Clinical trial NCT02373098
Conditions: Relapsing Remitting Multiple Sclerosis
Keywords: Fingolimod,cytokine,chemokine,RRMS
Sponsor: Novartis Pharmaceuticals
Start date: 2015-03-31; Completion date: 2017-01-18
Enrollment: 126 participants; Allocation: NON_RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
The main purpose of this study was to measure the serum levels of cytokines and chemokines that are known to increase during multiple sclerosis relapses.

Cytokines are a broad and loose category of small proteins that are important in cell signaling.

The second purpose of the study was to test the cytokine/chemokine changes measured in the 3rd and 6th months on the efficacy parameters.

Detailed description:

Arms: arm 1: None arm 2: Healthy volunteers with no intervention or drug administered.
Interventions: intervention 1: Fingolimod 0.5 mg
Intervention descriptions: intervention 1: 66 relapsing remitting MS (RRMS) patients were recruited. Patients who met all inclusion and none of the exclusion criteria were treated by Fingolimod 0.5 mg.

Protocol documents:
Prot_000.pdf: 


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Table of contents 
Table of contents ................................................................................................................. 2 
List of tables ........................................................................................................................ 4 
Glossary of terms ................................................................................................................. 6 
Protocol synopsis ................................................................................................................. 7 
1 
Introduction ....................................................................................................................... 11 
1.1 
Background ............................................................................................................ 11 
1.2 
Purpose .................................................................................................................. 12 
2 
Study objectives ................................................................................................................. 12 
2.1 
Primary and key secondary objectives .................................................................. 12 
2.2 
Secondary objectives ............................................................................................. 12 
 
 13 
3 
Investigational plan ........................................................................................................... 13 
3.1 
Study design........................................................................................................... 13 
3.2 
Rationale of study design ....................................................................................... 14 
3.3 
Rationale of dose/regimen, route of administration and duration of treatment ..... 15 
3.4 
Rationale for choice of comparator ....................................................................... 15 
3.5 
Purpose and timing of interim analyses/design adaptations .................................. 15 
3.6 
Risks and benefits .................................................................................................. 15 
4 
Population .......................................................................................................................... 15 
4.1 
Inclusion criteria .................................................................................................... 15 
4.2 
Exclusion criteria ................................................................................................... 16 
5 
Treatment ........................................................................................................................... 17 
5.1 
Protocol requested treatment ................................................................................. 17 
5.1.1 
Investigational treatment ....................................................................... 17 
5.1.2 
Additional study treatment .................................................................... 17 
5.2 
Treatment arms ...................................................................................................... 17 
5.3 
Treatment assignment, randomization ................................................................... 17 
5.4 
Treating the patient ................................................................................................ 17 
5.4.1 
Patient numbering ................................................................................. 17 
5.4.2 
Dispensing the investigational treatment .............................................. 17 
5.4.3 
Handling of study treatment .................................................................. 18 
5.4.4 
Administering study treatment .............................................................. 18 
5.4.5 
Permitted dose adjustments and interruptions of study treatment ........ 18 
5.4.6 
Rescue medication ................................................................................ 19 
5.4.7 
Concomitant treatment .......................................................................... 20 
5.4.8 
Prohibited Treatment ............................................................................. 20 


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5.4.9 
Discontinuation of study treatment and premature patient 
withdrawal ............................................................................................. 21 
5.4.10 
Study completion and post-study treatment .......................................... 22 
5.4.11 
Early study termination. ........................................................................ 22 
6 
Visit schedule and assessments ......................................................................................... 22 
6.1 
Information to be collected on screening failures.................................................. 24 
6.2 
Patient demographics/other baseline characteristics ............................................. 24 
6.3 
Treatment exposure and compliance ..................................................................... 24 
6.4 
Efficacy .................................................................................................................. 24 
6.4.1 
Efficacy assessment .............................................................................. 25 
6.4.2 
Appropriateness of efficacy assessments .............................................. 25 
6.5 
Safety ..................................................................................................................... 25 
6.5.1 
Laboratory evaluations .......................................................................... 25 
6.5.2 
Electrocardiogram (ECG) ..................................................................... 26 
6.5.3 
Optical Cohorence Tomography (OCT) ............................................... 26 
6.5.4 
Pregnancy and assessments of fertility ................................................. 27 
6.5.5 
Appropriateness of safety measurements .............................................. 27 
6.6 
Other assessments .................................................................................................. 27 
6.6.1 
Resource utilization ............................................................................... 27 
6.6.2 
Health-related Quality of Life ............................................................... 27 
6.6.3 
Pharmacokinetics .................................................................................. 27 
6.6.4 
Pharmacogenetics/pharmacogenomics ................................................. 27 
6.6.5 
Other biomarkers ................................................................................... 27 
7 
Safety monitoring .............................................................................................................. 27 
7.1.1 
Serious Adverse Events ......................................................................... 27 
7.1.2 
SAE Reporting to Ministry of Health and Ethical Committee ............. 29 
7.1.3 
Pregnancies ........................................................................................... 29 
7.1.4 
Contact information for Novartis Drug Safety department: ................. 29 
8 
Data review and database management ............................................................................. 29 
8.1 
Site monitoring ...................................................................................................... 29 
8.2 
Data collection ....................................................................................................... 30 
8.3 
Database management and quality control ............................................................ 30 
8.4 
Data Monitoring Committee .................................................................................. 31 
8.5 
Adjudication Committee ........................................................................................ 31 
9 
Data analysis ...................................................................................................................... 31 
9.1 
Analysis sets .......................................................................................................... 31 
9.2 
Patient demographics and other baseline characteristics ....................................... 31 


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9.3 
Treatments (study drug, rescue medication, other concomitant therapies, 
compliance) ............................................................................................................ 31 
9.4 
Analysis of the primary variable(s) ....................................................................... 31 
9.5 
Statistical model, hypothesis, and method of analysis .......................................... 31 
9.6 
Supportive analyses ............................................................................................... 31 
9.7 
Safety variables . .................................................................................................... 31 
9.8 
Sample size calculation .......................................................................................... 31 
9.9 
Interim analyses ..................................................................................................... 32 
10 Ethical considerations ........................................................................................................ 33 
10.1 
Regulatory and ethical compliance ........................................................................ 33 
10.2 
Informed consent procedures ................................................................................. 33 
10.3 
Responsibilities of the investigator and IRB/IEC .................................................. 33 
10.4 
Publication of study protocol and results............................................................... 33 
11 Protocol adherence ............................................................................................................ 34 
11.1 
Protocol Amendments ........................................................................................... 34 
12 References ......................................................................................................................... 34 
13 Appendix 1: 2005 Revisions to the McDonald diagnosis criteria for MS Guidelines 
from International Panel on the diagnosis of MS .............................................................. 36 
14 Appendix 2: Instructions for the Paced Auditory Serial Addition Test (PASAT) ............ 37 
15 Appendix 3: EDSS Assessment Criteria ........................................................................... 37 
 
List of tables 
Table 6-1 
Assessment schedule for MS Patients ................................................... 23 
Table 6-2 
Assessment schedule for Healthy Control Group ................................. 23 
 List of figures 
  Figures  1 
Study protocol (PASAT: Paced auditory serial addition task, 
SDMT: single digit modalities test, FACS: Flow assisted cell 
sorting, flow cytometry) ........................................................................ 14 


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List of abbreviations 
AE 
adverse event 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
b.i.d. 
twice a day 
CRF 
Case Report/Record Form (paper or electronic) 
CPO 
Country Pharma Organization 
CRO 
Contract Research Organization 
DS&E 
Drug Safety & Epidemiology 
ECG 
Electrocardiogram 
EDC 
Electronic Data Capture 
ICH 
International Conference on Harmonization of Technical Requirements for 
Registration of Pharmaceuticals for Human Use 
IEC 
Independent Ethics Committee 
i.v. 
intravenous 
IRB 
Institutional Review Board 
o.d. 
once a day 
p.o. 
oral 
SAE 
serious adverse event 
 
 


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Glossary of terms 
 
Assessment 
A procedure used to generate data required by the study 
Control drug 
Drugs(s) used as a comparator to reduce assessment bias, preserve 
blinding of investigational drug, assess internal study validity, and/or 
evaluate comparative effects of the investigational drug 
Enrollment 
Point/time of patient entry into the study; the point at which informed 
consent must be obtained (i.e. prior to starting any of the procedures 
described in the protocol) 
Epoch  
The planned stage of the subjects’ participation in the study. Each epoch 
serves a purpose in the study as a whole. Typical epochs are: 
determination of subject eligibility, wash-out of previous treatments, 
exposure of subject to treatment or to follow-up on subjects after treatment 
has ended. 
Investigational drug 
The drug whose properties are being tested in the study; this definition is 
consistent with US CFR 21 Section 312.3 and is synonymous with 
“investigational new drug” or “investigational medicinal product.” 
Investigational treatment 
All investigational drug(s) whose properties are being tested in the study 
as well as their associated treatment controls.  
This includes any placebos, any active controls, as well as approved drugs 
used outside of their indication/approved dosage or tested in a fixed 
combination.  
Investigational treatment generally does not include other treatments 
administered as concomitant background therapy required or allowed by 
the protocol when used within approved indication/dosage 
Medication number 
A unique identifier on the label of each investigational/study drug package 
in studies that dispense medication using an IRT system 
Subject Number  
A number assigned to each patient who enrolls into the study  
Part  
A subdivision of a single protocol into major design components. These 
parts often are independent of each other and have different populations or 
objectives. For example, a single dose design, a multiple dose design that 
are combined into one protocol, or the same design with different patient 
populations in each part. 
Period 
A subdivision of a cross-over study 
Premature patient withdrawal 
Point/time when the patient exits from the study prior to the planned 
completion of all investigational/study treatment administration and all 
assessments (including follow-up) 
Randomization number 
A unique identifier assigned to each randomized patient, corresponding to 
a specific treatment arm assignment 
Stop study participation 
Point/time at which the patient came in for a final evaluation visit or when 
study/investigational treatment was discontinued whichever is later 
Study drug/ treatment 
Any single drug or combination of drugs administered to the patient as part 
of the required study procedures; includes investigational drug (s), active 
drug run-ins or background therapy  
Study/investigational treatment 
discontinuation 
Point/time when patient permanently stops taking study/investigational 
treatment  for any reason; may or may not also be the point/time of 
premature patient withdrawal 
Variable 
Information used in the data analysis; derived directly or indirectly from 
data collected using specified assessments at specified time points 
 


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Protocol No. CFTY720DTR04 
Protocol synopsis 
  
Protocol 
number 
CFTY720DTR04 
Title 
Effects of Fingolimod (Gilenya®) on Cytokine and Chemokine Levels in 
Relapsing Remitting Multiple Sclerosis Patients 
Brief title 
 
