Tran, et al. e-24
Appendix e-1
METHODS
MRI.
Acquisitions
In the SAD study, acquisitions were performed at one center each at the VU Medical Center (VUMC), Amsterdam, The Netherlands, and at Glendale Adventist Medical Center (GAMC), Glendale, CA, USA. In the MAD study, all acquisitions were performed at Health Images, Englewood, CO, USA. Both conventional (T1-weighted gadolinium-enhancing, T2-weighted, proton density (PD)-weighted and 3-dimensional [3D] T1-weighted MRI), and nonconventional acquisitions (fluid attention inversion recovery [FLAIR], magnetization transfer [MT], diffusion-weighted imaging [DWI], and diffusion tensor imaging [DTI] MRI) were used.
In the SAD study, healthy volunteers received structural 1.5T-MRI (VUMC: Siemens Magnetom Sonata; GAMC: GE Signa HDxt), using a 2D FLAIR sequence (TR 9000 ms, TE 68 ms [VUMC] or 82.5 ms [GAMC], flip angle 150/90, in-plane resolution 0.98 × 0.98 mm, 3-mm slice thickness), and a 3D T1-weighted fast low angle shot (VUMC) or spoiled gradient-echo (GAMC) sequence with and without saturation pulse (TR 30/36 ms, TE 11 ms, flip angle 15/90, 0.98 × 0.98 mm in-plane resolution, 3-mm slice thickness). DWI was performed with single-shot spin-echo EPI in the axial plane (TR 3,400/10,000 ms, TE 122/82 ms, flip angle 90, 1.95 × 1.95 mm [VUMC] or 0.98 × 0.98 mm [GAMC] in-plane resolution, 5-mm slice thickness).
In the MAD study, participants with MS received structural 1.5T-MRI (GE Signa HDxt), using a 3D T1-weighted fast spoiled gradient-echo sequence (TR 11.1 ms, TE 5 ms, TI 450 ms, flip angle 20, 1.0 × 1.0 mm in-plane resolution, 1-mm slice thickness), 2-dimensional (2D) dual-echo PD/ T2 (TR 3,000 ms, TE 33/82.6 ms, flip angle 90, 46 contiguous axial slices of 3 mm, in-plane resolution 1.0 × 1.0 mm) and 2D spin-echo T1-weighted gadolinium-enhancing imaging (TR 467 ms, TE 20 ms, flip angle 90, 46 contiguous axial slices of 3 mm, in-plane resolution 1.0 × 1.0 mm). DWI was performed with single-shot spin-echo echo planar imaging (EPI) in the axial plane (TR 10,000 ms, TE 99.8 ms, flip angle 90, 0.98 × 0.98 mm in-plane resolution, 5-mm slice thickness) and MT imaging was acquired using a 3D T1-weighted spoiled gradient-echo sequence with and without saturation pulse (TR 35 ms, TE 9 ms, flip angle 15, 1.0 × 1.0 mm in-plane resolution, 3-mm slice thickness). DTI based on EPI was performed using 1 volume without directional weighting (i.e., b0) and 31 volumes with noncollinear diffusion gradients (i.e., 31 directions, b = 900 s/mm2, TR 15,000 ms, TE 105 ms, flip angle 90, 60 contiguous axial slices of 2.5 mm, in-plane resolution 0.94 × 0.94 mm).
Analysis
All analyses were performed at the Image Analysis Center (part of the VUMC). Conventional acquisitions and the FLAIR sequence were used for safety assessments. The DWI sequence was used for evidence of vascular damage (ischemic or hemorrhagic). T1 gadolinium-enhancing lesion counts, T1-hypointense lesion count, and lesion load were assessed on the T1-weighted post gadolinium MRI sequence. T2-hyperintense lesion counts and lesion load were assessed on the PD-weighted sequence, with the T2-weighted sequence as reference. All lesion identification and lesion load assessments were performed by trained personnel at the VU University Medical Center (Amsterdam) using Medical Image Processing, Analysis and Visualization (MIPAV) software (version 5.1.1).
