supplementary materials: the effects of dexpramipexole ... · gary pattee, m.d., june smith, and...

Supplementary Materials: The effects of dexpramipexole (KNS-760704)

in patients with amyotrophic lateral sclerosis

Authors and Institutions: Merit Cudkowicz1, Michael E. Bozik2, Evan W. Ingersoll2, Robert Miller3,

Hiroshi Mitsumoto4, Jeremy Shefner5, Dan H. Moore3, David Schoenfeld6, James L. Mather2, Donald

Archibald2, Mary Sullivan2, Craig Amburgey2, Juliet Moritz2 and Valentin K. Gribkoff2

1Neurology Clinical Trial Unit, Massachusetts General Hospital, Boston, MA

2Knopp Biosciences LLC, Pittsburgh, PA

3California Pacific Medical Center, San Francisco, CA

4Columbia University Medical Center, New York, NY

5SUNY Upstate Medical University, Syracuse, NY

6Massachusetts General Hospital, Boston, MA

Corresponding Author: Valentin K. Gribkoff, Ph.D. Knopp Biosciences LLC 2100 Wharton Street, Suite 615 Pittsburgh PA 15203 TEL: 203-605-9830 FAX: 412-488-8487 [email protected]

Nature Medicine doi:10.1038/nm.2579

Supplementary Materials Contents:

1.) Acknowledgements and list of contributors

2.) Supplementary Methods

3.) Supplementary tables:

Supplementary table 1. Schedule of Assessments for Part 1 of the Study.

Supplementary table 2. Schedule of Assessments for Part 2 of the Study.

Supplementary table 3. Regression tables for the linear mixed effects model – Part 1

Supplementary table 4. Regression tables for the linear mixed effects model – Part 2

Supplementary table 5. Summary of Deaths and Subjects Reporting Adverse Events in Parts 1 and 2 of the Study.

Supplementary table 6. Number of Subjects Reporting Common Treatment Emergent AEs (Reported in ≥ 10% of Subjects Receiving Active Study Drug) by Preferred Term in Decreasing Frequency (Part 1, Safety Population)

Supplementary table 7. Number of Subjects Reporting Common Treatment-Emergent AEs (Reported in ≥ 10% of Subjects Receiving either Treatment) by Preferred Term in Decreasing Frequency (Part 2, Safety Population)

Supplementary table 8. Subjects Experiencing Neutropenia in Study CL201.

Supplementary table 9. Comparison of Selected Pharmacokinetic Parameter Estimates for Healthy Volunteers (HV) and ALS Subjects

4.) Supplementary Figures

Supplemetary figure 1. The temporal progression of the ALSFRS-R total score for every individual subject in the indicated dose group enrolled in Part 1 of study CL-201.

Supplementary figure 2. The temporal progression of the ALSFRS-R total score for every individual subject in the indicated dose group enrolled in Part 2 of study CL-201.

5.) Supplementary materials literature references

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SUPPLEMENTARY MATERIALS

Acknowledgements

The authors wish to recognize the contributions of the following clinical investigators and coordinators,

who constitute the KNS-760704-CL201 Study Group (listed alphabetically by principal investigator):

Jinsy Andrews, M.D., Hiroshi Mitsumoto, M.D., Danielle Bell, and Kate Dalton; Columbia University Medical Center, New York, NY

Richard Barohn, M.D. and Maureen Walsh; University of Kansas Medical Center, Kansas City, KS

Mark Bromberg, M.D., Narneice Craven, and Summer Davis; University of Utah, Salt Lake City, Utah

Ted Burns, M.D. and Kristen Keller; University of Virginia, Charlottesville, VA Merit Cudkowicz, M.D., Darlene Sawicki, and Jennifer Berndt ; Massachusetts General

Hospital, Boston MA Peter Donofrio, M.D., Diana Davis, and Kay Artibee; Vanderbilt University Medical Center,

Nashville, TN Michael Graves, M.D., Juliana Albu, and Rebecca Alvarez; University of California at Los

Angeles, Los Angeles, CA Terry Heiman-Patterson, M.D., Sara Feldman, and Christine Barr ; Drexel University

College of Medicine, Philadelphia, PA Carlayne Jackson, M.D. and Pamela Kittrell; University of Texas Health Science Center San

Antonio, San Antonio TX Jonathan Katz, M.D., Robert Miller, M.D., Marguerite Engel, Giovanna Kushner, Dallas

Forshew, and Stacey Champion; California Pacific Medical Center, San Francisco, CA David Lacomis, M.D., Rebecca Molczan, and Danielle Rowlands; University of Pittsburgh

Medical Center, Pittsburgh, PA Gary Pattee, M.D., June Smith, and Kathy Ware; Neurology Associates, Lincoln, NE Alan Pestronk, M.D., Julaine Florence, Jeanine Schierbecker, Betsy Malkus, Pamela

