Clinical trial NCT03395223
Conditions: Acquired Methaemoglobinaemia
Keywords: Acquired Methaemoglobinemia Methylene Blue
Sponsor: Provepharm SAS
Start date: 2018-07-06; Completion date: 2020-08-31
Enrollment: 7 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This is an open label, uncontrolled, Phase 4 study including 10 patients who present in hospital/urgent care setting with acquired methemoglobinemia. The population may include pediatric and adult patients (males and females of all ages are included).

The study will run in both the EU and the US. The aim of the study is to confirm safety and efficacy of ProvayBlueTM for the treatment of acquired methemoglobinemia and has been requested by the US-FDA as a Post-Marketing requirements.

Detailed description:

Arms: arm 1: Methylene Blue 0.5% will be administered.

1 mg/kg will be administered intravenously over 5-30 minutes. If methemoglobin level remains above 30% or if clinical symptoms persist, give a repeat dose of up to 1 mg/kg one hour after the first dose.
Interventions: intervention 1: Methylene Blue
Intervention descriptions: intervention 1: Administration of Methylene Blue to treat acquired methaemoglobinaemia

Protocol documents:
SAP_003.pdf: 
D A T A  S U M M A R Y  P L A N  
Pr ot oc ol N u m ber:  
P V P -2 0 1 6 0 0 3  
Pr ot oc ol Title:    
O pe n L a bel C li nic al S t u d y t o E v al u ate t he S afet y 
a n d E ffic ac y of Pr o v a y Bl ueT M  ( Met h yle ne B l ue) 
f or t he T re at me nt of A c q uire d 
M et he m o gl o bi ne mi a  
D ate of Pr ot oc ol  
Fi n al Versi o n 3 , 1 0 A u g ust 2 0 1 7  
Pr ot oc ol N u m ber:  
H Q F- M E T H B- 2 0 1 8 0 0 1  
Pr ot oc ol Title:    
Use of Met h yle ne Bl ue i n Ac q uire d 
Met he m o gl o bi ne mi a: Pr os pecti ve O bser v ati o n al 
Re gistr y ( met H b)  
D ate of Pr ot oc ol  
1 3 A pril 2 0 1 8  
N C T # 0 3 3 9 5 2 2 3
D oc u me nt D ate:     
  3 0 A u g ust 2 0 2 1




 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 3  of 3 5  
 
T A B L E O F C O N T E N T S  
SI G N A T U R E S  ................................................................................................................................. 2 
T A B L E O F C O N T E N T S  ................................................................................................................. 3 
LI S T O F A B B R E VI A TI O N S A N D P H R A S E S  ............................................................................. 6 
1.  
I N T R O D U C TI O N A N D S C O P E ................................................................................. 7 
2.  
O V E R VI E W O F S T U D Y O BJ E C TI V E S A N D A S S E S S M E N T S .............................. 9 
2. 1.  
St u d y O bjecti ves a n d E n d p oi nts ................................................................................... 9 
2. 2.  
Data C ollecti o n  ........................................................................................................... 1 1 
2. 2. 1.  
H Q F -M E T H B - 2 0 1 8 0 0 1 .............................................................................................. 1 1 
2. 2. 1.  
P V P - 2 0 1 6 0 0 3 .............................................................................................................. 1 2 
3.  
D A T A P O O LI N G C O N SI D E R A TI O N S  ................................................................... 1 6 
3. 1.  
Data base Str u ct ure  ...................................................................................................... 1 6 
3. 2.  
A nal ysis P o p ulati o n .................................................................................................... 1 8  
3. 3.  
E n d p oi nts .................................................................................................................... 1 8  
3. 3. 1.  
Efficac y E n d p oi nts ...................................................................................................... 1 8  
3. 3. 2.  
Relati ve Ti me of Efficac y E n d p oi nts ......................................................................... 1 9  
3. 3. 3.  
Safet y E n d p oi nts ......................................................................................................... 1 9  
3. 4.  
Test H y p ot hesis a n d P V al ue J ustificati o n  ................................................................. 1 9 
3. 5.  
Pr oce d ures f or H a n dli n g Missi n g Data ....................................................................... 1 9  
3. 6.  
A nal ysis Ce nter  ........................................................................................................... 1 9 
3. 7.  
Defi niti o ns a n d Deri ve d Varia bles ............................................................................. 2 0  
3. 7. 1.  
Baseli ne  ....................................................................................................................... 2 0 
3. 7. 2.  
Patie nt Gr o u ps b y Met he m o gl o bi ne mia S us pecte d Ca usati ve A ge nt ........................ 2 0  
3. 7. 3.  
Patie nt Gr o u ps b y Met he m o gl o bi ne mia Se verit y ....................................................... 2 0  
4.  
D A T A S U M M A RI E S  ................................................................................................ 2 1 
4. 1.  
Dis p ositi o n  .................................................................................................................. 2 1 
4. 2.  
De m o gra p hic s a n d Baseli ne C haract eristics ............................................................... 2 1 
4. 3.  
Me dical Hist or y  .......................................................................................................... 2 2 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 4  of 3 5  
 
4. 4.  
Pr ot oc ol De viati o ns .................................................................................................... 2 2  
4. 5.  
Tre at me nt C o m plia nce  ................................................................................................ 2 2 
4. 6.  
Pri or a n d C o nc o mita nt Me dicati o ns ........................................................................... 2 2  
5.  
E F FI C A C Y A N A L Y SI S  ............................................................................................ 2 3 
5. 1.  
Treat me nt Effect o n met H b  ........................................................................................ 2 3 
5. 1. 1.  
Met H b Le vel at 1 - h o ur P ost D osi n g ........................................................................... 2 3 
5. 1. 2.  
Ti me W he n met H b L e vel Reac he d 5 0 % Re d u cti o n  ................................................... 2 4 
5. 1. 3.  
S u m mar y of met H b  ..................................................................................................... 2 4 
5. 1. 4.  
I n di vi d ual Pr ofile of met H b ........................................................................................ 2 5 
5. 2.  
C ha n ge i n Vital Si g ns ................................................................................................. 2 5  
5. 2. 1.  
Me dia n Vital Si g ns wit hi n 2 - H o ur P ost Treat me nt .................................................... 2 5 
5. 2. 2.  
Vital Si g ns N or mal/ A b n or mal Assess me nts  .............................................................. 2 6 
5. 2. 3.  
S u m maries of Vital Si g ns C ha n ge fr o m Baseli ne  ...................................................... 2 7 
5. 2. 4.  
I n di vi d ual Pr ofiles of Vital Si g ns ............................................................................... 2 7 
5. 3.  
Met he m o gl o bi ne mia Si g ns a n d S y m pt o ms ................................................................ 2 8  
5. 3. 1.  
S u m mar y of Si g ns a n d S y m pt o ms .............................................................................. 2 9  
5. 3. 2.  
I n di vi d ual Pr ofiles of Si g ns a n d S y m pt o ms ............................................................... 3 0 
6.  
S A F E T Y A N D T O L E R A BI LI T Y E V A L U A TI O N S  ................................................ 3 1 
6. 1.  
T otal E x p os ure ............................................................................................................ 3 1  
6. 2.  
A d verse E v e nts  ........................................................................................................... 3 1 
6. 3.  
Ot her Safet y Para meters  ............................................................................................. 3 2 
6. 3. 1.  
C ha n ges i n Ot her Bl o o d Gas Tests ............................................................................. 3 2  
6. 3. 2.  
C ha n ges i n La b orat or y T ests ...................................................................................... 3 2  
6. 3. 3.  
C ha n ges i n 1 2- Lea d E C G  ........................................................................................... 3 3 
7.  
A B L E O F C O N T E N T S O F P L A N N E D D A T A S U M M A RI E S  ............................... 3 4 
7. 1.  
S u m mar y Ta bles  ......................................................................................................... 3 4 
7. 2.  
Data Listi n gs  ............................................................................................................... 3 5 
 
 
 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 5  of 3 5  
 
 
I N-T E X T T A B L E S  
Ta ble 1:  O ver vi e w of St u d y O bjecti ves a n d E n d p oi nts .................................................................. 9 
Ta ble 2:  St u d y P V P- 2 0 1 6 0 0 3 Pr oce d ures..................................................................................... 1 4  
Ta ble 3:  E x pecte d S D T M D o mai ns f or t he P o ole d Data base ....................................................... 1 7  
Ta ble 4:  Vital Si g ns N or mal Ra n ges  ............................................................................................ 2 6 
Ta ble 5:  Bl o o d Gas Para meters of I nterest  ................................................................................... 3 2 
Ta ble 6:  Safet y La b orat or y Tests of I nterest ................................................................................. 3 3 
 
 
I N-T E X T FI G U R E S  
Fi g ure 1:  E xa m ple of I n di vi d ual Pr ofile of met H b Bef ore a n d After Treat me nt ......................... 2 5  
Fi g ure 2:  E xa m ple of I n di vi d ual Pr ofile of Bl o o d Press ure Bef ore a n d Aft er Treat me nt ............ 2 7  
Fi g ure 3:  E xa m ple of I n di vi d ual Pr ofile of Res pirat or y a n d Heart Rate Bef ore a n d After 
Treat me nt  .................................................................................................................... 2 8 
Fi g ure 4:  E xa m ple of I n di vi d ual Pr ofile of Met he m o gl o bi ne mia Si g ns a n d S y m pt o ms 
Bef ore a n d After Treat me nt  ........................................................................................ 3 0 
 
 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 6  of 3 5  
 
LI S T O F A B B R E VI A TI O N S  A N D P H R A S E S  
A b bre vi ati o n  
Defi niti o n  
A E  
a d verse e ve nt 
C R F  
case re p ort f or m 
D S P  
Data S u m mar y P la n 
E C G  
electr ocar di o gra m 
I C H 
I nter nati o nal C o nfere nce o n Har m o nizati o n 
met H b  
met he m o gl o bi n 
P C H G 
perce nt c ha n ge fr o m baseli ne  
P M R  
P ost- M ar keti n g Re q uire me nt 
P V P  
Pr o ve p har m, I nc.  
T E A E  
treat me nt-emer ge nt ad verse eve nt  
V S  
v ital si g ns 
 
 
 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 7  of 3 5  
 
1.  
IN T R O D U C TI O N A N D S C O P E  
Pr o va y Bl ue ™ ( met h yle ne bl ue) was a p pr o ve d u n der t he accelerate d a p pr o val re g ulati o n, 2 1 C F R 
3 1 4. 5 1 0. Pr o ve p har m was re q uire d t o c o n d uct “f urt her a de q uate a n d well-c o ntr olle d st u dies” t o 
verif y a n d descri be t he cli nical safet y a n d be ne fit of t he a ge nt i n t hera pe utic use f or t he treat me nt of 
ac q uire d met he m o gl o bi ne mia . T o f ulfill t his re q uire me nt, Pr o ve p har m has i nitiate d 2 st u dies a n d 
e nr oll me nt t o b ot h st u dies are cl ose d  at t his ti me.  
T he first st u d y, pr ot oc ol P V P- 2 0 1 6 0 0 3, title d “ O pe n L a bel C li nical St u d y t o E val uate t he S afet y 
a n d Efficac y of Pr o v a y Bl ue T M  ( Met h yle ne Bl ue) f or t he T reat me nt of A c q uire d 
Met he m o gl o bi ne mia, ” w as i nitiate d 1 9 Oct o ber 2 0 1 7; t he st u d y ha d e nr olle d a t otal of 7 patie nt s 
w he n t he e nr oll me nt w as cl ose d o n 3 1 A u g ust 2 0 2 0. T his st u d y is referre d t o hereafter as t he 
I nter ve nti o nal St u d y. Beca use of t h e sl o w pace of e nr oll me nt i n t his st u d y, d o u btless d ue t o t he 
pa ucit y of patie nts wit h t his ultra -rar e disease a vaila ble f or ass ess me nt, a sta n d -al o ne d ata bas e, 
as c o m m o nl y utilize d wit h cli nical trials, has n ot bee n set u p t o ca pt ure t he d ata; rat her, all 
cli nical data were ca pt ur e d o n a pa p er case re p ort f or m  ( C R F). 
T he sec o n d st u d y, pr ot oc ol H Q F- M E T H B - 2 0 1 8 0 0 1, title d “ Use of Met h yle ne Bl ue i n Ac q uire d 
Met he m o gl o bi ne mia: Pr os pecti ve O bser vati o nal Re gistr y ( met H b),” start e d Ma y 2 0 1 8, a n d 
e nr olle d 2 4 patie nt s bef ore cl os ure o n 3 1 A u g ust 2 0 2 0. T his st u d y is referre d t o hereafter as t he 
O bser vati o nal St u d y. T he O bser v ati o nal St u d y wa s e x pect e d t o last 2 years after t he 
c o m me nce me nt of e nr oll me nt i n u p t o a p pr o xi matel y 5 0 h os pital s yste ms. T his O bser vati o nal 
St u d y was desi g ne d t o s u p p ort l o w- b ur de n scree ni n g a n d data c ollecti o n , a n d t o e nr oll real-w orl d 
cases of ac q uir e d  met he m o gl o bine mia treate d wit h Pr o va y Bl ue T M  wit h o ut restricti o n ; t h us, st u d y 
sites were  m or e a me n a ble t o partici pat e i n t his pr o gra m t ha n i n t he I nter v e nti o nal St u d y. W he n 
e nr oll me nt was cl ose d , a p pr o xi matel y 9 0 i n di vi d ual h os pitals were  a p pr o ve d t o e nr oll patie nts 
i nt o t he O bser vati o nal St u d y; h o we ver, o nl y 2 4 patie nts were  e nr olle d, a gai n o wi n g t o t he rarit y 
of t he cli nical pres e ntati o n of i nterest . A n electr o nic data base h as bee n set  u p t o h o use data fr o m 
b ot h st u dies. 
Pr o ve p har m pla n s t o c o m bi ne t he data fr o m b ot h st u dies i n its re p ort t o F D A t o satisf y t he 
c o m pa n y’s first P ost- Mar keti n g Re q uire me nt ( P M R -3 0 6 5- 1) . T his  Data  S u m mar y Pla n ( D S P) is 
i nte n de d t o pr o vi de a tec h nical, detaile d ela b orati o n of t he met h o ds utilize d t o p o ol t h ose 2 
st u dies i nt o a si n gle data base a n d t o pre pare a n i nte grate d s u m mary of t he data wit h o ut 
i nfere ntial statistics. It is a ntici pate d t hat t he p o ole d res ults will be prese nte d as a series of 
i n di vi d ual patie nt  narrati ves wit h li mite d statistical a nal ysis , befitti n g t he o bser vati o nal nat ure of 
m ost of t he data. T he  o bj ecti ve of t his D S P is t o ass ure t hat t he met h o d ol o gies t o be use d f or 
s u m mar y are c o m plete, acc urat e, a n d c o nsiste nt.  
I n t he d e vel o p me nt of t his D S P, t he f oll o wi n g d oc u me nts were us e d: 
•  Pr ot oc ol P V P - 2 0 1 6 0 0 3: fi nal pr ot oc ol date d 1 0 A u g ust 2 0 1 7; C R F date d 0 4 Dece m b er 
2 0 1 7 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 8  of 3 5  
 
