#METABOLOMICS WORKBENCH u023968_20190207_183931 DATATRACK_ID:1618 STUDY_ID:ST001135 ANALYSIS_ID:AN001860 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 6210 TOF MS:INSTRUMENT_TYPE Other 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 automatic integration software MasterHands (Keio University, Tsuruoka, Japan) in MS:MS_COMMENTS order to obtain peak information including m/z, migration time for CE-TOFMS MS:MS_COMMENTS measurement (MT) and peak area. Signal peaks corresponding to isotopomers, MS:MS_COMMENTS adduct ions, and other product ions of known metabolites were excluded, and MS:MS_COMMENTS remaining peaks were annotated with putative metabolites from the HMT metabolite MS:MS_COMMENTS database based on their MTs and m/z values determined by TOFMS. The tolerance MS:MS_COMMENTS range for the peak annotation was configured at ±0.5 min for MT and ±10 ppm MS:MS_COMMENTS for m/z. In addition, peak areas were normalized against those of the internal MS:MS_COMMENTS standards and then the resultant relative area values were further normalized by MS:MS_COMMENTS sample amount. #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 Adenine 23.6 17.7 20.7 20.5 25.6 18.3 15.6 15.7 Adenosine 9.20 10.30 10.04 9.61 11.38 9.37 10.07 10.59 Alanine 12432 11711 9414 10703 9154 7941 4284 4301 Arginine 890 919 870 952 1059 948 1052 1105 Argininosuccinic acid 32.4 27.9 29.5 27.3 30.8 27.9 20.4 17.9 Asparagine 455 398 394 393 422 332 400 338 Aspartic acid 11047 12559 9616 11342 9440 9449 4934 5886 Beta-Alanine 390 370 333 376 402 362 362 340 Betaine 484 635 528 645 646 632 594 652 Betaine aldehyde 0.352 N.D. 0.537 0.006 0.712 N.D. N.D. N.D. Carnitine 599 635 593 647 586 597 548 574 Carnosine 9.46 9.97 8.61 9.08 9.15 9.90 9.32 9.86 Choline 159 197 147 191 193 157 177 204 cis-3-hydroxyproline 81.8 92.7 84.8 86.5 88.7 94.3 90.2 91.1 Citrulline 10.03 10.57 9.77 10.28 12.35 10.82 10.88 11.14 Creatine 4167 4497 4223 4705 5555 5035 5805 6347 Creatinine 5.25 5.50 4.89 7.15 7.48 6.07 6.83 6.22 Cystathionine 1791 2156 1840 2177 2063 2111 1941 2063 Cysteine 602 378 359 321 817 325 662 509 Gamma-Aminobutyric acid 8.14 7.77 8.84 7.50 9.47 6.84 9.76 7.86 Glutamic acid 55195 63135 53295 60239 61228 59639 58612 62625 Glutamine 54299 57601 51123 57098 60057 53072 48176 50866 Glutathione 18710 22947 14342 20757 22146 18541 22934 23263 Glycine 25088 24691 23745 25504 28075 24249 24921 25217 Guanosine 3.95 N.D. N.D. N.D. N.D. N.D. 4.17 N.D. Histidine 3697 3656 3350 4132 4099 3821 3985 4135 Homocysteine N.D. N.D. N.D. N.D. N.D. N.D. N.D. 2.97 Homoserine 13.6 12.9 13.2 15.2 N.D. 16.2 N.D. 12.7 Inosine N.D. N.D. N.D. N.D. N.D. 0.903 N.D. N.D. Isoleucine 6087 6197 5737 6492 7031 6292 6299 6688 Leucine 7514 8021 6994 8279 8565 7729 7793 8586 Lysine 2393 