Effects of Fingolimod (Gilenya®) on Cytokine and Chemokine Levels in 
Relapsing Remitting Multiple Sclerosis Patients 
Sponsor and 
Clinical Phase 
Novartis  
Phase IV 
Investigation 
type 
Drug 
Study type 
Interventional 
Purpose and 
rationale 
The main purpose of this study is to measure the serum levels of major Th1, Th2, 
Th17 and Treg cytokines as well as cytokines and chemokines that are known to 
be upregulated during multiple sclerosis relapses. Measurements will be 
performed at 3 time points; i.e. before treatment, 12±1 weeks and 24±2 weeks 
after fingolimod treatment. By this way, we will be able to test whether 
fingolimod treatment shifts the cytokine balance from Th1/Th17 to Th2/Treg and 
reduces the levels of inflammation related cytokines and chemokines. The basal 
levels of the cytokines will be compared with the cytokine levels of healthy 
controls. 
The second purpose of the study is to test the predictive value of 
cytokine/chemokine changes measured in the 3rd and 6th months on the EDSS, 
PASAT, SDMT, 9-hole peg test, timed 25-foot walk test and relapse rate 
observed after 6 months of treatment. 
This study design is not randomized or double blind, but it has no bias since the 
patients themselves will be their own controls if they fulfill the enrollment 
criteria.  
This study was designed since there is accumulating evidence that Th1 and Th17-
type immunities play major roles in multiple sclerosis pathogenesis in the last 
decade. By contrast, Th2 and Treg cytokines appear to counterbalance the 
production of Th1 and Th17 cytokines and thus play an ameliorating role in the 
natural course of multiple sclerosis. Not surprisingly, many multiple sclerosis 
medications exert their beneficial effects through modulation of these cytokines. 
The effects of fingolimod on cytokine production are largely unknown. So this 
study was designed to see to evaluate the effects of fingolimod on cytokine 
production. 
Primary 
Objective(s) 
and Key 
Secondary 
Objective 
Primary objective of this study is: 
-To evaluate the differences between serum cytokine and chemokine levels of 
healthy controls and multiple sclerosis patients. 


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Protocol No. CFTY720DTR04 
Secondary 
Objectives 
 
Secondary objective of this study is:  
-To test the predictive value of cytokine/chemokine changes measured in the 3rd 
and 6th months on the PASAT, SDMT, 9-Hole peg test, timed 25-foot walk, 
EDSS and relapse rate observed after 6 months of treatment. 
Study design 
This study is a multi-center, open label  study.  
Population 
80 relapsing remitting MS (RRMS) patients will be recruited according to the Mc 
Donald's criteria and 60 healthy controls will be recruited as the control group. 
All subjects will sign an informed consent that will be approved by the local 
Ethics Committee and the applicable health authorities prior to venipuncture. 
Basal EDSS, relapses during the last 2 years, paced auditory serial addition task 
(PASAT), 9-Hole peg test, timed 25-foot walk and single digit modalities test 
(SDMT) scores will be recorded before study entry. Serum and blood samples 
will be collected from each participant after evaluation for inclusion and 
exclusion criteria. Additional serum and blood samples at 2 different time points 
will be collected from MS patients at months 3 (±1 week) and 6 (±2 weeks) after 
starting fingolimod 0.5 mg/day po. treatment. 
Inclusion 
criteria 
1. Written informed consent must be obtained before any assessment is 
performed. 
2. RRMS patients aged between 18-65 years that fulfill following criteria: 
a) 
In non-treated/newly-diagnosed patients with two or more serious attacks 
within one year or three or more serious attacks within two years and one or 
multiple gadolinium-enhancing lesions in cranial MRI or with significantly 
increased T2 lesions compared to the previous MRI. 
b) 
In non-responders despite treatment with a beta-interferon or glatiramer 
acetate with adequate duration and dose, where: 
a. adequate treatment duration is at least 1 year and, 
b. non-responders are patients with no changes in attacks or increased 
number of attacks after one year of treatment, or with more severe attacks, or 
those with one or multiple contrast enhancing lesions in cranial MRI or where 
there is an increase in T2 lesions identified with successive MRI’s. 
c) 
In RRMS patients who are unable to tolerate first-line treatments due to 
adverse effects associated with parenteral administration. 
3. 
Last relapse of the patient should be at least 2 months before study entry. 
4. 
Last steroid dose of the patient should be at least 1 month before study 
entry. 
5. 
Last interferon or glatiramer acetate dose of the patient should be at least 1 
month before study entry. 


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6. 
Last cyclophosphamide, mitoxantrone, teriflunamide, natalizumab and  
azathioprine dose of the patient should be at least 3 months before study 
entry. 
Inclusion Criteria for Healthy Control  Group 
1. Healthy subjects  who don’t have any autoimmune disease  and aged 
>18. 
2. Written informed consent must be obtained before any assessment is 
performed. 
Exclusion 
criteria 
Patients fulfilling any of the following criteria are not eligible for inclusion in 
this study. No additional exclusions may be applied by the investigator, in order 
to ensure that the study population will be representative of all eligible patients. 
1. Patients with secondary progressive MS. 
2. Patients with known contraindications for fingolimod treatment. 
3. Other coexistent autoimmune diseases including Hashimoto thyroiditis, 
systemic lupus erythematosus, rheumatoid arthritis, psoriasis etc.  
4. Patients with any of the following cardiovascular conditions:  
• 
Resting heart rate < 45 bpm/min  
• 
Cardiac failure at any time during the first study visit (Class 
III as per NYHA classification) or significant heart disease 
as judged by the physician  
• 
Myocardial infarction during the last 6 months  
• 
History of Mobitz Type II grade 2 AV block  
• 
Past or current grade 3 AV block  
• 
Confirmed history of sick sinus syndrome or sino-atrial heart 
block  
• 
arrhythmia requiring current treatment with Class Ia drugs 
(disopyramid, procainamide, quinidine)  
• 
hypertension uncontrolled with medication 
5. History of malignancy of any organ system other than localized basal cell 
carcinoma of the skin. 
6. Pregnant or nursing (lactating) women 
7. Negative for varicella-zoster virus IgG antibodies at screening 
8. History of previous fingolimod therapy 
9.  Presence of any clinically active infections 
 
Investigational 
and reference 
therapy 
Fingolimod 


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Efficacy 
assessments 
EDSS, PASAT, SDMT, ,9-hole peg test, timed 25-foot walk and relapse rate  
Safety 
assessments 
This study does not include any planned safety analysis 
Other 
assessments 
Not Applicable 
Data analysis 
Data analysis will be performed after all of the data is collected from the enrolled 
patients. All MS patients and control group will be accepted as the analysis sets. 
Cytokine and chemokine levels will be analyzed cumulatively at the end of the 
study. The results will be recorded to the blood sampling logs and laboratory logs 
cumulatively.  
Key words 
RRMS, Fingolimod, Cytokine, Chemokine 
 


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1 
Introduction 
1.1 
Background  
Multiple sclerosis (MS) is the leading cause of neurological disability in young adults and the 
most common demyelinating disorder of the central nervous system. It is widely regarded as a 
chronic autoimmune disorder encompassing both inflammatory (myelin destruction) and 
degenerative (axonal loss) features. Disease onset is thought to be mediated by T lymphocytes 
that recognize myelin antigens, cross the blood brain barrier and drive the immune response 
against oligodendrocytes and myelin sheaths. MS typically presents in relapsing forms, either 
with a relapsing-remitting (RRMS) or secondary progressive (SPMS) course. Patients suffer 
acute self-limiting attacks (relapses) of neurological dysfunction followed by complete or 
incomplete 
remission. 
Between 
relapses, 
patients 
are 
either 
neurologically 
and 
symptomatically stable (RRMS) or continue to deteriorate in function unrelated to relapses 
(SPMS). Currently available therapies for patients with RRMS (interferon β and glatiramer 
acetate) have partial efficacy (about 30-40% reduction in relapse rate and at best a modest 
effect on disability progression). These agents are biologic parenteral treatments requiring 
injections associated with side effects (injection site reactions, flu-like symptoms). Therefore, 
there is a strong medical need for a safe and effective oral treatment of MS. 
Fingolimod is a novel, orally active, synthetic small molecule in clinical development for MS. 
Fingolimod is rapidly phosphorylated in vivo, and fingolimod-phosphate (fingolimod-P) acts 
as agonist of G protein-coupled receptors for sphingosine-1 phosphate (S1P). More 
particularly, fingolimod-P acts as ‘super agonist’ of the S1P1 receptor on thymocytes and 
lymphocytes, inducing internalization of that receptor. This renders these cells unresponsive 
to S1P1 signaling, thus depriving them of a signal necessary for egress from lymph nodes and 
secondary lymphoid tissues. The downstream result of this fingolimod induced interdiction of 
S1P1 signaling is a marked reduction in the number of both B and T lymphocytes in the 
intravascular compartment and a decrease in recirculation of these cells to extravascular 
compartments, including the CNS. In animal immunization models, fingolimod neither 
impaired T-cell activation, expansion, and memory, nor antibody production by B-cells. 
However, fingolimod will reduce the ability of an expanded number of activated effector 
lymphocytes to return to a site of inflammation or infection. 
These effects on lymphocytes are believed to be linked to the efficacy of FTY720 in reducing 
inflammatory activity as seen in animal models of MS as well as in patients with relapsing 
MS. However, these effects may also lead to potential immunosuppression and an increased 
risk for infections. This risk may be further enhanced with the concomitant use of other 
inmmunosuppressants including corticosteroids. 
Fingolimod has been shown to be highly active in a number of animal models for 
autoimmunity. In particular, fingolimod was highly effective both in a prophylactic and a 
therapeutic setting in several mouse and rat models of experimental autoimmune 
encephalomyelitis (EAE), an animal model of MS. 
The clinical experience with fingolimod is already extensive. To date, over 12000 subjects 
(healthy volunteers, transplant patients and MS patients) have been exposed to single or 
multiple doses of fingolimod. Based on this experience, pharmacodynamic effects ascribed to 


