concentration in patients with CTD. A total of 72 patients with CTD were recruited in the current study and genotyped for rs776746 in CYP3A5, which showed strong association with TAC concentration in patients with solid organ transplantation. The blood trough concentration of TAC after taking 3 mg per day was retrospectively obtained for each patient. As a result, allele A of rs776746 showed a significant association with a decreasing blood concentration of TAC (P=0.0038). Those who are carrying at least one copy of the A allele displayed decreased mean concentration of TAC by 31.0% compared with subjects with GG genotype. Rs776746 is associated with concentrations of TAC in patients with CTD.

Optimized methods for targeted peptide-based quantification of human uridine 5'-diphosphate-glucuronosyltransferases in biological specimens using liquid chromatography-tandem mass spectrometry.

The aim of this study was to optimize methods for quantifying 13 uridine 5'-diphosphate-glucuronosyltransferase (UGT) isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17) in human liver, intestinal, and kidney microsomes, and in recombinant human UGT-expressing insect cell membranes (rhUGTs) by targeted peptide-based quantification using liquid chromatography-tandem mass spectrometry. Production of targeted peptides was compared by combining three denaturing agents (urea, sodium deoxycholate, and octyl glucoside) and three denaturing temperatures (37°C, 60°C, and 95°C) followed by tryptic digestion for 2-20 hours. Denaturing conditions and digestion times yielding high production efficiency varied markedly among isoforms and specimens, indicating the importance of specific optimization. Each UGT isoform was quantified using the methods found to be optimal. The expression of 10 (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17), 6 (1A1, 1A3, 1A4, 1A10, 2B7, and 2B17), and 3 (1A6, 1A9, and 2B7) isoforms was detected in human liver, intestinal, and kidney microsomes, respectively, and levels were reproducible using multiple protocols. All isoforms were quantified in rhUGTs. Determining the levels of UGTs in human tissue specimens and those in rhUGTs is important for estimating the contribution of glucuronidation to body clearance based on in vitro-in vivo extrapolation.

Impact of genetic deficiencies of P-glycoprotein and breast cancer resistance protein on pharmacokinetics of aripiprazole and dehydroaripiprazole.

1. We investigated how deficiencies in P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) affect the pharmacokinetics of atypical antipsychotics aripiprazole and its active metabolite (dehydroaripiprazole) using normal Friend leukemia virus strain B (FVB) mice, BCRP knockout (Bcrp[-/-]) mice, and P-gp and BCRP triple knockout (Mdr1a/1b[-/-]Bcrp[-/-]) mice. 2. While plasma concentrations of aripiprazole and dehydroaripiprazole after oral administration were slightly higher in both Bcrp(-/-) and Mdr1a/1b(-/-)/Bcrp(-/-) mice than in normal FVB mice, the difference was not marked. The increase in absolute bioavailability (F) compared with normal mice (approximately 1.3-fold increase) was comparable between Bcrp(-/-) and Mdr1a/1b(-/-)/Bcrp(-/-) mice. This finding suggests that BCRP may be involved in the intestinal absorption of aripiprazole in mice, albeit with minimal contribution to absorption at best. 3. In contrast, the brain-to-plasma concentration ratio (Kp,brain) for aripiprazole and dehydroaripiprazole after oral administration was significantly higher in Mdr1a/1b(-/-)/Bcrp(-/-) mice than in normal mice, whereas Bcrp(-/-) mice exhibited Kp,brain values similar to those in normal mice. In addition, the Kp,brain values in Mdr1a/1b(-/-)/Bcrp(-/-) mice were not drastically different from those previously reported in Mdr1a/1b(-/-) mice, suggesting that brain penetration of aripiprazole and dehydroaripiprazole can be affected by P-gp, but with little synergistic effect of BCRP.

In vitro experimental system for evaluating inhibitory effect of investigational drugs on P-glycoprotein-mediated transcellular transport of tacrolimus (FK506).