•  Pr ot oc ol H Q F- M E T H B - 2 0 1 8 0 0 1: fi nal pr ot oc ol date d 1 3 A pril 2 0 1 9; C R F date d 0 4 Ma y 
2 0 1 8   
o  F or t he p ur p os e of c o m bi ni n g wit h data fr o m Pr ot oc ol P V P- 2 0 1 6 0 0 3, t he ori gi nal 
C R F date d 0 4 Ma y 2 0 1 8 was u p date d o n 2 7 O ct o b er 2 0 2 0   
 
T he pri nci ples i n t he f oll o wi n g g ui da nce d oc u m e nts ha ve bee n f oll o we d i n pre parati o n of t his 
D S P : 
•  I nter nati o nal C o nfere nce o n Har m o nizati o n (I C H) E 3 ( 1 9 9 5): Str uct ure a n d C o nte nt of 
Cli nical St u d y Re p orts 
•  I C H E 6 ( 1 9 9 6): G ui deli n e f or G o o d Cli nical Practice 
•  I C H E 9 ( 1 9 9 8): Statistical Pri nci ples f or Cli nical Trials 
I n t he e ve nt t h at a discre p a nc y is f o u n d bet wee n t he descri pti o ns i n t he st atistic al secti o n of 
t he pr ot oc ols a n d t his d oc u m e nt, t he descri pti o n i n t his d oc u me nt s u perse des t he 
descri pti o ns i n t he st atistic al secti o n of t he pr ot oc ols. 
A telec o nf ere n ce w as hel d o n 2 7 A pril 2 0 2 0 bet w ee n t he s p o ns or a n d t he a ge nc y. A 
p ost- meeti n g i nf or mati o n pac ka g e was s u b mitte d o n 0 1 Ma y  2 0 2 0, i n w hic h met H b data 
c ollecte d fr o m 1 3 patie nt s ( 3 fr o m P V P - 2 0 1 6 0 0 3 a n d 1 0 fr o m H Q F -M E T H B - 2 0 1 8 0 0 1) were 
s u m marize d a n d pr ese nte d t o t he a ge nc y. O n 0 9 A u g ust 2 0 2 0, t he a ge nc y c o nfir me d t hat t he 
c o m bi ne d data bas e wa s acce pta bl e t o be utilize d t o f ulfill t he p ost mar keti n g re q uire me nt N o. 1 
( P M R 1). T his D S P  was finalize d after i nc or p orati o n of t he details of t he m eeti n g disc ussi o n a n d 
s u bse q ue nt p ost-meeti n g c o m m u nicati o ns  bet wee n t he s p o ns or a n d t he a g e n c y . 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 9  of 3 5  
 
2.  
O V E R VI E W O F S T U D Y O B J E C TI V E S A N D A S S E S S M E N T S   
2. 1.  
St u d y O bjecti ves a n d E n d p oi nts  
A n o ver vie w of st u d y o bjecti ves a n d st u d y e n d p oi nts is pr o vi de d i n Ta ble 1 . T he O bser v ati o nal 
St u d y was desi g ne d t o mirr or  t he o bjecti ves, e n d p oi nts, a n d data c ollecti o n ele me nts of t he 
I nter ve nti o nal St u d y, wit h m o dificati o ns t o ( 1) reflect t he nat ure of o bser v ati o nal data, w here t he 
s pecific ti mi n g of pr oce d ures a n d meas ure me nts is dri ve n b y i nstit uti o nal sta n dar d care, n ot a 
st u d y pr ot oc ol, a n d ( 2) t o strea mli ne t he pr ot oc ol a n d st u d y pr oce d ures w here p ossi ble.  
T a ble 1:  O ver vie w of St u d y O bjecti ves a n d E n d p oi nts  
Pr ot oc ol 
N u m ber  
P V P -2 0 1 6 0 0 3  
(I nter ve nti o n al St u d y) 
H Q F -M E T H B -2 0 1 8 0 0 1   
( O bser v ati o n al St u d y) 
Pri m ar y 
O bjecti ve  
T o c o nfir m t hat Pr o va y Bl ue ™ after a 
si n gle a d mi nistrati o n is efficaci o us i n 
patie nts wit h ac q uire d 
met he m o gl o bi ne mia 
T o c o nfir m t hat Pr o va y Bl ue ™ 
is efficaci o us i n patie nts wit h 
ac q uire d m et h e m o gl o bi ne mia  
Sec o n d ar y 
O bjecti ves  
•  
T o e val uate t he efficac y of a si n gle 
d ose of 1 m g/ k g of Pr o va y Bl ue ™; 
•  
T o e val uate t he efficac y of a sec o n d 
d ose of 1 m g/ k g of Pr o va y Bl ue ™ t o 
f urt her re d uce met H b le vels w he n 
met H b is n ot f ull y re d uce d b y a si n gle 
d ose;  
•  
T o e val uate t he n or malizati o n of t he 
res pirat or y rate, heart rate a n d bl o o d 
press ure of patie nts w h o ac hie ve a 
re d ucti o n i n met H b le vel wit hi n 2 h o urs 
of recei vi n g t he first d ose of 
Pr o va y Bl ue ™; 
•  
T o c o nfir m t he safet y a n d t olera bilit y of 
Pr o va y Bl ue ™ i njecti o n i n patie nts wit h 
ac q uire d met he m o gl o bi ne mia. 
T o c o nfir m t he safet y a n d 
t olera bilit y of Pr o va y Bl ue™ 
i n patie nts wit h ac q uire d 
met he m o gl o bi ne mia  
St u d y 
P o p ul ati o n 
Patie nts w h o prese nt i n h os pital/ ur ge nt care 
setti n g dia g n ose d wit h ac q uire d 
met he m o gl o bi ne mia. T he p o p ulati o n ma y 
i ncl u de pe diatric a n d a d ult patie nts ( males 
a n d fe males of all a ges are i ncl u de d).  
Patie nts w h o are dia g n ose d i n 
a n y care setti n g wit h ac q uire d 
met he m o gl o bi ne mia. T he 
p o p ulati o n ma y i ncl u de pe diatric 
a n d a d ult patie nts ( males a n d 
fe males of all a ges are i ncl u de d). 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 0  of 3 5  
 
Pr ot oc ol 
N u m ber  
P V P - 2 0 1 6 0 0 3 
(I nter ve nti o n al St u d y) 
H Q F -M E T H B - 2 0 1 8 0 0 1  
( O bser v ati o n al St u d y) 
 
 
Pri m ar y Tre ate d Gr o u p:  At 
least te n ( 1 0) patie nt s w h o are 
treate d f or ac q uire d 
met he m o gl o bi ne mia t hat is 
s y m pt o matic (e g, e x hi biti n g 
slee pi ness, c ya n osis, dizzi ness) 
a n d/ or wit h meas ure d 
met he m o gl o bi n le vels > 3 0 % 
 
 
Sec o n d ar y Tre ate d Gr o u p:  U p 
t o ni net y ( 9 0) a d diti o nal 
as y m pt o matic patie nts w h o are 
treate d f or ac q uire d 
met he m o gl o bi ne mia wit h 
meas ure d met he m o gl o bi n le vels 
≤3 0 %  
S afet y 
Assess m e nts  
•  P h ysical e xa mi nati o n; 
•  Vital si g ns;  
•  He mat ol o g y a n d bi oc he mistr y 
•  Electr ocar di o gra m ( E C G);  
•  A d verse e ve nts ( A Es).  
Pre vale nce a n d nat ure of a n y 
a d verse e ve nts occ urri n g 
wit hi n 1 0 da ys of t he 
a d mi nistrati o n of 
Pr o va y Bl ue ™ 
Effic ac y 
E n d p oi nt s 
•  A re d ucti o n i n met H b b y 5 0 % wit hi n 
1 h o ur of d osi n g;  
•  C o nc o mita nt n or malizati o n of 
res pirat or y r ate wit hi n 2 h o urs of t he 
first d ose of Pr o va y Bl ue ™; 
•  C o nc o mita nt n or malizati o n of heart 
rate wit hi n 2 h o urs of t he first d ose of 
Pr o va y Bl ue ™; 
•  C o nc o mita nt n or malizati o n of 
diast olic bl o o d press ure ( D B P) wit hi n 
2 h o u rs of t he first d ose of 
Pr o va y Bl ue ™; 
•  C o nc o mita nt n or malizati o n of 
s yst olic bl o o d press ure ( S B P) wit hi n 
2 h o urs of t he first d ose of 
Pr o va y Bl ue ™. 
•  Ti me t o re d ucti o n a n d 
ma g nit u de of re d u cti o n i n 
met he m o gl o bi n after 
a d mi nistrati o n of 
Pr o va y Bl ue ™ f or 
treat me nt of ac q uire d 
met he m o gl o bi ne mia.  
•  Ti me t o n or malizati o n of 
t he res pirat or y rate, h eart 
rate, a n d bl o o d press ure 
after a d mi nistrati o n of 
Pr o va y Bl ue ™ f or 
treat me nt of ac q uire d 
met he m o gl o bi ne mia  
•  Pre vale nce a n d nat ure of 
a n y a d vers e e v e nts 
occ urri n g wit hi n 1 0 da ys 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 1  of 3 5  
 
Pr ot oc ol 
N u m ber  
P V P - 2 0 1 6 0 0 3 
(I nter ve nti o n al St u d y) 
H Q F -M E T H B - 2 0 1 8 0 0 1  
( O bser v ati o n al St u d y) 
of t he a d mi nistrati o n of 
Pr o va y Bl ue ™ 
•  Pre vale nce of ac q uire d 
met he m o gl o bi ne mia cases 
b y s us pecte d ca usal a ge nt  
•  Res ol uti o n of 
met he m o gl o bi ne mia -
relate d s y m pt o ms d uri n g 
t he i n de x h os pitalizati o n 
P K E n d p oi nt  
P K bl o o d dra ws were pla n ne d a n d 
plas ma c o nce ntrati o n s of met h yle ne bl ue 
a n d az ur e B wer e t o be d eter mi ne d. 
H o we ver, P K data w e re n ot c ollecte d d ue 
t o p o or P K bl o o d sa m ple ha n dli n g. 
T heref ore, t he P K e n d p oi nt will n ot be 
assesse d.  
N o ne 
 
2. 2.  
D at a C ollecti o n  
2. 2. 1.  
H Q F -M E T H B - 2 0 1 8 0 0 1 
Pr ot oc ol H Q F -M E T H B - 2 0 1 8 0 0 1 is desi g ne d t o be a l o w- b ur d e n mi ni mal -ris k O bser v ati o n al 
S t u d y; data c ollecti o n lar gel y is b y passi ve c hart re vie w, wit h f oll o w - u p b y tele p h o ne or p ostal 
mail a n d/ or usi n g me dical rec or ds w h ere a p plica bl e. At s o me  partici pati n g st u d y sites, f oll o w- u p 
of ac q uir e d met he m o gl o bi ne mia patie nts is sta n dar d care or p art of q u alit y i m pr o ve me nt eff orts, 
a n d s o me st u d y sites are o perati n g u n der a f ull wai ver of c o nse nt / ass e nt, base d o n l ocal I R B 
a p pr o vals.  
T o mi ni mize ris k a n d q ualif y f or t he m ost strea mli ne d st u d y pr oce d ures, t he O bser v ati o nal St u d y 
is electr o nic s o ur ce ( p a per f or ms ar e n ot re q uire d) a n d t he st u d y data are de-i de ntifie d t o t he 
HI P A A S afe - Har b or sta n dar d. N ota bl y, t he Safe- Har b or met h o d i n v ol ves c ollecti o n of relati ve 
“st u d y da ys” ( e g, “ St u d y Da y 1, 1 2: 4 5”) rat her t ha n a bs ol ute “cal e n dar d a ys” (e g, D ece m b er 3 1, 
2 0 1 9, 1 2: 4 5). Si g nifica ntl y, t he “st u d y da y” safe- h ar b or f or mat d oes n ot restrict a n y of t he 
pr o p ose d a nal ys es. I n o ur pr o p ose d a nal ys es, a pse u d o e nr oll me nt date wa s creat e d f or eac h 
patie nt  w he n t he patie nt’s case w as re gistere d i n t he data bas e .  
T o s u p p ort i nt uiti ve a n d c o nsiste nt prese ntati o n of t he data, St u d y Da y 1 H o ur 0 is defi ne d as t he 
da y a n d ti me of t he c o m pleti o n  of t he first m et h yle ne bl ue i nf usi o n, a n d t he ti mi n g of all st u d y 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 2  of 3 5  
 
