Augmenting Cerebral Blood Flow to Treat Established Multiple Sclerosis
Phase: PHASE2
Conditions: Multiple Sclerosis
Sponsor: The University of Texas Health Science Center, Houston

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 1 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Augmenting Cerebral Blood Flow to Treat Established Multiple Sclerosis 
 
NCT02466074 
 
Version Date: 06/27/2019 
 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 2 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
 
STUDY PROTOCOL 
 
PROTOCOL NUMBER: 
HSC-MS-14-0450 
PROTOCOL TITLE: 
Augmenting Cerebral Blood Flow to Treat Established Multiple Sclerosis 
VERSION: 
6.0 
DATE: 
April 1, 2019 
CORRESPONDING PRINCIPAL INVESTIGATOR: 
John A. Lincoln, MD, PhD, Associate Professor, UTHealth 
SITE PRINCIPAL INVESTIGATOR: 
John A. Lincoln, MD, PhD, Associate Professor, UTHealth 
SITE: 
McGovern Medical School, UTHealth 
COLLABORATORS: 
Léorah Freeman, MD, Assistant Professor, UTHealth 
Khader Hasan, PhD, Professor, UTHealth 
Refaat Gabr, PhD, Assistant Professor, UTHealth 
Jerry Wolinsky, MD, Emeritus Professor, UTHealth 
Ponnada Narayana, PhD, Professor, UTHealth 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 3 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
GENERAL INFORMATION 
 
 
Name and address of the person authorized to sign the protocol and amendments: 
John A. Lincoln, MD, PhD, |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 
Name and address of study monitor: 
James R. Jemelka, |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 
Name, title, address and telephone number(s) of the medical expert for the trial 
John A. Lincoln, MD, PhD, |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 
||||||||, 713-500-7016 
Name and title of the investigator(s) and sub-investigators responsible for the trial 
with address and phone number(s) 
John A. Lincoln, MD, PhD, |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 
||||||||, 713-500-7016 
Name and addresses of the clinical laboratories and/or other institutions involved in 
the trial 
N/a 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 4 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
TABLE OF CONTENTS 
1. 
BACKGROUND AND RATIONALE ............................................................................................... 5 
1.1. 
GENERAL INTRODUCTION .................................................................................................................................... 5 
1.2. 
RATIONALE AND JUSTIFICATION FOR THE STUDY .......................................................................................... 6 
A. 
RATIONALE FOR THE STUDY PURPOSE ................................................................................................................. 6 
B. 
RATIONALE FOR DOSES SELECTED ....................................................................................................................... 7 
C. 
RATIONALE FOR STUDY POPULATION ................................................................................................................... 7 
D. 
RATIONALE FOR STUDY DESIGN ............................................................................................................................. 7 
2. 
HYPOTHESIS AND OBJECTIVES .............................................................................................. 10 
2.1. 
HYPOTHESIS ....................................................................................................................................................... 11 
2.4. 
POTENTIAL RISKS AND BENEFITS ................................................................................................................... 13 
A. 
END POINTS - EFFICACY ....................................................................................................................................... 13 
B. 
END POINTS - SAFETY ........................................................................................................................................... 13 
3.1. 
LIST THE NUMBER OF SUBJECTS TO BE ENROLLED ..................................................................................... 13 
3.2. 
CRITERIA FOR RECRUITMENT .......................................................................................................................... 14 
3.3. 
INCLUSION CRITERIA ......................................................................................................................................... 14 
3.4. 
EXCLUSION CRITERIA ........................................................................................................................................ 14 
3.5. 
WITHDRAWAL CRITERIA .................................................................................................................................... 15 
3.6. 
SUBJECT REPLACEMENT .................................................................................................................................. 15 
4. 
TRIAL SCHEDULE ...................................................................................................................... 16 
5. 
STUDY DESIGN ...................................................................................................................... 19 
5.1. 
SUMMARY OF STUDY DESIGN ......................................................................................................................... 20 
6. 
METHODS AND ASSESSMENTS ............................................................................................... 21 
6.1. 
RANDOMIZATION AND BLINDING ...................................................................................................................... 24 
6.2. 
CONTRACEPTION AND PREGNANCY TESTING .............................................................................................. 24 
6.3. 
STUDY VISITS AND PROCEDURES .................................................................................................................. 25 
7. 
TRIAL MATERIALS ..................................................................................................................... 32 
7.1. 
DRUG SUBSTANCE: ACETAZOLAMIDE ............................................................................................................ 32 
7.2. 
DESCRIPTION OF DRUG PRODUCT ................................................................................................................. 34 
ACETAZOLAMIDE EXTENDED-RELEASE CAPSULES ...................................................................................................... 34 
7.3. 
STORAGE AND DRUG ACCOUNTABILITY ........................................................................................................ 40 
8. 
TREATMENT ............................................................................................................................... 40 
8.1. 
RATIONALE FOR SELECTION OF DOSE .......................................................................................................... 40 
8.2. 
STUDY DRUG FORMULATIONS ......................................................................................................................... 40 
8.3. 
STUDY DRUG ADMINISTRATION ...................................................................................................................... 40 
8.4. 
SPECIFIC RESTRICTIONS / REQUIREMENTS ................................................................................................. 40 
8.5. 
BLINDING .............................................................................................................................................................. 41 
8.6. 
CONCOMITANT THERAPY .................................................................................................................................. 41 
9. 
SAFETY MEASUREMENTS ........................................................................................................ 41 
9.1. 
DEFINITIONS ........................................................................................................................................................ 41 
9.2. 
COLLECTING, RECORDING AND REPORTING OF ADVERSE EVENTS ........................................................ 42 
9.3. 
SAFETY MONITORING PLAN ............................................................................................................................. 42 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 5 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
10. 
DATA ANALYSIS .................................................................................................................... 43 
10.1. 
DATA QUALITY ASSURANCE ............................................................................................................................ 43 
10.2. 
DATA ENTRY AND STORAGE ............................................................................................................................ 43 
11. 
SAMPLE SIZE AND STATISTICAL METHODS ...................................................................... 44 
12. 
ETHICAL CONSIDERATIONS................................................................................................. 45 
12.1. 
INFORMED CONSENT ......................................................................................................................................... 45 
12.2. 
IRB REVIEW ......................................................................................................................................................... 45 
12.3. 
CONFIDENTIALITY OF DATA AND PATIENT RECORDS ................................................................................. 45 
13. 
PUBLICATIONS ...................................................................................................................... 45 
14. 
RETENTION OF TRIAL DOCUMENTS ................................................................................... 45 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 5 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
STUDY PROTOCOL 
 
 
BACKGROUND AND RATIONALE 
General Introduction 
Multiple sclerosis (MS) is a chronic central nervous system disease affecting over 400,000 persons in 
the United States and over 2.5 million persons worldwide (Reingold, 2002). Clinical symptoms 
typically occur as a result of focal inflammation associated with demyelination and axonal damage. 
Symptoms can be transient for many patients during the initial stage of disease with some degree of 
improvement as inflammation subsides and tissue undergoes repair (Lassmann, 2011; Leary, 2005). 
Additionally, there is mounting evidence that inflammation might not be a prerequisite for tissue injury 
(Barnett et al., 2006; Barnett and Prineas, 2004; Narayana et al., 1998). Regardless of how tissue is 
initially injured, the degree of repair and corresponding resolution of clinical symptoms is often 
variable and not entirely understood. 
Magnetic resonance imaging (MRI) is commonly 
utilized to monitor lesion formation and evolution 
(Cotton et al., 2003; Meier and Guttmann, 2006; 
Narayana et al., 1998). The majority of new MS 
plaques initially enhance after administration of 
intravenous (IV) contrast, a hallmark of acute 
perivascular inflammation. These new plaques appear 
bright (hyperintense) on T2-weighted and frequently 
have a corresponding dark component (hypointense) 
on T1-weighted sequences (Cotton et al., 2003; Filippi, 
2000; Meier and Guttmann, 2006) [see figure 1]. Within 
months from their initial appearance as newly formed 
lesions, as much as 80% of the initial T2 lesion volume 
Figure 1: MS lesion appears as hyperintense 
resolves largely related to resolution of edema and 
(blue arrows) on T2-weighted and  
partial tissue repair (Cotton et al., 2003). By contrast, 
hypointense (red arrows) on T1-weighted 
up to 40% of lesions with initial T1 hypointense 
sequences respectively. 
components will evolve into persistent black holes 
(chronic area with low signal intensity on T1-weighted images) indicative of permanent tissue 
damage (Sahraian et al., 2010). Many studies have demonstrated a robust correlation between 
persistent black holes and clinical disability (Parry et al., 2002; Thaler et al., 2017). 
There are regional differences in cerebral perfusion within the normal human brain. Cerebral 
perfusion is altered in many disease states, including MS. Altered perfusion has been seen in 
patients with all MS phenotypes and early in relapsing-remitting disease (Helenius et al., 2003; 
Papadaki et al., 2013, 2012; Wuerfel et al., 2004). Changes in cerebral perfusion both in a given 
region and lesion area have been previously described in multiple sclerosis (Juurlink, 2013; Wuerfel 
et al., 2004). Our own work is consistent with others showing that areas within the brain that are 
most prone to hypoperfusion appear to be those with minimal collateral flow (Adhya et al., 2006; 
Helenius et al., 2003; Holland et al., 2012; Juurlink, 2013; Narayana et al., 2014; Wuerfel et al., 
2004). It is noteworthy that studies, including our own, have shown increased probability of lesion 
formation within areas corresponding to expected hypoperfused regions (Debernard et al., 2013; 
Holland et al., 2012; Narayana et al., 2014). Diminished regional perfusion and increased energy 
demand has also been suggested to potentiate axonal loss within lesions and contributes to the 
evolution of potentially irreversible and progressive tissue injury, visualized on serial MRI as 
persistent black holes (Trapp and Stys, 2009). Indeed, these changes appear to be dynamic and 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 6 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
associated with lesion evolution (Wuerfel et al., 2004). 
There are currently several potent anti-inflammatory medications available to treat patients with MS. 
All of these disease modifying therapies (DMTs) decrease the potential for new injury to varying 
degrees. Despite their anti-inflammatory properties, DMTs are not known to enhance cerebral 
perfusion. There are several medications currently available that are known to transiently enhance 
cerebral perfusion. Of these, acetazolamide (ACZ) is well tolerated and has a well-established safety 
profile (“Acetazolamide Tablets | Drug Summary | PDR.net,” n.d.; Lim et al., 2001; Stewart et al., 
2002). Computerized tomography (CT) or MRI perfusion imaging with IV ACZ challenge is a 
commonly utilized clinical tool in evaluating patients with chronic intracerebral occlusive disease and 
is a reliable predictor of critically reduced cerebral perfusion (Vagal et al., 2009). Acetazolamide is 
also currently utilized as an IV drug in the treatment of acute, closed-angle glaucoma, drug-induced 
edema or congestive heart failure and as an oral drug taken chronically in divided daily doses as 
adjunct treatment of chronic open-angle glaucoma, epilepsy, pseudotumor cerebri, chronic 
congestive heart failure, acute motion sickness, uric acid renal calculi and familial periodic paralyses. 
If cerebral hypoperfusion is important to the evolution of lesions then encouraging better tissue 
perfusion might reduce the evolution and persistence of black holes. 
Rationale and justification for the Study 
Rationale for the Study Purpose 
Focal regions of demyelination, variable axonal loss and gliosis characterize the pathologic damage 
that is the hallmark of MS. These lesions appear as T2 hyperintense when imaged by MRI. 
Diminished regional perfusion and increased energy demand within the MS lesion have been 
suggested to potentiate axonal loss. Axonal damage is often seen on MRI as an area of T1 
hypointensity (“black hole”) within the T2 hyperintense lesion (van Walderveen et al., 1998, 1999a). 
Indeed up to 90% of axons might be damaged within the area imaged as black hole (van Walderveen 
et al., 1999a).  Several reports have shown stronger correlations in MS patients between both 
general and system-specific clinical disability and black hole volume as compared to total T2 
hyperintense lesion volume (Caramanos et al., 2012; Giorgio et al., 2013; van Walderveen et al., 
1999a). 
There are twelve DMTs currently approved by the Food and Drug Administration (FDA) to treat 
patients with MS. All of these therapies impact the immune system and, in the aggregate, have been 
shown to decrease new inflammatory lesions (T2 hyperintense lesions) [see review by Tullman; 
(Tullman, 2013)]. Though we are able to decrease the potential for new injury, there are no 
medications that are shown to decrease the likelihood of black hole formation (lesion evolution) once 
the T2 lesion forms. If cerebral perfusion is an important contributor to tissue repair then MS lesions 
within hypoperfused areas might be more likely to develop permanent tissue loss. In this scenario, 
medications that encourage cerebral perfusion might alter the evolution of new MS plaques and 
diminish clinical disability progression. 
To test this hypothesis, we propose to utilize ACZ, an existing medication with a well-established 
safety and tolerability profile that has been shown to enhance cerebral perfusion (“Acetazolamide 
Tablets | Drug Summary | PDR.net,” n.d.; Lim et al., 2001; Stewart et al., 2002). To the best of our 
knowledge, ACZ has not been previously evaluated as a potential therapy in patients with MS. This 
study will determine if the novel application of long-term ACZ therapy in subjects with MS improves 
cerebral perfusion resulting in a smaller proportion of MS lesions that evolve to form the persistent 
black holes that reflect permanent tissue damage. Stage 1a of the study will define the magnitude of 
change in regional cerebral perfusion following acute IV ACZ therapy. Stage 1b will determine the 
degree to which short-term use of divided daily oral doses of ACZ improves global and regional 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 7 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
cerebral perfusion in subjects with MS. Finally, Stage 2 of the study will determine the effect of long- 
term oral ACZ on lesion evolution. 
Rationale for Doses Selected 
Studies have shown that ACZ administered as a single 1000 mg IV bolus transiently enhances 
cerebral perfusion in healthy subjects (Sullivan et al., 1987; Vagal et al., 2009). Subjects challenged 
with IV ACZ had between a 30% to 60% increase in cerebral blood flow, as measured by Xenon- 
enhanced computed tomography scan, within 15 minutes of IV bolus (Sullivan et al., 1987). Another 
study using ultrasonography demonstrated that enhanced perfusion was sustained for at least 30 
minutes with return to baseline measures within one hour (Hartkamp et al., 2012). This same dose 
and route of administration will be used in subjects with MS to determine the transient change to 
cerebral perfusion as quantified using MRI metrics as described in section 2.2. 
Though ACZ IV transiently enhances cerebral perfusion, longer-term changes would be required to 
potentially impact lesion evolution. Daily or divided-daily infusions necessitate chronic IV access, 
pose increased risk to the patient and are impractical. Oral ACZ has been shown to also increase 
cerebral perfusion in patients with other neurologic disorders (“Acetazolamide Tablets | Drug 
Summary | PDR.net,” n.d.) and might prove a practical and effective alternate chronic therapy for 
patients with MS. 
ACZ has been used in daily divided doses up to a maximum of 4000 mg per day to treat 
pseudotumor cerebri (NORDIC Idiopathic Intracranial Hypertension Study Group Writing Committee 
et al., 2014). Participants in Stage 1b of this study will be given escalating divided doses of oral ACZ 
up to 1500 mg per day to determine both the magnitude of change to cerebral perfusion with each 
dose and define the optimum dose(s) for additional studies. In the final stages of this study (stage 2a 
and 2b), subjects will be treated with ACZ, as determined in stage 1b, for six consecutive months to 
determine if long-term change to cerebral perfusion alters the evolution of MS plaques into persistent 
black holes. 
Rationale for Study Population 
Multiple sclerosis affects predominantly persons between 15 and 55 years of age with females being 
nearly four-times as likely to be diagnosed with disease as males (Koch-Henriksen and Sørensen, 
2010; Orton et al., 2006; Reingold, 2002). MS in the paediatric population is now better recognized 
and has been shown to differ from that in the adults in both clinical and radiologic presentation and 
evolution (Ghassemi et al., 2014; Renoux et al., 2007). 
This study will utilize female and male MS subjects between 18 and 55. 
Rationale for Study Design 
This study is designed as a prospective trial using a delayed-start design which is anticipated to lead 
to a larger randomized phase 3 clinical trial. Subjects will be evaluated prior to and after initiation of 
therapy to quantify the effect of therapy on lesion evolution. These initial pilot studies will determine 
the magnitude to which ACZ improves MRI measures of cerebral perfusion and alters the evolution of 
MS plaques. The studies outlined in this proposal will be useful not only in power calculation for the 
anticipated larger randomized clinical trial but also determine imaging characteristics of responders 
and non-responders, as it relates to treatment effect and identify the optimum outcome measures, 
including frequency at which measurements should be obtained. 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 8 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Many factors contribute to the formation and evolution of lesions in MS (Cotton et al., 2003; Meier 
and Guttmann, 2006; Narayana et al., 1998). As much as 60% of MS lesions resolve over six 
months while the remainder show evidence of persistent damage (Sahraian et al., 2010). In stage 2, 
MS subjects will be treated with oral ACZ for 24 and 48 consecutive weeks to determine if improved 
cerebral perfusion will result in improved tissue integrity within lesions. 
The formation of new MS lesion is best visualized on contrast-enhanced T1-weighted sequences and 
usually corresponds to new hyperintense area on T2-weighted MRI. To evaluate both existing and 
newly formed MS lesion, subjects will obtain both contrast-enhanced and non-contrast MRI. 
Stage 1a - Increases in cerebral perfusion after IV ACZ has been seen in healthy persons but not in 
subjects with MS. In addition to physiologic differences in regional cerebral perfusion, subjects with 
MS might have exaggerated differences secondary to disease-associated damage. This stage will 
determine the degree of change, temporal kinetics and difference between various regions in 
cerebral perfusion after IV ACZ in subjects with MS. 
Arterial spin labeling (ASL) is a non-invasive MRI technique where spins are tagged using a radio- 
frequency pulse to determine cerebral blood flow (CBF). Both continuous (CASL) and pulsed arterial 
spin (PASL) techniques have been utilized to measure CBF. CASL has the advantage of improved 
signal-to-noise and provides a clearer estimate of CBF though the technique is longer with greater 
demands on imaging hardware. PASL has the advantage of higher tagging efficiency and is a 
shorter technique though with greater associated noise. Pseudo-continuous ASL (pCASL) is a more 
recent technique that combines the merits of both CASL and PASL and largely overcomes previous 
disadvantages (Dai et al., 2008; Wong, 2007; Wu et al., 2007). pCASL technique has recently been 
further optimized to accurately and reproducibly quantify CBF (Aslan et al., 2010). Our lab has 
previously published on the use of pCASL to determine CBF in MS subjects (Narayana et al., 2014). 
Dynamic susceptibility contrast (DSC) MRI is a gadolinium contrast-based technique that can 
accurately and reproducibly quantify cerebral blood flow (CBF), cerebral blood volume (CBV) and 
mean transit time (MTT) of blood. There is a very small risk of an anaphylactic reaction to the 
injection of all commercially available paramagnetic contrast agents for the use in diagnostic and 
research imaging. There is a risk of the development of nephrogenic systemic fibrosis (NSF) 
following the administration of some gadolinium containing contrast agents to patients with moderate 
to severe renal disease and those on dialysis. Following recent recommendations of the American 
College of Radiology (see Kanal et al. AJR 188:1447, 2007), patients with renal disease are excluded 
from these studies. Standard precautions available for the management of such reaction are 
available within the UT Imaging Center where these studies will be performed. DSC techniques will 
provide information that cannot be obtained by using pCASL alone that is important to determine how 
blood flow is altered with ACZ. 
The degree of transient change to CBF, measured using pCASL, will be determined in MS subjects 
prior to and after administration of a 1000 mg IV bolus of ACZ. Time to peak effect and duration of 
action (not previously defined in MS subjects) will be monitored using pCASL at 15, 30, 120 and 240 
minutes from time of infusion. At the end of the kinetic study gadolinium will be injected and DSC 
measures of blood flow obtained. 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 9 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Stage 1b - Changes in cerebral perfusion will likely need to be sustained for prolonged periods to 
impact lesion evolution. Participants in this stage of the study will be given escalating divided doses 
of oral ACZ up to 4000 mg per day to determine both the magnitude of change to cerebral perfusion 
with maintenance daily oral ACZ therapy and to define the optimum dose(s) for additional studies. 
Subjects will undergo MRI using pCASL prior the start of each bi-monthly regimen of ACZ as well as 
on the day of drug discontinuation. The magnitude of change in CBF will be determined for various 
oral ACZ doses as well as for various cerebral regions 
 
