#METABOLOMICS WORKBENCH michaelsa93_20170809_132951_mwtab.txt DATATRACK_ID:1196 STUDY_ID:ST000845 ANALYSIS_ID:AN001368 PROJECT_ID:PR000600 VERSION 1 CREATED_ON August 10, 2017, 2:52 pm #PROJECT PR:PROJECT_TITLE Statin Immuno-Metabolomics in Asthma PR:PROJECT_TYPE Placebo-controled trial PR:PROJECT_SUMMARY Innovative and novel therapies are urgently needed for the treatment of patients PR:PROJECT_SUMMARY with severe asthma, especially those who are refractory to standard-of-care PR:PROJECT_SUMMARY bronchodilators and inhaled corticosteroids. The Zeki lab is investigating the PR:PROJECT_SUMMARY role of the mevalonate (MA) pathway, in the pathogenesis of airway inflammation PR:PROJECT_SUMMARY and remodeling. Although statins all inhibit HMGCR in the same manner in terms PR:PROJECT_SUMMARY of enzyme binding site, the statins’ varied physiochemical properties with PR:PROJECT_SUMMARY respect to their polarity (i.e. lipophilicity) result in very different immune PR:PROJECT_SUMMARY and lipid effects. The major significance of this work is to advance a new class PR:PROJECT_SUMMARY of inhaler therapies for asthma; the statins which work by an entirely different PR:PROJECT_SUMMARY mechanism than current ICS/LABA mainstays. Evidence suggests that statins may PR:PROJECT_SUMMARY have an additive benefit to corticosteroids in asthma, thereby confirming a PR:PROJECT_SUMMARY unique mechanism, namely via MVA pathway inhibition. This becomes particularly PR:PROJECT_SUMMARY important in the severe asthma population which is highly PR:PROJECT_SUMMARY corticosteroid-resistant, is poorly controlled with high exacerbation rates and PR:PROJECT_SUMMARY hospitalizations, and has the highest healthcare costs of all asthma phenotypes. PR:PROJECT_SUMMARY In essence, the potential public health impact of even an incremental PR:PROJECT_SUMMARY improvement in asthma symptom control cannot be underestimated. Even the PR:PROJECT_SUMMARY prevention of 1 asthma attack preserves lung function and reduces the adverse PR:PROJECT_SUMMARY personal and financial impact. This study aimed to determine if statin polarity PR:PROJECT_SUMMARY affects airway drug concentration and systemic drug absorption and to determine PR:PROJECT_SUMMARY the effect of inhaled statins on naïve airway immune cell populations and PR:PROJECT_SUMMARY alveolar-capillary membrane and epithelial barrier integrity in healthy rhesus PR:PROJECT_SUMMARY monkeys. In this particular component of the study, we investigated the PR:PROJECT_SUMMARY metabolic effects resulting from the use of statins in these healthy rhesus PR:PROJECT_SUMMARY monkeys. Specifically, the Newman lab analyzed for lipid mediator (oxylipin, PR:PROJECT_SUMMARY endocannabinoid, fatty acid, and nitro lipid) in lung and trachea tissue, PR:PROJECT_SUMMARY plasma, and BAL and bile acid changes in the lung and trachea tissue and plasma. PR:INSTITUTE University of California, Davis PR:DEPARTMENT Internal Medicine PR:LAST_NAME Zeki PR:FIRST_NAME Amir PR:ADDRESS 2825 J St. Suite 400 Sacramento, CA 95816 PR:EMAIL aazeki@ucdavis.edu PR:PHONE (916) 734-8230 #STUDY ST:STUDY_TITLE Statin Immuno-Metabolomics in Asthma (part III) ST:STUDY_TYPE Placebo-controled trial ST:STUDY_SUMMARY Innovative and novel therapies are urgently needed for the treatment of patients ST:STUDY_SUMMARY with severe asthma, especially those who are refractory to standard-of-care ST:STUDY_SUMMARY bronchodilators and inhaled corticosteroids. The Zeki lab is investigating the ST:STUDY_SUMMARY role of the mevalonate (MA) pathway, in the pathogenesis of airway inflammation ST:STUDY_SUMMARY and remodeling. Although statins all inhibit HMGCR in the same manner in terms ST:STUDY_SUMMARY of enzyme binding site, the statins’ varied physiochemical properties with ST:STUDY_SUMMARY respect to their polarity (i.e. lipophilicity) result