MT ratio (MTR) images were calculated from the 2 consecutive MT sequences, with and without the saturation pulse, with the formula:
MTR = (Mo – Ms) / Mo × 100
where Ms represents the signal intensity with the saturation pulse and Mo represents signal intensity without the saturation pulse. All pre-processing steps for DTI were performed using FSL4.1 (FMRIB Analysis Group, Oxford, UK), including motion- and eddy-current correction on images and gradient-vectors, followed by diffusion tensor fitting. Fractional anisotropy (FA) and mean diffusivity (MD) were derived for each voxel.
In MIPAV, six regions of interest (ROI) were placed at predefined locations within the normal-appearing white matter. The mean MTR percent unit, FA (scalar value between 0 and 1), and MD (in 10–5 mm²/sec) were assessed for each of the six ROI, both individually and combined. The mean MTR percent unit, mean FA, and mean MD were also assessed for new T1 gadolinium-enhancing and new T2-hyperintense lesions by first converting the lesion load ROIs to binary masks in MIPAV, then registering the original T1 and T2 images to the MTR, FA and MD images of the current, previous and follow-up visits, using the registration parameters to register the binary masks accordingly. Finally, the registered binary lesions masks were converted back to ROIs and overlaid over the corresponding MTR, FA and MD images. This was also done for preexisting T2-hyperintense lesions and preexisting T1-hypointense lesions.
Automated brain atrophy analysis was performed using the 3D T1-weighted sequence. Normalized brain volume and percentage brain volume changes were calculated with SIENA1 and SIENAX,1 respectively (parts of FSL;2 FMRIB Software Library, Oxford, UK). Normalized ventricular volume and percentage ventricular volume changes were calculated with modified version of SIENA and SIENAX software as described in detail elsewhere.3
REFERENCES
1. Smith SM, Zhang Y, et al. Accurate, robust and automated longitudinal and cross-sectional brain change analysis. NeuroImage 2002;17:479–489.
2. Smith SM, Jenkinson M, et al. Advances in functional and structural MR image analysis and implementation as FSL. NeuroImage 2004;23(S1):208–219.
3. Vrenken H, Vos EK, et al. Validation of the automated method VIENA: An accurate, precise, and robust measure of ventricular enlargement. Hum Brain Mapp 2014;35:1101–1110.
SUPPLEMENTARY FIGURES
Figure e-1. (A) Box plots of change from baseline at weeks 4 (all dose groups) and 8 (higher-dose groups only) for the magnetization transfer ratio of normal-appearing white matter in participants from the SAD study. (B) Example of regions of interest (colored circles) of normal-appearing white matter selected for magnetization transfer imaging in the brain of a healthy volunteer in the SAD study. (C) Box plots for changes from baseline to week 16 of the magnetization transfer ratio and fractional anisotropy of normal-appearing white matter in participants from the MAD study. Boxes represent the 25th to 75th percentiles; horizontal line in box interior = median; + = mean; □=outlier. MAD = multiple ascending dose; SAD = single ascending dose.
Figure e-2. Changes in magnetization transfer ratio (A, B) and fractional anisotropy (C, D) of preexisting T2-hyperintense lesions in participants with multiple sclerosis from the MAD study; box plots for changes from baseline to week 8 (A, C) or week 16 (B, D). Changes in magnetization transfer ratio (E, F) and fractional anisotropy (G, H) of preexisting T1-hypointense lesions in participants with multiple sclerosis from the MAD study; box plots for changes from baseline to week 8 (E, G) or week 16 (F, H). Boxes represent the 25th to 75th percentiles; horizontal line in box interior = median; + = mean; □ = outlier. MAD = multiple ascending dose; SAD = single ascending dose.
