Nobuyuki Amano

Takeda Pharmaceutical Company Limited, Ōsaka, Ōsaka, Japan

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Publications (14)43.75 Total impact

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    ABSTRACT: Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has emerged as a potential peripheral target for the treatment of obesity and metabolic disorders. We previously identified a novel series of N-phenylindoline-5-sulfonamide derivatives exemplified by 2 as potent and orally bioavailable MGAT2 inhibitors. Despite its attractive potency, further assessment revealed that this compound exhibited time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4). To remove the undesirable CYP3A4 TDI activity, structural modification was focused on the 2,4-difluoroaniline moiety on the basis of the assumption that this moiety would be involved in mechanism-based inhibition of CYP3A4 via oxidative metabolism. This led to the finding that the introduction of 4-chloro-2,6-difluoroaniline significantly improved CYP3A4 TDI risk. Further optimization resulted in the discovery of N-(4-chloro-2,6-difluorophenyl)-1-{5-[1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]pyrimidin-2-yl}-7-(2-oxopyrrolidin-1-yl)-2,3-dihydro-1H-indole-5-sulfonamide (27c) with potent MGAT2 inhibitory activity (IC50=7.8nM) and excellent ADME-Tox profiles including metabolic stability, oral bioavailability, and CYP3A4 TDI. In a mouse oral fat tolerance test, compound 27c effectively and dose-dependently suppressed the elevation of plasma triacylglycerol levels after oral administration at doses of 1 and 3mg/kg. We also discuss mitigation of the phototoxic liability of biaryl derivatives on the basis of the HOMO-LUMO gap hypothesis during the course of optimization efforts. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Bioorganic & Medicinal Chemistry 06/2015; DOI:10.1016/j.bmc.2015.06.003 · 2.95 Impact Factor
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    ABSTRACT: Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has attracted interest as a novel target for the treatment of obesity and metabolic diseases. Starting from a N-phenylbenzenesulfonamide derivative 1 with moderate potency for MGAT2 inhibition, we explored an effective location of the hydrophobic group at the 1-position to enhance MGAT2 inhibitory activity. Shifting the hydrophobic group to the adjacent position followed by introduction of a bicyclic central core to restrict the substituent orientation produced a N-phenylindoline-5-sulfonamide derivative 10b, which displayed much improved potency, with an IC50 value of 1.0 nM. This compound also exhibited excellent selectivity (greater than 30,000-fold) against related acyltransferases (MGAT3, DGAT1, DGAT2, and ACAT1). Subsequent optimization efforts were directed toward improving pharmacokinetic profiles, which resulted in the identification of 5-[(2,4-difluorophenyl)sulfamoyl]-7-(2-oxopyrrolidin-1-yl)-N-[4-(trifluoromethyl)phenyl]-2,3-dihydro-1H-indole-1-carboxamide (24d) endowed with potent MGAT2 inhibitory activity (IC50 = 3.4 nM) and high oral bioavailability (F = 52%, mouse). In a mouse oral fat tolerance test, oral administration of this compound effectively suppressed the elevation of plasma triacylglycerol levels.
    Journal of Medicinal Chemistry 04/2015; 58(9). DOI:10.1021/acs.jmedchem.5b00178 · 5.48 Impact Factor
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    ABSTRACT: We attempted to establish animal models to evaluate the effects of drug degradation in the stomach on oral bioavailability. In addition, we assessed the utilization of animal studies in determining the need for enteric-coated formulations. In order to control the gastric pH in rats and dogs, appropriate dosing conditions were investigated using pentagastrin and rabeprazole, which stimulate and inhibit gastric acid secretion. Using animals controlled for gastric acid secretion, the area under curve (AUC) ratios (AUC with rabeprazole/AUC with pentagastrin) of all compounds unstable under acidic conditions were evaluated. The AUC ratios of omeprazole and erythromycin, which are administered orally to humans, as enteric-coated tablets, were greater than 1.9 in the rats and dogs controlled for gastric acid secretion. On the contrary, the AUC ratios of clarithromycin, azithromycin, and etoposide (commercially available as a standard immediate-release form) were less than 1.3 each. In conclusion, in vivo models using rats and dogs were optimized to evaluate the effects of gastric acid on the oral bioavailability of drugs, and demonstrated that in vivo models can lead to a better understanding of the oral bioavailability, with respect to the formulation development. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.
