Circulating microRNA Profiles Identify a Patient Subgroup with High Inflammation and Severe Symptoms in Schizophrenia Experiencing Acute Psychosis.

Schizophrenia is a complex and heterogenous psychiatric disorder. This study aimed to demonstrate the potential of circulating microRNAs (miRNAs) as a clinical biomarker to stratify schizophrenia patients and to enhance understandings of their heterogenous pathophysiology. We measured levels of 179 miRNA and 378 proteins in plasma samples of schizophrenia patients experiencing acute psychosis and obtained their Positive and Negative Syndrome Scale (PANSS) scores. The plasma miRNA profile revealed three subgroups of schizophrenia patients, where one subgroup tended to have higher scores of all the PANSS subscales compared to the other subgroups. The subgroup with high PANSS scores had four distinctively downregulated miRNAs, which enriched 'Immune Response' according to miRNA set enrichment analysis and were reported to negatively regulate IL-1ß, IL-6, and TNFα. The same subgroup had 22 distinctively upregulated proteins, which enriched 'Cytokine-cytokine receptor interaction' according to protein set enrichment analysis, and all the mapped proteins were pro-inflammatory cytokines. Hence, the subgroup is inferred to have comparatively high inflammation within schizophrenia. In conclusion, miRNAs are a potential biomarker that reflects both disease symptoms and molecular pathophysiology, and identify a patient subgroup with high inflammation. These findings provide insights for the precision medicinal strategies for anti-inflammatory treatments in the high-inflammation subgroup of schizophrenia.

Quantitative analysis of an impact of P-glycoprotein on edoxaban's disposition using a human physiologically based pharmacokinetic (PBPK) model.

The purpose of this study was to evaluate the impact of P-glycoprotein (P-gp) efflux on edoxaban absorption in gastrointestinal tracts quantitatively by a physiologically based pharmacokinetic (PBPK) model constructed with clinical and non-clinical observations (using GastroPlus™ software). An absorption process was described by the advanced compartmental absorption and transit model with the P-gp function. A human PBPK model was constructed by integrating the clinical and non-clinical observations. The constructed model was demonstrated to reproduce the data observed in the mass-balance study. Thus, elimination pathways can be quantitatively incorporated into the model. A constructed model successfully described the difference in slopes of plasma concentration (Cp)-time curve at around 8 - 24 hr post-dose between intravenous infusion and oral administration. Furthermore, the model without P-gp efflux activity can reproduce the Cp-time profile in the absence of P-gp activity observed from the clinical DDI study results. Since the difference of slopes between intravenous infusion and oral administration also disappeared by the absence of P-gp efflux activity, P-gp must be a key molecule to govern edoxaban's PK behavior. The constructed PBPK model will help us to understand the significant contribution of P-gp in edoxaban's disposition in gastrointestinal tracts quantitatively.

DS-7080a, a Selective Anti-ROBO4 Antibody, Shows Anti-Angiogenic Efficacy with Distinctly Different Profiles from Anti-VEGF Agents.

Purpose: Neovascular age-related macular degeneration (nAMD) results from choroidal neovascularization (CNV) and causes severe vision loss. Intravitreal anti-vascular endothelial growth factor (VEGF) therapies have significantly improved therapeutic outcomes; however, a substantial number of patients experience disease progression. Roundabout 4 (ROBO4) has been reported to be a vascular-specific protein that stabilizes vasculature in ocular pathological angiogenesis. To explore ROBO4 targeting as a novel treatment against neovascularization, we generated a humanized anti-human ROBO4 antibody, DS-7080a, and evaluated its efficacy. Methods: ROBO4 mRNA in human whole eye cross-sections was examined by in situ hybridization. Human umbilical vein endothelial cell (HUVEC) migration was measured in the presence of VEGF, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), or conditioned medium of primary human retinal pigment epithelial (HRPE) cells. CNV was induced in cynomolgus monkeys by laser irradiation. Vascular leakage was measured by fluorescein angiography, and pathological changes were determined by histology. Results: ROBO4 mRNA was detected in choroidal vessels of nAMD patients. DS-7080a suppressed HGF- or bFGF-induced HUVEC migration in addition to that induced by VEGF. Further, HUVEC migration induced by HRPE-conditioned medium was inhibited by either DS-7080a or ranibizumab in a similar manner, and the combination of these showed further inhibition. In a laser-induced CNV monkey model, single intravitreous administration of 1.1 mg/eye of DS-7080a reduced the incidence of grade 4 leakage from 44.45% in control eyes to 1.85% (P < 0.05 by Dunnett's test). Conclusions: Anti-ROBO4 antibody DS-7080a suppressed HUVEC migration in a distinctly different fashion from anti-VEGF agents and improved laser-induced CNV in non-human primates. Translational Relevance: DS-7080a may be a novel treatment option for nAMD.