meas ure me nts is pres e nte d as  t he relati ve ti me el a pse d fr o m t his refere n ce p oi nt. T h us, a 
ne gati ve rel ati ve ti me i n dicates t hat a n e v e nt/assess me nt occ urre d pri or t o t he first i nf usi o n  
c o m pleti o n ti me, w hereas  a p ositi ve relati ve ti me i n dicates t hat a n e v e nt/assess me nt occ urre d 
after t he first i nf usi o n c o ncl usi o n ti me.  
T o gai n u n dersta n di n g a b o ut all real- w orl d cases of ac q uire d met he m o gl o bi ne mia treate d wit h 
Pr o va y Bl ue ™, a n d t o e n c o ura ge m ore sites t o partici pate b y ma ki n g t he e nr oll me nt criteria less 
restricti ve, e nr oll me nt of a “ Sec o n d ar y Treate d Gr o u p” was i ncl u de d i n t he O bser vati o nal St u d y. 
T hat is, t his st u d y gr o u ps e nr olle d patie nt s i nt o t w o s u b gr o u ps: Pri mar y cas es or Sec o n dar y 
cases.  
All pri mar y cases m ust f ulfill t he f oll o wi n g re q uire me nts:  
1.  Baseli ne met H b ≥3 0 %, O R  
2.  D oc u me nte d si g ns or s y m pt o ms of met he m o gl o bi ne mia wit h s u bse q ue nt assa y of 
met he m o gl o bi n le vels a b o ve t he i nstit uti onal u p per li mit of n or mal ( U L N ). F or t hese 
patie nt s, res p o nse t o t he q uesti o n ‘ Were t here a n y si g ns or s y m pt o ms j u d ge d t o be 
cli nicall y ass ociate d  wit h t he ac q uire d m et he m o gl o bi ne mia?’ m ust be a ‘ Y es’  
Data fr o m all  Pri mar y cases are f ull y m o nit ore d ; i nc o nsiste ncies are t o be q uerie d.  
T he Sec o n dar y Treate d Gr o u p pr o vi des relati vel y s parse data o n n o n- se v ere (as y m pt o matic wit h 
met he m o gl o bi n le vels < 3 0 %) ac q uir e d met he m o g lo bi ne mia cases t hat w ere n o net heless treat e d 
per cli nical j u d g me nt wit h Pr o va y Bl ue ™. As i de fr o m verificati o n of t he i ncl usi o n criteria, data 
i n t hese cases are n ot m o nit ore d.  
2. 2. 1.  
P V P - 2 0 1 6 0 0 3 
Pr ot oc ol P V P- 2 0 1 6 0 0 3 is a n o pe n-la b el, u nc o ntr olle d P hase 4 I nter v e nti o nal S t u d y of patie nts 
w h o prese nt i n h os pital/ ur ge nt care setti n g wit h ac q uire d met he m o gl o bi ne mia. T he p o p ulati o n 
ma y i ncl u de pe diatric a n d a d ult patie nts ( males a n d fe males of all a ges are i ncl u de d).  Eli gi ble 
patie nt s will recei ve a si n gle d ose of st u d y dr u g (t y picall y 1 m g/ k g of Pr o va y Bl ue ™)  treat me nt t o 
treat ac q uire d met he m o gl o bi ne mia res ulti n g i n a direct healt h be nefit. Patie nt s will be f oll o we d 
p ost treat me nt; safet y, efficac y, a n d P K d ata will be c ollecte d f oll o wi n g a pre defi ne d d ata 
c ollecti o n sc he d ule ( Ta bl e 2 ). 
T o create a h ar m o nize d electr o nic data b ase, d at a c ollecte d fr o m t his st u d y will be e ntere d i nt o  
t he electr o nic d ata c ollecti o n s yste m use d f or t he O bser vati o nal St u d y, a n d t he a bs ol ute date s of 
ser vice r ec or de d i n t his st u d y will be c o n verte d t o safe- h ar b or c o m plia nt “st u d y da ys” bef ore 
data ar e  e ntere d i nt o t he data base f or a n al yses . I n i nsta nces w here a s p ecifi c pr oce d ure/ dat a 
c ollecti o n ti me was n ot i nitiall y rec or de d o n t he p a per C R F, a data q uer y will be ge nerate d t o 
o btai n t he s pecific ti me fr o m t he site . All patie nt s fr o m t his pr ot oc ol are c o nsi dere d as Pri mar y 
cases.   


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 3  of 3 5  
 
T he ori gi nal C R Fs f or Pr ot oc ol P V P- 2 0 1 6 0 0 3 re q ueste d p osttreat me nt A E trac ki n g, b ut t h e 
St u d y Pr oce d ure C hart i n dicates t hat A Es are trac k e d pretreat me nt. T o be c o nsiste nt wit h t he 
O bser vati o nal St u d y , a n y A Es c ollecte d pri or t o st u d y dr u g treat me nt will b e i de ntifie d as N O T 
treat me nt-e mer ge nt e v e nts . N o n -treat me nt-e mer g ent A Es will be i ncl u de d i n t he data listi n g b ut 
will be e xcl u de d fr o m data s u m mar y (see Secti o n 6. 2  f or a d diti o nal details). 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 4  of 3 5  
 
T a ble 2 :  St u d y P V P- 2 0 1 6 0 0 3 Pr oce d ures  
 
 b. Patie nt s will b e f oll o we d u p wit h a visit t o ta k e place 5 t o 1 0 d a ys f oll o wi n g t h e last Pr o v a y Bl u e ™ a d mi nistrati o n a n d a tele p h o ne call t o ta ke place 1 0 t o 1 5 da ys  
     f oll o wi n g t he last Pr o va y Bl u e ™ a d mi nistrati o n. 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 5  of 3 5  
 
 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 6  of 3 5  
 
3.  
D A T A P O O LI N G C O N SI D E R A TI O N S   
A D at a I nte gr ati o n Pl a n ( DI P) will pr o vi de t he details of met h o ds t o c o m bi ne t he 2 st u dies i nt o 
a si n gle data bas e . T he f oll o wi n g pr o vi des t he ke y ele me nts of t he DI P.  
3. 1.  
D at a b ase Str uct ure  
It is t he i nte nti o n of Pr o v e p har m t o e nter select e d, perti ne nt C R F data el e me nts fr o m t he 
I nter ve nti o nal St u d y i nt o t he data base s er vi n g t he O bser vati o nal St u d y f or t he p ur p ose of 
pre pari n g t he d ata s u m mar y f or P M R- 3 0 6 5- 1. 
It is t her ef or e e x pected t h at certai n pr oce d ures will ha ve t o ta ke place bef ore t he C R F data 
ele me nts fr o m t he I nt er v e nti o nal St u d y ca n be e nt ere d i nt o t he electr o nic d ata base . T h os e 
pr oce d ur es i ncl u de b ut ar e n ot li mite d t o  c o n verti n g t he a bs ol ute cale n d ar d ates fr o m t he 
I n v esti gati o nal St u d y C R Fs t o t he relati ve st u d y d a ys use d i n t he data b ase. T he i nte grate d 
datasets will mi mic t he S D T M str uct ure a n d na mi n g c o n ve nti o n w he ne v er p ossi ble.  
I n st u d y P V P- 2 0 1 6 0 0 3, P K bl o o d dra ws were pla n ne d a n d plas ma c o n ce ntrati o n we re t o be 
dete r mi ne d. H o w e ver, P K data ar e n ot c ollect e d d ue t o p o or P K bl o o d sa m ple ha n dli n g. 
T heref ore, t he c orr es p o n di n g dataset ( P C) will n ot be create d. 
I n a sit uati o n w here a dat a fiel d t hat is pla n ne d i n o ne pr ot oc ol b ut n ot i n t he ot her pr ot oc ol, t he 
missi n g data will be mar ke d as ‘ N ot D o n e’ i n t he data base. F or i nsta nce, t h e O bser v ati o nal -
St u d y ( H Q F-M E T H B - 2 0 1 8 0 0 1) c ollects " Patie nt setti n g bef ore first prese nt ati o n", " Dia g n ose d 
b y", " Dia g n os is setti n g". T hese 3 q uesti o ns are n ot i ncl u de d i n t he I nter v e nti o nal St u d y. He nce, 
data val ue will be set t o ‘ N ot D o ne’ i n ‘ Patie nt setti n g bef ore first prese ntati o n’ dataset X D f or  
patie nt s fr o m t he I nterv e nti o nal St u d y ( P V P-- 2 0 1 6 0 0 3).  
 
 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 7  of 3 5  
 
T a ble 3 :  E x pecte d S D T M D o m ai ns f or t he P o ole d D at a b ase  
N a m e of D at a C ollecti o n Ele m e nts/ F or m  
S D T M D o m ai n  
De m o gra p hics (a ge, se x, race, et h nicit y) 
D M  
Wei g ht a n d hei g ht at e nr oll me nt 
V S  
Patie nt setti n g bef ore first prese ntati o n  
X D  
Past me dical hist or y  
M H  
Ac q uire d met he m o gl o bi ne mia dia g n osis, i ncl u di n g dia g n osis I C D- 1 0 C o de 
X D  
Met he m o gl o bi ne mia assess me nt a n d treat me nt bef ore recei vi n g st u d y dr u g 
X D  
Met he m o gl o bi ne mia si g ns a n d sy m pt o ms  
C E  
C o - o xi metr y bef ore a n d after Pr o va y Bl ue™ treat me nt 
L B  
Arterial bl o o d gas  
L B  
Pr o va y Bl ue ™ ad mi nistrati o n  
E X  
Ot her treat me nt f or met he m o gl o bi ne mia  
C M  
Vital si g ns  
V S  
E C G  
E G  
He mat ol o g y, bi oc he mistr y , a n d ot her la bs 
L B  
Me d icati o ns a n d pr oce d ures 
C M , P R 
H os pital co urse  
H O  
A d verse e ve nts  
A E  
F oll o w - u p o ver vie w a n d q uesti o nnaire s 
Q S  
 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 8  of 3 5  
 
3. 2.  
A n al ysis P o p ul ati o n  
T he f oll o wi n g a nal ysis sets will be i de ntifie d f or data s u m mar y . 
E nr olle d  A n al ysis Set : All patie nt s w h o si g n t he i nf or me d c o nse nt or ot h er wise meet all l ocal 
e nr oll me nt criteria f or t h e st u d y are c o nsi dere d t o ha ve e nr olle d.  
S afet y A n al ysis Set:   T h e safet y set will i ncl u de all e nr olle d patie nt s w h o are treate d wit h at 
least 1 Pr o v a yBl ue ™ i nf usi o n d ose. T h e safet y a n al ysis set will be use d f or safet y data s u m mar y. 
S pecificall y, t he saf et y p o p ulati o n will i ncl u de all treate d patie nt s fr o m t he I nter ve nti o nal St u d y 
a n d t he O bser v ati o nal St u d y.  
Effic ac y A n al ysis Set :  T he effi cac y a nal ysis set i ncl u des all safet y an al ysis set patie nts w h o 
ha ve at least 1 m et H b ass ess me nt bef ore a n d after Pr o va y Bl ue ™ i nf usi o n a n d w h o are Pri mar y 
cases. T his a n al ysis set will be use d f or efficac y e n d p oi nt e val uati o n.    
3. 3.  
E n d p oi nts  
3. 3. 1.  
Effic ac y E n d p oi nts  
T he e ffi cac y d ata s u m mar y will i ncl u de the f oll o wi n g e n d p oi nts: 
1)  C ha n ge fr o m bas eli ne i n m et H b after t h e first d ose of Pr o va yBl ue ™ i nf usi o n. 
T he pri mar y efficac y will be t he n u m ber ( %) patie nt s wit h ≥5 0 % re d ucti o n i n 
met H b  base d o n t he first a vaila ble p osttreat me nt assess me nt.  
•  T he f oll o wi n g are s ec o n d ar y e n d p oi nts t o s u p p ort t he pri mar y e n d p oi nt. 
i. C ha n ge i n met H b p osttreat me nt o ver ti me   
ii. Ti me w he n 5 0 % re d ucti o n i n met Hb is o bser ve d f or t he first ti me. 
2)  C ha n ge fr o m bas eli ne i n v it al si g ns ( bl o o d press ure, res pir at o r y r ate, a n d he art 
r ate) after t he first d ose of Pr o va yBl ue ™ i nf usi o n. 
•  T he sec o n d ar y efficac y will be t he n u m ber ( %) of patie nt s wit h c o nc o mita nt 
n or malizati o n of vital si g ns wit hi n 2 h o urs, i ncl usi ve, after t he e n d of i nf usi o n 
of t he first d ose of Pr o v a y Bl ue ™   
•  Ti me ( h o urs) w h e n t he first i nci de nce of n or mal vital si g ns  was o bser ve d i n 
patie nt s w h o ha d a b n or m al baseli ne vital si g ns 
3)  I n a d diti o n, 1 3 pres pecifie d cli nical si g ns a n d s y m pt o ms ass ociate d wit h met he m o gl o bi ne mia 
a n d t heir i nte nsit y (as Nati o nal Ca ncer I nstit ute C o m m o n Ter mi n ol o g y Criteria f or A d verse 
E ve nts [ N CI -C T C A E ] gra de) bef ore a n d after Pr o va y Bl ue ™ treat me nt are c ollecte d. T he 
prese nce/a bse n ce of t h ose si g ns a n d s y m pt o ms will be treate d as  a n ‘ot her ’ efficac y e n d p oi nt 
si nce decrease a n d/ or res ol uti o n of t h ose si g ns a n d s y m pt o ms ma y be c o nsi dere d s u p p orti ve 
of efficac y.  


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 1 9  of 3 5  
 
3. 3. 2.  
Rel ati ve Ti m e of Effic a c y E n d p oi nts  
A relati ve ti me fr a me will be use d t o dis pla y a n d t o s u m marize efficac y ass ess me nts. 
T he  st o p ti m e of t he first Pr o va y Bl ue T M  i nf usi o n will be use d t o deri ve rel ati ve ti me ( h o urs) 
f or e ac h p ost i nf usi o n m et H b a n d vit al si g ns assess me nt. After all eff o rts are m a de t o 
e ns ure t he c a pt ure of i nf usi o n st art a n d st o p ti me, w he n t he st o p ti me is missi n g , t he 
f oll o wi n g i m p ut ati o n r ule will be use d t o i m p ute t he missi n g st o p ti me.  
Missi n g st o p ti me will be i m p ute d wit h t he me dia n i nf usi o n d urati o n of t he efficac y 
p o p u lati o n. Mat he maticall y, t his i m p utati o n r ule ca n be e x presse d as  
St o p ti me =start ti me + me dia n i nf usi o n d urati o n  
W here t h e me dia n i nf usi o n d urati o n is deri ve d fr o m all patie nt s i n t he efficac y p o p ulati o n. 
Relati ve ti me of a n assess me nt ( h o urs) = Ti me of assess me nt – Ti me of st o p ti me of 1 st I nf usi o n. 
3. 3. 3.  
S afet y E n d p oi nts  
T he f oll o wi n g safet y e n d p oi nts will be s u m marize d: 
•  Pre vale nce a n d nat ure of treat me nt -e mer ge nt a d v erse e ve nts ( T E A E s), d efi ne d as all A E s 
wit h o nset date after t he first d ose of Pr o v a y Bl u e ™ i nf usi o n a n d wit hi n 1 0 d a ys of 
t he l ast d ose of Pr o v a y Bl ue ™ i nf usi o n.  
•  Res ults of cli nical la b orat or y tests bef ore a n d aft er  Pr o va y Bl ue ™ treat me nt. 
•  Res ults of E C G bef ore a n d after Pr o va y Bl ue ™ treat me nt. 
3. 4.  
Test H y p ot hesis a n d P V al ue J ustific ati o n  
N ot a p plica ble si nce n o i nfere ntial statistics will be pr o vi de d f or t his P M R.  W he ne v er it is 
a p pr o priate, 2-si de d 9 5 % c o nfi de nce i nter vals will be pr o vi de d f or esti mates.  
3. 5.  
Pr oce d ures f or H a n dli n g Missi n g D at a  
N o missi n g data i m p utati o n will be perf or me d f or safet y para meters. H o we ver, patie nt s wit h 
missi n g i nf usi o n st o p ti me will be assi g ne d a n i m p ute d st o p ti me i n or der t o deri ve t he efficac y 
e n d p oi nts ( See Secti o n 3. 3. 2  f or details).  
3. 6.  
A n al ysis Ce nter   
B ot h st u dies are m u ltice nter st u dies; he nce, i n vesti gati ve ce nter will be i de ntifie d as part of t he 
u ni q ue patie nt  i de ntificati o n. Data s u m maries will n ot be pre pare d b y ce nter u nless data w arra nt . 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 2 0  of 3 5  
 
