β -Hydroxysteroid Dehydrogenase Type 1 inhibition in Idiopathic Intracranial Hypertension: a double-blind randomized controlled trial.

Treatment options for idiopathic intracranial hypertension are limited. The enzyme 11 β hydroxysteroid dehydrogenase type 1 has been implicated in regulating cerebrospinal fluid secretion, and its activity is associated with alterations in intracranial pressure in idiopathic intracranial hypertension. We assessed therapeutic efficacy, safety and tolerability, and investigate indicators of in vivo efficacy of the 11 β -hydroxysteroid dehydrogenase type 1 inhibitor AZD4017 compared to placebo in idiopathic intracranial hypertension. A multicenter, UK, 16-week phase II randomized, double-blind, placebo-controlled trial of 12-weeks treatment with AZD4017 or placebo was conducted. Women aged 18 to 55 years with active idiopathic intracranial hypertension (>25cmH 2 O lumbar puncture opening pressure and active papilledema) were included. Participants received 400mg twice daily of oral AZD4017 compared to matching placebo over 12-weeks. The outcome measures were initial efficacy, safety and tolerability. The primary clinical outcome was lumbar puncture opening pressure at 12 weeks analysed by intention-to-treat. Secondary clinical outcomes were symptoms, visual function, papilledema, headache and anthropological measures. In vivo efficacy was evaluated in the central nervous system and systemically. 31 subjects (mean age 31.2 (SD=6.9) years and BMI 39.2 (SD=12.6) kg/m 2 ) were randomized to AZD4017 (n=17) or placebo (n=14). At 12 weeks, lumbar puncture pressure was lower in the AZD4017 group (29.7 cmH 2 O) compared with placebo (31.3 cmH 2 O), but the difference between groups was not statistically significant (mean difference: -2.8, 95% confidence interval: -7.1-1.5; p=0.2). An exploratory analysis assessing mean change in lumbar puncture pressure within each group found a significant decrease in the AZD4017 group (mean change: -4.3 cmH Nine transient drug-related adverse events were reported. One serious adverse event occurred in the placebo group (deterioration requiring shunt surgery). In vivo biomarkers of 11 β hydroxysteroid dehydrogenase type 1 activity (urinary glucocorticoid metabolites, hepatic prednisolone generation and CSF cortisone to cortisol ratios) demonstrated significant enzyme inhibition. This is the first phase 2 randomized controlled trial in idiopathic intracranial hypertension evaluating a novel therapeutic target. AZD4017 was safe, well-tolerated and inhibited 11 β -hydroxysteroid dehydrogenase type 1 activity in vivo . Possible clinical benefits were noted in this small cohort. A longer, larger study would now be of interest. optical SD:

The copyright holder for this preprint (which was not this version posted May 31, 2019. ; https://doi.org/10.1101/648410 doi: bioRxiv preprint Importantly, 11β-HSD1 expression and activity is dysregulated in obesity. (Sandeep et al., 2005) (Wake and Walker, 2004) Selective inhibitors of 11β-HSD1 have been developed as treatments for obesity, hepatic steatosis, metabolic syndrome and type 2 diabetes. (Boyle, 2008;Stefan et al., 2014) Based on these data, 11β-HSD1 could represent a therapeutic target for lowering CSF pressure. AZD4017 is a highly selective, fully reversible, competitive 11β-HSD1 inhibitor. It has been tested over short time intervals in healthy males (9 days), and abdominally obese subjects (10 days), and found to be safe and tolerable. ]-a, b, c, d, 2000.) The ability of AZD4017 to penetrate the blood-brain-barrier (BBB) is not established; however, the CP lies outside the BBB and consequently can be targeted directly following oral administration. (Davson, 1966;Eftekhari et al., 2015) We hypothesised that inhibition of 11β-HSD1 could be therapeutically beneficial in IIH. To test this we conducted a multicenter phase 2 double-blind, placebo-controlled RCT in IIH using the selective 11β-HSD1 inhibitor AZD4017, aiming to assess therapeutic efficacy, safety and tolerability, and investigate in vivo systemic and central nervous system efficacy.
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Study Conduct
The study was conducted from March 2014 to December 2016 in three UK hospitals. The National Research Ethics Committee York and Humber-Leeds West gave ethical approval (13/YH/0366). All patients provided written informed consent in accordance with the delaration of Helsinki. Detailed clinical trial methodology has been published. (Markey et al., 2017a)

