Clinical trial NCT02633397
Conditions: Sickle Cell Disease
Keywords: SCD Sickle Cell Disease Riociguat Adempas
Sponsor: Mark Gladwin
Start date: 2017-04-11; Completion date: 2022-05-04
Enrollment: 130 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The proposed study is a Phase 2 multi-center, randomized, double-blind, placebo-controlled, parallel groups study aimed to evaluate the safety, tolerability and the efficacy of riociguat compared with placebo in patients with sickle cell disease (SCD).

Detailed description:
This randomized study involves 12 weeks of treatment with riociguat pills or placebo pills, and a follow-up period of 30 days after treatment. The dose is adjusted every 2 weeks based on systolic blood pressure (SBP) and well-being assessed at that visit. Physical examinations, vital signs, blood tests and questionnaires will be performed at 2 week intervals during the double blinded study treatment. Echocardiogram, urine testing, six-minute walk distance and questionnaires will be assessed at the beginning and end of the treatment phase.

Arms: arm 1: Treatment Arm arm 2: Placebo Arm
Interventions: intervention 1: Riociguat intervention 2: Placebo
Intervention descriptions: intervention 1: Riociguat 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg and 2.5 mg three times a day for 12 weeks intervention 2: Matching placebo to riociguat 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg and 2.5 mg three times a day for 12 weeks

Protocol documents:
Prot_SAP_000.pdf: 
 
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STUDY TITLE 
A Phase IIb Randomized, Double-Blind, Placebo-Controlled Multi-Center Study to Assess the 
Safety, Tolerability, and  Efficacy of Riociguat in Patients with Sickle Cell Diseases  
(STERIO-SCD) 
Test drug: 
Riociguat 
Study purpose: 
Safety, tolerability and efficacy 
Clinical study phase: 
IIb 
Version no.: 
6.1 
Date: January 7, 2020 
IND # 
128277 
ClinicalTrials.gov registry 
number: 
NCT 02633397 
Protocol no.: 
PRO15110016 
Study Sponsor: 
Mark T. Gladwin, MD 
 
Lead Principal Investigator:   
Mark T. Gladwin, MD 
Professor of Medicine 
Chair, Department of Medicine 
Director, Heart, Lung, Blood and Vascular Medicine Institute 
University of Pittsburgh 
Phone: 412-648-3181 
 
 
 
 
 
 
 
 
 
The study will be conducted in compliance with the protocol, ICH-GCP and any applicable regulatory requirements. 
 
Confidential 
The information provided in this document is strictly confidential and is intended solely for the guidance of the clinical 
investigation.  Reproduction or disclosure of this document - whether in part or in full - to parties not associated with the clinical 
investigation, or its use for any other purpose, without the prior written consent of the Principal Investigator is not permitted. 
Throughout this document, symbols indicating proprietary names (, TM) are not displayed.  Hence, the appearance of product 
names without these symbols does not imply that these names are not protected. 
 


 
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PROTOCOL SIGNATURE PAGE 
Protocol Number: 
PRO15110016 
 
Protocol Title: 
A Phase IIb Randomized, Double-Blind, Placebo-Controlled Multi-
Center Study to Assess the Safety, Tolerability, and  Efficacy of 
Riociguat in Patients with Sickle Cell Diseases 
 
Version/Date: 
Version  6.1 /January 7, 2020 
 
 
 
 
   
SPONSOR’S APPROVAL 
 
The sponsor signatory agrees to the content of the final clinical study protocol as presented. 
 
Name: Mark T. Gladwin, MD 
Title: Lead Co- Principal Investigator, University of Pittsburgh 
 
 
Signature:  
 
Date:  
 
INVESTIGATOR AGREEMENT 
I confirm agreement to conduct the study in compliance with the protocol. 
 
I acknowledge that I am responsible for overall study conduct. I agree to personally conduct or 
supervise the described clinical study. 
 
I agree to ensure that all associates, colleagues and employees assisting in the conduct of the 
study are informed about their obligations. Mechanisms are in place to ensure that site staff 
receives the appropriate information throughout the study. 
 
 
Investigator Name: 
 
 
Site #: 
 
 
 
Investigator Signature:  
 
Date: 
 
 
 
 
 


 
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Synopsis 
Title 
A Phase IIb Randomized, Double-Blind, Placebo-Controlled 
Multi-Center Study to Assess the Safety, Tolerability, and 
Efficacy of Riociguat in Patients with Sickle Cell Disease 
(STERIO-SCD) 
Clinical study phase 
IIb 
Study objective(s) 
The purpose of this study is to evaluate the safety and 
tolerability of 12-weeks of treatment with riociguat versus 
placebo in high-risk patients with sickle cell disease, with 
additional evaluation for efficacy signal for improvement of 
blood pressure, exercise capacity, and/or proteinuria. 
Test Drug 
 
Name of active 
ingredient 
 
Dose(s)  
Route of administration 
Duration of treatment 
Riociguat  
Riociguat 
0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, and 2.5 mg three times a 
day (TID)  
Oral 
12 weeks 
Reference Drug 
 
Name of active 
ingredient 
 
Dose(s)  
Route of administration 
Duration of treatment 
      Placebo 
Not applicable 
Matching placebo to riociguat 0.5 mg, 1.0 mg, 1.5 mg, 2.0 
mg and 2.5 mg TID 
Oral 
12 weeks 
Indication 
Prevention or reduction of clinical complications of sickle cell 
disease 
Diagnosis and main criteria 
for inclusion 
 Age 18 years or older 
 Sickling disorder HbSS, HbSC, HbSbeta-thalassemia, 
HbSD, HbSO-Arab) documented by past medical history, 
hemoglobin electrophoresis or hemoglobin HPLC 
fractionation 
 At least one of the following findings: 
a. Systolic blood pressure ≥130 mm Hg 
b. Urine albumin to creatinine ratio > 300 mg/g 
c. TRV > 2.9 m/sec 
d. NT-proBNP level ≥ 160 pg/mL 
e. Urinalysis protein 1+ or higher 
 Willingness to provide a blood specimen for DNA analysis 


 
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 Stable therapy for three months if on hydroxyurea, 
vasodilator therapy, endothelin receptor antagonists, L-
glutamine, crizanlizumab, or voxelotor 
Study design 
Multi-center, randomized, double-blind, placebo-controlled, 
parallel groups study 
Type of control 
Placebo controlled 
Number of patients 
100 completed patients 
Study Endpoints 
Primary endpoints measure:   
Proportion of participants experiencing at least 1 treatment 
emergent SAE in the riociguat arm compared to the placebo 
arm. 
Secondary endpoints: 
 The frequency of SAE adjudicated by a designated 
committee of protocol investigators to be due to sickle 
cell related painful crisis in the riociguat group 
compared to the placebo group. 
 Overall incidences of treatment-emergent adverse 
events 
 Changes in pain intensity from baseline to Week 12 
using numerical pain score and the Brief Pain 
Inventory, and electronic daily pain diary piloted at 
selected sites. 
Main efficacy outcomes: 
 Changes in functional exercise capacity by assessing 6 
Minute Walk Distance Test with Borg 
Dyspnea/Fatigue Scale from baseline to Week 12     
 Changes in hemodynamic parameters including blood 
pressure as the main pharmacodynamic variable, and 
tricuspid regurgitant velocity, as well as other 
echocardiography markers of left ventricular diastolic 
dysfunction after 12 weeks of double blinded treatment 
with Riociguat compared with placebo. 
 Changes in the levels of plasma NT-proBNP from 
baseline to Week 12. 
 Changes in laboratory measures from baseline to Week 
12: 
o Renal: urine albumin to creatinine ratio, 
albuminuria stratified as microalbuminuria and 


 
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macroalbuminuria, estimated GFR, CKD stage 
based on albuminuria and GFR;  
o Hematologic:  Hemoglobin, LDH, reticulocyte 
count, white blood cell count, fetal hemoglobin 
 Incidences of Sickle Cell related clinical complications 
such as acute chest syndrome, priapism, new leg ulcer, 
new stroke, other new non-CNS thromboembolic event 
during the 12 week study treatment. 
Exploratory endpoints: 
 Maximum and trough plasma concentration (Cmax and 
Ctrough) 
 Biomarkers of oxidative stress: 3-Nitro-tyrosine, 
Malondialdehyde and 8-IsoPGF2a.  
 Biomarkers of NO-Pathway dysregulation: asymmetric 
dimethylarginine (ADMA), SDMA and cGMP. 
 Biomarkers of inflammation (C-reactive protein) and 
erythropoiesis (erythropoietin) 
 Changes in pain diary scores 
 
Plan for statistical analysis 
All variables will be analyzed descriptively with appropriate 
statistical methods: categorical variables by frequency tables 
and continuous variables by sample statistics (i.e. mean, 
standard deviation, minimum, median, quartiles and 
maximum).  A logistic regression is planned for the primary 
endpoint, and analysis of covariance (ANCOVA) analyses are 
planned for further endpoints.  
 
 
 


 
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Table of Contents 
  
 
Title page 
 
 
Synopsis 
 
 
1. Introduction………………………………………………………………………....................9 
 
2. Study objectives……………………………………………………………………………...20 
 
3. Lead Investigators and Coordinating Center Personnel ………………………………...…...21 
 
4. Study design………………………………………………………………………………..   22 
 
5. Study population……………………………………………………………………………..25 
 
6.Treatment[s]……………………………………………. ……………….…………….……..28 
 
7. Procedures and variables……………………………………………………………….…….32 
 
8. Statistical methods and determination of sample size…………………………………….….48 
 
9. Data handling and quality assurance…………………………………………………….…...52 
 
10. Premature termination of the study………………………………………………….……...54 
 
11. Ethical and legal aspects……………………………………………………………………54 
 
12. Reference list………………………………………………………………………………..57 
 
13. Appendices……………………………………………………………………………….….61 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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LIST OF ABBREVIATION 
 
Abbreviation 
Definition 
6MWD 
6-minute walking distance 
ADMA 
asymmetric dimethylarginine 
AE(s) 
adverse event(s) 
ANCOVA 
analysis of covariance 
ANOVA 
analysis of variance 
ATS 
American Thoracic Society 
BNP 
brain natriuretic peptide 
BP 
blood pressure 
CBC 
complete blood count 
cGMP 
cyclic guanosine monophosphate 
CFR 
code of federal regulation 
CKD 
chronic kidney disease 
Cl 
clearance 
CTCAE 
common terminology criteria for adverse events 
CTEPH 
chronic thromboembolic pulmonary hypertension 
DBP 
diastolic blood pressure 
DCC 
Data Coordinating Center 
DSMB 
data safety monitoring board 
EC 
Ethics Committee 
eCRF 
electronic case report form 
e.g. 
exempli gratia, for example 
eNOS 
endothelial NO synthase 
EOT 
end of treatment 
FDA 
Food and Drug Administration 
FRP 
females of reproductive potential 
g 
gram 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
HbSS 
hemoglobin S homozygote; sickle cell anemia; homozygous sickle cell disease 
HbSC 
hemoglobin S/hemoglobin C double heterozygote 
HbSbeta-thalassemia hemoglobin S/beta-thalassemia double heterozygote 
HbSD 
hemoglobin S/hemoglobin D double heterozygote 
HbSO-Arab 
hemoglobin S/hemoglobin O-Arab double heterozygote 
HPLC 
high performance liquid chromatography 
ICF 
informed consent form 


 
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Abbreviation 
Definition 
ICH 
International Conference on Harmonization 
IEC 
independent ethics committee 
IIP 
idiopathic interstitial pneumonia 
ILD 
interstitial pneumonia 
IPF 
idiopathic pulmonary fibrosis 
IRB 
institutional review board 
ITT 
intent to treat 
IND 
investigational new drug 
INR 
International Normalized Ratio 
IxRS 
interactive voice response system/ interactive web response system 
LDH 
lactate dehydrogenase 
MAP 
mean arterial pressure 
MD 
medical doctor 
Mg 
milligram 
mL 
Milliliter 
mmHg 
millimeter of mercury 
mPAP 
mean pulmonary artery pressure 
NADPH 
nicotinamide adenine dinucleotide phosphate 
NCT 
National Clinical Trial 
NO 
nitric oxide 
NT-proBNP 
N-terminal pro-hormone brain natriuretic peptide 
PD 
pharmacodynamics 
PDE5 
phosphodiesterase 5 
PDE9 
phosphodiesterase 9 
PK 
pharmacokinetic 
PVR 
pulmonary vascular resistance 
RHC 
right heart catheterization 
RVF 
right ventricular function 
RVSp 
right ventricular systolic pressure 
SAE(s) 
serious adverse event(s) 
SBP 
systolic blood pressure 
SDMA 
symmetric dimethylarginine 
SCD 
sickle cell disease 
sGC 
SNP 
soluble guanylate cyclase 
sodium nitroprusside 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 


 
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Abbreviation 
Definition 
TID 
ter in die; three times a day 
TRV 
tricuspid regurgitant velocity 
US(A) 
United States of America 
Vd 
Volume of distribution 
WHO 
World Health Organization 
 
1. 
INTRODUCTION 
1.1 
Background 
1.1.1  Basic pathobiology, incidence and prevalence of sickle cell disease 
Sickle cell disease (SCD) is a hereditary disorder of red blood cells that causes a wide variety of 
acute and chronic complications [1].  A single nucleotide mutation in the gene encoding the beta 
globin gene results in a single amino acid substitution in beta globin.  This seemingly tiny 
change in the hemoglobin protein results in sickle hemoglobin, which polymerizes, especially in 
the T conformation favored in deoxygenated sickle hemoglobin.  Sickle hemoglobin polymers 
induce remarkable rigidity and adhesiveness of the red cells that interferes with circulation in the 
microvasculature, leading to tissue ischemia and infarction, pain and end organ dysfunction.  
Precise population figures are not available, but the Centers for Disease Control in the United 
States Federal Government estimates[2]: 
 
SCD affects 90,000 to 100,000 Americans. 
 
SCD occurs among about 1 out of every 500 Black or African-American births. 
 
SCD occurs among about 1 out of every 36,000 Hispanic-American births. 
 
The World Health Organization estimates that 275,000 infants are born globally with SCD each 
year, 85% of those births in Africa, especially west Africa [3].  The remainder of the SCD births 
are distributed mainly among the United States, South America, Caribbean and Saudi Arabia-
India regions. 
1.1.2 Nitric oxide dysfunction in sickle cell disease 
Sickle erythrocytes are also prone to both extravascular and intravascular hemolysis. In recent 
years, work from our group and others has characterized mechanisms by which the products of 
hemolysis contribute to vascular dysfunction, especially compromising bioavailability of nitric 
oxide (NO) [4-11]. 
Nitric Oxide.  NO is arguably the most important endogenous regulator of vascular physiology.  
NO is produced in blood vessels by endothelial NO synthase (eNOS), a tetrahydrobiopterin-
dependent enzyme that converts arginine into NO plus citrulline.  NO regulates an entire 
biological program that favors the flow of blood in the vasculature; its deficiency results in 
vasoconstriction and activation of a pro-thrombotic, inflammatory state in blood vessels, 
especially arteries.  Vasculopathy due to decreased NO bioavailability predisposes to 
atherosclerosis in the general population, contributing to the risk of heart disease and stroke. 
Hemolysis-Endothelial Dysfunction Syndrome.  The products of hemolysis found in blood 
plasma from patients with sickle cell disease contribute to a vasculopathy featuring decreased 


 
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NO bioavailability [4-11] [12,13].  In addition, highly active oxidase pathways produce oxygen 
radicals that can further deplete NO [12-14].  These products in combination decrease NO 
bioavailability: 
 
Component 
Mechanistic Outcome 
Citation 
 Cell-free plasma hemoglobin 
 NO scavenging and oxidant stress 
[4] 
 Cell-free plasma arginase-1 
 L-arginine scavenging, decreased 
NO production and eNOS 
uncoupling 
 
[5] 
 Asymmetric 
dimethylarginine (ADMA) 
 eNOS inhibition and uncoupling 
[6-10] 
 Lactate dehydrogenase 
 Biomarker of associated plasma 
hemoglobin, arginase and ADMA 
[6,8,15] 
 Reactive oxygen species 
from xanthine oxidase and 
NADPH oxidase 
 NO scavenging and formation of 
peroxynitrite 
[12,14] 
 Decreased 
tetrahydrobiopterin 
 eNOS uncoupling 
[13,16] 
 
Each of these components serves as a biomarker of risk of pulmonary hypertension in patients 
with SCD.  The same pathobiology has also been implicated in several other complications of 
SCD, including relative systemic hypertension [17,18], proteinuria [19,20], chronic kidney 
disease [21-24], stroke [25], leg ulceration [11,26], and priapism [27,28].  Each of these 
complications is conceivably a consequence of vasculopathy in patients with SCD, much as they 
are in patients without SCD.  Each of these vasculopathic complications has been associated with 
clinical laboratory biomarkers of hemolysis, such as low total hemoglobin, high reticulocyte 
count, high serum lactate dehydrogenase and bilirubin [29,30].  This sickle cell vasculopathy has 
been associated with compromised response to exogenous NO donors in physiological assays of 
vasodilator function, indicating a state of NO resistance [4,31].  Collectively, our group has 
termed this constellation of vasculopathic features the Hemolysis-Endothelial Dysfunction 
Syndrome (Figure 1) [29].   
 