2603 2391 2713 2762 2422 2835 2929 Methionine 4961 5371 4642 5444 5333 5415 5387 5789 Ornithine 76.7 93.7 74.3 91.9 65.1 70.4 47.8 38.6 Oxidized glutathione 5703 5305 6472 5842 5502 5830 3560 3635 Phenylalanine 4984 5168 4691 5254 5549 5007 5165 5413 Proline 8283 8465 7272 7747 7335 6720 4165 4201 Putrescine 4.63 4.79 4.57 3.82 4.07 3.43 3.29 N.D. S-Adenosylhomocysteine 41.4 76.5 57.8 57.0 43.9 49.1 68.8 60.8 S-Adenosylmethionine 976 1008 870 1136 1084 1105 915 1097 Sarcosine N.D. 16.35 8.72 N.D. 17.85 3.07 13.53 14.62 Serine 20596 21174 18361 20581 21268 19113 16595 16974 Spermidine 8.07 8.56 8.03 8.71 8.85 9.14 5.47 7.54 Spermine 40.62 35.73 33.65 47.92 37.86 37.10 9.83 28.58 Threonine 31255 31006 28471 31658 33751 29901 27489 28305 Tryptophan 580.3 613.4 541.2 635.1 711.4 573.3 645.8 652.4 Tyrosine 5081 5351 4771 5518 5665 5200 5548 5557 Valine 8177 8421 7804 8862 9626 8897 9303 9781 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID HMDB ID Adenine C00147 HMDB00034 Adenosine C00212 HMDB00050 Alanine C00041,C00133,C01401 HMDB00161,HMDB01310 Arginine C00062,C00792 HMDB00517,HMDB03416 Argininosuccinic acid C03406 HMDB00052 Asparagine C00152,C01905,C16438 HMDB00168 Aspartic acid C00049,C00402,C16433 HMDB00191,HMDB06483 Beta-Alanine C00099 HMDB00056 Betaine C00719 HMDB00043 Betaine aldehyde C00576 HMDB01252 Carnitine C00318,C00487,C15025 HMDB00062 Carnosine C00386 HMDB00033 Choline C00114 HMDB00097 cis-3-hydroxyproline C01015,C01157 HMDB06055,HMDB00725 Citrulline C00327 HMDB00904 Creatine C00300 HMDB00064 Creatinine C00791 HMDB00562 Cystathionine C00542,C02291 HMDB00099 Cysteine C00097,C00736,C00793 HMDB00574,HMDB03417 Gamma-Aminobutyric acid C00334 HMDB00112 Glutamic acid C00025,C00217,C00302 HMDB00148,HMDB03339 Glutamine C00064,C00303,C00819 HMDB00641,HMDB03423 Glutathione C00051 HMDB00125 Glycine C00037 HMDB00123 Guanosine C00387 HMDB00133 Histidine C00135,C00768,C06419 HMDB00177 Homocysteine C00155,C05330 HMDB00742 Homoserine C00263 HMDB00719 Inosine C00294 HMDB00195 Isoleucine C00407,C06418,C16434 HMDB00172 Leucine C00123,C01570,C16439 HMDB00687 Lysine C00047,C00739,C16440 HMDB00182,HMDB03405 Methionine C00073,C00855,C01733 HMDB00696 Ornithine C00077,C00515,C01602 HMDB00214,HMDB03374 Oxidized glutathione C00127 HMDB03337 Phenylalanine C00079,C02057,C02265 HMDB00159 Proline C00148,C00763,C16435 HMDB00162,HMDB03411 Putrescine C00134 HMDB01414 S-Adenosylhomocysteine C00021 HMDB00939 S-Adenosylmethionine C00019 HMDB01185 Sarcosine C00213 HMDB00271 Serine C00065,C00716,C00740 HMDB00187,HMDB03406 Spermidine C00315 HMDB01257 Spermine C00750 HMDB01256 Threonine C00188,C00820 HMDB00167 Tryptophan C00078,C00525,C00806 HMDB00929 Tyrosine C00082,C01536,C06420 HMDB00158 Valine C00183,C06417,C16436 HMDB00883 METABOLITES_END #END