Publications by authors named "Takao Yamazaki"

23 Publications

  • Page 1 of 1

No Dose Adjustment for Isavuconazole Based on Age or Sex.

Antimicrob Agents Chemother 2019 06 24;63(6). Epub 2019 May 24.

Astellas Pharma Inc., Tokyo, Japan.

This phase 1, open-label, single-dose, parallel-group study evaluated the pharmacokinetics (PK) of isavuconazole after a single oral dose of the prodrug isavuconazonium sulfate in healthy nonelderly (age, 18 to 45 years) and elderly (age, ≥65 years) males and females. Overall, 48 subjects were enrolled in the study (=12 each in groups of nonelderly males and females and elderly males and females). All subjects received a single oral dose of 372 mg of isavuconazonium sulfate (equivalent to 200 mg isavuconazole). PK samples were collected for analysis of isavuconazole plasma concentrations from the predose time point up to 336 h postdose. Data were analyzed using population pharmacokinetic (PPK) analysis. The resulting PPK model included two compartments with Weibull absorption function as well as interindividual variability with respect to clearance, intercompartment clearance, volumes of central and peripheral compartments, and two Weibull absorption parameters, RA and KAMAX. The PPK analysis showed that elderly females had the highest exposure versus males (ratio of total area under the time-concentration curve [AUC], 138; 90% confidence interval [CI], 118 to 161) and versus nonelderly females (ratio of AUC, 147; 90% CI, 123 to 176). Higher exposures in elderly females were not associated with significant toxicity or treatment-emergent adverse events, as measured in this study. No dose adjustments appear to be necessary based on either age group or sex even with an increase in exposure for elderly females. (This study has been registered at ClinicalTrials.gov under registration no. NCT01657890.).
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http://dx.doi.org/10.1128/AAC.02629-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535513PMC
June 2019

Two Phase 1, Open-Label, Mass Balance Studies to Determine the Pharmacokinetics of C-Labeled Isavuconazonium Sulfate in Healthy Male Volunteers.

Clin Pharmacol Drug Dev 2018 02 27;7(2):207-216. Epub 2017 Jul 27.

Astellas Pharma Global Development Inc., Northbrook, IL, USA.

Isavuconazonium sulfate is the water-soluble prodrug of the active triazole isavuconazole. Two phase 1 studies were conducted to identify the metabolic profile and mass balance of isavuconazole and BAL8728 (inactive cleavage product). Seven subjects in study 1 (isavuconazole mass balance) received a single oral dose of [cyano- C]isavuconazonium sulfate corresponding to 200 mg isavuconazole. Six subjects in study 2 (BAL8728 mass balance) received a single intravenous dose of [pyridinylmethyl- C]isavuconazonium sulfate corresponding to 75 mg BAL8728. Pharmacokinetic parameters of radioactivity in whole blood and plasma and of isavuconazole and BAL8728 in plasma were assessed. Radioactivity ratio of blood/plasma, percentage of dose, and cumulative percentage of radioactive dose recovered in urine and feces for isavuconazole and BAL8728 were assessed. Metabolic profiling was carried out by high-performance liquid chromatography and mass spectrometry. Mean plasma isavuconazole pharmacokinetic parameters included apparent clearance (2.3 ± 0.7 L/h), apparent volume of distribution (301.8 ± 105.7 L), and terminal elimination half-life (99.9 ± 44.6 hours). In study 1, isavuconazole-derived radioactivity was recovered approximately equally in urine and feces (46.1% and 45.5%, respectively). In study 2, BAL8728-derived radioactivity was predominantly recovered in urine (96.0%). Isavuconazole (study 1) and M4 (cleavage metabolite of BAL8728; study 2) were the predominant circulating components of radioactivity in plasma.
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http://dx.doi.org/10.1002/cpdd.376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811773PMC
February 2018

Isavuconazole absorption following oral administration in healthy subjects is comparable to intravenous dosing, and is not affected by food, or drugs that alter stomach pH.

Int J Clin Pharmacol Ther 2016 Aug;54(8):572-80

Objective/methods: Two openlabel, single-dose, randomized crossover studies and one open-label, multiple-dose, parallel group study in healthy volunteers were conducted with the prodrug, isavuconazonium sulfate, to determine absolute bioavailability of the active triazole, isavuconazole (EudraCT 2007-004949-15; n = 14), and the effect of food (EudraCT 2007- 004940-63; n = 26), and pH (NCT02128893; n = 24) on the absorption of isavuconazole. Isavuconazonium sulfate 744 mg designed to deliver 400 mg of the active triazole isavuconazole was administered in the absolute bioavailability (oral or intravenous (IV) (2-hour infusion)) and food-effect studies (oral). In the pH-effect study, isavuconazonium sulfate 372 mg designed to deliver 200 mg of isavuconazole was administered orally three times daily (t.i.d.) for 2 days, followed by a single daily oral dose for 3 days, in the presence of steady state esomeprazole dosed orally at 40 mg/day.

Results: Isavuconazole was well tolerated in each study. Bioavailability: Geometric least squares mean ratios (GLSMR; oral/IV) for isavuconazole AUC∞, and Cmax were 98% (90% confidence interval (CI): 94, 101) and 78% (90% CI: 72, 85), respectively. Food-effect: GLSMR (fed/fasted) for AUC∞ and Cmax of isavuconazole in plasma were 110% (90% CI: 102, 118) and 92% (90% CI: 86, 98), respectively. Median tmax was 5 hours with food and 3 hours under fasted conditions. pH-effect: GLSMR for isavuconazole AUCtau and Cmax were 108% (90% CI: 89, 130) and 105% (90% CI: 89, 124), respectively.