 
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Figure 1.  Intravascular hemolysis reduces nitric oxide bioactivity. Nitric oxide is produced by 
isoforms of nitric oxide (NO) synthase, using the substrate L-arginine. Intravascular hemolysis 
simultaneously releases hemoglobin, arginase, and lactate dehydrogenase (LDH) from red cells into blood 
plasma. Cell-free plasma hemoglobin stoichiometrically inactivates NO, generating methemoglobin and 
inert nitrate (A). Plasma arginase consumes plasma L-arginine to ornithine, depleting its availability for 
NO production by vascular endothelial cells (B). LDH also released from the red cell into blood serum 
serves as a surrogate marker for the magnitude of hemoglobin and arginase release. NO is also consumed 
by reactions with reactive oxygen species (O2-) produced by the high levels of xanthine oxidase activity 
and NADPH oxidase activity seen in sickle cell disease, producing oxygen radicals like peroxynitrite 
(ONOO-)(C). The resulting decreased NO bioactivity in sickle cell disease is associated with pulmonary 
hypertension, priapism, leg ulceration, and possibly with non-hemorrhagic stroke. A similar pathobiology 
is seen in other chronic intravascular hemolytic anemias [29]. 
 
The most dramatic complication of this syndrome, pulmonary hypertension, occurs in 
approximately 10% of adults with SCD [24,32,33], and is associated with high risk of mortality 
[33,34].  It is associated with three important risk-defining variables that will be considered 
eligibility criteria for this trial:   
 Highly elevated echocardiographic tricuspid regurgitant velocity >2.9 m/s 
 Systolic hypertension 
 Proteinuria 
These have each been associated with poor prognosis in adults with SCD [17,21,35].  Each is 
hypothetically linked to the hemolysis-endothelial dysfunction syndrome and low NO 
bioavailability, making them attractive conditions to attempt therapeutic bypass of the NO 
pathway with an activator of soluble guanylyl cyclase.  Furthermore, our own results indicate 
that sGC stimulator Bay 41-8543 and sGC activator Bay 60-2770 can bypass NO scavenging 
reactions due to intravascular hemoglobin in rats (Figure 2) [36].   
 


 
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Figure 2.  Effect of pre-infusion of vasodilators on hemoglobin-induced vasoconstriction. (A) 
Experimental timeline. Rats were stabilized for 30min after surgery, and blood gases were drawn as 
indicated (BG1 and BG2). After determining a baseline (BL), vasodilators were infused, followed by 
infusion of purified human hemoglobin (8.1mM) until an end concentration of 175mg/kg was reached. 
MAP at T = 15, 30, and 45min after hemoglobin infusion is displayed. (B) Effect of pre-infusion with 
Ringer’s (n = 6) (–), SNP (0.4 g/kg/min, n = 7) (–), or sildenafil (10 g/kg/min, n = 8) (–) and bolus 
administration of BAY 41-8543 (10 g/kg, n = 8) (–) and BAY 60-2770 (1 g/kg, n = 9) (–) on basal MAP 
and peak MAP after infusion of hemoglobin. *Significantly different (p<0.05) from Ringer’s by two-way 
ANOVA with Bonferroni correction. (C) Area under the curve (AUC) values from the graphs in panel A 
expressed as percentage from Ringer’s values. *Significantly different (p<0.001) from Ringer’s by one-
way ANOVA with Bonferroni correction. SNP, sodium nitroprusside [36]. 
 
NO and cell adhesiveness in SCD.  A growing theme in SCD research is the role of cell 
adhesion, and this might be a potential therapeutic target of riociguat.  Under steady state 
conditions in patients and mice with SCD, many adhesion molecules and their counter-ligands 
are expressed at high levels on endothelium, erythrocytes, leukocytes and platelets, and also in 
plasma [37,38].  Increases in adhesiveness might be the precipitating cause of the intermittent 
vaso-occlusive pain crisis episodes that characterize the symptoms of SCD [39], and several 
pharmaceutical trials are testing approaches to inhibit adhesiveness.  Prominent approaches 
under investigation are to inhibit the adhesion receptor P-selectin with small molecules 
(rivipansel or pentosan polysulfate sodium)[40,41], or with a monoclonal blocking antibody [42].  
Preclinical studies suggest that increasing cGMP via PDE9 inhibition decreases adhesiveness of 
sickle cell blood leukocytes [43].  All of these promising results suggest a potential additional 
mechanism of action of riociguat for patients with SCD. 
1.1.3 Previous clinical trials of NO pathway drugs in SCD 
Sodium nitrite.  Nitrite is a prodrug for NO production by enzymatic activity by hemoglobin, 
myoglobin, xanthine oxidase and other hemoproteins, especially under low oxygen tension [44].  
In blood flow physiology studies conducted by our group, sodium nitrite infused in the brachial 
artery induced dose-dependent vasodilation in healthy volunteers and adults with SCD [45,46].  
However, no additional results of systemic sodium nitrite in SCD have been presented. 


 
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Inhaled nitric oxide.  A small pilot trial of inhaled NO suggested benefit in children with SCD 
receiving treatment in an emergency department for acute pain crisis [47].  This was perhaps 
surprising, since NO has a half-life of less than a second, and would not persist in the circulation 
from the lung to the systemic circulation.  In fact, we found no evidence of benefit in a 
randomized, double blind, placebo-controlled study involving 150 adolescents and adults with 
SCD hospitalized for acute pain crisis [48].   
 
Oral sildenafil.  This phosphodiesterase-5 inhibitor was already approved by the FDA for the 
treatment of penile erectile dysfunction (Viagra®) and pulmonary arterial hypertension 
(Revatio®).  Its mechanism of action involves inhibition of hydrolysis of cyclic GMP in tissues 
expressing its target PDE-5, mainly the penis and lung vasculature [49].  In a randomized 
controlled trial that enrolled adults and adolescents with SCD and elevated TRV and low 
exercise capacity, the Data Safety and Monitoring Board stopped the study prematurely due to an 
unexpected toxicity [50].  There was a 2.5-fold higher rate of hospitalization for acute pain in the 
sildenafil arm (45% for sildenafil, 22% for placebo, P=0.022).  In this highly truncated study, 
there was no evidence of treatment benefit, although only a small minority of study patients 
completed the trial.  Issues concerning the increased pain experienced by sildenafil recipients are 
addressed below. 
1.1.4 Pain and cGMP 
A substantial literature has emerged implicating cGMP as a pain sensitizing mechanism in the 
dorsal root ganglia of the spinal cord [51].  At present, this remains the most coherent hypothesis 
for the increased pain observed in SCD patients on sildenafil, which also increases cGMP.  In 
further support of this idea, sildenafil also amplified neuropathic pain in a rat model [52], 
although this is a very complex pathway that is incompletely understood [53,54].   
 
The track record of the cGMP-elevating agent sildenafil in promoting pain in patients with SCD 
suggests that any future trial of cGMP-elevating therapy in SCD should monitor closely for any 
evidence of increased pain as an adverse event.   
 
1.2 
Rationale of the Study 
Riociguat has a dual mode of action: it sensitizes soluble guanylate cyclase (sGC) to the body’s 
own nitric oxide (NO) and can also increase sGC activity in the absence of NO, causing 
vasorelaxation. 
Patients with sickle cell disease have a defect in sGC activation attributed to decreased NO 
bioavailability [55].  NO deficiency is compounded by several concurrent pathways active in 
sickle cell disease:  
 NO is scavenged by cell-free hemoglobin released from red blood cells as they lyse, 
and hemolysis is robust in sickle cell disease [4].   
 NO scavenging by reactive oxygen species produced by abundant activity of xanthine 
oxidase and other oxidases [12,14];  
 Depletion of the NO synthase substrate, arginine, by high plasma arginase activity, 
also released from red cells during hemolysis [5,56]; 


 
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 Inhibition of NO synthase by high plasma levels of its endogenous inhibitor, 
asymmetric dimethylarginine (ADMA), and by deficiency of its co-factor, 
tetrahydrobiopterin [6-10] 
Reduced sGC activation results in defects in vasodilation and in excessive activation of 
endothelial, neutrophil and platelet adhesion.  These all combine to contribute to (a) vascular 
occlusion which produces pain and organ injury, and (b) proliferative arteriopathy manifest 
primarily as pulmonary arterial hypertension, which is life limiting.  Adults with sickle cell 
disease with high systolic blood pressure have a higher risk of pulmonary hypertension, chronic 
kidney disease, ischemic stroke and death [17,18,57]. 
In a previous study of cGMP pathway manipulation in sickle cell disease using sildenafil, 
increased pain was a limiting adverse event, as discussed in the previous section [50].  Back pain 
is reported in the sildenafil package insert in 3-4% of sildenafil users (erectile dysfunction), and 
less frequently pain in the abdomen, chest, bone, eye or ear.  In pulmonary hypertension patients 
enrolled in the PATENT-1 and CHEST-1 studies, pain (especially chest pain, a known symptom 
of pulmonary hypertension patients) was reported more frequently even in the placebo users (5-
9%), but was no higher in the riociguat user (4-7%).  These results suggest that riociguat may not 
activate the same pain sensitization pathways that might be activated by sildenafil.     
The proposed clinical trial will recruit adults with sickle cell disease with systolic blood pressure 
(SBP) ≥130 mmHg, and/or macroalbuminuria as manifested by urine albumin to creatinine ratio 
of >300 mg/g, and/or TRV > 2.9 m/sec and/or urinalysis urine protein 1+ or higher, and/or serum 
amino-terminal pro-brain natriuretic peptide (NT-proBNP) as a cohort at high risk of sickle cell 
complications (See Table 1), and test safety and preliminary efficacy in reducing systemic blood 
pressure, albuminuria, TRV and NT-pro-BNP in an outpatient clinic setting.   
Rationale for adjustments to the initial protocol criteria for inclusion and exclusion are discussed 
below: 
 
*         Expand clinical results of macroalbuminuria & TRV acceptable window from 12 months  
to 24 months. 
- many patients have these results identifying them as higher risk patients in their chart 
from more than 12 months ago, and loosening the time window is expected to add 
eligible subjects without the delays of updating their laboratory and echocardiography 
testing. 
 
*         Add inclusion criteria to accept clinical result of NT-proBNP level ≥ 160 pg/mL within 
the past 24 months. 
- NT-proBNP is a clinically available laboratory marker of cardiac ventricular stress.  It 
has been established in adults with SCD to correlate with elevated TRV (R=0.50, P.001).  
NT-proBNP level of 160 pg/mL or greater independently predicts increased risk of death 
(21 deaths at 31 months’ median follow-up; risk ratio, 5.1; 95% confidence interval, 2.1-
12.5; P=.001; 19.5% absolute increase in risk of death).  These results from the NIH SCD 
cohort study were confirmed in the Multicenter Study of Hydroxyurea.  Elevated NT-
proBNP correlated with hemodynamic measurements on pulmonary artery 
catheterization, and with functional capacity indicated by shortened 6-minute walk 
distance [Machado RF, Anthi A, Steinberg MH, Bonds D, Sachdev V, Kato GJ, Taveira-
DaSilva AM, Ballas SK, Blackwelder W, Xu X, Hunter L, Barton B, Waclawiw M, 
Castro O, Gladwin MT; MSH Investigators. N-terminal pro-brain natriuretic peptide 


 
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levels and risk of death in sickle cell disease. JAMA. 2006 Jul 19;296(3):310-8. PubMed 
PMID: 16849664].   25% of adults with SCD have NT-proBNP level of 160 pg/mL or 
greater.  Although this population will overlap with other qualifying criteria in this 
protocol, in the frequent event of incomplete data in the medical record, this criterion will 
present an additional opportunity to identify high risk SCD patients from existing 
information in the medical record. 
 
*         Add inclusion criteria to accept clinical result of urinalysis protein 1+ or higher within the 
past 24 months 
- Similar to macroalbuminuria, urinalysis protein 1+ or higher identifies an adult SCD 
subpopulation with early renal disease, associated with elevated TRV.  The presence of 
proteinuria correlated with lower GFR and had a high positive predictive value (0.60) for 
elevated TRV in subjects with SCA (or HbS-beta-zero-thalassemia).  Proteinuria occurs 
in 20–30% of patients with SCD, although higher incidences have also been reported 
[19,58,59].  Again, although this population will overlap with other qualifying criteria in 
this protocol, in the frequent event of incomplete data in the medical record, this criterion 
will present an additional opportunity to identify high risk SCD patients from existing 
information in the medical record. 
 
*         Exclude patients with PH-IIP to reflect the current riociguat IB 
- Patients with pulmonary hypertension due to idiopathic interstitial pneumonia (PH-IIP), 
a more updated term for pulmonary fibrosis, did not benefit from riociguat in data in the 
riociguat investigator brochure.  Patients on this study with SCD who have the unlikely 
co-morbidity of PH-IIP will be excluded event, consistent with current updated labeling 
information [60]. 
 
*         Remove smoking from exclusion criteria 
- Smoking is not a contraindication in current riociguat labeling [60].  Tobacco smoking 
can increase riociguat elimination, thereby decreasing drug exposure, but is part of real 
world potential usage of riociguat.  Since opening of the study, 40-50% of SCD patients 
meeting inclusion criteria are smokers.  The removal of tobacco smoking exclusion may 
increase the eligible study pool by nearly double, and add valuable safety and preliminary 
efficacy information on this large subpopulation.    
 
*         Revise exclusion criteria to use Child-Pugh classification to assess the severity of hepatic 
impairment. This also is in line with current riociguat package insert and IB. 
- The current modification proposes to align with the current labeling: “not recommended 
in patients with severe (Child Pugh C) hepatic impairment [60].”  The current exclusion 
criterion of SCD patients with direct bilirubin level of 1.0 mg/dL or higher is excessively 
restrictive and will be eliminated in favor of the Child-Pugh C exclusion.  This will 
provide hepatic function standards consistent with the current label. 
 
Study Procedures: 
 
*         Add INR as one of the liver function tests 
 
- The INR is used to calculate the Child-Pugh class, as discussed above. 
 


 
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Table 1.  Associations of clinical and laboratory characteristics with systemic hypertension 
(systolic BP ≥130) in SCD patients.  Patients in the high SBP group have higher risk features 
demonstrated to predict higher mortality in sickle cell disease, including higher TRV, NT-
proBNP, serum creatinine, and proteinuria.  Results are from HbSS patients screened for the 
walk-PHaSST study.  HbSC patients are excluded from this table. 
 