 
Stage 2a –Safety/Tolerability & Kinetics of Treatment Effect 
This stage will continue to gather information about whether daily oral ACZ is tolerated and the 
kinetics of the treatment effect. Patients continue on the same dose for 12 consecutive weeks and 
undergo repeat imaging at predetermined intervals. Sustained treatment over a short duration will 
allow us to rapidly determine whether sustained treatment with ACZ can alter cerebral blood flow and 
will provide a go/no-go for future studies using this drug/paradigm. In this stage, all subjects will 
receive baseline pCASL and DSC MRI, will start oral ACZ continued for 12 consecutive weeks and 
undergo repeat imaging at predetermined intervals. MRI scans and clinical evaluations will be 
obtained every 6 weeks with contrast-based scans repeated at 12 weeks. As previously stated, DSC 
provides measurements of not only CBF but also blood volume (CBV) and transit time of blood 
(MTT). It is possible that in addition to changes in blood flow, the amount or rate of flow is altered with 
ACZ and may be an important mechanism to tissue repair. DSC techniques will provide information 
that cannot be obtained by using pCASL alone that might be important to determine how blood flow is 
altered with ACZ. 
In addition, we will use advanced MRI techniques to also quantify tissue oxygen extraction before 
and after ACZ therapy as well as determine the metabolic rate of oxygen consumption in various 
tissues. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 10 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Stage 2a –Safety/Tolerability & Kinetics of Treatment Effect 
This stage will continue to gather information about whether daily oral ACZ is tolerated and the 
kinetics of the treatment effect. Patients continue on the same dose for 12 consecutive weeks and 
undergo repeat imaging at predetermined intervals. Sustained treatment over a short duration will 
allow us to rapidly determine whether sustained treatment with ACZ can alter cerebral blood flow and 
will provide a go/no-go for future studies using this drug/paradigm. In this stage, all subjects will 
receive baseline pCASL and DSC MRI, will start oral ACZ continued for 12 consecutive weeks and 
undergo repeat imaging at predetermined intervals. MRI scans and clinical evaluations will be 
obtained every 6 weeks with contrast-based scans repeated at 12 weeks. As previously stated, DSC 
provides measurements of not only CBF but also blood volume (CBV) and transit time of blood (MTT). 
It is possible that in addition to changes in blood flow, the amount or rate of flow is altered with ACZ 
and may be an important mechanism to tissue repair. DSC techniques will provide information that 
cannot be obtained by using pCASL alone that might be important to determine how blood flow is 
altered with ACZ. 
In addition, we will use advanced MRI techniques to also quantify tissue oxygen extraction before and 
after ACZ therapy as well as determine the metabolic rate of oxygen consumption in various tissues. 
 
Stage 2b – Tachyphylaxis & Long-term Safety/Tolerability 
This stage is designed to determine if daily oral ACZ is tolerated and if tachyphylaxis might occur with 
long-term use for the specific formulation of oral ACZ that we will utilize. Some authors have reported 
attenuation of the initial increases in CBF with sustained use or oral ACZ though still showing a 
persistent increase in cerebral perfusion of between 15% - 20%, when compared to pre-treatment 
levels (Friberg et al., 1990). In this stage, all subjects will receive baseline pCASL and DSC MRI and 
will start oral ACZ. Patients will continue on 1000 mg BID dose for 24 consecutive weeks and undergo 
repeat imaging at predetermined intervals.  MRI scans and clinical evaluations will be obtained every 
6 weeks with contrast-based scans repeated every 12 weeks. Contrast will be administered every 12 
weeks to determine DSC measures of blood flow and whether new lesions have formed. As 
previously stated, DSC provides measurements of not only CBF but also blood volume (CBV) and 
transit time of blood (MTT). DSC techniques will provide information that cannot be obtained by using 
pCASL alone that might be important to determine how blood flow is altered with ACZ. 
Stage 3 - Many factors contribute to the formation and evolution of lesions in MS (Cotton et al., 2003; 
Meier and Guttmann, 2006; Narayana et al., 1998). As much as 60% of MS lesions resolve over six 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 11 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
months while the remainder show evidence of persistent damage (Sahraian et al., 2010). In this 
stage, all subjects will receive baseline pCASL and DSC MRI and will then be randomized to first 
receive either daily oral therapy with either “Treatment A”, extended-release ACZ capsules or 
“Treatment B”, placebo capsules for 24 consecutive weeks and undergo repeat imaging at 
predetermined intervals. MRI scans and clinical evaluations will be obtained every 6 weeks with 
contrast-based scans repeated every 12 weeks. Contrast will be administered every 12 weeks to 
determine DSC measures of blood flow and whether new lesions have formed and determine how 
blood flow is altered with ACZ. 
At the end of week 24, patients initially randomized to Treatment B will take 1000 mg extended- 
release ACZ capsules. Similar imaging and clinical evaluations will be performed for the second 24- 
week period. All subjects will discontinue drug at week 48 and obtain a follow-up clinical visit two 
weeks later. Based upon established pharmacokinetics, the half-life of drug is between 60-75 hours 
and one would expect insignificant levels within two weeks of discontinuation. 
To maintain blinding, patients, evaluating clinicians and MRI analysts will not be informed of treatment 
assignment or sequence of scans. 
We anticipate that sustained increases in regional cerebral perfusion over 6 months will result in 
improved tissue integrity as measured using diffusion tensor metrics and fewer MS lesions that evolve 
to persistent black holes. 
 
HYPOTHESIS AND OBJECTIVES 
Hypothesis 
The underlying hypothesis of this proposal is that long-term therapy with ACZ will improve cerebral 
perfusion in subjects with MS and this improved perfusion will result in improved tissue integrity in a 
proportion of MS lesions. To test this hypothesis, subjects will be treated with ACZ, a currently FDA 
approved agent previously shown in healthy controls to increase cerebral perfusion (Sullivan et al., 
1987; Vagal et al., 2009). Subjects will be scanned at various time points (see sections 4 and 6.3) 
and quantitative MRI metrics will be used to evaluate lesion evolution during and after discontinuation 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 12 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
of treatment. 
Primary & Key Secondary Objective 
Determine the degree to which divided daily doses of ACZ improves global and regional cerebral 
perfusion in subjects with MS. 
Study subjects will obtain serial MRI scans prior to, during and after therapy (see sections 4 and 6.3). 
Cerebral blood flow will be measured using an optimized pCASL technique as described by Aslan 
(Aslan et al., 2010). This will allow us to test the first component of our hypothesis that treatment with 
ACZ will result in increased regional and/or global perfusion. 
The primary endpoint of this study will be to determine if therapy with oral divided-dose ACZ 
administered each day for week-long period results in at least 15% increase in CBF. Studies have 
shown between 10% - 15% decrease in cerebral perfusion in patients with relapsing MS (Adhya et al., 
2006). Therefore, increases in CBF of at least 15% might result in change to lesion evolution. 
Baseline CBF measures will be compared to measures obtained at each dose escalation step using 
paired t test and Mann-Whitney test. 
Determine the effect of long-term oral ACZ on lesion evolution. The key secondary endpoint of this 
study is to determine if long-term use of oral daily divided-dose with ACZ improves tissue integrity in a 
proportion of MS lesions. 
MRI scans will be segmented using into white and gray matter, T2 and T1 lesion components and 
contrast-enhancing lesion component. Expert raters (I.V., S.D., L.F.), who are blinded to treatment 
arm (see section 6.1) will perform quality control of the automated segmentation and obtain 
quantitative measures of cerebral perfusion. The number and volume of T2 hyperintense and T1 
hypointense lesion will be determined using MRIAP. The ratio of T1 hypointense/T2 hyperintense 
lesion will be determined for baseline and each crossover period and correlated with CBF, CBV and 
MTT measures obtained at similar times using the Spearman rank correlation. 
Diffusion tensor imaging (DTI) is a reliable, validated technique that quantitatively measures 
molecular diffusivity within tissue and provides information about its microstructural integrity. DTI will 
be used to monitor tissue integrity and correlate change with increases in CBF following ACZ. 
Tertiary Objectives 
Several studies have quantitated transverse and longitudinal relaxation times within lesions and 
normal-appearing white matter and gray matter. These studies have shown that changes in both 
transverse and longitudinal relaxation can precede lesion formation and possibly better evaluate 
lesion evolution as compared to black hole volumes (Papadopoulos et al., 2010; van Walderveen et 
al., 1999a; Walsh et al., 2014; Warntjes et al., 2013). We will manually delineate regions of interest 
within lesions to obtain T2 and T1 relaxometry metrics. Lesions within areas with greatest change in 
CBF will be compared with those with least change. We anticipate a correlation between relaxometry 
measures and areas with varying CBF. 
This study will include analysis to determine if therapy with oral divided-dose ACZ impacts global 
measures of clinical disability. Change in clinical disability measures assessed using the multiple 
sclerosis functional composite scale (MSFC) and the expanded disability status scale (EDSS) will be 
correlated with CBF, CBV and MTT. In addition, patient reported clinical outcome measures will be 
assessed at each time point. As this trial uses a small sample size, we would not expect statistically 
significant treatment effects on clinical disability though this exploratory measure should be useful in 
power calculation for the anticipated larger, randomized clinical trial. 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 13 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
Potential Risks and Benefits: 
 
End Points - Efficacy 
Altered cerebral perfusion has been previously reported in patients with MS. Global, regional and 
lesional changes in perfusion have been documented and some have found associations between 
decreased regional CBF and T2 lesion volume (Ota et al., 2013). 
There are several currently approved treatments for MS that, in the aggregate, decrease the potential 
for formation of new MRI lesions. However, none have been clearly shown to impact the evolution of 
lesions, once formed. Several studies have shown that clinical disability correlates better with tissue 
integrity within black holes (Parry et al., 2002; Truyen et al., 1996; van Walderveen et al., 1999b). 
ACZ is a generally well tolerated therapy that has previously been shown to transiently increase 
cerebral perfusion. Long-term use of ACZ might more persistently improve global or regional CBF 
and could increase the proportion of lesions with improved tissue integrity. 
 
