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Cytokine and lipid metabolome effects of low-dose acetylsalicylic acid in critically ill patients with systemic inflammation: a pilot, feasibility, multicentre, randomised, placebo-controlled trial
Luca Cioccari*, Nora Luethi*, Thy Duong, Eileen Ryan, Salvatore L Cutuli, Patryck Lloyd-Donald, Glenn M Eastwood, Leah Peck, Helen Young, Suvi T Vaara, Craig J French, Neil Orford, Jyotsna Dwivedi, Yugeesh R Lankadeva, Michael Bailey, Gavin E Reid, Rinaldo Bellomo, (* equal first authors)
Crit Care Resusc 2020; 22 (3): 227-236
- Luca Cioccari* 1, 2, 3
- Nora Luethi* 1, 2
- Thy Duong 4
- Eileen Ryan 5
- Salvatore L Cutuli 1, 6
- Patryck Lloyd-Donald 1
- Glenn M Eastwood 1, 2
- Leah Peck 1
- Helen Young 1
- Suvi T Vaara 1
- Craig J French 2, 7
- Neil Orford 2, 8
- Jyotsna Dwivedi 9
- Yugeesh R Lankadeva 10
- Michael Bailey 2, 11
- Gavin E Reid 4, 5
- Rinaldo Bellomo 1, 2, 11
- (* equal first authors) 12
The trial was supported by the Intensive Care Trust Fund (Austin Hospital, Melbourne). The active and placebo drugs were sponsored by Bayer Pharmaceuticals, Germany. The funding bodies did not have input into the design, management or reporting of the trial.
OBJECTIVE: The systemic inflammatory response syndrome (SIRS) is a dysregulated response that contributes to critical illness. Adjunctive acetylsalicylic acid (ASA) treatment may offer beneficial effects by increasing the synthesis of specialised proresolving mediators (a subset of polyunsaturated fatty acid-derived lipid mediators).
DESIGN: Pilot, feasibility, multicentre, double-blind, randomised, placebo-controlled trial.
SETTING: Four interdisciplinary intensive care units (ICUs) in Australia.
PARTICIPANTS: Critically ill patients with SIRS.
INTERVENTIONS: ASA 100 mg 12-hourly or placebo, administered within 24 hours of ICU admission and continued until ICU day 7, discharge or death, whichever came first.
MAIN OUTCOME MEASURES: Interleukin-6 (IL-6) serum concentration at 48 hours after randomisation and, in a prespecified subgroup of patients, serum lipid mediator concentrations measured by mass spectrometry.
RESULTS: The trial was discontinued in December 2017 due to slow recruitment and after the inclusion of 48 patients. Compared with placebo, ASA did not decrease IL-6 serum concentration at 48 hours. In the 32 patients with analysis of lipid mediators, low-dose ASA increased the concentration of 15-hydroxyeicosatetraenoic acid, a proresolving precursor of lipoxin A4, and reduced the concentration of the proinflammatory cytochrome P-dependent mediators 17-HETE (hydroxyeicosatetraenoic acid), 18-HETE and 20-HETE. In the eicosapentaenoic acid pathway, ASA significantly increased the concentration of the anti-inflammatory mediators 17,18-DiHETE (dihydroxyeicosatetraenoic acid) and 14,15-DiHETE.
CONCLUSIONS: In ICU patients with SIRS, low-dose ASA did not significantly alter serum IL-6 concentrations, but it did affect plasma concentrations of certain lipid mediators. The ability to measure lipid mediators in clinical samples and to monitor the effect of ASA on their levels unlocks a potential area of biological investigation in critical care.
TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ACTRN 12614001165673).