Tran, et al. e-24
SUPPLEMENTARY TABLES
Table e-1. Baseline demographics and characteristics in healthy volunteers (SAD study) and participants with MS (MAD study)
SAD study, BIIB033 dose, mg/kgCharacteristic / Placebo (n = 18) / 0.1
(n = 6) / 0.3
(n = 6) / 1.0
(n = 6) / 3.0
(n = 6) / 10
(n = 6) / 30
(n = 6) / 60
(n = 6) / 100
(n = 6) / 3.0a
(n = 6)
Mean (SD) age, y / 38.4 (9.9) / 42.5 (12.9) / 40.7 (9.7) / 44.5 (10.1) / 36.2 (10.3) / 34.5 (4.4) / 30.5 (3.9) / 33.2 (11.4) / 33.0 (6.0) / 29.8 (4.0)
Female gender, n (%) / 2 (11) / 1 (17) / 1 (17) / 1 (17) / 1 (17) / 1 (17) / 0 / 0 / 0 / 0
Race, n (%)
White / 9 (50) / 6 (100) / 5 (83) / 6 (100) / 2 (33) / 5 (83) / 4 (67) / 2 (33) / 2 (33) / 2 (33)
Black / 6 (33) / 0 / 1 (17) / 0 / 2 (33) / 1 (17) / 1 (17) / 2 (33) / 4 (67) / 2 (33)
Other / 3 (17) / 0 / 0 / 0 / 2 (33) / 0 / 1 (17) / 2 (33) / 0 / 2 (33)
MAD study, BIIB033 dose, mg/kg
Characteristic / Placebo
(n = 15) / 0.3
(n = 4) / 1.0
(n = 4) / 3.0
(n = 4) / 10
(n = 4) / 30
(n = 4) / 60
(n = 4) / 100
(n = 4) / 100b
(n = 4)
Mean (SD) age, y / 48.8 (7.3) / 48.3 (9.3) / 50.8 (8.2) / 55.3 (1.7) / 40.5 (5.6) / 36.5 (11.6) / 54.5 (5.5) / 47.5 (10.1) / 44.3 (9.1)
Female gender, n (%) / 10 (67) / 4 (100) / 3 (75) / 4 (100) / 4 (100) / 4 (100) / 2 (50) / 2 (50) / 3 (75)
Race, n (%)
White / 14 (93) / 4 (100) / 3 (75) / 4 (100) / 4 (100) / 3 (75) / 4 (100) / 4 (100) / 4 (100)
Otherc / 1 (7) / 0 / 1 (25) / 0 / 0 / 1 (25) / 0 / 0 / 0
RRMS, n (%) / 10 (67) / 4 (100) / 4 (100) / 4 (100) / 4 (100) / 3 (75) / 3 (75) / 3 (75) / 4 (100)
SPMS, n (%) / 5 (33) / 0 / 0 / 0 / 0 / 1 (25) / 1 (25) / 1 (25) / 0
Mean (SD) time since diagnosis, y / 11.7 (10.1) / 9.8 (5.5) / 15.4 (13.9) / 6.5 (5.9) / 3.1 (2.5) / 9.3 (5.4) / 7.0 (5.8) / 9.1 (10.1) / 10.4 (10.7)
Mean (SD) EDSS score / 3.6 (1.4) / 2.5 (1.1) / 3.4 (0.9) / 2.8 (1.5) / 2.1 (0.3) / 3.3 (1.9) / 3.8 (1.2) / 3.1 (1.0) / 3.3 (1.2)
Received prior MS medication, n (%) / 5 (33) / 2 (50) / 3 (75) / 1 (25) / 1 (25) / 2 (50) / 2 (50) / 2 (50) / 1 (25)
Concomitant IFNß,
n (%) / 4 (27) / 1 (25) / 2 (50) / 1 (25) / 2 (50) / 2 (50) / 1 (25) / 2 (50) / 1 (25)
Concomitant GA,
n (%) / 8 (53) / 3 (75) / 2 (50) / 2 (50) / 2 (50) / 1 (25) / 2 (50) / 2 (50) / 3 (75)
Gd+ lesions, mean no. (SD) / 0 / 0.25 (0.50) / 0.25 (0.50) / 0 / 0 / 2.75 (5.50) / 0.50 (1.00) / 0.5 (1.00) / 0
No. of participants with Gd+ lesions (%) / 0 / 1 (25) / 1 (25) / 0 / 0 / 1 (25) / 1 (25) / 1 (25) / 0
T2-hyperintense lesions, mean no. (SD) / 42.6 (29.1) / 76.5 (44.6) / 38.0 (39.7) / 47.8 (27.3) / 22.8 (20.4) / 95.0 (48.8) / 38.3 (19.2) / 22.8 (30.9) / 59.0 (84.6)
T1-hypointense lesions, mean no. (SD) / 13.1 (9.5) / 30.3 (18.7) / 10.5 (14.9) / 13.8 (15.5) / 4.8 (3.0) / 30.8 (24.4) / 5.8 (4.3) / 3.5 (5.0) / 8.0 (13.4)
Abbreviations: EDSS = Expanded Disability Status Scale; GA = glatiramer acetate; Gd+ = gadolinium-enhancing; IFNβ = interferon beta; MAD = multiple ascending dose; MS = multiple sclerosis; RRMS = relapsing-remitting multiple sclerosis; SAD = single ascending dose; SD = standard deviation; SPMS = secondary progressive multiple sclerosis.