A Controlled Release System for Long-Acting Intravitreal Delivery of Small Molecules.

PURPOSE: The short half lives of small molecules in the vitreous requires frequent repeated intravitreal injections that are impractical for treatment of chronic eye diseases. We sought to develop a method for increasing the intravitreal half-life of small-molecule drugs. METHODS: We adapted a technology for controlled release of drugs from macromolecular carriers for use as a long-acting intravitreal delivery system for small molecules. As a prototype, a small molecule complement factor D inhibitor with an intravitreal half-life of 7 hours was covalently attached to a 4-arm PEG40kDa by a self-cleaving ß-eliminative linker with a cleavage half-life of approximately 1 week. RESULTS: After intravitreal injection in rabbits, the drug was slowly released in the vitreous, and equilibrated with the retina and choroid. The intravitreal half-life of the intact PEG-drug conjugate in the rabbit was 7 days, and that of the released drug was 3.6 days. We simulated the anticipated pharmacokinetics of the delivery system in human vitreous, and estimated that the half-life of a 4-arm PEG40kDa conjugate would be approximately 2 weeks, and that of the released drug would be approximately 5 days. CONCLUSIONS: We posit that a linker with a cleavage half life of 2 weeks would confer a half life of approximately 7 days to a released small molecule drug in humans, comparable to the half life of approved intravitreal injected macromolecular drugs. TRANSLATIONAL RELEVANCE: With this technology, a potent small molecule with an appropriate therapeutic window should be administrable by intravitreal injections in the human at once-monthly intervals.

Liver-selective distribution in rats supports the importance of active uptake into the liver via organic anion transporting polypeptides (OATPs) in humans.

Organic anion transporting polypeptide (OATP) 1B1 and 1B3 are key molecules that are involved in hepatic uptake related to drug elimination, and OATP-mediated drug interactions are of clinical concern. In this study, with an aim to determine a cutoff value for the potential involvement of OATP, we collected data on the distribution of 12 human OATP and 24 non-OATP radiolabeled substrates in rats. The OATP substrates exhibited a higher tissue-to-plasma ratio (Kp) in the liver than that in the other tissues. As an index of liver-specific distribution, a hepatic Kp ratio (the ratio of Kp in the liver to that in other tissues) was introduced, and a hepatic Kp ratio <10 was proposed as a criterion for excluding the involvement of OATP in vivo. Approximately 20% of the non-OATP substrates as well as 100% of the OATP substrates exceeded the cutoff value of 10; therefore, further in vitro transport studies will be required to decide whether to conduct clinical drug interaction studies. Since distribution studies are usually conducted in rats during drug development, the use of a hepatic Kp ratio is practical and could refine the current decision tree for selecting OATP substrates in the drug interaction guidance/guidelines.

Edoxaban transport via P-glycoprotein is a key factor for the drug's disposition.