In this study, an in vitro experimental system for evaluating the inhibitory effect of investigational drugs on the P-glycoprotein (P-gp, MDR1)-mediated transport of tacrolimus (FK506) was developed using LLC-PK1-MDR1 and LLC-PK1 wild-type (control) cells. The amount of tacrolimus (concentrations: 1 and 5 µm) transported into P-gp-expressing and control cells increased with time in both the apical-to-basal and basal-to-apical directions at incubation times ranging from 40 min to 2 h. The corrected apparent permeability (Papp) ratio, obtained by dividing the Papp ratio in P-gp-expressing cells by that in the control cells, ranged from 2.6 to 5.3, showing significant differences in the transport of tacrolimus between the P-gp-expressing cells and the control cells. This system was then subsequently used to examine the P-gp transport of tacrolimus in the presence of verapamil (30 µm), a model inhibitor for P-gp-mediated transport activity. The corrected Papp ratios in the absence and presence of verapamil were 6.9 and 0.8, respectively. Data derived in the present study suggest that our developed system has the ability to detect a sufficient difference in the P-gp transport of tacrolimus between P-gp-expressing and control cells, and we therefore believe our system to be suitable for use in evaluating the inhibitory effects of investigational drugs on the P-gp-mediated transport of tacrolimus.

Coordinated roles of pregnane X receptor and constitutive androstane receptor in autoinduction of voriconazole metabolism in mice.

The antifungal efficacy of voriconazole (VRC) differs among host species, with potent efficacy in humans but less in rodents. We investigated the possible involvement of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in the species-specific efficacy of VRC through pharmacokinetic analyses using genetically modified mice and primary human hepatocytes. VRC (30 mg/kg) was orally administered to wild-type, Pxr-null, Car-null, and Pxr- and Car-null (Pxr/Car-null) mice for 7 days. Hepatic VRC metabolism was significantly increased by VRC administration, and the elimination rates of plasma VRC were much higher on day 7 than on day 1 in wild-type mice. This autoinduction was also observed in Pxr-null and Car-null mice but not in Pxr/Car-null mice, suggesting coordinated roles of PXR and CAR in the autoinduction of VRC metabolism in mice. Hepatic Cyp3a11 mRNA levels were increased by VRC administration, hepatic metabolic activities for VRC were correlated with CYP3A activities, and the induced VRC metabolism was inhibited by ketoconazole (a CYP3A inhibitor). In primary human hepatocytes, VRC barely increased mRNA levels of CYP3A4 and CYP2B6 (human PXR/CAR target genes) at its therapeutic concentrations. In conclusion, these results suggest that VRC is metabolized mainly by CYP3A11 in mouse livers and that PXR- and CAR-mediated CYP3A11 induction, namely, autoinduction of VRC metabolism, is a primary reason for the ineffectiveness of VRC in mice. A limited ability of VRC to activate human PXR/CAR at its clinical concentration might explain the VRC efficacy in humans. Therefore, the ability to activate PXR/CAR might determine the VRC efficacy in different mammalian species.

Intestinal absorption mechanism of mirabegron, a potent and selective ß3-adrenoceptor agonist: involvement of human efflux and/or influx transport systems.

Mirabegron, a weak-basic compound, is a potent and selective ß3-adrenoceptor agonist for the treatment of overactive bladder. Mirabegron extended release formulation shows dose-dependent oral bioavailability in humans, which is likely attributable to saturation of intestinal efflux abilities leading to higher absorption with higher doses. This study evaluated the membrane permeability of mirabegron and investigated the involvement of human intestinal transport proteins in the membrane permeation of mirabegron. Transcellular transport and cellular/vesicular uptake assays were performed using Caco-2 cells and/or human intestinal efflux (P-glycoprotein [P-gp], breast cancer resistance protein [BCRP], and multidrug resistance associated protein 2 [MRP2]) and influx (peptide transporter 1 [PEPT1], OATP1A2, and OATP2B1) transporter-expressing cells, vesicles, or Xenopus laevis oocytes. The absorptive permeability coefficients of mirabegron in Caco-2 cells (1.68-1.83 × 10(-6) cm/s) at the apical and basal pH of 6.5 and 7.4, respectively, were slightly higher than those of nadolol (0.97-1.41 × 10(-6) cm/s), a low permeability reference standard, but lower than those of metoprolol and propranolol (both ranged from 8.49 to 11.6 × 10(-6) cm/s), low/high permeability boundary reference standards. Increasing buffer pH at the apical side from 5.5 to 8.0 gradually increased the absorptive permeation of mirabegron from 0.226 to 1.66 × 10(-6) cm/s, but was still less than the value in the opposite direction (11.0-14.2 × 10(-6) cm/s). The time- and concentration-dependent transport of mirabegron was observed in P-gp-expressing cells and OATP1A2-expressing oocytes with apparent Km values of 294 and 8.59 µM, respectively. In contrast, no clear BCRP-, MRP2-, PEPT1-, or OATP2B1-mediated uptake of mirabegron was observed in their expressing vesicles or cells. These findings suggest that mirabegron has low-to-moderate membrane permeability and P-gp is likely to be involved in its efflux into the lumen in the intestinal absorption process. The results also suggest that mirabegron could possibly be transported by intestinal influx transporters as well as simple diffusion.