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Protocol No. CFTY720DTR04 
fingolimod are: 1) a rapid and persistent reduction of the peripheral lymphocyte count that is 
reversible after treatment discontinuation, 2) a predictable reduction in heart rate that is 
maximal upon treatment initiation and attenuates over time under continued treatment, 3) a 
mild- to moderate increase in airway resistance early after treatment initiation. The molecular 
basis of these effects is well understood and compatible with the known mode of action of 
fingolimod via engagement of S1P receptors. 
An increased incidence of macular edema has been observed with fingolimod (in combination 
with cyclosporine A) in the renal transplantation studies. Regular ophthalmic monitoring in 
the ongoing clinical development program in MS patients has revealed cases of macular 
edema both prior to and after study drug initiation. The risk of macular edema in multiple 
sclerosis studies is currently under evaluation in the ongoing program, in which all patients 
are undergoing regular ophthalmology evaluations. 
There is accumulating evidence that Th1 and Th17-type immunities play major roles in 
multiple sclerosis pathogenesis in the last decade. By contrast, Th2 and Treg cytokines appear 
to counterbalance the production of Th1 and Th17 cytokines and thus play an ameliorating 
role in the natural course of multiple sclerosis. Not surprisingly, many multiple sclerosis 
medications exert their beneficial effects through modulation of these cytokines. The effects 
of fingolimod on cytokine production are largely unknown. 
1.2 
Purpose  
The main purpose of this study is to measure the serum levels of major Th1, Th2, Th17 and 
Treg cytokines as well as cytokines and chemokines that are known to be upregulated during 
multiple sclerosis relapses. Measurements will be performed at 3 time points; i.e. before 
treatment, 12±1 weeks and 24±2 weeks after fingolimod treatment. By this way, we will be 
able to test whether fingolimod treatment shifts the cytokine balance from Th1/Th17 to 
Th2/Treg and reduces the levels of inflammation related cytokines and chemokines. The basal 
levels of the cytokines will be compared with the cytokine levels of healthy controls. 
The second purpose of the study is to test the predictive value of cytokine/chemokine changes 
measured in the 3rd and 6th months on the EDSS, PASAT, SDMT, 9-hole peg test,timed 25-
foot walk test and relapse rate observed after 6 months of treatment. 
2 
Study objectives 
2.1 
Primary and key secondary objectives 
Primary objective of this study is: 
-To evaluate the differences between serum cytokine and chemokine levels of healthy controls 
and multiple sclerosis patients 
2.2 
Secondary objectives 
Secondary objective of this study is:  
-To test the predictive value of cytokine/chemokine changes measured in the 3rd and 6th 
months on the PASAT, SDMT, 9-Hole peg test, timed 25-foot walk, EDSS and relapse rate 
observed after 6 months of treatment. 




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Figure 1: Study protocol (PASAT: Paced auditory serial addition task, SDMT: single digit 
modalities test, FACS: Flow assisted cell sorting, flow cytometry) 
 
 
 
3.2 
Rationale of study design 
This study design is not randomized or double blind, but it has no bias since the patients 
themselves will be their own controls if they fulfill the enrollment criteria.  
This study was designed since there is accumulating evidence that Th1 and Th17-type 
immunities play major roles in multiple sclerosis pathogenesis in the last decade. By contrast, 
Th2 and Treg cytokines appear to counterbalance the production of Th1 and Th17 cytokines 
and thus play an ameliorating role in the natural course of multiple sclerosis. Not surprisingly, 
many multiple sclerosis medications exert their beneficial effects through modulation of these 
cytokines. The effects of fingolimod on cytokine production are largely unknown. So this 
study was designed to see to evaluate the effects of fingolimod on cytokine production. 
 


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3.3 
Rationale of dose/regimen, route of administration and duration 
of treatment 
The dose, dose regimen, titration scheme and tapering-off scheme, etc. are in accordance with 
product labeling.  
3.4 
Rationale for choice of comparator 
A comparator arm is not applicable for this study 
3.5 
Purpose and timing of interim analyses/design adaptations 
Not applicable 
3.6 
Risks and benefits  
The risk to subjects in this trial will be minimized by compliance with the inclusion/exclusion 
criteria, close clinical monitoring, and minimal study duration which will be planned as 1,5 
years enrollment time and 6 months follow up. 
4 
Population 
The study will include only 80 relapsing remitting MS patients and 60 healthy controls 
without any known autoimmune disease. RRMS patients will be followed for 6 months. 
4.1 
Inclusion criteria 
1. Written informed consent must be obtained before any assessment is performed. 
2. RRMS patients aged between 18-65 years that fulfill following criteria: 
d) In non-treated/newly-diagnosed patients with two or more serious attacks within one 
year or three or more serious attacks within two years and one or multiple gadolinium-
enhancing lesions in cranial MRI or with significantly increased T2 lesions compared to the 
previous MRI. 
e) In non-responders despite treatment with a beta-interferon or glatiramer acetate with 
adequate duration and dose, where: 
a. adequate treatment duration is at least 1 year and, 
b. non-responders are patients with no changes in attacks or increased number of 
attacks after one year of treatment, or with more severe attacks, or those with one or 
multiple contrast enhancing lesions in cranial MRI or where there is an increase in T2 
lesions identified with successive MRI’s. 
f) 
In RRMS patients who are unable to tolerate first-line treatments due to adverse 
effects associated with parenteral administration. 
3. 
Last relapse of the patient should be at least 2 months before study entry. 
4. 
Last steroid dose  of the patient should be at least 1 month before study entry. 


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5. 
Last interferon or glatiramer acetate dose of the patient should be at least 1 month 
before study entry. 
6. 
Last cyclophosphamide, mitoxantrone, teriflunamide, natalizumab and  azathioprine 
dose of the patient should be at least 3 months before study entry. 
 
Inclusion Criteria for Healthy Control  Group 
3. Healthy people who don’t have MS and aged >18. 
4. Written informed consent must be obtained before any assessment is performed. 
4.2 
Exclusion criteria 
Patients fulfilling any of the following criteria are not eligible for inclusion in this study. No 
additional exclusions may be applied by the investigator, in order to ensure that the study 
population will be representative of all eligible patients. 
1.Patients with secondary progressive MS. 
2.Patients with known contraindications for fingolimod treatment. 
3.Other coexistent autoimmune diseases including Hashimoto thyroiditis, systemic lupus 
erythematosus, rheumatoid arthritis, psoriasis etc.  
4.Patients with any of the following cardiovascular conditions:  
 
• Resting heart rate < 45 bpm/min  
• Cardiac failure at any time during the first study visit (Class III as per 
NYHA classification) or significant heart disease as judged by the 
physician  
• Myocardial infarction during the last 6 months  
• History of Mobitz Type II grade 2 AV block  
• Past or current grade 3 AV block  
• Confirmed history of sick sinus syndrome or sino-atrial heart block  
• arrhythmia requiring current treatment with Class Ia drugs (ajmaline, 
disopyramid, procainamide, quinidine)  
• hypertension uncontrolled with medication 
5.History of malignancy of any organ system other than localized basal cell carcinoma of 
the skin 
6.Pregnant or nursing (lactating) women7.Negative for varicella-zoster virus IgG 
antibodies at screening 
8.History of previous fingolimod therapy 


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9.Presence of any clinically active infections 
 
5 
Treatment 
5.1 
Protocol requested treatment 
5.1.1 
Investigational treatment 
Treatment will be 0.5 mg p.o fingolimod daily for the treatment arm. The study drug will be 
provided by Novartis . 
Control group will not receive any treatment. 
5.1.2 
Additional study treatment 
No additional treatment beyond investigational treatment is requested for this trial. 
5.2 
Treatment arms 
Not Applicable.  
5.3 
Treatment assignment, randomization  
Not Applicable.  
5.4 
Treating the patient 
5.4.1 
Patient numbering  
Each patient is uniquely identified by a Subject Number which is composed by the site 
number assigned by Novartis and a sequential number assigned by the investigator. Once 
assigned to a patient, the Subject Number will not be reused.  
Upon signing the informed consent form, the patient is assigned the next sequential number as 
given by the investigator using the next blank CRF book.  
5.4.2 
Dispensing the investigational treatment 
Each study site will be supplied by Novartis with investigational treatment in packaging of 
identical appearance. 
The investigational treatment packaging has a 2-part label. A unique medication number is 
printed on each part of this label which corresponds to the treatment. Investigator staff will 
select the investigational treatment to dispense to the patient using the visit number on the 
label. Immediately before dispensing investigational treatment to the patient, investigator staff 
will detach the outer part of the label from the packaging and affix it to the source document 
(Drug Label Form) for that patient’s unique patient number.  


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5.4.3 
Handling of study treatment 
5.4.3.1 
Handling of investigational treatment 
Investigational treatment must be received by a designated person at the study site, handled 
and stored safely and properly, and kept in a secured location to which only the investigator 
and designees have access. Upon receipt, all investigational treatment should be stored 
according to the instructions specified on the labels. Clinical supplies are to be dispensed only 
in accordance with the protocol. 
Medication labels will be in the local language and comply with the legal requirements of 
each country. They will include storage conditions for the investigational treatment but no 
information about the patient except for the visit  number. 
The investigator must maintain an accurate record of the shipment and dispensing of 
investigational treatment in a drug accountability log. Monitoring of drug accountability will 
be performed by the field monitor during site visits and at the completion of the trial. Patients 
will be asked to return all unused investigational treatment and packaging at the end of the 
study or at the time of discontinuation of investigational treatment.  
At the end of the study, and as appropriate during the course of the study, the investigator will 
return all unused investigational treatment, packaging, drug labels, and a copy of the 
completed drug accountability log to the Novartis monitor or to the Novartis address provided 
in the investigator folder at each site. 
Handling of other study treatment 
Not applicable. 
5.4.4 
Administering study treatment 
Prior to administration of the first dose of the study drug the investigator should reconfirm a 
list of concomitant medications taken by the patient. It is recommended not to initiate 
treatment with beta-blockers, calcium-channel blockers or digoxin within one week before or 
after the first dose of the study drug due to a possible additive effect on heart rate reduction. 
The first dose of the study drug must be administered under the supervision of the First Dose 
Administrator or designee. It is recommended that the first dose be administered in the 
morning time, before 12:00 noon. The patient will stay at the study center for a minimum of 6 
hours. Heart rate and blood pressure will be monitored hourly during the six hour stay and 
discharged if specific discharge criteria are met. Hourly monitoring will be extended if the 
discharge criteria are not met. Some patients may be required to return to the study center for 
6 hours following the 2nd dose. 
The investigator and/or study personnel should promote compliance by instructing the patient 
to take  one capsule of the study drug orally once daily with or without a meal, preferably at 
the same time each day and by stating that compliance is necessary for the patient’s safety and 
validity of the study. The patient should be instructed to contact the site if he/she is unable for 
any reason to take the study drug . 
5.4.5 
Permitted dose adjustments and interruptions of study treatment 
Dose adjustments will not be allowed, however drug interruptions will be allowed based on 


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the judgment of the Investigator. 
Conditions/events that may lead to the study drug interruptions based on investigator 
judgment and overall clinical assessment: 
• reported serious adverse event;  
• emergency medical condition, unplanned hospitalization, involving use of 
excluded concomitant medications;  
• abnormal laboratory value(s) or abnormal test or examination result(s) (e.g. PFT, liver 
function tests, ophthalmic findings etc.).  
• patient’s non-compliance.  
Should the patient interrupt the study drug, and should the investigator decide in agreement 
with the sponsor to re-initiate treatment with the study drug, depending on the duration of the 
interruption (see below), the first dose at re-start may need to take place under supervision of 
the First Dose Administrator in a similar manner as the first intake of the study medication. It 
is recommended not to initiate beta-blockers, calcium-channel blockers or digoxin treatment 
within one week before or after the day of re-initiation of the study drug administration due to 
a possible additive effect on heart rate reduction. 
When re-starting of study drug monitoring (as for first dose) is mandatory in the following 
cases: 
• The treatment lasted for less than 8 days and was interrupted for more than 1 day  
• The treatment lasted for 8 days or more and was interrupted for 8 days or more  
Re-start decision should be made on a case by case basis and should be discussed with the 
Medical Advisor at Novartis. A reason for the interruption of treatment and time of 
interruption should be appropriately documented in the source documents as well as in the 
Dosage Administration Record CRF. All required guidelines for the above maters are given as 
an appendix to this protocol 
 
5.4.6 
Rescue medication 
A standard course of corticosteroids (methylprednisolone) on an inpatient or outpatient basis 
is allowed for treatment of relapses as clinically warranted. 
Steroid treatment should consist of 3-5 days and up to 1,000 mg methylprednisolone/day. 
Standard of care procedures will be followed during treatment. 