Edoxaban (the free base of DU-176b), an oral direct factor Xa inhibitor, is mainly excreted unchanged into urine and feces. Because active membrane transport processes such as active renal secretion, biliary excretion, and/or intestinal secretion, and the incomplete absorption of edoxaban after oral administration have been observed, the involvement of drug transporters in the disposition of edoxaban was investigated. Using a bidirectional transport assay in human colon adenocarcinoma Caco-2 cell monolayers, we observed the vectorial transport of [(14)C]edoxaban, which was completely inhibited by verapamil, a strong P-glycoprotein (P-gp) inhibitor. In an in vivo study, an increased distribution of edoxaban to the brain was observed in Mdr1a/1b knockout mice when compared with wild-type mice, indicating that edoxaban is a substrate for P-gp. However, there have been no observations of significant transport of edoxaban by renal or hepatic uptake transporters, organic anion transporter (OAT)1, OAT3, organic cation transporter (OCT)2, or organic anion transporting polypeptide (OATP)1B1. Edoxaban exhibited no remarkable inhibition of OAT1, OAT3, OCT1, OCT2, OATP1B1, OATP1B3, or P-gp up to 30 µM; therefore, the risk of clinical drug-drug interactions due to any edoxaban-related transporter inhibition seems to be negligible. Our results demonstrate that edoxaban is a substrate of P-gp but not of other major uptake transporters tested. Because metabolism is a minor contributor to the total clearance of edoxaban and strong P-gp inhibitors clearly impact edoxaban transport, the P-gp transport system is a key factor for edoxaban's disposition.

Different hydrolases involved in bioactivation of prodrug-type angiotensin receptor blockers: carboxymethylenebutenolidase and carboxylesterase 1.

Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor blocker (ARB). We recently identified carboxymethylenebutenolidase homolog (CMBL) as the responsible enzyme for OM bioactivation in humans. In the present study, we compared the bioactivating properties of OM with those of other prodrug-type ARBs, candesartan cilexetil (CC) and azilsartan medoxomil (AM), by focusing on interspecies differences and tissue specificity. In in-vitro experiments with pooled tissue subcellular fractions of mice, rats, monkeys, dogs, and humans, substantial OM-hydrolase activities were observed in cytosols of the liver, intestine, and kidney in all the species tested except for dog intestine, which showed negligible activity, whereas lung cytosols showed relatively low activities compared with the other tissues. AM-hydrolase activities were well correlated with the OM-hydrolase activities. In contrast, liver microsomes exhibited the highest CC-hydrolase activity among various tissue subcellular fractions in all the species tested. As a result of Western blot analysis with the tissue subcellular fractions, the band intensities stained with anti-human CMBL and carboxylesterase 1 (CES1) antibodies well reflected OM- and AM-hydrolase activities and CC-hydrolase activity, respectively, in animals and humans. Recombinant human CMBL and CES1 showed significant AM- and CC-hydrolase activities, respectively, whereas CC hydrolysis was hardly catalyzed with recombinant carboxylesterase 2 (CES2). In conclusion, OM is bioactivated mainly via intestinal and additionally hepatic CMBL not only in humans but also in mice, rats, and monkeys, while CC is bioactivated via hepatic CES1 rather than intestinal enzymes, including CES2. AM is a substrate for CMBL.

Role of the highly conserved threonine in cytochrome P450 2E1: prevention of H2O2-induced inactivation during electron transfer.

A highly conserved threonine in the I-helix of cytochrome P450s has been suggested to play an important role in dioxygen activation, a critical step for catalytic turnover. However, subsequent studies with some P450s in which this highly conserved threonine was replaced by another residue such as alanine showed that significant catalytic activities were still retained when the variants were compared with the wild type enzymes. These results make the role of this residue unclear. We provide data here that suggest a novel role for this highly conserved threonine (Thr303) in the function of P450 2E1. We found that the P450 2E1 T303A mutant undergoes rapid autoinactivation in the reconstituted system during catalytic turnover when the electrons are provided by NADPH. This inactivation was much faster than that of the wild type P450 2E1 and was prevented by catalase. Both the P450 2E1 wild type and T303A mutants produce hydrogen peroxide during the incubations. The inactivation was accompanied by heme destruction with part of the heme becoming covalently attached to protein. The heme destruction was prevented by catalase or by the presence of substrate. Interestingly, this inactivation occurred much more rapidly in the presence of both an electron transfer system and hydrogen peroxide externally added to the enzyme. This accelerated inactivation during catalytic turnover was also found with a 2B4 T302A mutant, which corresponds to 2E1 T303A. Our results suggest that the conserved threonine in these P450s prevents rapid autoinactivation during the catalytic cycle and that this residue may be highly conserved in P450s since it allows them to remain catalytically active for longer periods of time.