    Journal of Pharmaceutical Sciences 02/2015; DOI:10.1002/jps.24401 · 3.01 Impact Factor
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    ABSTRACT: Using structure-based drug design, we identified and optimized a novel series of pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced solid malignancies.
    Bioorganic & medicinal chemistry letters 02/2013; 23(12). DOI:10.1016/j.bmcl.2013.02.083 · 2.33 Impact Factor
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    ABSTRACT: To date, the in vitro-in vivo correlation (IVIVC) of P-glycoprotein (P-gp)-mediated drug-drug interaction (DDI) at the blood-brain barrier (BBB) in rats indicated that the cutoff value to significantly affect the brain penetration of digoxin was [I,(unbound)/K(i)] of 1, where I,(unbound) is the unbound plasma concentration of P-gp inhibitors. Based on the IVIVC in rats, we speculated that clinically used P-gp inhibitors do not cause DDI at the human BBB, because none of the compounds studied was [I,(unbound)/K(i)]>1 at therapeutic doses. Recently, positron emission tomography studies with P-gp substrates such as [(11)C]verapamil, [(11)C]N-desmethyl loperamide, and [(11)C]loperamide together with potent P-gp inhibitors have indicated that increases in the influx rate constant for brain entry were observed in humans. Therefore, we aimed to retrospectively analyze the results of P-gp-mediated DDIs with in vitro P-gp inhibition assays and to confirm the appropriate cutoff value. In vitro P-gp inhibition assays using verapamil, N-desmethyl loperamide, and loperamide as P-gp probe substrates were performed in human MDR1-expressing LLC-PK1 cells. The efflux ratios decreased in the presence of P-gp inhibitors and the Ki of tariquidar was 10 nmol/L regardless of probe substrates. Taking the in vitro K(i) and unbound plasma concentrations in clinical DDI studies together, the criterion [I,(unbound)/K(i)] of 1 was an appropriate cutoff limit to observe significant P-gp-mediated DDI at the BBB in humans. On the other hand, no significant DDI was observed in cases where [I,(unbound)/K(i)] was less than 0.1. This criterion was comparable to the previous IVIVC result in rats.
    Drug metabolism and disposition: the biological fate of chemicals 01/2013; 41(4). DOI:10.1124/dmd.112.049577 · 3.33 Impact Factor
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    Bioorganic & Medicinal Chemistry Letters 12/2012; 22(23):7272. DOI:10.1016/j.bmcl.2012.09.098 · 2.33 Impact Factor
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    ABSTRACT: Spiro-pyrazolidinedione derivatives without quaternary chiral center were discovered by structure-based drug design and characterized as potent acetyl-CoA carboxylase (ACC) inhibitors. The high metabolic stability of the spiro-pyrazolo[1,2-a]pyridazine scaffold and enhancement of the activity by incorporation of a 7-methoxy group on the benzothiophene core successfully led to the identification of compound 4c as an orally bioavailable and highly potent ACC inhibitor. Oral administration of 4c significantly decreased the values of the respiratory quotient in rats, indicating the stimulation of fatty acid oxidation.
    Bioorganic & medicinal chemistry letters 05/2012; 22(14):4769-72. DOI:10.1016/j.bmcl.2012.05.062 · 2.33 Impact Factor
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    ABSTRACT: Spiro-lactone (S)-1 is a potent acetyl-CoA carboxylase (ACC) inhibitor and was found to be metabolically liable in human hepatic microsomes. To remove one of the risk factors in human study by improving the metabolic stability, we focused on modifying the spiro-lactone ring and the benzothiophene portion of the molecule. Spiro-imide derivative 8c containing a 6-methylthieno[2,3-b]pyridine core exhibited potent ACC inhibitory activity and favorable pharmacokinetic profiles in rats.