3. 7.  
Defi niti o ns a n d Deri ve d V ari a bles  
3. 7. 1.  
B aseli ne   
Baseli ne will be defi ne d as t he last meas ure me nt bef ore t h e start of t he first i nf usi o n . Baseli ne 
rec or ds will be mar ke d f or all efficac y a n d safet y (la b orat or y, E C G, vital si gn s, a n d cli nical si g ns 
a n d s y m pt o ms) rec or ds i n t he data base.  
T here w as a sit uati o n w h ere a b aseli ne met H b m eas ure me nt ha d t h e e xact ti me of t he first d ose 
i nf usi o n start ti me; t he data w ere q u erie d a n d c o nfir me d.   
3. 7. 2.  
P atie nt  Gr o u ps  b y Met he m o gl o bi ne mi a S us pecte d C a us ati ve A g e nt  
Patie nt s are gr o u pe d i n 2 s u bsets base d o n t heir met he m o gl o bi ne mia s us pecte d ca usati ve a ge nt: 
Gr o u p 1 i ncl u des all pati e nt s t hat m et H b was ass o ciate d wit h Da ps o ne us e, a n d Gr o u p 2 i ncl u des 
all n o n- Da ps o ne- ca us e d met H b. T he differe ntiati o n is pri maril y base d o n  
•  t he fre q u e nc y wit h w hic h Da ps o ne is e x pecte d t o be i de ntifie d as t he ca usati ve a ge nt f or 
i n d uci n g met he m o gl o bi ne mia, a n d  
•  t he l o n g half-life of Da ps o ne a n d its meta b olite da ps o ne h y dr o x yla mi ne, w hic h li kel y 
res ults i n o n g oi n g pr o d ucti o n of met H b t hr o u g h t he ac ute treat me nt p has e, i n c o ntrast t o 
s h ort-acti n g i nciti n g a ge nts  s uc h as a m yl nitrate, w here n or m al p h ysi ol o gic c o n versi o n of 
met H b t o he m o gl o bi n ma y be gi n s h ortl y after e x p os ure.  
Patie nt  gr o u p will be use d f or data s u m mar y t a bles.   
3. 7. 3.  
P atie nt  Gr o u ps b y Met he m o gl o bi ne mi a Se verit y  
I n t he O bser v ati o nal St u d y, all e nr olle d patie nt s are classifie d as eit her a Pri mar y cas e or a 
Sec o n dar y case ( S ee Secti o n 2. 2. 1 ). T he patie nt s mar ke d as Pri mar y cases are m ore se vere case s 
a n d t he data are m o nit ore d, w hereas t he patie nts m ar ke d as Sec o n dar y cas es are  less se v ere a n d, 
asi de fr o m verificati o n of t he i ncl usi o n criteria, dat a i n t hese cas es are  n ot m o nit ore d.  
All patie nt s fr o m t he I nter ve nti o nal St u d y are cl assifie d as Pri mar y cas es. 
Sec o n dar y cases are e xcl u de d fr o m t he efficac y a nal ysis set (t he effi cac y a nal ysis set i ncl u des 
t he Pri mar y cases t h at ha ve baseli ne a n d at least 1 p ost baseli ne met H b meas ure me nt, See 
Secti o n  3. 2 ). Alt h o u g h all patie nt s are i ncl u de d i n t he safet y a n al ysis set, d ue t o t he differe nce i n 
data m o nit ori n g bet wee n t he Pri mar y a n d Sec o n d ar y cases, t he s afet y d ata will be a nal yze d 
t wice: o ne set will i ncl u de patie nt s fr o m t he Pri mar y cases, a n d t he sec o n d set will i ncl u de all 
patie nt s. 
Patie nt  t y pe ( Pri mar y vs Sec o n dar y) will be clearl y fla g ge d i n t he i n di vi d ual patie nt  data listi n g s 
as well as t he pr ofile fi g ures.  


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 2 1  of 3 5  
 
4.  
D A T A S U M M A RI E S  
It is a ntici pate d t hat t he p o ole d res ults will be pres e nte d as a s eries of i n di vi d ual patie nt  
descri pti ve narrati ves wit h li mite d statistical a nal ysis . T he narrati ves will als o i de ntif y iss ues i n 
i n di vi d ual patie nt past me dical hist or y, e x p os ures, a n d c o m or bi dities t hat ma y i m pact t he 
c o nsi derati o n a n d i nter pr etati o n  of “ n or mal ” vital si g ns a n d relate d para met ers.  
W he n a p pr o priate, s u m m ar y ta bles will als o be pre pare d t h at will i ncl u de descri pti ve statistics, 
s uc h as sa m ple size, m ea n, sta n dar d de viati o n, mi ni m u m, me dia n, a n d ma xi m u m f or c o nti n u o us 
varia bles a n d n u m b er ( %) of patie nts f or cate g orical varia bles. D ata s u m maries will pr o vi de 
descri pti ve statistics f or all patie nt s, patie nt s wit h p ossi ble ca usati ve a g e nt of Da ps o ne, a n d 
patie nt s wit h n o n- Da ps o ne ca usati ve a ge nts.   
All data i n t he p o ole d dat a base will ha ve a patie nt  le vel data listi n g.  
4. 1.  
Dis p ositi o n  
A s u m mar y ta ble (T a ble 1 4. 1. 1 ) will pr o vi de fre q ue nc y c o u nts f or all patie nts e nr olle d, patie nt s 
i n t he safet y an al ysis set, patie nt s i n t he efficac y a nal ysis set, a n d 1 0- da y p osttreat me nt stat us 
(ali ve, deceas e d, u n k n o w n, l ost t o f oll o w- u p). A s u m mar y of dis p ositi o n will be pr o vi de d o verall 
a n d b y pr ot oc ol ( P V P - 2 0 1 6 0 0 3 vs H Q F-M E T H B - 2 0 1 8 0 0 1). 
Dis p ositi o n data will be pr o vi de d as a d ata listi n g ( Listi n g 1 6. 2. 1).  
4. 2.  
De m o gr a p hics a n d B aseli ne C h ar acteristics  
T he de m o gra p hi c s u m mar y will i ncl u de descri pti v e statistics f or a ge , s e x, race, et h nicit y, wei g ht, 
hei g ht, a n d b o d y mass i n de x ( B MI ) at b aseli ne.  
Baseli ne c har acteristics a n d patie nt  p o p ulati o n c haracteristics will als o i ncl u de t he f oll o wi n g 
st u d y i nf or mati o n: 
1)  Patie nt setti n g bef ore first prese ntati o n  
2)  Ac q uire d met he m o gl o bi ne mia dia g n osis: 
a. Dia g n osis b y  
b.  Dia g n osis setti n g  
c. S us pecte d ca us ati ve a ge nt  
d.  E x p os ure t o a ge nt  
3)  Met he m o gl o bi ne mia assess me nts a n d treat me nt at baseli ne:  
a. A n y si g ns a n d s y m pt o ms j u d ge d t o be cli nicall y ass ociate d wit h t he ac q uire d 
met he m o gl o bi ne mia ( yes/ n o)? 
b.  Are c o - o xi metr y res ults a vaila ble fr o m t he i n de x h os pitalizati o n ( yes/ n o)? 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 2 2  of 3 5  
 
c. Are arterial bl o o d gas res ults a vaila ble fr o m t he i n de x h os pitalizati o n 
( yes/ n o)? 
d.  Baseli ne met H b le vel  
e. Was Pr o va y Bl ue ™ a d mi nistere d bef ore e nr oll me nt t o t he st u d y ( yes / n o)? 
f. Were a n y ot her tr eat me nt s e m pl o ye d t o miti gate t he ac q uire d 
met he m o gl o bi ne mia ( yes/ n o)? 
De m o gra p hics a n d baseli ne c har acteristics will be ta b ulate d f or t he s afet y a nal ysis set 
(T a bles 1 4. 1. 2. 1 a n d 1 4. 1. 2. 2) a n d efficac y a nal ysis set (T a ble 1 4. 1. 2. 3 ) wit h n o f or mal 
i nfere ntial tests. De m o gr a p hic data will be pr o vi d e d as a dat a listi n g (Listi n g  1 6. 2. 2). 
4. 3.  
Me dic al Hist or y  
All me dical hist or y data  ca pt ure d  will be liste d wit h o ut a n y f or mal s u m maries  ( Listi n g 1 6. 2. 3).  
4. 4.  
Pr ot oc ol De vi ati o ns  
A n y cli nicall y rele v a nt  pr ot oc ol de viati o n will be i de ntifie d a n d descri be d i n t he cli nical st u d y 
re p ort wit h o ut a n y f or m al s u m mar y. 
4. 5.  
Tre at me nt C o m pli a nce  
N ot a p plica ble. 
4. 6.  
Pri or a n d C o nc o mit a nt Me dic ati o ns  
All pri or a n d c o nc o mita nt me dicati o ns will be liste d wit h o ut a n y f or mal s u m m aries 
( Listi n g 1 6. 2. 6). 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 2 3  of 3 5  
 
5.  
E F FI C A C Y A N A L Y SI S  
Efficac y data listi n g s b y i n di vi d ual patie nt  will be pre pare d f or all effi cac y e n d p oi nts, i ncl u di n g 
t he o bser ve d v al ue a n d d eri ve d varia bles ( Listi n g 1 6. 2. 8, Listi n g 1 6. 2. 9, Listi n g 1 6. 2. 1 3). 
Efficac y a nal ysis will  be base d o n t he patie nts i ncl u de d i n t he effi cac y a nal ysis set.  
5. 1.  
Tre at me nt Effect o n  met H b   
M et H b meas ur e me nt s ca n be o btai ne d via c o -o xi metr y fr o m eit her arteri al or ve n o us bl o o d gas 
s peci me ns. Data will be p o ole d a n d s orte d b y ti me of meas ure me nt ta ke n f or a nal ysis . T he 
f oll o wi n g para meters will be deri ve d t o ai d disc ussi o n of treat me nt effect o n t he m et H b 
re d ucti o n. 
5. 1. 1.  
M et H b Le vel at 1 - h o ur P ost D osi n g 
T he ti mi n g of p ost d osi n g met H b le vel assa ys is i n c o nsiste nt  i n cli nical practice a n d may n ot be 
o btai ne d u ntil h o urs after t he c o ncl usi o n of first d ose i nf usi o n i n t he e mer g e nc y de p art me nt.  It 
s h o ul d be n ote d t hat, c o nsiste nt wit h s uc h practice, t he O bser vati o nal St u d y d oes n ot ha ve a 
pres pecifi e d sc he d ul e f or met H b p ost d osi n g c ollecti o n. M et H b le vel at 1 - h o ur p ost d osi n g will be 
deri ve d usi n g li near i nter p olati o n. T his met h o d will be referre d t o as bac k w ar d li near 
i nter p olati o n if t he first p ost d osi n g met H b was aft er 1 h o ur, or f or w ar d li near i nter p olati o n if t he 
first p ost d osi n g met H b was bef or e 1 h o ur. T his met h o d will be mat he maticall y e x presse d as 
f oll o ws. 
Ste p 1: calc ulate r ate of met H b c ha n ge p er mi n utes)   
  
= ( met H b _ 1 - b aseli ne) / ti me of met H b _ 1 i n mi n utes  
Ste p 2:  
met H b _ 1 hr = bas eli ne + 6 0 *[( met H b _ 1 - b aseli ne) / (ti me of met H b _ 1 i n mi n utes)]  
w here  
•  baseli ne = bas eli ne met H b defi ne d as t he l ast met H b bef ore t he first d ose of st u d y 
dr u g)  
•  met H b _ 1 = first p ost treat me nt met H b o bser vati o n  
•  c o nsta nt ( 6 0) c o n v erte d t he ti me u nits fr o m mi n utes t o h o urs 
F or e x a m ple, a patie nt  h a s met H b of 7 4. 4 0 at baseli ne a n d 1 5. 7 0 at 7 0 mi n utes 
p ost d osi n g. T he esti mate d met H b _ 1 hr is 2 4. 0 8 5 7 1.  
Perce nt c h a n ge fr o m bas eli ne ( P C H G) at 1- h o ur p ost d osi n g ca n be esti mate d as f oll o ws.  
P C H G ( % ) at 1- h o ur p ost d osi n g = 1 0 0 *[ ( met H b _ 1-baseli ne)  / ti me of met H b _ 1 i n 
mi n utes]/ baseli ne  


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 2 4  of 3 5  
 
T he P C H G f or t he a b o v e e xa m ple is - 6 7. 6 3 %. 
 
P C H G at 1 - h o ur = 1 0 0 * {[ 6 0 *( 1 5. 7- 7 4. 4)/ 7 0] / 7 4. 4 } 
D ue t o t he m at he m atic al pr o pert y of t his deri v ati o n, a p atie nt  c o ul d ne ver ac hie ve 5 0 % re d ucti o n 
at 1- h o ur p ost d osi n g if t he first p ost d osi n g met H b w as t a ke n after 2 h o urs. He nce, t his deri v ati o n 
will be a p plie d o nl y t o t h ose p atie nt s w h o h a d first p ost d osi n g met H b bef ore 2 h o ur s. 
5. 1. 2.  
Ti m e W he n m et H b Le vel Re ac he d 5 0 % R e d ucti o n    
T o esti mate t he ti me w he n a 5 0 % re d u cti o n w o ul d be o btai ne d, t he f oll o wi n g li near i nter p olati o n 
will be a p plie d.  
T 5 0 ( mi n utes after first d ose) = ( b aseli ne/ 2) / [ 6 0 *( baseli ne - met H b _ 1) / ti me of met H b _ 1 i n 
mi n utes]  
T 5 0 f or t he a b o v e e xa m ple is 4 4 mi n utes.  
T 5 0 = ( 7 4/ 2) / {[( 7 4. 4- 1 5. 7)/ 7 0] / 7 4. 4 } = 3 7. 2/ 0. 8 3 8 5 7 1 4 2 8 = 4 4. 3 6 
T he u nits f or T 5 0 will be c o n verte d t o h o urs b y T 5 0 / 6 0 i n t he dataset.  
D ue t o t he m at he m atic al pr o pert y of t his deri v ati o n, a p atie nt  c o ul d ne ver ac hie ve 5 0 % re d ucti o n 
at 1- h o ur p ost d osi n g if t he first p ost d osi n g met H b w as t a ke n after 2 h o urs . He nce, t his deri v ati o n 
will be a p plie d o nl y t o t h ose p atie nt s w h o h a d first p ost d osi n g met H b bef ore 2 h o ur s. 
 