Interindividual variability of carboxymethylenebutenolidase homolog, a novel olmesartan medoxomil hydrolase, in the human liver and intestine.

Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor antagonist. OM is rapidly converted into its active metabolite olmesartan by multiple hydrolases in humans, and we recently identified carboxymethylenebutenolidase homolog (CMBL) as one of the OM bioactivating hydrolases. In the present study, we further investigated the interindividual variability of mRNA and protein expression of CMBL and OM-hydrolase activity using 40 individual human liver and 30 intestinal specimens. In the intestinal samples, OM-hydrolase activity strongly correlated with the CMBL protein expression, clearly indicating that CMBL is a major contributor to the prodrug bioactivation in human intestine. The protein and activity were highly distributed in the proximal region (duodenum and jejunum) and decreased to the distal region of the intestine. Although there was high interindividual variability (16-fold) in both the protein and activity in the intestinal segments from the duodenum to colon, the interindividual variability in the duodenum and jejunum was relatively small (3.0- and 2.4-fold, respectively). In the liver samples, the interindividual variability in the protein and activity was 4.1- and 6.8-fold, respectively. No sex differences in the protein and activity were shown in the human liver or intestine. A genetically engineered Y155C mutant of CMBL, which was caused by a single nucleotide polymorphism rs35489000, showed significantly lower OM-hydrolase activity than the wild-type protein although no minor allele was genotyped in the 40 individual liver specimens.

The inactivation of human CYP2E1 by phenethyl isothiocyanate, a naturally occurring chemopreventive agent, and its oxidative bioactivation.

Phenethylisothiocyanate (PEITC), a naturally occurring isothiocyanate and potent cancer chemopreventive agent, works by multiple mechanisms, including the inhibition of cytochrome P450 (P450) enzymes, such as CYP2E1, that are involved in the bioactivation of carcinogens. PEITC has been reported to be a mechanism-based inactivator of some P450s. We describe here the possible mechanism for the inactivation of human CYP2E1 by PEITC, as well as the putative intermediate that might be involved in the bioactivation of PEITC. PEITC inactivated recombinant CYP2E1 with a partition ratio of 12, and the inactivation was not inhibited in the presence of glutathione (GSH) and not fully recovered by dialysis. The inactivation of CYP2E1 by PEITC is due to both heme destruction and protein modification, with the latter being the major pathway for inactivation. GSH-adducts of phenethyl isocyanate (PIC) and phenethylamine were detected during the metabolism by CYP2E1, indicating formation of PIC as a reactive intermediate following P450-catalyzed desulfurization of PEITC. Surprisingly, PIC bound covalently to CYP2E1 to form protein adducts but did not inactivate the enzyme. Liquid chromatography mass spectroscopy analysis of the inactivated CYP2E1 apo-protein suggests that a reactive sulfur atom generated during desulfurization of PEITC is involved in the inactivation of CYP2E1. Our data suggest that the metabolism of PEITC by CYP2E1 that results in the inactivation of CYP2E1 may occur by a mechanism similar to that observed with other sulfur-containing compounds, such as parathion. Digestion of the inactivated enzyme and analysis by SEQUEST showed that Cys 268 may be the residue modified by PIC.

Paraoxonase 1 as a major bioactivating hydrolase for olmesartan medoxomil in human blood circulation: molecular identification and contribution to plasma metabolism.

Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor antagonist. The OM-hydrolyzing enzyme responsible for prodrug bioactivation was purified from human plasma through successive column chromatography and was molecularly identified through N-terminal amino acid sequencing, which resulted in a sequence of 20 amino acids identical to that of human paraoxonase 1 (PON1). Two recombinant allozymes of human PON1 (PON1(192QQ) and PON1(192RR)) were constructed and were clearly demonstrated to hydrolyze OM; hydrolysis by the latter allozyme was slightly faster than that by the former. In addition, we evaluated the contribution of PON1 to OM bioactivation in human plasma. Enzyme kinetic studies demonstrated that OM was hydrolyzed more effectively by the recombinant PON1 proteins than by purified albumin. The OM-hydrolyzing activities of the recombinant PON1 proteins and diluted plasma were greatly reduced in the absence of calcium ions. Immunoprecipitation with anti-PON1 IgG completely abolished the OM-hydrolyzing activity in human plasma, whereas the activity was partially inhibited with anti-albumin IgG. The distribution pattern of the OM-hydrolyzing activity in human serum lipoprotein fractions and lipoprotein-deficient serum was examined and showed that most of the OM-hydrolyzing activity was located in the high-density lipoprotein fraction, with which PON1 is closely associated. In conclusion, we identified PON1 as the OM-bioactivating hydrolase in human plasma on a molecular basis and demonstrated that PON1, but not albumin, plays a major role in OM bioactivation in human plasma.

PEGylation of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) results in decreased uptake into rats and human liver.

OBJECTIVES: Our aim was to investigate the effect of PEGylation on the uptake of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) into rat liver, kidney and spleen, and human liver. METHODS: Copolymer of polyethyleneglycol allylmethylether and maleamic acid sodium salt with OCIF (poly(PEG)-OCIF) (0.5 mg/kg) was administered to rats and the concentrations of poly(PEG)-OCIF in the liver, kidney and spleen at 15 min after administration were measured by ELISA. For human liver uptake, the liver perfusion of OCIF and (3)H-labelled poly(PEG)-OCIF was conducted using fresh human liver block. KEY FINDINGS: The tissue uptake of poly(PEG)-OCIF in rats was significantly lower compared with that of OCIF. In fresh human liver perfusion, (3)H-poly(PEG)-OCIF was rarely taken up into the liver. On the other hand, more than 50% of the perfused OCIF was taken up. CONCLUSIONS: PEGylation of OCIF using poly(PEG) dramatically suppressed the uptake of OCIF into human liver as well as into rat liver and could be a promising approach for improving the pharmacokinetic and pharmacological effects of OCIF in the clinical setting.

Human carboxymethylenebutenolidase as a bioactivating hydrolase of olmesartan medoxomil in liver and intestine.

Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in the liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin, to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys(132) of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics.

Inhibitory effects of angiotensin receptor blockers on CYP2C9 activity in human liver microsomes.

We investigated the inhibitory effects of the angiotensin receptor blockers (ARBs), candesartan, irbesartan, losartan, losartan active metabolite (EXP-3174), olmesartan, telmisartan and valsartan (0.3-300 microM), on the CYP2C9 activity in human liver microsomes using (S)-(-)-warfarin as a typical CYP2C9 substrate. Except for olmesartan and valsartan, these ARBs inhibited the activity of 7-hydroxylation of (S)-(-)-warfarin with IC50 values of 39.5-116 microM. Of six synthetic derivatives of olmesartan, five compounds which possess either alkyl groups or a chloro group at the same position as that of the hydroxyisopropyl group in olmesartan inhibited CYP2C9 activity with IC50 values of 21.7-161 microM. Olmesartan and the olmesartan analogue, RNH-6272, both having a hydroxyisopropyl group, showed no inhibition, indicating that the hydrophilicity of this group greatly contributes to the lack of CYP2C9 inhibition by these two compounds. A three-dimensional model for docking between EXP-3174 and CYP2C9 indicated that the chloro group of EXP-3174 is oriented to a hydrophobic pocket in the CYP2C9 active site, indicating that the lipophilicity of the group present in ARBs at the position corresponding to that of the hydroxyisopropyl group in olmesartan is important in inhibiting CYP2C9 activity.

Tissue distribution of humanized anti-human Fas monoclonal antibody (R-125224) based on fas antigen-antibody reaction in collagen-induced arthritis monkeys.