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Apart from its well known anti-inflammatory properties owing to, at least in part, its irreversible inhibition of cyclooxygenase (COX)-1, ASA also modulates COX-2 activity. 8
The primary objective of this randomised controlled trial was to test the feasibility and safety of ASA treatment as well as its biological effects on conventional inflammatory markers in critically ill patients with SIRS. The secondary objective was to analyse SPMs in low volumes of human plasma in a clinical setting using modern mass spectrometry methods. 20
Study design, screening and randomisationWe conducted a multicentre, double-blind, randomised, placebo-controlled trial across four interdisciplinary intensive care units (ICUs) in Australia. All ICU admissions with two or more positive SIRS criteria were assessed for eligibility. Eligible patients were adults (18 years or older) who were expected to stay in the ICU for at least 48 hours. We excluded patients transferred to ICU after cardiac surgery or neurosurgery, those with active bleeding, coagulopathy or other contraindications to ASA, and patients already taking ASA for primary or secondary prevention. Details of the inclusion and exclusion criteria are provided in Table S1 (Online Appendix). Eligible patients were randomly assigned in a 1:1 ratio to receive either ASA 100 mg or placebo administered twice daily via oral route or nasogastric tube. Randomisation was performed by the site investigators using sealed opaque envelopes. Blinding regarding the trial regimen was ensured by supply of the study drugs as visually identical tablets. All site investigators, patients and treating clinicians were unaware of the trial-group assignment and sequence. Study drugs were continued until ICU discharge, up to a maximum of 7 days, or until the development of any exclusion criteria. All other aspects of patient care were at the discretion of the treating clinicians.
Outcome measuresThe primary outcome was serum IL-6 level at 48 hours after randomisation. Secondary outcomes included serum levels of other inflammatory biomarkers (IL-10, E-selectin and C-reactive protein) at 48 hours, length of ICU and hospital stay, duration of mechanical ventilation, and vital status at ICU and hospital discharge. Exploratory outcomes included plasma PUFA-derived lipid mediator levels, which were measured once daily over the first 72 hours in one centre (Austin Hospital). Safety outcomes included the requirement for blood transfusions and the frequency of renal replacement therapy during the intervention period.
Blood sampling and processingFor batch analysis of IL-6, IL-10 and E-selectin, blood samples were obtained at the time of randomisation and after 48 hours. An additional plasma sample was taken daily for the first 72 hours in the subgroup of patients included in the lipid mediator analysis. Blood was collected from the central venous or arterial catheter using a BD Vacutainer system (Z Serum Sep Clot Activator and 3E 3K-EDTA, respectively; BD Diagnostics, Franklin Lakes, NJ, USA). For biomarker analysis, blood samples were centrifuged at 5000 rpm for 5 minutes immediately after collection, and then the supernatant serum and/or plasma was aspirated into 2 mL Eppendorf Tubes and stored directly at –80°C.
Analysis of IL-6, IL-10 and E-selectin levelsQuantification of serum IL-6, IL-10 and E-selectin levels was performed by enzyme-linked immunosorbent assays (ELISAs) using commercially available kits as per the manufacturer’s instructions (Thermo Fisher Scientific, Vienna, Austria). All tests were performed in duplicate. Reactions were developed using chromogen (TMB substrate; Invitrogen, Carlsbad, CA, USA) at 100 µL/well, and then stopped by the addition of 100 µL/well stop solution (2 M H2SO4) and immediately read (λ = 450 nm and 550 nm) on a spectrophotometer (Synergy H1 Hybrid Microplate Reader, BioTek Instruments, Winooski, Vt, USA). The intra-assay variation for all serum cytokine and E-selectin ELISAs was < 10%.
Extraction and analysis of SPMsAfter thawing on ice, 0.4 mL of plasma spiked with internal standards was quenched with 1.2 mL of ice-cold methanol (containing 0.01% butylated hydroxytoluene) and left to equilibrate for 15 minutes to allow protein precipitation. The samples were then centrifuged for 5 minutes at 10 000 rpm, and the supernatant was diluted into 4.8 mL of Milli-Q water. Lipid mediators were then enriched by using solid-phase extraction (96-well plates, Trajan Scientific and Medical, Melbourne, Australia). The wells were conditioned with 0.5 mL of methanol and then 0.5 mL of Milli-Q water before samples were loaded. Next, the wells were washed twice with 0.5 mL of 5% methanol and eluted with 0.4 mL of methanol. The eluants were dried to completion under a gentle stream of nitrogen. Samples were reconstituted in 40 µL of methanol (containing 0.01% butylated hydroxytoluene), and then 10 µL of reconstituted sample was injected for reversed-phase high-performance liquid chromatography (HPLC) electrospray ionisation tandem mass spectrometry analysis (Shimadzu LC/MS 8060 triple quadrupole mass spectrometer, coupled with a Nexera HPLC system; Shimadzu, Kyoto, Japan). Mass spectrometry scans were performed using positive and negative ionisation mode polarity switching, based on a method previously developed by Yamada and colleagues for the Method Package for Lipid Mediators for Shimadzu, 21
Statistical analysisThe trial aimed to include 120 patients to reach a > 90% power to demonstrate a significant difference in IL-6 levels. All analyses were performed on an intention-to-treat basis. In the case of missing data, we reported the number of available observations and did not use imputation. Proportions were compared using the χ2 or Fisher exact test. Continuous variables are presented as medians with interquartile ranges (IQRs) and compared using the Mann–Whitney test. After logarithmic transformation, plasma biomarkers were analysed using the two-way RM-ANOVA (repeated measures analysis of variance) to test for the fixed effects of time, treatment allocation and treatment by time interaction. Analyses were performed using STATA/SE 14.0 (StataCorp, College Station, Tex, USA) and SAS 9.4 (SAS Institute, Cary, NC, USA). Unless otherwise stated, a two-sided P value of 0.05 was used to indicate statistical significance. No adjustments were made for multiple comparisons.