Conclusions: Orally administered isavuconazonium sulfate effectively delivers isavuconazole, as evidenced by the fact that oral isavuconazole is bioequivalent to the IV formulation. Dose adjustments are not required when switching between oral and IV formulations, regardless of food or drugs that increase gastric pH.
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http://dx.doi.org/10.5414/CP202434DOI Listing
August 2016

Pharmacokinetic and Pharmacodynamic Evaluation of the Drug-Drug Interaction Between Isavuconazole and Warfarin in Healthy Subjects.

Clin Pharmacol Drug Dev 2017 Jan 4;6(1):86-92. Epub 2016 Aug 4.

Astellas Pharma Global Development, Inc, Northbrook, IL, USA.

This phase 1 trial evaluated pharmacokinetic and pharmacodynamic interactions between the novel triazole antifungal agent isavuconazole and warfarin in healthy adults. Multiple doses of isavuconazole were administered as the oral prodrug, isavuconazonium sulfate (372 mg 3 times a day for 2 days loading dose, then 372 mg once daily thereafter; equivalent to isavuconazole 200 mg), in the presence and absence of single doses of oral warfarin sodium 20 mg. Coadministration with isavuconazole increased the mean area under the plasma concentration-time curves from time 0 to infinity of S- and R-warfarin by 11% and 20%, respectively, but decreased the mean maximum plasma concentrations of S- and R-warfarin by 12% and 7%, respectively, relative to warfarin alone. Mean area under the international normalized ratio curve and maximum international normalized ratio were 4% lower in the presence vs absence of isavuconazole. Mean warfarin area under the prothrombin time curve and maximum prothrombin time were 3% lower in the presence vs absence of isavuconazole. There were no serious treatment-emergent adverse events (TEAEs), and no subjects discontinued the study due to TEAEs. All TEAEs were mild in intensity. These findings indicate that coadministration with isavuconazole has no clinically relevant effects on warfarin pharmacokinetics or pharmacodynamics.
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http://dx.doi.org/10.1002/cpdd.283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5298089PMC
January 2017

Pharmacokinetic Evaluation of CYP3A4-Mediated Drug-Drug Interactions of Isavuconazole With Rifampin, Ketoconazole, Midazolam, and Ethinyl Estradiol/Norethindrone in Healthy Adults.

Clin Pharmacol Drug Dev 2017 Jan 25;6(1):44-53. Epub 2016 Jul 25.

Astellas Pharma Global Development, Inc, Northbrook, IL, USA.

This report describes the phase 1 trials that evaluated the metabolism of the novel triazole antifungal isavuconazole by cytochrome P450 3A4 (CYP3A4) and isavuconazole's effects on CYP3A4-mediated metabolism in healthy adults. Coadministration of oral isavuconazole (100 mg once daily) with oral rifampin (600 mg once daily; CYP3A4 inducer) decreased isavuconazole area under the concentration-time curve (AUC ) during a dosing interval by 90% and maximum concentration (C ) by 75%. Conversely, coadministration of isavuconazole (200 mg single dose) with oral ketoconazole (200 mg twice daily; CYP3A4 inhibitor) increased isavuconazole AUC from time 0 to infinity (AUC ) and C by 422% and 9%, respectively. Isavuconazole was coadministered (200 mg 3 times daily for 2 days, then 200 mg once daily) with single doses of oral midazolam (3 mg; CYP3A4 substrate) or ethinyl estradiol/norethindrone (35 μg/1 mg; CYP3A4 substrate). Following coadministration, AUC increased 103% for midazolam, 8% for ethinyl estradiol, and 16% for norethindrone; C increased by 72%, 14%, and 6%, respectively. Most adverse events were mild to moderate in intensity; there were no deaths, and serious adverse events and adverse events leading to study discontinuation were rare. These results indicate that isavuconazole is a sensitive substrate and moderate inhibitor of CYP3A4.
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http://dx.doi.org/10.1002/cpdd.285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5298035PMC
January 2017

Pharmacokinetic Assessment of Drug-Drug Interactions of Isavuconazole With the Immunosuppressants Cyclosporine, Mycophenolic Acid, Prednisolone, Sirolimus, and Tacrolimus in Healthy Adults.

Clin Pharmacol Drug Dev 2017 Jan 25;6(1):76-85. Epub 2016 Jul 25.

Astellas Pharma Global Development, Inc, Northbrook, IL, USA.

This report summarizes phase 1 studies that evaluated pharmacokinetic interactions between the novel triazole antifungal agent isavuconazole and the immunosuppressants cyclosporine, mycophenolic acid, prednisolone, sirolimus, and tacrolimus in healthy adults. Healthy subjects received single oral doses of cyclosporine (300 mg; n = 24), mycophenolate mofetil (1000 mg; n = 24), prednisone (20 mg; n = 21), sirolimus (2 mg; n = 22), and tacrolimus (5 mg; n = 24) in the presence and absence of clinical doses of oral isavuconazole (200 mg 3 times daily for 2 days; 200 mg once daily thereafter). Coadministration with isavuconazole increased the area under the concentration-time curves (AUC ) of tacrolimus, sirolimus, and cyclosporine by 125%, 84%, and 29%, respectively, and the AUCs of mycophenolic acid and prednisolone by 35% and 8%, respectively. Maximum concentrations (C ) of tacrolimus, sirolimus, and cyclosporine were 42%, 65%, and 6% higher, respectively; C of mycophenolic acid and prednisolone were 11% and 4% lower, respectively. Isavuconazole pharmacokinetics were mostly unaffected by the immunosuppressants. Two subjects experienced elevated creatinine levels in the cyclosporine study; most adverse events were not considered to be of clinical concern. These results indicate that isavuconazole is an inhibitor of cyclosporine, mycophenolic acid, sirolimus, and tacrolimus metabolism.
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http://dx.doi.org/10.1002/cpdd.284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5298005PMC
January 2017

Pharmacokinetic Interaction Between Isavuconazole and a Fixed-Dose Combination of Lopinavir 400 mg/Ritonavir 100 mg in Healthy Subjects.