Study Population
Women (18-55 years) were eligible if they had a clinical diagnosis of active IIH meeting the updated, modified Dandy criteria (ICP>25cmH 2 O and active papilledema) and normal brain imaging (including magnetic resonance venography or CT with venography) at recruitment (for detailed eligibility criteria see Suppl. Table 1). (Friedman et al., 2013;Markey et al., 2017b)

Study design
A 16-week phase II, double-blind placebo-controlled RCT with a 12-week dosing duration and 4-week follow-up off drug.

Randomization and Blinding
Participants were allocated to either drug or placebo. Patients were allocated a trial number randomly by phone, using block-of-6 randomization. Participants and investigators were masked to treatment allocation during the trial.

Intervention
An oral selective 11β-HSD1 inhibitor, AZD4017, at 400mg twice daily for 12-weeks, compared to a matched placebo. Trial dosing was added to existing therapy for IIH, other drugs were maintained at a fixed dose throughout the study. certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Clinical assessments
The primary outcome for clinical efficacy was the difference in ICP between AZD4017 and placebo, as measured by LP at 12 weeks. Secondary outcomes included: IIH symptoms, visual function (visual acuity (VA) measured using LogMAR (log of the minimum angle of resolution), perimetric mean deviation (PMD) using Humphrey 24-2 central threshold automated perimetry and contrast sensitivity assessed by MARs charts (Mars Perceptrix, USA)), papilledema, headache associated disability and anthropological measures. Papilledema was evaluated using spectral domain optical coherence tomography (OCT; Spectralis, Heidelberg Engineering) to quantify the peripapillary retinal nerve fibre layer (RNFL) average and maximal values. Papilledema was graded from fundus photographs by three masked neuroophthalmologists using the Frisén classification (0 denotes no papilledema to grade 5 severest papilledema). (Frisen, 1982) Headache was evaluated through the headache impact test-6 disability questionnaire (HIT-6), headache severity (verbal rating score 0 to 10), frequency (days per month), duration and analgesic use (days per month). (Bayliss et al., 2003) Pill counting at each visit determined drug compliance.
In the original grant application and early versions of the protocol, the primary outcome measure was stated as the change in ICP between baseline and 12 weeks. Following adoption of the study by the Birmingham Clinical Trials Unit, the primary outcome was changed to ICP at 12 weeks, with adjustment for baseline ICP in the analysis. This change was made blind to any data analysis. certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Glucocorticoid and AZD4017 blood and CSF levels
Samples were collected and stored at -80ºC. Cortisol and cortisone levels in serum and CSF were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the University of Birmingham, as previously described. (Hassan-Smith et al., 2015;Long et al., 2016) Plasma and CSF AZD4017 levels were quantified by an external laboratory (Alderley Analytical, Knutsford, UK).

In vivo hepatic 11β-Hydroxysteroid Dehydrogenase activity
Inhibition of hepatic 11β-HSD1 activity was informed by measuring first-pass metabolism of 10mg of oral prednisone to prednisolone. Serum prednisone and prednisolone were measured certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted May 31, 2019.

In vitro adipose 11β-Hydroxysteroid Dehydrogenase inhibition by AZD4017
Subcutaneous and omental adipose explants (1-2g in triplicate) were obtained from IIH patients undergoing bariatric surgery. Samples were incubated with either 2000nM, 200nM or 20nM of AZD4017 and 100nM of cortisone alongside three controls (without AZD4017) for 24 hours.