End Points - Safety 
ACZ is generally well tolerated with few reported adverse events (AEs). Transient perioral numbness 
has been reported in patients receiving bolus of IV ACZ. Hypersensitivity reactions, including diffuse 
pruritus, hives and shortness of breath have been reported with repeat administration of either IV or 
oral medication. A complete list of AEs is available in the package insert (see section 7) and will be 
provided to subjects with the informed consent. 
Subjects will have face-to-face clinical evaluations at each scheduled visit (see section 4), will have 
monthly telephone or email contacts during the course of study and encouraged to report any AE 
occurring with drug at any point during the study. Any subject who is unable to tolerate study 
medication will be withdrawn from the study. 
During the dose escalation, in addition to issues of tolerability, subjects will be monitored at each 
transition point for the following specific serious AEs: paraesthesia, change in taste, accelerated 
change in blood pressure or fluid retention. Any subject with serious AEs will be withdrawn from the 
study. Patients with persistent serious AEs will be treated using standard medical practice. 
 
 
STUDY POPULATION 
 
List the number of subjects to be enrolled. 
This exploratory study is designed to evaluate the long-term effects of oral ACZ on CBF in adult 
subjects with MS. We will enroll 100 MS subjects between the age of 18 and 55 without history of 
cerebrovascular disease or glaucoma. 
There are several studies suggesting that MS lesions in the pediatric population differ from that 
typically seen in the adult population (Ghassemi et al., 2014; Renoux et al., 2007). To minimize 
potential confounding variables, all stages of the study will recruit only adult subjects with MS. 
All subjects will be recruited from patients seen at the MS clinic located at the University of Texas 
Professional building (UTP) Neurology practice||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||. 
Information about the study will be available on the UT Neurology web site and in print format at the 
UTP MS clinic. 
Informed consent will be obtained from each participant prior to entry and, for subjects who elect to 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 14 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
participate in multiple parts of the study, prior to each stage. 
Criteria for Recruitment 
Subjects who wish to participate in either stage of the study will be evaluated by the principle 
investigator to determine if they meet inclusion and exclusion criteria. A thorough medical history will 
be performed to determine if any pre-existing cardiovascular disease or glaucoma is present. 
Subjects whose history is unknown will be encouraged to obtain evaluation by a primary care 
physician and eye-care specialist prior to recruitment. In addition, available blood tests, performed 
within six months of study entry, will be reviewed to determine if any renal or hepatic dysfunction is 
present. 
Potential subjects with historical or laboratory evidence to suggest dysfunction in one of the above 
clinical systems will be excluded from study participation. As the rate of these comorbidities is low in 
a young population, we do not anticipate large numbers of failures at this stage of screening. 
Inclusion Criteria 
Patients who meet all of the following inclusion criteria during the screening period will be eligible for 
enrolment in this study: 
1. Age between 18 and 55 years, inclusive. 
2. Diagnosis of relapsing forms of multiple sclerosis using revised McDonald criteria (Polman et al., 
2011). 
3. Stable on any FDA-approved disease modifying therapy (DMT). The term “stable” implies that the 
subject has not had change in therapy for any reason for the six months prior to study entry. 
4. 
5. EDSS 0 – 6.0 inclusive (Kurtzke, 1983). 
6. Understood and signed written informed consent, obtained prior to the study subject undergoing 
any study-related procedure, including screening tests. 
Exclusion Criteria 
Patients who meet any of the following exclusion criteria during the screening period will not be 
eligible for enrollment in this study: 
1. Known hypersensitivity to sulfonamides or derivatives 
2. Known history of renal or hepatic disease, cerebrovascular disease including stroke, transient 
ischemic attack, myocardial infarction, angina or congestive heart failure 
3. Evidence to suggest hyponatremia or hypokalemia, marked kidney dysfunction defined as 
creatinine greater than 2.0 mg/dL or liver disease dysfunction defined as aspartate 
aminotransferase (AST) or alanine aminotransferase (ALT) greater than three-fold upper limit of 
normal (ULN). 
4. Evidence to suggest suprarenal gland failure. 
5. Evidence of hyperchloremic acidosis. 
6. Initiation of new immunosuppressant treatment after the subject becomes protocol-eligible (except 
for corticosteroids) or enrolment in a concurrent trial. 
7. Prior treatment with mitoxantrone, natalizumab, rituximab, methotrexate, cladribine, 
cyclophosphamide or within 6 months of initiation of study. 
8. Subjects with any history of cytopenia. 
9. History of pulmonary obstruction or emphysema. 
10. Active hepatitis B or hepatitis C infection or evidence of cirrhosis. 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 15 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
11. Human immunodeficiency virus (HIV) positivity. 
12. Uncontrolled diabetes mellitus defined as HbA1c > 8% and/or requiring intensive management. 
13. Uncontrolled viral, fungal, or bacterial infection (excluding asymptomatic bacteriuria). 
14. Any condition that, in the opinion of the investigators, would jeopardize the ability of the subject to 
tolerate treatment with ACZ. 
15. Prior history of malignancy. 
16. Positive pregnancy test or inability or unwillingness to use effective means of birth control. 
Effective birth control is defined as: 
• 
Refraining from all acts of vaginal intercourse (abstinence) 
• 
Consistent use of birth control pills 
• 
Tubal sterilization or male partner who has undergone vasectomy 
• 
Placement of an IUD (intrauterine device) 
• 
Use, with every act of intercourse, of a diaphragm with contraceptive jelly and/or condoms with 
contraceptive foam 
17. Presence of metallic objects implanted in the body that would preclude the ability of the subject to 
safely have MRI exams. 
18. Psychiatric illness, mental deficiency, or cognitive dysfunction making compliance with treatment 
or informed consent impossible. 
 
Withdrawal Criteria 
 
Patients can be withdrawn at any time if the investigator concludes that it would be in the patients’ 
best interest for any reason. 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, or fail to return for 
visits, or become lost to follow up for any other reason. Protocol violations should not lead to patient 
withdrawal unless they indicate a significant risk to the patient’s safety. If premature patient 
withdrawal occurs for any reason, the principle investigator must determine the primary reason for a 
patient’s premature withdrawal from the study and record this information on the Unanticipated 
Problems Tracking Log. 
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 withdrawal), the investigator will show "due 
diligence" by documenting in the source documents steps taken to contact the patient, e.g., dates of 
telephone calls, registered letters, etc. In the case of death, a patient will be considered withdrawn. 
 
Subject Replacement 
Patients who prematurely withdraw will not be replaced. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 16 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
TRIAL SCHEDULE 
 
Visit Schedule: 
Stage 1a – IV ACZ in MS Subjects 
Total of 10 subjects (5 healthy and 5 MS patients) 
Day -7 
Screen 
0 min 
15 min 
30 min 
120 min 
240 min 
Treatment 
1 gm IV bolus ACZ administered 
immediately after ‘0 min’ pCASL scan 
H & P 
X 
CBC, CMP 
X 
Urine pregnancy 
X 
pCASL MRI 
X 
X 
X 
X 
X 
DSC MRI 
X 
 
Stage 1b –Oral ACZ dose-escalation in MS Subjects 
Up to a Total of 10 subjects 
Day -7 
Screen 
Wk 0 
Visit 1 
Wk 2 
Visit 2 
Wk 4 
Visit 3 
Wk 6 
Visit 4 
Wk 8 
Visit 6 
Treatment 
500 mg 
PO BID 
1000 
mg PO 
BID 
1500 
mg PO 
BID 
2000 
mg PO 
BID 
H & P 
X 
X 
X 
X 
X 
X 
CBC, CMP 
X 
X 
Urine 
pregnancy* 
X 
pCASL MRI 
X 
X 
X 
X 
X 
MSFC 
X 
X 
X 
X 
X 
EDSS 
X 
X 
X 
X 
X 
Blood Sample 
X 
X 
X 
X 
X 
* Female participants who have already been evaluated for pregnancy will not be 
retested unless otherwise indicated 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 17 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
• 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 18 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 19 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
 
Stage 2a – Safety/Tolerability & Kinetics of Treatment Effect 
• 
Up to a Total of 10 subjects 
Wk -4 
Screen 
Wk 0 
Visit 1 
Wk 6 
Visit 2 
Wk 12 
Visit 3 
Wk 14 
Visit 4 
Treatment 
1000 mg ACZ BID 
d/c ACZ 
H & P 
X 
X 
X 
X 
X 
CBC, CMP 
X 
X 
X 
X 
Urine 
pregnancy* 
X 
DSC MRI 
X 
X 
pCASL 
MRI 
X 
X 
X 
X 
MSFC 
X 
X 
X 
X 
EDSS 
X 
X 
X 
X 
MFIS, 
MSWS-12 
X 
X 
X 
X 
Blood 
Sample 
X 
X 
X 
X 
* Female participants who have already been evaluated for pregnancy will not be retested 
unless otherwise indicated 
- 
Additional CBC,CMP will be performed if measured ALT/AST > 1X ULN or if other 
alterations above baseline 
- 
EDSS – Expanded disability status scale 
- 
MSFC – Multiple Sclerosis Functional Composite Scale 
 
 
 
 
 
Stage 2b – Tolerability & Tachyphylaxis of ACZ in MS Subjects 
• 
Up to a Total of 10 subjects 
Wk -1 
Screen 
Wk 0 
Visit 1 
Wk 6 
Visit 2 
Wk 12 
Visit 3 
Wk 18 
Visit 4 
Wk 24 
Visit 5 
Treatment 
ACZ at dose identified in 
Stage 1b 
H & P 
X 
X 
X 
X 
X 
X 
CBC, CMP 
X 
X 
X 
X 
X 
Urine 
pregnancy* 
X 
DSC MRI 
X 
X 
X 
pCASL 
MRI 
X 
X 
X 
X 
X 
MSFC 
X 
X 
X 
X 
X 
EDSS 
X 
X 
X 
X 
X 
MFIS, 
MSWS-12 
X 
X 
X 
X 
X 
Blood 
Sample 
X 
X 
X 
X 
X 
* Female participants who have already been evaluated for pregnancy will not be retested unless 
otherwise indicated 
- 
Additional CBC,CMP will be performed if measured ALT/AST > 1X ULN or if other 
alterations above baseline 
- 
EDSS – Expanded disability status scale 
- 
MSFC – Multiple Sclerosis Functional Composite Scale 

HSC-MS-14-0450, Version No 6, April 1, 2019
 
 
 
 
Stage 3 – RCT; Long-term therapy with ACZ in MS Subjects 
• 
Delayed-start design with oral therapy “Treatment A” (ACZ) or “Treatment B” (placebo) 
• 
Total of 60 subjects 
Wk -1 
Screen 
Wk 
0 
Visit 1 
Wk 6 
Visit 
2 
Wk 
12 
Visit 
3 
Wk 
18 
Visit 
4 
Wk 
24 
Visit 
5 
Wk 
30 
Visit 
7 
Wk 
36 
Visit 
8 
Wk 
42 
Visit 
9 
Wk 
48 
Visit 10 
Wk 
50 
Visit 
11 
Treatment 
Randomize 
to ACZ or 
placebo 
d/c 
treatment 
End 
Study 
H & P 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
CBC, CMP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urine 
pregnancy* 
X 
DSC MRI 
X 
X 
X 
X 
X 
pCASL 
MRI 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
MSFC 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
EDSS 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
MFIS, 
MSWS-12 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Blood 
Sample 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
* Female participants who have already been evaluated for pregnancy will not be retested unless otherwise 
indicated 
- 
Additional CBC,CMP will be performed if measured ALT/AST > 1X ULN or if other alterations 
above baseline 
- 
EDSS – Expanded disability status scale 
- 
MSFC – Multiple Sclerosis Functional Composite Scale 
 
STUDY DESIGN 
 
Summary of Study Design 
 
The initial stages of this study are designed as a proof of principle to show that ACZ therapy improves 
cerebral perfusion in MS subjects. The final stage of this study is a prospective crossover design to 
evaluate the potential for ACZ to persistently enhance cerebral perfusion and impact the evolution of 
T2 hyperintense MS lesions into permanent black holes. This pilot study is anticipated to lead to a 
larger randomized phase 3 clinical trial. 
Participants will be recruited from the MS patient population seen at the UTP practice. The MS Clinic 
is a National MS Society affiliated comprehensive and coordinated center for MS care and a member 
of the Consortium of MS Centers. This clinic evaluates approximately 2700 MS patients per year. 
Given the safety and tolerability of study drug, we anticipate recruitment of two MS subjects each 
month for all stages of this study. 
 
Stage 1a - This stage will determine the degree of change, temporal kinetics and difference between 
various regions in cerebral perfusion after IV ACZ in subjects with MS. 
 
 
Protocol Number: 
Page 20 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 21 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
 
 
 
 
 
 
 
In total, this portion of the study is anticipated to be complete within 2.5 months. ACZ therapy and all 
image acquisition will be completed on the same day for subjects enrolled in stage 1a. 
As all subjects will be treated with IV ACZ, the image analyst will be unblinded during this stage. 
Cerebral perfusion measures will be determined by S.D. 
Stage 1b - Participants in this stage of the study will be given escalating divided doses of oral ACZ up 
to 4000 mg per day to determine both the magnitude of change to cerebral perfusion with 
maintenance daily oral ACZ therapy and to define the optimum dose(s) for additional studies. Stage 
1b and 2 of the study will utilize extended release oral ACZ, distributed by Hermann Research 
pharmacy and self-administered by the subject. 
 