in very different immune ST:STUDY_SUMMARY and lipid effects. The major significance of this work is to advance a new class ST:STUDY_SUMMARY of inhaler therapies for asthma; the statins which work by an entirely different ST:STUDY_SUMMARY mechanism than current ICS/LABA mainstays. Evidence suggests that statins may ST:STUDY_SUMMARY have an additive benefit to corticosteroids in asthma, thereby confirming a ST:STUDY_SUMMARY unique mechanism, namely via MVA pathway inhibition. This becomes particularly ST:STUDY_SUMMARY important in the severe asthma population which is highly ST:STUDY_SUMMARY corticosteroid-resistant, is poorly controlled with high exacerbation rates and ST:STUDY_SUMMARY hospitalizations, and has the highest healthcare costs of all asthma phenotypes. ST:STUDY_SUMMARY In essence, the potential public health impact of even an incremental ST:STUDY_SUMMARY improvement in asthma symptom control cannot be underestimated. Even the ST:STUDY_SUMMARY prevention of 1 asthma attack preserves lung function and reduces the adverse ST:STUDY_SUMMARY personal and financial impact. This study aimed to determine if statin polarity ST:STUDY_SUMMARY affects airway drug concentration and systemic drug absorption and to determine ST:STUDY_SUMMARY the effect of inhaled statins on naïve airway immune cell populations and ST:STUDY_SUMMARY alveolar-capillary membrane and epithelial barrier integrity in healthy rhesus ST:STUDY_SUMMARY monkeys. In this particular component of the study, we investigated the ST:STUDY_SUMMARY metabolic effects resulting from the use of statins in these healthy rhesus ST:STUDY_SUMMARY monkeys. Specifically, the Newman lab analyzed for lipid mediator (oxylipin, ST:STUDY_SUMMARY endocannabinoid, fatty acid, and nitro lipid) in lung and trachea tissue, ST:STUDY_SUMMARY plasma, and BAL and bile acid changes in the lung and trachea tissue and plasma. ST:INSTITUTE USDA ST:DEPARTMENT Obesity and metabolism research unit ST:LAST_NAME Newman ST:FIRST_NAME John ST:ADDRESS 430 West Health Sciences Dr. Davis, Ca, 95616 ST:EMAIL John.Newman@ars.usda.gov ST:PHONE (530) 752-1009 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Macaca mulatta SU:TAXONOMY_ID 9544 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - PLAZ-14 Treatment:Control | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-32 Treatment:Control | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-23 Treatment:Control | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-09 Treatment:Simvastatin | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-19 Treatment:Simvastatin | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-26 Treatment:Simvastatin | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-04 Treatment:Control | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-34 Treatment:Control | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-08 Treatment:Control | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-25 Treatment:Simvastatin | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-13 Treatment:Simvastatin | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-06 Rep Avg. Treatment:Simvastatin | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-29 Treatment:Control | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-11 Treatment:Control | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-05 Treatment:Control | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-10 Treatment:Simvastatin | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-21 Treatment:Simvastatin | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-35 Treatment:Simvastatin | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-33 Treatment:Control | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-31 Treatment:Control | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-20 Treatment:Control | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-24 Treatment:Pravastatin | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-36 Treatment:Pravastatin | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-22 Treatment:Pravastatin | Day:Day 0 SUBJECT_SAMPLE_FACTORS - PLAZ-18 Treatment:Control | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-15 Treatment:Control | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-17 Treatment:Control | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-27 Treatment:Pravastatin | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-02 Treatment:Pravastatin | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-07 Rep Avg. Treatment:Pravastatin | Day:Day 8 SUBJECT_SAMPLE_FACTORS - PLAZ-12 Treatment:Control | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-01 Treatment:Control | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-30 Treatment:Control | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-28 Treatment:Pravastatin | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-16 Treatment:Pravastatin | Day:Day 12 SUBJECT_SAMPLE_FACTORS - PLAZ-03 Treatment:Pravastatin | Day:Day 12 #COLLECTION CO:COLLECTION_SUMMARY Monkeys were treated with placebo or Provastatin for 12 days. Further, after the CO:COLLECTION_SUMMARY wash out period animals were treated with Simvastatin for 12 days. Plasma was CO:COLLECTION_SUMMARY collected at day 0, 8 and 12 of each treatment. CO:SAMPLE_TYPE Blood CO:BLOOD_SERUM_OR_PLASMA Plasma #TREATMENT TR:TREATMENT_SUMMARY Monkeys were treated (by inhalation) with placebo or Provastatin for 12 days. TR:TREATMENT_SUMMARY Further, after the wash out period animals were treated with Simvastatin for 12 TR:TREATMENT_SUMMARY days. Plasma was collected at day 0, 8 and 12 of each treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Oxylipins, endocannabinoids, bile acids and fatty acids were isolated from an SP:SAMPLEPREP_SUMMARY aliquot of 20μl plasma in Eppendorf tubes. The tube was spiked was 5ul of SP:SAMPLEPREP_SUMMARY OxyEndo Fusion, 10μl spike of Bile Acid SSTD Solutions, and 5μl of BHT/EDTA SP:SAMPLEPREP_SUMMARY solution. A total of 100μl CUDA/PHAU in methanol was added to the tube, before SP:SAMPLEPREP_SUMMARY it was capped, vortexed, and centrifuged (3 min, 10,000 RCF, room temp). It was SP:SAMPLEPREP_SUMMARY then placed on wet ice for 5 min. Next it was spin filtered with Millapore spin SP:SAMPLEPREP_SUMMARY filters (0.1 µm, Millipore, Billerica, MA) at 8 ºC, 4500 RPM for 3 min, before SP:SAMPLEPREP_SUMMARY being transferred to 2 mL LC-MS amber vials. Extracts were stored at -20ºC SP:SAMPLEPREP_SUMMARY until analysis by UPLC-MS/MS. The internal standard was used to quantify the SP:SAMPLEPREP_SUMMARY recovery of surrogate standards. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Aquity C18 BEH 1.7µm 100mm x 2.1mm column CH:FLOW_GRADIENT See protocol/methods file CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 60 °C CH:SOLVENT_A 0.1% Formic Acid CH:SOLVENT_B 0.1% Formic Acid in Acetonitrile CH:INTERNAL_STANDARD See protocol/methods file CH:RETENTION_TIME See protocol/methods file CH:SAMPLE_INJECTION 5 µL CH:ANALYTICAL_TIME 16 min CH:WEAK_WASH_SOLVENT_NAME 20% methanol, 10% isopropanol CH:WEAK_WASH_VOLUME 600 µL CH:STRONG_WASH_SOLVENT_NAME 50:50 Acetonitrile:Methanol CH:STRONG_WASH_VOLUME 600 µL #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Newman AN:OPERATOR_NAME Kamil Borkowski AN:DETECTOR_TYPE API 6500 QTrap (AB Sciex, Framingham, MA, USA) AN:SOFTWARE_VERSION AB Sciex MultiQuant version 3.0.2 AN:DATA_FORMAT .mzML #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex 6500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Concentration (nM) MS_METABOLITE_DATA_START Samples PLAZ-14 PLAZ-32 PLAZ-23 PLAZ-09 PLAZ-19 PLAZ-26 PLAZ-04 PLAZ-34 PLAZ-08 PLAZ-25 PLAZ-13 PLAZ-06 