5. 1. 3.  
S u m m ar y of m et Hb 
T he treat me nt effect o n m et H b will be e val uate d base d o n c ha n ge i n met H b fr o m baseli ne. 
Beca use of t h e wi de r a n g e i n assess me nt ti mes, t he s u m mar y ta ble will i ncl u de o nl y 2 ti me 
p oi nts: Baseli ne (last met H b bef or e treat me nt) a n d pre dicte d m et H b at 1- h o ur p ostt reat me nt. T he 
ta ble will pr o vi de descri pti ve statistics f or t he o bser ve d met H b le vel, c ha n g e fr o m baseli ne, a n d 
perce nt c ha n ge fr o m bas eli ne, i ncl u di n g sa m ple size, mea n, sta n d ar d d e viati o n, a n d 2-si de d 9 5 % 
c o nfi de nce i nter vals f or t he mea n, mi ni m u m, me dia n, a n d ma x i m u m. N u m ber ( %) patie nt s wit h 
≥5 0 % met H b re d ucti o n 1 h o ur p osttreat me nt will be i de ntifie d . (T a ble 1 4. 2. 1. 1 ). T his ta ble will 
als o i ncl u de pre dict e d ti me w he n met H b 5 0 % re d ucti o n w o ul d occ ur a n d ti me w he n t he first 
met H b is obtai ne d.  
A sec o n d s u m mar y ta b le (T a ble 1 4. 2. 1. 2 ) will pr o vi de met H b le vel descri pti ve s u m mar y at 
baseli ne a n d met H b le v el of t he first f oll o w -u p p osttreat me nt f or all patie nts i n t he efficac y 
a nal ysis set . I n t his ta ble, t he le vel of met H b at t he first f oll o w- u p a n d ti me w he n t he first f oll o w-
u p met H b i s ta ke n will be s u m marize d.  


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 2 5  of 3 5  
 
5. 1. 4.  
I n di vi d u al Pr ofile of met H b  
I n di vi d ual pr ofiles of met H b o ver ti me will be pre pare d f or eac h patie nt . All m et Hb assess me nts 
will be i ncl u de d . T he pr ofile will fla g if t h e patie nt  is a P ri mar y case or a S ec o n dar y cas e.  
T he c ha n g e fr o m bas eli ne i n m et H b will als o be gra p hicall y prese nte d , s h o wi n g t he c ha n ge fr o m 
baseli ne b y ti me ( h o urs) f or eac h patie nt  (Fi g ure 1 4. 2. 1. 3)  as ill ustrate d i n Fi g ure 1 .  
Fi g ure 1:  E x a m ple of I n di vi d u al Pr ofile of m et H b Bef ore a n d After T re at me nt  
 
H o ur 0 is w he n t he first d ose of Pr o va y Bl ue T M  i nf usi o n is c o m plete d a n d t he d otte d vertical 
refere nce li ne is 1 h o ur after t he e n d of i nf usi o n. 
5. 2.  
C h a n ge i n Vit al Si g ns   
Vital si g ns  c ha n ge fr o m baseli ne, i ncl u di n g bl o o d press ure, res pirat or y rate, a n d heart rate, will 
be deri ve d f or eac h patie nt. Baseli ne is t he last vital si g n meas ure me nt bef ore a patie nt  recei ves 
t he first i nf usi o n of Pr o va y Bl ueT M .  
5. 2. 1.  
Me di a n Vit al Si g ns wit hi n 2 -H o ur P ost Tre at me nt    
Vital si g ns meas ure me nts, i ncl u di n g s yst olic bl o o d press ure, diast olic bl o o d press ure, h eart r ate 
a n d res pirat or y rate, wer e ta ke n at vari o us ti me p oi nts a n d fre q ue nc y. I n or d er t o s y nc hr o nize t he 
meas ure me nt s, all vital si g ns meas ures ta k e n wit hi n 2 h o urs will be p o ole d wit hi n eac h 
i n di vi d ual patie nt  dataset t o deri ve t he me dia n f or t he patie nt .  




 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 2 7  of 3 5  
 
5. 2. 3.  
S u m m aries of Vit al Si g ns C h a n ge fr o m B aseli ne 
A s u m mar y ta ble will i ncl u de V S meas ure me nt a n d c ha n ge fr o m baseli ne b y relati ve ti me fra me 
wit h descri pti ve statistics of sa m ple size, mea n, sta n dar d de vi ati o n, 2-si de d 9 5 % c o nfi de n ce 
i nter vals, q uartiles ( 2 5 % tile, 5 0 % tile [me dia n ], a n d 7 5 % tile), mi ni m u m, a n d ma xi m u m. T he 
ta ble will als o i ncl u de n u m ber ( %) p atie nt s w h ose vital si g ns  are wit hi n t he n or mal ra n ge 
(T a ble 1 4. 2. 2. 1 ). N u m ber ( %) p atie nt s w h o ha d at least 1 i nci de nce of n or mal vital si g ns  wit hi n 
t he 2 h o urs f oll o wi n g t he c o m pleti o n of first d ose Pr o va y Bl ueT M  i nf usi o n will als o be prese nte d.  
C ha n ge i n S p O 2 will be assesse d i n 3 cate g ories, b ase d o n ± 2 % r ule as f oll o ws  
1)  I n crease d if t he differ e nce ( p ost baseli ne – bas eli ne) is > 2  
2)  N o C ha n ge if t he diff ere nce ( p ost baseli ne – b aseli ne) is wit hi n ± 2  
3)  Decr ease d if t h e differe n ce ( p ost baseli ne – b aseli ne) is < 2  
5. 2. 4.  
I n di vi d u al Pr ofiles of Vit al Si g ns 
T he c ha n g e fr o m bas eli ne i n vital si g ns  will als o be gra p hicall y prese nte d , s h o wi n g t he c ha n ge 
b y ti me ( h o urs) f or eac h patie nt  (Fi g ure 1 4. 2. 2. 2 ), as ill ustrate d i n Fi g ure 2:   E xa m ple of 
I n di vi d ual Pr ofile of Bl o o d Press ure Bef ore a n d After Treat me nt a n d Fi g ure 3 . T he pr ofile  will 
fla g if t he patie nt  is a Pri mar y cas e or a Sec o n dar y case.  
Fi g ure 2:  E x a m ple of I n di vi d u al Pr ofile of Bl o o d Press ure Bef o re a n d After Tre at me nt  
 
 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 2 8  of 3 5  
 
Fi g ure 3:  E x a m ple of I n di vi d u al Pr ofile of Res pir a t or y a n d He art R ate Bef ore a n d After 
Tre at m e nt  
 
 
5. 3.  
Met he m o gl o bi ne mi a Si g ns a n d S y m pt o ms  
T he f oll o wi n g 1 3 si g ns a n d s y m pt o ms t hat ma y be ass ociate d wit h met he m o gl o bi ne mia will be 
c ollecte d. D ecr ease a n d/ or res ol uti o n of t hese s y m pt o ms ma y be c o nsi dere d s u p p orti ve of 
efficac y.   T his assess me nt is a p plica ble o nl y f or t h e Pri mar y cas e. 
1.  Gra yis h - bl ue s ki n disc ol orati o n 
2.  C ya n osis 
3.  D ys p nea 
4.  Hea dac he  
5.  Fati g ue 
6.  Wea k n ess  
7.  Dizzi ness  
8.  S y nc o pe 
9.  C o ma 
1 0.  Seiz ures  


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 2 9  of 3 5  
 
1 1.  De press e d C N S  
1 2.  Arr h yt h mias 
1 3.  Meta b olic aci d osis  
Beca use t he t er m s ‘Gr a yis h - bl ue s ki n disc ol orati o n’ a n d ‘ C ya n osis’ are esse ntiall y t he sa me 
me dicall y s pea ki n g, t he s e 2 ter ms will be c olla pse d w he n t he a n al ysis dataset is creat e d fr o m t he 
C R F data base.  If b ot h b o xes o n t he C R F are c hec ke d wit h i de ntical ti me, t he rec or d wit h hi g hest 
i nte nsit y gra de will be fla g ge d t o be use d f or data s u m mar y; all rec or ds will be dis pla ye d i n data 
listi n g.  
Ot her n ote d si g ns a n d s y m pt o ms will als o be c ollecte d (as ‘ Ot her’). V er bati m te xt of ‘ Ot her’ 
si g ns a n d s y m pt o ms will be re vie w e d,  a n d si milar s y m pt o ms will be ma n uall y gr o u pe d i nt o a 
gr o u p ter m w he n t he a nal ysis dataset is b uilt . T he gr o u pe d ter m will be la b ele d as t he M o difie d 
ter m i n t he a nal ysis datas et. F urt her, it is u n derst o o d t hat s o me of t hese si g ns a n d s y m pt o ms ma y 
be i nc o nsiste ntl y detecte d (s uc h as c y a n osis i n a d ar kl y pi g me nte d patie nt) or i nc o nsiste ntl y 
classifie d/se ve rit y-rate d (s uc h as “ de presse d C N S”). 
Data c ollecti o n will i ncl u de se verit y ass ess me nt per N CI -C T C A E gra de a n d ti me of 
o bser vati o ns. I n s o me i nci de nces, a s y m pt o m c o ul d be re p ort e d wit h o ut se verit y. T h ose 
i nci de nces will be dis pla ye d as ‘ Y,’ i n dicati n g t hat t he s y m pt o m is prese nt wit h u n k n o w n gra de.  
O bser vati o ns fr o m si g ns a n d s y m pt o ms assess me nts will be gr o u pe d i nt o t he f oll o wi n g ti me 
peri o ds: 
•  Bef ore Tr eat me nt  
•  P ost treat me nt: ≤2 4 h o urs 
•  P ost treat me nt: 2 4- 4 8 h o urs 
•  ' P osttreat me nt: 4 8- 7 2 h o urs 
•  P ost treat me nt: 7 2- 9 6 h o urs 
•  P ost treat me nt: 9 6- 1 2 0 h o urs 
•  P ost treat me nt: > 1 2 0 h o urs 
5. 3. 1.  
S u m m ar y of Si g ns a n d S y m pt o ms  
S u m mar y ta bles ( T a ble 1 4. 3. 3. 1. 1, T a ble 1 4. 3. 3. 1. 2) will be pre pare d t o pr o vi de n u m ber ( %) of 
patie nt s w h o ha d a gi ve n si g n/s y m pt o m prese nt.  T he perce nta ge will be cal c ulate d  usi n g 2 
differe nt d e n o mi nat ors:  
•  R e p orte d: t he de n o mi nat or will be t he n u m ber of patie nt s w h o ha d assess me nt wit hi n 
eac h ti me peri o d  


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 3 0  of 3 5  
 
•  All Patie nt s: t he de n o mi nat or will be t he t otal n u m ber of patie nts i n eac h patie nt gr o u p i n 
t he ta ble.  
5. 3. 2.  
I n di vi d u al Pr ofiles of Si g ns a n d S y m pt o ms  
Eac h s y m pt o m a n d its se verit y will be dis pla ye d via scatter b u b ble pl ots f or eac h p atie nt (Fi g ure 
1 4. 3. 3. 2 ), s h o wi n g t he o bser ve d gr a de a n d relati ve ti me ( X) t o t he first i nf usi o n. T he first ti me 
w he n a s y m pt o m is j u d ge d t o be res ol ve d will als o be dis pla ye d. Data wit h assess me nt ti me will 
be i ncl u de d i n t he gra p hi c a n d all data ( wit h a n d wit h o ut ti me) will be i ncl u de d i n t he listi n g. 
T his fi g ure is ill ustrate d i n Fi g ure 4 .  
All 1 2 pres pecifie d si g ns a n d s y m pt o ms will be i de ntifie d i n t he pr ofile Y-a xis, t he 
si g n/s y m pt o m N CI-C T C A E gra de will be dis pla ye d if prese nt e d at a gi ve n ti me ( X -a xis). A n y 
ot her s y m pt o ms will als o be dis pla ye d usi n g gr o u pe d ter m w hic h will be re vie we d a n d fi nalize d 
base d o n ver b ati m  ter m bef ore d ata bas e l oc k.   
Fi g ure 4:  E x a m ple of I n di vi d u al Pr ofile of Met he m o gl o bi ne mi a Si g ns a n d S y m pt o ms 
Bef ore a n d Aft er Tre at m e nt  
 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 3 1  of 3 5  
 
6.  
S A F E T Y A N D T O L E R A BI LI T Y E V A L U A TI O N S  
Safet y data s u m maries will be base d o n t he safet y a nal ysis set . H o we v er,  t h e data will be 
ta b ulate d t wice: t he first t a ble will i ncl u de Pri mar y cases o nl y a n d t he s ec o n d ta ble will i ncl u de 
all patie nt s. Safet y data listi n g b y i n di vi d ual patie nt  will be pre pare d f or all safet y e n d p oi nts.  
6. 1.  
T ot al E x p os ure  
N u m ber of d oses recei ve d a n d i nf usi o n d urati o n will be ta b ulate d ( T a ble 1 4. 3. 1. 1 a n d 
T a ble 1 4. 3. 1. 2 ). Please n ote t hat a n y patie nt  wit h o ut a rec or de d i nf usi o n st o p ti me will ha ve a 
missi n g i nf usi o n d urati o n i n t his s u m mar y, alt h o u g h t he missi n g i nf usi o n st o p ti me is t o be 
i m p ute d i n or der t o deri v e t he relati ve ti me fra me f or efficac y e n d p oi nts (see Secti o n  3. 3. 2 ). 
6. 2.  
A d verse E ve nts  
A d verse e ve nt s re p orte d after t he first d ose of Pr o va y Bl ue ™ i nf usi o n start ti me a n d wit hi n 1 0 
da ys after t h e last d ose of Pr o va y Bl ue ™ i nf usi o n will be c o nsi dere d treat m e nt- e mer g e nt a d verse 
e ve nts ( T E A Es). A n y A Es t hat d o n ot ha ve o nset ti me will be i ncl u de d as T E A Es if re p ort e d.  
Patie nt s w h o di d n ot ha ve a n y A Es will be verifie d via s o urce d o c u me ntati o n.  
T he Me dical Di cti o nar y f or Re g ulat or y Acti vities (Versi o n  2 3) will be us e d t o classif y all A Es 
wit h res pect t o s yste m or ga n class a n d preferre d ter m . All a d verse e ve nts will be liste d.  
T he f oll o wi n g s u m mar y ta bles will be pr o d uce d f or t he T E A Es. All data s u m maries will pr o vi de 
n u m ber ( %) p atie nts as w ell as t otal n u m ber of e v e nts i n eac h cat e g or y  b y p atie nt  gr o u p. Eac h 
s u m mar y will be pr o d uce d i n 2 sets: o ne i ncl u des t he Pri mar y cas es a n d o ne i ncl u des all patie nt s 
i n t he safet y a n al ysis set .  
1.  a t o pli ne s u m mar y of T E A Es ( T a ble 1 4. 3. 2. 1. 1, T a ble 1 4. 3. 2. 1. 2 ) 
2.  a s u m mar y ta ble b y pr eferre d ter m i n des ce n di n g or der of t otal i nci de n ce 
(T a ble 1 4. 3. 2. 2 . 1, T a ble 1 4. 3. 2. 2. 2) 
3.   a s u m mar y ta ble b y s yst e m or ga n class a n d preferre d ter m (T a ble 1 4. 3. 2. 3. 1, 
T a ble 1 4. 3. 2. 3. 2 ) 
4.  a detaile d s u m mar y ta ble b y s yste m or ga n class, preferre d t er m a n d se v erit y 
(T a ble 1 4. 3. 2. 4. 1, T a ble 1 4. 3. 2. 4. 2 ) 
5.  a detaile d s u m mar y ta ble b y s yste m or ga n class, preferre d t er m a n d relati o ns hi p 
(T a ble 1 4. 3. 2. 5. 1, T a ble 1 4. 3. 2. 5. 2 ) 
6.  a ta ble of seri o us T E A Es b y s yste m or ga n class a n d preferre d t er m ( T a ble 1 4. 3. 2. 6. 1, 
T a ble 1 4. 3. 2. 6. 2 ) 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 3 2  of 3 5  
 
6. 3.  
Ot her S afet y P ar a meters  
6. 3. 1.  
C h a n ges i n Ot her  Bl o o d G as Tests 
I n a d diti o n t o met H b, res ults fr o m 1 1 ot her tests o btai ne d via bl o o d gas a nal ysis wit h or wit h o ut 
c o- o xi metr y ar e c ollecte d  (T a ble 5 ). I n di vi d ual pr ofiles of t he c o - o xi metr y res ults will be 
pre par e d  (Fi g ure 1 4. 3. 4 ), s h o wi n g t he o bser ve d v al ue ( Y) a n d rel ati ve ti me ( X) t o t he first 
i nf usi o n. Data wit h assess me nt ti me will be i ncl u de d i n t he gra p hic dis pla y; all data ( wit h a n d 
wit h o ut assess me nt ti me) will be i ncl u de d i n t he data listi n g  ( Listin g 1 6. 2. 1 2).  
 