Patient Characteristics 
Sys BP < 130 mm Hg 
Sys BP≥130 mm Hg 
 
 
n 
median (IQR)2 
n 
median (IQR)2 
p4 
 
 
 
N (%1) 
400 (80.0) 
100 (20.0) 
 
 
 
 
Demographics and Vital Status 
 
 
 
 
 
 
Age 
400 
33 (24-44) 
100 
41 (31-51) 
<0.0001 
 
Male gender, N(%) 
400 
187 (46.8) 
100 
58 (58.0) 
0.04 
 
Deaths, N(%) 
376 
14 (3.7) 
96 
4 (4.2) 
0.84 
 
Follow-up time, months 
376 
29.3 (25.3-33.7) 
96 
28.4 (23.8-33.6) 
0.36 
 
BMI 
394 
22.9 (20.7-25.4) 
98 
23.6 (21.0-27.0) 
0.04 
 
O2 Sat 
397 
97 (95-99) 
100 
97 (94-98) 
0.07 
 
Respiratory rate 
394 
18 (16-20) 
98 
18 (16-20) 
0.10 
 
Pulse pressure 
400 
48 (42-55) 
100 
61 (54-70) 
<0.0001 
 
MAP 
400 
81 (76-86) 
100 
95 (91-101) 
<0.0001 
Clinical Measures 
 
 
 
 
 
 
Hydroxyurea, current use, N(%) 
400 
175 (43.8) 
100 
50 (50.0) 
0.26 
 
On chronic transfusion therapy, N(%) 
399 
61 (15.3) 
100 
11 (11.0) 
0.28 
 
 20+ lifetime transfusions, N(%) 
400 
160 (40.0) 
100 
39 (39.0) 
0.86 
 
100+ lifetime transfusions, N(%) 
400 
52 (13.0) 
100 
14 (14.0) 
0.79 
 
Has chronic pain, N(%) 
399 
144 (36.1) 
100 
40 (40.0) 
0.47 
 
Acute pain rating 
377 
9 (7-10) 
96 
9 (7-10) 
0.80 
 
Had mild pain in last year, N(%) 
394 
251 (63.7) 
100 
75 (75.0) 
0.03 
 
 
Mild episodes in last year, n3 
251 
10 (3-30) 
75 
6 (2-36) 
0.23 
 
Had moderate pain in last year, N(%) 
396 
263 (66.4) 
100 
71 (71.0) 
0.38 
 
 
Moderate episodes in last year, n3 
263 
7 (3-20) 
71 
5 (2-15) 
0.12 
 
Had severe pain in last year, N(%) 
400 
166 (41.5) 
100 
53 (53.0) 
0.04 
 
 
Severe episodes in last year, n3 
166 
3 (2-6) 
53 
3 (1-5) 
0.40 
 
Had extremely severe pain in last year, N(%) 
400 
210 (52.5) 
100 
57 (57.0) 
0.42 
 
 
Extremely severe episodes in last year, n3 
210 
2 (1-4) 
57 
2 (1-4) 
0.67 
 
Six Minute Walk Distance 
395 
437 (385-504) 
97 
445 (378-510) 
0.88 
Echocardiographic Measures 
 
 
 
 
 
 
TRV, m/sec 
361 
2.5 (2.3-2.7) 
88 
2.6 (2.4-2.9) 
0.006 
 
TRV ≥ 2.5 m/sec, N(%) 
361 
220 (60.9) 
88 
62 (70.5) 
0.10 
 
TRV ≥ 2.7 m/sec, N(%) 
361 
137 (38.0) 
88 
43 (48.9) 
0.06 
 
TRV ≥ 3.0 m/sec, N(%) 
361 
34 (9.4) 
88 
18 (20.5) 
0.004 
 
LV Lat E/Ea ratio 
362 
6.33 (5.17-8.11) 
90 
6.67 (5.36-8.00) 
0.32 
 
Abnormal LV function, N(%) 
395 
20 (5.1) 
99 
7 (7.1) 
0.43 
 
LV ejection fraction 
386 
61 (60-66) 
98 
62 (59-67) 
0.79 
 
 
 
 
 
 
 
Laboratory Measures 
 
 
 
 
 
 
NT-proBNP (pg/mL) 
370 
71 (29-147) 
94 
110 (41-284) 
0.003 
 
NT-proBNP ≥ 160, N(%) 
370 
81 (21.9) 
94 
38 (40.4) 
0.0002 
 
Ferritin 
361 
273.6 (109.4-
684.4) 
89 
344.4 (154.9-
786.2) 
0.21 
 
Ferritin ≥ 1000, N(%) 
361 
66 (18.3) 
89 
19 (21.3) 
0.51 
 
Fetal Hemoglobin  
355 
6.0 (2.5-11.9) 
90 
8.6 (3.3-14.1) 
0.12 
 
HbF ≥ 5%, N(%) 
355 
207 (58.3) 
90  
58 (64.4) 
0.29 
 
HbF ≥ 7%, N(%) 
355 
158 (44.5) 
90 
49 (54.4) 
0.09 
 
Hemolytic Component 
333 
0.52 (-0.34-1.43) 
87 
0.68 (-0.54-
1.72) 
0.50 
 
Absolute Reticulocyte Count (thousands/uL) 
368 
245 (165-344) 
95 
249 (160-325) 
0.91 
 
Reticulocytes (%) 
369 
9.0 (6.3-12.9) 
92 
9.4 (5.9-14.1) 
0.78 


 
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Patient Characteristics 
Sys BP < 130 mm Hg 
Sys BP≥130 mm Hg 
 
 
n 
median (IQR)2 
n 
median (IQR)2 
p4 
 
 
 
 
Hemoglobin (g/dL) 
391 
8.8 (7.7-9.8) 
98 
8.6 (7.6-10.0) 
0.67 
 
Hematocrit (%) 
392 
25.3 (22.0-28.3) 
98 
25.1 (21.4-29.2) 
0.58 
 
MCHC (g/dL) 
389 
34.6 (33.5-35.6) 
98 
34.6 (33.7-35.5) 
0.77 
 
MCV (fL) 
391 
91.0 (85.0-99.9) 
97 
93.8 (87.5-
100.4) 
0.13 
 
Platelets (thousands/uL) 
391 
367 (288-459) 
98 
355 (263-434) 
0.15 
 
RBC (millions/uL) 
391 
2.7 (2.3-3.2) 
98 
2.7 (2.2-3.1) 
0.23 
 
WBC (thousands/uL) 
392 
9.7 (7.6-12.2) 
98 
9.3 (6.7-12.5) 
0.57 
 
ANC (thousands/uL) 
385 
4.9 (3.5-6.6) 
96 
5.3 (3.1-7.1) 
0.59 
 
Neutrophils (%) 
308 
53.4 (44.8-60.1) 
81 
54.0 (45.5-61.9) 
0.47 
 
Albumin (mg/dL) 
391 
4.3 (3.9-4.5) 
98 
4.1 (3.8-4.4) 
0.01 
 
Alkaline Phosphatase (IU/L) 
392 
90 (69-120) 
97 
93 (71-136) 
0.41 
 
ALT (IU/L) 
393 
22 (16-33) 
98 
25 (18-34) 
0.15 
 
AST (IU/L) 
382 
41 (31-56) 
95 
48 (29-67) 
0.24 
 
AST (standardized unit normal) 
382 
-0.07 (-0.50-
0.62) 
95 
0.12 (-0.58-
0.99) 
0.26 
 
CO2  (mEq/L) 
392 
24 (22-26) 
98  
24 (22-26) 
0.76 
 
BUN (mg/dL) 
393 
10 (7-27) 
97 
14 (8-28) 
0.03 
 
Creatinine (mg/dL) 
394 
0.7 (0.6-0.8) 
98 
0.9 (0.6-1.2) 
<0.0001 
 
LDH (IU/L) 
360 
416 (306-598) 
93 
426 (291-636) 
0.80 
 
LDH (standardized unit normal) 
360 
-0.02 (-0.53-
0.66) 
93 
0.14 (-0.45-
1.07) 
0.21 
 
Total Bilirubin (mg/dL) 
392 
2.8 (1.9-4.2) 
98 
2.6 (1.6-3.6) 
0.20 
 
Calcium (mEq/L) 
384 
9 (9-10) 
97 
9 (9-10) 
0.15 
 
Chloride (mEq/L) 
394 
106 (104-107) 
98 
105 (104-108) 
0.28 
 
Magnesium (mEq/L) 
379 
0.86 (0.78-0.90) 
93 
0.86 (0.82-0.93) 
0.09 
 
Phosphate/Phosphorus  (mEq/L) 
369 
4.1 (3.6-4.8) 
95 
4.3 (3.6-5.2) 
0.46 
 
Potassium (mEq/L) 
391 
4.4 (4.1-4.7) 
97 
4.5 (4.2-4.8) 
0.12 
 
Sodium (mEq/L) 
394  
138 (137-140) 
98 
139 (137-140) 
0.38 
 
Total Protein (mg/dL) 
388 
7.8 (7.3-8.2) 
97 
7.5 (7.2-8.0) 
0.02 
 
Urine microalbumin (mg/G creatinine) 
310 
23 (7-120) 
85 
72 (9-774) 
0.012 
 
Urine albumin >30 mg/G creatinine, N (%) 
micro and macroalbuminuria  
310 
143 (46.1) 
85 
47 (55.3) 
0.14 
 
Urine albumin >300 mg/G creatinine, N (%) 
macroalbuminuria 
310 
44 (14.2) 
85 
27 (31.8) 
0.0002 
 
Protein in Urine, N positive(%) 
380 
112 (29.5) 
94 
50 (53.2) 
<0.0001 
 
Protein Level in Urine 
95 
0.0 (0.0-1.0) 
42 
1.0 (0.0-2.0) 
0.02 
 
1.2.1 Implications for clinical trial design 
The first priority research question for riociguat in SCD is whether this drug is tolerated in 
patients with SCD.  The primary outcome measure in the trial of riociguat proposed in this 
protocol will be safety and tolerability, focused on measuring overall incidence of treatment 
emergent SAE in the riociguat arm compared to the placebo arm.  Secondary outcome measures 
will evaluate the changes in pain intensity and potential signal of efficacy in vasodilator effect on 
blood pressure, estimated pulmonary artery pressure, and proteinuria. Additional outcome 
measures will include adverse events attributable to SCD, which hypothetically will be reduced 
by riociguat, thereby serving simultaneously as a pilot study to identify variables with a signal of 
improvement with sGC stimulation.  All of the selected outcome variables have potential for 
improvement with sGC stimulation.  
 


 
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1.3 
Riociguat 
1.3.1 Mechanism of action 
Soluble guanylate cyclase (sGC) is an important regulator in the cardiovascular system and is present 
in vascular cells and platelets. The endogenous stimulus of the sGC is endothelial-cell-derived nitric 
oxide (NO), which acts in a paracrine fashion. In the underlying smooth muscle cells and in platelets 
NO activates the sGC, resulting in increased intracellular cyclic guanosine monophosphate (cGMP) 
concentrations (which induce vasorelaxation), inhibition of cell proliferation and migration as well as 
inhibition of platelet adhesion and aggregation [55]. 
 
Riociguat is a direct stimulator of sGC in vitro and in vivo which is independent from NO, the 
endogenous activator of the enzyme. Moreover, in the presence of NO, it enhances the effects of NO. 
1.3.2 Preclinical 
The toxicological program included studies to investigate the systemic toxicity as well as 
exaggerated pharmacodynamic effects after single and repeated administration up to 26 weeks in rats 
and up to 52 weeks in dogs, reproductive toxicity studies, genotoxicity studies and carcinogenicity 
studies as well as studies addressing specific questions (phototoxicity studies, toxicity of impurities, 
mechanistic investigation and studies in juvenile animals).  The toxicological program was designed 
to support the chronic use of riociguat in humans. 
 
The toxicological profile after repeated administration was characterized by effects secondary to the 
intracellular cGMP increase.  In rats, the toxicological profile of riociguat was characterized by 
hemodynamic effects and sequelae thereof, as well as by changes on bone metabolism and 
morphology in juvenile and adolescent rats.  The bone findings are considered to be related to the 
pharmacological mechanism of action, as the NO-sGC-cGMP pathway is involved in bone 
metabolism.  As the findings were restricted to areas undergoing rapid turnover during bone growth, 
the findings in fast growing animals are not considered relevant for the adult patient population. 
 
In experimental animals, riociguat treatment has revealed no impact on fertility and early embryonic 
development.   Riociguat was negative in a battery of in vitro and in vivo genotoxicity tests and 
revealed no evidence for a genotoxic risk. 
 
In 2-year lifetime bioassays in rats and mice, no drug-related neoplasms were observed under 
riociguat treatment, and an absence of a carcinogenic risk for humans is deduced. 
 
In summary, single and repeat-dose toxicity studies in rats, mice and dogs, which fulfill the 
requirements for non-clinical safety evaluation, revealed no unexpected toxicity of riociguat.  
 
The non-clinical safety profile is characterized by exaggerated pharmacological activity of riociguat 
subsequent to an increase of intracellular cGMP levels. Studies on genotoxicity, juvenile toxicity and 
carcinogenicity did not yield any specific concern.  
 
Developmental toxicity revealed an increase of cardiac malformations in rats. This effect is seen to 
be due to a mechanism of action related to hemodynamic and potential anti-proliferative effect on the 
developing heart. In rats administered riociguat orally (1, 5, 25 mg/kg/day) throughout 
organogenesis, an increased rate of cardiac ventricular-septal defect was observed at the highest dose 
tested. The highest dose produced evidence of maternal toxicity (reduced body weight). Post-
implantation loss was statistically significantly increased from the mid-dose of 5 mg/kg/day. Plasma 


 
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exposure at the lowest dose is approximately 0.15 times that in humans at the maximally 
recommended human dose (MRHD) of 2.5 mg three times a day based on area under the time-
concentration curve (AUC). Plasma exposure at the highest dose is approximately 3 times that in 
humans at the MRHD while exposure at the mid-dose is approximately 0.5 times that in humans at 
the MRHD. In rabbits given doses of 0.5, 1.5 and 5 mg/kg/day, an increase in spontaneous abortions 
was observed starting at the middle dose of 1.5 mg/kg, and an increase in resorptions was observed at 
5 mg/kg/day. Plasma exposures at these doses were 5 times and 15 times the human dose at MRHD 
respectively. Because riociguat was shown to have teratogenic effects when administered to animals; 
the drug is only available to females in the US through a restricted REMS (Risk Evaluation and 
Mitigation Strategies) program. 
1.3.3 Clinical experience summary 
Phase II studies in patients with pulmonary hypertension demonstrated that sGC stimulation as 
exerted by riociguat has the expected positive hemodynamic effects in this disease by reducing mean 
pulmonary artery pressure and pulmonary vascular resistance and increasing cardiac output at doses 
≥1 mg in single-dose studies.  
 
 
Riociguat administered at doses of 1 to 2.5 mg tid over 12 weeks exerted significant, strong, 
and favorable effects on pulmonary hemodynamics and functional capacity in patients with 
PAH or CETPH. This was supported by echocardiographic data, NT-proBNP, and WHO 
functional class assessment. These improvements were maintained over 4.5 years of follow-
up with continuous riociguat treatment.  
 
Administration of riociguat at doses of 0.5 to 2.0 mg as a titration regimen for 16 weeks did 
not result in statistically significant improvements in mean pulmonary arterial pressure 
(PAPmean) as compared to placebo in patients with symptomatic PH associated with left 
ventricular systolic dysfunction. However, treatment in the 2.0 mg target-dose arm (in 
comparison to placebo) resulted in consistent improvements in other hemodynamic 
parameters measured invasively and by echocardiography (left ventricular ejection fraction) 
(phase II Study 14308 LEPHT).  
 
Phase III studies in patients with PH demonstrated that  
 
Administration of riociguat at doses of 1.0-2.5 mg (titration regimen) for 16 weeks results in 
a statistically significant and clinically meaningful improvement in 6MWD as compared to 
placebo in patients with CTEPH or recurrent or persisting PH after surgical treatment.  
Between baseline and week 16 an improvement in 6MWD was seen in the riociguat 1.0-2.5 
mg group (mean increase of 38.9 m) as compared to a small change in 6MWD in the placebo. 
The treatment comparison demonstrated a statistically significant improvement in 6MWD for 
the riociguat 1.0-2.5 mg group compared to the placebo group in the ITT analysis set 
(p<0.0001) [56]. 
 
Administration of riociguat at doses of 1.0-2.5 mg (titration regimen) for 12 weeks results in 
a statistically significant and clinically meaningful improvement in 6MWD as compared to 
placebo in patients with symptomatic PAH.  The improvement in 6MWD was seen in both 
riociguat treatment groups: mean increase of 29.6 m in the riociguat 1.0-2.5 mg group and 
31.1 m in the riociguat 1.0-1.5 mg group, as compared to a small change in 6MWD in the 
placebo group (mean –5.6 m, median 8.5 m) The treatment comparison demonstrated a 
statistically significant improvement in 6MWD for the riociguat 1.0-2.5 mg group as 
compared to the placebo group in the ITT analysis set (p<0.001) [57]. 
 
Administration of riociguat with an individual dose titration regimen (1.0-2.5 mg) to a 
background treatment of a stable dose of sildenafil 20 mg tid appeared to be generally safe. 