Treatment and data collection for stage 1b will take 2.5 months for each enrolled subject. As many as 
two subjects can be evaluated during the month and we would anticipate that this portion of the study 
can be completed within twelve months. 
Image analysis will be performed once each subject completes treatment. Cerebral perfusion 
measures will be determined for each dose by the same unblinded analyst (S.D.). 
Stage 2a - This stage will continue to gather information about whether daily oral ACZ is 
tolerated and the kinetics of the treatment effect. Patients continue on the same dose for 12 
consecutive weeks and undergo repeat imaging at predetermined intervals. Sustained treatment 
over a short duration will allow us to rapidly determine whether sustained treatment with ACZ 
can alter cerebral blood flow and will provide a go/no-go for future studies using this 
drug/paradigm. Patients will obtain MRI scans and clinical evaluations every 6 weeks with 
contrast-based scans repeated at 12 weeks. 
 
 
 
 
 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 22 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
 
 
Stage 2b - This stage will determine long-term tolerability and if tachyphylaxis is present with 
oral ACZ. Patients will continue on the same dose for 24 consecutive weeks and undergo repeat 
imaging at predetermined intervals. MRI scans and clinical evaluations will be obtained every 6 
 
weeks with contrast-based scans repeated every 12 weeks. 
 
 
Stage 3 - This stage is a prospective delayed-start design to evaluate the potential for ACZ to 
persistently enhance cerebral perfusion and impact the evolution of MS lesions. This pilot study 
is anticipated to lead to a larger randomized phase 3 clinical trial. 
Subjects will obtain both contrast-enhanced and non-contrast MRI to fully evaluate both existing 
and newly formed MS lesions. In addition, contrast based analysis of cerebral perfusion offers 
measurements of CBV and MTT of blood, data not available with pCASL alone. DSC techniques 
will provide information that cannot be obtained by using pCASL alone that might be important 
to determine how blood flow is altered with ACZ. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 23 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
Subjects will be randomized to either ACZ or placebo first. Both subjects and raters (both clinical 
and imaging) will be blinded to treatment arm. Image analysis will be performed at the end of 
study. Each subject scan will be assigned a random five-digit number. Experienced imaging 
analysts (I.V., S.D., L.F.), blinded to treatment arm, will obtain manual and semi-automatic 
quantitative measures of global and regional cerebral blood flow, T2 lesion volume, T1 lesion 
volume, T1-gadolinium (Gd) positive lesion volume, gray and white matter volume and global 
and regional atrophy. We will compare quantitative imaging measures between treatments. 
METHODS AND ASSESSMENTS 
Patient recruitment, consenting and database procedures – All subjects for the study will be 
specifically recruited from the Neurology clinic located in the UTP building. Recruitment of all subjects 
will initially be sequential, based upon planned follow-up visits to the Neurology Clinic. Subsequent 
adjustments to patient recruitment might be introduced based upon periodic evaluation of evolving 
data that might dictate a more focused approach within certain patient groups. We anticipate that 
patient volunteers can be identified and consented on the day of their regularly scheduled Neurology 
Clinic visit. Thus, the first level of potential bias in the subjects studied will simply be that of the 
stochastic nature of the scheduled visits. However, there may be additional selection characteristics 
placed on recruitment of patient volunteers for the studies, should age or gender characteristics of 
the MS patients not be well reflected in the accumulating group. 
As patients consent for the study they will be given a unique patient study code number that will be 
used to identify them in a database specifically constructed for the study. Primary consent will 
concentrate on the nature of the study, a description of the database and their consent to be re- 
contacted by study personnel for future participation in the study. The primary consent will include a 
brief description of advanced brain MRI techniques that will be utilized. Any patient contacted later 
for their interest in other parts of this trial will then be re-consented, with the next procedure and its 
potential risks described in appropriate detail. All necessary patient demographics will be collected 
for entry into the database by a study coordinator/data entry manager (James Jemelka). 
Data handling and storage – The MRI-Analysis Center (MRI-AC), previously directed by Dr. Wolinsky 
and currently directed by me, has extensive experience with the management of large amounts of 
deidentified patient related data and images gathered for both local research studies and national 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 24 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
and international studies on thousands of subjects, each with multiple sessions. All data is stored on 
the MRI-AC macpdbs1 server physically secured in the University Professional Tower Data Center 
located within virtual Zone 100 (contains servers with protected health information as well as other 
sensitive or critical data; no access may be initiated directly from the Internet to Zone 100; all servers 
in Zone 100 are geographically located in secure areas). All workstations physically located in the 
MRI-AC are confined to Zone 40 (contains the bulk of the computing resources geographically 
distributed throughout the UTHSCH campus; no access may be initiated directly from the Internet into 
Zone 40; protected health information must be encrypted; rules for traffic initiated form Zone 40 to 
other areas: Internet is allowed, Zone 20 is allowed, Zone 100 is restricted to defined servers and 
protocols). Only individuals who can authenticate to the VPN appliance connected to Zone 40 and 
have appropriate authorization privileges may access MRI-AC resources from the Internet. Port 
configurations of the firewalls separating the Zones are managed by the UTHSCH Information 
Security Staff. Port configuration is managed by a firewall database in accordance with firewall 
policies and procedures. The MRI-AC Director is the information owner of the macpdbs1 server and 
the workstations. The steward of the macpdbs1 server is the Data Center Operations and Services 
(DCOS) division of the UTHSCH Information Technology Department. The steward of the MRI-AC 
workstations is the Medical School Information Technology (MSIT) Office. We will utilize these 
resources to manage and store all data and digital images generated as part of this project. 
Randomization and Blinding 
During stage 3 of this study, subjects will be randomized to either first receive ACZ or placebo. 
Both subjects and raters (clinical and image analysts) will be blinded to treatment arm. 
MRI scans for each subject will be assigned a unique non-contiguous random ID using a 5-digit 
random number generator and all imaging raters (I.V., S.D., L.F.) will be blinded to treatment 
arm. A semi-automated, previously validated tissue segmentation software (FreeSurfer v5.3, 
https://surfer.nmr.mgh.harvard.edu/) will be used to determine T2 hyperintense (lesion) and T1 
hypointense (black hole) volumes. A separate expert rater (K.H.) will review and analyse DTI 
images. An expert rater (I.V), blinded both to treatment arm and sequence of scan acquisition, 
will perform quality control checks of software segmentation. 
Image analysis to determine regional and global CBF as well as atrophy will be performed by 
additional qualified expert raters (S.D., L.F., K.H.) who are blinded to the treatment arm. 
Contraception and Pregnancy Testing 
Female participants must use effective means of birth control for the duration of the study. 
Effective birth control is defined as refraining from all acts of vaginal intercourse (abstinence), 
consistent use of birth control pills, tubal sterilization or male partner who has undergone 
vasectomy, placement of an IUD (intrauterine device), or use of a diaphragm with contraceptive 
jelly and/or condoms with contraceptive foam with every act of intercourse. 
Study participants will receive urine pregnancy tests prior to ACZ and as scheduled in the trial 
schedule (see section 4). 
 
 
 
 
 
 
 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 25 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Study Visits and Procedures 
 
Stage 1a: 
 
Screening Visit – Subjects will receive a complete medical and neurological examination. In addition, 
all subjects will obtain CBC, CMP and, if female, urine pregnancy test. 
Subjects will receive non-contrast MRI brain with scout, 3DT1 and pCASL sequences immediately 
prior to infusion of IV ACZ. The scout sequence is used to establish proper subject orientation and 
slice prescription and requires less than 5 minutes. The 3DT1 is a rapid sequence that allows for 
proper registration between scans and will be obtained after the scout images and again if the patient 
is removed from the MRI gantry.  Finally, pCASL sequences will be performed to determine CBF. 
The group of sequences including pCASL will collectively require about 12 minutes. 
Subjects will receive 1 gram bolus of IV ACZ and a second scan using pCASL sequences will be 
obtained within 15 minutes of infusion. Subjects will remain in the MRI gantry and receive a third 
scan to determine CBF at 30 minutes from time of infusion. Subjects will then be removed from the 
MRI gantry and allowed a break. 
Subjects will again be placed on the MRI gantry and scout, 3DT1 and pCASL sequences obtained 
within 120 minutes from time of ACZ infusion. Subjects will then be removed from the MRI gantry 
and allowed a break. A final scan will be performed using the same sequences within 240 minutes 
from time of ACZ infusion. 
 
 
Stage 1b: 
 
 
 
 
 
 
 
 
 
 
 
Screening Visit – Subjects will receive a complete medical and neurological examination. In addition, 
all subjects will obtain CBC, CMP and, if female, urine pregnancy test. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 26 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Visit 1 – Subjects will receive non-contrast MRI brain using sequences previously described to 
ensure proper orientation and registration (scout, 3DT1), sequences necessary for segmentation 
using MRIAP (2D dual echo, 2D fluid-attenuated inversion recovery [FLAIR]) and pCASL sequences 
to determine CBF at baseline. Subjects will begin taking 250 mg oral ACZ twice daily and continue 
for two weeks. Subjects will be encouraged to call the PI with any adverse events (see sections 2.4b 
and 7.1.2) while on study drug. 
Visit 2 – Prior to dose-escalation, subjects will receive non-contrast MRI brain sequences as 
described above to measure effect of previous oral daily divided dose of ACZ on CBF. Upon 
completion of MRI, subjects will begin taking 500 mg oral ACZ twice daily and continue for two 
weeks. Subjects will be encouraged to call the PI with any adverse events (see sections 2.4b and 
7.1.2) while on study drug. 
Visit 3 – Prior to dose-escalation, subjects will receive non-contrast MRI brain sequences as 
described above to measure effect of previous oral daily divided dose of ACZ on CBF. Upon 
completion of MRI, subjects will begin taking 1000 mg oral ACZ twice daily and continue for two 
weeks. Subjects will be encouraged to call the PI with any adverse events (see sections 2.4b and 
7.1.2) while on study drug. 
Visit 4 – Prior to dose-escalation, subjects will receive non-contrast MRI brain sequences as 
described above to measure effect of previous oral daily divided dose of ACZ on CBF. Upon 
completion of MRI, subjects will begin taking 1500 mg oral ACZ twice daily and continue for two 
weeks. Subjects will be encouraged to call the PI with any adverse events (see sections 2.4b and 
7.1.2) while on study drug. 
Visit 5 – Prior to dose-escalation, subjects will receive non-contrast MRI brain sequences as 
described above to measure effect of previous oral daily divided dose of ACZ on CBF. Upon 
completion of MRI, subjects will begin taking 2000 mg oral ACZ twice daily and continue for two 
weeks. Subjects will be encouraged to call the PI with any adverse events (see sections 2.4b and 
7.1.2) while on study drug. 
Visit 6 – Subjects will receive non-contrast MRI brain sequences as described above to measure 
effect of previous oral daily divided dose of ACZ on CBF. Upon completion of MRI, subjects will 
discontinue use of ACZ. 
 
Stage 2a: 
c) 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 27 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
 
Screening Visit – Subjects will receive a complete medical and neurological examination and 
obtain screening EDSS evaluation. In addition, all subjects will obtain CBC, CMP and, if female, 
urine pregnancy test. 
Visit 1 – Subjects will receive MRI brain with and without gadolinium contrast utilizing the 
following sequences in the order listed: 
Scout and 3D T1-weighted images to ensure proper orientation and registration 
2D dual echo, 3D FLAIR necessary for segmentation 
Muti-shell DWI (DTI sequences) 
A single shot 2D echo-planar imaging (EPI) for pCASL-based measures of CBF 
2D T1-weighted images prior to Gd administration 
DSC sequence for measures of CBF, CBV, MTT 
2D T1-weighted images after Gd administration 
In addition to segmenting MS lesion (T2 hyperintense), we are able to segment T1 pre- and 
post-Gd lesion volumes. Images obtained at the site at UT are routinely subject to review by a 
radiologist who is not further involved in image analysis. 
Visit 2 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
without gadolinium contrast utilizing the sequences described above except for DSC sequence. 
Visit 3 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
with and without gadolinium contrast utilizing the sequences described above. 
Upon completion of the MRI scan, all subjects will be instructed to discontinue ACZ treatment. 
Visit 4 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
without gadolinium contrast utilizing the sequences described above except for DSC sequence. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 28 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
a. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 29 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Stage 2b: 
 
 
Screening Visit – Subjects will receive a complete medical and neurological examination and 
obtain screening EDSS evaluation. In addition, all subjects will obtain CBC, CMP and, if female, 
urine pregnancy test. 
Visit 1 – Subjects will receive MRI brain with and without gadolinium contrast utilizing the MRI 
sequences as detailed in Stage 2a. 
An MRI scan with the sequences listed above as well as an MSFC & EDSS evaluation by a 
blinded rater (L.F.) will be obtained prior to treatment to determine baseline values. Patients will 
also complete subjective disability outcome measures MFIS and MSWS-12. Upon completion of 
the baseline scan, all subjects will start ACZ therapy and will continue for 24 weeks. 
Subjects will be contacted by telephone or email monthly to document tolerability and 
encouraged to report any AE occurring with drug at any point during the study. 
Visit 2 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
without gadolinium contrast utilizing the sequences described above except for DSC sequence. 
Visit 3 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
with and without gadolinium contrast utilizing the sequences described above. 
Visit 4 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
without gadolinium contrast utilizing the sequences described above except for DSC sequence. 
Visit 5 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
with and without gadolinium contrast utilizing the sequences described above. 
Upon completion of the MRI scan, all subjects will be instructed to discontinue treatment. 
 
 
Stage 3: Screening Visits and Procedures 
 
The screening visit will occur within one week of study entry. Subjects with relapsing forms of 
multiple sclerosis without evidence of other neurologic or cardiovascular disease will be 
recruited for this study. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 30 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Subjects will provide a complete medical history and undergo a complete medical and 
neurological exam. Blood tests (complete blood counts [CBC] and complete metabolic panel 
[CMP]) will be obtained from every study subject. Female subjects will also be given a urine 
pregnancy test during the screening visit. In addition, all subjects will undergo a screening 
MSFC and EDSS (Kurtzke, 1983). 
All subjects will be required to discontinue use of herbal supplements for the duration of the 
study. Finally, both male and female subjects will be counselled on the use of contraceptive 
methods (see section 6.2) during the study. 
 
 
Screening Visit – Subjects will receive a complete medical and neurological examination and 
obtain screening EDSS evaluation. In addition, all subjects will obtain CBC, CMP and, if female, 
urine pregnancy test. 
 
