Supplementary webappendix - TheLancet.com

Supplementary webappendix
This webappendix formed part of the original submission and has been peer reviewed.
We post it as supplied by the authors.
Supplement to: Selmaj K, Li DKB, Hartung H-P, et al. Siponimod for patients with
relapsing-remitting multiple sclerosis (BOLD): an adaptive, dose-ranging, randomised,
phase 2 study. Lancet Neurol 2013; published online June 11. http://dx.doi.org/10.1016/
S1474-4422(13)70102-9.
This online publication has been corrected. The corrected version first appeared at
thelancet.com/neurology on July 16, 2013.

ONLINE APPENDIX
Steering committee
K Selmaj, Department of Neurology, Medical Academy of Lodz, Poland; MS Freedman, Multiple Sclerosis Research Clinic, The Ottawa Hospital, Ottawa, ON, Canada;
HP Hartung, Department of Neurology, Heinrich Heine University, Dusseldorf, Germany; L Kappos, Department of Neurology, University Hospital Basel, Basel,
Switzerland; DK Li, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada; P Rieckmann, Department of Neurology, Sozialstiftung Bamberg
Hospital, Bamberg, Germany; O Stüve, University of Texas Southwestern Medical Center, Dallas, TX, USA; X Montalban, Unit of Clinical Neuroimmunology, Vall
d´Hebron University Hospital, Barcelona, Spain.
Patients
Interferon-beta or glatiramer acetate treatment had to have been stopped at least 3 months before randomisation.
Detailed cardiac exclusion criteria
The following cardiovascular conditions were excluded: history or presence of stable or unstable ischaemic heart disease, myocardial infarction, myocarditis, or
cardiomyopathy; history of Raynaud’s disease; cardiac failure at time of screening and/or at baseline (class II–IV, according to New York Heart Association classification) or
any severe cardiac disease as determined by the investigator; history of cardiac arrest; history of symptomatic bradycardia; resting pulse rate 440 msec on screening electrocardiogram (ECG) before randomisation; history or presence of symptomatic
arrhythmia or arrhythmia requiring treatment or being otherwise of clinical significance; arterial hypertension, uncontrolled by medication; condition requiring treatment with
medication that impairs cardiac conduction (e.g. β blockers, verapamil-type and diltiazem-type calcium-channel blockers, or cardiac glycosides); history of syncope of
suspected cardiac origin; history of catheter ablation.
Study design and randomisation
Randomisation was stratified by age (18–20, 21–30, 31–40, 41–50, and ≥51 years) and sex for cohorts 1 and 2 separately to ensure a balanced distribution of patients for
these parameters across treatment arms. Within each stratum, a country was dynamically assigned to an available block: for cohort 1, each block had a size of four; for cohort
2, each block had a size of 9.
Siponimod and placebo were to be taken once daily, preferably at the same time every day after a meal; it was recommended that during dose initiation, study drug was
administered before 12:00 pm.
For cohort 2 patients, a titration scheme was implemented for study drug initiation via a protocol amendment. In cohort 1, five cases of second-degree AV block associated
with symptomatic bradycardia occurred (siponimod 2 mg, three patients; siponimod 10 mg, two patients) among a total of 187 patients exposed to study medication until that
point. All events occurred or started to occur within 2 hours of the first dose of study drug and all patients made a full recovery within 24 hours of receiving the first dose of
study drug; therefore, a dose-titration scheme was implemented before study commencement for cohort 2. Owing to the low doses used in cohort 2, only patients receiving
siponimod 1·25 mg underwent dose titration, which was achieved over 5 days (day 1, 0·25 mg; day 2, 0·25 mg; day 3, 0·5 mg; day 4, 0·75 mg; day 5, 1·25 mg); patients
1

andomised to siponimod 0·25 mg received 0·25 mg from day 1. This dosing scheme was chosen based on healthy volunteer data, showing that a starting dose of 0·25 mg or
less daily was expected to have minimal cardiac effects. In addition, mobile cardiac telemetry (MCT) during the dose-titration period was included to enhance surveillance
for potential cardiac conduction events.