Townsend, and Charlie Wulf; Washington University, St. Louis, MO Yvonne Rollins, M.D., Bjorn Oskarsson, M.D., Elizabeth Whitethorn, Catherine McKenna,

and John Cumming; University of Colorado Health Sciences Center, Aurora, CO Jeffrey Rothstein, M.D. and Richard M. Kimball; Johns Hopkins University, Baltimore, MD Stacy Rudnicki, M.D. and Deborah Fewell; University of Arkansas for Medical Sciences,

Little Rock, AR Khema Sharma, M.D., Julie Steele, and Don Koggan, M.D.; University of Miami, Miami, FL Jeremy Shefner, M.D., Ph.D. and Mary Lou Watson; SUNY Upstate Medical University,

Syracuse, NY

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Zachary Simmons, M.D. and Helen (Beth) Stephens; Penn State Milton S. Hershey Medical Center, Hershey, PA

Michael Weiss, M.D., Sharon Downing, John Lin, and Joanna Haug ; University of Washington Medical Center, Seattle, WA

Supplementary Methods

Enrollment Criteria

Eligible subjects were aged 21 to 80 years; had familial or sporadic ALS according to the “possible” to

“definite” criteria for ALS diagnosis,1;2 were 24 months from ALS symptom onset; and had upright

vital capacity (VC) >65% of predicted for age, height, and gender.

Time from symptom onset was an original element of this protocol relative to previous studies in ALS.

Many ALS studies enroll subjects with significantly longer times from symptom onset.3-6 The effect this

has on the study population is to ensure a relatively equally represented cross-section of progression

phenotypes, as compared with studies that allow longer times from symptom onset and are consequently

over-represented by subjects with slowly progressive disease.

Concomitant riluzole was permitted if subjects were on a stable dose for at least 60 days; subjects not

taking riluzole were also eligible.

Study Conduct and Visit Schedule

The first subject was dosed on April 9, 2008 and the final subject completed 24 weeks of double-blind

active treatment in Part 2 on August 21, 2009.

Clinic visits in Part 1 (see Supplemental Table 1) were performed at baseline (pre-dose), Day 1 (post-

dose), and Weeks 1, 2, 4, 8, and 12 (or end-of-study if a subject discontinued prematurely). Safety

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evaluations were performed at every visit and clinical status was assessed at all visits except for Weeks

1 and 2. At baseline and Week 12, plasma and CSF samples were obtained from a subset of patients who

consented to provide them for exploratory analyses of protein surrogate markers of disease progression.

A subset of subjects also participated in a pharmacokinetic sub-study (see Supplemental Materials).

Clinic visits in Part 2 (see Supplemental Table 2) were scheduled at baseline of placebo washout (i.e.,

the Part 1, Week 12 visit), Week 4 (baseline of active treatment), and Weeks 2, 4, 8, 16, and 24 of active

treatment. At Weeks 12 and 20 of active treatment, evaluations of clinical status and AEs were

performed by a qualified health care professional over the telephone. If subjects discontinued the study

prior to the active treatment Week 24 visit, vital status or date of death were determined by periodic

follow-up contacts through the end of the study. At baseline and Week 24 of active treatment, plasma

samples were obtained for exploratory analyses of protein surrogate markers of disease progression.

Dose Selection

We have previously found that dexpramipexole is highly orally bioavailable and renally excreted in

humans7 and, in animals, enters the brain at high levels (unpublished results). Therefore, doses were

administered orally as 2 tablets of 12.5 mg, 37.5 mg, or 75 mg dexpramipexole or placebo twice a day.

Linear Mixed Effects Slopes Model

The following is a description of the linear mixed effects slopes model method used for analysis of

efficacy variables. This model fits a random intercept and slope to each subject. The fixed effects are

time, treatment, and a time-treatment interaction. The latter term measures the extent to which the rate of

change differs among treatment groups and was used to test the overall null hypothesis that the slopes of

all 4 treatment groups were equal and to construct pairwise comparisons between each dose group and

placebo. The estimated coefficient of time (the slope, or rate of change) and its 95% CI was provided

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for each treatment group. As pre-specified in the Statistical Analysis Plan, unstructured covariance was

used along with standard SAS default options (as detailed in the SAS/STAT User’s Guide, Version 8,

Volume 3 from the SAS Institute, Inc., 1999): the estimation method was REML, the residual variance

method was profile, the fixed effects SE method was model-based, and the degrees of freedom method

was containment. Linear decline of the ALSFRS-R over time has been shown previously,8 though a

recent study suggests that decline may be curvilinear.9 Linearity in this sample was determined by

testing the significance of a quadratic time term in the model. The regression tables for the mixed

effects models of ALSFRS-R total scores from Part 1 and Part 2 have been provided below (see

Supplemental Tables 3 and 4). The longitudinal graphs of the raw ALSFRS-R total score data have also

been provided (see Supplemental Figs. 1 and 2).