Visit 1 – Subjects will be randomized to receive Treatment A or Treatment B first for the initial 24 
weeks. Subjects will receive MRI brain with and without gadolinium contrast utilizing MRI 
sequences as detailed in Stage 2a. 
Subjects will be contacted by telephone or email monthly to document tolerability and 
encouraged to report any AE occurring with drug at any point during the study. 
Visit 2 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
without gadolinium contrast utilizing the sequences described above except for DSC sequence. 
Visit 3 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
with and without gadolinium contrast utilizing the sequences described above. 
Visit 4 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 31 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
without gadolinium contrast utilizing the sequences described above except for DSC sequence. 
Visit 5 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
with and without gadolinium contrast utilizing the sequences described above. 
Upon completion of the MRI scan, subjects previously treated with Treatment B will start 
Treatment A and all subjects will continue on Treatment A for the remaining 24 weeks of the 
study. 
Visit 6 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
with and without gadolinium contrast utilizing the sequences described above. 
Visit 7 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
without gadolinium contrast utilizing the sequences described above except for DSC sequence. 
Visit 8 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
with and without gadolinium contrast utilizing the sequences described above. 
Visit 9 – Subjects will receive a complete medical and neurological examination, MSFC & EDSS 
evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) and will 
be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI brain 
without gadolinium contrast utilizing the sequences described above except for DSC sequence. 
Visit 10 – Subjects will receive a complete medical and neurological examination, MSFC & 
EDSS evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) 
and will be asked questions to ascertain tolerability to daily therapy. All subjects will obtain MRI 
brain with and without gadolinium contrast utilizing the sequences described above. Upon 
completion of the brain MRI, all subjects will be instructed to discontinue assigned treatment. 
Visit 11 – Subjects will receive a complete medical and neurological examination, MSFC & 
EDSS evaluation as well as patient-reported clinical disability measures (MFIS & MSWS-12) 
and will be asked questions to ascertain any other AEs after discontinuation of therapy. All 
subjects will obtain MRI brain without gadolinium contrast utilizing the sequences described 
above except for DSC sequence. 
Subjects will be contacted by telephone or email monthly to document tolerability and 
encouraged to report any AE occurring with drug at any point during the study. 
For any part of the study, a subject whose CBC or CMP is not at or near baseline values will 
repeat these blood tests. 
a. Final Study Visit: 
The final study visit will be used to document any potential adverse event associated with 
treatment arm, not previously documented. 
 
b. Post Study Follow-up and Procedures 
Subjects will be referred to their primary care physician for continued monitoring as directed 
should there be persistent adverse events from use of study drug. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 32 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
c. Discontinuation Visit and Procedures 
Subjects may withdraw from study for any reason and will not be replaced. Subjects who 
choose to withdraw from the study will be encouraged to complete the end-of-study visit. 
Patients with intolerable paraesthesia, change in taste, accelerated change in blood 
pressure or fluid retention will be withdrawn from the study. Patients with persistent AEs will 
be referred to their primary care physician for continued monitoring and treatment using 
standard or care practices. 
TRIAL MATERIALS 
. Drug Substance: Intravenous Acetazolamide 
Package Insert for Intravenous Acetazolamide: 
AcetaZOLAMIDE for Injection USP 
For Intravenous Use. 
Rx ONLY. 
DESCRIPTION 
Acetazolamide, an inhibitor of the enzyme carbonic anhydrase, is a white to faintly yellowish white 
crystalline, odorless powder, weakly acidic, very slightly soluble in water and slightly soluble in 
alcohol. The chemical name for acetazolamide is N-(5-Sulfamoyl-1,3,4-thiadiazol-2yl)-acetamide and 
has the following structural formula: M.W. 222.24 C4H6N4O3S2 
Acetazolamide is available for intravenous use, and is supplied as a sterile powder requiring 
reconstitution. Each vial contains acetazolamide sodium equivalent to 500 mg of acetazolamide. The 
bulk solution is adjusted to pH 9.6 using sodium hydroxide and, if necessary, hydrochloric 
acid prior to lyophilization. 
CLINICAL PHARMACOLOGY 
Acetazolamide is a potent carbonic anhydrase inhibitor, effective in the control of fluid secretion (e.g., 
some types of glaucoma), in the treatment of certain convulsive disorders (e.g., epilepsy) and in the 
promotion of diuresis in instances of abnormal fluid retention (e.g., cardiac edema). Acetazolamide is 
not a mercurial diuretic. Rather, it is a nonbacteriostatic sulfonamide possessing a chemical structure 
and pharmacological activity distinctly different from the bacteriostatic sulfonamides. 
Acetazolamide is an enzyme inhibitor that acts specifically on carbonic anhydrase, the enzyme that 
catalyzes the reversible reaction involving the hydration of carbon dioxide and the dehydration of 
carbonic acid. In the eye, this inhibitory action of acetazolamide decreases the secretion of aqueous 
humor and results in a drop in intraocular pressure, a reaction considered desirable in cases of 
glaucoma and even in certain nonglaucomatous conditions. Evidence seems to indicate that 
acetazolamide has utility as an adjuvant in the treatment of certain dysfunctions of the central nervous 
system (e.g., epilepsy). Inhibition of carbonic anhydrase in this area appears to retard abnormal 
paroxysmal, excessive discharge from central nervous system neurons. The diuretic effect of 
acetazolamide is due to its action in the kidney on the reversible reaction involving hydration of carbon 
dioxide and dehydration of carbonic acid. The result is renal loss of HCO3 ion, which carries out 
sodium, water, and potassium. Alkalinization of the urine and promotion of diuresis are thus effected. 
Alteration in 
ammonia metabolism occurs due to increased reabsorption of ammonia by the renal tubules as a 
result of urinary alkalinization. 
INDICATIONS AND USAGE 
For adjunctive treatment of: edema due to congestive heart failure; drug-induced edema; 
centrencephalic epilepsies (petit mal, unlocalized seizures); chronic simple (open-angle) glaucoma, 
secondary glaucoma, and preoperatively in acute angle-closure glaucoma where delay of surgery 
desired in order to lower intraocular pressure. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 33 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
CONTRAINDICATIONS 
Acetazolamide therapy is contraindicated in situations in which sodium and/or potassium blood serum 
levels are depressed, in cases of marked kidney and liver disease or dysfunction, in suprarenal gland 
failure, and in hyperchloremic acidosis. It is contraindicated in patients with cirrhosis because of the 
risk of development of hepatic encephalopathy. 
Long-term administration of acetazolamide is contraindicated in patients with chronic noncongestive 
angle-closure glaucoma since it may permit organic closure of the angle to occur while the worsening 
glaucoma is masked by lowered intraocular pressure. 
WARNINGS 
Fatalities have occurred, although rarely, due to severe reactions to sulfonamides including Stevens- 
Johnson syndrome, toxic epidermal necrolysis, fulminant hepatic necrosis, agranulocytosis, aplastic 
anemia, and other blood dyscrasias. Sensitizations may recur when a sulfonamide is readministered 
irrespective of the route of administration. If signs of hypersensitivity or other serious reactions occur, 
discontinue use of this drug. Caution is advised for patients receiving concomitant high-dose aspirin 
an acetazolamide, as anorexia, tachypnea, lethargy, coma and death have been reported. 
PRECAUTIONS 
General 
Increasing the dose does not increase the diuresis and may increase the incidence of drowsiness 
and/or paresthesia. Increasing the dose often results in a decrease in diuresis. Under certain 
circumstances, however, very large doses have been given in conjunction with other diuretics in order 
to secure diuresis in complete refractory failure. 
Information for Patients 
Adverse reactions common to all sulfonamide derivatives may occur: anaphylaxis, fever, rash 
(including erythema multiforme, Stevens-Johnson syndrome, toxic epidermal necrolysis), crystalluria, 
renal calculus, bone marrow depression, thrombocytopenic purpura, hemolytic anemia, leukopenia, 
pancytopenia and agranulocytosis. Precaution is advised for early detection of such reactions and the 
drug should be discontinued and appropriate therapy instituted. 
In patients with pulmonary obstruction or emphysema where alveolar ventilation may be impaired, 
acetazolamide which may precipitate or aggravate acidosis, should be used with caution. 
Caution is advised for patients receiving concomitant high-dose aspirin and acetazolamide, as 
anorexia, tachypnea, lethargy, coma and death have been reported (see WARNINGS). 
Laboratory Tests 
To monitor for hematologic reactions common to all sulfonamides, it is recommended that a baseline 
CBC and platelet count be obtained on patients prior to initiating acetazolamide therapy and at regular 
intervals during therapy. If significant changes occur, early discontinuance and institution of 
appropriate therapy are important. Periodic monitoring of serum electrolytes is recommended. 
Carcinogenesis, Mutagenesis, Impairment of Fertility 
Long-term studies in animals to evaluate the carcinogenic potential of acetazolamide have not been 
conducted. In a bacterial mutagenicity assay, acetazolamide was not mutagenic when evaluated with 
and without metabolic activation. The drug had no effect on fertility when administered in the diet to 
male and female rats at a daily intake of up to 4 times the recommended human dose of 1000 mg in a 
50 kg individual. 
Pregnancy: Teratogenic Effects: Pregnancy Category C 
Acetazolamide, administered orally or parenterally, has been shown to be teratogenic (defects of the 
limbs) in mice, rats, hamsters and rabbits. There are no adequate and well-controlled studies in 
pregnant women. Acetazolamide should be used in pregnancy only if the potential benefit justifies the 
potential risk to the fetus. 
Nursing Mothers 
Because of the potential for serious adverse reaction in nursing infants from acetazolamide, a decision 
should be made whether to discontinue nursing or to discontinue the drug taking into account the 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 34 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
importance of the drug to the mother. 
Description of Oral ACZ Drug Product 
Manufactured by: 
Emcure Pharmaceuticals USA, Inc. 
21-B Cotters Lane 
East Brunswick, NJ 08816 
Distributed by: 
Zydus Pharmaceuticals USA Inc. 
Princeton, NJ 08534 
Repackaged by: 
American Health Packaging 
Columbus, Ohio 43217 
 
Package Insert 
ACETAZOLAMIDE- acetazolamide capsule, extended release 
American Health Packaging 
 
 
Acetazolamide Extended-Release Capsules 
Rx Only 
 
DESCRIPTION 
Acetazolamide extended-release capsules are an inhibitor of the enzyme carbonic anhydrase. 
Acetazolamide is a white to faintly yellowish white crystalline, odorless powder, weakly acidic, very 
slightly soluble in water and slightly soluble in alcohol. The chemical name for acetazolamide is N-(5- 
Sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide and has the following chemical structure: 
MW 222.24 
C4H6N4O3S2 
Each acetazolamide extended-release capsule intended for oral administration contains 500 mg of 
acetazolamide. In addition, each capsule contains the following inactive ingredients: ammonio 
methacrylate copolymer dispersion type A and B, FD&C yellow #6, gelatin, microcrystalline cellulose, 
sodium lauryl sulfate, talc and titanium dioxide. The capsule is printed with black pharmaceutical ink 
which contains black iron oxide as a coloring agent. 
 
CLINICAL PHARMACOLOGY 
Acetazolamide is a potent carbonic anhydrase inhibitor, effective in the control of fluid secretion (e.g., 
some types of glaucoma), in the treatment of certain convulsive disorders (e.g., epilepsy) and in the 
promotion of diuresis in instances of abnormal fluid retention (e.g., cardiac edema). 
Acetazolamide is not a mercurial diuretic. Rather, it is a non-bacteriostatic sulfonamide possessing a 
chemical structure and pharmacological activity distinctly different from the bacteriostatic 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 35 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
sulfonamides. 
Acetazolamide is an enzyme inhibitor that acts specifically on carbonic anhydrase, the enzyme that 
catalyzes the reversible reaction involving the hydration of carbon dioxide and the dehydration of 
carbonic acid. In the eye, this inhibitory action of acetazolamide decreases the secretion of aqueous 
humor and results in a drop in intraocular pressure, a reaction considered desirable in cases of 
glaucoma and even in certain non-glaucomatous conditions. Evidence seems to indicate that 
acetazolamide has utility as an adjuvant in treatment of certain dysfunctions of the central nervous 
system (e.g., epilepsy). Inhibition of carbonic anhydrase in this area appears to retard abnormal, 
paroxysmal, excessive discharge from central nervous system neurons. The diuretic effect of 
acetazolamide is due to its action in the kidney on the reversible reaction involving hydration of carbon 
dioxide and dehydration of carbonic acid. The result is renal loss of HCO3 ion, which carries out 
sodium, water, and potassium. Alkalinization of the urine and promotion of diuresis are thus affected. 
Alteration in ammonia metabolism occurs due to increased reabsorption of ammonia by the renal 
tubules as a result of urinary alkalinization. 
Acetazolamide extended-release capsules provide prolonged action to inhibit aqueous humor 
secretion for 18 to 24 hours after each dose, whereas tablets act for only eight to 12 hours. The 
prolonged continuous effect of acetazolamide permits a reduction in dosage frequency. 
Plasma concentrations of acetazolamide peak from three to six hours after administration of 
acetazolamide extended-release capsules, compared to one to four hours with tablets. Food does not 
affect bioavailability of acetazolamide extended-release capsules. 
Placebo-controlled clinical trials have shown that prophylactic administration of acetazolamide at a 
dose of 250 mg every eight to 12 hours (or a 500 mg controlled-release capsule once daily) before 
and during rapid ascent to altitude results in fewer and/or less severe symptoms of acute mountain 
sickness (AMS) such as headache, nausea, shortness of breath, dizziness, drowsiness, and fatigue. 
Pulmonary function (e.g., minute ventilation, expired vital capacity, and peak flow) is greater in the 
acetazolamide treated group, both in subjects with AMS and asymptomatic subjects. The 
acetazolamide treated climbers also had less difficulty in sleeping. 
 
INDICATIONS AND USAGE 
For adjunctive treatment of: chronic simple (open-angle) glaucoma, secondary glaucoma, and 
preoperatively in acute angle-closure glaucoma where delay of surgery is desired in order to lower 
intraocular pressure. Acetazolamide extended-release capsules are also indicated for the prevention 
or amelioration of symptoms associated with acute mountain sickness despite gradual ascent. 
 
CONTRAINDICATIONS 
Hypersensitivity to acetazolamide or any excipients in the formulation. Since acetazolamide is a 
sulfonamide derivative, cross sensitivity between acetazolamide, sulfonamides and other sulfonamide 
derivatives is possible. 
Acetazolamide therapy is contraindicated in situations in which sodium and/or potassium blood serum 
levels are depressed, in cases of marked kidney and liver disease or dysfunction, in suprarenal gland 
failure, and in hyperchloremic acidosis. It is contraindicated in patients with cirrhosis because of the 
risk of development of hepatic encephalopathy. 
Long-term administration of acetazolamide is contraindicated in patients with chronic non-congestive 
angle-closure glaucoma since it may permit organic closure of the angle to occur while the worsening 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 36 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
glaucoma is masked by lowered intraocular pressure. 
 