Study procedures
Study visits
Brain magnetic resonance
imaging (MRI)*
Vital signs
Screening, baseline, days 1, 2 (cohort 2 only), and 7, and monthly for 3 (cohort 2) or 6 (cohort 1) months
Screening, baseline, and monthly for 3 (cohort 2) or 6 (cohort 1) months
Screening, baseline, days 1, 2 (cohort 2 only), and 7, and monthly for 3 (cohort 2) or 6 (cohort 1) months
ECG Baseline and dose initiation at 2, 4, and 6 hours post-dose on day 1 for cohort 1 and days 1, 2, and 7 for cohort 2
Holter ECG (over 24 hours)
Screening and on day 1, month 3, and month 6 for cohort 1; screening and on day 1 at month 3 and for at least 6 hours on days 2 and 7 for
cohort 2
*Perceptive Informatics ® , Waltham, MA, USA provided image analysis, with the University of British Columbia MS/MRI Research Group being the central MRI readers.
Monitoring of physical condition included general, dermatological, and ophthalmic examinations, chest X-ray or chest high-resolution computer tomography, and lung
function tests.
For patients in cohort 1, the first dose of study drug was administered at the study centre and the patient stayed under observation for a minimum of 6 hours. Cohort 2 patients
were monitored at the study centre for a minimum of 6 hours after dose administration on days 1, 2, and 7. All patients were discharged after 6 hours of monitoring if specific
discharge criteria were met. For cohort 2 patients, in addition to standard ECG and Holter ECG monitoring, continuous ECG telemetry via MCT was performed during dose
titration at sites where it was feasible.
Relapse was defined as new or worsening neurological symptoms occurring at least 30 days after the onset of a preceding relapse and lasting at least 24 hours without fever
or infection. A relapse was confirmed by the independent evaluating physician (examining neurologist) when it was accompanied by either: an increase in Expanded
Disability Status Scale (EDSS) score of at least 0·5 point, or an increase of 1 point on two different functional systems of the EDSS, or an increase of 2 points on one of the
functional systems.
MRI scans included T1-weighted images before and after administration of Gd contrast medium (0·1 mmol/kg), T2-weighted (T2 and proton density) images, and fluid
attenuated inversion recovery images. Prior to the start of the study, a radiologist and technician from each centre received an MRI manual from the imaging CRO, outlining
technical implementation, image quality requirements, and MRI administrative procedures. Each site was asked to program the MRI scanner for use in the study and perform
and submit a dummy scan to assess the image quality and to evaluate the compatibility of the electronic data carrier. Once the dummy run was accepted, all the parameter
settings for the study-specific MRI sequences remained unchanged for the duration of the study. Each MRI scan performed for the study was reviewed by a local
neuroradiologist.
2

Multiple comparison procedures with modelling methodology
The primary endpoint was assessed using a multiple comparison procedure with modelling techniques (MCP-mod) methodology 1 adapted for lesion count data. MCP-mod
methodology offers a formal framework to model dose–response relationships, while explicitly taking into account model uncertainty.
Pre-defined candidate dose–response models covering the shape of the possible dose–response curve were: linear model; E max model (with ED 50 =1 mg); Hill-E max model 1
(with ED 50 =2 mg and Hill coefficient=2); Hill-E max model 2 (with ED 50 =3 mg and Hill coefficient=3); exponential model (with δ=3·633).
Because the drug effect was expressed as a percentage reduction in the monthly number of CUALs, and the model used to describe the lesion count invoked a log-link, these
five candidate dose–response models were expressed in the log scale. For interpretability, the models were then transformed on to the percentage reduction scale.