    Bioorganic & medicinal chemistry letters 04/2012; 22(11):3643-7. DOI:10.1016/j.bmcl.2012.04.047 · 2.33 Impact Factor
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    ABSTRACT: It has been reported that in vivo biliary clearance can be predicted using sandwich-cultured rat and human hepatocytes. The predicted apparent biliary clearance (CL(bile, app)) from sandwich- cultured rat hepatocytes (SCRH) based on medium concentrations correlates to in vivo CL(bile, app) based on plasma concentrations of angiotensin II receptor blockers (ARBs), HMG-CoA reductase inhibitors (statins), β-lactam antibiotics, and topotecan. However, the predicted biliary clearance from SCRH was 7- to 300-fold lower than in vivo biliary clearance. We speculated that the process of biliary excretion might not have been evaluated using sandwich-cultured hepatocytes. To evaluate this issue, intrinsic biliary clearance (CL(bile, int)) based on intracellular compound concentrations was evaluated to investigate the in vitro-in vivo correlation of this process among ARBs, statins, β-lactam antibiotics, and topotecan. Intrinsic biliary clearance in SCRH correlated to in vivo values obtained by constant intravenous infusion of six compounds, but not rosuvastatin and cefmetazole, to rats. Moreover, differences between SCRH and in vivo CL(bile, int) (0.7-6-fold) were much smaller than those of CL(bile, app) (7-300-fold). Therefore, in vivo CL(bile, int) is more accurately reflected using SCRH than CL(bile, app). In conclusion, to predict in vivo biliary clearance more accurately, CL(bile, int) should be evaluated instead of CL(bile, app) between SCRH and in vivo.
    Drug metabolism and disposition: the biological fate of chemicals 12/2011; 40(3):602-9. DOI:10.1124/dmd.111.042101 · 3.33 Impact Factor
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    ABSTRACT: Highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for measuring digoxin (DGX), a typical P-glycoprotein probe, in human plasma, rat plasma, and rat brain. We extracted DGX and deuterium-labeled DGX (as internal standard) from sample fluids under basic conditions using acetonitrile and sodium chloride-saturated 0.1 mol/L sodium hydroxide. The upper organic layer was diluted with distilled water, and the resulting solution was injected into an LC/MS/MS system in negative ionization mode. Chromatographic separation was achieved on a C(18)-ODS column in the gradient mobile phase, which comprised 0.05% (w/v) ammonium carbonate (pH 9.0) and methanol at a flow rate of 0.7 mL/min. Regardless of the type of biological matrix, intra-day and inter-day validation tests demonstrated good linearity of calibration curves within ranges of 0.1-10 ng/mL for plasma and 0.5-50 ng/g for rat brain and gave excellent accuracy and precision of quality control samples at 4 concentration levels. Unlike existing methods, our approach uses negative ionization to avoid competitive adduct formation of DGX. Our method showed higher sensitivity and wider applicability to various types of biological matrices than existing methods. Our method will support clinical and preclinical investigation of in vivo P-glycoprotein functionality using DGX.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 10/2011; 879(32):3837-44. DOI:10.1016/j.jchromb.2011.10.031 · 2.69 Impact Factor
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    ABSTRACT: The decision tree to determine whether the P-glycoprotein (P-gp)/multidrug resistance protein 1 (MDR1)-mediated drug-drug interaction (DDI) study is recommended has been proposed by the International Transporter Consortium. We, therefore, designed an in vitro P-gp inhibition assay and determined the appropriate risk criteria for P-gp-mediated DDI at the drug discovery stage. Effects of P-gp inhibitors on digoxin transport across a monolayer of MDR1-expressing cells were examined. The IC(50) (half-maximal inhibitory concentration) values generated from the efflux ratio (ER) were smaller than those generated from basolateral-to-apical directional apparent permeability. The difference in IC(50) values was kinetically described in a compartment model analysis. This analysis indicated that ER is a highly sensitive parameter that can be used for the degree of P-gp inhibition. Considering IC(50) values and the increase in digoxin exposure in clinical DDI studies, the risk criteria of [I(2)]/IC(50) = 30 ([I(2)], theoretically maximal gastrointestinal concentration) was the optimal cutoff value to predict a clinically relevant DDI. We also investigated whether the IC(50) value itself is applicable to assess the DDI risk. In conclusion, compounds with IC(50) values less than 2 μM exhibit high risk for P-gp-mediated DDIs. However, compounds with IC(50) values greater than or equal to 2 μM are inconclusive because clinical doses should be considered for the precise DDI risk assessment.