Criteria for Part 1 Exploratory Failure Analyses

Failure analyses for Part 1 ALSFRS-R and VC data used the change from baseline to endpoint with the

last observation carried forward (LOCF) for subjects who discontinued in Part 1. In Part 1, the LOCF

method was appropriate for exploratory analyses because of the very small number of drop-outs and the

absence of deaths.

For the exploratory failure analyses, failure was defined as a decrease of 6 or more points on the

ALSFRS-R total score as used in a previous study.10 For VC, failure was defined as a decrease of 20%

or more in VC over the 12-week period. These criteria represent approximately twice the generally

observed average decline in these measures over a 3-month period.

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Joint Rank Analysis

When deaths are unevenly distributed between the treatment groups, the mixed effects slopes model

may not adequately account for the effect of deaths in the estimate of the treatment effect. For this

reason, a joint rank analysis of change from baseline in ALSFRS-R and death was conducted. This

analysis may also be described as a Combined Assessment of Function and Survival (CAFS). In this

analysis, a subject’s score is calculated by comparing each subject to every other subject in the trial,

setting a score of +1 if the outcome was better than the subject being compared, -1 if worse and 0 if tied.

For the comparison, if 2 subjects complete the study, their comparison score will be based on the change

from baseline of the ALSFRS-R values at the end of the study; a subject who dies earlier than the

comparator subject will be given a comparison score of -1; for subjects who discontinue early for

reasons other than death, their comparison to each other subject will be based on the comparison of their

change from baseline of the ALSFRS-R values at the latest time-point at which they both have an

ALSFRS-R value. The subject’s score will then be calculated by summing up their comparison to all

the other subjects in the study and then the subject is given a rank based on these summed scores. In

general, these comparisons will result in (1) subjects who die being assigned the worst scores (ranks)

and being ranked according to the time of death; and (2) subjects who survive and complete the study

being ranked more favorably than subjects who die and then according to their endpoint ALSFRS-R

change value, with special handling to rank early discontinuations as described above.

Covariate Selection for Part 2 Exploratory Analysis of Joint Rank

The covariates used in this exploratory analysis of Part 2 efficacy (ANCOVA on the joint ranks) data

were baseline ALSFRS-R score, time from symptom onset, site of disease onset, and riluzole use. The

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first 3 covariates were selected as variables that were predictive of the joint rank by using a stepwise

selection regression procedure with criteria (p=0.15) for entry and (p=0.15) to stay; concomitant riluzole

use was selected based on a modest imbalance among treatment groups at baseline.

Exploratory Analysis of ALSFRS-R slopes in Part 2

An exploratory analysis involving the imputation of a score of zero for the first post-death visit for

subjects who died in Part 2 was conducted as one means of examining the impact of death within the

analysis of ALSFRS-R slopes. This was not a prespecified analysis, but has been used in literature

reports of other ALS studies.6 Linearity was rejected in the ALSFRS-R mixed model with imputed

zeros, but the slopes were presented based on the linear model for purposes of comparability with other

linear slopes results because the treatment effect (p-value and percent difference) was almost identical to

the results of a model that included a quadratic term.

Safety Results

No per-treatment-group differences were observed in the incidence of vital sign, ECG, or laboratory

abnormalities that met pre-specified criteria for potential clinical significance in either part of the study.

Five subjects (2 in Part 1 and 3 in Part 2) receiving 300 mg/day experienced reversible National Cancer

Institute (NCI) Common Terminology Criteria for Adverse Events (CTC) Grade 2, asymptomatic (4) or

Grade 4, febrile (1) neutropenia; there was no recurrence in 3 of 4 subjects who resumed active

treatment including the Grade 4 case (see details below).

Part 1

A summary of subjects reporting AEs in Part 1 is shown in Supplemental Table 5. A total of 92 of 102

(90.2%) subjects reported at least 1 AE in Part 1, and the number of subjects reporting at least 1 AE was

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similar across treatment groups. Two subjects had AEs leading to discontinuation, and 5 subjects

reported serious AEs, none of which were judged by the investigator to be related to treatment with the

study drug. There were no deaths during Part 1 of the study. AEs reported in ≥10% of subjects are

shown in Supplemental Table 6. There were no statistically significant differences for any AE for the

overall chi-square test comparing the 4 dose groups, and there were no statistically significant

differences between placebo and the high dose group or between placebo and the pool of all Active

Treatment for any AE (Fisher’s exact test). Most or all reports of headache, nausea, and laboratory AEs

across all treatment groups were in subjects who were concomitantly taking riluzole.

Part 2

Placebo Washout Period. During the 4-week placebo washout period there were no differences among

groups in the incidence or patterns of AEs reported. The only AE for which incidence in all subjects

exceeded 10% was fall (11%). A total of 5 subjects reported an SAE, none of which were considered to

be treatment-related. Three (3%) subjects died during the placebo washout period (2 subjects had

received 50 mg and 1 subject had received placebo in Part 1; all were ALS-related), and 1 subject

underwent a tracheostomy (received 300 mg in Part 1).

