The effect of intravenous interleukin-1 receptor antagonist on inflammatory mediators in cerebrospinal fluid after subarachnoid haemorrhage: a phase II randomised controlled trial

This was a two-centre (Salford Royal NHS Foundation Trust, Salford, and Addenbrooke’s Hospital, Cambridge, UK), randomised, double-blind, placebo-controlled trial. Ethical and appropriate competent authority approvals were obtained. The inclusion and exclusion criteria are shown in Table 1.

Informed consent was obtained from the patient or the patient’s representative. Demographic and clinical details were recorded. SAH severity was graded clinically using the World Federation of Neurosurgical Societies (WFNS) score [21] and radiologically using the Fisher grade [22]. The randomisation service was provided by an independent third-party professional company, Sealed Envelope (London, UK; http://​www.​sealedenvelope.​com). After consent the patient was randomised, baseline clinical assessment was performed, and CSF (2 mL) and plasma (5 mL) samples were taken. This was immediately followed by administration of placebo or IL-1Ra. IL-1Ra was injected as Kineret, a recombinant, non-glycosylated form of human IL-1Ra [23]. An IV bolus of 500 mg, given over 1 minute, was immediately followed by a 10 mg/kg/hr infusion over 24 hours. CSF and plasma samples were taken at 6, 12, 24 (end of infusion), 36, 48, and 72 hours. Adverse event reporting was carried out according to Medicines and Healthcare Products Regulatory Agency (MHRA) and sponsor regulations.

Blood samples were collected into tubes containing ethylenediaminetetraacetic acid (EDTA) and centrifuged at 2,000 g at 4°C for 15 minutes. Plasma was frozen at −70°C. The CSF was sampled from the patient’s external ventricular drain (EVD), after discarding the first 2 mL, and processed as for plasma. IL-1Ra concentrations were measured by enzyme-linked immunosorbent assay (ELISA), as described elsewhere [19]. Monocyte chemoattractant protein-1 (MCP-1), IL-1β, IL-6, IL-8, IL-10, and tumour necrosis factor-alpha (TNF-α) concentrations in plasma and CSF, and CRP in plasma, were measured using Luminex bead technology (Luminex, Austin, TX, USA). Bio-Plex COOH beads (Bio-Rad Laboratories, Hemel Hempstead, UK) were coupled to Pelikine anti-IL-1β (catalogue number, Cat: M9334) anti-IL-6 (Cat: M191602), anti-IL-8 (Cat: M191802), anti-IL-10 (Cat: M191002), or anti-TNF-α (Cat: M192302) monoclonal antibodies (Mast Group, Bootle, UK), R&D anti-IL-1α (Cat: 840201) or anti-MCP-1 (Cat: 840204) antibodies (R&D Systems, Minneapolis, MN, USA), or Biodesign anti-CRP monoclonal antibody (Biodesign anti-CRP; Cat: M86842M, clone C2), using the Bio-Plex amine coupling kit (Cat: 171–406001). Detection antibodies were Pelikine anti-IL-1β (Cat: M193404), anti-IL-6 (Cat: M191604), anti-IL-8 (Cat: M191804), and anti-TNF-α (Cat: M192304) from Mast Group, anti IL-1α (Cat: M193404) or anti-MCP-1 (Cat: 840205) from R&D Systems. The plasma IL-6, IL-8, IL-10, IL-1β, MCP-1, and TNF-α assays were conducted as a 7-plex, using 15% horse serum, 5% bovine serum, and 1% mouse serum in high performance ELISA buffer (HPE; Pelikine) as a diluent. CSF assays were conducted as a 4-plex (IL-1α, IL-1β, IL-10, and TNF-α) in HPE with 1% horse serum, or a 3-plex (IL-6, IL-8, and MCP-1) in HPE. All standards were calibrated against current National Institute for Biological Standards and Control (NIBSC, South Mimms, UK) standards. Plasma CRP was measured in a single-plex competitive assay, with 10% horse serum, 5% bovine serum, and 1% mouse serum diluent in Tris-buffered saline. The competitor was CRP (P100-0; SCIPAC, Sittingbourne, UK) that had been biotinylated with Pierce EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce, Rockford, IL, USA). The assay was calibrated with respect to NIBSC human CRP (NIBSC; 85/506). Binding of biotinylated CRP was assessed, following addition of R-phycoerythrin streptavidin (Jackson ImmunoResearch Laboratories Inc, Stratech, Newmarket, UK; Cat: 016-110-084), using a Bio-Plex 200 system. Multiplex assay diluents were matched to plasma or CSF, with three or four quality controls (QCs) for each analyte. The assay performance across the assays in this study is provided in Table 2, in terms of sensitivity and inter-assay coefficient of variation (CV) for these QCs.

