#METABOLOMICS WORKBENCH u023968_20190207_183931 DATATRACK_ID:1618 STUDY_ID:ST001135 ANALYSIS_ID:AN001861 PROJECT_ID:PR000760 VERSION 1 CREATED_ON February 13, 2019, 5:53 pm #PROJECT PR:PROJECT_TITLE Antidiabetic and cardiovascular beneficial effects of a liver-localized PR:PROJECT_TITLE mitochondrial uncoupler PR:PROJECT_SUMMARY Inducing mitochondrial uncoupling (mUncoupling) is an attractive therapeutic PR:PROJECT_SUMMARY strategy for treating metabolic diseases because it leads to calorie-wasting by PR:PROJECT_SUMMARY reducing the efficiency of oxidative phosphorylation (OXPHOS) in mitochondria. PR:PROJECT_SUMMARY Here we report a safe mUncoupler, OPC-163493, which has unique pharmacokinetic PR:PROJECT_SUMMARY characteristics. OPC-163493 shows a good bioavailability upon oral PR:PROJECT_SUMMARY administration and primarily distributed to specific organs: the liver and PR:PROJECT_SUMMARY kidneys, avoiding systemic toxicities. It exhibitsinsulin-independent PR:PROJECT_SUMMARY antidiabetic effects in multiple animal models of type I and type II diabetes PR:PROJECT_SUMMARY and antisteatotic effects in fatty liver models. These beneficial effects can be PR:PROJECT_SUMMARY explained by the improvement of glucose metabolism and enhancement of energy PR:PROJECT_SUMMARY expenditure by OPC-163493 in the liver. Moreover, OPC-163493 treatment lowered PR:PROJECT_SUMMARY blood pressure, extended survival, and improved renal function in the rat model PR:PROJECT_SUMMARY of stroke/hypertension, possibly by enhancing NO bioavailability in blood PR:PROJECT_SUMMARY vessels and reducing mitochondrial ROS production. OPC-163493 is a PR:PROJECT_SUMMARY liver-localized/targeted mUncoupler that ameliorates various complications of PR:PROJECT_SUMMARY diabetes. PR:INSTITUTE Otsuka Pharmacetical Co., Ltd. PR:LAST_NAME Kanemoto PR:FIRST_NAME Naohide PR:ADDRESS 463-10 Kagasuno Kawauchi-cho, Tokushima, Tokusima, 770-0865, Japan PR:EMAIL Kanemoto.Naohide@otsuka.jp PR:PHONE +81-88-665-2126 #STUDY ST:STUDY_TITLE Different dose exposure of OPC-163493 on HepG2 cells (part-I) ST:STUDY_TYPE Compound dosage test ST:STUDY_SUMMARY Metabolomics analysis were on 8 samples of HepG2 cells that were treated with ST:STUDY_SUMMARY compound OPC-163493 (DMSO control, 1, 3, or 10µM; each n=2) exposure for 30 ST:STUDY_SUMMARY min. ST:INSTITUTE Otsuka Pharmaceuticals ST:LAST_NAME Kanemoto ST:FIRST_NAME Naohide ST:ADDRESS 463-10 Kagasuno Kawauchi-cho Tokushima 771-0192, Japan ST:EMAIL Kanemoto.Naohide@otsuka.jp ST:PHONE 81-03-6717-1400 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Not applicable SU:CELL_STRAIN_DETAILS HepG2 cells #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - OPC-0uM-1 Treatment:- Duration (min)=30; Cell line=HepG2 cell SUBJECT_SAMPLE_FACTORS - OPC-0uM-5 Treatment:- Duration (min)=30; Cell line=HepG2 cell SUBJECT_SAMPLE_FACTORS - OPC-1uM-2 Treatment:1 