Double-Blind Active Treatment Period. See Supplemental Table 5 for a summary of deaths and AEs in

Part 2. A total of 87 of 92 (95%) subjects in the randomized active treatment phase of Part 2 reported at

least 1 AE, and the number of subjects reporting at least 1 AE was similar across the 2 treatment groups.

AEs occurring in ≥10% of subjects in either treatment group are shown in Supplemental Table 7. Dry

mouth and insomnia rates were higher in the 300 mg group (Fisher’s exact test p=0.026 and p=0.01,

respectively). None of these differences were considered clinically significant. Twelve subjects (9 in the

50 mg group, and 3 in the 300 mg group) died during the double-blind active treatment period prior to

what would have been their target date for a Part 2 active treatment Week 24 visit. Seven of the 12

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deaths were recorded as due to respiratory failure or ALS disease progression; 1 death was due to ileus

(50 mg), 1 death was due to sudden death (50 mg), and the cause of death was not available for 3 deaths

(2 in the 50 mg group and 1 in the 300 mg group, for which only the date of death was captured in post-

study follow-up contacts). Sixteen subjects, 11 (23%) in the 50 mg group and 5 (11%) in the 300 mg

group, had SAEs, 2 of whom had events that were considered to be treatment-related. There were 2

suspected unexpected serious adverse reactions (SUSARs) reported during the 24-week double-blind

active treatment period: neutropenia (300 mg) and sudden death (50 mg).

Neutropenia

Five (5) cases of neutropenia occurred during Study CL201, including one case that was categorized as

an NCI CTC Grade 4 event. During CL201, 4 of the 5 subjects were re-challenged with dexpramipexole.

Three were successfully re-challenged without recurrence of neutropenia (including the subject with

Grade 4 neutropenia), completed dosing in the study and elected to enroll into the follow-on open-label

extension study (CL211). A fourth subject had a recurrence of asymptomatic Grade 2 neutropenia

following re-challenge, leading to that subject’s discontinuation from the study. A fifth subject had a

single neutrophil value (1.43 x103/uL) that met the specified threshold of Grade 2 neutropenia at the last

visit in Part 2 of the study; the subject elected not to enroll in the open-label CL211 study and,

consequently, was not re-challenged with dexpramipexole. A repeat test performed 20 days after the

termination visit indicated that neutrophils had returned to within the normal range (2.54 x103/uL).

Details of the 5 subjects with neutropenia events in CL201 are shown in Supplemental Table 8.

Clinical Laboratory Assessments

The following clinical laboratory parameters were assessed throughout the study:

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Hematology: CBC with differential (neutrophils, lymphocytes, monocytes, eosinophils, basophils),

platelet count, red blood cell count (RBC), hemoglobin concentration (Hgb), hematocrit (Hct), mean

corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin

concentration (MCHC), and red cell distribution width (RDW).

Chemistries and Lipids: serum potassium, blood urea nitrogen (BUN), creatinine, glucose, uric acid,

calcium, phosphorus, sodium, chloride, total protein, albumin, globulin, creatinine kinase (CK), a

lipid profile (total cholesterol, high-density lipoprotein [HDL], low-density lipoprotein [LDL], and

triglycerides) and the following liver tests: aspartate aminotransferase (AST), alanine

aminotransferase (ALT), total bilirubin, and alkaline phosphatase. A serum pregnancy test was also

obtained monthly in women of childbearing potential.

Urinalysis: glucose, ketones, bilirubin, urobilinogen, protein, and WBC.

Pharmacokinetic Sub-Study

Subjects who consented to participate in the PK sub-study had blood samples collected at specified

times over the course of an 8 hour period during Part 1 of the study. The day for these serial blood

draws was to be scheduled prior to enrolling in Part 2 of the study and after the subject had completed at

least 7 days of consecutive dosing with study drug, but the timing was otherwise left to the discretion of

the site and patient for convenience. Blood samples for the measurement of the plasma concentration of

KNS-760704 were collected before (pre-dose) and 15, 30, and 45 minutes and 1, 1.5, 2, 2.5, 3, 4, 6, and

8 hours post-dose.

Of the 102 patients who were randomized in Study KNS-760704-CL201, a total of 23 patients

participated in the PK sub-study at 8 of the 20 enrolling US study centers. Of those 23, 3 patients

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received placebo, 8 patients received 25 mg Q12H, 8 patients received 75 mg Q12H, and 4 patients

received 150 mg Q12H.

Pharmacokinetics were linear across this range of doses. Steady-state was achieved before study Day 10,

the earliest PK study day, consistent with the observed elimination half-life of 6.6 hours to 8.2 hours.