Clin Pharmacol Drug Dev 2017 Jan 20;6(1):93-101. Epub 2016 Jul 20.

Astellas Pharma Global Development, Inc, Northbrook, IL, USA.

This phase 1, open-label study evaluated the pharmacokinetic effects of coadministration of the antifungal agent, isavuconazole (administered as its water-soluble prodrug isavuconazonium sulfate), with the antiretroviral agent lopinavir/ritonavir in healthy adults. In part 1, 13 subjects were randomized to 2 arms to receive multiple doses of oral isavuconazole 100 mg either alone or with lopinavir/ritonavir 400/100 mg. In part 2, a different group of 55 subjects were randomized to 3 arms to receive multiple doses of oral isavuconazole 200 mg, either alone or with lopinavir/ritonavir 400/100 mg, or to receive oral lopinavir/ritonavir 400/100 mg alone. Mean area under the concentration-time curve (AUC) following the last dose (AUC ) and C of isavuconazole increased by 113% and 96% in part 1 and by 96% and 74% in part 2 in the presence vs absence of lopinavir/ritonavir, respectively. Mean AUC and C of lopinavir were 27% and 23% lower, and mean AUC and C of ritonavir were 31% and 33% lower in the presence vs absence of isavuconazole, respectively. Mild to moderate gastrointestinal disorders were the most common adverse events experienced. These findings indicate that coadministration of lopinavir/ritonavir with isavuconazole can decrease the exposure of lopinavir/ritonavir and increase the exposure of isavuconazole. Patients should be monitored for reduced antiviral efficacy if these agents are coadministered.
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http://dx.doi.org/10.1002/cpdd.282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297880PMC
January 2017

Pharmacokinetic Effects of Isavuconazole Coadministration With the Cytochrome P450 Enzyme Substrates Bupropion, Repaglinide, Caffeine, Dextromethorphan, and Methadone in Healthy Subjects.

Clin Pharmacol Drug Dev 2017 Jan 15;6(1):54-65. Epub 2016 Jul 15.

Astellas Pharma Global Development, Inc, Northbrook, IL, USA.

This report describes phase 1 clinical trials performed to assess interactions of oral isavuconazole at the clinically targeted dose (200 mg, administered as isavuconazonium sulfate 372 mg, 3 times a day for 2 days; 200 mg once daily [QD] thereafter) with single oral doses of the cytochrome P450 (CYP) substrates: bupropion hydrochloride (CYP2B6; 100 mg; n = 24), repaglinide (CYP2C8/CYP3A4; 0.5 mg; n = 24), caffeine (CYP1A2; 200 mg; n = 24), dextromethorphan hydrobromide (CYP2D6/CYP3A4; 30 mg; n = 24), and methadone (CYP2B6/CYP2C19/CYP3A4; 10 mg; n = 23). Compared with each drug alone, coadministration with isavuconazole changed the area under the concentration-time curves (AUC ) and maximum concentrations (C ) as follows: bupropion, AUC reduced 42%, C reduced 31%; repaglinide, AUC reduced 8%, C reduced 14%; caffeine, AUC increased 4%, C reduced 1%; dextromethorphan, AUC increased 18%, C increased 17%; R-methadone, AUC reduced 10%, C increased 3%; S-methadone, AUC reduced 35%, C increased 1%. In all studies, there were no deaths, 1 serious adverse event (dextromethorphan study; perioral numbness, numbness of right arm and leg), and adverse events leading to study discontinuation were rare. Thus, isavuconazole is a mild inducer of CYP2B6 but does not appear to affect CYP1A2-, CYP2C8-, or CYP2D6-mediated metabolism.
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http://dx.doi.org/10.1002/cpdd.281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297975PMC
January 2017

Pharmacokinetic Interactions Between Isavuconazole and the Drug Transporter Substrates Atorvastatin, Digoxin, Metformin, and Methotrexate in Healthy Subjects.

Clin Pharmacol Drug Dev 2017 Jan 15;6(1):66-75. Epub 2016 Jul 15.

Astellas Pharma Global Development, Northbrook, IL, USA.