Statistical analyses (not reported in main manuscript)
Interim analysis
To select additional doses for cohort 2, six candidate profiles approximating the anticipated shape of the dose–response curve (low, high, linear, low-high, low-linear, E max )
were pre-specified (see Online Appendix figure 1). The correlation between each of these candidate profiles (see Online Appendix table 1) and the cohort 1 mean number of
CUALs (estimated per dose level using a negative binomial regression model) was calculated. The best candidate profile was identified as the one associated with the
strongest correlation. The interim analysis was performed by an independent statistician otherwise not involved in the conduct of the study; the results were kept confidential
and presented to a Data Monitoring Committee independent of Novartis. The Data Monitoring Committee reviewed the interim analysis and confirmed the selection of the
pre-defined candidate profile and the associated pre-defined cohort 2 doses.
Primary outcome analyses
The null hypothesis of a flat dose–response relationship for the percentage reduction in the number of monthly CUALs versus placebo was tested at a one-sided significance
level of 2·5% (α=0·025).
The primary and secondary endpoints were analysed using the full analysis set according to the intention-to-treat principle (all patients assigned a randomisation number who
received at least one dose of study medication and had no protocol deviation). Safety endpoints were assessed in the safety set.
The Bayesian longitudinal model used for the primary outcome measure was designed for an estimation of the dose–response curve specifically at month 3, without direct
inclusion of the month 1 data. This analysis was necessary because the raw CUAL count data by month and dose (and a separately fitted negative binomial regression)
revealed a clear differentiation from placebo at months 2 and 3 but not at month 1. The resulting siponimod dose–response curve was summarised by a plot of the posterior
median estimate and associated 95% confidence intervals and by the dose achieving an ED 50 in CUAL count versus placebo.
3

Secondary endpoint analyses
Secondary endpoints were analysed using the full analysis set (all patients in the full analysis set who completed at least 2 months of treatment and had no major protocol
deviation). Safety endpoints were assessed in the safety set (all patients who received at least one dose of study medication and had no protocol deviation).
The number of monthly new/newly enlarged T2 lesions at 3 and 6 months was estimated using a negative binomial generalised estimating equation (GEE) regression model
accounting for repeated measures on each patient, adjusted for treatment group × month (the month number of each lesion count measurement) interaction, using the log link.
Number of monthly CUAL at 3 months, number of monthly new and all Gd-enhancing T1 lesions, and number of monthly new Gd-enhancing T1 lesions in patients with
high disease activity at baseline were estimated using a negative binomial GEE regression model accounting for repeated measures on each patient, adjusted for baseline
number of Gd-enhancing T1 lesions and treatment group × month (the month number of each lesion count measurement) interaction, using the log link.
Proportions of patients were calculated using a weighted logistic regression model adjusted for treatment group and baseline number of Gd-enhancing T1 lesions.
Annualised relapse rate (ARR) was estimated from the group-level ARR using a negative binomial model regression model, adjusted for treatment group and baseline
number of relapses in the previous 2 years, with log time on study in years as the offset variable, using the log link. Group-level ARR was calculated as the total number of
relapses for all patients in the treatment group divided by the total number of days on study for all patients in the group and multiplied by 365·25 to obtain the annual rate.
4

Results (not reported in main manuscript)
Efficacy
After confirmation of a significant dose–response relationship with the E max model, the Bayesian longitudinal model was chosen as the best-fit model, because this allowed
for estimation of the dose–response curve specifically at month 3 (see Online Appendix figure 1). Based on the Bayesian longitudinal dose–response curve, the dose
providing a 50% reduction in number of CUALS versus placebo was estimated to be 0·51 mg (95% confidence interval 0·19–1·34).