Estimates of clearance and volume of distribution were similar across dosage groups. Likewise, the time

to maximum concentrations (Tmax) was similar across treatment groups at 1.77, 1.82, and 1.70 hours

for the 25, 75, and 150 mg Q12H groups, respectively. The maximum concentration (Cmax) and

estimate of total exposure (AUC) increased proportionately with dose. Supplemental Table 9 shows

pharmacokinetic parameter estimates for healthy volunteers versus ALS subjects. The parameter

estimates for total plasma clearance uncorrected for bioavailability (CL/F), volume of distribution

uncorrected for bioavailability (Vz/F), and half-life (t½) are comparable between the two populations.

There was no evidence of drug accumulation in any dose group once steady state had been reached.


Supplementary Table 1. Schedule of Assessments for Part 1 of the Study.

Event Screening1 Baseline2,3

(pre-dose)

Day 1 (post-dose)

Week 1 Week 2 Week 4 Week 8 Week 12

(Part 2 Baseline) Early

Termination

Informed consent ×

Inclusion/Exclusion review × ×

Medical history ×

ALS Diagnosis History ×

Physical examination, including weight × × × ×

Neurological examination ×

Vital signs4 × ×5 ×6 × × × × × ×

ECG (12-lead) × ×5 ×6 × × × × × × Labwork (Serum Chemistries, CBC, Urinalysis) × × × × × × × ×

Serum Pregnancy Test (US female patients of child bearing potential)

× × × × × ×

Lithium screen × × × × ×

Clinical Status Assessments

McGill SIS × × × × × ×

ALSFRS-R × × × × × ×

Upright slow vital capacity (SVC) × × × × × ×

Supine slow vital capacity (SVC) × × ×

Cerebrospinal Fluid Collection × × × Plasma sample for protein biomarker analysis × × ×

Collection of concomitant Medication use × × ×

Collection of Adverse Events × × Calculate Drug Compliance and Dispense Study Medication ×8 × × ×

Study Drug Administration ×7 1. Screening procedures had to be completed within 21 days prior to Day 1. 2. Baseline procedures did not need to be repeated if the screening visit was performed within the baseline acceptance window (7 days prior to first dose for all procedures, except for the collection of CSF and plasma

protein samples which can be collected up to14 days prior to Day 1). 3. Baseline procedures could have been completed on the same day as study Day 1. 4. Vital signs will be measured after the patient has been rested for five minutes (systolic and diastolic systemic blood pressure in mmHg, respiratory rate/minute, heart rate/minute, and temperature). 5. Pre-dose vitals signs and ECG are collected on Day 1, prior to first dose of study drug. 6. Day 1, post-dose vitals signs and ECG collected approximately 2 hours post-dose (±20 minutes). 7. First dose of study drug will be taken after all Baseline procedures have been completed. 8. Dispense study medication only

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Placebo Washout 24-Week Double-Blind Active Treatment Period (Relative Week Number Shown)

Event Baseline 1

(Part 1 Week 12)

Week 4 (pre-dose)

Week 0 (post-dose)

Week 2

Week 4

Week 8

Week 12

Week 16

Week 20

Week 24

Early Termination

Physical examination, including weight × × × × × × ×

Vital signs2 × × ×3 × × × × × ×

ECG (12-lead) × × ×3 × × × × × × Labwork (Serum Chemistries, CBC, Urinalysis)

× × × × × × × ×

Serum Pregnancy Test (US female patients of child bearing potential) × × × × × × ×

Lithium screen × × × × × × ×

Clinical Status Assessments ×

McGill SIS × × × × ×4 × ×4 × ×

ALSFRS-R × × × × ×5 × ×5 × ×

Upright slow vital capacity (SVC) × × × × × × × Plasma sample for protein biomarker analysis

× ×6 ×

Collection of concomitant Medication use × × × ×

Collection of Adverse Events × × × × Calculate Drug Compliance and Dispense Study Medication

× × × × × ×7 ×7

Study Drug Administration × ×8 ×

Living Status Assessment9 ×

Supplementary Table 2. Schedule of Assessments for Part 2 of the Study. Note that for the 24-week Double-Blind Active Treatment (DBAT) Period the Relative Week Number is shown as a Column Label.

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1. Part 1, Week 12 assessments did not need to be repeated at Baseline Part 2. 2. Vital signs were measured after the patient had been rested for five minutes (systolic and diastolic systemic blood pressure in mmHg, respiratory rate/minute, heart rate/minute, and temperature). 3. Vital Signs and ECG were collected approximately 2 hours post-dose (±20 minutes). 4. McGill SIS at DBAT Week 12 and Week 20 were performed via phone contact by a certified study team member. 5. ALSFRS-R at DBAT Week 12 and Week 20 were performed via phone contact by a certified study team member. 6. Plasma for protein biomarker analysis collected at Week 28 only. 7. DBAT Week 24 and Early Termination (calculate drug compliance only). 8. Week 4 study drug taken after all Part 2 Week 4 pre-dose procedures have been completed. 9. Living Status Assessment to be collected via telephone every three months after patient completes the study (i.e., after DBAT Week 24 or early termination).