T a ble 5 :  Bl o o d G as P ar a m eters of I nterest   
P ar a m eter ( U nits)  
D at a P a nel  
Arteri al Bl o o d G as  
C o -O xi metr y  
1  
T otal He m o gl o bi n ( g/ d L)  
Yes  
Yes  
2  
S O 2/ Sa O2 ( %) 
N o  
Yes  
3  
C 2 C T ( %)  
N o  
Yes  
4  
O x y H b ( %)  
Yes  
Yes  
5  
de o x y H b ( %)  
N o  
Yes  
6  
C O H b ( %)  
N o  
Yes  
7  
p H  
Yes  
Yes  
8  
Pa O 2 
Yes  
N o  
9  
Pa C O 2 
Yes  
N o  
1 0  
H C O 3 
Yes  
N o  
1 1  
Sa O 2 
Yes  
N o  
A b bre viati o ns: S O 2 = o x y ge n sat urati o n ; Sa O 2 = arterial o x y ge n sat urati o n ; O 2 C T = ox y ge n c o nte nt ; 
O x y H b  = o x y ge nate d he m o gl o bi n; de o x y H b = d e o x y he m o gl o bi n ; C O H b = car b o x y he m o gl o bi n ; p H = h y dr o ge n 
i o ns ( H +) i n bl o o d; Pa O2 = partial press ure of o x y ge n; Pa C O 2 = partial press ure of  car b o n di o xi de ; H C O 3 = 
Bicar b o nate.  
 
6. 3. 2.  
C h a n ges i n L a b or at or y Tests 
S electe d saf et y la b orat or y tests of i nterest will be  c ollecte d (s ee Ta bl e 6 ). All la borat or y  res ults 
were fr o m l ocal la b orat or y . Alt h o u g h ot her la borat or y test s o utsi de t his list c o ul d be c ollecte d, 
f or t he p ur p ose of t he dat a s u m mar y, t he res ults of la borat or y tests c ollecte d i n ‘ Ot her La bs’ will 
o nl y be a vaila ble i n t he listi n g ( Listi n g 1 6. 2. 1 1. 3).  
 
 


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 3 3  of 3 5  
 
 
T a ble 6 :  S afet y L ab or at or y Tests of I nterest   
He m at ol o g y  
Bi oc he mistr y  
He m o gl o bi n  
Gl uc ose  
Platelet C o u nt  
S o di u m  
R e d B l o o d C ell C o u nt  
P otassi u m  
W hite B l o o d C ell C o u nt  
C hl ori de  
 
Urea  
 
Calci u m  
 
I n or ga nic P h os p h or us 
 
Creati ni ne  
La b orat or y  res ults will be gra p hicall y dis pla ye d b y patie nt , s h o wi n g t he o bser ve d val u e ( Y) a n d 
relati ve ti me ( X) after t he first d ose of Pr o va y Bl ue™ I nf usi o n ( Fi g ure 1 4. 2. 5 f or he m at ol o g y, 
Fi g ure 1 4. 3. 6 f or C he mistr y ).  
 
6. 3. 3.  
C h a n ges i n 1 2- Le a d E C G  
Res ults of E C G are t o be c ollecte d bef ore a n d aft er Pr o va y Bl ueT M  treat me nt, i ncl u di n g P Q 
i nter val, Q R S d urati o n, Q T i nter val, Q Tc i nter val a n d o verall i nter pretati o n ( n or mal; a b n or mal 
n ot cli nicall y si g nifica nt; a b n or mal cli nicall y si g nifica nt ).  
N u m ber ( %) patie nts i n eac h  res ults i nter pretati o n cate g or y at eac h ti me p oi nt will be ta b ulate d at 
eac h pla n n e d ti me p oi nt ( T a ble 1 4. 3. 7. 1, T a ble 1 4. 3. 7. 2). T he P Q i nter val, Q R S d urati o n, Q T 
i nter val, a n d Q T c i nter val will n ot be ta b ulate d, b ut a data listi n g will be pr o vi de d  
( Listi n g 1 6. 2. 1 4).  


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 3 4  of 3 5  
 
7.  
A B L E O F C O N T E N T S O F P L A N N E D D A T A S U M M A RI E S   
7. 1.  
S u m m ar y T a bles  
N u m ber  
T y pe  
Title  
A n al ysis Set  
1 4. 1. 1  
Ta ble  
Patie nt  E nr oll me nt a n d A nal ysis Set a n d P ost 
Treat me nt F oll o w -u p Stat us  
E nr olle d A nal ysis Set  
1 4. 1. 2. 1  
Ta ble  
De m o gra p hics a n d Baseli ne C haracteristics  
Safet y A nal ysis Set - Pri mar y Cases  
1 4. 1. 2. 2  
Ta ble  
De m o gra p hics a n d Baseli ne C haracteristics  
Safet y A nal ysis Set - All Patie nt s 
1 4. 1. 2. 3  
Ta ble  
De m o gra p hics a n d Baseli ne C haracteristics  
Efficac y A nal ysis Set  
1 4. 2. 1. 1  
Ta ble  
Pre dicte d Met H b Le vel 1 H o ur P ost First I nf usi o n 
i n Patie nts wit h First Met H B Dra w n Wit hi n 
2  H o urs of D osi n g  
Efficac y A nal ysis Set  
1 4. 2. 1. 2  
Ta ble  
S u m mar y of 1st F oll o w -u p Met H b  Le vel 
Re gar dless W he n t he Assess m e nt Was Ta ke n  
Efficac y A nal ysis Set  
1 4. 2. 1. 3  
Fi g ure  
I n di vi d ual Pr ofile of C ha n ge fr o m Baseli ne i n 
Met H b Bef ore a n d After Treat me nt  
Safet y A nal ysis Set - All Patie nt s 
1 4. 2. 2. 1  
Ta ble  
S u m mar y of Vital Si g ns at Baseli ne a n d Wit hi n 
2  H o urs P ost First I nf usi o n  
Efficac y A nal ysis Set  
1 4. 2. 2. 2  
Fi g ure  
I n di vi d ual Pr ofile of C ha n ge fr o m Baseli ne i n Vital 
Si g ns Bef ore a n d After Treat me nt  
Safet y A nal ysis Set - All Patie nt s 
1 4. 2 . 3. 1 
Ta ble  
S u m mar y of Met he m o gl o bi ne mia Si g ns a n d 
S y m pt o ms Bef ore a n d After Treat me nt  
Efficac y A nal ysis Set  
1 4. 2 . 3.2 
Fi g ure  
Scatter Pl ots S h o wi n g I n di vi d ual Pr ofile of 
Met he m o gl o bi ne mia Si g ns a n d S y m pt o ms Bef ore 
a n d After Treat me nt 
Efficac y A nal ysis Set  
1 4. 3. 1. 1  
Ta ble  
St u d y Dr u g I nf usi o ns  
Safet y A nal ysis Set - Pri mar y Cases  
1 4. 3. 1. 2  
Ta ble  
St u d y Dr u g I nf usi o ns  
Safet y A nal ysis Set - All Patie nt s 
1 4. 3. 2. 1. 1  
Ta ble  
T o pli ne S u m mar y of Treat me nt E mer ge nt A d verse 
E ve nt  
Safet y A nal ysis Set - Pri mar y Cases  
1 4. 3. 2. 1. 2  
Ta ble  
T o pli ne S u m mar y of Treat me nt E mer ge nt A d verse 
E ve nt  
Safet y A nal ysis Set - All Patie nt s 
1 4. 3. 2. 2. 1  
Ta ble  
All T E A E b y Preferre d Ter m Dis pla ye d t he M ost 
C o m m o nl y Re p orte d E ve nt First  
Safet y A nal ysis Set - Pri mar y Cases  
1 4. 3. 2. 2. 2  
Ta ble  
All T E A E b y Preferre d Ter m Dis pla ye d t he M ost 
C o m m o nl y Re p orte d E ve nt First  
Safet y A nal ysis Set - All Patie nt s 
1 4. 3. 2. 3. 1  
Ta ble  
All T E A E b y S O C a n d Preferre d Ter m  
Safet y A nal ysis Set - Pri mar y Cases  
1 4. 3. 2. 3. 2  
Ta ble  
All T E A E b y S O C a n d Preferre d Ter m  
Safet y A nal ysis Set - All Patie nt s 
1 4. 3. 2. 4. 1  
Ta ble  
All T E A E b y S O C, Preferre d Ter m a n d I nte nsit y  
Safet y A nal ysis Set - Pri mar y Cases  
1 4. 3. 2. 4. 2  
Ta ble  
All T E A E b y S O C, Preferre d Ter m a n d I nte nsit y  
Safet y A nal ysis Set - All Patie nt s 
1 4. 3. 2. 5 . 1 
Ta ble  
All T E A E b y S O C, Preferre d Ter m a n d 
Relati o ns hi p t o St u d y Dr u g  
Safet y A nal ysis Set - Pri mar y Cases  
1 4. 3. 2. 5. 2  
Ta ble  
All T E A E b y S O C, Preferre d Ter m a n d 
Relati o ns hi p t o St u d y Dr u g  
Safet y A nal ysis Set - All Patie nts 
1 4. 3. 2. 6. 1  
Ta ble  
All Seri o us T E A E b y S O C a n d Preferre d Ter m  
Safet y A nal ysis Set - Pri mar y Cases  


 
Pr ot oc ol P V P - 2 0 1 6 0 0 3 & H Q F-M E T H B - 2 0 1 8 0 0 1 
Data S u m mar y Pla n  
3 0 A u g ust 2 0 2 1 
 
 
P a g e 3 5  of 3 5  
 
1 4. 3. 2. 6. 2  
Ta ble  
All Seri o us T E A E b y S O C a n d Preferre d Ter m  
Safet y A nal ysis Set - All Patie nt s 
1 4. 3. 3 
Fi g ure  
I n di vi d ual Pr ofile of Bl o o d Gas Res ults Bef ore a n d 
After Treat me nt  
Safet y A nal ysis Set - All Patie nt s 
1 4. 3. 4 
Fi g ure  
I n di vi d ual Pr ofile of He mat ol o g y Test Res ults 
Bef ore a n d After Treat me nt  
Safet y A nal ysis Set - All Patie nt s 
1 4. 3. 5 
Fi g ure  
I n di vi d ual Pr ofile of Bi oc he mistr y Test Res ults 
Bef ore a n d After Treat me nt  
Safet y A nal ysis Set - All Patie nt s 
1 4. 3. 6 . 1 
Ta ble  
S u m mar y of 1 2 -Lea d E C G Res ults b y 
I nter pretati o n Cate g or y a n d Ti me p oi nt 
Safet y A nal ysis Set - Pri mar y Cases  
1 4. 3. 6 . 2 
Ta ble  
S u m mar y of 1 2 -Lea d E C G Res ults b y 
I nter pretatio n Cate g or y a n d Ti me p oi nt  
Safet y A nal ysis Set - All Patie nt s 
 
 
7. 2.  
D at a Listi n gs  
N u m ber  
Title  
A n al ysis Set  
1 6. 2. 1  
Dis p ositi o n a n d A nal ysis P o p ulati o n  
I T T A nal ysis Set  
1 6. 2. 2  
De m o gra p hics  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 3  
Me dical Hist or y  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 4  
Met he m o gl o bi ne mia Assess m e nt a n d Dia g n osis  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 5  
St u d y Dr u g A d mi nistrati o n  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 6  
Pri or a n d C o nc o mita nt Me dicati o ns  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 7  
Ot her Pr oce d ures D uri n g St u d y  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 8  
Met H b Deri ve d Efficac y Para meters  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 9  
Vital Si g ns a n d C ha n ge fr o m Baseli ne  
Safet y A nal ys is Set - All Patie nt s 
1 6. 2. 1 0  
All A d verse E ve nts Re p orte d i n t he St u d y  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 1 1. 1  
St u d y I de ntifie d He mat ol o g y Test Res ults  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 1 1. 2  
St u d y I de ntifie d Bi oc he mistr y Test Res ults  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 1 1. 3  
Res ults fr o m Ot her La b Tests C ollecte d  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 1 2  
Bl o o d Gas Para meters Res ults  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 1 3  
Met he m o gl o bi ne mia Si g ns a n d S y m pt o ms  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 1 4  
1 2 -Lea d E C G Res ults  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 1 5  
F oll o w -u p  
Safet y A nal ysis Set - All Patie nt s 
1 6. 2. 1 6  
Listi n g of H os pital C o urse  
Safet y A nal ysis Set - All Patie nt s 
 

Prot_004.pdf: 
Provepharm SAS 
CLINICAL STUDY PROTOCOL – CONFIDENTIAL 
MEBIPAM - MEthylene Blue In Patients with Acquired Methemoglobinemia 
Protocol Number: 
PVP-2016003 
Investigational New Drug (IND) 
Number: 
IND: 118,156 
Study Medication: 
ProvayBlue™ methylene blue 
Title: 
Open label clinical study to evaluate the safety and efficacy 
of ProvayBlueTM (methylene blue) for the treatment of 
acquired methemoglobinemia 
Version and Date: 
Final Version 3 dated 10 August 2017 
Sponsor: 
Provepharm SAS 
22 Rue Marc Donadille 
13013 Marseille, France 
Medical Monitor: 
Sponsor’s Contact: 
Confidentiality Statement: 
This document contains information which is the property of Provepharm SAS and therefore is provided to you as 
strictly confidential information for your review. It is understood that this information cannot and will not be 
disclosed to others without written approval from Provepharm SAS. 
P R O V E N C E  T E C H N O L O G I E S  G R O U P
NCT#03395223




Protocol PVP-2016003 
ProvayBlueTM for acquired methemoglobinemia 
Page 3 of 67 
FINAL V3 10-AUG-2017 – US 
CONFIDENTIAL 
2. 
PROTOCOL AGREEMENT PAGE 
I confirm that I have read and that I understand this protocol, the Investigator Brochure (IB), and 
all other product information provided by Provepharm SAS (the Sponsor). I agree to conduct this 
study in conformity with this protocol and also in accordance with the ethical principles that have 
their origin in the Declaration of Helsinki, the E6 ICH Harmonized Tripartite Guideline for Good 
Clinical Practice, all applicable laws and regulations, including, without limitation, data privacy 
laws and regulations, and the regulatory requirements for reporting serious adverse events defined 
in Section 15.3 of this protocol. 
 
