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Supplemental data belonging to:
The selective sirtuin 1 activator SRT2104 reduces endotoxin-induced cytokine release and coagulation activation in humans
Anne Jan van der Meer1, Brendon P. Scicluna1, Perry D. Moerland2, Jiang Lin3, Eric W. Jacobson4, George P. Vlasuk4 and Tom van der Poll1
1Center for Experimental Molecular Medicine (CEMM), Academic Medical Center, Amsterdam, the Netherlands. 2Bioinformatics Laboratory, Department of Clinical Epidemiology, Academic Medical Center,Amsterdam, the Netherlands. 3GlaxoSmithKline, 5 Moore Drive, RTP, North Carolina, United States, 4Sirtris, A GSK Company, 200 Technology Square, Cambridge, MA, United States.
Word counts: text: 1214
Numbers: figures: 3; tables: 2 ; references: 4
2 Supplemental Materials and Methods
Design and subjects of human endotoxemia study
The study was approved by the institutional scientific and ethics committees, and registered under NCT01014117 (clinicaltrials.gov). Written informed consent was obtained from all subjects. Twenty-four healthy, non-smoking, Caucasian male volunteers (mean (± SE) age 22.9 ± 0.5 years) completed the study. Medical history, physical examination, hematological and biochemical screening and electrocardiogram were all normal. The study was randomized, double-blind, placebo-controlled and consisted of three treatment arms (N = 8 per arm): (1) Oral SRT2104 (2 gram/day) for seven consecutive days; (2) placebo on Days 1- 6 and SRT2104 (2 gram) on Day 7; (3) placebo for seven consecutive days. All subjects attended the Clinical Research Unit of the Academic Medical Center on the evening prior to the first dosing of SRT2104 or placebo and left on Day 2. The subjects self-administered test material orally for the next five days and returned to the Clinical Research Unit on the evening of Day 6; subjects remained in the unit until Day 8. After an overnight fast study drug (placebo or SRT2104) was taken orally approximately 15 minutes following consumption of a standardized meal on all dosing days. On Day 7, all subjects received intravenous lipopolysaccharide (Escherichia coli LPS, lot #118884; U.S. standard reference endotoxin, kindly provided by the National Institutes of Health, Bethesda, MD) 4 ng/kg body weight 3 hours after dosing with SRT2104/placebo. Aural temperature, blood pressure, heart rate, and respiratory rate were measured at half-hour intervals (Philips V24C, Eindhoven, the Netherlands). Clinical symptoms such as headache, chills, nausea, and myalgia were recorded throughout the study period using a graded scale (0, absent; 1, mild; 2, moderate; 3, severe).
3 Blood collection
Blood was collected from a cannulated forearm vein. For pharmacokinetic analyses blood was collected on both the first and the last dosing days 30 minutes before and 15 minutes, 0.5, 1, 2, 3, 4, 8, 12 and 24 hours after SRT2104/placebo dosing. For analyses of LPS-induced inflammatory responses, blood was obtained 3 hour before LPS injection, directly before LPS administration (t = 0 h) and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, and 21 hours thereafter. For leukocyte and differential counts, blood was drawn into sterile 4.5-ml tubes containing EDTA-K3. Heparinized plasma (for measurement of SRT2104 plasma levels and C-reactive protein (CRP), EDTA-K3 anticoagulated plasma (cytokines, chemokines and elastase) or citrated plasma (coagulation and fibrinolysis) was obtained by immediate centrifugation (4°C, 15 min, 3000 rpm) and stored at –20°C until assayed. For genome wide transcription analysis, blood was collected 30 minutes before the first SRT2104/placebo dosing, 30 minutes before last SRT2104/placebo dosing and 4 hours after LPS injection in PAXgene tubes (Qiagen, Venlo, the Netherlands).
RNA preparation and genome-wide transcriptional profiling
Whole blood total RNA was isolated by means of the Qiacube (Qiagen) automated PAXgene blood RNA kit protocol in accordance to the manufacturer’s instructions. Total RNA yield and integrity (RIN>7.0) were assessed by spectrophotometry (NanoDrop ND-1000) and by bioanalysis (Agilent 2100 Bioanalyzer). Synthesis, amplification and purification of anti- sense RNA was performed by using the Illumina TotalPrep RNA Amplification Kit (Ambion art. No. AM-IL1791) following the Illumina Sentrix Array Matrix expression protocol at ServiceXS (Leiden, the Netherlands). A total of 750 ng biotinylated cRNA was hybridized onto the HumanHT-12v3 Expression BeadChip (Illumina), which contains 48,804 probes covering 27,455 well-established RefSeq annotated genes and 12,837 UniGene annotated
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genes. The raw scan data were read using the beadarray package (version 1.16.0),1 using the R statistical package (version 2.11.0; R Foundation for Statistical Computing, Vienna, Austria).
Illumina’s default pre-processing steps were performed using beadarray. Briefly, estimated background was subtracted from the foreground for each bead. For replicate beads, outliers greater than 3 median absolute deviations from the median were removed and the average signal was calculated for the remaining intensities. For each probe a detection score was calculated by comparing its average signal with the summarized values for the negative control probes. Log-transformation was applied to summarized data in order to remove mean- variance relationship in intensities. Resulting data were then quantile normalized.2 Quality control was performed both on bead level and on bead summary data. Using the arrayQualityMetrics package (v2.6.0) on the normalized bead summary data, one outlier array was detected. This array was discarded and the data was renormalized. Probes were reannotated using the package illuminaHumanv3BeadID.db from Bioconductor. Gene ontology overrepresentation was assessed by using the Database for Annotation, Visualization, and Integrated Discovery (DAVID).3;4 The array datasets are available for download at the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) under the ac- cession number GSE48119.