a Subcutaneous dose.
b Single dose, shorter infusion time (80 minutes).
c Not white or black.
Table e-2. Summary of shifts from baseline in hematology and blood chemistry in healthy volunteers (SAD study) and participants with MS (MAD study)
Variable / Placebo (n = 18) / 0.1
(n = 6) / 0.3
(n = 6) / 1.0
(n = 6) / 3.0
(n = 6) / 10
(n = 6) / 30
(n = 6) / 60
(n = 6) / 100
(n = 6) / 3.0a
(n = 6)
Hematology
Hemoglobin
Shift to low / 4/17 / 0/6 / 1/6 / 1/6 / 2/6 / 2/5 / 3/5 / 3/6 / 2/4 / 5/6
Shift to high / 0/18 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 2/5 / 0/6
Red blood cell count
Shift to low / 2/18 / 0/6 / 1/6 / 2/5 / 0/6 / 1/5 / 2/6 / 2/6 / 2/5 / 2/6
Shift to high / 0/18 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6
White blood cell count
Shift to low / 1/18 / 0/6 / 1/6 / 1/6 / 1/6 / 0/6 / 1/6 / 0/5 / 1/6 / 0/6
Shift to high / 1/18 / 0/6 / 1/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 1/6
Platelet count
Shift to low / 0/18 / 0/6 / 0/6 / 1/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6
Shift to high / 0/18 / 0/6 / 1/5 / 1/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 1/6
Total absolute neutrophil count
Shift to low / 2/18 / 0/5 / 1/6 / 3/6 / 1/6 / 0/6 / 1/6 / 0/5 / 1/6 / 0/6
Shift to high / 1/18 / 0/6 / 1/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 1/6
Lymphocytes
Shift to low / 1/18 / 0/6 / 1/6 / 1/6 / 1/6 / 0/6 / 1/6 / 0/6 / 0/6 / 0/6
Shift to high / 1/18 / 0/6 / 0/5 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6
Monocytes
Shift to low / 1/18 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 1/6 / 1/6 / 1/6
Shift to high / 0/18 / 0/6 / 2/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 1/6
Blood Chemistry
Total protein
Shift to low / 5/17 / 0/6 / 0/6 / 0/6 / 1/6 / 2/6 / 3/6 / 6/6 / 3/6 / 4/6
Shift to high / 0/18 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6
Albumin
Shift to low / 3/17 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 1/6 / 2/6 / 0/4
Shift to high / 0/18 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6
Alkaline phosphatase
Shift to low / 1/17 / 0/6 / 0/6 / 0/6 / 0/6 / 0/5 / 1/6 / 0/4 / 0/6 / 0/6
Shift to high / 0/18 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6
Alanine aminotransferase
Shift to low / 1/15 / 0/6 / 0/6 / 0/6 / 2/6 / 2/3 / 3/4 / 3/5 / 2/6 / 1/3
Shift to high / 2/18 / 0/6 / 1/6 / 0/6 / 1/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6
Aspartate aminotransferase
Shift to low / 0/18 / 0/6 / 0/6 / 0/6 / 0/6 / 0/5 / 1/6 / 0/6 / 0/6 / 1/3
Shift to high / 1/18 / 0/6 / 1/6 / 0/6 / 0/6 / 2/6 / 0/6 / 1/6 / 3/6 / 0/6
Total bilirubin
Shift to low / 0/18 / 0/6 / 0/6 / 0/6 / 1/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6
Shift to high / 2/18 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 1/6 / 2/6 / 1/6 / 1/6
Creatinine
Shift to low / 2/18 / 1/6 / 0/6 / 0/4 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6 / 0/6