Absorption, metabolism and excretion of darexaban (YM150), a new direct factor Xa inhibitor in humans.

1. The absorption, metabolism and excretion of darexaban (YM150), a novel oral direct factor Xa inhibitor, were investigated after a single oral administration of [(14)C]darexaban maleate at a dose of 60 mg in healthy male human subjects. 2. [(14)C]Darexaban was rapidly absorbed, with both blood and plasma concentrations peaking at approximately 0.75 h post-dose. Plasma concentrations of darexaban glucuronide (M1), the pharmacological activity of which is equipotent to darexaban in vitro, also peaked at approximately 0.75 h. 3. Similar amounts of dosed radioactivity were excreted via faeces (51.9%) and urine (46.4%) by 168 h post-dose, suggesting that at least approximately half of the administered dose is absorbed from the gastrointestinal tract. 4. M1 was the major drug-related component in plasma and urine, accounting for up to 95.8% of radioactivity in plasma. The N-oxides of M1, a mixture of two diastereomers designated as M2 and M3, were also present in plasma and urine, accounting for up to 13.2% of radioactivity in plasma. In faeces, darexaban was the major drug-related component, and N-demethyl darexaban (M5) was detected as a minor metabolite. 5. These findings suggested that, following oral administration of darexaban in humans, M1 is quickly formed during first-pass metabolism via UDP-glucuronosyltransferases, exerting its pharmacological activity in blood before being excreted into urine and faeces.

T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription.

T helper type 1 (Th1) development is facilitated by interrelated changes in key intracellular factors, particularly signal transducer and activator of transcription (STAT)4, T-bet, and GATA-3. Here we show that CD4+ cells from T-bet-/- mice are skewed toward Th2 differentiation by high endogenous GATA-3 levels but exhibit virtually normal Th1 differentiation provided that GATA-3 levels are regulated at an early stage by anti-interleukin (IL)-4 blockade of IL-4 receptor (R) signaling. In addition, under these conditions, Th1 cells from T-bet-/- mice manifest IFNG promotor accessibility as detected by histone acetylation and deoxyribonuclease I hypersensitivity. In related studies, we show that the negative effect of GATA-3 on Th1 differentiation in T-bet-/- cells arises from its ability to suppress STAT4 levels, because if this is prevented by a STAT4-expressing retrovirus, normal Th1 differentiation is observed. Finally, we show that retroviral T-bet expression in developing and established Th2 cells leads to down-regulation of GATA-3 levels. These findings lead to a model of T cell differentiation that holds that naive T cells tend toward Th2 differentiation through induction of GATA-3 and subsequent down-regulation of STAT4/IL-12Rbeta2 chain unless GATA-3 levels or function is regulated by T-bet. Thus, the principal function of T-bet in developing Th1 cells is to negatively regulate GATA-3 rather than to positively regulate the IFNG gene.

Conclusive identification of the oxybutynin-hydrolyzing enzyme in human liver.

The aim of this study was to conclusively determine the enzyme responsible for the hydrolysis of oxybutynin in human liver. Hydrolysis in human liver microsomes (HLMs) and human liver cytosol (HLC) followed Michaelis-Menten kinetics with similar K(m) values. In recombinant human carboxylesterase (CES)-expressing microsomes, CES1 was much more efficient than CES2 and yielded a K(m) value more comparable with that found in HLMs or HLC than did CES2. A correlation analysis using a set of individual HLMs, in which both CESs acted independently showed that the hydrolysis rate of oxybutynin, correlated significantly with a CES1 marker reaction, clopidogrel hydrolysis, but not with a CES2 marker reaction, irinotecan (CPT-11) hydrolysis. Chemical inhibition studies using bis-(p-nitrophenyl) phosphate, clopidogrel, nordihydroguaiaretic acid, procainamide, physostigmine, and loperamide revealed that the effects of these compounds in HLMs, HLC, and recombinant CES1-expressing microsomes were similar, whereas those in CES2-expressing microsomes were clearly different. These results strongly suggest that CES1, rather than CES2, is the principal enzyme responsible for the hydrolysis of oxybutynin in human liver.