Statistical analysis
The sample size of eight subjects per group provides 80% power to detect a statistically significant difference in a given efficacy parameter if an effect size of 1.5 (effect size = difference in means divided by common standard deviation) is obtained and a two group t-test with common variance is employed with alpha = 0.05. Values presented are given as mean ± SE unless otherwise stated. All analyses were summarized by treatment group. For all endpoints, area-under-the curve (AUC) for the time from start of study drug on the LPS
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challenge day to 12 hours post LPS injection (AUC -3 to 12 hours) was calculated using the linear trapezoidal rule: the sum of the areas between each chronological pair of assessments (using observed times). The primary analysis was done by repeated measures analysis of variance (RM-ANOVA). All p-values listed in the Results section are based on this primary analysis by RM-ANOVA unless stated otherwise. The secondary analysis was done on the AUC (-3 to 12 hours) by one-way ANOVA model with treatment as a factor. Analysis of CRP was done by an analysis of covariance (ANCOVA) model, fitting change from baseline as the dependent variable, treatment as a fixed effect, and baseline as a covariate. Comparison of the treatment groups was tested using two-sided alpha level of 0.05 . All statistical analyses were performed using SAS, version 9.2 (SAS Institute, Cary, NC, USA). Differential gene expression was assessed using moderated t-tests and F-tests using the limma package and Benjamini-Hochberg false discovery rate corrected p-values.
6 References
1. Dunning MJ, Smith ML, Ritchie ME, Tavare S. beadarray: R classes and methods for Illumina bead-based data. Bioinformatics. 2007;23:2183-2184.
2. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.
Bioinformatics. 2003;19:185-193.
3. Huang dW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat.Protoc. 2009;4:44-57.
4. Huang dW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37:1- 13.
7 Supplemental Figure legends and tables
Figure S1: LPS-induced fibrinolytic response. Mean (± SE) total tissue type plasminogen activator (tPA) (A) and plasminogen activator inhibitor type I (PAI-1) (B) after LPS
administration (4ng/kg i.v., t= 0 h). SRT2104 or placebo was given daily for 7 days prior to LPS administration (last dose at -3h before LPS injection). NS = not significant. Black square:
Placebo for 7 days. Blue dot: placebo for 6 days followed by single dose SRT2104 (-3h before LPS). Red triangle:SRT2104 for 7 days.
Figure S2: LPS-induced leukocyte responses. Mean (± SE) total leukocyte count (A), neutrophil count (B) elastase-α1-antitrypsin levels (C) after LPS administration (4ng/kg i.v.
t = 0 h). SRT2104 or placebo was given daily for 7 days prior to LPS administration (last dose at -3h before LPS injection). NS = not significant. Black square: Placebo for 7 days. Blue dot:
placebo for 6 days followed by single dose SRT2104 (-3h before LPS). Red triangle:
SRT2104 for 7 days.
Figure S3: Volcano plot analysis of the endotoxin-induced transcriptional response. Blue circles denote probes for which the log2 transformed fold-change is greater than 2. BH adjusted p-value, Benjamini-Hochberg false discovery rate corrected nominal p-value of the moderated t test.
8 Supplemental Table 1: SRT2104 plasma levels Time (hours
after ingestion )
SRT-2104 single dose (Day 7) (ng/ml)
SRT-2104 multiple dose (Day 1) (ng/ml)
SRT-2104 multiple dose (Day 7) (ng/ml)
0 0 0 99.0 ± 129.4
0.25 0 0.1 ± 0.3 95.6 ± 124.4
0.5 6.5 ± 5.4 16.6 ± 32.6 105.1 ± 128.6
1 125.2 ± 125.7 144.9 ± 346.2 212.1 ± 220.7
2 444.5 ± 278.5 286.4 ± 325.5 406.7 ± 391.8
3 496.3 ± 274.1 401.0 ± 277.9 495.0 ± 507.8
4 399.2 ± 208.3 320.7 ± 263.8 445.8 ± 374.7
8 179.9 ± 167.0 215.9 ± 290.7 243.5 ± 260.5
12 111.7 ± 84.9 102.5 ± 121.8 150.2 ± 153.2
24 61.8 ± 38.4 46.0 ± 45.9 94.0 ± 88.2
Data are means ± standard deviation . Time indicates hours after ingestion of SRT2104. LPS was administered 3 hours after SRT2104 ingestion, i.e., at the expected tmax.
9 Supplemental Table 2: Pharmacokinetic data
SRT-2104 single dose (Day 7) (ng/ml)
SRT-2104 multiple dose (Day 1) (ng/ml)
SRT-2104 multiple dose (Day 7) (ng/ml) Cmax (ng/ml) 560.4 ± 251.9 515.2 ± 380.0 593.7 ± 527.3
tmax (hour) 3.4 ± 2.0 3.3 ± 1.9 2.9 ± 1.1
AUC (ng/ml.hr) 4032.3 ± 2208.4 3559.5 ± 3217.2 5045.8 ± 4835.0
t½ (hour) 13.7 ± 4.5 10.8 ± 3.0 16.0 ± 6.3
Data are means ± standard deviation .