Ethics approval and patient consentThe study protocol was approved by the ethics committees of Austin Health HREC/14/Austin/219) and each of the participating institutions. Written informed consent was obtained from all participants or their legal representatives.
Patient flow and recruitmentBetween March 2015 and December 2017, we screened 985 patients for eligibility and randomly assigned 50 patients to one of the study groups (a recruitment rate of 1.5 participants per month). The trial was discontinued prematurely, on 31 December 2017, due to slow patient recruitment and insufficient funding. The most frequent reasons for exclusion were that the patient was already taking ASA (259/985, 26.3%) and that the patient had a high risk of bleeding or a coagulopathy (244/985, 24.8%). The patient flow through the study is summarised in Figure 1. Of the 50 patients assigned to a study group, one patient in each group revoked consent, leaving a total of 48 patients (23 in the ASA group and 25 in the placebo group) for the final intention-to-treat analysis. With 48 patients included, the study had > 80% power to demonstrate a 30% relative difference in the primary outcome.
Patient characteristicsOverall, 40 patients (83.3%) fulfilled the Sepsis-2 criteria and, of those, 18 (45.0%) presented with septic shock. There were no significant differences in median (IQR) Acute Physiology and Chronic Health Evaluation (APACHE) III scores (53 [47–62] v 52 [42–64], respectively), numbers of patients receiving vasopressor therapy or other baseline characteristics between the ASA and the placebo group. Baseline demographics and clinical characteristics of patients, according to treatment allocation, are shown in Table 1.
All 48 patients received the assigned trial regimen. The median (IQR) time from randomisation to commencement of the study drug was 59 (11–115) minutes in the ASA group and 30 (15–65) minutes in the placebo group (P = 0.75). Median duration of study treatment was 4 (2–6) days in the ASA group and 6 (3–7) days in the placebo group (P = 0.11) and patients received a median of 7 (4–12) and 10 (5–14) ASA and placebo doses (P = 0.11), respectively (Online Appendix, table S2).
Primary and secondary outcomesSeven patients were discharged from the ICU in less than 48 hours. Therefore, 41 patients (85.4%; 19 in the ASA group and 22 in the placebo group) had one complete set of blood samples taken at baseline and one at 48 hours after assignment to a study group. In these patients, serum concentrations of IL-6, IL-10 and E-selectin at 48 hours were not significantly different between study groups (Table 2). Furthermore, we found no differences in C-reactive protein levels at 48 hours, ICU length of stay, duration of mechanical ventilation, ICU mortality and hospital mortality between the groups.
Anti-inflammatory and proresolving lipid mediatorsFor comprehensive lipid mediator analysis, we analysed 262 blood samples from 32 patients (15 in the ASA group and 17 in the placebo group) using mass spectrometry. We detected 81 different specific lipid mediators derived from three major bioactive essential fatty acid pathways (AA, EPA and DHA) (Online Appendix, figures S1 and S2).
In the AA pathway, low-dose ASA increased the concentration of the proresolving mediator 15-HETE (hydroxyeicosatetraenoic acid; P = 0.03 for treatment by time interaction), and reduced the concentration of the proinflammatory, cytochrome P-dependent mediators 17-HETE (P = 0.046), 18-HETE (P = 0.01) and 20-HETE (P = 0.01) (Figure 2).
In the EPA pathway, ASA transiently increased the concentration of the anti-inflammatory mediators 14,15-DiHETE (dihydroxyeicosatetraenoic acid; P = 0.04 for treatment by time interaction) and 17,18-DiHETE (P = 0.03) (Figure 2). We found no significant differences in the abundance of metabolites of DHA.