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Use of any oral tapering is not permitted. 
The use of steroid therapy should be recorded in the related section of CRF. 
Investigators should consider the added immunosuppressive effects of corticosteroid therapy 
and increase vigilance regarding infections during such treatment and in the weeks following 
administration. 
Patients should be reminded of the importance of reporting any signs or symptoms of an 
infection during treatment. Special consideration should be given to symptoms or signs of 
herpes simplex or zoster reactivation (e.g. lancinating pain, skin lesions) and appropriate 
antiviral therapy (e.g acyclovir, valcyclovir) should be promptly initiated and continued for up 
to 30 days after stopping high dose steroid treatment. An infectious disease specialist may be 
consulted to guide such therapy if needed. 
Should a patient develop any neurological symptoms or signs, unexpected for MS in the 
opinion of the investigator or accelerated neurological deterioration, the investigator should 
immediately schedule an MRI and follow the “Guidance on monitoring of patients with 
symptoms or signs of neurological deterioration inconsistent with MS”. Steroids should not be 
taken prior to conducting the unscheduled MRI. 
If the patient is currently under treatment with corticosteroids, the blood sample should be 
obtained at least 1 month after the end of the corticosteroid treatment. 
5.4.7 
Concomitant treatment 
The investigator should instruct the patient to notify the study site about any new medications 
he/she takes after the patient was enrolled into the study.  
5.4.8 
Prohibited Treatment 
Use of the following treatments is NOT allowed concomitantly with the study drug during the 
course of the study: (use can only be considered if the study drug is permanently 
discontinued): 
- 
Immunosuppressive medication (e.g. cyclosporine, azathioprine, methotrexate, 
cyclophosphamide, mitoxantrone, cladribine);  
- 
Other concomitant medications: immunoglobulins, monoclonal antibodies 
(including natalizumab), INF- ß, glatiramer acetate, adrenocorticotropic hormone (ACTH). 
The administration of any live or live attenuated vaccines (including for measles) is 
prohibited while patients are receiving study drug and for 3 months after study drug 
discontinuation. They may be administered, thereafter, once there is confirmation that 
lymphocyte counts are in the laboratory normal range. Use of the following treatments is 
allowed to manage potential adverse reactions associated with the study drug:  
i. 
anticholinergics (atropine s.c. or i.v.) for treatment of symptomatic 
bradycardia as the first line treatment, up to 3 mg/day;  
ii. 
beta-agonists/sympathomimetics (dopamine drip 5-20μg/kg/min or 
epinephrine drip 2- 10 μg/min) for treatment of non-responsive bradycardia.  
Standard short course of corticosteroids (methylprednisolone i.v.) is allowed for 
treatment of relapses.  


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The medications allowed for treatment of adverse reactions and relapses are not considered 
study supplies, and therefore, need to be supplied by the study site. The investigator should 
instruct the patient to notify the study site about any new medications he/she takes after the 
start of the study drug.  
5.4.9 
Discontinuation of study treatment and premature patient withdrawal 
Patients may voluntarily withdraw from the study for any reason at any time. They may be 
considered withdrawn if they state an intention to withdraw, fail to return for visits, or become 
lost to follow-up for any other reason. 
If premature withdrawal occurs for any reason, the investigator must make every effort to 
determine the primary reason for a patient’s premature withdrawal from the study and record 
this information on the Study Completion CRF  
The investigator should discontinue study treatment for a given patient or withdraw the 
patient from study if, on balance, he/she believes that continuation would be detrimental to the 
patient’s well-being. 
Study drug must be discontinued for a given patient if the investigator determines that 
continuing it would result in a significant risk for that patient. The following conditions/events 
may be considered sufficient to support a decision about the study drug discontinuation in 
individual cases: 
• serious adverse event (e.g. cardiac failure, diagnosed malignancy) 
• abnormal laboratory value(s) including liver function tests or abnormal test result(s) 
(e.g. PFTs, MRI and ophthalmic findings). See below and Appendix 7 and 8 for 
guidance for safety monitoring  
• withdrawal of consent (eg., in case the patient does not consent to continue in the 
study after a confirmed relapse or disability progression)  
• pregnancy  
• use of prohibited medications, listed in Section 5.1 and 6.6.5  
• adverse events  
• protocol violation  
• unsatisfactory therapeutic effect  
• patient’s condition no longer requires study treatment  
• administrative problems (e.g. patient’s non-compliance)  
In addition, the following conditions based on the Guidance on safety and ophthalmic 
monitoring should result in study drug discontinuation:  
- Hepatic Increase in ALT > 5 x ULN Increase in AST > 5 x ULN  
- New neurological symptoms accompanied by MRI findings unexpected for 
MS  
- Diagnosis of macular edema  


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- Decrease in visual acuity with abnormal OCT (increase in central foveal 
thickness of > 20% compared to screening or cystic changes in the macula). 
 
 
Patients who discontinue study drug should not be considered withdrawn from the study, 
unless one of the conditions listed above have been met. The sponsor should be notified about 
any decision regarding discontinuation of the study medication. Patients who discontinue the 
study drug should be treated according to the best standard of care. In addition to scheduled 
visits, patients who discontinue study drug due to adverse events or abnormalities on safety 
monitoring tests must be followed up with additional visits as needed in order to confirm the 
resolution of abnormalities. A Study Drug Discontinuation CRF should be completed, giving 
the date and primary reason for stopping the study drug. 
For patients who are lost to follow-up (i.e. those patients whose status is unclear because they 
fail to appear for study visits without stating an intention to withdraw), the investigator should 
show "due diligence" by documenting in the source documents steps taken to contact the 
patient, e.g. dates of telephone calls, registered letters, etc. 
Patients who are prematurely withdrawn from the study will be replaced by an equal number 
of newly enrolled patients. 
5.4.10 
Study completion and post-study treatment 
The investigator must provide follow-up medical care for all patients who are prematurely 
withdrawn from the study, or must refer them for appropriate ongoing care.  
5.4.11 
Early study termination. 
The study can be terminated at any time for any reason by Novartis. Should this be necessary, 
the patient should be seen as soon as possible and treated for a prematurely withdrawn patient. 
The investigator may be informed of additional procedures to be followed in order to ensure 
that adequate consideration is given to the protection of the patient’s interests. The 
investigator will be responsible for informing IRBs and/or ECs of the early termination of the 
trial. 
 
 
6 
Visit schedule and assessments 
Table 6-1 and Table 6-2 lists all of the assessments and indicates with an “x” when the visits 
are performed. 
 
 
 
 


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Table 6-1 
Assessment schedule for MS Patients 
Visit 
1 
2 
3 
Day 
0  
90±7 
180±14 
Obtain Informed Consent 
X 
 
 
Evaluation of Inclusion/Exclusion Criteria 
X 
 
 
Demgraphic and Previous Data 
X 
 
 
Other Concomitant Diseases 
X 
 
 
MS and Treatment History 
X 
 
 
Assessment of the MS Relapses Within Last 2 Years 
X 
 
 
Recording of Relapses 
 
 
X 
Pregnancy Test (Urine) 
X 
 
 
Varicella-Zoster Ab 
X 
 
 
Hematology * 
 X** 
X 
X 
Clinical Chemistry * 
 X** 
X 
X 
 
 
 
 
ECG 
X 
 
 
Optical Cohorence Tomography 
X  
X 
 
EDSS Measurements 
X 
 
X 
PASAT Measurements 
X 
 
X 
Symbol Digit Modalities Test (SDMT) Measurements 
X 
 
X 
9-Hole Peg Test  
X 
 
X 
25 Foot Walk Test 
X 
 
X 
Collecting Serum and Blood Samples for Biomarker Research 
X 
X 
X 
Dispense Study Medication 
X 
X 
 
First Dose Administration 
X 
 
 
End of the Study Visit  
 
 
X 
 
* 
* Hematology: Leukocytes, Hemoglobin, Absolute Neutrophilis, Absolut, Lymphocyte, Platelet  
Clinical Chemistry: AST, ALT, BUN, Creatinine, Total Cholesterol, Tryglicerides, LDL-K, HDL-K, TSH,ST4, 
Glucose, HbA1c  
** If the patient has hematology and clinical chemistry results within the last 1month, the tests do not 
have to be repeated.  
Table 6-2 
Assessment schedule for Healthy Control Group 
Visit 
1 
2 
3 
Day 
0  
90±7 
180±14 
Obtain İnformed Consent 
X 
 
 
Evaluation of Inclusion/Exclusion Criteria 
X 
 
 
Collecting Serum and Blood Samples  
X 
 
 
 