This article summarizes 4 phase 1 trials that explored interactions between the novel, triazole antifungal isavuconazole and substrates of the drug transporters breast cancer resistance protein (BCRP), multidrug and toxin extrusion protein-1 (MATE1), organic anion transporters 1/3 (OAT1/OAT3), organic anion-transporting polypeptide 1B1 (OATP1B1), organic cation transporters 1/2 (OCT1/OCT2), and P-glycoprotein (P-gp). Healthy subjects received single doses of atorvastatin (20 mg; OATP1B1 and P-gp substrate), digoxin (0.5 mg; P-gp substrate), metformin (850 mg; OCT1, OCT2, and MATE1 substrate), or methotrexate (7.5 mg; BCRP, OAT1, and OAT3 substrate) in the presence and absence of clinical doses of isavuconazole (200 mg 3 times a day for 2 days; 200 mg once daily thereafter). Coadministration with isavuconazole increased mean area under the plasma concentration-time curves (90% confidence interval) of atorvastatin, digoxin, and metformin to 137% (129, 145), 125% (117, 134),  and 152% (138, 168) and increased mean maximum plasma concentrations to 103% (88, 121), 133% (119, 149), and 123% (109, 140), respectively. Methotrexate parameters were unaffected by isavuconazole. There were no serious adverse events. These findings indicate that isavuconazole is a weak inhibitor of P-gp, as well as OCT1, OCT2, MATE1, or a combination thereof but not of BCRP, OATP1B1, OAT1, or OAT3.
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http://dx.doi.org/10.1002/cpdd.280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297980PMC
January 2017

ASP9853, an inhibitor of inducible nitric oxide synthase dimerization, in combination with docetaxel: preclinical investigation and a Phase I study in advanced solid tumors.

Cancer Chemother Pharmacol 2016 Mar 25;77(3):549-58. Epub 2016 Jan 25.

Sarah Cannon Research Institute/Tennessee Oncology, PLLC, Nashville, TN, USA.

Purpose: ASP9853 is an inhibitor of inducible nitric oxide (NO) synthase (iNOS) dimerization, which results in decreased NO production. Here, we report preclinical pharmacology of ASP9853 and the impact of ASP9853 in combination with a taxane on tumor volume in vivo. In addition, a Phase I open-label study of ASP9853 plus docetaxel was conducted to assess this combination in patients with advanced solid tumors.

Methods: The preclinical efficacy of ASP9853 in combination with a taxane was studied in tumor-bearing mice. In the clinic, patients with solid tumors that had progressed or failed to respond to previous therapies were treated with once-daily ASP9853 in combination with docetaxel once every 3 weeks to assess safety and tolerability and to determine the maximum tolerated dose (MTD) and the recommended Phase II dose (RP2D) of the combination.

Results: ASP9853 in combination with docetaxel showed greater tumor growth inhibition than docetaxel alone against non-small lung cancer xenografts. Twenty patients were treated with ASP9853 and docetaxel. Five patients experienced neutropenic dose-limiting toxicities. Owing to overall toxicity that limited further dose escalation, the ASP9853 concentrations predicted for efficacy, based on the preclinical data, were not achieved. Due to toxicity and lack of clear efficacy, the study was terminated without determination of MTD or RP2D.

Conclusions: Inhibition of iNOS by ASP9853 in combination with docetaxel was not tolerable and resulted in the possible potentiation of neutropenia. Manipulation of the iNOS pathway, with or without chemotherapy, appears to be more complicated than initially expected.
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http://dx.doi.org/10.1007/s00280-016-2967-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767605PMC
March 2016

Decomposition of cyclohexane ion induced by intense femtosecond laser fields by ion-trap time-of-flight mass spectrometry.

J Chem Phys 2016 Jan;144(2):024313

Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Decomposition of cyclohexane cations induced by intense femtosecond laser fields at the wavelength of 800 nm is investigated by ion-trap time-of-flight mass spectrometry in which cyclohexane cations C6H12 (+) stored in an ion trap are irradiated with intense femtosecond laser pulses and the generated fragment ions are recorded by time-of-flight mass spectrometry. The various fragment ion species, C5Hn (+) (n = 7, 9), C4Hn (+) (n = 5-8), C3Hn (+) (n = 3-7), C2Hn (+) (n = 2-6), and CH3 (+), identified in the mass spectra show that decomposition of C6H12 (+) proceeds efficiently by the photo-irradiation. From the laser intensity dependences of the yields of the fragment ion species, the numbers of photons required for producing the respective fragment ions are estimated.
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http://dx.doi.org/10.1063/1.4939769DOI Listing
January 2016

Comparison of the safety, tolerability, and pharmacokinetics of fidaxomicin in healthy Japanese and caucasian subjects.

Clin Drug Investig 2015 Jun;35(6):375-84

Astellas Pharma Inc., 2-5-1 Nihonbashi-Honcho, Chuo-ku, Tokyo, 103-8411, Japan,

Background And Objectives: Fidaxomicin treatment of Clostridium difficile infection is known to produce minimal systemic exposure, as the antibacterial (antibiotic) remains primarily in the gut. In this randomized, double-blind, placebo-controlled study, the safety, tolerability, and pharmacokinetics of single and multiple ascending doses of fidaxomicin were evaluated in healthy Japanese and Caucasian subjects.

Methods: Thirty-six healthy subjects were randomly assigned in a 3:1 ratio to receive either fidaxomicin or placebo. Cohort 1 (100 mg) and Cohort 2 (200 mg) comprised 12 Japanese subjects each and Cohort 3 (200 mg) comprised 12 Caucasian subjects. Subjects received a single dose of the study drug on Day 1 and received multiple doses for 10 days after a wash-out period.

Results: After multiple 200 mg dosing of fidaxomicin, both mean maximum plasma concentrations (C max) in Japanese (8.7 ± 5.3 ng/mL) and Caucasian (7.0 ± 3.7 ng/mL) subjects and the area under the concentration-time curve (AUC) were higher in Japanese subjects (58.5 ± 36.7 ng·h/mL) than in Caucasian subjects (37.6 ± 15.7 ng·h/mL), although variation in both groups was large. The mean fecal concentrations of fidaxomicin in Japanese and Caucasian subjects were 2669 and 2181 μg/g, respectively. The possibly study drug-related adverse events were diarrhea (n = 1), feeling hot (n = 1), and hypersomnia (n = 2), which were mild in severity.