Rep Avg. PLAZ-29 PLAZ-11 PLAZ-05 PLAZ-10 PLAZ-21 PLAZ-35 PLAZ-33 PLAZ-31 PLAZ-20 PLAZ-24 PLAZ-36 PLAZ-22 PLAZ-18 PLAZ-15 PLAZ-17 PLAZ-27 PLAZ-02 PLAZ-07 Rep Avg. PLAZ-12 PLAZ-01 PLAZ-30 PLAZ-28 PLAZ-16 PLAZ-03 Factors Treatment:Control | Day:Day 0 Treatment:Control | Day:Day 0 Treatment:Control | Day:Day 0 Treatment:Simvastatin | Day:Day 0 Treatment:Simvastatin | Day:Day 0 Treatment:Simvastatin | Day:Day 0 Treatment:Control | Day:Day 8 Treatment:Control | Day:Day 8 Treatment:Control | Day:Day 8 Treatment:Simvastatin | Day:Day 8 Treatment:Simvastatin | Day:Day 8 Treatment:Simvastatin | Day:Day 8 Treatment:Control | Day:Day 12 Treatment:Control | Day:Day 12 Treatment:Control | Day:Day 12 Treatment:Simvastatin | Day:Day 12 Treatment:Simvastatin | Day:Day 12 Treatment:Simvastatin | Day:Day 12 Treatment:Control | Day:Day 0 Treatment:Control | Day:Day 0 Treatment:Control | Day:Day 0 Treatment:Pravastatin | Day:Day 0 Treatment:Pravastatin | Day:Day 0 Treatment:Pravastatin | Day:Day 0 Treatment:Control | Day:Day 8 Treatment:Control | Day:Day 8 Treatment:Control | Day:Day 8 Treatment:Pravastatin | Day:Day 8 Treatment:Pravastatin | Day:Day 8 Treatment:Pravastatin | Day:Day 8 Treatment:Control | Day:Day 12 Treatment:Control | Day:Day 12 Treatment:Control | Day:Day 12 Treatment:Pravastatin | Day:Day 12 Treatment:Pravastatin | Day:Day 12 Treatment:Pravastatin | Day:Day 12 CA 32.9 103 ND 549 156 43.3 60.6 81.2 39.9 722 76 27.1 108 257 2.8 119 34.7 15.3 443 59.7 33.2 98.2 6580 15.7 57.3 1660 1590 182 139 42.8 13.7 64.1 1.51 57.9 24.2 9.36 CDCA 115 576 34.4 344 769 135 461 389 741 763 2120 153 1560 1190 66.2 221 143 103 426 294 182 163 11900 ND 320 2410 3330 791 2320 166 58 237 66.7 162 284 103 TCA 335 278 4.35 325 80.4 4690 12.8 68.4 0.458 36.9 ND 37.45 241 97.5 57.1 23.6 ND 125 80.2 238 34.7 175 1140 632 11.8 168 5.43 19.3 57 249.5 75.8 367 8.04 44.3 25.2 309 TCDCA 487 546 25.5 535 276 4180 83.4 388 33.7 135 41 112.5 613 281 219 71.8 5.1 117 128 530 131 444 2360 995 78.8 899 31 132 546 421.5 97.5 889 67.3 217 113 651 GCA 174 105 ND 410 187 2320 41 317 45.6 114 ND 183 226 42.1 60.6 18 ND 113 22.6 213 8.75 66.8 1430 730 39.6 562 ND 53.6 368 1345 35.8 184 ND 38.6 6.73 519 GCDCA 404 250 24.8 604 463 1540 194 928 327 356 430 498 442 163 333 107 27.5 140 98.9 338 103 184 1370 842 416 1600 90.8 435 2660 1840 98.7 442 79.5 312 127 935 DCA 197 598 253 162 274 875 121 250 594 328 1210 1375 755 1490 679 375 1130 1130 256 970 858 291 2860 677 241 678 1470 1030 1150 1620 363 171 248 172 651 537 LCA 230 299 314 197 748 417 154 167 718 296 1110 855.5 523 747 743 468 1690 758 474 729 599 366 1430 449 396 293 963 369 1090 556 416 278 346 215 1010 385 TDCA 313 161 21.8 358 25.5 4880 49.1 205 37.1 89.2 12.1 233 410 206 420 39.5 15.4 678 35.9 341 99.6 411 717 2060 55.2 631 33.2 186 358 1990 63 457 56.1 340 97.6 2680 GDCA 176 53.6 22 289 56.6 1910 70.8 416 224 162 327 888 199 104 354 86.4 67.2 437 19.8 160 63.4 125 335 1710 177 624 56.5 349 2210 6295 58.1 117 33.3 327 86.2 2600 GLCA 143 51.4 115 158 79.7 267 52.1 158 250 139 618 376.5 108 127 288 416 386 185 130 138 51.3 236 262 444 223 104 137 292 659 829.5 53.5 69.6 104 245 153 160 GHDCA 12.7 11.5 12.2 25 12.4 16.4 12.1 13.8 14.5 13 16 19.8 13.2 11.9 13.8 12.3 13.9 14.6 11.8 13.8 12.2 14.1 11.4 23.5 13.2 15.7 11.8 16.2 20.6 32.2 11.6 12.8 11.9 20.2 12.5 15.7 UDCA 1.24 39.7 3.05 4.43 12.3 3.63 0.78 3.24 7.59 2.62 29.1 7.43 20.2 39.3 1.95 10.6 4.9 1.67 5.25 7.63 2.68 1.28 98.6 0.378 3.07 17.7 36.3 49.3 26.7 5.755 7.8 2.78 0.895 3.29 16.3 2.68 TUDCA 5.13 11.5 2.56 64.8 5.29 39.8 1.68 8.04 2.03 5.81 1.96 4.565 6.18 7.07 3.86 3.94 1.91 4.4 2.44 8.97 2.35 24.3 20.3 16.9 1.73 9.74 1.79 7.12 6.24 13.6 2.65 16.5 2.09 12.4 3.79 10.8 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CA CDCA TCA TCDCA GCA GCDCA DCA LCA TDCA GDCA GLCA GHDCA UDCA TUDCA METABOLITES_END #END