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Patients should be seen for all visits on the designated day with an allowed “visit window” of 
“7” days at visit 2 and “14” days at visit 3 , or as close to it as possible, or as shortly after it as 
possible, or as shortly before it as possible, etc. 
At a minimum, patients will be contacted for safety evaluations during the 30 days following 
the last study visit or following the last administration of study treatment if there are post-
treatment follow-up visits (whichever is later), including a final contact at the 30-day point. 
Documentation of attempts to contact the patient should be recorded in the source 
documentation.  
6.1 
Information to be collected on screening failures 
Since the study is aiming to collect serum samples from the MS patients and healthy 
volunteers, screening visit is not applicable. The investigators will assess the 
inclusion/exclusion criteria in visit 1 and the patients who are not eligible for the study will 
not give any serum samples. There is no information to be collected for screen failures. 
6.2 
Patient demographics/other baseline characteristics 
Data of patient demographics and baseline characteristics will be collected from all patients 
that include: date of birth and sex. Relevant medical history/current medical condition data 
includes data until the start of study drug. If there is, concomitant dieases will be recorded.  In 
addition, MS history and MS treatment will be recorded. 
At Visit 1, an ECG will be performed to ensure that the patients continue to meet inclusion 
criteria prior to taking the first dose of fingolimod. After 6 hours of the first dose, the ECG 
will be repeated. Should any of the cardiac inclusion criteria is met based on the ECG results, 
the patient will not be allowed to participate in the study. ECGs will be printed out and will be 
kept at the study site as a source document. 
6.3 
Treatment exposure and compliance 
In order to collect accurate information about the study drug exposure, the following records 
should be maintained for each  patient: records of study medication dispensed and returned, 
dosages administered and intervals between visits.  
Compliance will be assessed by the investigator and/or study personnel at each visit using 
capsule counts and information provided by the patient. A monitor will perform and document 
drug accountability during site visits and at the end of the study. 
Patients who discontinue study medication for 1 month totally will be considered withdrawn. 
6.4 
Efficacy 
General definition of relapse: Appearance of a new neurological abnormality or worsening of 
previously stable or improving pre-existing neurological abnormality, separated by at least 30 
days from onset of a preceding clinical demyelinating event. The abnormality must be present 
for at least 24 hours and occur in the absence of fever (<37.5C) or infection. 
 


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Severity of MS relapse 
Mild relapse 
Moderate relapse 
Severe relapse 
EDSS increase of 0.5 point 
EDSS increase of 1 or 2 
points 
Exceeding Moderate criteria 
Or 
or 
or 
1 point FS change in one to 
three systems 
2-point FS change in one or 
two systems 
Exceeding Moderate criteria 
 
or 
or 
 
1-point change in four or 
more systems 
Exceeding Moderate criteria 
Reference: Panitch, H (2002) 
 
Expanded Disability Status Scale (EDSS) 
EDSS is a scale for assessing neurologic impairment in MS (Kurtzke 1983) including (1) a 
series of scores in each of eight functional systems, and (2) the EDSS steps (ranging from 0 
(normal) to 10 (death due to MS). The functional systems are Visual, Brain Stem, Pyramidal, 
Cerebellar, Sensory, Bowel & Bladder, Cerebral and Other functions. It is recommended that 
fatigue is not included in the Cerebral score of the EDSS. 
EDSS will be assessed by the investigator  at each site, who has successfully completed 
his/her certification requirements. EDSS assessments are scheduled at baseline and at the end 
of the 6 monthly treatments on Visit 3. 
6.4.1 
Efficacy assessment  
MS Relapses and EDSS is the key efficacy assessment criteria. Efficacy assessment will be 
performed at the end of the study.  
6.4.2 
Appropriateness of efficacy assessments  
Measurements are the standard key efficacy criteria for MS. 
6.5 
Safety 
This study does not include any planned safety analysis. 
6.5.1 
Laboratory evaluations 
Blood samples will be collected from each patient and healthy control after evaluation for 
inclusion and exclusion criteria.  Additional blood samples at 2 different time points will be 
collected from MS patients at months 3 (±1 week) and 6 (±2 weeks) after starting fingolimod 
0.5 mg/day po. treatment. Serum samples will be sent to the central laboratory (DETAE) for  
flow cytometry  assay on the same day they are collected. Serum samples will be stored at -
20oC at local centers for a longest duration of two week and kept frozen at -80oC 
 


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 until assayed. The 
levels of IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, IL-23, IFN-γ, TNF-α, VLA4, CCL2, 
CCL5, CXCR3, CXCL13 will be measured by ELISA according to the manufacturer’s 
protocols. Optical density (OD) will be measured at 450 nm and cytokine/chemokine 
concentrations (pg/ml) will be calculated by referring to a standard curve.  
 
In order to perform the flow cytometry assay freshly purified peripheral blood mononuclear 
cells (PBMC) from venous blood will be stimulated for 24 h by a combination of phorbol 
ester (PMA, 50 ng/ml) and Ca2+ Ionophore (Ionomycin, 250 ng/ml). The combination of 
these two stimuli will be used to achieve the strongest stimulus for intracytoplasmic cytokine 
secretion. Monensin will be added at a final concentration of 1 mM. After stimulation, the 
cells will be washed with PBS and will be stained with anti-CD4, anti-CD8, anti-CD25, and 
then fixed and permeabilized. After washing with PBS, the cells will be stained with PE-
conjugated anti-IL-4, anti-IL-6, anti-IL-9, anti-IL-10, anti-IL-13, anti-IL-17, anti-IFN-γ, anti-
TNF-α for 30 min at room temperature and analyzed by flow cytometry. 
Cytokine and chemokine levels will be analyzed cumulatively at the end of the study. The 
results will be recorded to the blood sampling logs and laboratory logs cumulatively. 
6.5.1.1 
Hematology 
Hematology parameters will be collected at screening and each scheduled visit and will 
include:  WBC count, absolute lymphocytes, platelet, absolute neutrophil count,and 
hemoglobin. 
6.5.1.2 
Clinical chemistry 
Blood samples will be collected at screening (Visit 1) and at each scheduled visit  to verify a 
patient’s eligibility and will include but not limited to the following parameters:  
AST, ALT, BUN, Creatinine, Total Cholesterol, Tryglicerides, LDL-K, HDL-K, TSH,ST4, 
Glucose, HbA1c  
 
6.5.2 
Electrocardiogram (ECG) 
A standard 12 lead ECG will be performed at baseline. The ECG will be performed before the 
intake of the first dose of the study drug and after 6 hours of the intake of the study drug. 
Interpretation of the tracing must be made by a qualified physician and documented on the 
ECG section of the CRF. Each ECG tracing should be labeled with the study and subject 
number, date, and kept in the source documents at the study site. Clinically significant 
abnormalities should also be recorded on the Medical History/Adverse event CRF page. 
Clinically significant findings must be discussed with the Novartis Medical Monitor prior to 
enrolling the patient in the study. 
6.5.3 
Optical Cohorence Tomography (OCT) 
An optical coherence tomography for the eyes will be performed in the baseline and in Visit 
2. The tracing of the OCT must be made by a qualified physician and documented on the OCT 
section of the CRF. Each OCT tracing should be labeled with the study and subject number, 
date, and kept in the source documents at the study site. Clinically significant abnormalities 


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should also be recorded on the Medical History/Adverse event CRF page. Clinically 
significant findings must be discussed with the Novartis Medical Monitor prior to enrolling 
the patient in the study. 
 
6.5.4 
Pregnancy and assessments of fertility 
All pre-menopausal women who are not surgically sterile will have a urine pregnancy test. A 
positive urine pregnancy test requires immediate interruption of study drug until serum β-hCG 
is performed and found to be negative. If positive, the patient must be discontinued from the 
trial. 
6.5.5 
Appropriateness of safety measurements 
The safety assessments selected are standard for this indication/patient population. 
6.6 
Other assessments 
For clinical measures; EDSS and relapses will be recorded after 6 months of treatment. EDSS 
scoring will also be performed at study entry. PASAT, 9-Hole Peg test, timed 25-foot walk 
and SDMT tests will be performed only at study entry and termination after 6 months. 
6.6.1 
Resource utilization 
Not Applicable 
6.6.2 
Health-related Quality of Life 
Not Applicable 
6.6.3 
Pharmacokinetics 
Not Applicable 
6.6.4 
Pharmacogenetics/pharmacogenomics 
Not Applicable 
6.6.5 
Other biomarkers 
Not Applicable 
7 
Safety monitoring  
7.1.1 
Serious Adverse Events  
A Serious Adverse Event (SAE)  is defined as an event which: 
•  is fatal or life-threatening 
•  results in persistent or significant disability/incapacity 
•  constitutes a congenital anomaly/birth defect 
•  requires inpatient hospitalization or prolongation of existing hospitalization, unless 
hospitalization is for: 


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• routine treatment or monitoring of the studied indication, not associated with any 
deterioration in condition 
•  elective or pre-planned treatment for a pre-existing condition that is unrelated to the 
indication under study and has not worsened since the start of study drug 
•  treatment on an emergency outpatient basis for an event not fulfilling any of the 
definitions of a SAE given above and not resulting in hospital admission 
• social reasons and respite care in the absence of any deterioration in the patient’s general 
condition 
• is medically significant, i.e., defined as an event that jeopardizes the patient or may 
require medical or surgical intervention to prevent one of the outcomes listed above 
 
Unlike routine safety assessments, SAEs are monitored continuously and have special 
reporting requirements; see Section 
 
To ensure patient safety, every SAE, regardless of suspected causality, occurring after the 
patient has provided informed consent and until 30 days after the patient has stopped study 
participation must be reported to Novartis within 24 hours of learning of its occurrence.  
Any SAEs experienced after this 4-week period should only be reported to Novartis if the 
investigator suspects a causal relationship to the study drug.   
Recurrent episodes, complications, or progression of the initial SAE must be reported as 
follow-up to the original episode, regardless of when the event occurs. This report must be 
submitted within 24 hours of the investigator receiving the follow-up information. An SAE 
that is considered completely unrelated to a previously reported one should be reported 
separately as a new event. 
Information about all SAEs is collected and recorded on the Serious Adverse Event Report 
Form.  The investigator must assess the relationship to study drug, complete the SAE Report 
Form in English, and send the completed, signed form by fax within 24 hours to the local 
Novartis Novartis Drug Safety and Epidemiology Department.  The telephone and telefax 
number of the contact persons in the local department of Novartis Integrated Medical Safety 
are listed in the investigator folder provided to each site and is also listed below. The original 
copy of the SAE Report Form and the fax confirmation sheet must be kept with the case 
report form documentation at the study site. 
Follow-up information is sent to the same person to whom the original SAE Report Form was 
sent, using a new SAE Report Form stating that this is a follow-up to the previously reported 
SAE and giving the date of the original report. The follow-up information should describe 
whether the event has resolved or continues, if and how it was treated, and whether the patient 
continued or withdrew from study participation.  
If the SAE is not previously documented in the Investigator’s Brochure or Package Insert 
(new occurrence) and is thought to be related to the Novartis study drug, a Novartis Drug 
Safety and Epidemiology Department associate may urgently require further information from 
the investigator for Health Authority reporting. Novartis may need to issue an Investigator 


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Notification (IN) to inform all investigators involved in any study with the same drug that this 
SAE has been reported.  
7.1.2 
SAE Reporting to Ministry of Health and Ethical Committee 
Local Novartis Turkey Drug Safety Department will report all SAEs to Ministry of Health 
within 7 days of receipt by Novartis. SAEs are also reported to relevant Ethics Committee 
simultaneously in the same frame. 
Suspected Unexpected Serious Adverse Events will be collected and reported to Ministry of 
Health and to relevant Ethics Committee every 6 months as a line listing. 
Annual Safety Report will be submitted to Ministry of Health and to relevant Ethics 
Committee on an annual basis. 
 