Conclusions: In both Japanese and Caucasian subjects, fidaxomicin demonstrated similarly minimal systemic absorption, and was mainly excreted in feces. Fidaxomicin was safe and well-tolerated in all subjects.
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http://dx.doi.org/10.1007/s40261-015-0291-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449367PMC
June 2015

Chronic treatment with novel GPR40 agonists improve whole-body glucose metabolism based on the glucose-dependent insulin secretion.

J Pharmacol Exp Ther 2013 Sep 12;346(3):443-52. Epub 2013 Jul 12.

Pharmacology Research Laboratories, Drug Discovery Research, Astellas Pharma Inc, Tsukuba, Japan.

GPR40 is a free fatty acid receptor that has been shown to regulate glucose-dependent insulin secretion. This study aimed to discover novel GPR40 agonists and investigate the whole-body effect on glucose metabolism of GPR40 activation using these novel GPR40 agonists. To identify novel GPR40-specific agonists, we conducted high-throughput chemical compound screening and evaluated glucose-dependent insulin secretion. To investigate the whole-body effect on glucose metabolism of GPR40 activation, we conducted repeat administration of the novel GPR40 agonists to diabetic model ob/ob mice and evaluated metabolic parameters. To characterize the effect of the novel GPR40 agonists more deeply, we conducted an insulin tolerance test and a euglycemic-hyperinsulinemic clamp test. As a result, we discovered the novel GPR40-specific agonists, including AS2034178 [bis{2-[(4-{[4'-(2-hydroxyethoxy)-2'-methyl[1,1'-biphenyl]-3-yl]methoxy}phenyl)methyl]-3,5-dioxo-1,2,4-oxadiazolidin-4-ide} tetrahydrate], and found that its exhibited glucose-dependent insulin secretion enhancement both in vitro and in vivo. In addition, the compounds also decreased plasma glucose and HbA1c levels after repeat administration to ob/ob mice, with favorable oral absorption and pharmacokinetics. Repeat administration of AS2034178 enhanced insulin sensitivity in an insulin tolerance test and a euglycemic-hyperinsulinemic clamp test. These results indicate that improvement of glucose-dependent insulin secretion leads the improvement of whole-body glucose metabolism chronically. In conclusion, AS2034178 and other GPR40 agonists may become useful therapeutics in the treatment of type 2 diabetes mellitus.
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http://dx.doi.org/10.1124/jpet.113.206466DOI Listing
September 2013

Discovery of a novel neuroprotective compound, AS1219164, by high-throughput chemical screening of a newly identified apoptotic gene marker.

Eur J Pharmacol 2011 Nov 1;669(1-3):7-14. Epub 2011 Aug 1.

Pharmacology Research Laboratories, Astellas Pharma Inc., 5-2-3, Tokodai Tsukuba, Ibaraki 300-2698, Japan.

We have reported that tacrolimus (FK506), an immunosuppressive drug, and diclofenac, a non-steroidal anti-inflammatory drug, possess different modes of neuroprotective action. FK506 suppresses only thapsigargin-induced apoptosis in neuroblastoma SH-SY5Y cells while diclofenac reverses tunicamycin-induced as well as thapsigargin-induced apoptosis. The aim of this study is to discover novel compounds that exert neuroprotective properties by using the transcriptional response of a newly identified gene, which was regulated by both FK506 and diclofenac, as a surrogate screening marker in high-throughput chemical screening and characterize the compounds in comparison with FK506 and diclofenac. Using a microarray with 4504 human cDNAs and quantitative RT-PCR, two genes as apoptotic markers, transmembrane protein 100 (TMEM100) and limb-bud and heart (LBH), were identified because the thapsigargin-induced elevations in their mRNA levels were reversed by both FK506 and diclofenac. A luciferase reporter assay with a TMEM100 promoter region was applied to high-throughput chemical screening. AS1219164, {3-[(E)-2-{5-[(E)-2-pyridin-4-ylvinyl]pyridin-3-yl} vinyl]aniline}, suppressed thapsigargin-induced transactivation of the TMEM100 gene and reversed thapsigargin-induced increases in TMEM100 and LBH mRNA levels in SH-SY5Y cells, similar to the effects of FK506 and diclofenac. Furthermore, AS1219164 protected against SH-SY5Y cell death induced by four apoptotic agents including thapsigargin, similar to diclofenac, but was more potent than diclofenac, while FK506 only showed protective effects against thapsigargin-induced cell death. In conclusion, a novel neuroprotecitve compound, AS1219164, was discovered by high-throughput chemical screening using a reporter assay with the TMEM100 gene promoter regulated by both FK506 and diclofenac. Reporter assay using the promoter region of a gene under pharmacological and physiological transcriptional regulation would be well suit for use in high-throughput chemical screening.
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http://dx.doi.org/10.1016/j.ejphar.2011.07.027DOI Listing
November 2011

AS1907417, a novel GPR119 agonist, as an insulinotropic and β-cell preservative agent for the treatment of type 2 diabetes.

Biochem Biophys Res Commun 2010 Oct 15;400(4):745-51. Epub 2010 Sep 15.

Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki 305-8585, Japan.