WARNINGS 
Fatalities have occurred, although rarely, due to severe reactions to sulfonamides including Stevens- 
Johnson syndrome, toxic epidermal necrolysis, fulminant hepatic necrosis, anaphylaxis, 
agranulocytosis, aplastic anemia, and other blood dyscrasias. Sensitizations may recur when a 
sulfonamide is readministered irrespective of the route of administration. If signs of hypersensitivity or 
other serious reactions occur, discontinue use of this drug. 
Caution is advised for patients receiving concomitant high-dose aspirin and acetazolamide, as 
anorexia, tachypnea, lethargy, metabolic acidosis, coma, and death have been reported. 
 
PRECAUTIONS 
 
General 
Increasing the dose does not increase the diuresis and may increase the incidence of drowsiness 
and/or paresthesia. Increasing the dose often results in a decrease in diuresis. Under certain 
circumstances, however, very large doses have been given in conjunction with other diuretics in order 
to secure diuresis in complete refractory failure. 
 
Information for Patients 
Adverse reactions common to all sulfonamide derivatives may occur: anaphylaxis, fever, rash 
(including erythema multiforme, Steven-Johnson syndrome, toxic epidermal necrolysis), crystalluria, 
renal calculus, bone marrow depression, thrombocytopenic purpura, hemolytic anemia, leukopenia, 
pancytopenia, and agranulocytosis. Caution is advised for early detection of such reactions and the 
drug should be discontinued and appropriate therapy instituted. 
In patients with pulmonary obstruction or emphysema where alveolar ventilation may be impaired, 
acetazolamide which may precipitate or aggravate acidosis should be used with caution. Gradual 
ascent is desirable to try to avoid acute mountain sickness. If rapid ascent is undertaken and 
acetazolamide is used, it should be noted that such use does not obviate the need for prompt descent 
if severe forms of high altitude sickness occur, i.e., high altitude pulmonary edema (HAPE) or high 
altitude cerebral edema. 
Caution is advised for patients receiving concomitant high-dose aspirin and acetazolamide, as 
anorexia, tachypnea, lethargy, metabolic acidosis, coma, and death have been reported (see 
WARNINGS). 
Both increases and decreases in blood glucose have been described in patients treated with 
acetazolamide. This should be taken into consideration in patients with impaired glucose tolerance or 
diabetes mellitus. 
Acetazolamide treatment may cause electrolyte imbalances, including hyponatremia and 
hypokalemia, as well as metabolic acidosis. Therefore, periodic monitoring of serum electrolytes is 
recommended. Particular caution is recommended in patients with conditions that are associated with, 
or predispose a patient to, electrolyte and acid/base imbalances, such as patients with impaired renal 
function (including elderly patients; see PRECAUTIONS, PRECAUTIONS, Geriatric Use), patients 
with diabetes mellitus, and patients with impaired alveolar ventilation. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 37 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Some adverse reactions to acetazolamide, such as drowsiness, fatigue, and myopia, may impair the 
ability to drive and operate machinery. 
 
Laboratory Tests 
To monitor for hematologic reactions common to all sulfonamides, it is recommended that a baseline 
CBC and platelet count be obtained on patients prior to initiating acetazolamide therapy and at regular 
intervals during therapy. If significant changes occur, early discontinuance and institution of 
appropriate therapy are important. Periodic monitoring of serum electrolytes is recommended. 
Drug Interactions 
Aspirin: 
See WARNINGS 
Acetazolamide modifies phenytoin metabolism with increased serum levels of phenytoin. This may 
increase or enhance the occurrence of osteomalacia in some patients receiving chronic phenytoin 
therapy. Caution is advised in patients receiving chronic concomitant therapy. By decreasing the 
gastrointestinal absorption of primidone, acetazolamide may decrease serum concentrations of 
primidone and its metabolites, with a consequent possible decrease in anticonvulsant effect. Caution 
is advised when beginning, discontinuing, or changing the dose of acetazolamide in patients receiving 
primidone.Because of possible additive effects with other carbonic anhydrase inhibitors, concomitant 
use is not advisable. 
Acetazolamide may increase the effects of other folic acid antagonists. 
Acetazolamide decreases urinary excretion of amphetamine and may enhance the magnitude and 
duration of their effect. 
Acetazolamide reduces urinary excretion of quinidine and may enhance its effect. 
Acetazolamide may prevent the urinary antiseptic effect of methenamine. 
Acetazolamide increases lithium excretion and the lithium may be decreased. 
Acetazolamide and sodium bicarbonate used concurrently increases the risk of renal calculus 
formation. 
Acetazolamide may elevate cyclosporine levels. 
 
Drug/Laboratory Test Interactions 
Sulfonamides may give false negative or decreased values for urinary phenolsulfonphthalein and 
phenol red elimination values for urinary protein, serum non-protein, and serum uric acid. 
Acetazolamide may produce an increased level of crystals in the urine. 
Acetazolamide interferes with the HPLC method of assay for theophylline. Interference with the 
theophylline assay by acetazolamide depends on the solvent used in the extraction; acetazolamide 
may not interfere with other assay methods for theophylline. 
 
Carcinogenesis, Mutagenesis, Impairment of Fertility 
Long-term studies in animals to evaluate the carcinogenic potential of acetazolamide has not been 
conducted. In a bacterial mutagenicity assay, acetazolamide was not mutagenic when evaluated with 
and without metabolic activation. 
The drug had no effect on fertility when administered in the diet to male and female rats at a daily 
intake of up to 4 times the recommended human dose of 1000 mg in a 50 kg individual. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 38 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
Pregnancy 
Teratogenic Effects 
Pregnancy Category C: 
Acetazolamide, administered orally or parenterally, has been shown to be teratogenic (defects of the 
limbs) in mice, rats, hamsters, and rabbits. There are no adequate and well-controlled studies in 
pregnant women. Acetazolamide should be used in pregnancy only if the potential benefit justifies the 
potential risk to the fetus. 
 
Nursing Mothers 
Because of the potential for serious adverse reactions in nursing infants from acetazolamide, a 
decision should be made whether to discontinue nursing or to discontinue the drug taking into account 
the importance of the drug to the mother. Acetazolamide should only be used by nursing women if the 
potential benefit justifies the potential risk to the child. 
 
Pediatric Use 
The safety and effectiveness of acetazolamide extended-release capsules in pediatric patients below 
the age of 12 years have not been established. Growth retardation has been reported in children 
receiving long-term therapy, believed secondary to chronic acidosis. 
 
Geriatric Use 
Metabolic acidosis, which can be severe, may occur in the elderly with reduced renal function. 
In general, dose selection for an elderly patient should be cautious, usually starting at the low end of 
the dosing range, reflecting the greater frequency of decreased hepatic, renal, or cardiac function, and 
concomitant disease or other drug therapy. 
 
ADVERSE REACTIONS 
Body as a whole: 
Headache, malaise, fatigue, fever, pain at injection site, flushing, growth retardation in children, flaccid 
paralysis, anaphylaxis. 
Digestive: 
Gastrointestinal disturbances such as nausea, vomiting, diarrhea. 
Hematological/Lymphatic: 
Blood dyscrasias such as aplastic anemia, agranulocytosis, leukopenia, thrombocytopenic purpura, 
melena. 
Hepato-biliary disorders: 
Abnormal liver function, cholestatic jaundice, hepatic insufficiency, fulminant hepatic necrosis 
Metabolic/Nutritional: 
Metabolic acidosis, electrolyte imbalance, including hypokalemia, hyponatremia, osteomalacia with 
long-term phenytoin therapy, loss of appetite, taste alteration, hyper/hypoglycemia 
Nervous: 
Drowsiness, paresthesia (including numbness and tingling of extremities and face), depression, 
excitement, ataxia, confusion, convulsions dizziness 
Skin: 
Allergic skin reactions including urticaria, photosensitivity, Stevens-Johnson syndrome, toxic 
epidermal necrolysis 
Special senses: 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 39 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Hearing disturbances, tinnitus, transient myopia 
Urogenital: 
Crystalluria, increased risk of nephrolithiasis with long-term therapy, hematuria, glycosuria, renal 
failure polyuria 
 
OVERDOSAGE 
No specific antidote is known. Treatment should be symptomatic and supportive. 
Electrolyte imbalance, development of an acidotic state, and central nervous system effects might be 
expected to occur. Serum electrolyte levels (particularly potassium) and blood pH levels should be 
monitored. 
Supportive measures are required to restore electrolyte and pH balance. The acidotic state can 
usually be corrected by the administration of bicarbonate. 
Despite its high intraerythrocytic distribution and plasma protein binding properties, acetazolamide 
may be dialyzable. This may be particularly important in the management of acetazolamide 
overdosage when complicated by the presence of renal failure. 
 
DOSAGE AND ADMINISTRATION 
 
Glaucoma: 
The recommended dosage is 1 capsule (500 mg) two times a day. Usually 1 capsule is administered 
in the morning and 1 capsule in the evening. It may be necessary to adjust the dose, but it has usually 
been found that dosage in excess of 2 capsules (1 g) does not produce an increased effect. The 
dosage should be adjusted with careful individual attention both to symptomatology and intraocular 
tension. In all cases, continuous supervision by a physician is advisable. 
In those unusual instances where adequate control is not obtained by the twice-a-day administration 
of acetazolamide extended-release capsules, the desired control may be established by means of 
acetazolamide (tablets or parenteral). Use tablets or parenteral in accordance with the more frequent 
dosage schedules recommended for these dosage forms, such as 250 mg every four hours, or an 
initial dose of 500 mg followed by 250 mg or 125 mg every four hours, depending on the case in 
question. 
 
Acute Mountain Sickness: 
Dosage is 500 mg to 1000 mg daily, in divided doses using tablets or extended-release capsules as 
appropriate. In circumstances of rapid ascent, such as in rescue or military operations, the higher 
dose level of 1000 mg is recommended. It is preferable to initiate dosing 24 to 48 hours before ascent 
and to continue for 48 hours while at high altitude, or longer as necessary to control symptoms. 
 
HOW SUPPLIED 
Acetazolamide Extended-Release Capsules, 500 mg are white to off-white pellets filled in empty hard 
gelatin capsules with orange opaque cap imprinted with "EP" in black ink and white opaque body 
imprinted with "107" in black ink and are supplied as follows: 
Unit dose packages of 30 (3x10) NDC 68084-401-21 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 40 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Storage and Drug Accountability 
 
Acetazolamide tablets will be stored at room temperature (between 68oF to 77oF) as suggested by the 
manufacturer. Intravenous formulation of ACZ will be stored as lyophilized powder at the temperature 
specified above in the Hermann Research pharmacy. Lyophilized powder will be reconstituted prior to 
infusion by the pharmacy.  All study medication will be stored at the Hermann Research pharmacy 
and dispensed as needed during study. 
 
TREATMENT 
Rationale for Selection of Dose 
 
The ACZ challenge test, using 1000 mg IV ACZ, is a commonly utilized clinical tool in evaluating 
patients with chronic intracerebral occlusive disease and is a reliable predictor of critically reduced 
cerebral perfusion (Vagal et al., 2009). Oral doses as high as 4000 mg daily have been previously 
used in the NORDIC clinical trial (NORDIC Idiopathic Intracranial Hypertension Study Group Writing 
Committee et al., 2014). 
Participants in this study will be treated with the highest tolerated dose of oral ACZ extended release 
capsules shown to exert the greatest effect on cerebral perfusion. This dose will also be utilized for an 
anticipated subsequent large phase 3 trial to determine clinical efficacy. 
 
Study Drug Formulations 
 
FDA approved extended release ACZ tablets from American Health will be used for “Treatment A”. 
Commercially available placebo capsules manufactured by American Health composed entirely of 
lactose monohydrate filler that will be used for “Treatment B”. 
 
Study Drug Administration 
 
“Treatment A” will be obtained directly from American Health and stored at MHHS pharmacy at 
Memorial Hermann Hospital. “Treatment B” is commercially available directly from American Health 
and will be shipped to MHHS pharmacy, shipped to CPHS pharmacy and stored. For stage 3, 
patients will be given either “Treatment A” or “Treatment B”, based upon randomization, in sufficient 
quantities for each study visit. At the end of each study visit, patients will be asked to return any 
unused capsules. Unused capsules will be counted and stored in a secure facility at the McGovern 
Medical School, UTHealth. 
 
Subjects with known hypersensitivity to sulfonamides or derivative drugs will be excluded from study 
participation (see section 3.4 for complete exclusion criteria). Subjects will be informed of common 
adverse reactions to sulfonamides including anaphylaxis, fever, rash (including erythema multiforme, 
Stevens-Johnson syndrome, toxic epidermal necrolysis), crystalluria, renal calculus, bone marrow 
depression, thrombocytopenic purpura, hemolytic anemia, leukopenia, pancytopenia and 
agranulocytosis. The drug will be discontinued and appropriate therapy instituted for any of these 
adverse reactions. 
 
Specific Restrictions / Requirements 
 
Subjects will be required to discontinue use of any frequently used over the counter medication, 
vitamin or other supplement one week prior to treatment with ACZ and for the duration of the study. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 41 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Blinding 
 
Stage 1a to 2b are unblinded stages. Stage 3 is the only double-blinded portion of this study. Both 
subjects and raters will remain blinded to treatment randomization.Common AE such as paresthesias, 
particularly a “tingling” feeling in the extremities, and gastrointestinal disturbances such as nausea, 
vomiting and diarrhea typically occur early in treatment and might be more pronounced in subjects 
initially randomized to ACZ. Subjects will be encouraged to avoid disclosing any AE so as to maintain 
the blind for clinical rater(s). Imaging rater(s) will not have direct contact with study subjects and no 
potential for unblinding is present. 
Patients with intolerable paraesthesia, change in taste, accelerated change in blood pressure or fluid 
retention will be withdrawn from the study. Patients with persistent AEs will be referred to their primary 
care physician for continued monitoring and treatment using standard or care practices. 
 
Concomitant therapy 
 
Subjects will be encouraged to refrain from any over the counter medication, vitamin or other 
supplement including concomitant use of high-dose aspirin for analgesia. Subjects will be warned that 
an increased potential for serious AE has been reported in patients taking both ACZ and high-dose 
aspirin. 
Subjects will be required, as stated in inclusion criteria (see section 3.3,) to be stable on any FDA- 
approved DMT. 
 