7.1.3 
Pregnancies 
To ensure patient safety, each pregnancy in a patient on study drug must be reported to 
Novartis within 24 hours of learning of its occurrence. The pregnancy should be followed up 
to determine outcome, including spontaneous or voluntary termination, details of the birth, 
and the presence or absence of any birth defects, congenital abnormalities, or maternal and/or 
newborn complications.  
Pregnancy should be recorded on a Clinical Trial Pregnancy Form and reported by the 
investigator to the local Novartis Drug Safety and Epidemiology Department. Pregnancy 
follow-up should be recorded on the same form and should include an assessment of the 
possible relationship to the Novartis study drug of any pregnancy outcome. Any SAE 
experienced during pregnancy must be reported on the SAE Report Form. 
7.1.4 
Contact information for Novartis Drug Safety department: 
 
 
 
Tel: 
 
Novartis Drug Safety Fax: 
 
 
Novartis Drug Safety Mailbox: 
 
8 
Data review and database management 
8.1 
Site monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, a Novartis 
representative will review the protocol and CRFs with the investigators and their staff. During 
the study, the field monitor will visit the site regularly to check the completeness of patient 
records, the accuracy of entries on the CRFs, the adherence to the protocol and to Good 
Clinical Practice, the progress of enrollment, and to ensure that study treatment is being 


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stored, dispensed, and accounted for according to specifications. Key study personnel must be 
available to assist the field monitor during these visits. 
The investigator must maintain source documents for each patient in the study, consisting of 
case and visit notes (hospital or clinic medical records) containing demographic and medical 
information, laboratory data, electrocardiograms, and the results of any other tests or 
assessments. All information on CRFs must be traceable to these source documents in the 
patient's file. [Data not requiring a separate written record will be defined before study start 
and will be recorded directly on the CRFs.] The investigator must also keep the original 
informed consent form signed by the patient (a signed copy is given to the patient). 
The investigator must give the monitor access to all relevant source documents to confirm 
their consistency with the CRF entries. Novartis monitoring standards require full verification 
for the presence of informed consent, adherence to the inclusion/exclusion criteria, 
documentation of SAEs, and of data that will be used for all primary variables. Additional 
checks of the consistency of the source data with the CRFs are performed according to the 
study-specific monitoring plan. No information in source documents about the identity of the 
patients will be disclosed. 
8.2 
Data collection 
Designated investigator staff must enter the information required by the protocol onto the 
Novartis CRFs that are printed on 3-part, non-carbon-required paper. Field monitors will 
review the CRFs for completeness and accuracy and instruct site personnel to make any 
required corrections or additions. The CRFs are forwarded to the Medical Documents 
Reception Center of Novartis [or CRO working on behalf of Novartis] by field monitors or by 
the investigational site, with one copy being retained at the investigational site. Once the 
CRFs are received by Novartis [or CRO working on behalf of Novartis], their receipt is 
recorded, the original copy is placed in Central Files, and the non-carbon-required copy is 
forwarded to the responsible Data Management staff for processing.   
8.3 
Database management and quality control 
Data from the CRFs are entered into the study database by Novartis Data Management staff 
using single data entry. Verification is performed manually by a separate member of the Data 
Management staff by comparing the Case Report Form to the data entered into the database. 
Data from the CRFs are entered into the study database by Contract Research Organization 
(CRO) staff following their own internal standard operating procedures that have been 
reviewed and approved by Novartis. 
Subsequently, the entered data are systematically checked by Data Management staff, using 
error messages printed from validation programs and database listings. Other errors or 
omissions are entered on Data Query Forms, which are returned to the investigational site for 
resolution. The signed original and resolved Data Query Forms are kept with the CRFs at the 
investigator site, and a copy is sent to Novartis so the resolutions can be entered into the 
database. Quality control audits of all key safety and efficacy data in the database are made 
prior to locking the database. 
Adverse events will be coded using the Medical dictionary for regulatory activities 
(MedDRA) terminology. 


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Laboratory samples will be processed centrally and the results will be sent electronically to 
Novartis (or a designated CRO). 
8.4 
Data Monitoring Committee 
Not required. 
8.5 
Adjudication Committee 
Not required. 
 
9 
Data analysis  
9.1 
Analysis sets  
Data analysis will be performed after all of the data is collected from the enrolled patients. All 
MS patients and control group will be accepted as the analysis sets.  
9.2 
Patient demographics and other baseline characteristics   
Data collected for patient demographics and the baseline characteristics will be summarized 
by descriptive statistics with summary tables. 
9.3 
Treatments (study drug, rescue medication, other concomitant 
therapies, compliance)  
Data collected for treatments will be summarized by descriptive statistics with summary 
tables. 
9.4 
Analysis of the primary variable(s) 
Serum IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, IL-23, IFN-γ, TNF-α, VLA4, CCL2, 
CCL5, CXCR3, CXCL13 levels, PBMC intracellular IL-4, IL-6, IL-9, IL-10, IL-13, IL-17, 
IFN-γ, TNF-α levels, EDSS, PASAT, 9-Hole peg test, timed 25-foot walk, SDMT scores and 
relapse rates will be analyzed. 
9.5 
Statistical model, hypothesis, and method of analysis 
There is no special hypothesis planned for this study. 
9.6 
Supportive analyses  
Not Applicable 
9.7 
Safety variables . 
Adverse and serious advers events and pregnancies will be summarized.  
9.8 
Sample size calculation 
 


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Due to the lack of previous studies using all of the cytokines and chemokines and 
multitudinous analyses, it is difficult to estimate a precise sample size for our study. Despite 
this, our previous publications indicate that the basal IL-17 concentration is normally 
distributed with a mean of 32 pg/ml (±stdev of 15 pg/ml) in RRMS patients. If the true 
difference in the mean concentration of matched pairs is 7 pg/ml, we will need to study 50 
pairs of subjects to be able to reject the null hypothesis with a power of 90%. The Type I error 
probability associated with this test of this null hypothesis is 0,05.  
With assumption of drop out rate of 20% minimum 60 RRMS patients and 60 healthy control  
subjects should enroll to the study. Due to expected high rate of screen failure patients, we 
decided to screen 80 RRMS patients in order to reach 60 eligible RRMS subjects for the 
study. 
 
9.9 
Interim analyses  
Not Applicable 


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10 
Ethical considerations 
10.1 
Regulatory and ethical compliance 
This clinical study was designed and shall be implemented and reported in accordance with 
the ICH Harmonized Tripartite Guidelines for Good Clinical Practice, with applicable local 
regulations (including European Directive 2001/20/EC, US Code of Federal Regulations Title 
21, and Japanese Ministry of Health, Labor, and Welfare), and with the ethical principles laid 
down in the Declaration of Helsinki. 
10.2 
Informed consent procedures 
Eligible patients may only be included in the study after providing written (witnessed, where 
required by law or regulation), IRB/IEC-approved informed consent, or, if incapable of doing 
so, after such consent has been provided by a legally acceptable representative of the patient. 
In cases where the patient’s representative gives consent, the patient should be informed about 
the study to the extent possible given his/her understanding. If the patient is capable of doing 
so, he/she should indicate assent by personally signing and dating the written informed 
consent document or a separate assent form. Informed consent must be obtained before 
conducting any study-specific procedures (i.e. all of the procedures described in the protocol). 
The process of obtaining informed consent should be documented in the patient source 
documents.  
Novartis will provide to investigators in a separate document a proposed informed consent 
form that complies with the ICH GCP guideline and regulatory requirements and is 
considered appropriate for this study. Any changes to the proposed consent form suggested by 
the investigator must be agreed to by Novartis before submission to the IRB/IEC, and a copy 
of the approved version must be provided to the Novartis monitor after IRB/IEC approval. 
10.3 
Responsibilities of the investigator and IRB/IEC 
The protocol and the proposed informed consent form must be reviewed and approved by a 
properly constituted Institutional Review Board/Independent Ethics Committee/Research 
Ethics Board (IRB/IEC) before study start. A signed and dated statement that the protocol and 
informed consent have been approved by the IRB/IEC must be given to Novartis before study 
initiation. Prior to study start, the investigator is required to sign a protocol signature page 
confirming his/her agreement to conduct the study in accordance with these documents and all 
of the instructions and procedures found in this protocol and to give access to all relevant data 
and records to Novartis monitors, auditors, Novartis Clinical Quality Assurance 
representatives, designated agents of Novartis, IRBs/IECs, and regulatory authorities as 
required. If an inspection of the clinical site is requested by a regulatory authority, the 
investigator must inform Novartis immediately that this request has been made. 
10.4 
Publication of study protocol and results 
Novartis assures that the key design elements of this protocol will be posted in a publicly 
accessible database such as clinicaltrials.gov. In addition, upon study completion and 


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finalization of the study report the results of this trial will be either submitted for publication 
and/or posted in a publicly accessible database of clinical trial results. 
11 
Protocol adherence 
Investigators ascertain they will apply due diligence to avoid protocol deviations. Under no 
circumstances should the investigator contact Novartis or its agents, if any, monitoring the 
trial to request approval of a protocol deviation, as requests to approve deviations will not be 
granted. 
This protocol defines the study objectives, the study procedures and the data to be collected 
on study participants.  Under no circumstances should an investigator collect additional data 
or conduct any additional procedures for any research related purpose involving any 
investigational drugs.  
If the investigator feels a protocol deviation would improve the conduct of the study this must 
be considered a protocol amendment, and unless such an amendment is agreed upon by 
Novartis and approved by the IRB/IEC it cannot be implemented. All significant protocol 
deviations will be recorded and reported in the CSR. 
11.1 
Protocol Amendments 
Any change or addition to the protocol can only be made in a written protocol amendment that 
must be approved by Novartis, Health Authorities where required, and the IRB/IEC. Only 
amendments that are required for patient safety may be implemented prior to IRB/IEC 
approval. Notwithstanding the need for approval of formal protocol amendments, the 
investigator is expected to take any immediate action required for the safety of any patient 
included in this study, even if this action represents a deviation from the protocol. In such 
cases, Novartis should be notified of this action and the IRB/IEC at the study site should be 
informed within 10 working days or less, if required by local regulations.  
12 
References 
Kürtüncü M, Tüzün E, Türkoğlu R, Petek-Balcı B, Içöz S, Pehlivan M, Birişik Ö, Ulusoy C, 
Shugaiv E, Akman-Demir G, Eraksoy M.Effect of short-term interferon-β treatment on 
cytokines in multiple sclerosis: significant modulation of IL-17 and IL-23. Cytokine. 2012 
Aug;59(2):400-2. doi: 10.1016/j.cyto.2012.05.004. Epub 2012 May 30. 
Serpero LD, Filaci G, Parodi A, Battaglia F, Kalli F, Brogi D, Mancardi GL, Uccelli A, 
Fenoglio D. Fingolimod Modulates Peripheral Effector and Regulatory T Cells in MS 
Patients. J Neuroimmune Pharmacol. 2013 May 7. [Epub ahead of print]. 
Commodaro AG, Pedregosa JF, Peron JP, Brandão W, Rizzo LV, Bueno V. The imbalance 
between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection. Clinics 
(Sao Paulo). 2012 Jul;67(7):805-13. 
Mehling M, Lindberg R, Raulf F, Kuhle J, Hess C, Kappos L, Brinkmann V. Th17 central 
memory T cells are reduced by FTY720 in patients with multiple sclerosis. Neurology. 2010 
Aug 3;75(5):403-10. 