Statistical Analysis
Analysis of the clinical data was based on the full analysis set according to the statistical analysis plan (Supplemental document). Analysis was conducted using intention-to-treat with data from all available randomized participants used. The primary comparison was between AZD4017 versus placebo at 12 weeks. The majority of data was continuous, so groups were compared using linear regression models with baseline measurements included as a covariate in the model. IIH symptom data was binary and was analyzed using log-binomial models with baseline symptom included as a covariate in the model. The primary analysis of visual data included data from both eyes, using a linear mixed model with participant included as a random effect. We also analyzed data from the most affected eye at baseline as defined by certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted May 31, 2019. ; https://doi.org/10.1101/648410 doi: bioRxiv preprint multiple comparisons made. Clinical data was analyzed using SAS (version 9.4) and STATA (version 14).
Analysis of laboratory data was performed using SPSS (version 24, IBM, New York, USA). An unpaired t-test was used for normally distributed data (Mann-Whitney U test for non-parametric data). For related groups, either the paired t-test or Wilcox signed-rank test was used for parametric and non-parametric data respectively. We reported means and standard deviations (medians and ranges for non-parametric data).

Sample Size
To detect a difference between groups of 14% in ICP (assuming a standard deviation of 10% for ICP) with 90% power and two-sided alpha=0.05, required 12 participants per group. Allowing for 20% drop-out, we aimed to recruit 30 participants.

Data availability
The trial is registered at Clinicaltrials.gov NCT02017444; European Clinical Trials Database (EudraCT Number: 2013-003643-31). The data that support the findings of this study are available from the corresponding author, upon reasonable request. certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Baseline characteristics demonstrate a cohort of IIH patients with active disease were recruited (Table 1).

Clinical Outcomes Primary Clinical Outcome
At 12 weeks, the mean ICP was 29.

Secondary Clinical Outcomes
At weeks 12 and 16, there were no statistically significant differences between the two treatment groups in IIH symptoms (Suppl. Table 2). At 12 and 16 weeks, the Humphrey Visual Field Analyzer PMD (worst eye) was not significantly different between groups (adjusted mean difference at 12 weeks: 0.3dB, 95% CI: -2.0-2.7, p=0.8) ( Figure 2D, E, F; Table 2, Suppl. Table   3). However, within group analysis showed that the PMD improved from -6.1 dB (SD=5.4) at baseline to -3.4 dB (SD=3.2) (mean change 2.7 dB (SD=4.3), p=0.04) at 12 weeks in the AZD4017 group and from -3.4 dB (SD=6.8) to -2.2 dB (SD=3.1) (mean change 0.3 dB (SD=6.0), p=1.0) in the placebo group. There were also no statistically significant differences certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
All headache outcomes were not statistically significantly different between AZD4017 and placebo at weeks 12 or 16 (Suppl. Table 4). There were also no statistically significant differences in any of the anthropological outcomes (BMI, waist:hip ratio).

Safety and Tolerability
Study medication was well tolerated with participants in both arms taking on average 98% of the total 168 study medication doses (mean doses taken were 164 (range 146 -168) and 165 (range 158-168) in the AZD4017 and placebo group respectively). There were no participant withdrawals due to adverse effects. Nine adverse events (in 6 participants) were deemed related to AZD4017, none were serious and 3 were due to non-clinically relevant fluctuations in serum cortisol. Adverse events are shown in Suppl. Table 5. One serious adverse event was reported in the placebo arm and deemed unrelated (fulminant deterioration in IIH necessitating CSF shunting one day post-randomization).
No differences were noted between treatment groups for the safety blood tests (Suppl. Table 6).
As expected, there was a rise in the HPA stimulatory hormone, ACTH, over 12 weeks in the AZD4017 group (mean difference at 12 weeks: 12.36ng/l, 95%CI: -0.03-24.74). There was no difference in serum cortisol, testosterone or androstenedione, although serum DHEAS, a marker of adrenal androgen production was higher at 12 weeks in the AZD4017 group (mean difference certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

In vivo assessments
Blood and CSF levels of AZD4017 and glucocorticoids AZD4017 concentrations were detected in the serum after one week of treatment and sustained at week 12 (n=6). The presence of AZD4017 in the CSF was 0.5% that of the serum (Suppl. Serum and CSF cortisol and cortisone were examined in the placebo and AZD4017 groups at baseline and at 12 weeks. The serum cortisol:cortisone ratio was not significantly different between arms at weeks 0 (p=0.6) and 12 (p=0.5) ( Figure 3A). The CSF cortisol:cortisone ratio did not differ between arms at baseline (p=0.9); however, at week 12 there was a significant decrease in the CSF cortisol:cortisone in the AZD4017 group compared to placebo (p=0.002), and the AZD4017 group between baseline and 12 weeks (p=0.03) ( Figure 3B), implying local 11β-HSD1 activity can regulate CSF glucocorticoid exposure.