After 3 months of treatment, all siponimod doses except for 0·25 mg significantly reduced the number of all monthly Gd-enhancing lesions versus placebo (relative reduction
vs placebo: siponimod 10 mg, 72%; siponimod 2 mg, 60%; siponimod 1·25 mg, 85%; siponimod 0·5 mg, 55%; siponimod 0·25 mg, 45%). After 6 months of treatment,
reductions in the number of all monthly Gd-enhancing lesions versus placebo were significant for all doses (siponimod 10 mg, 91%; siponimod 2 mg, 82%; siponimod
0·5 mg, 83%).
The proportions of patients who were free from CUALs were consistently higher in all siponimod treatment groups compared with placebo up to month 3 and month 6
(Online Appendix figure 2); this reached significance for siponimod 2 mg at month 3 and at month 6, and siponimod 1·25 mg and 0·25 mg at month 3.
In patients with high disease activity at baseline (≥2 Gd-enhancing T1 lesions), a significant effect of all siponimod doses, except for 0·25 mg, was observed on the number of
new monthly Gd-enhancing lesions at month 3 (10 mg, 87%; 2 mg, 84%; 1·25 mg, 95%; 0·5 mg, 73%; 0·25 mg, 50%). At month 6, significant effects for all siponimod doses
in cohort 1 were observed (10 mg, 79%; 2 mg, 76%; 0·5 mg, 77%) (Online Appendix figure 3).
5

Safety
One patient (siponimod 10 mg) was reported to have an adverse event of macular oedema, which resolved following study drug discontinuation; the patient had a history of
bilateral uveitis and optic neuritis of the right eye.
One death occurred in cohort 2 (siponimod 1·25 mg). On day 29 of the study, the patient, a 43-year-old man who was an active smoker and had a family history of coronary
artery disease, experienced severe chest pain, which persisted, leading to discontinuation from the study on day 52. The patient’s death was reported 27 days after treatment
discontinuation. The cause of death was determined by the coroner to be acute myocardial insufficiency – extensive stenosis was observed on post-mortem examination.
A causal relationship between siponimod and the chest pain, which occurred on study drug, cannot be ruled out; the outcome of death, however, may have been related to
factors other than siponimod, which is thought to be eliminated from the body ~6 days following drug discontinuation.
One patient (siponimod 10 mg), a 42-year-old man who was an active smoker, experienced a myocardial infarction. Bradycardia and junctional escape rhythm was detected
on day 1 but no adverse events (AEs) were reported; first-degree AV block was detected on day 90. The patient had a history of hypertension, and antihypertensive
medication was adjusted during the study for worsening of the condition. The patient completed the study, but approximately 45 days after the last dose of study medication,
the patient complained of upper abdominal symptoms; 2 days later he was hospitalised and treated for the event. He was discharged 7 days later.
Intention overdose occurred in a 39-year-old male with a history of reactive depression on day 20: the patient took 41 tablets of study medication in an attempted suicide and
later drove himself to the hospital. The patient did not experience bradycardia, had a normal white blood count (7.26x109 /L) and had an increase of transaminases (ALT at
90 U/l, AST at 52 U/l). The study medication was permanently discontinued due to the intentional overdose on the same day (day 20).
Benign intracranial hypertension occurred in a 46-year-old female. The patient started having symptoms of a MS relapse and an episode of optic neuritis on day 56 and
discontinued study medication on day 58 as she no longer wanted to continue study medication. On day 59 she was diagnosed with a confirmed MS relapse (with
photophobia and eye pain). On day 68, her left eye was swollen shut and her right eye vision was worse; on day 70 she was diagnosed with probable shingles. On day 72,
the patient was diagnosed with pseudotumour cerebri and the MRI report indicated that there was mild progression of demyelination. There was no evidence of venous
thrombosis. This DMC’s ophthalmologist concluded that the patient’s pseudotumour cerebri may be a co-incidental condition, medication-related (including but not limited
to siponimod), or related to a co-existing medical condition.
In cohort 1, bradycardia AEs and first-degree AV block AEs were reported in four patients in siponimod treatment groups in cohort 1 (three with 10 mg and one with
0·5 mg). In the 0·25 mg group in cohort 2, Holter monitoring on days 1, 2, and 7 revealed first-degree AV block in two patients treated with siponimod 0·25 mg (vs two
patients receiving placebo).
