Early efficacy trial of anakinra in corticosteroid-resistant autoimmune inner ear disease

BACKGROUND. Autoimmune inner ear disease (AIED) is a rare disease that results in progressive sensorineural hearing loss. Patients with AIED initially respond to corticosteroids; however, many patients become unresponsive to this treatment over time, and there is no effective alternative therapy for these individuals. METHODS. We performed a phase I/II open-label, single-arm clinical trial of the IL-1 receptor antagonist anakinra in corticosteroid-resistant AIED patients. Given that the etiology of corticosteroid resistance is likely heterogeneous, we used a Simon 2-stage design to distinguish between an unacceptable (= 30%) response rate to anakinra therapy. Subjects received 100 mg anakinra by subcutaneous injection for 84 days, followed by a 180-day observational period. RESULTS. Based on patient responses, the Simon 2-stage rule permitted premature termination of the trial after 10 subjects completed the 84-day drug period, as the target efficacy for the entire trial had been achieved. Of these 10 patients, 7 demonstrated audiometric improvement, as assessed by pure tone average (PTA) and word recognition score (WRS). In these 7 responders, reduced IL-1 beta plasma levels correlated with clinical response. Upon discontinuation of treatment, 3 subjects relapsed, which correlated with increased IL-1 beta plasma levels. CONCLUSION. We demonstrated that IL-1 beta inhibition in corticosteroid-resistant AIED patients was effective in a small cohort of patients and that IL-1 beta plasma levels associated with both clinical hearing response and disease relapse. These results suggest that a larger phase II randomized clinical trial of IL-1 beta inhibition is warranted

Alternate Splicing of Interleukin-1 Receptor Type II (IL1R2) In Vitro Correlates with Clinical Glucocorticoid Responsiveness in Patients with AIED

Autoimmune Inner Ear Disease (AIED) is poorly characterized clinically, with no definitive laboratory test. All patients suspected of having AIED are given glucocorticoids during periods of acute hearing loss, however, only half initially respond, and still fewer respond over time

Clinical History of patients treated with prednisone.

<p>“Prior tx?”: although all had prior rapid declines in hearing requiring prednisone therapy, none were treated in a 3 month period prior to enrollment. Several patients had occasional vestibular symptoms, however, these symptoms did not coincide with periods of hearing fluctuation. Serology was performed at Immco Diagnostics. NA = not available: serologic testing at Immco could not be performed as these tests were not covered b y the patient's insurance carrier. Audiometric change shown represents the change between the average of the post-treatment pure tone thresholds from the pre-treatment pure tone average threshold. NS = not significant: average post-treatment hearing changes of less than 5 dB are not felt to represent significant changes. All declines in hearing are reported as 0 dB. NR = clinical non-responder; R = clinical responder.</p

Clinical History of AIED and control patients undergoing cochlear implantation.

<p>PTA: Pure tone average of the audiogram at 500, 1000, 2000 and 4000 Hz, (although 4000 Hz is not traditionally included, this information is important to speech perception for cochlear implantees and therefore PTA represents these four frequencies for these patients). NP: Not Performed: control subjects did not have serology for AIED performed as there was no clinical indication for such studies, HINT: Hearing in Noise Test; IT-MAIS: Infant-Toddler Meaningful Auditory Integration Scale, LVA: Large Vestibular Aqueduct; Cx26: connexin 26 gene mutation. Superscript numbers in column 1 refer to inclusion of the patient in data sets for the below figures: 1: included in analysis for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005293#pone-0005293-g001" target="_blank">figure 1</a>; 2b & c: included in analysis for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005293#pone-0005293-g002" target="_blank">figure 2b or 2c</a>. In one patient, all RNA was used for microarray analysis (79 y/o female, control).</p

Expression of IL1R2 in cochlear implant recipients.

<p>2A Diagram of IL1R2 mRNA depicting regions detected by microarray probes and Q-RT-PCR primers. At position 1117, an alternate splice site has been described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005293#pone.0005293-Liu1" target="_blank">[25]</a> that induces a stop codon. 2B Q-RT-PCR for 3 control subjects and 5 AIED subjects comparing autologous perilymph stimulated to unstimulated PBMC for both the shorter soluble IL1R2 (sIL1R2) and the longer membrane bound form of Il1R2 (mIL1R2). Results were dramatically different and statistically significant for mIL1R2 (as seen with an asterix (*)), using a Mann Whitney test (p = 0.03) (in stat). 2C ELISA for soluble IL-1R2 (sIL1R2). Supernatants from 16-hour PBMC cultures from 3 AIED patients and 2 controls were used to determine the level of soluble receptor. Stimulus conditions were no stimulus (white bar), pneumococcal stimulus (grey bar) or autologous cochlear perilymph (black bar). Relative amounts were set at one for the unstimulated condition as this data set included 2 children (one AIED, one control). sIL-1R2 levels are less in children however, a similar patterns of expression were observed. Standard deviations are shown.</p

Differential expression of IL1R2 by microarray in response to autologous cochlear perilymph in patients undergoing cochlear implantation.

<p>1A Comparison of RNA expression using the Affymetrix U133A 2.0 chip to compare cultured PBMC of controls (CTRL) and AIED patients for the following conditions: either unstimulated (UNTX), stimulated with 23-valent pneumococcal vaccine (PNEUMO) or autologous cochlear perilymph (C). Only 10 genes (panel A) were significantly different (p<0.05) when a threshold of 2 and a Benjamini & Hochberg correction was applied (Genesifter, VixXlabs). Only one of them unique to the cochlear fluid stimulated condition IL1R2 (affy ID 205403), P<0.05. Array data in 1A can be viewed at <a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">www.ncbi.nlm.nih.gov/geo/</a> using the user “Vambutas” with a password “daniella” to view series “GSE4277” containing 18 arrays from the six patients with “1” next to their description in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005293#pone-0005293-t001" target="_blank">table 1</a>. 1B A comparative analysis of IL1R2 expression in these arrays is seen in panel 1B, with standard deviations shown.</p

Pre-treatment induced expression of mIL1R2 in response to dexamethasone correlates with post-treatment clinical hearing restoration.

<p>3A 18 patients with presumptive AIED and sudden declines in hearing were enrolled, blood drawn prior to treatment, and treated with 60 mg prednisone/day×7 days and then tapered. Pre-treatment PBMC were divided and incubated with either no stimulus or dexamethasone (8 µg/ml is shown). IL1R2 mRNA was measured by Q-RT-PCR. Post-treatment audiograms were obtained between 7–14 days later. Audiometric improvement (Δ) was measured as the average of 250, 500, 1,2,& 4 kHz. Responders (R) all returned to prior baseline hearing. Pre-treatment levels of mIL-1R2 Q-RT-PCR (expressed as fold change over baseline) predicted steroid responsiveness in AIED (P<0.0001, Mann Whitney test). 3B Pre and post treatment mIL1R2 levels in a subset of clinical responders and non-responders. Pre-treatment responders have no basal mIL1R2 expression in PBMC (unstim) at 45 cycles (in replicate samples), compared with non-responders that have substantially higher basal levels. Although PBMC from both responders and non-responders demonstrate increased mIL1R2 RNA expression in response to in vitro dexamethasone stimulation (dex stim), responders are exquisitely sensitive. The concentration of dexamethasone shown for the PBMC stimulation experiments was 20 ug/ml, however, 4 ug/ml and 8 ug/ml produced identical results, suggesting maximal stimulation even at 4 ug/ml. 3C Despite clear differences in mIL1R2 expression patterns, sIL1R2 expression is differences between responders and non-responders are minimal suggesting alternate splicing in clinical responders.</p