The primary outcome measure was the area under the curve (AUC) for CSF IL-6 concentration, between 6 and 24 hours from the start of the infusion, adjusted for baseline. Values were log-transformed before analysis and adjustment was achieved by subtraction of the baseline value from values at all time points prior to calculation of AUC. Comparisons between the two treatment groups were made using Student’s t-test. Similar analyses were carried out for IL-1α, IL-1β, IL-8, IL-10, MCP-1, TNF-α, and CRP. Statistical analyses were performed with Stata version 12 (StataCorp, College Station, TX, USA). The target sample size was intended to include 16 participants per treatment group, including replacement of participants in whom it was not possible to obtain all samples up to 24 hours from the start of infusion. This sample size gave 80% power at the 5% significance level to detect differences of one standard deviation in outcomes between treatment groups, similar to that seen for ischaemic stroke patients in our previous study [19].

A total of 254 patients with SAH were admitted to the centres between June 2009 and July 2010. Of these, 56 patients had a confirmed aneurysm and required EVD within 72 hours of ictus. A summary flow chart of screening and recruitment is shown in Figure 1. Overall, 18 patients were recruited into the trial: seven patients received placebo, six patients received IL-1Ra, and five patients were withdrawn from the study prior to randomisation. One patient who received placebo did not complete the 24-hour infusion and analyses were based on the 12 patients in whom the infusion was completed (six placebo, six IL-1Ra). A total of 38 potentially eligible patients were not recruited. Table 3 summarises the baseline characteristics of each treatment group. There were no adverse or serious adverse events attributable to the study drug (Table 4).

The mean CSF IL-6 concentration, prior to infusion was 1,001 pg/mL (range 42 to 2,698 pg/mL). The mean plasma IL-6 concentration in all patients at this time point was 69 pg/mL (range 3 to 469 pg/mL). The CSF IL-6 concentrations reduced over the first 24 hours from the start of infusion in all six of the active group and in three of the six controls (Figure 2a). A similar pattern occurred for plasma IL-6 concentrations, with reductions for five of the six active compared with two of the five control participants (Figure 2b). Samples of CSF were not available for three participants at 48 hours and five participants at 72 hours, and plasma samples were not available for two participants at 72 hours, but the 72-hour CSF and plasma IL-6 kinetics are shown for the remaining participant samples in Figure 3.

Plasma and CSF values for IL-1β, IL-1α, and TNF-α were near or below the limits of detection for most samples. Details of AUC from 6 to 24 hours for other outcome measures, both unadjusted (raw) and adjusted for baseline, are given in Table 5. The baseline-adjusted AUC difference for IL-6 in CSF was 12.8, which is essentially a difference of one standard deviation (12.3) for the combined data. This is consistent with the difference anticipated from the power calculation, for the numbers recruited. Similarly, the baseline-adjusted AUC difference for IL-6 in plasma was 18.6, compared with one standard deviation for the combined data (17.2). Reductions in AUC for CSF and plasma IL-8 and IL-10 were also seen, although again the differences were not significant. Plasma CRP was not affected by IL-1Ra over the 6- to 24-hour period, but reduced over the following 24 hours in the IL-Ra cohort, relative to placebo (Figure 3b).

Although the study was not designed or powered to measure clinical outcome, the Glasgow Outcome Score (GOS) at 6 months was obtained from the clinical notes and reported for each patient who received placebo or IL-1Ra in the study (Table 6). There was no evidence of difference between treatment groups.