No difference was observed in mean changes in blood pressure among siponimod treatment groups compared with placebo at month 3. At month 6, a slight increase in mean
arterial blood pressure was observed in siponimod 2 mg dose (change from baseline to month 6, mean±SD: 10 mg, –1·37±7·80; 2 mg, 2·27±5·33; 0·5 mg, –1·10±7·2).
Increases in bilirubin above 1·5 × the upper limit of normal were uncommon and equally distributed between active treatment and placebo groups (Online Appendix table 3);
Hy’s law criteria were not met in any patients.
6

Drug exposure pharmacokinetics
Geometric mean siponimod plasma trough concentrations by treatment and visit are shown in Online Appendix table 4 and indicate dose-proportional exposure to siponimod,
with greater exposure at the higher doses assessed. Trough concentration was used as a measure of exposure in place of area under the curve between 0 and 24 hours
(AUC 0–24 ) because the concentration–time profile over 24 hours for siponimod is relatively flat and trough blood level serves as a good marker of steady-state exposure.
Discussion (not reported in the main manuscript)
The BOLD extension study (ClinicalTrials.gov Identifier: NCT01185821) will investigate the effects of dose titration across the doses studied in the BOLD core study.
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ONLINE APPENDIX FIGURES AND TABLES
Figure 1: Candidate relative effect dose–response profiles
The candidate dose–response profiles were pre-specified to approximate the shapes observed for the estimated dose–response coefficients from the negative binomial
regression fit at the interim analysis. Black dots represent doses studied in cohort 1 for each of the candidate profiles; open circles show the doses selected for cohort 2.
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Figure 2: Proportions of patients free from new MRI activity (CUALs) up to 3 and 6 months (full analysis set)
Patients free from new MRI activity are defined as patients free from CUALs over the time period specified (i.e. without evidence of any new Gd-enhancing T1 lesions and
without evidence of new/newly enlarged T2 lesions). Proportions and p values are for treatment comparison of siponimod versus placebo calculated using a weighted logistic
regression model. N = number of patients with at least one post-baseline MRI scan. N value for placebo shows number of patients included in the analysis at 3/6 months,
respectively.
CUAL, combined unique active lesion; MRI, magnetic resonance imaging.
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Figure 3: Estimated number of new monthly Gd-enhancing lesions at month 3 and month 6 in patients with high disease activity at baseline (full analysis set)
Analysed using a negative binomial generalised estimating equation regression model.
p values = pair-wise comparisons of active treatment versus placebo (i.e. lesion ratios).
n = number of patients with at least one scan up to 3 months or 6 months.
n value for placebo shows number of patients included in the analysis at 3 or 6 months, respectively.
High baseline disease activity is defined as at least two Gd-enhancing T1 lesions.
NA, not available due to design of study
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Table 1: Correlation between observed effects of siponimod 10 mg, 2 mg and 0·5 mg on number of CUALs and candidate dose–response profiles
Low-high Low-linear E max Low High Linear
0·938 0·859 0·979 0·594 0·957 0·742
Observed effects of siponimod 10 mg, 2 mg and 0·5 mg doses on number of CUALs estimated using a negative binomial model.
CUAL, combined unique active lesion.
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Table 2: Incidence of selected day 1 Holter findings in cohort 1 according to study group (safety population)
Siponimod
Events, n (%)
10 mg
n=50
2 mg
n=49
0·5 mg
n=43
Placebo
n=45
n 48 48 39 43
Second-degree (Mobitz I) AV block 5 (10·4) 6 (12·5) 2 (5·1) 1 (2·3)
Second-degree (Mobitz II, 2:1) AV block 3 (6·3) 2 (4·2) 0 0
First-degree AV block 2 (4·2) 2 (4·2) 0 0
Junctional escape rhythm 2 (4·2) 1 (2·1) 0 0
AV dissociation 2 (4·2) 0 0 0
AV, atrioventricular.