 
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However, during the long-term extension phase, 8 of 17 patients discontinued due to AEs (3 
deaths and 5 discontinuations due to AEs) during an average treatment period of 
approximately 10 months. The limited information on efficacy from the small sample, both 
during the double-blind main phase and the open-label long-term extension, did not indicate a 
signal of efficacy and clinical benefit. Therefore, the overall benefit-risk balance for the 
combination is not positive and combined use of riociguat with sildenafil is contraindicated 
[58]. 
1.3.4 Safety profile 
The safety data described below reflect exposure to riociguat in two, randomized, double blind, 
placebo-controlled trials in patients with inoperable or recurrent/persistent CTEPH (CHEST-1) and 
treatment naive or pre-treated PAH patients (PATENT-1). The population (riociguat: n = 490; 
Placebo: n = 214) was between the age of 18 and 80 years [59]. 
 
The safety profile of riociguat in patients with inoperable or recurrent/persistent CTEPH (CHEST 1) 
and treatment naive or pre-treated PAH (PATENT 1) were similar. Therefore, adverse drug reactions 
(ADRs) identified from the 12 and 16 week placebo-controlled trials for PAH and CTEPH 
respectively were pooled, and those occurring more frequently on riociguat than placebo (≥3%) are 
displayed below. 
 
Table 2. Adverse Reactions Occurring More Frequently (≥3%) on riociguat than Placebo (Pooled 
from CHEST 1 and PATENT 1) 
 
Adverse Reactions  
Riociguat %  
(n=490)  
Placebo %  
(n=214)  
Headache  
27  
18  
Dyspepsia and 
Gastritis  
21  
8  
Dizziness  
20  
13  
Nausea  
14  
11  
Diarrhea  
12  
8  
Hypotension  
10  
4  
Vomiting  
10  
7  
Anemia (including 
laboratory parameters)  
7  
2  
Gastroesophageal 
reflux disease  
5  
2  
Constipation  
5  
1  
 
2. 
STUDY OBJECTIVES 
The purpose of this study is to evaluate the safety and tolerability of 12-weeks of treatment with 
riociguat versus placebo in high-risk patients with sickle cell disease, with additional evaluation 
for efficacy signal for improvement of blood pressure, exercise capacity and/or proteinuria. 
2.1 
Primary Hypothesis 
We hypothesize that riociguat will be safely tolerated by adults with sickle cell disease. 
 


 
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The alternative hypothesis is that a difference in the proportion of participants experiencing at 
least 1 treatment-emergent severe adverse event will be seen between study arms.  This reflects a 
superiority trial design, similar to other Phase 2 safety and tolerability studies.  
 
The proposed clinical trial will recruit adults with sickle cell disease with systolic blood pressure 
(SBP) ≥130 mmHg, and/or macroalbuminuria as manifested by urine albumin to creatinine ratio 
of >300 mg/g, TRV > 2.9 m/sec, NT-proBNP level > 160 pg/mL, and/or urinalysis protein 1+ or 
higher as a cohort at high risk of sickle cell complications.  The study will enroll 100 adults with 
sickle cell disease to complete the protocol.  
2.2 
Secondary Hypothesis 
Riociguat will yield a signal of improved outcome in adults with sickle cell disease, with 
potential improvements in systolic blood pressure, albuminuria, echocardiography TRV, serum 
NT-proBNP or six minute walk distance, known predictors of early mortality in adults with 
sickle cell disease. 
 
3. 
LEAD INVESTIGATORS AND COORDINATING CENTER PERSONNEL 
 
Lead Principal Investigator:   
Mark T. Gladwin, MD 
Professor of Medicine 
Chair, Department of Medicine 
Director, Heart, Lung, Blood and Vascular Medicine 
Institute 
University of Pittsburgh 
Phone: 412-648-3181 
 
Director of Regulatory Affairs:  
Nydia Chien, MSN, RN, CCRC 
Heart, Lung, Blood and Vascular Medicine Institute 
University of Pittsburgh 
Phone: 412-647-2791 
 
Data Coordinating Center:   
Kaleab Z. Abebe, PhD  
Center for Clinical Trials & Data Coordination  
University of Pittsburgh 
Phone: 412-692-2023 
 


 
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Echocardiography Core Lab: 
 
 
 
Biospecimen Repository Core 
Lab: 
 
 
 
 
External Data Evaluation Bodies: 
John Gorcsan, MD 
Washington University, Dept. of Cardiology 
660 S. Euclid Ave. Campus Box 8086 
St. Louis, MO 63110 
Phone (314) 273-2255  
 
Yingze Zhang, PhD, Director 
University of Pittsburgh, Dept. of Medicine 
Division on Pulmonary, Allergy and Critical Care Medicine 
NW 907 Montefiore 
3459 Fifth Avenue 
Pittsburgh, PA 15213 
Phone: (412) 647-2515 
 
Institutional Data Safety Review Board (IDSMB) 
Clinical & Translational Science Institute 
University of Pittsburgh 
Phone: 412-864-3472 
All other study personnel not included in this section are identified in a separate personnel list 
(not part of this clinical study protocol) as appropriate. This list will be updated as needed; an 
abbreviated version with personnel relevant for the participating centers will be available in each 
center’s investigator site file. 
A complete list of all participating centers and their investigators, as well as all required 
signature documents, will be maintained in the Coordinating Center study file. 
4. 
STUDY DESIGN 
4.1 
Classification and Methodological Design 
This is a Phase 2b multi-center, randomized, double-blind, placebo-controlled, parallel groups 
study aimed to evaluate the safety, tolerability and the efficacy of riociguat compared with 
placebo in patients with SCD. 
4.2 
Study Design Overview 
The study plans to enroll patients with a clinical diagnosis of SCD for a total of 100 adult 
patients to complete the protocol. 
 
The study will consist of a screening epoch, a 12 week double blinded treatment epoch, and a 
follow-up epoch of 30 days post treatment. Following completion of screening and baseline 
assessment, patients who meet all the inclusion and none of the exclusion criteria will be 
randomized on a 1:1 basis to receive either placebo or riociguat at the starting dose of 1.0 mg 
TID (three times a day) for 12 weeks.  
 
The dose will be titrated every 2 weeks (+/-3 days) based on  monitoring of subject’s systolic 
blood pressure (SBP) and well-being assessed at that visit (see Figure 3). 
 


 
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PK/PD data will be collected on a subgroup of approximately 25 subjects at select sites. To 
preserve the subject blind, PK subjects will receive one dose of study medication (1 mg 
riociguat/placebo) per their randomized assignment.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Following the starting dose of 1.0 mg TID (minimum dose interval - approximately 6 hrs; 
maximum dose interval - approximately 10 hrs), the subsequent dose modification will not exceed 
±0.5 mg TID increments in 2-week intervals (+/- 3 days) to 1.5 mg, 2.0 mg, and 2.5 mg TID, 
resulting in a maximal total daily dose of 7.5 mg. Patients will be maintained on lower doses if 
higher doses are not tolerated (minimum lowest possible dose is 0.5 mg TID, total daily dose 1.5 
mg). If the patient does not tolerate the lowest possible riociguat dose (0.5 mg TID), he/she must 
be withdrawn from the study. 
 
At each biweekly titration visit, peripheral SBP will be measured and signs/symptoms of 
hypotension assessed.  The individual patient dose will be assigned based on the following dose 
titration algorithm (see Figure 4).   Date/time of when the subject took their last dose of riociguat 
will be recorded.  
 SBP ≥ 95 mmHg and no signs or symptoms of hypotension, dosage should be increased by 
0.5 mg TID 
 SBP < 95 mmHg and no signs or symptoms of hypotension, dosage should be maintained 
 SBP < 95 mmHg, and any signs or symptoms of hypotension associated with dizziness, 
lightheadedness or syncope, the current dose should be decreased by 0.5 mg TID 
 
 
 
 
 
 
 
Screening 
Randomizatio
 
 
 
 
2.5 mg TID 
 
 
 
 
2.0 mg TID 
 
 
 
 
1.5 mg TID 
 
 
 
 
1.0 mg TID 
 
 
 
 
 
 
0.5 mg TID 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
Placebo 
Riociguat 
Figure 3. Schematics of Study Design 
Week2 
Week 4 
Week 6 
Week 8 
Week 10 
Week 12 
Week 0 
End of treatment  
safety follow-up 
Sham titration
Titration Phase 
Maintenance Phase 


 
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In the SCD population, as acute painful episodes and other associated clinical complications may 
frequently occur requiring hospitalizations or interruptions of study treatment, dose titration may 
be delayed, but every effort should be made to keep titrations delays to a maximum of 2 weeks. 
It is allowed in case of hospitalizations or for safety reasons, to suspend the next up-titration step 
and to maintain, or reduce, the dose. It is also permissible to up-titrate the dose for a patient if a 
down-titration has previously taken place. Dose reduction at any time is also permissible at the 
discretion of the investigator in the event the subject experiences symptomatic hypotension or for 
other safety reasons. 
 
At the end of the dose titration phase with the target goal of the maximal dose of 2.5 mg TID 
(total daily dose of 7.5 mg), no further increase in dose will be allowed. The established 
individual dose will then be taken as the “optimal dose” to be administered for the remainder of 
the 12-week study period. 
 
To assure blinding of the treatment arms, patients allocated to the placebo group will undergo a 
sham titration from Baseline Visit onwards following the rules of the individual dose titration 
scheme. 
 
Safety monitoring including physical examinations, adverse event assessment, vital signs to 
include sitting blood pressure will be conducted at 2 week intervals during the double blinded 
study treatment. Safety lab assessment will be done at four week intervals during the treatment 
phase. Safety and tolerability, especially pain, will be assessed using several specific patient 
reported outcome instruments. Functional outcomes measuring the changes in Six Minute Walk 
Distance and Borg Dyspnea/Fatigue Scale will be assessed from baseline to Week 12. 
 
 
  Increase 
0.5 mg 
TID 
 
 
 
 
 
 
 
 
Decrease 
0.5 mg 
TID 
                                            MAINTAIN 
 CU                                             CURRENT DOSE 
SBP ≥ 95 mmHg 
No signs or symptoms of 
hypotension 
SBP < 95 mmHg 
No signs or symptoms of 
hypotension 
 
Figure 4. Dose Titration Algorithm 
SBP < 95 
mmHg 
Signs or symptoms 


 
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All randomized patients, regardless of whether they completed study treatment through Week 12 
or discontinued study treatment prior to Week 12, will be asked to return for a safety follow-up 
visit (approximately 30 days after stop of study medication intake). 
4.3 
Dose Interruptions 
If a dose is missed, the patient should continue with the next regularly scheduled dose. 
 
If during the trial an interruption takes place that lasts longer than 14 days (42 consecutive missed 
doses), restarting the study medication again is not allowed (respective patients must be withdrawn 
from the trial). Up to 14 days of missed doses, the patient can restart study drug at the original 
starting dose of 1.0 mg and follow the regular dose titration every two weeks as appropriate. In 
case of interruptions shorter than 3 days (9 doses), it is at the discretion of the investigator if the 
study medication can be restarted at the same dose or at 0.5 mg less than the last dose, with 
resumption of dose titration every two weeks as appropriate.   
 
Hospitalization 
Study visit data should be collected whenever possible if the subject is hospitalized. The decision 
to continue the study drug during hospitalization is at the discretion of the PI. If mechanisms are 
in place at the site to allow patients to receive the study medication while hospitalized, the 
protocol titration schedule should be followed.  
4.4 
Randomization 
Patients will be randomly allocated to the riociguat arm, or the Placebo arm using a web 
randomization system. The randomization will be done in a 1:1 ratio in accordance with a 
computer-generated random code provided by the Data Coordinating Center (DCC) at the 
Baseline visit. The randomization is planned as a block randomization according to study site 
and will occur at Baseline. Subjects will begin taking the study medication the morning after the 
Baseline visit.  
 
To obtain PK/PD data, approximately 25 patients enrolled at select sites will participate in 
PK/PD testing in order to obtain at least 13 evaluable samples sets. PK/PD subjects will have a 
trough sample drawn with their baseline labs. After Baseline assessments are completed, subjects 
will take one dose of riociguat 1 mg/placebo by mouth (based on their randomization 
assignment) followed by blood sampling at any one time point from 20 minutes to 3 hours post 
dose. Times of dosing and sampling will be recorded. The DCC will work with the outside 
laboratory providing the PK/PD analysis to confirm the presence of at least 13 evaluable 
samples. If data is found to be insufficient, PK/PD sampling may be re-opened until 13 is 
reached.    
 
5. 
STUDY POPULATION 
The patient population for the clinical trial is adults with sickle cell disease with high systolic 
blood pressure, and/or macroalbuminuria as manifested by urine albumin to creatinine ratio >300 
mg/g, and/or TRV > 2.9 m/sec randomized between two arms of patients on placebo and patients 
on riociguat.  Some patients may meet more than one criterion. 
5.1 
Eligibility 


 
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5.1.1 Inclusion criteria 
Patients must fulfill each of the following criteria: 
 Age ≥ 18 years. 
 Sickling disorder (HbSS, HbSC, HbSbeta-thalassemia, HbSD, HbSO-Arab) documented 
by medical history, hemoglobin electrophoresis, or hemoglobin HPLC fractionation. 
 At least one of the following findings: 
   a. Systolic blood pressure ≥130 mm Hg on at least two occasions at least 1 day apart (one 
of these may be by past medical history and one is a current reading [at screening]), 
   b. Macroalbuminuria as manifested by urine albumin to creatinine ratio > 300 mg/g. A 
clinical result within the past 24 months is acceptable to determine eligibility,  
   c. Tricuspid regurgitant velocity (TRV) > 2.9 m/sec measured by echocardiography. A 
clinical echocardiogram done within the past 24 months while the patient is in his/her 
stable disease state is acceptable to determine eligibility. 
d. NT-proBNP level ≥ 160 pg/mL. A clinical result within the past 24 months is acceptable 
to determine eligibility. 
e. Urinalysis protein 1+ or higher. A clinical result within the past 24 months is acceptable 
to determine eligibility. 
 Females of reproductive potential (FRP) must have a negative, pre-treatment pregnancy 
test. Post-menopausal women (defined as no menses for at least 1 year or post-surgical 
from bilateral oophorectomy/hysterectomy) are not required to undergo a pregnancy test.  
 Females of reproductive potential must use acceptable methods of contraception. 
Subjects may choose one highly effective form of contraception (intrauterine devices 
[IUD], contraceptive implants or tubal sterilization) or a combination of methods 
(hormone method with a barrier method or two barrier methods). If a partner’s 
vasectomy is the chosen method of contraception, a hormone or barrier method must be 
used along with this method. Adequate contraception is required beginning at the signing 
of the informed consent form until one month after the last dose of the study medication.  
 Patients must be willing to provide a blood sample for DNA analysis. 
5.1.2 Exclusion criteria 
Patients will be excluded from the study if any of the following criteria are met: 
 
 Pregnant or breastfeeding women 
 Patients with severe hepatic impairment defined as Child Pugh C 
 End stage renal disease requiring dialysis  
 Patients with eGFR <30 mL/min/1.73m, where GFR is estimated based on CKD-epi 
equation;   
 Patients on phosphodiesterase type 5 inhibitors (PDE-5) (such as sildenafil, tadalafil, 
vardenafil) and nonspecific PDE inhibitors (such as dipyridamole or theophylline) or 
nitrates. 


 
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 Patients on strong cytochrome P450 (CYP) and P-glycoprotein 1(P-gp)/BCRP inhibitors 
such as systemic azole antimycotics (e.g., ketoconazole, itraconazole), or HIV protease 
inhibitors (such as ritonavir). 
 Patients on St. John’s Wort. 
 If patients are taking antihypertensive drugs, hydroxyurea, endothelin receptor 
antagonists, L-glutamine, crizanlizumab, or voxelotor prior to enrollment, they are 
excluded until the dose level is stable for at least three months. Subjects may return for 
repeat screening after dose is stable for 3 months. The decision to repeat the Screening 
labs is at the discretion of the PI.   
 Systolic blood pressure < 95 mm Hg at Screening or Baseline Visit before randomization. 
 Current enrollment in an investigational new drug trial. Patients are eligible for 
enrollment 30 days after the last dose of an investigational drug has been received. 
 Evidence of positive qualitative urine drug test at screening for cocaine, phencyclidine 
(PCP), heroin, or amphetamines within three months prior to enrollment. Positive 
findings in subjects prescribed medications for treatment of pain and/or anxiety should 
not be considered evidence of positive qualitative urine drug test.  
 Patients who have recently (last six months) experienced serious bleeding from the lung, 
or have undergone a bronchial arterial embolization procedure.  
 