Principal Investigator 
 
Title and Name: 
 
Hospital Name: 
 
 
 
Signature: 
 
Date : 
 


Protocol PVP-2016003 
ProvayBlueTM for acquired methemoglobinemia 
Page 4 of 67 
FINAL V3 10-AUG-2017 – US                                                                                           CONFIDENTIAL 
3. 
SYNOPSIS 
Study title: 
Open label clinical study to evaluate the safety and efficacy of 
ProvayBlueTM (methylene blue) for the treatment of acquired 
methemoglobinemia 
Protocol number: 
PVP-2016003 
Type of study: 
Phase 4 
Sponsor: 
Provepharm SAS 
Co-ordinating 
Investigator: 
Study medication: 
ProvayBlue™ (methylene blue injection USP) 
Dose to be studied: 
1 mg/kg 
Dosage form: 
Each ProvayBlue™, 10 mL single-dose ampule contains 50 mg 
Proveblue® methylene blue and water for injection q.s. Each mL of 
solution contains 5 mg methylene blue and water for injection q.s. 
Route: 
ProvayBlue™ is a sterile solution intended for intravenous (IV)
administration. 
Dosing regimen: 
ProvayBlue™ has been approved by the US FDA to be administered 
intravenously at a dose of 1 mg/kg over 5-30 minutes and if the 
methemoglobin (metHb) level remains above 30% or if clinical 
symptoms persist, to give a repeat dose of up to 1 mg/kg 1 hour after 
the first dose. 
Depending on the clinical status of the patient, additional doses may be 
administered at the discretion of the Investigator. The data on these 
additional doses will be recorded in the study Case Report Form (CRF).
If ProvayBlue™ is administered as an infusion, it MUST be diluted in 
5% dextrose in water (D5W) only. Dilution in saline will result in 
precipitation of the product and must not be administered. 
 
Study centers: 
The study will include sites which will be based in France, Germany, 
UK and the USA. 
Study objectives: 
Primary Objective 
• To confirm that ProvayBlueTM after a single administration is 
efficacious in patients with acquired methemoglobinemia. 
Secondary Objectives 
• To evaluate the efficacy of a single dose of 1 mg/kg of 
ProvayBlue™; 
• To evaluate the efficacy of a second dose of 1 mg/kg of 
ProvayBlue™ to further reduce metHb levels when metHb is 
not fully reduced by a single dose; 


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• To evaluate the normalization of the respiratory rate, heart rate 
and blood pressure of patients who achieve a reduction in 
metHb level within 2 hours of receiving the first dose of 
ProvayBlue™; 
• To confirm the safety and tolerability of ProvayBlue™ injection 
in patients with acquired methemoglobinemia. 
Exploratory Objective 
• To collect sparse pharmacokinetic (PK) data in patients. 
Study design: 
This is an open label, uncontrolled Phase 4 study of 10 patients who 
present 
in 
hospital/urgent 
care 
setting 
with 
acquired 
methemoglobinemia. The population may include pediatric and adult 
patients (males and females of all ages are included).  
The aim of the study is to confirm safety and efficacy of ProvayBlueTM
for the treatment of acquired methemoglobinemia. 
This study comprises of an analysis of data for patients presenting with 
acquired methemoglobinemia and requiring ProvayBlueTM. The 
analysis itself will focus on clinical outcome of treatment with 
ProvayBlueTM alone. If possible, depending on the number of patients, 
the data will be summarized by age, sex and body weight.  
Study population: 
Patients who present in hospital/urgent care setting diagnosed with 
acquired methemoglobinemia. The population may include pediatric 
and adult patients (males and females of all ages are included).  
Diagnosis and main 
criteria for inclusion: Inclusion Criteria 
1. Pediatric or adult patients (males and females of all ages are 
included) diagnosed with acquired methemoglobinemia and receiving 
treatment with ProvayBlue™ as per the treating physician’s diagnosis 
and hospital standard of care. 
Acquired 
methemoglobinemia 
is 
defined 
as 
a 
level 
of 
methemoglobinemia >30% or ≤30% in case of clinical symptoms (e.g. 
sleepiness, cyanosis, dizziness, etc.). 
2. Written informed consent obtained prior to any data collection 
(retrospective and prospective) for this study and study specific 
assessments. 
Exclusion criteria  
1. Known severe hypersensitivity reactions to methylene blue or any 
other thiazine dye; 
2. Known deficiency in glucose-6-phosphate dehydrogenase (G6PD)
due to the risk of hemolytic anemia as well as lack of therapeutic effect; 
3. Known deficiency in Nicotinamide Adenine Dinucleotide Phosphate 
Hydrogen (NADPH) reductase.  


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4. Known use of selective serotonin reuptake inhibitors (SSRIs), 
serotonin 
and 
norepinephrine 
reuptake 
inhibitors (SNRIs), 
MonoAmine Oxidase (MAO) inhibitors or drugs metabolised via CYP 
isoenzymes anticipated during the treatment phase of the study. 
5. Women who refuse to stop breastfeeding for up to 8 days after 
receiving the last dose of ProvayBlueTM. 
Total sample size: 
Because this is an ultra-orphan indication, the enrollment will be 
10 patients, in adult and pediatric population. 
Based on evaluation of historical data, the potential enrollment for this 
study is estimated to be 1/3 patient per site per year (i.e., one patient per 
site over the study period of approximately 32 months). Thus it is 
expected that 10 patients may be enrolled during the 32-months-study.
A sample size of 10 will provide at least 80% power to detect a 90% 
response rate and exclude a response rate less than 50%. 
As this is an open label study with only one treatment there will be no 
randomization and no blinding of subjects. Study numbers will be 
allocated to subjects in the order in which they are enrolled into the 
study and by site. 
Number of planned 
visits: 
The trial includes the following visits:  
• Screening and enrolment 
• Study Treatment 
- 
H0: ProvayBlueTM Dose 1 
- 
H1: 1 hour after the end of ProvayBlue™ infusion.
Administration of ProvayBlueTM Dose 2 (if applicable)  
- 
H2: 2 hours after the end of ProvayBlue™ Dose 1 infusion
- 
H3: 2 hours after the end of ProvayBlue™ Dose 2 infusion
(if applicable) 
• H24 (24 hours after the end of ProvayBlue™ Dose 1 infusion) 
• Hospital Discharge (if earlier than H24) 
• Follow up visit (5 -10 days following the last ProvayBlue™ 
administration) 
• Follow up call (10 -15 days following the last ProvayBlue™ 
administration) 
Efficacy parameters: 
The following efficacy parameters will be assessed: 
• A reduction in metHb by 50% within 1 hour of dosing; 


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• Concomitant normalization of respiratory rate within 2 hours of 
the first dose of ProvayBlue™; 
• Concomitant normalization of heart rate within 2 hours of the 
first dose of ProvayBlue™; 
• Concomitant normalization of diastolic blood pressure (DBP)
within 2 hours of the first dose of ProvayBlue™; 
• Concomitant normalization of systolic blood pressure (SBP)
within 2 hours of the first dose of ProvayBlue™. 
Pharmacokinetic 
parameters: 
• PK samples will be analyzed for methylene blue and azure B. 
PK samples are to be taken at the discretion of the Investigator based 
on the condition of the patient. The time blood sample(s) are taken 
should be recorded, but there is no absolute time window for the PK 
samples. Suggested PK sampling time points are as follows: 
• Approximately 1 hour after the end of each ProvayBlue™ 
infusion (i.e. Dose 1, Dose 2 and any additional dose if 
applicable); 
• Prior to Dose 2 ProvayBlue™ infusion (if applicable);  
• Approximately 24 hours post the end of the last ProvayBlue™ 
infusion, or just prior to discharge from the clinic if earlier than 
24 hours. 
Safety parameters: 
The following safety parameters will be assessed: 
• Physical examination; 
• Vital signs; 
• Hematology and biochemistry; 
• Electrocardiogram (ECG); 
• Adverse events (AEs). 
Statistical methods: 
Statistical analyses will be performed by Orion under the authority of 
the Sponsor. Statistical analyses will be carried out using SAS®, 
Version 9.3 or later, SAS Institute, Cary, North Carolina, USA. 
The statistical methods for this study will be described in a detailed 
statistical analysis plan (SAP), which will be finalized prior to database 
lock. Any deviations from the planned analysis as described in the SAP 
will be justified and recorded in the final clinical study report. 
 


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4. 
TABLE OF CONTENTS 
1. 
SIGNATURE PAGE .............................................................................................. 2 
2. 
PROTOCOL AGREEMENT PAGE ................................................................... 3 
3. 
SYNOPSIS .............................................................................................................. 4 
4. 
TABLE OF CONTENTS....................................................................................... 8 
5. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS................... 12 
6. 
INTRODUCTION ................................................................................................ 14 
6.1. 
RATIONALE FOR THE STUDY ......................................................................... 14 
6.2. 
ACQUIRED METHEMOGLOBINEMIA ............................................................ 14 
6.3. 
CLINICAL SYMPTOMS ASSOCIATE WITH METHEMOGLOBINEMIA ..... 15 
6.4. 
TREATMENT OF ACQUIRED METHEMOGLOBINEMIA ............................. 15 
6.5. 
PROVAYBLUETM ................................................................................................. 15 
6.6. 
RISK-BENEFIT ASSESSMENT .......................................................................... 16 
7. 
STUDY OBJECTIVES AND ENDPOINTS ...................................................... 17 
7.1. 
STUDY OBJECTIVES .......................................................................................... 17 
7.1.1. 
Primary Objective .................................................................................................. 17 
7.1.2. 
Secondary Objectives ............................................................................................. 17 
7.1.3. 
Exploratory Objective ............................................................................................ 17 
7.2. 
STUDY ENDPOINTS ........................................................................................... 17 
7.2.1. 
Primary Efficacy Endpoint..................................................................................... 17 
7.2.2. 
Secondary Efficacy Endpoints ............................................................................... 17 
7.2.3. 
Safety Endpoints .................................................................................................... 18 
7.2.4. 
Pharmacokinetic (PK) Endpoints ........................................................................... 18 
8. 
INVESTIGATIONAL PLAN.............................................................................. 19 
8.1. 
SUMMARY OF STUDY DESIGN ....................................................................... 19 
8.2. 
DISCUSSION OF TRIAL DESIGN...................................................................... 20 
8.3. 
TRIAL TIMETABLE (STUDY PERIODS).......................................................... 21 
9. 
SELECTION AND WITHDRAWAL OF PATIENTS..................................... 22 


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9.1. 
INFORMED CONSENT ....................................................................................... 22 
9.2. 
INCLUSION CRITERIA ....................................................................................... 23 
9.3. 
EXCLUSION CRITERIA...................................................................................... 23 
9.4. 
ASSIGNMENT OF PATIENT NUMBER ............................................................ 23 
9.5. 
PREMATURE WITHDRAWAL OF PATIENTS FROM STUDY AND 
REPLACEMENT POLICY ................................................................................... 23 
9.5.1. 
Discontinuation Criteria ......................................................................................... 23 
9.5.2. 
Replacement Policy................................................................................................ 24 
10. 
STUDY MEDICATION ...................................................................................... 25 
11. 
CONCOMITANT THERAPY/MEDICAL MANAGEMENT OF 
ADVERSE EVENTS (AEs) ................................................................................. 26 
11.1. 
RECORDING OF PREVIOUS CONCOMITANT THERAPY ............................ 26 
11.2. 
PRECAUTIONS FOR USE, INTERACTIONS WITH OTHER MEDICINAL 
PRODUCTS AND OTHER FORMS OF INTERACTION OF THE IMP ........... 26 
11.3. 
MEDICATIONS THAT MUST NOT BE ADMINISTERED .............................. 28 
11.4. 
GUIDANCE ON MEDICATIONS TO BE ADMINISTERED/PATIENT 
MANAGEMENT FOR EXPECTED ADVERSE EVENTS (AEs) ...................... 28 
11.4.1. 
Hypersensitivity ..................................................................................................... 28 
11.4.2. 
Hemolytic Anemia ................................................................................................. 28 
12. 
SCHEDULE OF ASSESSMENTS ..................................................................... 29 
12.1. 
SCREENING AND ENROLMENT ...................................................................... 29 
12.2. 
TREATMENT ....................................................................................................... 30 
12.3. 
DISCHARGE ......................................................................................................... 32 
12.4. 
FOLLOW-UP......................................................................................................... 32 
12.5. 
WITHDRAWAL/PREMATURE STUDY DISCONTINUATION ...................... 32 
13. 
ASSESSMENTS OF EFFICACY ....................................................................... 34 
13.1. 
SPECIFICATION OF EFFICACY PARAMETERS ............................................ 34 
13.2. 
METHODS AND TIMING FOR ASSESSING, RECORDING AND 
ANALYSING EFFICACY PARAMETERS ........................................................ 34 
14. 
ASSESSMENTS OF PHARMACOKINETIC (PK) ......................................... 35 
15. 
ASSESSMENTS OF SAFETY ............................................................................ 36 
15.1. 
SPECIFICATION OF SAFETY PARAMETERS ................................................ 36 