 
SAFETY MEASUREMENTS 
Definitions 
The descriptions and grading scales found in the NCI Common Terminology Criteria for Adverse 
Events v 3.0 (CTCAE; published June 10, 2013) will be utilized for AE reporting. 
 
Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment 
- 
Probable – The AE is likely related to the study treatment 
- 
Possible – The AE may be related to the study treatment 
- 
Unlikely – The AE is doubtfully related to the study treatment 
- 
Unrelated – The AE is clearly NOT related to the study treatment 
 
 
AEs should be recorded and graded 1 to 5 according to the CTCAE grades provided below: 
Grade 1 = Mild adverse event 
Grade 2 = Moderate adverse event 
Grade 3 = Severe and undesirable adverse event 
Grade 4 = Life-threatening or disabling adverse event 
Grade 5 = Death 
 
For additional information, consult the National Cancer Institute’s Common Terminology Criteria for 
Adverse Events v3.0 (published June 10, 2003) at the following URL: 
http://ctep.cancer.gov/reporting/ctc.html 
 
An AE is defined as any untoward medical occurrence in a patient administered a pharmaceutical 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 42 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
product. Adverse events are to be recorded in the clinical chart. Severity, action taken, and 
relationship will be recorded as noted in the chart. 
Serious Adverse Event (SAE) is defined as any untoward medical occurrence resulting in 
hospitalization or additional medical intervention in a patient administered a pharmaceutical product. 
SAE is to be recorded in the clinical chart. Severity, action taken, and relationship will be recorded as 
noted in the chart. 
A comprehensive list of AEs provides previously reported and/or potential AEs associated with ACZ. 
Common AEs are in bold. They include paresthesias, particularly a “tingling” feeling in the 
extremities, hearing dysfunction or tinnitus, loss of appetite, taste alteration and gastrointestinal 
disturbances such as nausea, vomiting, dyspepsia and diarrhea, polyuria, and occasional 
instances of drowsiness/fatigue and confusion. Metabolic acidosis and electrolyte imbalance may 
occur. Transient myopia has been reported though this condition invariably subsides upon diminution 
or discontinuance of the medication. 
Other occasional adverse reactions include urticaria, melena, hematuria, glycosuria, hepatic 
insufficiency, flaccid paralysis, photosensitivity and convulsions. 
Collecting, Recording and Reporting of Adverse Events 
Recording AEs – The treating physician will be the principal investigator (John Lincoln). The treating 
physician will oversee subject management including all assessments and treatment of adverse 
events and disease relapses should they occur. The treating physician will make decisions regarding 
determining relatedness and grading severity of all AEs. The treating physician will be blinded to 
treatment arm unless necessary to assess safety concern. 
Maintaining documentation and reporting AEs – The clinical coordinator (James Jemelka) will assist in 
subject management, maintain documentation including all subject case report forms (unanticipated 
problems tracking log) and will submit all reports to the IRB per UTSHC-Houston policy 
Safety Monitoring Plan 
This pilot study will determine the extent to which improved cerebral perfusion from oral ACZ therapy 
reduces the evolution of MS lesion into permanent black hole. Subjects will be monitored, as per 
protocol, by the treating physician for clinical MS activity. However, to our knowledge there are no 
case reports or other studies to suggest that therapy with ACZ will contribute to new lesion formation 
or clinical relapse. As such, we do not anticipate an increase in relapse frequency secondary to ACZ. 
Subjects will be monitored by the treating physician for AEs and serious AEs (see section 9.1). The 
frequency and grade of AEs will be documented and treatment arm(s) adjusted or discontinued as 
needed. Subjects can withdraw consent for study at any point and for any reason during the trial. 
ACZ is a well-established FDA approved therapy for various acute and chronic diseases. Patients 
generally tolerate drug without serious AEs. We have not observed any serious AE in our experience 
with IV or oral ACZ and do not anticipate any serious AE with oral ACZ used in this trial. 
To ensure safety to participants, an independent 3-person DSMB made up of two individuals with 
expertise in vascular neurology and one with statistical or epidemiological background will be 
coordinated by the UTHealth CCTS Regulatory Group. The 3-member board will meet biannually to 
review enrolment, evaluate reasons for screen failure, detail protocol deviations and treatment 
duration for participants. In addition, they will review all adverse events. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 43 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
DATA ANALYSIS 
Data Quality Assurance 
 
Each investigator in this study is experienced in the scientific method and have participated in or 
independently conducted clinical trials. All data during stage 2 of this study will be gathered by raters 
blinded to treatment. 
 
Data Entry and Storage 
 
As patients consent for the study they will be given a unique patient study code number that will be 
used to identify them in a database specifically constructed for the study. All necessary patient 
demographics will be collected for entry into the database by a study coordinator/data entry manager 
(James Jemelka). Information gathered on paper will be transferred to REDCap (Research Electronic 
Data CAPture), an electronic data capture software package available to academic institutions. 
The MRI-Analysis Center (MRI-AC), directed by Dr. Wolinsky, has extensive experience with the 
management of large amounts of de-identified patient related data and images gathered for both local 
research studies and national and international studies on thousands of subjects, each with multiple 
sessions. All data is stored on the MRI-AC macpdbs1 server physically secured in the University 
Professional Tower Data Center located within virtual Zone 100 (contains servers with protected 
health information as well as other sensitive or critical data; no access may be initiated directly from 
the Internet to Zone 100; all servers in Zone 100 are geographically located in secure areas). All 
workstations physically located in the MRI-AC are confined to Zone 40 (contains the bulk of the 
computing resources geographically distributed throughout the UTHSCH campus; no access may be 
initiated directly from the Internet into Zone 40; protected health information must be encrypted; rules 
for traffic initiated form Zone 40 to other areas: Internet is allowed, Zone 20 is allowed, Zone 100 is 
restricted to defined servers and protocols). Only individuals who can authenticate to the VPN 
appliance connected to Zone 40 and have appropriate authorization privileges may access MRI-AC 
resources from the Internet. Port configurations of the firewalls separating the Zones are managed by 
the UTHSCH Information Security Staff. Port configuration is managed by a firewall database in 
accordance with firewall policies and procedures. The MRI-AC Director is the information owner of the 
macpdbs1 server and the workstations. The steward of the macpdbs1 server is the Data Center 
Operations and Services (DCOS) division of the UTHSCH Information Technology Department. The 
steward of the MRI-AC workstations is the Medical School Information Technology (MSIT) Office. We 
will utilize these resources to manage and store all data and digital images generated as part of this 
project. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 44 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
SAMPLE SIZE AND STATISTICAL METHODS 
Studies have shown between 10% - 15% decrease in cerebral perfusion in patients with relapsing MS 
(Adhya et al., 2006). Therefore, increases in CBF of at least 15% might result in change to lesion 
evolution. 
The primary aim of the proposed study is to evaluate the change in global CBF with ACZ, so the 
primary outcome is the change in global CBF after 24 weeks relative to pre-treatment baseline. Based 
on the preliminary data, we expect to see a 15% difference between the ACZ treatment and placebo 
groups on the improvement in global cerebral perfusion with standard deviation (SD) of 30%. We ran 
Monte Carlo simulations under a linear mixed effect model (same model as planned for analysis) and 
calculated a needed total sample size of n=50 to detect a group difference of 0.5 SD at the 24 weeks 
(phase 1) assuming a within-subject correlation of 0.5, a significance level of 0.05, and 90% power. 
We hypothesize a similar group difference in phase 2 (ACZ-ACZ group would not change between 
weeks 24 and 48 but delayed-start group would improve) and similar power to detect this effect. With 
50 patients, for Aim 1.2, we will have 80% power to detect an average correlation as low as 0.39 
between CBF and MD or FA. Considering a dropout rate of 18%, we need to recruit 60 participants. 
Aims 2 are mainly exploratory and parameters derived from this study will be used to help design 
future larger scale confirmatory studies. 
All analyses will be intent-to-treat. To summarize the data, descriptive statistics will be used. 
Demographics and baseline characteristics at randomization will be summarized for each treatment 
arm. Generalized linear mixed models will be used to analyze all outcomes and will include treatment 
group and time, group-by-time interaction, and disease duration (stratifying variable) as covariates, 
and a random intercept to account for within-patient correlation. 
For the primary outcome, we will focus on comparing the treatment groups in phase 1. A linear mixed 
model will be used to compare the estimated change in global CBF between baseline and week 24 in 
the ACZ and delayed-start groups. A secondary analysis will study ACZ treatment effect on CBF in 
different regions by treating regions as clusters in the linear mixed model. If normality of CBF outcome 
does not hold, an appropriate transformation or different outcome distribution will be used. 
For the secondary endpoint at 48 weeks (phase 2), a similar mixed model will be used to compare 
changes in global CBF between weeks 24 and 48 weeks in the early-start and delayed-start groups. 
The model will include the same terms as for phase 1. 
Secondary outcomes considers repeated measures of MD or FA at different time in different regions. 
Linear mixed models will be used to evaluate changes in MD and FA from baseline (24 weeks for 
delayed-start) to 24 weeks (48 weeks for delayed-start) after ACZ therapy. These changes will be 
compared between regions with and without increased CBF (>15% difference from baseline). Terms 
in the model will be as for CBF with the addition of increased CBF (yes/no) for the region. Mixed 
models will also be used to compare MD and FA changes between white matter areas with and 
without lesions. 
Logistic or linear mixed models will be used to evaluate change from baseline to after 24 week ACZ 
therapy (using phase 1 and phase 2 data) in the tertiary composite change outcome. Models will 
include the same terms as for analyses of the primary outcome. We will evaluate associations 
between changes in clinical outcomes and changes in CBF, CBV, and MTT by including each of these 
variables (one at a time) as a time-varying covariate. 
As this trial uses a small sample size, we would not expect statistically significant treatment effects on 
clinical disability though this exploratory measure should be useful in power calculation for the 
anticipated larger, randomized clinical trial. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 45 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 
ETHICAL CONSIDERATIONS 
Informed Consent 
 
The informed consent form is a method of providing information regarding the trial to a prospective 
participant and allows for an informed decision about participation in the study. All participants (or 
their legally acceptable representative) must read, sign, and date a consent form before participating 
in the study, taking study drug, and/or undergoing any study-specific procedures. If a participant does 
not speak and read English, the consent materials will be translated into the Spanish. 
The informed consent form will be updated or revised whenever important new safety information is 
available, whenever the protocol is amended, and/or whenever any new information becomes 
available that may affect a subject’s participation in the trial. 
A copy of the informed consent form will be given to a prospective participant for review. The principle 
investigator (J.L.), in the presence of a witness, will review the consent form, discuss potential AEs 
associated with ACZ therapy and, when necessary, discuss the imaging protocol to be utilized and 
answer questions. The participant will be informed that their participation is voluntary and that they 
may withdraw from the study at any time, for any reason. 
 
IRB review 
 
This protocol and the associated informed consent documents must be submitted to the IRB for 
review and approval. 
 
Confidentiality of Data and Patient Records 
 
As patients consent for the study they will be given a unique patient study code number that will be 
used to identify them in a database specifically constructed for the study. All necessary patient 
demographics will be collected for entry into the database by a study coordinator/data entry manager 
(James Jemelka). 
Results of blood and urine tests obtained during the study period will also be coded to the patient and 
collected for entry into the database by a study coordinator/data entry manager. The study PI will 
review test results solely to determine subject exclusion or removal from the study and to document 
laboratory-specific AE. 
 
PUBLICATIONS 
 
The data or results of the completed study may become the subject of, or contained within, a refereed, 
reputable peer review journal. 
 
RETENTION OF TRIAL DOCUMENTS 
 
Records for all participants, including CRFs, all source documentation (containing evidence to study 
eligibility, history and physical findings, laboratory data, results of consultations, etc.) as well as IRB 
records and other regulatory documentation will be collected by the study coordinator/data entry 
manager (James Jemelka) and PI (John Lincoln) and will be stored on-site at UT Health. The PI will 
be the sole custodian of this information. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 46 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 47 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
CLINICAL PROTOCOL – REFERENCES 
 