 Pulmonary hypertension associated with Idiopathic Interstitial Pneumonias 
 Medical disorder, condition, or history that in the investigator’s judgment would impair 
the patient's ability to participate or complete this study or render the patient to be 
inappropriate for enrollment. 
5.2 
Withdrawal of Patients from Study 
5.2.1 Withdrawal 
Patients must be withdrawn from the trial (treatment and procedures) for the following reasons:  
 Pregnancy   
 Patient withdraws consent from study treatment and study procedures.  A patient must be 
removed from the trial at his/her own request or at the request of his/her legally 
acceptable representative.  At any time during the trial and without giving reasons, a 
patient may decline to participate further.  The patient will not suffer any disadvantage as 
a result. 
 Patient is lost to follow-up. 
 Death 
 If, in the investigator's opinion, continuation of the trial would be harmful to the patient's 
well-being. 
 At the specific request of the sponsor (e.g., detection of a new significant safety concern 
related to the study drug). 


 
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 Occurrence of AEs or intercurrent diseases which the investigator judges unacceptable 
for continuation of participation in the study. 
 In case no further dose reduction is possible and the patient does not tolerate the lowest 
possible riociguat dose (0.5 mg TID). 
 
 If treatment is interrupted for more than 42 (14 days) consecutive missed doses during 
any dose titration phase. (Patient is able to restart study drug at 1.0 mg after being off 
study drug up for up to 14 days.)  
Patients may be withdrawn from the study for the following reasons: 
 The patient is non-compliant with study drug, trial procedures, or both 
 Development of an intercurrent illness or situation which would, in the judgment of the 
investigator, significantly affect assessments of clinical status and trial endpoints. 
 Use of illicit drugs or other substances that may, in the opinion of the investigator, have a 
reasonable chance of contributing to toxicity or otherwise skewing trial result. 
 Occurrence of adverse drug reactions, which have from the investigator’s point of view, a 
negative impact on the patient’s individual risk-benefit ratio. (Investigators are obliged to 
reassess the patient’s individual risk-benefit ratio on a continuous basis. Factors like 
anticipated treatment effect, progression of underlying disease, occurrence of side effects 
and alternative treatment options have to be considered.) 
 Pertinent non-compliance with the conditions for the trial or instructions by the 
investigator. 
 Participation in another clinical trial requiring study medication 
Any patient removed from the trial will remain under medical supervision until discharge or 
transfer is medically acceptable. 
In all cases, the reason for withdrawal must be recorded in the CRF. 
5.2.2  Screen failures/dropouts 
A patient who discontinues study participation prematurely for any reason is defined as a 
“dropout” if the patient has already been randomized and received at least one dose of study 
drug. 
A patient who, for any reason (e.g., failure to satisfy the selection criteria), terminates the study 
before the time point used for the definition of “dropout” (see above) is regarded a “screening 
failure.” 
5.2.3  Replacement 
Randomized patients who withdraw prematurely will not be replaced. 
 
6. 
TREATMENT[S]   
6.1 
Treatments to be Administered 


 
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Riociguat immediate release (IR) tablets are available in dose strengths of 0.5 mg, 1.0 mg, 1.5 
mg, 2.0 mg, and 2.5 mg. Riociguat IR tablets of all dose strengths are round tablets with 6 mm 
diameter.  
Riociguat and placebo will be self-administered TID (approximately 6 to 10 hours apart), as 
film-coated tablets with or without food. 
Riociguat and placebo will be supplied as film-coated tablets packaged in high-density 
polyethylene bottles. All tablet formulations will be identical in appearance (size, shape, color) 
and smell. The packaging and labeling will be designed to maintain blinding to the Investigator’s 
team and to patients. 
6.2 
Treatment Assignment 
A patient number (a unique identification number) will be assigned when a patient is evaluated 
for inclusion into the study. Patients who complete all screening procedures and meet all 
eligibility criteria for the study will be allocated using a web-based randomization system in a 
1:1 ratio.  
6.2.1  Dosing and administration 
 
Riociguat 
Dosage: 
0.5, 1 mg, 1.5 mg, 2 mg, 2.5 mg (individual dose titration) 
Route of administration: 
Oral 
Frequency of administration: 
TID 
 
 
Placebo 
Dosage: 
0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg (sham dose titration) 
Route of administration: 
Oral 
Frequency of administration: 
TID 
All subjects will begin taking study medication the AM after the Baseline visit. The starting 
dosage is 1.0 mg taken by mouth 3 times a day with or without food. If systolic blood pressure 
remains greater than 95 mmHg and the patient has no signs or symptoms of hypotension, up-
titrate the dose by 0.5 mg taken three times a day. If systolic blood pressure remains less than 95 
mmHg and no signs or symptoms of hypotension, dosage should be maintained. Dose increases 
should be 2 weeks apart (+/-3 days). The dose can be increased to the highest tolerated dosage, 
up to a maximum of 2.5 mg taken three times a day. SBP < 95 mmHg and no signs or symptoms 
of hypotension, dosage should be maintained. 
Dose reduction at any time is permissible at the discretion of the investigator in the event the 
participant experiences symptomatic hypotension or for other safety reasons. In the event down 
titration is required between scheduled study visits, subjects should remain at the lower dose 
ordered until their next scheduled study visit. Up titration should not be considered until it is 
determined the subject is tolerating the current dose for at least two weeks (+/- 3 days). The 
lowest dose permissible is 0.5 mg, and participants who cannot tolerate 0.5 mg should be 
withdrawn from the trial.  
 


 
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6.3 
Blinding and Unblinding 
The study will be conducted as a double blind trial. In general, patients and investigators will 
remain blinded until study database lock and authorization of data release. 
Unblinding by drug safety personnel of the sponsor 
In compliance with applicable regulations, in the event of a SUSARs, the patient’s treatment 
code will usually be unblinded before reporting to the health authorities, ethic committees and 
investigators (see Section 7.6.3.4). 
Emergency unblinding by the investigator  
Unblinding may occur for emergency purposes only.  Investigators should note that the 
occurrence of a serious adverse event or progressive disease should not routinely precipitate the 
immediate unblinding of the label.  The Investigator is required to promptly document and 
explain to the Sponsor any premature unblinding of the study drug within 24 hours of 
unblinding.  
6.3.1 Accountability 
Study drugs will be stored at the investigational site in accordance with Good Clinical Practice 
(GCP) and Good Manufacturing Practices (GMP) requirements, and will be inaccessible to 
unauthorized personnel.  
The investigator, or a responsible party designated by the investigator, must maintain a careful 
record of the inventory and disposition of the investigational drug products. 
6.3.2   Destruction and return 
At the end of the study, unused supplies of riociguat and placebo will be destroyed according to 
site institutional policies. Destruction will be documented in the Drug Accountability Record 
Form. Written instructions on medication destruction will be made available to affected parties 
as applicable. 
6.4 
Treatment Compliance 
An adequate record of receipt, distribution, and destruction of all study drugs must be kept in the 
form of a Drug Accountability Form.  
Patient compliance with the treatment and protocol includes willingness to comply with all 
aspects of the protocol, and to have blood collected for all safety evaluations.  At the discretion 
of the principal investigator, a patient may be discontinued from the trial for non-compliance 
with follow-up visits or study drug. 
Study drug compliance will be calculated by taking the amount of drug ingested divided by the 
amount the subject should have ingested and multiply by 100. Overall compliance with study 
drug intake will be assessed during data analysis.  
6.5 
Prior and Concomitant Therapy 
All medication that is considered necessary for the patient’s welfare, and which is not expected 
to interfere with the evaluation of the study treatment, may be given at the discretion of the 
investigator.   


 
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All medications (including contrast media) taken within 30 days prior to the start of the study 
and during the study must be recorded in the in the eCRF (including start/stop dates, dose 
frequency, route of administration, and indication). 
6.5.1   Permitted medications 
 Hydroxyurea: Hydroxyurea is  an FDA approved drug for the treatment of sickle cell 
disease. There is no expected drug interaction or additive side effect between riociguat 
(mainly vasodilation) and hydroxyurea (principally myelosuppression).  We plan to allow 
enrollment of subjects already on a stable dose of hydroxyurea as well as subjects not 
taking hydroxyurea.  In this way, riociguat will be tested for its effect both independent 
of hydroxyurea and in combination with hydroxyurea.  To a limited extent, hydroxyurea 
can act as an NO donor, and the safety of its coadministration with riociguat will be an 
important safety determination of this study.  
 L-glutamine: L-glutamine was approved by the FDA on July 7, 2017 to treat sickle cell 
disease. There is no expected drug interaction or additive side effect between riociguat 
and L-glutamine. We plan to allow enrollment of subjects already on a stable dose of L-
glutamine. It may also be used concurrently with or without hydroxyurea.   
 Crizanlizumab: Approved by the FDA on November 15, 2019 to reduce the frequency of 
VOC in adults with sickle cell disease. There is no expected drug interaction or additive 
side effect between riociguat and crizanlizumab. We plan to allow enrollment of subjects 
already on a stable dose of crizanlizumab. It may also be used concurrently with or 
without hydroxyurea.   
 Voxelotor: Approved by the FDA on November 25, 2019 to treat sicke cell disease. 
There is no expected drug interaction or additive side effect between riociguat and 
voxelotor. We plan to allow enrollment of subjects already on a stable dose of voxelotor. 
It may also be used concurrently with or without hydroxyurea.   
 Clinically indicated and stably dosed FDA approved medications will be permissible 
except for PDE5 inhibitors, P450 (CYP) and P-glycoprotein 1(P-gp)/BCRP inhibitors, 
and oral nitrates.   
 Systemic vasodilators, endothelin receptor antagonists, and prostacyclin derivatives will 
be permissible. However, the dosage of vasodilators, endothelin receptor antagonists, and 
prostacyclin derivatives need to be stable for at least three months prior to enrollment and 
preferably their dosage should remain stable throughout the 12-week study treatment 
period.  
 Treatment with non-conventional therapies (e.g., herbs [with the exception of St. John’s 
Wort], acupuncture) and vitamin/mineral supplements is acceptable provided that, in the 
opinion of the investigator, such treatment will not interfere with the trial endpoints. 
 Patients may receive standard of care for any underlying illness. 
6.5.2   Restricted medications 
Co-administration of riociguat with the following concomitant medications is NOT allowed: 
 Nitrates: Nitrates or NO donors (such as amyl nitrite) in any form (e.g., nitrates, 
molsidomine, sodium nitroprusside) because of hypotension 


 
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 PDE inhibitors: PDE-5-inhibitors (such as sildenafil, tadalafil, vardenafil) and 
nonspecific PDE inhibitors (such as dipyridamole or theophylline) because of 
hypotension 
 Arginine supplements 
7. 
PROCEDURES AND VARIABLES 
7.1.1 Study flowchart 
See Appendix A for Schedule of Procedures. 
7.1.2 Timing of assessments 
Outlined below are study procedures that will be performed at screening, during the study 
treatments, and at follow-up.   
 
Visit 1 (Screening) 
 
 Obtain written informed consent. 
 Demographics, and review of medical history for inclusion and exclusion criteria 
 Concomitant medications 
 Vital Signs  
 Clinical laboratory evaluations including CBC with differential, reticulocyte count, hepatic  
panel to include total bilirubin, INR (International Normalized Ratio), renal chemistry, and 
LDH (INR may be used from the patient’s medical record within 3 months of screening visit)  
 Urine or serum pregnancy test for women of child bearing potential 
 Electronic pain diary (piloted only at selected sites) 
 
Re-screening: It is permitted to re-screen each subject once. The investigator will use clinical 
judgment to determine if any of the screening procedures need to be repeated or if re-consenting 
is required. Additional re-screening may be allowed only upon approval by the Coordinating 
Center. 
 
Visit 2 Baseline (Week 0)  
 
The Baseline assessments may be done in two visits to accommodate subject/site schedule 
limitations). The Baseline visit may occur from 0-4 weeks post screening, and may also be 
combined with the Screening visit procedures.  
 
 Confirm systolic blood pressure eligibility  
 Randomization 
 Comprehensive Medical History 
 Concomitant medications  
 Review of Symptoms 
 Complete physical examination to include vital signs, body weight, and height.  
 Brief Pain Inventory, ASCQ-Me, PROMIS Assessments 
 Clinical laboratory evaluations including CBC with differential, reticulocyte count, hepatic 
chemistry to include total/direct bilirubin, albumin, renal chemistry, and LDH  


 
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 Urinalysis 
 Urine albumin and  urine creatinine concentrations 
 Urine or serum pregnancy test for women of child bearing potential 
 Research blood sample for  hemoglobin HPLC or electrophoresis and ferritin will be 
processed at the site’s clinical lab 
 Research blood sample for biomarkers analysis, NT-proBNP, DNA, PK/PD (if appropriate) 
will be drawn at the clinical site and sent to the Coordinating Center Core Lab for processing 
 Research blood sample for genotyping for alpha-thalassemia and BCL11a rs766432 
 Baseline research echocardiogram  
 6 Minute Walk Test with Borg Dyspnea/Fatigue Scale 
 Electronic pain diary (piloted only at select sites) 
 Record results of RHC if performed at standard of care 
 PK/PD studies (performed only on approximately 25 patients enrolled at select sites) 
 Dispensing study drugs and instructions for use and storage. Study medication should be 
started the AM after the baseline visit. 
 
Visit 3 (Week 2) 
 
 Treatment compliance 
 Physical examination  
 Vital signs 
 Titration of study drug dose 
 Dispensing study drugs 
 Concomitant medications 
 Review of Symptoms/AE assessment 
 Electronic pain diary (piloted only at select sites) 
 
Visit 4 (Week 4) 
 
 Treatment compliance 
 Physical examination  
 Vital signs 
 Clinical laboratory evaluations including CBC with differential, reticulocyte count, hepatic 
chemistry to include total/direct bilirubin, albumin, renal chemistry, and LDH  
 Urine or serum pregnancy test for women of child bearing potential 
 Titration of study drug dose 
 Dispensing study drugs 
 Concomitant medications 
 Review of Symptoms/AE assessment 
 Electronic pain diary (piloted only at select sites) 
 
Visit 5 (Week 6) 
 
 Treatment compliance 
 Physical examination  
 Vital signs 


 
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 Titration of study drug dose 
 Dispensing study drugs 
 Concomitant medications 
 Review of Symptoms/AE assessment 
 Electronic pain diary (piloted only at select sites) 
 
Visit 6 (Week 8) 
 
 Treatment compliance 
 Physical examination  
 Vital signs 
 Clinical laboratory evaluations including CBC with differential, reticulocyte count, hepatic 
chemistry to include total/direct bilirubin, albumin, renal chemistry, and LDH  
 Urine or serum pregnancy test for women of child bearing potential 
 Titration of study drug dose 
 Dispensing study drugs 
 Concomitant medications 
 Review of Symptoms/AE assessment 
 Electronic pain diary (piloted only at select sites) 
 
Visit 7 (Week 10) 
 
 Treatment compliance 
 Physical examination  
 Vital signs 
 Titration of study drug dose 
 Dispensing study drugs 
 Concomitant medications 
 Review of Symptoms/AE assessment 
 Electronic pain diary (piloted only at select sites) 
 
Visit 8 End of Treatment Visit (Week 12) 
 
 Treatment compliance 
 Physical examination  
 Vital signs and body weight 
 Clinical laboratory evaluations including CBC with differential, reticulocyte count, hepatic  
chemistry to include total/direct bilirubin, albumin, renal chemistry, and LDH 
 Research blood sample for hemoglobin HPLC or electrophoresis and ferritin will be 
processed at the site’s clinical lab 
 Urinalysis  
 Urine albumin and urine creatinine concentrations 
 Urine or serum pregnancy test for women of child bearing potential 
 Research blood samples for biomarkers analysis and NT-proBNP will be drawn at the 
clinical site and sent to the Coordinating Center Core Lab for processing 
 Research echocardiogram  


 
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 6 minute walk test with Borg Dyspnea/Fatigue Scale 
 Brief Pain Inventory, ASCQ-Me, PROMIS Assessments 
 Concomitant medications 
 Review of Symptoms/AE assessment 
 Electronic pain diary (piloted only at select sites) 
 Record results of RHC if performed as standard of care after the baseline visit 
 
Visit 9 (Week 13)  
 
 Phone call follow-up 
 Concomitant medications 
 Review of Symptoms/AE assessment 
 
 Final Visit (30 days (+/-3 days) after Week 12 Visit) 
 
 Physical examination  
 Vital signs 
 Urine or serum pregnancy test for women of child bearing potential 
 Concomitant medications 
 Review of Symptoms/AE assessment 
 
7.1.3    Early Discontinuation of Study Treatment and/or Early Withdrawal from Study 
 
If for whatever reason a subject was withdrawn from the study drug treatment prior to 
completing Week 12, the subject should be encouraged to remain in the study and return to the 
clinic to complete end of treatment assessments as indicated at Visit 8 within 3 days of 
discontinuing study drug if possible, as well as scheduled telephone follow-up Visit 9 (one week 
post discontinuation of study drug) and Final Visit 10 (30 days post discontinuation of study 
drug) in order to collect important safety information. 
 