Supplementary Table 3. Regression tables for the linear mixed effects model – Part 1

Mixed Model Analysis for ALSFRS-R Total Score – Intent-to-Treat Population

Dimensions Covariance Parameters 4 Columns in X 10 Columns in Z Per Subject 2 Subjects 102 Max Obs Per Subject 4 Number of Observations Number of Observations Read 399 Number of Observations Used 399 Number of Observations Not Used 0 Iteration History Iteration Evaluations -2 Res Log Like Criterion 0 1 2585.63745668 1 2 1952.29932218 0.00008860 2 1 1952.24255833 0.00000035 3 1 1952.24234436 0.00000000 Convergence criteria met. Covariance Parameter Estimates Cov Parm Subject Estimate UN(1,1) USUBJID 27.6537 UN(2,1) USUBJID 2.0790 UN(2,2) USUBJID 1.5232 Residual 2.0582 Fit Statistics -2 Res Log Likelihood 1952.2 AIC (smaller is better) 1960.2 AICC (smaller is better) 1960.3 BIC (smaller is better) 1970.7 Null Model Likelihood Ratio Test DF Chi-Square Pr > ChiSq 3 633.40 <.0001 Solution for Fixed Effects Effect Group Part 1 Estimate Standard Error DF t Value Pr > |t| Intercept 37.2920 1.0389 98 35.89 <.0001

TRT1P 150 mg 1.7770 1.4833 196 1.20 0.2324

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TRT1P 300 mg 1.3110 1.4837 196 0.88 0.3780

TRT1P 50 mg -0.04397 1.5317 196 -0.03 0.9771

TRT1P Placebo 0 . . . .

time -1.2782 0.2740 97 -4.66 <.0001

time*TRT1P 150 mg 0.1134 0.3905 196 0.29 0.7718

time*TRT1P 300 mg 0.4007 0.3974 196 1.01 0.3146

time*TRT1P 50 mg -0.6064 0.4076 196 -1.49 0.1385

time*TRT1P Placebo 0 . . . .

TRT1P is the treatment group during Part 1

The Estimate for effect “TRT1P” is the difference from the placebo intercept; i.e. the intercept for the 150mg group is 37.2920 + 1.7770; the intercept for the placebo group is 37.2920 + 0

The estimate for the effect “Time” is the slope in the placebo group

“*” represents interaction terms

The Estimate for effect “time*TRT1P” is the difference from the placebo slope; i.e. the slope for the 150mg group is -1.2782 + 0.1134; the slope for the placebo group is -1.2782 + 0

Type 3 Tests of Fixed Effects Effect Num DF DEN DF F Value Pr > F

TRT1P 3 196 0.75 0.5259

time 1 97 83.02 <.0001

time*TRT1P 3 196 2.05 0.1087

P=0.5259 for TRT1P indicates that the treatment group intercepts are not different.

P<0.0001 indicates that the slope of the placebo group is different from zero.

P=0.1087 is a test of the global null hypothesis that the slopes are equal in all four treatment groups. Estimates Label Slope Estimate Standard Error DF t Value Pr > |t|

150 mg -1.1648 0.2782 196 -4.19 <.0001

300 mg -0.8776 0.2878 196 -3.05 0.0026

50 mg -1.8846 0.3018 196 -6.25 <.0001

Placebo -1.2782 0.2740 196 -4.66 <.0001

150 mg vs. Placebo 0.1134 0.3905 196 0.29 0.7718

300 mg vs. Placebo 0.4007 0.3974 196 1.01 0.3146

50 mg vs. Placebo -0.6064 0.4076 196 -1.49 0.1385

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Supplementary Table 4. Regression tables for the linear mixed effects model – Part 2

Mixed Model Analysis for ALSFRS-R Total Score – Intent-to-Treat Population

Dimensions Covariance Parameters 4 Columns in X 6 Columns in Z Per Subject 2 Subjects 90 Max Obs Per Subject 7

Number of Observations Number of Observations Read 560 Number of Observations Used 560 Number of Observations Not Used 0 Iteration History Iteration Evaluations -2 Res Log Like Criterion 0 1 3948.49909473 1 3 2813.20447215 0.01459059 2 1 2799.66427212 0.00524507 3 1 2794.08407526 0.00081041 4 1 2793.28666756 0.00003117 5 1 2793.25824122 0.00000007 6 1 2793.25817999 0.00000000 Convergence criteria met. Covariance Parameter Estimates Cov Parm Subject Estimate UN(1,1) SUBJID 60.7093 UN(2,1) SUBJID 1.7574 UN(2,2) SUBJID 0.6280 Residual 3.1803 Fit Statistics -2 Res Log Likelihood 2793.3 AIC (smaller is better) 2801.3 AICC (smaller is better) 2801.3 BIC (smaller is better) 2811.3 Null Model Likelihood Ratio Test DF Chi-Square Pr > ChiSq 3 1155.24 <.0001

