#METABOLOMICS WORKBENCH michaelsa93_20170809_135138 DATATRACK_ID:1197 STUDY_ID:ST000846 ANALYSIS_ID:AN001370 PROJECT_ID:PR000600 VERSION 1 CREATED_ON August 10, 2017, 2:57 pm #PROJECT PR:PROJECT_TITLE Statin Immuno-Metabolomics in Asthma 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 IV) 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:LABORATORY Newman's Lab 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 - Zeki R 74 Tissue:Right Lung | Treatment:Control SUBJECT_SAMPLE_FACTORS - Zeki R 28 Repl Avg. Tissue:Right Lung | Treatment:Control SUBJECT_SAMPLE_FACTORS - Zeki R 48 Tissue:Right Lung | Treatment:Control SUBJECT_SAMPLE_FACTORS - Zeki R 68 Tissue:Right Lung | Treatment:Simvastatin SUBJECT_SAMPLE_FACTORS - Zeki R 40 Tissue:Right Lung | Treatment:Simvastatin SUBJECT_SAMPLE_FACTORS - Zeki R 95 Tissue:Right Lung | Treatment:Simvastatin SUBJECT_SAMPLE_FACTORS - Zeki L 74 Tissue:Left Lung | Treatment:Control SUBJECT_SAMPLE_FACTORS - Zeki L 48 Tissue:Left Lung | Treatment:Control SUBJECT_SAMPLE_FACTORS - Zeki L 28 Repl Avg. Tissue:Left Lung | Treatment:Control SUBJECT_SAMPLE_FACTORS - Zeki L 95 Tissue:Left Lung | Treatment:Simvastatin SUBJECT_SAMPLE_FACTORS - Zeki L 40 Tissue:Left Lung | Treatment:Simvastatin SUBJECT_SAMPLE_FACTORS - Zeki L 68 Tissue:Left Lung | Treatment:Simvastatin SUBJECT_SAMPLE_FACTORS - Zeki T 74 Tissue:Trachea | Treatment:Control SUBJECT_SAMPLE_FACTORS - Zeki T 48 Tissue:Trachea | Treatment:Control SUBJECT_SAMPLE_FACTORS - Zeki T 28 Repl Avg. Tissue:Trachea | Treatment:Control SUBJECT_SAMPLE_FACTORS - Zeki T 40 Tissue:Trachea | Treatment:Simvastatin SUBJECT_SAMPLE_FACTORS - Zeki T 68 Tissue:Trachea | Treatment:Simvastatin SUBJECT_SAMPLE_FACTORS - Zeki T 95 Tissue:Trachea | Treatment:Simvastatin #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. Tissue were CO:COLLECTION_SUMMARY collected upone animals termination at day 12 of Simvastatin treatment CO:SAMPLE_TYPE Tissue CO:TISSUE_CELL_IDENTIFICATION Trachea and lung tissue #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. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Oxylipins, endocannabinoids, bile acids and fatty acids were isolated using a SP:SAMPLEPREP_SUMMARY Waters Ostro Sample Preparation Plate (Milford, MA). Lung and trachea samples SP:SAMPLEPREP_SUMMARY were aliquoted (~40-60mg) into 2mL polypropylene tubes and spiked with a 5 µL SP:SAMPLEPREP_SUMMARY anti-oxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL SP:SAMPLEPREP_SUMMARY 1000nM analytical deuterated surrogates. A total of 50 µL of methanol, 550µL SP:SAMPLEPREP_SUMMARY isopropanol w/ 10mM ammonium formate & 1% formic acid and 100 uL water were SP:SAMPLEPREP_SUMMARY added and the tube was placed in a Geno/Grinder for 30 sec before being SP:SAMPLEPREP_SUMMARY centrifuged at 10,000g for 5 min at room temp. The supernate was then SP:SAMPLEPREP_SUMMARY transferred into the plate wells and samples were eluted into glass inserts SP:SAMPLEPREP_SUMMARY containing 10 μL 20% glycerol by applying a vacuum at 15 Hg for 10 min. Eluent SP:SAMPLEPREP_SUMMARY was dried by speed vacuum for 35 min at the medium BP setting, before switching SP:SAMPLEPREP_SUMMARY to an aqueous setting for an additional 35 min. Once dry, samples were SP:SAMPLEPREP_SUMMARY re-constituted with the internal standard 1-cyclohexyl ureido, 3-dodecanoic acid SP:SAMPLEPREP_SUMMARY (CUDA) and 1-Phenyl 3-Hexadecanoic Acid Urea (PHAU) at 100 nM (50:50 MeOH:CAN), SP:SAMPLEPREP_SUMMARY vortexed 1 min, transferred to a spin filter (0.1 