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Protocol No. CFTY720DTR04 
Liao JJ, Huang MC, Goetzl EJ. Cutting edge: Alternative signaling of Th17 cell development 
by sphingosine 1-phosphate. J Immunol. 2007 May 1;178(9):5425-8. 
Johnson TA, Lapierre Y, Bar-Or A, Antel JP. Distinct properties of circulating CD8+ T cells 
in FTY720-treated patients with multiple sclerosis. Arch Neurol. 2010 Dec;67(12):1449-55. 
Yopp AC, Fu S, Honig SM, Randolph GJ, Ding Y, Krieger NR, Bromberg JS. FTY720-
enhanced T cell homing is dependent on CCR2, CCR5, CCR7, and CXCR4: evidence for 
distinct chemokine compartments. J Immunol. 2004 Jul 15;173(2):855-65. 
Cinamon G, Matloubian M, Lesneski MJ, Xu Y, Low C, Lu T, Proia RL, Cyster JG. 
Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone. 
Nat Immunol. 2004 Jul;5(7):713-20. 
Ledgerwood LG, Lal G, Zhang N, Garin A, Esses SJ, Ginhoux F, Merad M, Peche H, Lira 
SA, Ding Y, Yang Y, He X, Schuchman EH, Allende ML, Ochando JC, Bromberg JS. The 
sphingosine 1-phosphate receptor 1 causes tissue retention by inhibiting the entry of 
peripheral tissue T lymphocytes into afferent lymphatics. Nat Immunol. 2008 Jan;9(1):42-53. 
Zhang ZY, Zhang Z, Schluesener HJ. FTY720 attenuates lesional interleukin-17(+) cell 
accumulation in rat experimental autoimmune neuritis. Neuropathol Appl Neurobiol. 2009 
Oct;35(5):487-95. 
Sharma N, Akhade AS, Qadri A. Sphingosine-1-phosphate suppresses TLR-induced CXCL8 
secretion from human T cells. J Leukoc Biol. 2013 Apr;93(4):521-8. 
 
 
 
 
 
 
 
 
 
 
 
 
 


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13 
Appendix 1: 2005 Revisions to the McDonald diagnosis 
criteria for MS Guidelines from International Panel on the 
diagnosis of MS 
(McDonald 2001,Polman 2005,) 
Clinical Presentation 
Additional Data Needed for MS Diagnosis 
Two or more attacks; objective 
clinical evidence of 2 or more lesions 
Nonea 
Two or more attacks; objective 
clinical evidence of 1 lesion 
Dissemination in space, demonstrated by: MRIb 
or 
Two or more MRI-detected lesions consistent with MS 
plus positive CSFc 
or 
Await further clinical attack implicating a different site 
One attack; objective clinical 
evidence of 2 or more lesions 
Dissemination in time, demonstrated by: MRId 
or 
Second clinical attack 
One attack; objective clinical 
evidence of 1 lesion 
(monosymptomatic presentation; 
clinically isolated syndrome) 
Dissemination in space, demonstrated by: MRIb 
or 
Two or more MRI-detected lesions consistent with MS 
plus positive CSFc 
and 
Dissemination in time, demonstrated by: MRId 
or 
Second clinical attack 
 
If the criteria indicated are fulfilled, the diagnosis is multiple sclerosis (MS) 


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aNot additional tests are required; however, if tests [magnetic resonance imaging (MRI), 
cerebral spinal fluid (CSF)] are undertaken and are negative, extreme caution should be taken 
before making a diagnosis of MS. Alternative diagnoses must be considered. There must be 
not better explanation for the clinical picture. 
bMRI demonstrated of space dissemination must fulfill the criteria listed below. 
cPositive CSF determined by oligoclonal bands detected by established methods (preferably 
isoelectric focusing) different from any such bands in serum or by a raised IgG index. 
dMRI demonstration of time dissemination must fulfill the criteria listed below. 
14 
Appendix 2: Instructions for the Paced Auditory Serial 
Addition Test (PASAT) 
 
The PASAT is a measure of cognitive function that specifically assesses auditory information 
processing speed and flexibility, as well as calculation ability. The PASAT is the last measure 
administered at each visit. It is presented on audio CD to control the rate of stimulus 
presentation. Single digits are presented every 3 seconds and the patient must add each new 
digit to the one immediately prior to it. The test result is the number of correct sums given 
(out of 60 possible). To minimize familiarity with stimulus items in clinical trails and other 
serial studies, two alternate forms have been developed; the order of these should be 
counterbalanced across testing sessions. 
MATERIALS NEEDED 
CD player, audio CD with PASAT stimuli, clipboard, PASAT Record Forms 
15 
Appendix 3: EDSS Assessment Criteria 
EXPANDED DISABILITY STATUS SCALE 
 
0= normal neurological exam (all FS grade 0) 
1.0 = no disability, minimal signs in one FS (one FS grade 1)  
1.5 = no disability, minimal signs in more than one FS (more than one FS grade 1) 
2.0 = minimal disability in one FS (one FS grade 2, others 1 or 0)  
2.5 = minimal disability in two FS (two FS grade 2, others 1 or 0) 


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3.0 = moderate disability in one FS (one FS grade 3, others 1 or 0) though fully ambulatory; 
or mild disability in three or four FS (three/four FS grade 2, others 1 or 0) though fully 
ambulatory 
3.5 = fully ambulatory but with moderate disability in one FS (one FS grade 3) and mild 
disability in one or two FS (one/two FS grade 2) and others 0 or 1;or fully ambulatory with 
two FS grade 3 (others 0 or 1);or fully ambulatory with five FS grade 2 (others 0 or 1) 
4.0 = ambulatory without aid or rest for 3500 meters; up and about some 12 hours a day 
despite relatively severe disability consisting of one FS grade 4 (others 0 or 1)or combinations 
of lesser grades exceeding limits of previous steps 
4.5 = ambulatory without aid or rest for 3300 meters; up and about much of the day, 
characterised by relatively severe disability usually consisting of one FS grade 4 and 
combination of lesser grades exceeding limits of previous steps 
5.0 = ambulatory without aid or rest for 3200 meters (usual FS equivalents include at least one 
FS grade 5, or combinations of lesser grades usually exceeding specifications for step 4.5) 
5.5 = ambulatory without aid or rest 3100 meters 
6.0 = unilateral assistance (cane or crutch) required to walk at least 100 meters with or 
without resting 
6.5 = constant bilateral assistance (canes or crutches) required to walk at least 20 meters 
without resting 
7.0 = unable to walk 5 meters even with aid, essentially restricted to wheelchair; wheels self 
and transfers alone; up and about in wheelchair some 12 hours a day 
7.5 = unable to take more than a few steps; restricted to wheelchair; may need some normal 
neurological exam (all FS grade 0) no disability, minimal signs in one FS (one FS grade 1)no 
disability, minimal signs in more than one FS (more than one FS grade 1) minimal disability 
in one FS (one FS grade 2, others 0 or 1) minimal disability in two FS (two FS grade 2, others 
0 or 1)moderate disability in one FS (one FS grade 3, others 0 or 1) though fully help in 
transferring and in wheeling self 
8.0 = essentially restricted to bed or chair or perambulated in wheelchair, but out of bed most 
of day; retains many self-care functions; generally has effective use of arms 
8.5 = essentially restricted to bed much of the day; has some effective use of arm(s); retains 
some self-care functions 
9.0 = helpless bed patient; can communicate and eat 
9.5 = totally helpless bed patient; unable to communicate effectively or eat/swallow  
10.0 = death due to MS 

SAP_001.pdf: 
BIYOMARKER 
Statistical Analysis Plan 
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STATISTICAL ANALYSIS PLAN 
Version 2.0 
 
 
 
EFFECTS OF FINGOLIMOD (GILENYA®) ON CYTOKINE AND CHEMOKINE 
LEVELS IN RELAPSING REMITTING MULTIPLE SCLEROSIS PATIENTS 
 
 
 
 
STUDY NUMBER: FTY720/Fingolimod  
STUDY NAME: BIYOMARKER 
 
 
 


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Statistical Analysis Plan Signature Page 
Version 2.0  
Sponsor 
Protocol 
Study title 
: 
NOVARTIS 
: 
[CFTY720DTR04] / NCT02373098 
: 
Effects of Fingolimod (Gilenya®) on Cytokine and Chemokine 
Levels in Relapsing Remitting Multiple Sclerosis Patients 
, Bio., M.Sc. 
30.11.2017 
Author 
Date 
Reviewed by 
Date 
, M.D. 
04.12.2017 
Approved by 
Date 


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TABLE OF CONTENTS 
    
1 
ABBREVIATIONS ...................................................................................................................................................... 4 
2 
BACKGROUND ........................................................................................................................................................... 5 
3 
OBJECTIVES OF THE STUDY ................................................................................................................................ 5 
3.1. 
Primary Objective ................................................................................................................................................ 5 
3.2. 
Secondary Objective ........................................................................................................................................... 5 
4 
STUDY DESIGN .......................................................................................................................................................... 5 
5 
ANALYZED POPULATIONS ................................................................................................................................... 7 
5.1. 
Sample Size Calculation .................................................................................................................................... 7 
6 
ANALYSIS PLAN ........................................................................................................................................................ 7 
6.1 
Demographics .................................................................................................................................................. 7 
6.2 
CLINICAL DATA ............................................................................................................................................... 8 
6.3 
LABORATORY TESTS .................................................................................................................................... 8 
6.3.1 
Clinical Biochemistry ........................................................................................................................... 8 
6.3.2 
Hematology .............................................................................................................................................. 8 
6.4 
ASSESSMENTS OF SCALE ............................................................................................................................ 9 
6.5 
ASSESSMENT OF CYTOKINE AND CHEMOKINE LEVELS ............................................................... 9 
6.5.1 
ELISA .......................................................................................................................................................... 9 
6.5.2 
Flow Cytometry ..................................................................................................................................... 9 
6.6 
SAFETY ASSESSMENT .................................................................................................................................. 9 
 