If a subject cannot or is unwilling to attend any clinic visit(s), at a minimum, the site staff should 
make every effort to make phone contact with the subject to obtain information concerning the 
subject’s health status including adverse events and concomitant medications for the visit 8, 9 
and 10 forms. 
 
If a subject decides to completely withdraw from the study (refuses any further study 
participation or contact), all efforts should be made to complete and report the observations prior 
to withdrawal. 
7.2 
Safety Evaluation and Procedures 
7.2.1 Medical history 
Medical history findings (i.e. previous diagnoses, diseases or surgeries) meeting all criteria listed 
below will be collected: 
 
Indication 
 
Start before signing of the informed consent  


 
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 
Considered relevant to the study 
Detailed instructions on the differentiation between (i) medical history and (ii) adverse events 
can be found in Section 7.6.3.1. 
7.2.2  Physical examination 
A physical examination that includes a review of the following body systems will be performed 
at each clinic visit beginning at Baseline: 
 
General Appearance 
 
Skin 
 
Head, Ears, Eyes, Nose, Throat 
 
Spine/Neck/Thyroid 
 
Respiratory 
 
Cardiovascular 
 
Abdomen 
 
Nervous System  
 
Musculoskeletal 
Any abnormalities or changes in intensity from the baseline assessment noted during the review 
of body systems should be documented and reported on the appropriate eCRF. If a new clinically 
significant finding (i.e., not noted at baseline) occurs at subsequent examination, an AE form 
must be completed. In addition, resolution of any abnormal findings during the study will be 
noted in the medical record and/or the eCRF if clinically significant. 
7.2.3  Vital signs 
Blood pressure, heart rate, and respirations will be measured after the subject has been at rest for 
10 minutes in a sitting position. Height and weight will be measured at the Baseline Visit. At 
Visit 8, weight measurement will be repeated. 
7.2.4 Pain assessments 
Brief Pain Inventory 
The Brief Pain Inventory will be used as a tool to measure the intensity of pain and interference 
of pain as well as to query the patient about pain relief, pain quality, and patient’s perception of 
the cause of pain during the study treatment at Baseline and Week 12. 
 
Adult Sickle Cell Quality of Life Measurement Information System 
The Adult Sickle Cell Quality of Life Measure (ASCQ-Me) is an NIH-funded multicenter 
network that has enrolled over 500 patients in developing a patient reported outcome specific to 
sickle cell disease. The measure has been adapted to a version that can be administered by 
computer [61,62].  ASCQ-Me is the first disease-specific partner of the NIH Roadmap Patient-
Reported Outcomes Measurement Information System (PROMIS)[63-65].  ASCQ-Me will be 
accompanied by the ten-item Global Health form from PROMIS.  These are not yet validated 
endpoints accepted by the FDA, but the FDA has encouraged more data to be developed with 
them to potentially validate future use as endpoints for drug approval.   


 
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Patients will report outcomes using the sickle cell-specific patient reported outcome instrument, 
ASCQ-Me, and PROMIS at Baseline and Week 12.  Whenever possible, the site staff will 
provide study subjects a secure web link that will allow subjects t to enter their responses directly 
from a computer, tablet computer, or smart phone without additional intervention from the site 
staff.  The ASCQ-Me computer version has been designed and tested for direct user input by 
sickle cell patients using computers, tablets or smart phones. Subjects may complete paper 
versions of the assessments if internet access is not feasible.  
 
Electronic Pain Diary 
The electronic pain diary will be piloted only at select sites in approximately fifteen patients. 
This is a secure, password-protected electronic pain diary housed on a secure password-protected 
server (Fig. 5). The adaptable design allows the application to be accessed using a smartphone, 
computer, or tablet. The mobile web site has been chosen over an “app” so that the application is 
platform independent (i.e., users can use an iPhone, Android, or any other platform).  The pain 
diary allows patients to enter medications taken at home.  Content validity has been validated 
and its development and initial end-user reviews have been published in the Clinical Journal of 
Pain [66].   


 
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Figure 5.  Representative screen shots from the online sickle cell disease electronic pain 
diary.  This pain diary has been developed in an iterative process, customized to the needs of 
sickle cell disease [66]. 
7.2.5 Laboratory Parameters 
All clinical laboratory assays will be performed locally at each participating site according to the 
laboratory’s normal procedures. Clinical lab results drawn within the +/- 3 day window for the 
subjects standard care may be accepted for the study labs.  Lab assays may be repeated once to 
rule out lab error, before determining if results are normal or abnormal. It is recognized that in 
SCD lab values that are out of the reference range may in fact be baseline for that individual. 
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whether the result is considered to be clinically significant or not. Out of range results not 
considered to be clinically significant by the investigator will not be considered abnormal for the 
purposes of this study. The laboratory parameters are outlined below. For details on the timing of 
blood and urine sampling, refer to Appendix A. 
 
 
Hematology: Complete Blood Count (CBC): Hemoglobin, hematocrit, red blood cell 
(RBC), mean corpuscular hemoglobin concentration (MCHC), mean cell volume (MCV), 
reticulocyte counts, white blood cell (WBC) count, platelet count, and ANC), reticulocyte 
counts 
 
Liver function tests: AST, ALT, albumin, total protein, total bilirubin, direct bilirubin, INR, 
alkaline phosphatase, LDH 
 
Renal function tests: potassium, sodium, chloride, bicarbonate, blood urea nitrogen (BUN), 
and creatinine 
 
Urinalysis  
 
Urine albumin and creatinine concentrations 
 
Pregnancy Test: Urine or serum pregnancy test will be performed only for women of child 
bearing potential at screening, prior to receiving study treatment (Baseline), and at 4 week 
intervals. There is site to site variability in preference for urine or serum pregnancy test. 
Some institutions require a serum pregnancy test, which is equal or superior in sensitivity to 
the urine pregnancy test. Adding the option of a serum pregnancy test allows for flexibility 
per site institutional policies.  
7.3 Efficacy Evaluation and Procedures 
7.3.1 Tricuspid regurgitant velocity by echocardiography 
Increased tricuspid regurgitant velocity (TRV) is shown to link to high incidences of unexplained 
deaths in adult SCD patients with cardiopulmonary complications. TRV and echocardiography 
markers of left ventricular diastolic dysfunction will be assessed using a non-invasive 
transthoracic echocardiography at baseline and the end of double blinded study treatment at 
Week 12 according to the Echo Manual.   
 
Other routine echocardiographic measures will include LV volumes and ejection fraction, LV 
mass and LV mass index, and left atrial volume. Mitral inflow recordings will be obtained with 
the pulsed Doppler sample volume at the level of the mitral leaflet tips during maximal opening 
in diastole. Tissue Doppler recordings will also be measured with sample volumes at both the 
septal mitral annulus and lateral mitral annulus. The ratio of early diastolic LV inflow velocity to 
lateral mitral annular velocity will be calculated as an estimate of LV filling pressure. TRV will 
be used to estimate pulmonary artery systolic pressures. 
 
The de-identified echocardiograms will be sent to the Echo Core Lab at Washington University 
in St. Louis, MO for central reading.  Echocardiography will be performed at baseline and Week 
12 on all randomized subjects. Readers will be blinded to randomization assignment. 
7.3.2 Optional right heart catheterization 
Right heart catheterization is not required but may be considered as standard of care at discretion 
of the local investigator. It could be considered for patients who have both TRV >2.7 m/s and 6 
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Baseline and Week 12.  If the patient has had right heart catheterization up to 12 months prior to 
enrollment, these results could be collected at time of enrollment. 
 
Table 3. Right Heart Catheterization Parameters 
 
Directly measured parameters 
Value 
Unit 
Mean right atrial pressure (RAPmean) 
 
mmHg 
Right Ventricular Systolic Pressure (RVSP) 
 
mmHg 
Right Ventricular Diastolic Pressure (RVDP) 
 
mmHg 
Systolic, diastolic, and mean pulmonary arterial 
pressure (PAPsyst, PAPdiast, mPAP) 
 
mmHg 
Pulmonary capillary wedge pressure (PCWP) 
 
mmHg 
Heart rate (HR) 
 
beats per minute 
Systolic, diastolic, and mean systemic arterial 
pressure (SBP, DBP, SAPmean) 
 
mmHg 
Cardiac output (CO); average of 3 measurements, 
performed and calculated by CO 
device/thermodilution methodology; 5 
measurements in case of atrial fibrillation 
 
L/min 
Mixed venous oxygen saturation rate (SvO2) 
 
% 
 
7.3.3 Six (6) Minute Walk Distance (6MWD) Test 
The 6MWD Test must be performed at Baseline and Week 12.  Per the American Thoracic Society 
(ATS) Guideline, the 6MWD Test will be carried out indoors, along a long, flat, straight, enclosed 
corridor with hard surface that is seldom traveled. The walking course must be 30 meters in length, 
but not less than 25 meters. The length of the corridor and turnaround points should be marked. 
Patients will be instructed to walk alone, not run, from one end to the other end of the walking 
course, at their own pace, while attempting to cover as much ground as possible in 6 min.  
7.3.4 The Borg Dyspnea/Fatigue Scale 
The Borg Scale will be measured in conjunction with the 6MWD Test at Baseline and Week 12. 
The scale will be used to measure the level of severity of breathlessness perceived by the patient 
before and after 6MWD test. The severity is measured on a 10 point scale with 0 = nothing at all 
and 10 = maximum severity of breathlessness. The Borg Scale will be explained to the patients 
before starting the 6MWD Test. Patients will be asked to rank their exertion at the end of the 
6MWD Test. The rate of exertion will be given according to the following scale ranging from 0 to 
10. 
7.3.5 Biomarkers and Repository  
At Baseline and Week 12, blood will be collected in specific tubes provided by the Coordinating 
Center. The details of tube type and sample processing will be provided in the lab manual for this 
protocol.   
 
N-terminal prohormone of brain natriuretic peptide (NT-proBNP) 
 


 
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NT-proBNP, a protein generated by the heart, is elevated in SCD patients suffering from 
congestive heart failure and pulmonary hypertension. NT-proBNP will be evaluated at Baseline 
and Week 12. NT-proBNP will be quantified in plasma. Samples will be drawn before intake of 
study drugs. Further details on collection, labeling, storage, and shipping of samples are provided 
in a separate laboratory manual.  
 
Additional exploratory biomarkers 
The following additional exploratory plasma biomarkers known to play important roles in 
prognosis of sickle cell disease will be quantified at Baseline and Week 12.  
 
- Biomarkers of oxidative stress: 3-Nitro-tyrosine, Malondialdehyde and 8-IsoPGF2a.  
- Biomarkers of NO-Pathway dysregulation: asymmetric dimethylarginine (ADMA), 
SDMA and cGMP. 
- Biomarkers of inflammation (C-reactive protein) and erythropoiesis (erythropoietin) 
 
Biospecimen Repository 
 
Any remaining blood will be stored indefinitely at the University of Pittsburgh’s Core 
Biospecimen Repository for future research. Samples will be stored without identifiers.  
 
7.4 
Pharmacokinetics / Pharmacodynamics Studies  
A small subgroup of patients will be used to obtain at least 13 evaluable sample sets for PK/PD 
of riociguat in SCD.  We will look at drug concentrations at two timepoints for each patient – 
approximately at Cmax and at expected time of trough.  Data analysis will be conducted using 
non-compartmental methods.  
 
Pharmacokinetics studies, coupled with the individual patient’s systolic blood pressure as the 
pharmacodynamic parameter, will be performed in a subset of approximately 25 patients at select 
sites. PK/PD subjects will be given one dose of riociguat/placebo (based on randomization 
assignment) by mouth at the Baseline Visit. The plasma concentrations of riociguat will be 
determined using a sparse sampling approach. Blood samples of 4 mL each for PK analysis will 
be collected at two time points: (1) at trough (before a study medication tablet is taken) and (2) at 
any time between 20 minutes and 3 hours post dose. Time of dose administration and sample 
collection will be recorded. Vital signs and blood pressure will be measured and monitored 
closely at each time points. 
 
Details on collection, labeling, storage, and shipping of samples are provided in a separate 
laboratory manual.  Riociguat samples will be sent to the outside lab for PK/PD analysis. Should 


 
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data be insufficient, PK/PD sampling may be re-opened in order to obtain the minimal samples 
sets required for adequate data.  
7.5 
Genotyping for α-Thalassemia and BCL11a 
Exploratory genotyping for α-Thalassemia and BCL11a is planned to better understand the 
relationships between the pathophysiology and specific genetic polymorphisms, and its 
association with fetal hemoglobin levels and pain crises in adults with SCD.  
 
A one-time 5 mL of blood sample will be obtained from each patient at Baseline. De-identified 
blood samples will be shipped to and stored at University of Pittsburgh Sickle Cell Disease 
biospecimen repository for analysis. Alpha globin gene sequence analysis will be performed to 
identify nondeletional point mutations. SNaPshot PCR, capillary electrophoresis and cycle 
sequencing will be used for the genotyping of common single nucleotide polymorphisms (SNPs) 
at the BCL11a gene known to influence HbF levels and associate with pain crisis rate in SCD 
patients. 
7.6 
Data Safety Monitoring Plan 
7.6.1 Data Safety and Monitoring Board 
An independent Data Safety and Monitoring Board (DSMB) will be created to review this study. 
The DSMB will comprise a panel of experts with relevant experience and will periodically 
review safety subject enrollment data and the supporting documentation. None of the DSMB 
members will be affiliated with this study or have a conflict of interest.  
 
In order to allow ongoing safety monitoring during the conduct of the study, DSMB will receive 
unblinded safety data. If significant safety issues arise in between scheduled meetings, ad hoc 
meetings will be scheduled to review the data. The DSMB will also review the interim analysis 
after 50 subjects have been enrolled and received study drugs; however, enrollment will not be 
suspended pending DSMB review. Based on the safety implications of the data, the DSMB may 
recommend modification or termination of the study. 
7.6.2 Data Safety and Monitoring Plan 
The DSMB will meet at periodic intervals during the course of the study. The DSMB will be 
expected to convene as needed, but not less than every 6 months to review the progression of the 
study including subject enrollment, protocol compliance, and adverse event reports. The DSMB 
will conduct interim monitoring of accumulating data from research activities to assure the 
continued safety of human subjects, relevance and appropriateness of the study, and the integrity 
of research data. 
 
Apart from the DSMB mentioned above, the study investigator has primary responsibility for the 
oversight of the data and safety monitoring at each participating site.  An independent medical 
monitor will review related adverse events and serious adverse events reported from all sites. 
Subject recruitment and retention as well as adverse events and unanticipated problems will be 
monitored locally at each site as well as centrally by the Data Coordinating Center.  In addition 
to DSMB meetings, the DCC will report adverse events on a monthly basis to the Steering 
Committee for this study.       