µm, Millipore, Billerica, SP:SAMPLEPREP_SUMMARY MA), centrifuged for 3 min at 6ºC at <4500g (rcf), before being transferred to SP:SAMPLEPREP_SUMMARY 2 mL LC-MS amber vials. Extracts were stored at -20ºC until analysis by SP:SAMPLEPREP_SUMMARY UPLC-MS/MS. The internal standard was used to quantify the recovery of surrogate SP:SAMPLEPREP_SUMMARY 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 0.25 mL/min CH:COLUMN_TEMPERATURE 60 °C CH:SOLVENT_A 0.1% acetic acid CH:SOLVENT_B 90% ACN / 10% IPA CH:INTERNAL_STANDARD See protocol/methods file CH:RETENTION_TIME See protocol/methods file CH:SAMPLE_INJECTION 5 µL CH:ANALYTICAL_TIME 20 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 #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex 6500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Concentration (nM) MS_METABOLITE_DATA_START Samples Zeki R 74 Zeki R 28 Repl Avg. Zeki R 48 Zeki R 68 Zeki R 40 Zeki R 95 Zeki L 74 Zeki L 48 Zeki L 28 Repl Avg. Zeki L 95 Zeki L 40 Zeki L 68 Zeki T 74 Zeki T 48 Zeki T 28 Repl Avg. Zeki T 40 Zeki T 68 Zeki T 95 Factors Tissue:Right Lung | Treatment:Control Tissue:Right Lung | Treatment:Control Tissue:Right Lung | Treatment:Control Tissue:Right Lung | Treatment:Simvastatin Tissue:Right Lung | Treatment:Simvastatin Tissue:Right Lung | Treatment:Simvastatin Tissue:Left Lung | Treatment:Control Tissue:Left Lung | Treatment:Control Tissue:Left Lung | Treatment:Control Tissue:Left Lung | Treatment:Simvastatin Tissue:Left Lung | Treatment:Simvastatin Tissue:Left Lung | Treatment:Simvastatin Tissue:Trachea | Treatment:Control Tissue:Trachea | Treatment:Control Tissue:Trachea | Treatment:Control Tissue:Trachea | Treatment:Simvastatin Tissue:Trachea | Treatment:Simvastatin Tissue:Trachea | Treatment:Simvastatin 1-OG 3530 963.5 1590 891 928 1280 923 4490 1815 1340 1250 1150 2440 307 566 2190 1200 4640 2-OG 19500 5070 10400 4260 3400 5980 5700 23800 11750 10700 5440 4930 7750 693 1115 4620 3860 8510 1-LG 1560 734 1700 570 1430 635 1160 4620 1715 1010 1380 946 1920 189 500.5 1040 951 2070 2-LG 25700 7750 18300 5650 12900 5700 13600 13000 16500 13200 19500 8420 5220 1070 1165 3040 2930 2800 1-AG 615 157.5 287 287 260 234 351 1430 556.5 343 364 467 452 51.4 121.9 283 144 515 2-AG 25200 3475 11400 4750 4980 8290 5500 28400 18450 9630 9730 7540 4600 399 828.5 3120 1150 4860 PEA 140 233.5 336 153 159 367 204 283 252.7 291 212 284 330 380 240.5 256 288 239 SEA 725 613.5 635 168 297 538 299 603 611.5 608 396 336 362 323 261 448 274 340 OEA 99.2 68.1 69.7 36 50.7 66.6 29.4 83.9 86.9 104 58.9 58.4 35.6 20.6 16.6 40.5 18.7 28.3 LEA 32.2 13.5 28.4 18.8 19.2 25.3 11.8 94.4 20.5 31.8 16.4 20.5 27.9 9.3 12.4 13.2 8.9 21.2 aLEA 1 0.3 0.8 0.3 0.4 0.7 0.4 2.2 0.6 0.9 0.5 0.5 0.3 0.2 0.2 0.2 0.1 0.3 Dihomo GLA EA 4.1 1.3 2.2 0.6 1.2 1.7 1.5 3 2.6 2.8 1.7 1.7 2.1 0.9 0.4 1.1 0.6 0.6 AEA 11.8 4.3 8.3 2.4 3.7 7.8 3.7 38.5 7.5 11.3 4.1 7.3 9.8 2.4 2.8 4.3 2.8 5.5 DEA 8.9 2.5 4.6 1.8 2.1 4.4 2.5 21.7 5.4 6.2 2.5 3.3 9.6 1.4 1.3 4.6 2.2 5.3 DHEA 6.2 3.2 2.8 0.9 3.8 5.1 4 14.5 7.4 5.4 4.7 2.8 10.2 2.6 2.5 2.1 2.4 3.4 PGF2a EA 0.3 0.4 0.2 0.5 0.3 0.2 0.2 0.3 0.3 0.5 0.3 0.4 0.3 0.3 0.3 0.1 0.3 0.3 NO-Gly 7.8 3.9 2 3.1 4.4 4 2.1 3.5 4.6 5.2 6.5 4.5 1.5 0.5 0.9 4.5 0.9 2 PGF3a PGE2 1G PGF2a 1G PGE2 EA PGD2 EA 15-HETE EA 11(12)-EpETre EA NA-Gly MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 1-OG 2-OG 1-LG 2-LG 1-AG 2-AG PEA SEA OEA LEA aLEA Dihomo GLA EA AEA DEA DHEA PGF2a EA NO-Gly PGF3a PGE2 1G PGF2a 1G PGE2 EA PGD2 EA 15-HETE EA 11(12)-EpETre EA NA-Gly METABOLITES_END #END