Duration (min)=30; Cell line=HepG2 cell SUBJECT_SAMPLE_FACTORS - OPC-1uM-6 Treatment:1 Duration (min)=30; Cell line=HepG2 cell SUBJECT_SAMPLE_FACTORS - OPC-3uM-3 Treatment:3 Duration (min)=30; Cell line=HepG2 cell SUBJECT_SAMPLE_FACTORS - OPC-3uM-7 Treatment:3 Duration (min)=30; Cell line=HepG2 cell SUBJECT_SAMPLE_FACTORS - OPC-10uM-4 Treatment:10 Duration (min)=30; Cell line=HepG2 cell SUBJECT_SAMPLE_FACTORS - OPC-10uM-8 Treatment:10 Duration (min)=30; Cell line=HepG2 cell #COLLECTION CO:COLLECTION_SUMMARY Culture medium of HepG2 cells in 100-mm dish was aspirated and cells were washed CO:COLLECTION_SUMMARY twice by 5% mannitol solution, and then processed with sampleprep steps. CO:SAMPLE_TYPE HepG2 cells CO:STORAGE_CONDITIONS Room temperature #TREATMENT TR:TREATMENT_SUMMARY HepG2 cells were seeded into a 100mm dish the day before OPC-163493 treatment. TR:TREATMENT_SUMMARY OPC-163493 treatments (DMSO control, 1, 3, or 10uM, each n=2) were performed for TR:TREATMENT_SUMMARY 30min in FBS-free DMEM with high glucose (25mM). TR:TREATMENT_DOSE 0uM, 1uM, 3uM, 10uM TR:TREATMENT_DOSEDURATION 30 min TR:CELL_MEDIA MEM with Earle`s salts, L-Glutamine and Non-Essiontial Amino Acids, 10% fetal TR:CELL_MEDIA bovine serum, and 1mM sodium pyruvate solution #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Culture medium of HepG2 cells in 100-mm dish was aspirated and cells were washed SP:SAMPLEPREP_SUMMARY twice by 5% mannitol solution, and then the cells were treated with methanol. SP:SAMPLEPREP_SUMMARY The cell extract was treated with Mili-Q water containing internal standard and SP:SAMPLEPREP_SUMMARY filtered with 5-kDa cutoff filter. The filtrate was centrifugally concentrated SP:SAMPLEPREP_SUMMARY and re-suspended in 50 µL of Milli-Q water. SP:PROCESSING_STORAGE_CONDITIONS Room temperature SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION 50 uL Mili-Q #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY capillary electrophoresis was connected with time-of-flight mass spectrometry CH:CHROMATOGRAPHY_SUMMARY (CE-TOFMS) for cation analysis and tandem mass spectrometry (CE-MS/MS) for CH:CHROMATOGRAPHY_SUMMARY anion. CH:CHROMATOGRAPHY_TYPE CE CH:INSTRUMENT_NAME Agilent 7100 CE CH:COLUMN_NAME Fused silica capillary, i.d. 50 µm × 80 cm #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS The spectrometer was scanned from m/z 50 to 1,000. Peaks were extracted using MS:MS_COMMENTS MasterHands, automatic integration software (Keio University, Tsuruoka, MS:MS_COMMENTS Yamagata, Japan) and MassHunter Quantitative Analysis B.04.00 (Agilent MS:MS_COMMENTS Technologies) in order to obtain peak information including m/z, peak area, and MS:MS_COMMENTS migration time (MT). Signal peaks were annotated according to the HMT metabolite MS:MS_COMMENTS database based on their m/z values with the MTs. The peak area of each MS:MS_COMMENTS metabolite was normalized with respect to the area of the internal standard and MS:MS_COMMENTS metabolite concentration was evaluated by standard curves with three-point MS:MS_COMMENTS calibrations using each standard compound. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Concentration (pmol/1000000 cells) MS_METABOLITE_DATA_START Samples OPC-0uM-1 OPC-0uM-5 OPC-1uM-2 OPC-1uM-6 OPC-3uM-3 OPC-3uM-7 OPC-10uM-4 OPC-10uM-8 Factors Treatment:- Treatment:- Treatment:1 Treatment:1 Treatment:3 Treatment:3 Treatment:10 Treatment:10 2,3-Diphosphoglyceric acid 7.85 15.14 5.76 10.46 3.94 5.24 N.D. 1.23 2-hydroxyglutaric acid 152 139 126 132 133 126 123 117 2-Phosphoglyceric acid 5.52 10.74 6.58 7.06 7.56 9.46 7.22 9.25 3',5' cyclic AMP 198 209 183 224 210 228 176 215 3',5' cyclic GMP 0.0640 0.1106 0.0461 0.0829 0.0726 0.0859 0.1020 0.0820 3-hydroxy-3-methylglutaryl-CoA 4.20 3.39 4.39 4.57 6.98 5.30 9.58 8.91 3-Phosphoglyceric acid 62.2 139.6 71.0 99.2 87.3 117.1 68.5 103.2 6-Phosphogluconic acid 140.1 131.3 113.9 114.8 125.2 104.2 87.3 102.6 Acetyl-CoA N.D. 0.272 0.221 0.373 0.335 N.D. N.D. N.D. Adenylsuccinic acid 1.43 1.37 1.09 1.34 1.09 1.07 1.06 1.09 ADP 854 834 813 1054 1113 1133 1739 1723 ADP-ribose 3.44 2.38 2.41 2.27 2.98 2.42 1.51 2.19 AMP 212 180 178 194 296 211 455 418 ATP 11381 10615 10386 11184 11699 11217 10336 10957 Cis-Aconitic acid N.D. 4.035 7.518 2.567 7.254 7.278 5.601 0.769 Citric acid 1079 1079 1144 1188 1494 1309 1126 1138 Coenzyme A 129.2 175.8 40.9 140.6 199.7 115.4 269.2 291.7 Dihydroxyacetone phosphate 372 414 319 387 270 266 105 153 Folic acid 3.76 5.31 4.51 5.08 4.61 3.88 3.50 4.62 Fructose-1,6-biphosphate 480 421 371 383 345 239 157 234 Fructose-1-phosphate 619 609 564 608 632 663 506 558 Fructose-6-phosphate 174 184 154 175 168 202 161 181 Fumaric acid 237.93 210.38 196.12 228.96 210.49 108.79 8.75 4.56 Galactose-1-phosphate 23.8 24.0 20.1 14.3 25.6 20.0 19.5 14.9 GDP 136 123 123 144 158 160 200 220 Glucose-1-phosphate 33.83 26.85 5.61 20.71 9.18 29.87 14.34 24.08 Glucose-6-phosphate 540 543 478 612 557 604 529 618 Glyceraldehyde-3-phosphate 19.84 31.77 17.01 22.82 11.38 14.03 N.D. 4.13 Glycerol 3-phosphate 2794 2663 2797 2770 2945 2880 3305 3292 GMP 36.8 35.1 34.8 42.9 52.2 41.7 78.6 91.2 GTP 2243 2209 1935 2190 2254 2172 1769 1879 IMP 50.4 46.2 41.9 49.9 46.7 52.5 70.8 81.3 Ketoisovaleric acid 119.1 83.6 91.0 120.4 140.1 111.6 116.8 118.0 Lactic acid 13208 8323 10483 11054 14632 11156 14799 13892 Malic acid 2041 1689 1631 1496 1499 1232 606 656 Malonyl-CoA 0.407 0.371 N.D. 0.308 0.344 0.342 0.280 0.278 Mevalonic acid 0.356 0.894 0.370 N.D. 0.450 0.439 N.D. N.D. N-acetylglutamic acid 64.0 52.8 69.2 64.7 77.7 69.3 86.1 78.8 NAD+ 2521 2277 2300 2481 2599 2567 2484 2571 NADH 150.2 186.7 122.8 135.8 91.4 91.5 65.0 71.3 NADP+ 423 370 374 422 441 394 391 451 NADPH 22.6 46.5 25.8 35.4 19.6 42.9 19.5 24.0 