R-125224 is a novel humanized anti-human Fas monoclonal antibody prepared from HFE7A, which is a monoclonal mouse IgG anti-Fas antibody, by grafting the mouse complementarity-determining regions to human IgG, presently being developed as a drug for treatment of rheumatoid arthritis. In the present study, we investigated the tissue distribution of radioactivity in cynomolgus monkeys with collagen-induced arthritis at the arm joint (CIA monkeys) after intravenous administration of (125)I-labeled R-125224 ((125)I-R-125224). At 168 h after administration, we observed a high radioactivity in the bone marrow, thymus, lungs, liver, adrenals, spleen, ovaries, axillary lymph node and mesenteric lymph node compared to the radioactivity in the plasma. These tissues and organs in human are reported to express Fas antigen, strongly suggesting a specific binding of (125)I-R-125224 to Fas antigen in cynomolgus monkeys. Semi-micro autoradioluminograms of arm joint showed that radioactivity is detected in pharmacological site, such as the bone marrow and articular cavity at 168 h. The kinetics in binding of R-125224 to activated monkey lymphocytes and hepatocytes was also investigated. K(d) values of activated lymphocytes and hepatocytes were 1.51+/-0.08 and 0.60+/-0.11 nM, respectively, which were similar to those values in human lymphocytes and hepatocytes, demonstrating that R-125224 cross-reacts with the monkey Fas antigen.

In SCID mice with transplanted joint tissues from rheumatism patients, a model mice of human rheumatoid arthritis, anti-human fas antibody (R-125224) distributes specifically to human synovium.

PURPOSE: We investigated the tissue distribution of a humanized anti-human Fas monoclonal antibody, R-125224, in SCID mice transplanted with synovial tissues from patients with rheumatoid arthritis (SCID-HuRAg mice). The binding kinetics of R-125224 was also determined, using isolated human synovial cells. MATERIALS AND METHODS: Tissue distribution was assessed at 1, 24 and 168 h after intravenous administration of (125)I-R-125224 to SCID-HuRAg mice (0.4 mg/kg). The in vitro binding of (125)I-R-125224 to isolated human synovial cells was investigated. RESULTS: After intravenous administration of (125)I-R-125224 to SCID-HuRAg mice, the radioactivity distributed to various tissues at 1 h. Thereafter, the radioactivity in the tissues gradually decreased except for the transplanted synovial tissues, in which the radioactivity increased in a time-dependent manner, and at 168 h, the tissue/plasma concentration ratio was about 1. The in vitro binding affinity of (125)I-R-125224 to human synovial cells was high with a dissociation constant of 1.32 +/- 0.62 nM and the binding was inhibited by non-labeled R-125224 in a concentration-dependent manner. CONCLUSION: R-125224, a candidate compound for treating rheumatoid arthritis, specifically distributed to the pharmacological target site, human synovium transplanted in SCID mice, with high affinity.

OATP1B1, OATP1B3, and mrp2 are involved in hepatobiliary transport of olmesartan, a novel angiotensin II blocker.

Hepatic uptake and biliary excretion of olmesartan, a new angiotensin II blocker, were investigated in vitro using human hepatocytes, cells expressing uptake transporters and canalicular membrane vesicles, and in vivo using Eisai hyperbilirubinemic rats (EHBR), inherited multidrug resistance-associated protein (mrp2)-deficient rats. The uptake by human hepatocytes reached saturation with a Michaelis constant (K(m)) of 29.3 +/- 9.9 microM. Both Na(+)-dependent and Na(+)-independent uptake of olmesartan by human hepatocytes were observed. The uptake by Na(+)-independent human liver-specific organic anion transporters OATP1B1 and OATP1B3 expressed in Xenopus laevis oocytes was also saturable, with K(m) values of 42.6 +/- 28.6 and 71.8 +/- 21.6 microM, respectively. The Na(+)-dependent taurocholate-cotransporting polypeptide expressed in HEK 293 cells did not transport olmesartan. The cumulative biliary excretion in EHBR was one-sixth compared with that in Sprague-Dawley rats. ATP-dependent uptake of olmesartan was observed in both human canalicular membrane vesicles (hCMVs) and MRP2-expressing vesicles. An MRP inhibitor, MK-571 ([[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid) completely inhibited the uptake of olmesartan by hCMVs. In conclusion, the hepatic uptake and biliary excretion of olmesartan are mediated by transporters in humans. OATP1B1 and OATP1B3 are involved in hepatic uptake, at least in part, and MRP2 plays a dominant role in the biliary excretion.