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Table 3: Selected laboratory measures according to study group (safety population)
Siponimod
10 mg
n=50
2 mg
n=49
1·25 mg
n=42
0·5 mg
n=43
0·25 mg
n=51
Placebo
n=61
Haematology measures a
Lymphocyte count, × 10 9 /L
Baseline, mean ± SD 1·82 ± 0·58 1·91 ± 0·59 1·85 ± 0·39 1·83 ± 0·53 1·85 ± 0·65 1·84 ± 0·56
Day 7, mean ± SD 0·47 ± 0·45 0·57 ± 0·37 0·76 ± 0·37 1·00 ± 0·40 1·48 ± 0·52 1·86 ± 0·56
Month 3, mean ± SD 0·31 ± 0·19 0·43 ± 0·37 0·60 ± 0·32 0·85 ± 0·41 1·38 ± 0·58 1·82 ± 0·62
Month 6, mean ± SD 0·35 ± 0·30 0·48 ± 0·41 0·91 ± 0·49 1·81 ± 0·59
Liver function measures b
Alanine aminotransferase, n (%)
>3–5 × ULN 2 (4·3) 2 (4·3) 1 (2·4) 0 1 (2·0) 0
>5–20 × ULN 0 0 0 0 1 (2·0) 0
>20 × ULN 0 0 0 0 0 0
Aspartate transaminase, n (%)
>3 × ULN 0 0 0 0 0 0
Gamma glutamyltransferase, n (%)
>2.5–5 × ULN 4 (8·5) 7 (14·9) 1 (2·4) 2 (4·7) 2 (4·0) 0
>5–20 × ULN 1 (2·1) 1 (2·1) 0 0 1 (2·0) 0
>20 × ULN 0 0 0 0 0 0
Bilirubin, n (%)
>1·5–3 × ULN 0 1 (2·1) 0 0 1 (2·0) 2 (3·3)
>3 × ULN 0 0 0 0 0 0
a n=44 (siponimod 10 mg), 45 (siponimod 2 mg), 37 (siponimod 1·25 mg), 40 (siponimod 0·5 mg), 39 (siponimod 0·25 mg), 56 (placebo) for baseline and day 7;
n=34 (siponimod 10 mg), 41 (siponimod 2 mg), 37 (siponimod 1·25 mg), 35 (siponimod 0·5 mg), 46 (siponimod 0·25 mg), 56 (placebo) for month 3; n=24 (siponimod
10 mg), 35 (siponimod 2 mg), 31 (siponimod 0·5 mg), 31 (placebo) for month 6.
b n=47 (siponimod 10 mg), 47 (siponimod 2 mg), 42 (siponimod 1·25 mg), 43 (siponimod 0·5 mg), 50 (siponimod 0·25 mg), 61 (placebo).
SD, standard deviation; ULN, upper limit of normal.
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Table 4: Geometric mean (coefficient of variation; CoV) siponimod plasma trough concentrations by treatment and visit (pharmacokinetic analysis set a )
Visit
Geometric mean
(CoV), (%)
10 mg
n=50
2 mg
n=49
Siponimod
1·25 mg
n=42
0·5 mg
n=43
0·25 mg
n=51
Month 1 114 (52) 22·6 (54) 12·8 (46) 6·05 (52) 2·68 (40)
Month 3 113 (51) 21·7 (48) 11·4 (49) 5·28 (60) 2·43 (57)
Month 6 52·7 (530 b ) 23·7 (58) NA 5·17 (66) NA
a Patients in the siponimod groups with one evaluable pharmacokinetic sample on at least one of the scheduled visits.
b High variability due to deviations in the timing of planned pharmacokinetic sample collection relative to drug intake.
NA, not available due to design of study.
Reference
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