G-protein-coupled receptor (GPR) 119 is involved in glucose-stimulated insulin secretion (GSIS) and represents a promising target for the treatment of type 2 diabetes as it is highly expressed in pancreatic β-cells. Although a number of oral GPR119 agonists have been developed, their inability to adequately directly preserve β-cell function limits their effectiveness. Here, we evaluated the therapeutic potential of a novel small-molecule GPR119 agonist, AS1907417, which represents a modified form of a 2,4,6-tri-substituted pyrimidine core agonist, AS1269574, we previously identified. The exposure of HEK293 cells expressing human GPR119, NIT-1 cells expressing human insulin promoter, and the pancreatic β-cell line MIN-6-B1 to AS1907417, enhanced intracellular cAMP, GSIS, and human insulin promoter activity, respectively. In in vivo experiments involving fasted normal mice, a single dose of AS1907417 improved glucose tolerance, but did not affect plasma glucose or insulin levels. Twice-daily doses of AS1907417 for 4weeks in diabetic db/db, aged db/db mice, ob/ob mice, and Zucker diabetic fatty rats reduced hemoglobin A1c levels by 1.6%, 0.8%, 1.5%, and 0.9%, respectively. In db/db mice, AS1907417 improved plasma glucose, plasma insulin, pancreatic insulin content, lipid profiles, and increased pancreatic insulin and pancreatic and duodenal homeobox 1 (PDX-1) mRNA levels. These data demonstrate that novel GPR119 agonist AS1907417 not only effectively controls glucose levels, but also preserves pancreatic β-cell function. We therefore propose that AS1907417 represents a new type of antihyperglycemic agent with promising potential for the effective treatment of type 2 diabetes.
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http://dx.doi.org/10.1016/j.bbrc.2010.08.141DOI Listing
October 2010

Neuroprotective efficacy of the peroxisome proliferator-activated receptor delta-selective agonists in vitro and in vivo.

J Pharmacol Exp Ther 2007 Mar 13;320(3):1087-96. Epub 2006 Dec 13.

Neuroscience Discovery Research, Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as ligand-modulated transcription factors that regulate gene expression in many important biological processes. The PPARdelta subtype has the highest expression in the brain and is postulated to play a major role in neuronal cell function; however, the precise physiological roles of this receptor remain to be elucidated. Herein, we show that the high-affinity PPARdelta agonists L-165041 [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid] and GW501516 [2-methyl4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-triazol-5-yl)-methylsulfanyl)phenoxy acetic acid] protect against cytotoxin-induced SH-SY5Y cell injury in vitro and both ischemic brain injury and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in vivo. In the SH-SY5Y studies, treatment with L-165041 or GW501516 significantly and concentration-dependently attenuated cell death following thapsigargin, 1-methyl-4-phenylpyridinium, or staurosporine exposure, with the extent of damage correlated with the level of caspase-3 inhibition. In the transient (90 min) middle cerebral artery occlusion model of ischemic brain injury in rats, i.c.v. infusion of L-165041 or GW501516 significantly attenuated the ischemic brain damage measured 24 h after reperfusion. Moreover, the PPARdelta agonists also significantly attenuated MPTP-induced depletion of striatal dopamine and related metabolite contents in mouse brain. These results demonstrate that subtype-selective PPARdelta agonists possess antiapoptotic properties in vitro, which may underlie their potential neuroprotective potential in in vivo experimental models of cerebral ischemia and Parkinson's disease (PD). These findings suggest that PPARdelta agonists could be useful tools for understanding the role of PPARdelta in other neurodegenerative disorders, as well as attractive therapeutic candidates for stroke and neurodegenerative diseases such as PD.
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http://dx.doi.org/10.1124/jpet.106.115758DOI Listing
March 2007

CyclinB2 and BIRC5 genes as surrogate biomarkers for neurite outgrowth in SH-SY5Y subclonal cells.

Neuropharmacology 2006 Jun 30;50(8):1041-7. Epub 2006 Mar 30.

Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan.

Neurite outgrowth plays a key role in neuronal development and regeneration, and is the hallmark assay for the effects of neurotrophic factors such as nerve growth factor (NGF). However, measuring neurite outgrowth is a slow and resource-intensive process. We therefore wanted to identify surrogate biomarkers for neurite outgrowth activity by gene expression analysis in SH-O10 cells, a subclone of the human SH-SY5Y neuroblastoma cell line but with much higher NGF-induced neurite outgrowth activity. Microarray analysis identified seven genes where mRNA levels were changed. NGF-induced decreases in levels of two genes, CyclinB2 and BIRC5, were confirmed by quantitative real-time RT-PCR. Levels of NGF-induced decreases in CyclinB2 and BIRC5 mRNA in several SH-SY5Y subclones with different neurite outgrowth responses correlated with their neurite outgrowth activities. Decreases in CyclinB2 and BIRC5 mRNA induced by FK506 or retinoic acid, both of which exert potentiation of NGF-induced neurite outgrowth effects but with different mechanisms, also correlated with their neurite outgrowth activities. In conclusion, decreasing levels of CyclinB2 and BIRC5 mRNA strongly correlate with neurite outgrowth activities in terms of NGF-related effect in SH-SY5Y subclonal cells, and have potential to become quantitative surrogate biomarkers for measuring NGF-related neurite outgrowth.
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http://dx.doi.org/10.1016/j.neuropharm.2006.02.004DOI Listing
June 2006

Diclofenac, a non-steroidal anti-inflammatory drug, suppresses apoptosis induced by endoplasmic reticulum stresses by inhibiting caspase signaling.

Neuropharmacology 2006 Apr 4;50(5):558-67. Epub 2006 Jan 4.