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Solution for Fixed Effects Effect Group Part 2 Estimate Standard Error DF t Value Pr > |t|

Intercept 34.1648 1.1899 88 28.71 <.0001

TRT2PN 50 mg -0.3039 1.6646 380 -0.18 0.8552

TRT2PN 300 mg 0

TIME -1.0208 0.1357 88 -7.52 <.0001

TIME*TRT2PN 50 mg -0.2628 0.1944 380 -1.35 0.1772

TIME*TRT2PN 300 mg 0

TRT2PN is the treatment group during Part 2

The Estimate for effect “TRT2PN” is the difference from the 300mg group intercept; i.e. the intercept for the 50mg group is 34.1648 – 0.3039; the intercept for the 300mg group is 34.16480 + 0

Time is the time of the ALSFRS-R observation in months

The estimate for the effect “Time” is the slope in the 300mg group

* represents interaction terms

The Estimate for effect “time*TRT2PN” is the difference from the 300mg group slope; i.e. the slope for the 50mg group is -1.0208 – 0.2628; the slope for the 300mg group is -1.0208 + 0

Type 3 Tests of Fixed Effects Effect Num DF Den DF F Value Pr > F

TRT2PN 1 380 0.03 0.8552

TIME 1 88 140.56 <.0001

TIME*TRT2PN 1 380 1.83 0.1772

Estimates Label Slope Estimate Standard Error DF t Value Pr > |t|

50 mg -1.2836 0.1391 380 -9.22 <.0001

300 mg -1.0208 0.1357 380 -7.52 <.0001

Dif 0.2628 0.1944 380 1.35 0.1772

Label: Dif is difference in slopes: 300 mg treatment group minus 50 mg treatment group

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Supplementary Table 5. Summary of Deaths and Subjects Reporting Adverse Events in Parts 1 and 2 of the Study.

Part 1 Part 2*

Parameter PBO

(N=27) 50 mg (N=23)

150 mg (N=26)

300 mg (N=26)

50 mg (N=48)

300 mg (N=44)

Deaths: N (%) 0 0 0 0 9 (19%) 3 (7%)

Subjects with ≥1 AE: N(%)

25 (93%) 19 (83%) 25 (96%) 23 (88%) 46 (96%) 41 (93%)

AEs leading to D/C: N (%)

1 (4%) 0 0 1 (4%) 1 (2%) 1 (2%)

Serious AEs: N(%) 0 2 (9%) 0 3 (12%) 11 (23%) 5 (11%)

* 24-week double-blind active treatment period.

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Supplementary Table 6. Number of Subjects Reporting Common Treatment Emergent AEs (Reported in ≥ 10% of Subjects Receiving Active Study Drug) by Preferred Term in Decreasing Frequency (Part 1, Safety Population)

Active Treatment

Dexpramipexole (total daily dose)

Number of Events

Placebo (N=27)

Overall (N=75)

50 mg (N=23)

150 mg (N=26)

300 mg (N=26)

Subjects with ≥1 TEAE 25 (92.6%) 67 (89.3%) 19 (82.6%) 25 (96.2%) 23 (88.5%)

Fall 4 (14.8%) 24 (32.0%) 6 (26.1%) 11 (42.3%) 7 (26.9%)

Muscular Weakness 6 (22.2%) 18 (24.0%) 5 (21.7%) 7 (26.9%) 6 (23.1%)

Post-Lumbar Puncture Syndrome 7 (25.9%) 14 (18.7%) 4 (17.4%) 5 (19.2%) 5 (19.2%)

Headache 5 (18.5%) 10 (13.3%) 1 (4.3%) 3 (11.5%) 6 (23.1%)

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Supplementary Table 7. Number of Subjects Reporting Common Treatment-Emergent AEs (Reported in ≥ 10% of Subjects Receiving either Treatment) by Preferred Term in Decreasing Frequency (Part 2, Safety Population)

Preferred Term

50 mg/day (N=48)

300 mg/day (N=44)

Subjects with ≥1 treatment-emergent adverse event

46 (96%) 41 (93%)

Fall 11 (23%) 11 (25%)

Muscular weakness 13 (27%) 7 (16%)

Constipation 8 (17%) 11 (25%)

Salivary hypersecretion 8 (17%) 5 (11%)

Depression 6 (13%) 5 (11%)

Dyspnea 5 (10%) 6 (14%)

Dysarthria 4 (8%) 5 (11%)

Dysphagia 5 (10%) 4 (9%)

Headache 3 (6%) 6 (14%)

Dry mouth 1 (2%) 7 (16%)

Oedema peripheral 7 (15%) 1 (2%)

Upper respiratory tract infection 5 (10%) 3 (7%)

Anxiety 2 (4%) 5 (11%)

Sinusitis 2 (4%) 5 (11%)

Cough 1 (2%) 5 (11%)

Insomnia 0 6 (14%)

Muscle spasticity 5 (10%) 1 (2%)

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Supplementary Table 8. Subjects Experiencing Neutropenia in Study CL201.