In vivo systemic 11β-Hydroxysteroid Dehydrogenase activity
The urinary (5αTHF+THF):THE glucocorticoid metabolite ratio reflective of systemic 11β-HSD1 activity was significantly reduced in AZD4017 vs. placebo groups at week 1 (0.16±0.04 versus 0.90±0.36, p<0.0001) and week 12 (0.27±0.29 versus 0.90±0.28; p<0.0001). By contrast, the ratios did not differ between the two treatment groups at baseline (p=0.6) and 4 weeks after certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Hepatic 11β-Hydroxysteroid Dehydrogenase activity
The placebo group had robust capacity to generate prednisolone following oral prednisone at both baseline and after 12 weeks. The baseline prednisolone generation curve for the AZD4017 group was indistinguishable from the placebo curve; however at 12 weeks the AZD4017 group were essentially unable to generate prednisolone ( Figure 3D,E), indicating effective inhibition of hepatic 11β-HSD1 activity. Area under the curve analysis of the mean time points at 12 weeks showed significantly impaired prednisolone generating capacity for AZD4017 vs. placebo (228±99 vs 1738±142; p<0.0001), an 85.9% reduction (p<0.0001) in overall prednisolone generating capacity after 12 weeks ( Figure 3D,E). There was no correlation between the change in the area under the curve for prednisolone and ICP (r=0.1; p=0.8) or PMD (r=0.4; p=0.2).