Pharmacology Research Labs, Astellas Pharma Inc., Tsukuba, Ibaraki 300-2698, Japan.

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used in the treatment of inflammation and pain. In many reports, NSAIDs have induced apoptosis in a variety of cell lines such as colon cancer cells. On the other hand, more recently a few reports have found that NSAIDs protect against apoptosis. Here we investigate endoplasmic reticulum (ER)-stress-induced apoptosis of neuronal cells. The aim of this study is to examine the involvement of NSAIDs, in particular diclofenac, on ER-stress-induced apoptosis of human neuroblastoma SH-SY5Y cells. Diclofenac significantly suppressed SH-SY5Y cell death induced by two types of ER-stress-inducing agents: thapsigargin, an inhibitor of Ca2+-ATPase on the endoplasmic reticulum membrane, and tunicamycin, a glycosylation blocker. Other NSAIDs, such as indomethacin, ibuprofen, aspirin, and ketoprofen, also suppressed ER-stress-induced SH-SY5Y cell death. The dose-dependent anti-apoptotic effect of diclofenac did not correlate with the reduction of prostaglandin release. Administration of prostaglandin E2, which was a primary product of arachidonic metabolism, showed no effects against anti-apoptotic effects produced by diclofenac. Thapsigargin and tunicamycin each significantly activated caspase-3, -9, and -2 in the intrinsic apoptotic pathway in SH-SY5Y cells. Diclofenac suppressed the activation of caspases induced by both ER stresses. Thapsigargin and tunicamycin decreased the mitochondrial membrane potential in SH-SY5Y cells. Diclofenac suppressed the mitochondrial depolarization induced by both ER stresses. Diclofenac inhibited ER-stress-induced apoptosis of SH-SY5Y cells by suppressing the activation of caspases in the intrinsic apoptotic pathway. This is the first report to find that diclofenac has protective effects against ER-stress-induced apoptosis.
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http://dx.doi.org/10.1016/j.neuropharm.2005.10.016DOI Listing
April 2006

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor.

J Neurochem 2005 Sep 30;94(5):1264-76. Epub 2005 Jun 30.

Pharmacology, Pharmakinetics Research Laboratories, Astellas Pharma Inc., Ibaraki, Japan.

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.
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http://dx.doi.org/10.1111/j.1471-4159.2005.03273.xDOI Listing
September 2005

Protective effect of FK506 against apoptosis of SH-SY5Y cells correlates with regulation of the serum inducible kinase gene.

Biochem Pharmacol 2005 May;69(10):1473-81

Advanced Technology Platform Research Laboratory, Fujisawa Pharmaceutical Co. Ltd. 5-2-3, Tokodai, Tsukuba, Ibaraki 300-2698, Japan.

Recently, we established an in vitro model of apoptosis induced by exposure of neuroblastoma SH-SY5Y cells to thapsigargin, an endoplasmic reticular calcium-ATPase inhibitor, and demonstrated that FK506 (tacrolimus) protected against apoptosis. The purpose of this paper was to investigate a possible correlation between the protective effect of FK506 against apoptosis and the regulation of the serum inducible kinase (SNK) and fibroblast growth factor inducible kinase (FNK) genes-which are polo-like kinases expressed abundantly in the brain by FK506. Thapsigargin increased the mRNA level of SNK and FNK in SH-SY5Y cells. FK506 inhibited the increase in SNK mRNA but not FNK mRNA. Deletion analysis of the SNK promoter showed that the promoter site, which was regulated by thapsigargin and FK506 in a calcineurin-dependent manner, is a cAMP response element (CRE)/activating transcription factor (ATF)-like element located 84 base pairs (bp) proximal to the transcriptional initiation site. Although transcription of the SNK gene was also regulated by tunicamycin, etoposide, or staurosporine, FK506 did not show any effects on these regulations. We recently reported that FK506 did not protect against apoptosis induced by these agents. These results indicate that the induction of SNK mRNA by thapsigargin in SH-SY5Y cells is regulated by FK506 via an inhibition of calcineurin at the transcriptional stage, and the transcriptional regulation of the SNK gene by FK506 was well correlated with the protective effect of the compound against apoptosis. Thus, transcriptional regulation of the SNK gene may be a biological marker for analysis of apoptosis of SH-SY5Y cells.
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http://dx.doi.org/10.1016/j.bcp.2005.02.019DOI Listing
May 2005

Suppressive effects of FR167653, an inhibitor of p38 mitogen-activated kinase, on calreticulin mRNA expression induced by endoplasmic reticulum stresses.

Eur J Pharmacol 2004 Jan;484(2-3):147-56

Advanced Technology Platform Research Laboratory, Fujisawa Pharmaceutical Co. Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan.