N-carbamoylaspartic acid 33.38 31.23 24.89 26.31 19.43 18.88 7.03 8.03 Oxoglutaric acid 947 781 618 830 1645 1276 1753 2171 Phosphocreatine 3001 3144 2518 3005 2458 2748 1352 1745 Phosphoenolpyruvic acid 22.6 53.1 25.4 37.1 32.5 46.7 27.0 40.5 Phosphoribosyl pyrophosphate 180.2 165.1 188.1 186.9 225.9 177.1 72.1 73.3 Pyruvic acid 239 254 187 429 238 261 139 299 Ribose-1-phosphate 53.6 47.7 39.4 48.8 45.7 43.3 39.2 49.2 Ribose-5-phosphate 45.35 31.24 24.26 37.94 25.17 28.07 9.15 20.33 Ribulose-5-phosphate 69.4 34.1 36.9 53.4 41.3 40.1 31.4 34.1 Sedoheptulose 7-phosphate 38.7 44.5 37.8 44.2 43.8 55.6 57.2 44.5 Succinic acid 234 182 207 216 279 194 314 341 UDP-glucose 2029 1734 1617 1755 1802 1629 1366 1402 XMP 3.84 3.60 3.25 3.53 3.53 3.07 2.69 3.25 Xylulose-5-phosphate 162.1 157.4 140.9 112.7 80.5 107.1 48.5 88.5 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID HMDB ID 2,3-Diphosphoglyceric acid C01159 HMDB01294 2-hydroxyglutaric acid C02630,C01087,C03196 HMDB00606,HMDB00694 2-Phosphoglyceric acid C00631 HMDB03391 3',5' cyclic AMP C00575 HMDB00058 3',5' cyclic GMP C00942 HMDB01314 3-hydroxy-3-methylglutaryl-CoA C00356 HMDB01375 3-Phosphoglyceric acid C00197 HMDB00807 6-Phosphogluconic acid C00345 HMDB01316 Acetyl-CoA C00024 HMDB01206 Adenylsuccinic acid C03794 HMDB00536 ADP C00008 HMDB01341 ADP-ribose C00301 HMDB01178 AMP C00020 HMDB00045 ATP C00002 HMDB00538 Cis-Aconitic acid C00417 HMDB00072 Citric acid C00158 HMDB00094 Coenzyme A C00010 HMDB01423 Dihydroxyacetone phosphate C00111 HMDB01473 Folic acid C00504 HMDB00121 Fructose-1,6-biphosphate C00354 HMDB01058 Fructose-1-phosphate C01094,C02976 HMDB01076 Fructose-6-phosphate C05345,C00085 HMDB00124 Fumaric acid C00122 HMDB00134 Galactose-1-phosphate C00446 HMDB00645 GDP C00035 HMDB01201 Glucose-1-phosphate C00103 HMDB01586 Glucose-6-phosphate C00668,C01172,C00092 HMDB01401 Glyceraldehyde-3-phosphate C00118,C00661 HMDB01112 Glycerol 3-phosphate C00093 HMDB00126 GMP C00144 HMDB01397 GTP C00044 HMDB01273 IMP C00130 HMDB00175 Ketoisovaleric acid C00141 HMDB00019 Lactic acid C00186,C00256,C01432 HMDB00190,HMDB01311 Malic acid C00149,C00497,C00711 HMDB00156,HMDB00744 Malonyl-CoA C00083 HMDB01175 Mevalonic acid C00418,C02104 HMDB00227 N-acetylglutamic acid C00624 HMDB01138 NAD+ C00003 HMDB00902 NADH C00004 HMDB01487 NADP+ C00006 HMDB00217 NADPH C00005 HMDB00221 N-carbamoylaspartic acid C00438 HMDB00828 Oxoglutaric acid C00026 HMDB00208 Phosphocreatine C02305 HMDB01511 Phosphoenolpyruvic acid C00074 HMDB00263 Phosphoribosyl pyrophosphate C00119 HMDB00280 Pyruvic acid C00022 HMDB00243 Ribose-1-phosphate C00620 HMDB01489 Ribose-5-phosphate C00117 HMDB01548 Ribulose-5-phosphate C00199,C01101 HMDB00618 Sedoheptulose 7-phosphate C05382 HMDB01068 Succinic acid C00042 HMDB00254 UDP-glucose C00029 HMDB00286 XMP C00655 HMDB01554 Xylulose-5-phosphate C00231,C03291 HMDB00868 METABOLITES_END #END