Acetazolamide Tablets | Drug Summary | PDR.net [WWW Document], n.d. URL 
http://www.pdr.net/drug-summary/acetazolamide-tablets?druglabelid=667&id=637 (accessed 
2.12.14). 
Adhya, S., Johnson, G., Herbert, J., Jaggi, H., Babb, J.S., Grossman, R.I., Inglese, M., 2006. Pattern of 
hemodynamic impairment in multiple sclerosis: Dynamic susceptibility contrast perfusion MR 
imaging at 3.0 T. NeuroImage 33, 1029–1035. doi:10.1016/j.neuroimage.2006.08.008 
Aslan, S., Xu, F., Wang, P.L., Uh, J., Yezhuvath, U.S., van Osch, M., Lu, H., 2010. Estimation of 
labeling efficiency in pseudocontinuous arterial spin labeling. Magn. Reson. Med. Off. J. Soc. 
Magn. Reson. Med. Soc. Magn. Reson. Med. 63, 765–771. doi:10.1002/mrm.22245 
Barnett, M.H., Henderson, A.P.D., Prineas, J.W., 2006. The macrophage in MS: just a scavenger after 
all? Pathology and pathogenesis of the acute MS lesion. Mult. Scler. Houndmills Basingstoke 
Engl. 12, 121–132. 
Barnett, M.H., Prineas, J.W., 2004. Relapsing and remitting multiple sclerosis: pathology of the newly 
forming lesion. Ann. Neurol. 55, 458–468. doi:10.1002/ana.20016 
Caramanos, Z., Francis, S.J., Narayanan, S., Lapierre, Y., Arnold, D.L., 2012. Large, nonplateauing 
relationship between clinical disability and cerebral white matter lesion load in patients with 
multiple sclerosis. Arch. Neurol. 69, 89–95. doi:10.1001/archneurol.2011.765 
Cotton, F., Weiner, H.L., Jolesz, F.A., Guttmann, C.R.G., 2003. MRI contrast uptake in new lesions in 
relapsing-remitting MS followed at weekly intervals. Neurology 60, 640–646. 
Dai, W., Garcia, D., de Bazelaire, C., Alsop, D.C., 2008. Continuous flow-driven inversion for arterial 
spin labeling using pulsed radio frequency and gradient fields. Magn. Reson. Med. Off. J. Soc. 
Magn. Reson. Med. Soc. Magn. Reson. Med. 60, 1488–1497. doi:10.1002/mrm.21790 
Debernard, L., Melzer, T.R., Van Stockum, S., Graham, C., Wheeler-Kingshott, C.A., Dalrymple-Alford, 
J.C., Miller, D.H., Mason, D.F., 2013. Reduced grey matter perfusion without volume loss in 
early relapsing-remitting multiple sclerosis. J. Neurol. Neurosurg. Psychiatry. doi:10.1136/jnnp- 
2013-305612 
Filippi, M., 2000. Enhanced magnetic resonance imaging in multiple sclerosis. Mult. Scler. Houndmills 
Basingstoke Engl. 6, 320–326. 
Friberg, L., Kastrup, J., Rizzi, D., Jensen, J.B., Lassen, N.A., 1990. Cerebral blood flow and end-tidal 
PCO2 during prolonged acetazolamide treatment in humans. Am. J. Physiol. 258, H954-959. 
Ghassemi, R., Narayanan, S., Banwell, B., Sled, J.G., Shroff, M., Arnold, D.L., Canadian Pediatric 
Demyelinating Disease Network, 2014. Quantitative determination of regional lesion volume and 
distribution in children and adults with relapsing-remitting multiple sclerosis. PloS One 9, 
e85741. doi:10.1371/journal.pone.0085741 
Giorgio, A., Stromillo, M.L., Bartolozzi, M.L., Rossi, F., Battaglini, M., De Leucio, A., Guidi, L., Maritato, 
P., Portaccio, E., Sormani, M.P., Amato, M.P., De Stefano, N., 2013. Relevance of hypointense 
brain MRI lesions for long-term worsening of clinical disability in relapsing multiple sclerosis. 
Mult. Scler. J. 20, 214–219. doi:10.1177/1352458513494490 
Hartkamp, N.S., Hendrikse, J., van der Worp, H.B., de Borst, G.J., Bokkers, R.P.H., 2012. Time course 
of vascular reactivity using repeated phase-contrast MR angiography in patients with carotid 
artery stenosis. Stroke J. Cereb. Circ. 43, 553–556. doi:10.1161/STROKEAHA.111.637314 
Helenius, J., Perkiö, J., Soinne, L., Østergaard, L., Carano, R.A.D., Salonen, O., Savolainen, S., Kaste, 
M., Aronen, H.J., Tatlisumak, T., 2003. Cerebral hemodynamics in a healthy population 
measured by dynamic susceptibility contrast MR imaging. Acta Radiol. Stockh. Swed. 1987 44, 
538–546. 
Holland, C.M., Charil, A., Csapo, I., Liptak, Z., Ichise, M., Khoury, S.J., Bakshi, R., Weiner, H.L., 
Guttmann, C.R.G., 2012. The relationship between normal cerebral perfusion patterns and 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 48 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
white matter lesion distribution in 1,249 patients with multiple sclerosis. J. Neuroimaging Off. J. 
Am. Soc. Neuroimaging 22, 129–136. doi:10.1111/j.1552-6569.2011.00585.x 
Juurlink, B.H.J., 2013. The evidence for hypoperfusion as a factor in multiple sclerosis lesion 
development. Mult. Scler. Int. 2013, 598093. doi:10.1155/2013/598093 
Koch-Henriksen, N., Sørensen, P.S., 2010. The changing demographic pattern of multiple sclerosis 
epidemiology. Lancet Neurol. 9, 520–532. doi:10.1016/S1474-4422(10)70064-8 
Kurtzke, J.F., 1983. Rating neurologic impairment in multiple sclerosis: an expanded disability status 
scale (EDSS). Neurology 33, 1444–1452. 
Lassmann, H., 2011. Review: The architecture of inflammatory demyelinating lesions: implications for 
studies on pathogenesis: Active lesions in multiple sclerosis. Neuropathol. Appl. Neurobiol. 37, 
698–710. doi:10.1111/j.1365-2990.2011.01189.x 
Leary, S.M., 2005. Multiple sclerosis: diagnosis and the management of acute relapses. Postgrad. Med. 
J. 81, 302–308. doi:10.1136/pgmj.2004.029413 
Lim, L.-L., Foldvary, N., Mascha, E., Lee, J., 2001. Acetazolamide in Women with Catamenial Epilepsy. 
Epilepsia 42, 746–749. doi:10.1046/j.1528-1157.2001.33600.x 
Meier, D.S., Guttmann, C.R.G., 2006. MRI time series modeling of MS lesion development. 
NeuroImage 32, 531–537. doi:10.1016/j.neuroimage.2006.04.181 
Narayana, P.A., Doyle, T.J., Lai, D., Wolinsky, J.S., 1998. Serial proton magnetic resonance 
spectroscopic imaging, contrast-enhanced magnetic resonance imaging, and quantitative lesion 
volumetry in multiple sclerosis. Ann. Neurol. 43, 56–71. doi:10.1002/ana.410430112 
Narayana, P.A., Zhou, Y., Hasan, K.M., Datta, S., Sun, X., Wolinsky, J.S., 2014. Hypoperfusion and T1- 
hypointense lesions in white matter in multiple sclerosis. Mult. Scler. Houndmills Basingstoke 
Engl. 20, 365–373. doi:10.1177/1352458513495936 
NORDIC Idiopathic Intracranial Hypertension Study Group Writing Committee, Wall, M., McDermott, 
M.P., Kieburtz, K.D., Corbett, J.J., Feldon, S.E., Friedman, D.I., Katz, D.M., Keltner, J.L., 
Schron, E.B., Kupersmith, M.J., 2014. Effect of acetazolamide on visual function in patients with 
idiopathic intracranial hypertension and mild visual loss: the idiopathic intracranial hypertension 
treatment trial. JAMA J. Am. Med. Assoc. 311, 1641–1651. doi:10.1001/jama.2014.3312 
Orton, S.-M., Herrera, B.M., Yee, I.M., Valdar, W., Ramagopalan, S.V., Sadovnick, A.D., Ebers, G.C., 
Canadian Collaborative Study Group, 2006. Sex ratio of multiple sclerosis in Canada: a 
longitudinal study. Lancet Neurol. 5, 932–936. doi:10.1016/S1474-4422(06)70581-6 
Ota, M., Sato, N., Nakata, Y., Ito, K., Kamiya, K., Maikusa, N., Ogawa, M., Okamoto, T., Obu, S., Noda, 
T., Araki, M., Yamamura, T., Kunugi, H., 2013. Abnormalities of cerebral blood flow in multiple 
sclerosis: a pseudocontinuous arterial spin labeling MRI study. Magn. Reson. Imaging 31, 990– 
995. doi:10.1016/j.mri.2013.03.016 
Papadaki, E.Z., Mastorodemos, V.C., Amanakis, E.Z., Tsekouras, K.C., Papadakis, A.E., Tsavalas, 
N.D., Simos, P.G., Karantanas, A.H., Plaitakis, A., Maris, T.G., 2012. White matter and deep 
gray matter hemodynamic changes in multiple sclerosis patients with clinically isolated 
syndrome. Magn. Reson. Med. Off. J. Soc. Magn. Reson. Med. Soc. Magn. Reson. Med. 68, 
1932–1942. doi:10.1002/mrm.24194 
Papadaki, E.Z., Simos, P.G., Panou, T., Mastorodemos, V.C., Maris, T.G., Karantanas, A.H., Plaitakis, 
A., 2013. Hemodynamic evidence linking cognitive deficits in clinically isolated syndrome to 
regional brain inflammation. Eur. J. Neurol. Off. J. Eur. Fed. Neurol. Soc. 
doi:10.1111/ene.12338 
Papadopoulos, K., Tozer, D.J., Fisniku, L., Altmann, D.R., Davies, G., Rashid, W., Thompson, A.J., 
Miller, D.H., Chard, D.T., 2010. TI-relaxation time changes over five years in relapsing-remitting 
multiple sclerosis. Mult. Scler. Houndmills Basingstoke Engl. 16, 427–433. 
doi:10.1177/1352458509359924 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 49 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Parry, A., Clare, S., Jenkinson, M., Smith, S., Palace, J., Matthews, P.M., 2002. White matter and 
lesion T1 relaxation times increase in parallel and correlate with disability in multiple sclerosis. J. 
Neurol. 249, 1279–1286. doi:10.1007/s00415-002-0837-7 
Polman, C.H., Reingold, S.C., Banwell, B., Clanet, M., Cohen, J.A., Filippi, M., Fujihara, K., Havrdova, 
E., Hutchinson, M., Kappos, L., Lublin, F.D., Montalban, X., O’Connor, P., Sandberg-Wollheim, 
M., Thompson, A.J., Waubant, E., Weinshenker, B., Wolinsky, J.S., 2011. Diagnostic criteria for 
multiple sclerosis: 2010 Revisions to the McDonald criteria. Ann. Neurol. 69, 292–302. 
doi:10.1002/ana.22366 
Reingold, S.C., 2002. Prevalence estimates for MS in the United States and evidence of an increasing 
trend for women. Neurology 59, 294; author reply 294-295. 
Renoux, C., Vukusic, S., Mikaeloff, Y., Edan, G., Clanet, M., Dubois, B., Debouverie, M., Brochet, B., 
Lebrun-Frenay, C., Pelletier, J., Moreau, T., Lubetzki, C., Vermersch, P., Roullet, E., Magy, L., 
Tardieu, M., Suissa, S., Confavreux, C., Adult Neurology Departments KIDMUS Study Group, 
2007. Natural history of multiple sclerosis with childhood onset. N. Engl. J. Med. 356, 2603– 
2613. doi:10.1056/NEJMoa067597 
Sahraian, M.A., Radue, E.-W., Haller, S., Kappos, L., 2010. Black holes in multiple sclerosis: definition, 
evolution, and clinical correlations. Acta Neurol. Scand. 122, 1–8. doi:10.1111/j.1600- 
0404.2009.01221.x 
Stewart, W.C., Halper, L.K., Johnson-Pratt, L., Polis, A., Hartenbaum, D., 2002. Tolerability and efficacy 
of dorzolamide versus acetazolamide added to timolol. J. Ocul. Pharmacol. Ther. Off. J. Assoc. 
Ocul. Pharmacol. Ther. 18, 211–220. doi:10.1089/108076802760116133 
Sullivan, H.G., Kingsbury, T.B., 4th, Morgan, M.E., Jeffcoat, R.D., Allison, J.D., Goode, J.J., McDonnell, 
D.E., 1987. The rCBF response to Diamox in normal subjects and cerebrovascular disease 
patients. J. Neurosurg. 67, 525–534. doi:10.3171/jns.1987.67.4.0525 
Thaler, C., Faizy, T.D., Sedlacik, J., Holst, B., Stürner, K., Heesen, C., Stellmann, J.-P., Fiehler, J., 
Siemonsen, S., 2017. T1 Recovery Is Predominantly Found in Black Holes and Is Associated 
with Clinical Improvement in Patients with Multiple Sclerosis. AJNR Am. J. Neuroradiol. 38, 
264–269. doi:10.3174/ajnr.A5004 
Trapp, B.D., Stys, P.K., 2009. Virtual hypoxia and chronic necrosis of demyelinated axons in multiple 
sclerosis. Lancet Neurol. 8, 280–291. doi:10.1016/S1474-4422(09)70043-2 
Truyen, L., van Waesberghe, J.H., van Walderveen, M.A., van Oosten, B.W., Polman, C.H., Hommes, 
O.R., Adèr, H.J., Barkhof, F., 1996. Accumulation of hypointense lesions (“black holes”) on T1 
spin-echo MRI correlates with disease progression in multiple sclerosis. Neurology 47, 1469– 
1476. 
Tullman, M.J., 2013. A review of current and emerging therapeutic strategies in multiple sclerosis. Am. 
J. Manag. Care 19, S21-27. 
Vagal, A.S., Leach, J.L., Fernandez-Ulloa, M., Zuccarello, M., 2009. The Acetazolamide Challenge: 
Techniques and Applications in the Evaluation of Chronic Cerebral Ischemia. Am. J. 
Neuroradiol. 30, 876–884. doi:10.3174/ajnr.A1538 
van Walderveen, M.A., Barkhof, F., Pouwels, P.J., van Schijndel, R.A., Polman, C.H., Castelijns, J.A., 
1999a. Neuronal damage in T1-hypointense multiple sclerosis lesions demonstrated in vivo 
using proton magnetic resonance spectroscopy. Ann. Neurol. 46, 79–87. 
van Walderveen, M.A., Kamphorst, W., Scheltens, P., van Waesberghe, J.H., Ravid, R., Valk, J., 
Polman, C.H., Barkhof, F., 1998. Histopathologic correlate of hypointense lesions on T1- 
weighted spin-echo MRI in multiple sclerosis. Neurology 50, 1282–1288. 
van Walderveen, M.A., Truyen, L., van Oosten, B.W., Castelijns, J.A., Lycklama à Nijeholt, G.J., van 
Waesberghe, J.H., Polman, C., Barkhof, F., 1999b. Development of hypointense lesions on T1- 
weighted spin-echo magnetic resonance images in multiple sclerosis: relation to inflammatory 
activity. Arch. Neurol. 56, 345–351. 

Protocol Number: HSC-MS-14-0450, Version No 6, April 1, 2019 
Page 50 
IRB NUMBER: HSC-MS-14-0450 
IRB APPROVAL DATE: 06/27/2019 
 
Walsh, A.J., Blevins, G., Lebel, R.M., Seres, P., Emery, D.J., Wilman, A.H., 2014. Longitudinal MR 
imaging of iron in multiple sclerosis: an imaging marker of disease. Radiology 270, 186–196. 
doi:10.1148/radiol.13130474 
Warntjes, J.B.M., Engström, M., Tisell, A., Lundberg, P., 2013. Brain characterization using normalized 
quantitative magnetic resonance imaging. PloS One 8, e70864. 
doi:10.1371/journal.pone.0070864 
Wong, E.C., 2007. Vessel-encoded arterial spin-labeling using pseudocontinuous tagging. Magn. 
Reson. Med. Off. J. Soc. Magn. Reson. Med. Soc. Magn. Reson. Med. 58, 1086–1091. 
doi:10.1002/mrm.21293 
Wu, W.-C., Fernández-Seara, M., Detre, J.A., Wehrli, F.W., Wang, J., 2007. A theoretical and 
experimental investigation of the tagging efficiency of pseudocontinuous arterial spin labeling. 
Magn. Reson. Med. Off. J. Soc. Magn. Reson. Med. Soc. Magn. Reson. Med. 58, 1020–1027. 
doi:10.1002/mrm.21403 
Wuerfel, J., Bellmann-Strobl, J., Brunecker, P., Aktas, O., McFarland, H., Villringer, A., Zipp, F., 2004. 
Changes in cerebral perfusion precede plaque formation in multiple sclerosis: a longitudinal 
perfusion MRI study. Brain J. Neurol. 127, 111–119. doi:10.1093/brain/awh007