 
 


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1 
ABBREVIATIONS 
AE 
Adverse Event 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase 
CCL2 
Chemokine (C-C motif) ligand 2 
CXCL13 
Chemokine (C-X-C motif) ligand 13 
CXCR3 
Chemokine (C-X-C motif) receptor 3 
ELISA 
Enzyme-linked Immunosorbent Assay 
EDSS 
Expanded Disability Status Scale 
GCP 
Good Clinical Practice 
ICH 
International Conference on Harmonisation of Technical Requirements for 
Registration of Pharmaceuticals for Human Use 
MS 
Multiple Sclerosis 
PASAT 
Paced Auditory Serial Addition Test 
RRMS 
Relapsing-remitting Multiple Sclerosis 
SAE 
Serious Adverse Event 
SDMT 
Symbol Digit Modalities Test 
TNF 
Tumor Necrosis Factor 
VLA 
Very Late Antigen 
WBC 
White Blood Cell 
 
 


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2 
BACKGROUND 
The purpose of this statistical plan is to define the tables, lists, data summaries and graphs to be 
prepared and to determine the statistical methods to be used for the analysis of the results of the 
study titled, “Effects of Fingolimod (Gilenya®) on Cytokine and Chemokine Levels in 
Relapsing Remitting Multiple Sclerosis Patients”. 
This statistical analysis plan has been prepared in pursuant to and in line with ICH-GCP 
principles and protocol no. CFTY720DTR04.   
3 
OBJECTIVES OF THE STUDY 
3.1. PRIMARY OBJECTIVE 
The primary objective of this study is to evaluate the differences between serum cytokine and 
chemokine levels of healthy controls and multiple sclerosis patients.  
3.2. SECONDARY OBJECTIVE 
The secondary objective of this study is to test the predictive value of cytokine/chemokine 
changes measured in the 3rd and 6th months on the PASAT, SDMT, 9-Hole peg test, timed 25-
foot walk, EDSS and relapse rate observed after 6 months of treatment.  
4 
STUDY DESIGN 
This study is a multi center, open label  study.  
80 relapsing remitting MS (RRMS) patients will be recruited according to the Mc Donald's 
criteria and 60 healthy controls will be recruited as the control group. All subjects will sign an 
informed consent that will be approved by the local Ethics Committee and the applicable health 
authorities prior to venipuncture. Basal EDSS, relapses during the last 2 years, paced auditory 
serial addition task (PASAT), 9-Hole peg test, timed 25-foot walk and single digit modalities 
test (SDMT) scores will be recorded before study entry. Serum and blood samples will be 
collected from each participant after evaluation for inclusion and exclusion criteria. Additional 
serum and blood samples at 2 different time points will be collected from MS patients at months 
3 (±1 week) and 6 (±2 weeks) after starting fingolimod 0.5 mg/day po. treatment (Figure 1). 
Serum samples will be stored at -20oC at local centers for a longest duration of two weeks and 
kept frozen at -80oC 
 


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 until assayed. The levels of IL-4, IL-6, IL-8, IL-9, IL-10, 
IL-13, IL-17, IL-23, IFN-γ, TNF-α, VLA4, CCL2, CCL5, CXCR3, CXCL13 will be measured 
by ELISA according to the manufacturer’s protocols. Optical density (OD) will be measured at 
450 nm and cytokine/chemokine concentrations (pg/ml) will be calculated by referring to a 
standard curve.  
Whole blood samples will be dispatched 
 
 In order to perform flow cytometry assay for intracellular 
cytokine measurement PBMC will be isolated freshly. 
EDSS and relapses will be recorded after 6 months of treatment. PASAT, 9-hole peg test, timed 
25-foot walk and SDMT's will be performed only at study entry and termination after 6 months 
(Figure 1). 
 
Figure 1: Study protocol (PASAT: Paced auditory serial addition task, SDMT: single digit 
modalities test, FACS: Flow assisted cell sorting, flow cytometry) 
 
 


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5 
ANALYZED POPULATIONS 
The study will include only 80 relapsing remitting MS patients and 60 healthy controls without 
any known autoimmune disease. RRMS patients will be followed for 6 months.  
5.1.   SAMPLE SIZE CALCULATION 
Due to the lack of previous studies using all of the cytokines and chemokines and multitudinous 
analyses, it is difficult to estimate a precise sample size for our study. Despite this, our previous 
publications indicate that the basal IL-17 concentration is normally distributed with a mean of 
32 pg/ml (±stdev of 15 pg/ml) in RRMS patients. If the true difference in the mean 
concentration of matched pairs is 7 pg/ml, we will need to study 50 pairs of subjects to be able 
to reject the null hypothesis with a power of 90%. The Type I error probability associated with 
this test of this null hypothesis is 0,05.  
With assumption of drop out rate of 20% minimum 60 RRMS patients and 60 healthy control  
subjects should enroll to the study. Due to expected high rate of screen failure patients, we 
decided to screen 80 RRMS patients in order to reach 60 eligible RRMS subjects for the study. 
6 
ANALYSIS PLAN 
Data analysis will be performed after all of the data is collected from the enrolled patients. All 
MS patients and control group will be accepted as the analysis sets. 
Cytokine and chemokine levels will be analyzed cumulatively at the end of the study. The 
results will be recorded to the blood sampling logs and laboratory logs cumulatively.   
6.1 
DEMOGRAPHICS 
• Gender: Subjects, male and female, in both groups (Control & MS patients) will be 
expressed separately as n and %.  
• Age: Age data for subjects in the “Control” and in the “MS patients” groups will be 
tabulated by calculating n, mean, standard deviation, minimum, maximum and median.   
 
 
 


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6.2 
CLINICAL DATA 
• Previous MS treatment of the patients will be expressed as n and %. 
• Age of onset of MS of the patients will be tabulated by calculating n, mean, standard 
deviation, minimum, maximum and median.  
• Duration of MS (years) of the patients will be tabulated by calculating n, mean, standard 
deviation, minimum, maximum and median.  
• MS relapses in the previous 2 years of the patients will be tabulated by calculating n, 
mean, standard deviation, minimum, maximum and median. 
• MS relapses requiring corticosteroids in the previous 2 years of the patients will be 
tabulated by calculating n, mean, standard deviation, minimum, maximum and median. 
• MS relapses in the previous 1 year of the patients will be tabulated by calculating n, mean, 
standard deviation, minimum, maximum and median.  
• MS relapses requiring corticosteroids in the previous 1 year of the patients will be 
tabulated by calculating n, mean, standard deviation, minimum, maximum and median. 
• Duration of treatments (months) of the patients will be tabulated by calculating n, mean, 
standard deviation, minimum, maximum and median. 
• Relapse rates of the patients will be evaluated. 
6.3 
LABORATORY TESTS 
6.3.1 Clinical Biochemistry 
Patients’ AST, ALT, BUN, Creatinine, Total Cholesterol, Tryglicerides, LDL-C, HDL-C, 
TSH, ST4, Glucose and HbA1c values will be tabulated by calculating mean and standart 
deviation and compared as Visit 1 vs. Visit 2 and Visit 1 vs. Visit 3 by using appropriate 
statistical test. Wilcoxon test will be used in the dependent variables with non-normal 
distributions whereas Paired sample t test will be used in the dependent variables with normal 
distributions. 
6.3.2 Hematology 
Patients’ WBC, Absolute Lymphocyte Count, Platelet, Absolute Neutrophil Count and 
Hemoglobin values will be tabulated by calculating mean and standart deviation and compared 
as Visit 1 vs. Visit 2 and Visit 1 vs. Visit 3 by using appropriate statistical test. Wilcoxon test 
will be used in the dependent variables with non-normal distributions whereas Paired sample t 
test will be used in the dependent variables with normal distributions. 


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6.4 
ASSESSMENTS OF SCALE 
Patients’ EDSS, PASAT, SDMT, 9-hole peg test and timed 25-foot walk test values will be 
tabulated by calculating mean and standart deviation and compared as Visit 1 vs. Visit 3 by 
using appropriate statistical test. Wilcoxon test will be used in the dependent variables with 
non-normal distributions whereas Paired sample t test will be used in the dependent variables 
with normal distributions. 
6.5 
ASSESSMENT OF CYTOKINE AND CHEMOKINE LEVELS 
6.5.1 ELISA 
Subjects’ IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, IL-23, IFN-γ, TNF-α, VLA4, CCL2, 
CCL5, CXCR3 and CXCL13 values will be tabulated by calculating mean and standart error 
and compared as Control group vs. Visit 1 (MS patients), Visit 1 (MS patients) vs. Visit 2 (MS 
patients), Visit 1 (MS patients) vs. Visit 3 (MS patients) and Control group vs. Visit 3 (MS 
patients) by using appropriate statistical test. Mann Whitney U test with significance level of 
0.05 was used for the independent variables since non-normal distributions had been observed. 
Wilcoxon test was used for the dependent variables with non-normal distributions. For the 
independent variables with normal distributions Independent Sample t test was used whereas 
Paired sample t test was used for the dependent variables with normal distributions. 
6.5.2 Flow Cytometry 
Subjects’ IL-4, IL-6, IL-9, IL-10, IL-13, IL-17, IFN-γ and TNF-α values will be tabulated by 
calculating mean and standart error and compared as Control group vs. Visit 1 (MS patients), 
Visit 1 (MS patients) vs. Visit 2 (MS patients), Visit 1 (MS patients) vs. Visit 3 (MS patients) 
and Control group vs. Visit 3 (MS patients) by using appropriate statistical test. Mann Whitney 
U test with significance level of 0.05 was used for the independent variables since non-normal 
distributions had been observed. Wilcoxon test was used for the dependent variables with non-
normal distributions. For the independent variables with normal distributions Independent 
Sample t test was used whereas Paired sample t test was used for the dependent variables with 
normal distributions. 
6.6 
SAFETY ASSESSMENT 
Incidence of AEs, incidence of SAEs and number of patients who died or experienced other 
serious or clinically significant events will be tabulated.