 
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The IND Sponsor is responsible to comply with the local regulation and legislation for adverse 
events reporting. 
All patients who receive at least one dose of study treatment will be valid for the safety analysis.  
All observations pertinent to the safety of the study treatment will be recorded and included in 
the final report. 
All AEs whether considered drug-related or not, will be reported with a diagnosis, start/stop 
dates, action taken, whether treatment was discontinued, any corrective measures taken, 
outcome, and other possible causes.  For all events, the relationship to treatment and the intensity 
of the event will be determined by the investigator. 
Safety variables may include but are not limited to the following: laboratory changes in vital 
signs (blood pressure, heart rate, respiratory rate, and temperature) and, in some instances, 
changes in chest x-ray images, as produced at the investigator’s discretion (e.g., for evaluation 
for pneumonia). 
7.6.3 Adverse events 
Patients should be monitored for side effects of all concomitant medications regardless of the 
path of drug elimination. Investigators should refer to the Safety Information section of the 
current IB for riociguat, including the DCSI (development core safety information), for the 
expected side effects of riociguat.  As with any agent, there is always the potential for 
unexpected AEs, including hypersensitivity reactions.  The IB will be updated if any new 
relevant safety data are obtained. 
Therapeutic monitoring should be performed following dose selection of riociguat in a manner 
consistent with the local clinical standard of care.   
All concomitant medications must be recorded.  
Patients must be carefully monitored for AEs.  This monitoring also includes clinical laboratory 
tests.  Adverse events should be assessed in terms of their seriousness, intensity, and relationship 
to the study drug, AEs will be collected from the time of enrollment until the study visit at 30±2 
days following discontinuation of the study drug. 
7.6.3.1 Definitions 
Definition of adverse event (AE) 
In a clinical study, an AE is any untoward medical occurrence (i.e. any unfavorable and 
unintended sign [including abnormal laboratory findings], symptom or disease) in a patient or 
clinical investigation patient after providing written informed consent for participation in the 
study.  Therefore, an AE may or may not be temporally or causally associated with the use of a 
medicinal (investigational) product. 
A surgical procedure that was planned prior to the start of the study by any physician treating the 
patient should not be recorded as an AE (however, the condition for which the surgery is 
required may be an AE if the condition worsens compared to baseline). 
 Conditions that started before signing of informed consent and for which no symptoms or 
treatment are present until signing of informed consent are recorded as medical history 
(e.g., seasonal allergy without acute complaints). 


 
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 Conditions that started before signing of informed consent and for which symptoms or 
treatment are present after signing of informed consent, at unchanged intensity, are 
recorded as medical history (e.g., allergic pollinosis). 
 Conditions that started or deteriorated after signing of informed consent will be 
documented as adverse events. 
Definition of serious adverse event (SAE) 
An SAE is classified as any untoward medical occurrence that, at any dose, meets any of the 
following criteria (a – f): 
a. Results in death. 
b. Is life-threatening. 
The term ‘life-threatening’ in the definition refers to an event in which the patient was at 
risk of death at the time of the event, it does not refer to an event which hypothetically 
might have caused death if it were more severe. 
c. Requires inpatient hospitalization or prolongation of existing hospitalization. 
A hospitalization or prolongation of hospitalization will not be regarded as an SAE if at 
least one of the following exceptions is met: 
- The admission results in a hospital stay of less than 12 hours. 
- The admission is pre-planned (i.e., elective or scheduled surgery arranged prior to 
the start of the study). 
- The admission is not associated with an AE (e.g., social hospitalization for purposes 
of respite care). 
However, it should be noted that invasive treatment during any hospitalization may 
fulfill the criterion of ‘medically important’ and as such may be reportable as an SAE 
dependent on clinical judgment.  In addition, where local regulatory authorities 
specifically require a more stringent definition, the local regulation takes precedence. 
d. Results in persistent or significant disability / incapacity. 
Disability means a substantial disruption of a person’s ability to conduct normal life’s 
functions. 
e. Is a congenital anomaly / birth defect. 
f. Is another medically important serious event as judged by the investigator. 
7.6.3.2 Classifications for adverse event assessment 
The following classifications will be used: 
 Seriousness 
 Intensity 
As an alternative to the grading system described in the standard text below (mild, 
moderate, severe), other systems for intensity may be used (e.g., CTCAE, Grade 1 to 
Grade 5).  If used, this needs to be stated and definitions of the grades should be 
provided. If applicable, a “translation” between the CTCAE system and the standard 
system of intensity grading may have to be provided. 


 
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 Attribution 
 Study treatment action 
 Other specific treatment of AE 
 Outcome 
All AEs will be assessed and documented by the investigator according to the categories detailed 
below. 
7.6.3.2.1 Seriousness 
For each AE, the seriousness must be determined according to the criteria given in 
Section 7.6.3.1. 
7.6.3.2.2 Intensity 
The severity of the AE is assessed by the investigator and classified as: 
Mild 
Moderate 
Severe 
7.6.3.2.3  Attribution 
Attribution is an assessment of the relationship between the AE and the medical intervention. 
After naming and grading the event, the clinical investigator must assign an attribution to the AE 
using the following attribution categories: 
 
RELATIONSHIP 
ATTRIBUTION 
DESCRIPTION 
Unrelated to investigational 
agent/intervention 
Unrelated  
The AE is clearly NOT related 
to the intervention  
Unlikely  
The AE is doubtfully related to 
the intervention  
Related to investigational 
agent/intervention  
Possible  
The AE may be related to the 
intervention  
Probable  
The AE is likely related to the 
intervention  
Definite  
The AE is clearly related to the 
intervention  


 
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Factors to be considered in assessing the relationship of the AE to study treatment include: 
- The temporal sequence from drug administration:  The event should occur after the drug is 
given.  The length of time from drug exposure to event should be evaluated in the clinical 
context of the event. 
- Recovery on drug discontinuation (de-challenge), recurrence on drug re-introduction (re-
challenge): 
- Patient’s response after de-challenge or patients response after re-challenge should be 
considered in the view of the usual clinical course of the event in question. 
- Underlying, concomitant, intercurrent diseases:   
Each event should be evaluated in the context of the natural history and course of the disease 
being treated and any other disease the patient may have. 
- Concomitant medication or treatment:  
The other drugs the patient is taking or the treatment the patient receives should be examined 
to determine whether any of them may be suspected to cause the event in question. 
- The pharmacology and pharmacokinetics of the study treatment:  
The pharmacokinetic properties (absorption, distribution, metabolism and excretion) of the 
study treatment, coupled with the individual patient’s pharmacodynamics should be 
considered. 
7.6.3.2.4 Action taken with study treatment  
Any action on study treatment to resolve the AE is to be documented using the categories listed 
below. 
The study treatment action should be recorded separately for each study treatment. 
־ 
Drug withdrawn 
־ 
Drug interrupted 
־ 
Dose not changed 
־ 
Dose increased 
־ 
Not applicable 
־ 
Unknown 
7.6.3.2.5 Other specific treatment(s) of adverse events 
־ 
None 
־ 
Other 


 
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7.6.3.2.6 Outcome 
The outcome of the AE is to be documented as follows:  
־ 
Recovered/resolved 
־ 
Recovering/resolving 
־ 
Recovered/resolved with sequelae 
־ 
Not recovered/not resolved 
־ 
Fatal 
־ 
Unknown 
7.6.3.3 Assessments and documentation of adverse events 
Each serious adverse event must be followed up until resolution or stabilization, by submission 
of updated reports to the designated person. An isolated laboratory abnormality that is assigned 
grade 4, according to CTCAE definition, is not reportable as an SAE; unless the investigator 
assesses that the event meets standard ICH criteria for an SAE. CTCAE grade 4 baseline 
laboratory abnormalities that are part of the disease profile should not be reported as an SAE, 
specifically when they are allowed or not excluded by the protocol inclusion/exclusion criteria.   
7.6.3.4 Reporting of serious adverse events and unanticipated problems 
Each serious adverse event must be followed up until resolution or stabilization, by submission 
of updated reports to the designated person. An isolated laboratory abnormality that is assigned 
grade 4, according to CTCAE definition, is not reportable as an SAE; unless the investigator 
assesses that the event meets standard ICH criteria for an SAE.  CTCAE grade 4 baseline 
laboratory abnormalities that are part of the disease profile should not be reported as an SAE, 
specifically when they are allowed or not excluded by the protocol inclusion/exclusion criteria.   
 
When required, and according to local law and regulations, serious adverse events must be 
reported to the Ethics Committee and Regulatory Authorities.  
 
All serious adverse events should be reported to the Coordinating Center. The investigator will 
then submit a detailed written report to the Coordinating Center. The DSMB, all investigators, 
and IRB will also be promptly notified in accordance with the respective policies and 
procedures. 
7.6.3.5 Expected adverse events 
For this study, the applicable reference document is the most current version of the investigator’s 
brochure (IB) / summary of product characteristics. 
Overview listings of frequent events that have occurred so far in the clinical development are 
shown in the current IB.  If relevant new safety information is identified, the information will be 
integrated into an update of the IB and distributed to all participating sites. 
The expectedness of AEs will be determined by Bayer according to the applicable reference 
document and according to all local regulations. 


 
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7.6.4 Adverse event of special safety interest: Pregnancy 
Pregnancy has been defined as an event of special interest for riociguat; therefore the investigator 
must report to the Coordinating Center any pregnancy occurring in a study patient, during the 
patient’s participation in this study or within one month of discontinuing riociguat. The report 
should be submitted within the same timelines as an SAE, although a pregnancy per se is not 
considered an SAE.   
The outcome of the pregnancy should be followed up whenever possible, and any abnormal 
outcome of the mother or the child should be reported. In the event that study drug exposure 
occurs in a pregnancy via a male study participant, efforts should be made to obtain information 
on any abnormality of pregnancy course or outcome.  
8. 
STATISTICAL METHODS AND DETERMINATION OF SAMPLE SIZE 
8.1 
Analysis Sets 
A randomized patient will be valid for safety and efficacy analyses regardless of whether study 
drug was administered. Results will be analyzed according to intention-to-treat (ITT).  
8.2 
Variables/Endpoints 
8.2.1 Primary variables/endpoints 
The primary endpoints measure:  The proportion of participants who experience at least 1 
treatment-emergent SAE, which is defined as those occurring after the baseline visit (i.e. after 
initiation of study drug), adjudicated by a designated committee of protocol investigators and 
outside experts.  This endpoint will monitor the safety and tolerability of riociguat in patients 
with sickle cell disease. 
8.2.2 Secondary variables endpoints 
Secondary outcomes will include more detailed safety measures that evaluate whether riociguat 
increases pain signals: 
 The frequency of SAE adjudicated by a designated committee of protocol investigators 
and outside experts to be due to sickle cell related painful crisis in the riociguat group 
compared to the placebo group, excluding study drug or disease independent SAE (e.g., 
accidents) 
 Overall incidences of treatment-emergent adverse events 
 Changes in pain intensity from baseline to Week 12 using numerical pain score and the 
Brief Pain Inventory 
Efficacy measures will be among the secondary variables to evaluate signals related to the 
pulmonary and systemic vascular tone: 
 Changes in functional exercise capacity by assessing 6 Minute Walk Distance test from 
baseline to Week 12 (primary efficacy outcome) 
 Changes in hemodynamic parameters including blood pressure as the main 
pharmacodynamic variable, and tricuspid regurgitant velocity, echocardiography markers 
of left ventricular diastolic dysfunction after 12 weeks of double blinded treatment with 
riociguat compared with placebo 


 
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 Changes in the levels of plasma NT-proBNP from baseline to Week 12 
 Changes in the Modified Borg Dyspnea/Fatigue Scale from baseline to Week 12 
 Changes in laboratory measures from baseline to Week 12 (1) Renal: urine albumin to 
creatinine ratio, albuminuria stratified as microalbuminuria and macroalbuminuria, 
estimated GFR, CKD stage based on albuminuria and GFR; (2) Hematologic:  
Hemoglobin, LDH, reticulocyte count, white blood cell count, fetal hemoglobin 
 Incidences of sickle cell disease-related clinical complications such as acute chest 
syndrome, priapism, new leg ulcer, new stroke, other new non-CNS thromboembolic 
event during the 12 week study treatment. 
8.2.3 Exploratory variables/endpoints 
Exploratory endpoints: 
 Maximum and trough plasma concentration (Cmax and Ctrough) 
 Changes from baseline to week 12 in biomarkers of oxidative stress: 3-Nitro-tyrosine, 
Malondialdehyde and 8-IsoPGF2a  
 Changes from baseline to week 12 in biomarkers of NO-Pathway dysregulation: 
asymmetric dimethylarginine (ADMA), SDMA and cGMP. Changes in pain diary scores 
 Changes from baseline to week 12 in biomarkers of inflammation (C-reactive protein) 
and erythropoiesis (erythropoietin) 
 Changes from baseline to week 12 in electronic daily pain diary 
8.3 
Statistical and Analytical Plans 
All primary and secondary outcomes will be described using sample means or proportions along 
with 90% confidence intervals within and between study arms.  Suitable transformations will be 
considered for continuous variables if there are substantial departures from normality.  
Demographic and baseline characteristics will be compared between the study arms using two-
sample t-tests or chi-square tests, as appropriate.  Any covariates that are significantly associated 
with study arm will be included as relevant covariates in the primary safety and efficacy analyses 
only if they are highly prognostic of the outcome measures. All hypothesis testing will be 
conducted with two-tailed, 10% type I error.   
Attrition & Missing Data: We will make every effort to minimize attrition and missing data in 
the study.  We will record reasons for drop-out and investigate any relationship with study arm.  
We will also compare baseline characteristics between participants who do and do not complete 
the study.  With regard to missing data, we will investigate the mechanism and degree of 
missingness using established techniques.[67]  If the amount of missingness is great, we will 
compare various imputation techniques to see how robust the overall inferences are. 
8.3.1 Safety analysis 
The safety analysis will be performed in the population of patients valid for ITT. 
 
SAEs 
The null hypothesis, as it relates to the safety analysis, is that there will be no difference in the 
proportion of participants who experience at least 1 treatment-emergent SAE between study 


 
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arms. Logistic regression will be used to compare the proportion of participants experiencing at 
least 1 treatment-emergent SAE by study arm, adjusting for clinical site.  We will also 
investigate the incidence of treatment-emergent SAEs by treatment group using a stratified log-
rank test. SAEs are considered to be treatment-emergent if they have started or worsened after 
first dose of study medication up to 7 days after end of study treatment. The incidence of AEs 
during follow-up (i.e. AEs occurring more than 7 days after end of study medication) will be 
tabulated separately. 
 
Mortality during the 12 week study treatment period will be summarized descriptively. Any 
deaths in the study treatment period will be listed, with day of death relative to start and stop 
of study drug and cause of death. 
 
The eligibility criteria of this clinical trial will enrich for a population of adults with sickle cell 
disease at high risk for death.  Observational studies indicate that for adult SCD patients with 
elevated TRV, mortality can be as high as 40% at 45 months [21].  This translates to a crude 
mortality rate of 0.009 per patient-month.  On this study, we will have approximately 100 
patients each enrolled for approximately 3.5 months, or 350 patient-months in aggregate.  Even 
with no treatment, we could anticipate up to 3 deaths for this cohort while on study.  This 
baseline mortality rate will need to be considered by the regulatory bodies for appropriate 
perspective.  SCD adults with systemic hypertension or proteinuria are similarly at risk for 
mortality [17,18]. 
 
Pain intensity 
A secondary safety measure will be the Brief Pain Inventory (BPI) and numerical pain score 
measured at 12 weeks.  We will utilize an analysis of covariance (ANCOVA) for each outcome 
adjusting for the baseline measure and including predictors for study arm and clinical site.  The 
utilization rate of acute care facilities will be compared between study arms using Poisson 
regression as a function of study arm and clinical site.      
 
Laboratory data 
The safety evaluation of laboratory data will include: 
 listings of laboratory data out of normal range 
 incidence rates of treatment-emergent laboratory values outside of normal range by 
treatment group 
 incidence rates of pre-specified laboratory data abnormalities by treatment group 
 descriptive analysis of continuous laboratory parameters, and their changes from baseline 
by visit and treatment group 
 
Vital signs 
Vital signs (systolic BP, diastolic BP, and heart rate) analysis will be summarized by visit and 
treatment group. 
8.3.2 Efficacy analysis 
6MWD 
The evaluation of the primary efficacy outcome will be based on change from baseline in 
6MWD using analysis of covariance (ANCOVA) with baseline 6MWD, treatment arm and 
clinical site as covariates. The main comparison in this analysis will be the difference in 6MWD 


 
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between the riociguat arm and placebo using a two-sided test of the relevant beta coefficient at 
the 5% level of significance. Effect sizes and 90% confidence intervals of treatment difference 
will also be calculated. The above approaches assume 6MWD will be approximately normally 
distributed. The residuals from the models will be tested for normality using the Shapiro-Wilk 
test. Should this be significant at the 5% level, suitable transformations will be investigated (i.e. 
natural log) for the outcome or comparisons on 6MWD will be made using the nonparametric 
Wilcoxon test.  
 