Several endoplasmic reticulum chaperones are simultaneously transactivated in response to various forms of endoplasmic reticulum stresses. Calreticulin is one such chaperone. We here show that the compound FR167653 [1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate] suppresses the transactivation of calreticulin following endoplasmic reticulum stress. FR167653, like SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole], has been reported to inhibit p38 mitogen-activated kinase (p38 MAPK). In this study, FR167653 concentration-dependently inhibited the up-regulation of the calreticulin mRNA level following an endoplasmic reticulum stress induced by thapsigargin in human embryonic kidney 293 (HEK293) cells and rat phechromocytoma PC12 cells. The compound concentration-dependently suppressed the transactivation of luciferase by thapsigargin in a reporter assay with a calreticulin promoter-luciferase conjugated reporter vector. SB203580 also significantly suppressed the transactivation of calreticulin by thapsigargin. Therefore, FR167653 regulated the mRNA expression of calreticulin at the transcriptional level without affecting the stability of the mRNA, as well as via inhibition of p38 MAPK activated by thapsigargin. FR167653 also inhibited the transactivation of calreticulin stimulated by two other endoplasmic reticulum stress inducers, tunicamycin and A23187. Moreover, the inhibitory action of the compound on the transactivation was observed in other cell lines. The calreticulin promoter region includes three sequential cis-acting endoplasmic reticulum stress response elements (ERSEs). As each of these ERSEs was sequentially deleted, there was an increasing loss of the transactivation by thapsigargin or tunicamycin. FR167653 inhibited the transactivation in all the reporter plasmid constructs containing the calreticulin promoter region with an ERSE/ERSEs. In conclusion, FR167653 is the first compound shown to inhibit the transactivation of calreticulin following various endoplasmic reticulum stresses. The suppressive effects of the compound were considered to be due to an inhibition of the signaling leading to ERSEs activation in the calreticulin promoter region via an inhibition of p38 MAPK, which is activated by endoplasmic reticulum stresses.
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http://dx.doi.org/10.1016/j.ejphar.2003.11.037DOI Listing
January 2004

Detailed in vitro pharmacological analysis of FK506-induced neuroprotection.

Neuropharmacology 2003 Sep;45(3):394-403

Advanced Technology Platform Research Laboratory, Fujisawa Pharmaceutical Co., Ltd., 5-2-3, Tokodai, 300-2698, Tsukuba, Ibaraki, Japan.

FK506, a calcineurin inhibitor, shows potent neuroprotective effects in animal models such as those of stroke and neurodegenerative diseases. However, the mechanism underlying these neuroprotective effects is unclear. In this study, an in vitro model, in which FK506 protected the cells against cell death, was established and analyzed in detail by pharmacological experiments. Thapsigargin (TG), an inhibitor of endoplasmic reticulum calcium-ATPase, induced SH-SY5Y cell death. FK506 concentration-dependently protected the cells from this type of death. In contrast, FK506 did not suppress SH-SY5Y cell death caused by the following molecules: tunicamycin (TM), an inhibitor of N-linked glycosylation; etoposide (Eto), a topoisomerase II inhibitor; and staurosporine (STS), a phospholipid/calcium-dependent protein kinase inhibitor. Additionally, FK506 did not inhibit TG-induced cell death in either SK-N-MC or HeLa cell lines. FK506 completely inhibited caspase-3 activation and apoptosis caused by TG in a concentration-dependent manner, but not that caused by TM, Eto, and STS. TG did not activate caspase-3 in SK-N-MC cells, although it slightly activated caspase-3 in HeLa cells. FK506 did not change caspase-3 activity in either SK-N-MC or HeLa cell lines. Cyclosporin A, another calcineurin inhibitor, showed the same results as FK506 in this study, whereas rapamycin, an immunosuppressant not associated with calcineurin activity, did not have any effect in this context. Thus, the suppressive effects of FK506 on cell death are specific to SH-SY5Y cells treated with TG and are caused by the inhibition of calcineurin and subsequent suppression of caspase-3 activation. Therefore, an in vitro system using SH-SY5Y cells treated with TG could provide a model reflective of certain aspects of the neuroprotective activity of FK506.
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http://dx.doi.org/10.1016/s0028-3908(03)00168-0DOI Listing
September 2003

Discovery of novel inhibitors of inducible nitric oxide synthase.

J Pharm Pharmacol 2002 Aug;54(8):1141-5

Advanced Technology Platform Research Laboratory, Fujisawa Pharmaceutical Co., Ltd, 5-2-3, Tokodai, Tusukuba, Ibaraki 300-2698, Japan.

We have discovered three compounds, 5-chloro-1,3-dihydro-2H-benzimidazol-2-one (FR038251), 1,3(2H,4H)-isoquinolinedione (FR038470) and 5-chloro-2,4(1H,3H)-quinazolonedione (FR191863), which show inhibition of inducible nitric oxide synthase (iNOS). The iNOS inhibitory activity of the compounds was examined in comparison with that of aminoguanidine, a representative iNOS inhibitor. FR038251, FR038470 and FR191863 inhibited mouse iNOS, with IC50 values of 1.7, 8.8 and 1.9 microM, respectively, in an in-vitro enzyme assay. These inhibitory activities are comparable with that of aminoguanidine (IC50 = 2.1 microM). The three compounds had iNOS selectivity 38- and 8-times, > 11- and 3-times, and 53- and 3-times compared with rat neuronal NOS and bovine endothelial NOS, respectively. These compounds concentration-dependently inhibited NO production in intact RAW264.7 mouse macrophages stimulated by lipopolysaccharide (LPS)/interferon-gamma. At 100 microM, FR038251, FR038470, FR191863 and aminoguanidine showed 81, 44, 54 and 78% inhibition of NO production, respectively. In an in-vivo experiment, FR038251, FR038470, FR191863 and aminoguanidine inhibited NO production in LPS-treated mice by 68, 40, 5 and 68% at an oral dose of 100 mg kg-1. The in-vivo inhibitory activity of FR038251 was almost identical to that of aminoguanidine. In conclusion, the three FR compounds are iNOS inhibitors with novel structures and may be candidate compounds leading to discovery of more iNOS inhibitors in the future.
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http://dx.doi.org/10.1211/002235702320266325DOI Listing
August 2002
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