Subject #003-003

Subject #002-105

Subject #004-096

Subject #007-063

Subject #010-101

Original onset of neutropenia

CL201 Part 1 Week 12




CL201 Part 2

Termination Visit

CL201 Part 1 treatment assignment

300 mg/day 300 mg/day 50 mg/day Placebo Placebo

CL201 Part 2 treatment assignment

300 mg/day 50 mg/day 300 mg/day 300 mg/day 300 mg/day

CL211 treatment assignment

NA 300 mg/day 300 mg/day 300 mg/day NA

Neutrophil count at CL201 baselinea 2.26 x103/uL 2.27 x103/uL 4.13 x103/uL 5.07 x103/uL b 2.70 x103/uL b

Neutrophil count at nadir

1.18 x103/uL 1.07 x103/uL 1.06 x103/uL 6c 1.43 x103/uL

NCI CTC Grade Grade 2 Grade 2 Grade 2 Grade 4 Grade 2

Concomitant riluzole Yes No Yes No No

Signs/symptoms abated on drug withdrawal

Yes Yes Yes Yes Yes

Recurrence on re-challenge

Yes No No No NA

Disposition Discontinued in

CL201 Part 2

Active in CL211

Active in CL211

ALS related death during

CL211

Completed Part 2, chose not to enter CL211

Outcome of neutropenia

Reversible on withdrawal of

study drug

Reversible on interruption of

study drug


study drug


study drug

Reversible on discontinuation of study drug

a Part 1 Baseline unless noted b Part 2 Week 4 (Part 2 active treatment baseline) c Reported as 1% of WBC 600 µL

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Supplementary Table 9. Comparison of Selected Pharmacokinetic Parameter Estimates for Healthy Volunteers (HV) and ALS Subjects

Study KNS-760704-CL001a KNS-760704-CL002b KNS-760704-

CL201 Population HV HV HV ALS Doses (mg/day) 50, 150, 300 50, 100, 150 25, 75, 150 Regimen Single dose Single dose Q12H Q12H

Parameterc CL/F (mL/min) 492→527 571→665 437→510 365→427 Vz/F (L) 258→311 307→373 255→349 224→256 t½ (h) 6.40→6.96 6.36→6.69 6.87→8.05 6.63→8.23

cKNS-760704-CL001 was an ascending single-dose study in healthy volunteers. cKNS-760704-CL002 was an ascending single- and multiple-dose study in healthy volunteers. cValues reported are the range of the mean values across the doses


Supplementary Figure 1:

Figure Legend: Each panel represents the temporal progression of the ALSFRS-R total score for every individual subject in the indicated dose group enrolled in Part 1 of study CL-201.

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Supplemental Figure 2:

Figure Legend: Each panel represents the temporal progression of the ALSFRS-R total score for every individual subject in the indicated dose group enrolled in Part 2 of study CL-201.


SUPPLEMENTARY MATERIAL LITERATURE REFERENCES

(1) Brooks BR. El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial "Clinical limits of amyotrophic lateral sclerosis" workshop contributors. J Neurol Sci 1994;124 Suppl:96-107.

(2) Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 2000;1:293-299.

(3) Bensimon G, Lacomblez L, Meininger V. A controlled trial of riluzole in amyotrophic lateral sclerosis. ALS/Riluzole Study Group. N Engl J Med 1994;330:585-591.

(4) Miller RG, Bouchard JP, Duquette P et al. Clinical trials of riluzole in patients with ALS. ALS/Riluzole Study Group-II. Neurology 1996;47:S86-S90.

(5) Gordon PH, Moore DH, Miller RG et al. Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a phase III randomised trial. Lancet Neurol 2007;6:1045-1053.

(6) Miller R, Bradley W, Cudkowicz M et al. Phase II/III randomized trial of TCH346 in patients with ALS. Neurology 2007;69:776-784.

(7) Bozik ME, Mather JM, Kramer WH, Gribkoff VK, Ingersoll EW. Safety, tolerability, and pharmacokinetics of KNS-760704 (dexpramipexole) in healthy adult subjects. J Clin Pharmacol. In press.

(8) Traynor BJ, Zhang H, Shefner JM, Schoenfeld D, Cudkowicz ME. Functional outcome measures as clinical trial endpoints in ALS. Neurology 2004;63:1933-1935.

(9) Gordon PH, Cheng B, Salachas F et al. Progression in ALS is not linear but is curvilinear. J Neurol 2010.

(10) Aggarwal SP, Zinman L, Simpson E et al. Safety and efficacy of lithium in combination with riluzole for treatment of amyotrophic lateral sclerosis: a randomised, double-blind, placebo-controlled trial. Lancet Neurol 2010;9:481-488.

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