    Journal of Pharmaceutical Sciences 09/2011; 100(9):4013-23. DOI:10.1002/jps.22652 · 3.01 Impact Factor
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    ABSTRACT: The magnitude of P-glycoprotein [(P-gp)/multidrug resistance protein 1 (MDR1)]-mediated drug-drug interaction (DDI) at the blood-brain barrier (BBB) in rats was estimated by in vitro-in vivo correlation (IVIVC). In in vitro studies, rat Mdr1a-expressing LLC-PK1 cells were examined for the evaluation of P-gp inhibitory activity using digoxin as a P-gp probe substrate. The in vitro K(i) value was calculated using a modified corrected flux ratio that reflects the P-gp function. In in vivo studies, digoxin with or without P-gp inhibitors was administered to rats by constant intravenous infusion to evaluate the effect of P-gp inhibition on digoxin transport to the brain under steady-state conditions. In the presence of elacridar, the brain-to-plasma concentration ratio (K(p,brain)) of digoxin was approximately 14 times the control value. However, no significant change in the K(p,brain) was observed in the presence of clinically used P-gp inhibitors, with the exception of cyclosporine A. A positive correlation was found between the in vivo K(p,brain) of digoxin and [I(,unbound)/K(i)] (where I(,unbound) is the unbound plasma concentration of P-gp inhibitors). Compounds with [I(,unbound)/K(i)] values of >1 increased K(p,brain) of digoxin in rats. In summary, we used a quantitative approach to evaluate the impact of P-gp-mediated DDI at the rat BBB. We successfully established the IVIVC, which indicated the potential DDI in the presence of potent P-gp inhibitors. On the basis of the IVIVC in rats and K(i) values in human MDR1, we speculated that clinically used P-gp inhibitors do not cause DDI at the human BBB, because none of the compounds studied showed [I(,unbound)/K(i)] values of >1 at therapeutic doses.
    Drug metabolism and disposition: the biological fate of chemicals 10/2010; 39(1):8-14. DOI:10.1124/dmd.110.035774 · 3.33 Impact Factor
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    T Nishimura · Y Kato · N Amano · M Ono · Y Kubo · Y Kimura · H Fujita · A Tsuji
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    ABSTRACT: The oral bioavailability of some therapeutic agents is markedly lower in cynomolgus monkeys than in humans. We investigated small-intestinal absorption of the P-glycoprotein (P-gp) substrates etoposide and digoxin in monkeys to clarify the influence of efflux transport on their intestinal permeability. The pharmacokinetics of etoposide and digoxin was examined in monkeys and rats after oral and intravenous administration. Intestinal permeability and segmental differences in permeability were investigated with an Ussing-type chamber. The bioavailability of etoposide was 12.9 and 13.9% in monkeys and rats, respectively. Total body clearance of etoposide in monkeys was much less than hepatic blood flow, suggesting that the bioavailability would be limited at intestinal absorption. Marked vectorial transport of etoposide in the secretory direction was observed in rats, especially in the lower small intestine, and segmental differences were consistent with the distribution of P-gp expression. Vectorial transport was minimal in monkey small intestine. Our kinetic analysis indicated that P-gp contributes little to the intestinal permeability of etoposide and digoxin in monkeys, and apical uptake is rate-limiting. Low bioavailability of etoposide in monkeys is due to poor intestinal uptake resulting from low influx from the apical side, rather than secretion via P-gp.
    Pharmaceutical Research 08/2008; 25(11):2467-76. DOI:10.1007/s11095-008-9658-4 · 3.95 Impact Factor
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    ABSTRACT: Oral bioavailability of some drugs is substantially lower in cynomolgus monkeys than in various other species, including humans. In the present study, midazolam was used as a model drug to investigate the reason for the lower bioavailability in these monkeys. The bioavailability of midazolam after oral administration was minimal in monkeys and rats, being only 2.1 and 1.1%, respectively. In monkeys, this low bioavailability could not be explained simply in terms of a hepatic first-pass effect. To examine the roles of intestinal metabolism and transport, we evaluated apical-to-basal and basal-to-apical transport of midazolam, and the formation of metabolites in small intestinal tissues using an Ussing-type chamber. The values of mucosal extraction ratio were estimated to be 0.97, 0.93, and 0.89 during apical-to-basal transport in the upper, middle, and lower small intestine of monkeys, respectively, whereas the corresponding values for rats were close to zero, indicating that extensive metabolism of midazolam occurs, particularly in the upper region of the small intestine in monkeys, but not rats. Interestingly, formation of the metabolites was much greater during transport in the apical-to-basal direction than in the basal-to-apical direction, and this could be well explained by a mathematical model based on the assumption that extensive metabolism is associated with the uptake process of midazolam from the apical cell surface. Thus, we conclude that an asymmetric distribution of metabolic activity in the small intestine, leading to extensive metabolism during uptake from the apical cell surface, accounts for the minimal oral bioavailability of midazolam in cynomolgus monkeys.
    Drug Metabolism and Disposition 09/2007; 35(8):1275-84. DOI:10.1124/dmd.106.013037 · 3.33 Impact Factor