Other Efficacy measures 
Changes from baseline to Week 12 will also be assessed between study arms on hemodynamic 
parameters, levels of plasma NT-proBNP, the Borg Dyspnea/Fatigue Scale, laboratory measures, 
and Sickle Cell related complications.  Continuous outcomes will be analyzed as above, using 
ANCOVA with the baseline outcome, study arm, and clinical site as covariates.  Logistic 
regression with similar covariates will be conducted for binary outcomes.  Finally, incidence of 
clinical complications will be compared between study arms using Poisson regression.  
8.3.3 Exploratory analysis 
The exploratory variables will be summarized using appropriate statistical methods: categorical 
variables by frequency tables and continuous variables by sample statistics. The 95% confidence 
intervals will be provided based on either, ANCOVA or stratified Wilcoxon test or Mantel-
Haenszel weights, stratified by region, depending on the nature and distribution of the data. 
 
Pharmacokinetic analysis 
Due to the limited number of plasma concentrations, PK parameters will be estimated by fitting a 
compartmental model derived from richer datasets in healthy volunteers. 
PK parameters to be estimated will include: 
 
 
CL  
concentration time curve (AUC) 
Total plasma clearance  
 
  
Vd  
Volume of distribution  
 
 
 
PK parameters will be summarized using descriptive statistics (geometric means, standard 
deviations, coefficients of variation, sample size [N], minimum, maximum and median). Figures 
will be created to display mean and individual (observed and model-predicted) analyte 
concentration-time curves. 
 
Additional analyses will be performed as deemed necessary upon review of the data. 
8.4 
Planned Interim Analyses 
In addition to periodic safety monitoring, an interim analysis will be performed on the primary 
outcome of treatment-emergent SAEs after the first 50 patients have completed study follow up.  
A statistician who is independent of the study will conduct the interim analysis and report the 
unblinded results to the independent data and safety monitoring board (DSMB). Assuming an 
overall two-sided, type I error of 10%, an O’Brien-Fleming (O-F) spending function will be used 
to determine the stopping rule for the primary outcome.  If the stopping criteria is met (absolute 


 
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value of O-F test statistic > 2.538) during the interim analysis, follow up may be halted.  
Otherwise, the remaining 50 patients will be recruited and followed.  Use of the O’Brien-
Fleming spending function allows the overall type I error to remain at 10% while still accounting 
for the interim peek at the data.   
8.5       Determination of Sample Size 
Sample size and power considerations were based on detectable effects in the previous walk-
PHASST trial, mainly an unanticipated safety signal that showed an increased frequency of pain 
events in the sickle cell patients on sildenafil compared to the placebo group. This will be a 
group-sequential design, utilizing an O’Brien-Fleming alpha-spending function with an overall 
potential sample size of 100 and a planned interim analysis using available data for the first 50 
patients. If an O-F stopping boundary of 2.538 (alpha=0.011) is not exceeded, enrollment will 
continue for the remaining 50 patients. The primary analysis will be conducted with an 8.9% 
type I error rate.  
 
We present detectable effect sizes for a group-sequential parallel arm design with 1:1 allocation 
ratio of 100 participants.  
 
For the primary safety outcome of treatment-emergent SAEs, an absolute difference of 24% 
(95% CI: 3.1%, 44.9%) was seen in the walk-PHASST trial, favoring the placebo arm in a 
superiority trial design typical of other safety and tolerability studies. With an overall sample 
size of 100 patients completing the 12 week visit, we will have approximately 82% power to 
detect differences in the safety outcome similar to that of the walk-PHASST trial. In fact, the 
study would have sufficient power (at least 80%) to detect absolute differences as small as 0.23, 
assuming 22% of placebo participants experience at least 1 SAE. If only 70% of randomized 
participants complete the 12 week visit, our power to detect differences seen in walk-PHASST 
reduces to approximately 68%; however we would have at least 80% to detect absolute 
differences as small as .28.  
  
We have performed additional power analyses based upon the results of the PATENT-1 trial of riociguat 
to evaluate the likelihood, as part of the secondary outcomes, of detecting efficacy signals in this 
riociguat trial.  The following analyses present only a frame of reference, and are not used to determine 
the study size of this trial.  In terms of detecting an efficacy signal such as 6MWD and MAP, 100 total 
patients will provide at least 80% power to detect changes of 42.6 m and 6.5 mmHg, respectively. These 
scenarios assume an overall type I error rate of 10%. 
 
We will have approximately 50 participants randomized to the riociguat arm. As a result, we will have 
93% and > 99% power to detect within-arm changes of 30 m and 9 mmHg for 6MWD and MAP, 
respectively. These meaningful differences were seen in the PATENT-1 trial of riociguat.   
 
9. 
DATA HANDLING AND QUALITY ASSURANCE 
9.1 Data Recording 
Data may be entered directly into the eCRF. However, if direct data entry is not possible, sites 
may print and record data on the paper CRF for later data entry into the web-based system.   
9.2 Monitoring 


 
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In accordance with applicable regulations, GCP, Coordinating Center personnel will contact the 
site prior to the start of the study to review with the site staff the protocol, study requirements, 
and their responsibilities to satisfy regulatory, ethical, and sponsor’s requirements. When 
reviewing data collection procedures, the discussion will also include identification and 
documentation of source data items.  
The sponsor/designee will monitor the site activity to verify that the: 
 Data are authentic, accurate and complete 
 Safety and rights of patients are being protected 
 Study is conducted in accordance with the currently approved protocol (including study 
treatment being used in accordance with the protocol) 
 Any other study agreements, GCP, and all applicable regulatory requirements are met. 
9.3 Data Management 
The Data Coordinating Center will oversee all aspects of data management. With the 
consultation of the DCC, the PI and coordinator will develop an operations manual to 
standardize all procedures and staff training in areas such as patient recruitment, measurement, 
assessment, and data entry, management, and security. 
The DCC will create an electronic System for Data Management (eSYSDM), based on detailed 
study protocols and requirements that includes an electronic case report form and a tracking 
system, with the capability to incorporate EHR. The eSYSDM is developed using .NET 2.0 to 
create the interface and SQL for the database.  The DCC will work closely with investigative 
team and other study personnel to ensure that protocols are being followed, data integrity and 
confidentiality are maintained, and that the data contains a minimum amount of missing data. All 
study files residing in designated network folders will be backed-up daily and archived weekly. 
The weekly archived files are maintained for 1 year until the data are erased. All study subjects 
will be assigned unique study identifiers that will appear on all data collection instruments, 
electronic media, documents, and files used in the statistical analysis and manuscript preparation. 
Only authorized team members will have access to personal information needed for tracking and 
informed consent. Other data quality assurance measures will include detailed documentation of 
computer operations and data editing procedures and regular meetings with project staff to 
review any changes in procedure. The DCC also has specific data quality measures that will be 
implemented.  These include data verification, built in data validation mechanisms such as logic 
and out of range data checks, and repeated evaluation of the data collection and entry process. 
9.4 Audit and Inspection 
To ensure compliance with GCP and regulatory requirements, a member of the Coordinating 
Center may arrange to conduct an audit to assess the performance of the study at the study site 
and of the study documents originating there. The investigator/institution will be informed of the 
audit outcome. 
In addition, inspections by regulatory health authority representatives and IEC(s)/IRB(s) are 
possible. The investigator should notify the Coordinating Center immediately of any such 
inspection. The investigator/institution agrees to allow the auditor or inspector direct access to all 
relevant documents and allocate his/her time and the time of his/her staff to the auditor/inspector 


 
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to discuss findings and any issues. Audits and inspections may occur at any time during or after 
completion of the study. 
9.5 Archiving 
Essential documents shall be archived safely and securely in such a way that ensures that they 
are readily available upon authorities’ request. 
Patient files will be archived according to local regulations and in accordance with the maximum 
period of time permitted by the hospital, institution or private practice. Where the archiving 
procedures do not meet the minimum timelines required by the Coordinating Center, alternative 
arrangements must be made to ensure the availability of the source documents for the required 
period. 
The investigator/institution notifies the Coordinating Center if the archival arrangements change 
(e.g., relocation or transfer of ownership). The investigator site file is not to be destroyed without 
the sponsor’s approval. The contract with the investigator/institution will contain all regulations 
relevant for the study center. 
10. 
 PREMATURE TERMINATION OF THE STUDY 
 If risk-benefit ratio becomes unacceptable owing to, for example, 
- Safety findings from this study (e.g., SAEs) 
- Results of any interim analysis 
- Results of parallel clinical studies 
- Results of parallel animal studies (on e.g., toxicity, teratogenicity, carcinogenicity or 
reproduction toxicity). 
 If the study conduct (e.g., recruitment rate; drop-out rate; data quality; protocol 
compliance) does not suggest a proper completion of the trial within a reasonable time 
frame. 
The investigator has the right to close his/her center at any time. 
For any of the above closures, the following applies: 
 Closures should occur only after consultation between involved parties. 
 All affected institutions (e.g., IEC(s)/IRB(s); competent authority(ies); study center; head 
of study center) must be informed as applicable according to local law. 
 In case of a partial study closure, ongoing patients, including those in post study follow-
up, must be taken care of in an ethical manner. 
Details for individual patient's withdrawal can be found in Section 5.2.1. 
11. 
ETHICAL AND LEGAL ASPECTS 
11.1 
Ethical and Legal Conduct of the Study 
The procedures set out in this protocol, pertaining to the conduct, evaluation, and documentation 
of this study, are designed to ensure that the investigator abide by Good Clinical Practice (GCP) 


 
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guidelines and under the guiding principles detailed in the Declaration of Helsinki.  The study 
will also be carried out in keeping with applicable local law(s) and regulation(s). 
Documented approval from appropriate IEC(s)/IRBs will be obtained for all participating centers 
before start of the study, according to GCP, local laws, regulations and organizations.  When 
necessary, an extension, amendment or renewal of the EC/IRB approval must be obtained and 
also forwarded to the Coordinating Center. 
Strict adherence to all specifications laid down in this protocol is required for all aspects of study 
conduct; the investigator may not modify or alter the procedures described in this protocol.   
Modifications to the study protocol will not be implemented by the investigator without 
discussion and agreement by the Coordinating Center.  However, the investigator may 
implement a deviation from, or a change of, the protocol to eliminate an immediate hazard(s) to 
the trial patients without prior IEC/IRB/Bayer approval/favorable opinion.  As soon as possible, 
the implemented deviation or change, the reasons for it and if appropriate the proposed protocol 
amendment should be submitted to the IEC/IRB/head of medical institution.  Any deviations 
from the protocol must be explained and documented by the investigator. 
The Principal Investigator is responsible for the conduct of the clinical trial at the site in 
accordance with Title 21 of the Code of Federal Regulations and/or the Declaration of Helsinki.  
The Principal Investigator is responsible for personally overseeing the treatment of all study 
patients.  The Principal Investigator must assure that all study site personnel, including sub-
investigators and other study staff members, adhere to the study protocol and all FDA/GCP 
regulations and guidelines regarding clinical trials both during and after study completion. 
The Principal Investigator at each institution or site will be responsible for assuring that all the 
required data will be collected and properly documented. 
11.2 
Patient Information and Consent 
Each patient will have ample time and opportunity to ask questions and will be informed about 
the right to withdraw from the study at any time without any disadvantage and without having to 
provide reasons for this decision. 
Only if the patient voluntarily agrees to sign the informed consent form and has done so, may 
he/she enter the study.  Additionally, the investigator and other information provider (if any) will 
personally sign, date and time the form.  The patient will receive a copy of the informed consent 
form. 
The signed informed consent statement is to remain in the investigator site file or, if locally 
required, in the patient’s note/file of the medical institution. 
In the event that informed consent is obtained on the date that baseline study procedures are 
performed, the study record or patient’s clinical record must clearly show that informed consent 
was obtained prior to these procedures. 
The informed consent form and any other written information provided to patients will be 
revised whenever important new information becomes available that may be relevant to the 
patient’s consent, or there is an amendment to the protocol that necessitates a change to the 
content of the patient information and / or the written informed consent form.  The investigator 
will inform the patient consenter of changes in a timely manner and will ask the patient to 
confirm his/her participation in the study by signing the revised informed consent form.  Any 


 
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revised written informed consent form and written information must receive the IEC/IRB’s 
approval in advance of use. 
11.3 
Publication Policy 
The details of publication policy between Bayer and the sponsor are provided in the contract.  
The Steering Committee will help guide publications from this study. The Principal Investigators 
at the Coordinating Center should ensure that the information regarding the study be publicly 
available on the internet at www.clinicaltrials.gov. 
11.4 
Confidentiality 
All records identifying the patient will be kept confidential and, to the extent permitted by the 
applicable laws and/or regulations, will not be made publicly available. 
Should direct access to medical records require a waiver or authorization separate from the 
patient’s statement of informed consent, it is the responsibility of the Investigator to obtain such 
permission in writing from the appropriate individual. 
 


 
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Appendix A: Schedule of Procedures 
Study Epoch 
Screening 
Double-Blinded Study Treatment Phase 
Telephone 
Follow-up 
Final Visit 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
Week on Study Intervention 
Week 
-0 ~ -4 
Baseline 
Week 
0 
Week 
2 
Week 
4 
Week 
6 
Week 
8 
Week 
10 
Week 
12  
or ED 
Week 
13  
or ED  
Week 16 
or 30 days 
post 
treatment 
Assessment Window 
Study Procedure 
 
- 
± 3d  
± 3d 
± 3d  
± 3d  
± 3d 
± 3d  
± 3 d 
± 3 d 
Informed consent 
x 
 
 
 
 
 
 
 
 
 
Demographics and Review of 
Eligibility  
x 
 
 
 
 
 
 
 
 
 
Comprehensive Medical history 
 
x 
 
 
 
 
 
 
 
 
Review of Inclusion & 
Exclusion criteria 
x 
x 
 
 
 
 
 
 
 
 
Physical exam 
 
x 
x 
x 
x 
x 
x 
x 
 
x 
Height and weight #  
 
x 
 
 
 
 
 
x 
 
 
Vital signs 
x 
x 
x 
x 
x 
x 
x 
x 
 
x 
Randomization 
 
x 
 
 
 
 
 
 
 
 
Dispense study drug 
 
x 
x 
x 
x 
x 
x 
 
 
 
Titration of study drug dose 
 
 
x 
x 
x 
x 
x 
 
 
 
Urine or serum pregnancy test ¥ 
x 
x 
 
x 
 
x 
 
x 
 
x 
Child Pugh with INR± 
x 
 
 
 
 
 
 
 
 
 
Lab evaluation± 
x 
x 
 
x 
 
x 
 
x 
 
 
NT-proBNP 
 
x 
 
 
 
 
 
x 
 
 
Ferritin ±
 
x 
 
 
 
 
 
x 
 
 
Hemoglobin HPLC±
 
x 
 
 
 
 
 
x 
 
 
U/A & urine albumin & creatinine  
£  
x 
 
 
 
 
 
x 
 
 
Blood samples for biomarkers 
 
x 
 
 
 
 
 
x 
 
 
Blood sample for genotyping 
 
x 
 
 
 
 
 
 
 
 
PK Sampling/PD * 
 
x 
 
 
 
 
 
 
 
 
Echocardiogram 
≠  
x 
 
 
 
 
 
x 
 
 
Record Results of RHC Ω 
 
A 
 
 
 
 
 
A 
 
 
6MWD Test 
 
x 
 
 
 
 
 
x 
 
 
Borg Dyspnea/Fatigue Scale 
 
x 
 
 
 
 
 
x 
 
 
Brief Pain Inventory 
 
x 
 
 
 
 
 
x 
 
 
ASCQ-Me/PROMIS 
 
x 
 
 
 
 
 
x 
 
 
Electronic Pain diary § 
S 
R 
S 
R 
S 
R 
S 
R 
 
 
Treatment compliance 
 
 
x 
x 
x 
x 
x 
x 
 
 
Concomitant medications 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
ROS/AE assessment 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
X=Required        A=When available        §  Piloted only at  select sites (S= set up diary & R= review diary)    ED=Early Discontinuation 
#   Only weight measurement will be repeated at Visit 8 
¥   For women of childbearing potential only 
*   Only in approximately 25 subjects enrolled at select sites 
£   A clinical U/A protein result or urine albumin to creatinine ratio result obtained within the past 24 months is acceptable at screening to 
determine macroalbuminuria eligibility. 
     UA, urine albumin and urine creatinine will be done on all subjects at Baseline and Week 12. 
≠   A clinical echocardiogram performed within the past 24 months when the patient is in his/her stable disease state is acceptable at 
screening to determine TRV eligibility. A research echocardiogram will be performed on all randomized subjects at Baseline and at 
Week 12.  
Ω  RHC is not required but record results if performed as standard of care 
±  Labs that will be drawn and processed at the site’s clinical lab