A humanized anti-human Fas antibody, R-125224, induces apoptosis in type I activated lymphocytes but not in type II cells.

Fas-mediated apoptosis plays an important role in the immune system, including the elimination of autoreactive lymphoid cells. The Fas-mediated signaling pathway is classified into two types, type I and type II, in human lymphoid cell lines. We investigated whether a humanized anti-human Fas mAb, R-125224, has cell selectivity in induction of apoptosis. R-125224 induced apoptosis in H9 cells, SKW6.4 cells and activated human lymphocytes when cross-linked with anti-human IgG. On the other hand, R-125224 did not induce apoptosis in HPB-ALL cells, Jurkat cells or human hepatocytes. By analysis of death-inducing signaling complex formation, it was demonstrated that R-125224 induced apoptosis selectively in type I cells but not in type II cells. Type I cells also expressed more Fas and had more Fas-clustering activity than type II cells. Moreover, co-localization of these clusters and GM1, which is an sphingoglycolipid associated with lipid rafts, was detected. It was also shown that R-125224 treatment could reduce the number of activated human CD3+Fas+ cells in a SCID mouse model in vivo. Thus, we demonstrated that R-125224 induces apoptosis specifically in type I cells in vitro and in vivo.

Pharmacological and pharmacokinetic study of olmesartan medoxomil in animal diabetic retinopathy models.

A close relationship between the renin-angiotensin system and the pathophysiology of diabetic retinopathy has been suggested, several angiotensin II type 1 receptor (angiotensin AT1 receptor) antagonists being effective in animal models. Therefore, we examined the efficacy of an angiotensin AT1 receptor antagonist, olmesartan medoxomil (CS-866), in animal retinopathy models. In diabetic stroke-prone spontaneously hypertensive (SHRSP) rats, 4-week treatment with CS-866 prevented the elongation of oscillatory potential peaks dose-dependently which almost normalized at 3 mg/kg/day. Next, in oxygen-induced retinopathy mice, CS-866 at 1 mg/kg significantly prevented the retinal neovascularization. In these animal models, plasma concentrations of CS-866 were comparable to the in vitro IC50 value of the angiotensin AT1 receptor. In summary, our data demonstrated that CS-866 was effective in early and late stage retinopathy models through the inhibition of the angiotensin AT1 receptor. These findings suggest the possibility of CS-866 as a therapeutic agent for diabetic retinopathy.

Evidence for the involvement of a pulmonary first-pass effect via carboxylesterase in the disposition of a propranolol ester derivative after intravenous administration.

The disposition kinetics of O-butyryl propranolol (butyryl-PL), a model compound containing an ester moiety, after intravenous administration was compared with that of PL in rats and beagle dogs. Rats showed only 30% conversion of butyryl-PL to PL up to 2 h after dosing, whereas dogs showed nearly complete conversion within 10 min after administration. The CL(total) of butyryl-PL in rats was 5.8 l/h/kg and that in dogs was 65.6 +/- 18.6 l/h/kg, both of which were greater than hepatic blood flow. The in vivo conversion from butyryl-PL to PL in the rat could be explained on the basis of the hydrolysis characteristics in the liver and blood. The in vitro hydrolysis data and the in vivo data after intra-arterial administration clearly demonstrated that the extremely high CL(total) of butyryl-PL in dogs was dependent on first-pass hydrolysis in the lung in addition to hydrolysis at a blood flow-limited rate in the liver and kidney. The availability of butyryl-PL after passage through the lung was 50%. Furthermore, the isoform of carboxylesterase involved in the pulmonary hydrolysis of butyryl-PL in the dog was identified as D1, a CES-1 group enzyme. However, butyryl-PL was not recognized as a substrate by CES-1 family carboxylesterases, which are present at high levels in the rat lung (RH-1) and kidney (RL-1). These findings indicate that extrahepatic metabolism, especially in the lung, is important in the disposition of drugs containing an ester moiety after intravenous administration and that the substrate specificity of carboxylesterase isozyme distinguishes from others.