Safety outcomesNone of the participants experienced clinically relevant bleeding and there was no difference in proportion or number of participants receiving blood transfusions in the ICU (Table 2).
Key findingsIn this multicentre randomised placebo-controlled trial of critically ill patients with SIRS, low-dose ASA did not reduce levels of classic inflammatory biomarkers, including IL-6, compared with placebo. Because the study was stopped prematurely, it was underpowered and therefore did not provide sufficient evidence to support or refute the primary hypothesis. However, we detected a wide array of complex lipid mediators of inflammation using mass spectrometry. ASA had a measurable effect on lipid mediators derived from the AA and EPA acid pathways, increasing the concentrations of individual proresolving and anti-inflammatory mediators, and decreasing the levels of specific proinflammatory and vasoregulatory mediators.
Relationship to previous studiesObservational data suggest that prehospital use of low-dose ASA is associated with reduced mortality in sepsis 6, 7, 23, 24, 25, 26
Until recently, the presumed beneficial effects of ASA have been attributed to its anti-inflammatory actions, as it is a negative regulator of prostaglandin-induced cytokine production. 30, 31
Early detection and quantification of specific lipid mediators of inflammation using mass spectrometry has the potential to provide real-time information on changes in inflammatory status. However, although the technology for lipid mediator analysis has improved in terms of measurement specificity and sensitivity, 15, 21
To our knowledge, only one study to date has measured SPMs in critically ill patients using liquid chromatography mass spectrometry techniques. 37
In our study, low-dose ASA significantly increased the concentration of the proresolving mediator 15-HETE, increased the concentration of the anti-inflammatory mediators 14,15-DiHETE and 17,18-DiHETE, and decreased the level of the proinflammatory mediators 20-HETE, 17-HETE and 18-HETE. Such non-COX-mediated effects of ASA are increasingly recognised 38
We did not detect any significant differences in levels of 14-HDHA (hydroxydocosahexaenoic acid; maresin precursor), 17-HDHA (protectin precursor), resolvin D1, resolvin E2 or resolvin E3. Besides, we did not detect measurable levels of ASA-triggered (acetylated-COX-dependent) lipid mediators such as 15(R)-HETE and 5(S),6(S)-lipoxin A4. These discrepancies to other studies may be due to several reasons. First, owing to rigorous exclusion criteria, our patients were less ill than those in other trials, as reflected by lower APACHE III scores. Second, we used a different dose of ASA (200 mg per day, as compared with 81 mg, 39, 40, 41
Implications of the findingsThe slow recruitment rate of 1.5 patients per month was primarily due to the exclusion of a significant proportion of patients with pre-existing ASA treatment or with evidence of coagulopathy. Of note, we observed no episode of considerable bleeding. Thus, our observations indicate that future trials of ASA in critically ill patients could, therefore, consider less stringent exclusion criteria. Following other reports, 44
Strengths and limitationsTo our knowledge, this is the first randomised placebo-controlled trial providing a comprehensive assessment of the effects of ASA in critically ill patients with severe inflammation. All the enrolled patients received the allocated intervention of ASA or placebo, which were administered less than 1 hour after randomisation. We measured classic inflammatory markers such as C-reactive protein levels, white cell counts and inflammatory cytokine levels, and compared them to specific lipid mediators of inflammation, using advanced, cutting-edge technology. The multicentre, double-blind design of the trial increased the robustness of our findings.
Our study had, however, some limitations. The most important limitation was the small sample size due to slow recruitment and early termination, which decreased our power to detect a difference in the primary outcome. However, we were able to demonstrate apparent changes in key lipid mediators of inflammation in a small subset of patients. Our screening failure rate is in line with that of other larger randomised controlled trials of ASA. 29
ConclusionsIn patients admitted to the ICU with SIRS, of which more than 80% were septic, low-dose ASA did not significantly alter IL-6 concentrations at 48 hours compared with placebo. However, ASA increased the concentration of specific proresolving and anti-inflammatory lipid mediators and decreased the concentration of proinflammatory mediators. Our findings provide additional data on the complex lipid mediator system in systemic inflammation, and support further investigations outside the more commonly measured cytokine system.
Acknowledgements: We thank the trial site research staff for their support with randomisation and data collection: Samantha Bates and Rebecca Morgan, Footscray Hospital, Western Health; and Tania Elderkin†, University Hospital Geelong († Deceased March 2019).