Adipose 11β-Hydroxysteroid Dehydrogenase activity
While AZD4017 effectively inhibited hepatic 11β-HSD1, we were unable to show impaired capacity to generate cortisol from cortisone in explanted subcutaneous adipose biopsies. At baseline and following 12 weeks of oral AZD4017, there was no significant change in total cortisol vs. placebo (9.0±5.6 vs 12.4±4.9 nmol; p=0.3) or percentage conversion of cortisone to cortisol (23±14 vs 27±18 %; p>0.99) ( Figure 3F). However, AZD4017 was able to significantly certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted May 31, 2019. ; https://doi.org/10.1101/648410 doi: bioRxiv preprint 1 7 inhibit 11β-HSD1 activity when added to ex vivo adipose explants from subcutaneous and omental depots. 20nM AZD4017 significantly impaired conversion of cortisone to cortisol (>70% vs. control), 200nM onwards was sufficient to effectively block cortisol generation, particularly in the subcutaneous depot ( Figure 3G,H). certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Discussion
We report the first phase 2 RCT assessing 11β-HSD1 inhibitor AZD4017 for the treatment of IIH. We have shown some possible clinical benefit for AZD4017, and that it was well tolerated and safe. We found evidence for effective in vivo 11β-HSD1 inhibition.
Our primary hypothesis stated that 11β-HSD1 inhibition in IIH patients would reduce CSF secretion and lower ICP while being safe and tolerable following 12 weeks of treatment. ICP was the primary clinical outcome measure, representing the hallmark of the disease driving clinical sequelae. At 12 weeks, although ICP was lower in the AZD4017 group compared to placebo, the difference between groups was not statistically significant. Exploratory analyses of the mean change within groups found a significant improvement in ICP in the AZD4017 group between baseline and 12 weeks, but not in the placebo group. Previous trials have noted that ICP reduction below the cut off of 25 cmH 2 O is not universally required to translate into resolution of IIH clinical features. (Sinclair et al., 2010b) A minimal clinically important change in ICP in IIH has not been determined, and establishing one would be useful for future trials.
The visual field perimetric assessment is another clinically meaningful measure and has been selected as the primary outcome measures in previous IIH trials. We found no difference between groups in PMD at 12 weeks, however, there was significant improvement over time in the AZD4017 arm but not in the placebo arm. This may reflect the pragmatic recruitment of all degrees of PMD at enrolment whilst other trials have enrolled a selected cohort (e.g. -2 to -5 dB). (Wall et al., 2014) Additionally, his small trial was not powered to determine significant in the secondary outcome measures. certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted May 31, 2019. ; https://doi.org/10.1101/648410 doi: bioRxiv preprint 9 Headache is a key disabling feature in IIH. (Mulla et al., 2015) We did not detect differences between the groups in any of the headache assessments at 12 weeks, although data from the patient completed HIT-6 favoured the AZD4017 group. Evaluating the effect of AZD4017 on headache measures over a longer treatment duration would be of interest.
Previous trials showed that 11β-HSD1 inhibition leads to adaptive changes in HPA stimulatory hormone ACTH and the adrenal androgen precursor DHEA. Our data support these findings, but with no significant change in downstream effector hormones (cortisol and testosterone).
In vivo evaluation of our patients demonstrated that AZD4017 was a highly effective systemic and hepatic 11β-HSD1 inhibitor, in line with previous studies using11β-HSD1 inhibitors in humans (Schwab et al., 2017) , (Courtney et al., 2008). Systemic efficacy may modify metabolic aspects of IIH with indirect benefits on ICP. (Hornby et al., 2018) While AZD4017 effectively inhibited 11β-HSD1 when applied to subcutaneous and omental adipose tissue explants, we were unable to prove inhibition in vivo, and propose that 11β-HSD1 activity recovers over the assay period once removed from AZD4017, a reversible competitive inhibitor.
Blood-brain-barrier AZD4017 penetrance was low, with levels in the CSF 0.5% those of plasma levels, but were associated with reduced CSF cortisol:cortisone ratio suggesting that 11β-HSD1 may contribute to cortisol availability in the CSF. The unchanged serum cortisol:cortisone is likely reflective of HPA axis set-point. certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Limitations
We were unable to directly evaluate 11β-HSD1 inhibition at the choroid plexus, the tissue responsible for CSF secretion. We have evaluated efficacy of other IIH drugs using rodent ICP monitoring models, (Botfield et al., 2017;Scotton et al., 2018) but AZD4017 is only effective in humans and primates, thus limiting our ability to evaluate its action in rodent models. The trial duration was likely too short. A duration of 12 weeks was chosen for evaluation of safety and tolerability and represented the longest duration of dosing to date with AZ4017. This may not have been sufficient for meaningful evaluation of clinical outcomes with other IIH RCTs evaluating drugs over a 6 month period. (Committee et al., 2014) The enrolment criteria for the study were deliberately broad allowing inclusion of a spectrum of IIH patients with active disease and ensuring generalizability of results; however, this did not allow evaluation in disease subgroups such as those with mild visual loss vs. those with severe irreversible visual loss.
Finally, the sample size (31 participants) is small which may have limited meaningful evaluation of clinical measures and the trial was not design to establish significant changes in the secondary clinicaloutcome measures.

Conclusions
This is the first phase II study evaluating the novel pharmacological therapy AZD4017 in IIH.
We demonstrate safety, tolerability and provide strong in vivo evidence for effective 11β-HSD1 inhibition. There was a significant reduction in ICP in the AZD4017 and not the placebo group certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted May 31, 2019. ; https://doi.org/10.1101/648410 doi: bioRxiv preprint 1 over the treatment duration (exploratory within group analysis); however, the primary analysis evaluating the difference between groups at 12 weeks did not reach statistical significance. The data suggest that 11β-HSD1 inhibition may have utility for reducing the effects and consequences of raised ICP in patients with IIH. Further evaluation of these therapeutic strategies in this disabling disease, for which few useful medical options exist, would be worthwhile.
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted May 31, 2019. certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted May 31, 2019. ; https://doi.org/10.1101/648410 doi: bioRxiv preprint 1  Number of patients with better, same or worse average OCT RNFL height at 12 weeks. Data is presented as mean ± 95% confidence index. *<0.05, ** < 0.01. adipose (H) Omental adipose 11β-HSD1 activity (cortisol production from cortisone) after 24 certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted May 31, 2019. ; https://doi.org/10.1101/648410 doi: bioRxiv preprint All measures shown in the table are of worst eye. Negative values in the adjusted mean difference between treatment arms favour AZD4017. C.I: confidence interval; OCT: optical coherence tomography; RNFL: retinal nerve fibre layer; SD: standard deviation.

Supplemental files
Supplemental File 1: Additional data certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.