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15.2. 
METHODS AND TIMING FOR ASSESSING, RECORDING AND 
ANALYSING SAFETY PARAMETERS ............................................................. 36 
15.2.1. 
Physical Examination ............................................................................................. 36 
15.2.2. 
Vital Signs .............................................................................................................. 36 
15.2.3. 
Biochemistry .......................................................................................................... 37 
15.2.4. 
Hematology ............................................................................................................ 37 
15.2.5. 
Methemoglobin ...................................................................................................... 38 
15.2.6. 
12-lead Electrocardiogram (ECG) ......................................................................... 38 
15.2.7. 
Adverse Events (AEs) ............................................................................................ 38 
15.3. 
ADVERSE EVENT (AE) REPORTING ............................................................... 38 
15.3.1. 
Definition of an Adverse Event (AE) .................................................................... 38 
15.3.2. 
Definition of a Serious Adverse Events (SAE) ...................................................... 39 
15.3.3. 
Recording and Reporting of Adverse Events (AEs) .............................................. 40 
16. 
EVALUATION OF RESULTS ........................................................................... 46 
16.1. 
SAMPLE SIZE AND STUDY POWER ............................................................... 46 
16.2. 
STATISTICAL METHODS .................................................................................. 46 
16.2.1. 
General Considerations .......................................................................................... 46 
16.2.2. 
Analysis Sets .......................................................................................................... 47 
16.3. 
EVALUATION OF BASELINE DATA ............................................................... 47 
16.4. 
EVALUATION OF EFFICACY ........................................................................... 47 
16.5. 
EVALUATION OF SAFETY ............................................................................... 47 
16.6. 
EVALUATION OF PHARMACOKINETICS (PK) ............................................. 47 
16.7. 
INTERIM ANALYSIS .......................................................................................... 47 
17. 
OBLIGATIONS OF THE PRINCIPAL INVESTIGATOR (PI) .................... 48 
17.1. 
INDEPENDENT ETHICS COMMITTEE/ INSTITUTIONAL REVIEW 
BOARD (IEC/IRB) ................................................................................................ 48 
17.2. 
REGULATORY BODY APPROVAL .................................................................. 49 
17.3. 
INFORMED CONSENT ....................................................................................... 49 
17.4. 
CASE REPORT FORMS AND SOURCE DOCUMENT VERIFICATION ....... 49 
17.5. 
CONFIDENTIALITY ............................................................................................ 50 
17.6. 
STAFF INFORMATION AND RESPONSIBILITIES ......................................... 50 
17.7. 
ESSENTIAL DOCUMENT RETENTION ........................................................... 50 
18. 
STUDY MANAGEMENT ................................................................................... 52 
18.1. 
MONITORING ...................................................................................................... 52 
18.2. 
QUALITY ASSURANCE (QA) AND QUALITY CONTROL (QC) .................. 52 
18.2.1. 
Quality Control (QC) ............................................................................................. 52 
18.2.2. 
Quality Assurance (QA)......................................................................................... 52 


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18.2.3. 
Audit....................................................................................................................... 52 
18.3. 
DATA QUERY PROCESS ................................................................................... 53 
18.4. 
PROTOCOL DEVIATIONS/AMENDMENTS .................................................... 53 
18.5. 
DISCONTINUATION OF THE STUDY .............................................................. 54 
18.6. 
PUBLICATIONS ................................................................................................... 54 
19. 
REFERENCES ..................................................................................................... 55 
20. 
APPENDICES ...................................................................................................... 57 
APPENDIX 1: 
US PACKAGE INSERT OF PROVAYBLUE™.................................... 58 
APPENDIX 2: 
STUDY FLOW CHART ......................................................................... 65 
APPENDIX 3: 
ESTIMATES OF 1% OF THE PATIENT’S BLOOD VOLUME .......... 67 
LIST OF TABLES 
Table 6-1: 
Signs and Symptoms Related to Blood Levels of Methemoglobin 
(metHb) ...................................................................................................... 15 
Table 15-1: 
Relationship of the Adverse Event (AE) to Study Medication .................. 42 
Table 15-2: 
Definition of Severity of Adverse Events (AEs) ........................................ 43 
 
LIST OF FIGURES 
Figure 8-1: 
Study Design .............................................................................................. 20 
 


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5. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
ADR 
Adverse Drug Reaction 
ADL 
Activities of Daily Living 
AE 
Adverse Event 
BP 
Blood Pressure 
°C 
Degrees Celsius 
CI 
Confidence Interval 
CRA 
Clinical Research Associate 
CRF 
Case Report Form 
CTC 
Common Toxicity Criteria 
CYP 
Cytochrome 
DBP 
Diastolic Blood Pressure 
ECG 
Electrocardiogram 
EMA 
European Medicine Agency  
FAS 
Full Analysis Set 
FDA 
Food and Drug Administration 
FWA 
Federal Wide Assurance 
g 
Gram(s) 
G6PD 
Glucose-6-phosphate Dehydrogenase 
GCP 
Good Clinical Practice 
IB 
Investigator Brochure 
ICH 
International Conference on Harmonisation 
IEC 
Independent Ethics Committee 
IND 
Investigational New Drug  
IRB 
Institutional Review Board 
ITT 
Intent-to-Treat 
IV 
Intravenous 
Kg 
Kilogram(s) 
LMB 
Leucomethylene Blue 
LPLV 
Last Patient Last Visit  
MAO 
MonoAmine Oxidase 
MB 
Methylene Blue 
MedDRA 
Medical Dictionary for Regulatory Activities 
metHb 
Methemoglobin 
mg 
Milligram(s) 


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µl 
Microliter(s) 
mL 
Milliliter(s) 
mmHg 
Millimeters of Mercury 
NADPH 
Nicotinamide Adenine Dinucleotide Phosphate Hydrogen 
NCI 
National Cancer Institute 
PI 
Principal Investigator 
PICD 
Patient Informed Consent Document 
PK 
Pharmacokinetics 
PP 
Per-Protocol 
QA 
Quality Assurance 
QC 
Quality Control 
QP 
Qualified Person 
qs 
The Amount which is Enough (Quantum Satis) 
RA 
Regulatory Authority 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SBP 
Systolic Blood Pressure 
SD 
Standard Deviation 
SNRIs 
Serotonin and Norepinephrine Reuptake Inhibitors 
SPC 
Summary of Product Characteristics 
SSRIs 
Serotonin Reuptake Inhibitors  
SUSAR 
Suspected, Unexpected Serious Adverse Reaction 
t½ 
Elimination half life 
TEAE 
Treatment-emergent Adverse Event 
UK 
United Kingdom 
US 
United States 
USP 
United States Pharmacopeial Convention 
WHO 
World Health Organization 
 


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6. 
INTRODUCTION 
6.1. 
RATIONALE FOR THE STUDY 
ProvayBlueTM1 (methylene blue injection USP) was approved on 08 April 2016 by the United 
States (US) 
Food and Drug Administration (FDA) for the treatment of 
acquired 
methemoglobinemia under accelerated approval (see APPENDIX 1: 
US Package Insert of 
ProvayBlue™).  
As a post-marketing requirement, Provepharm is required to conduct a clinical study to evaluate 
the safety and efficacy of ProvayBlueTM for the treatment of acquired methemoglobinemia. The 
minimal efficacy endpoints should include achieving a 50% reduction in methemoglobin (metHb) 
within 1 hour of the first dose of ProvayBlueTM in addition to normalization of the respiratory rate, 
heart rate and blood pressure within 2 hours of the first dose of ProvayBlueTM. 
ProvayBlueTM will be administered as per the APPENDIX 1: 
US 
Package 
Insert 
of 
ProvayBlue™. 
In this open label, prospectively designed clinical study to evaluate the safety and efficacy of 
ProvayBlueTM for the treatment of acquired methemoglobinemia, ProvayBlueTM will be 
administered to patients who present in hospital/urgent care setting with acquired 
methemoglobinemia. ProvayBlueTM will be administered as per the treating physician’s diagnosis 
and hospital standard of care. ProvayBlue™ has been approved by the US FDA to be administered 
intravenously at a dose of 1 mg/kg over 5-30 minutes and if the metHb level remains above 30% 
or if clinical symptoms persist, to give a repeat dose of up to 1 mg/kg 1 hour after the first dose 
(see APPENDIX 1: 
US Package Insert of ProvayBlue™ for dosing guidance).  
The primary objective in this study is to confirm the efficacy of ProvayBlueTM after a single 
administration in patients with acquired methemoglobinemia. 
6.2. 
ACQUIRED METHEMOGLOBINEMIA 
Methemoglobinemia is generally caused by an abnormal increase in blood concentration of an 
altered form of hemoglobin, metHb, where one or several iron molecules are found oxidized in the 
ferric state (Fe3+), instead of the normal ferrous form (Fe2+). This modification makes the hem 
group unable to bind to a molecule of oxygen. In addition, the change in conformation of the 
metHb structure reinforces the oxygen affinity with the remaining ferrous hems, increasing the 
impairment in the oxygen delivery to tissues (Hersh 2004; Lunenfeld and Kane 2004; Beutler 
2005; Kane et al. 2007; Umbreit 2007). 
Methemoglobinemia is characterized by a reduced ability of the blood to carry oxygen, due to 
lower than normal levels of hemoglobin (Beutler 2005; Hersh 2004). Methemoglobinemia can be 
inherited, but is more commonly acquired following exposure to toxic agents (drugs or 
environmental toxins). 
 
 
1 “ProvayBlue™” is the name of the medicinal product registered in the USA. The approved name in European Union 
for this product is “Methylthioninium chloride Proveblue”. 


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6.3. 
CLINICAL SYMPTOMS ASSOCIATE WITH METHEMOGLOBINEMIA 
Clinical symptoms of methemoglobinemia vary according to the level of metHb in the blood. 
MetHb is found at a normal range of 1%, as a fraction of the total hemoglobin species (Hersh 2004; 
Lunenfeld and Kane 2004).  
Symptoms typically are proportional to the level of metHb as shown in Table 6-1 below. 
Table 6-1: 
Signs and Symptoms Related to Blood Levels of Methemoglobin (metHb) 
Level of Methemoglobin 
Symptoms/Outcome 
<3% 
No symptoms 
3-15% 
Grayish-blue skin discoloration only (most notably on mucus membranes) 
15-30% 
Cyanosis, chocolate-colored blood 
30-50% 
Dyspnea, headache, fatigue, weakness,  dizziness, syncope 
50-70% 
Coma, seizures, depressed central nervous system, arrhythmias, metabolic 
acidosis 
>70% 
Death 
Source: Do Nascimento 2008 
 
6.4. 
TREATMENT OF ACQUIRED METHEMOGLOBINEMIA 
Currently, a consensus of opinion exists among the medical community regarding use of 
methylene blue (MB) in cases of methemoglobinemia (Martindale 2007; Therapeutic Drugs 1999). 
MB allows the rapid conversion of metHb into normal hemoglobin and rapid improvement of 
methemoglobinemia symptoms. The broad use of MB in this indication is documented by case 
reports available in the literature and is the current standard of care. 
6.5. 
PROVAYBLUETM 
ProvayBlueTM is an oxidation-reduction agent indicated for the treatment of pediatric and adult 
patients with acquired methemoglobinemia (refer to APPENDIX 1: 
US Package Insert of 
ProvayBlue™).  
Methylene blue is a water soluble thiazine dye that promotes a non-enzymatic redox conversion 
of metHb to hemoglobin. In situ, methylene blue is first converted to leucomethylene blue (LMB) 
via nicotinamide adenine dinucleotide phosphate (NADPH) reductase. It is the LMB molecule 
which then reduces the ferric iron of metHb to the ferrous state of normal hemoglobin. 
The efficacy of ProvayBlueTM was assessed on the basis of a metHb decrease of at least 50% within 
1 hour after intravenous (IV) administration of 1–2 mg/kg ProvayBlueTM (or a bioequivalent 
formulation) in 6 patients identified by retrospective chart review or literature search. All 
6 patients had a decrease in metHb by at least 50% within 1 hour after treatment. 


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An additional 41 cases of treatment of methemoglobinemia with a methylene blue class product 
were identified in the published literature. Of these 41 patients, 37 (90%) had a metHb decrease of 
at least 50% within 1 hour after intravenous administration of the methylene blue class product. 
In a combined analysis of all 47 patients treated intravenously with ProvayBlueTM (or a 
bioequivalent formulation) or with another methylene blue class product, there was no difference 
in response rate by dose. The metHb decreased by at least 50% within 1 hour of infusion for 15/17 
(88%) of patients treated with 1 mg/kg, 12/13 (92%) treated with 2 mg/kg and 16/17 (94%) treated 
with a different dose or for those whose dose was not reported. 
Refer to APPENDIX 1: 
US Package Insert of ProvayBlue™ for adverse reactions, clinical 
pharmacology and non-clinical toxicology information associate with ProvayBlueTM. 
6.6. 
RISK-BENEFIT ASSESSMENT 
ProvayBlueTM will be administered to the study patients to treat acquired methemoglobinemia 
resulting in a direct health benefit.  
Refer to APPENDIX 1: 
US Package Insert of ProvayBlue™ for the risks associated with the 
use of ProvayBlueTM. 
Entering a patient who requires methylene blue treatment, especially minors, into the protocol is 
associated with minimal incremental risk to the patient. The treating physician/Investigator and 
other treating health care professionals should continuously monitor the patient to ensure that the 
degree of burden and risk to the patient is minimized. 
Extreme caution should be exercised when administering to newborns and infants below the age 
of 3 months due to lower concentrations of NADPH-methemoglobin reductase necessary for 
reducing methemoglobin to hemoglobin, making these infants more susceptible to 
methemoglobinemia produced by high doses of methylthioninium chloride.  
The approximate volume of blood planned for collection from each patient over the course of the 
study presents no undue risk to the patients, nor does the collection of additional blood in the event 
an indwelling cannula is utilized (for wasting to ensure clean sample) or for recheck of safety labs, 
if deemed necessary by the Investigator. 
As per regulations and guidelines, a limited volume of blood will be drawn from each patient 
during this study. Blood drawn from pediatric patients will not exceed the recommended maximum 
blood sampling volume for children in clinical trials which should not exceed 1% of total blood 
volume in one draw and 3% of total blood volume within a 4-week period (European Medicines 
Agency [EMA] guideline). Refer to APPENDIX 3: estimates of 1% of the patient’s blood volume 
for the maximum volume of blood that can be drawn per patient. The number of attempts of venous 
punctures in children and adolescents will be restricted to a maximum of 3 attempts.  
The safety data collected by this protocol (i.e., physical examination, vital signs, laboratory 
analysis [including metHb], and adverse event [AE]) are adequate to protect the patient’s safety 
and should detect all expected treatment-emergent AEs (TEAEs). 
 


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7. 
STUDY OBJECTIVES AND ENDPOINTS 
7.1. 
STUDY OBJECTIVES 
7.1.1. 
Primary Objective 
• To confirm that ProvayBlueTM after a single administration is efficacious in patients with 
acquired methemoglobinemia. 
7.1.2. 
Secondary Objectives 
• To evaluate the efficacy of a single dose of 1 mg/kg of ProvayBlue™; 
• To evaluate the efficacy of a second dose of 1 mg/kg of ProvayBlue™ to further reduce 
metHb levels when metHb is not fully reduced by a single dose; 
• To evaluate the normalization of the respiratory rate, heart rate and blood pressure of 
patients who achieve a reduction in metHb level within 2 hours of receiving the first dose 
of ProvayBlue™; 
• To confirm the safety and tolerability of ProvayBlue™ injection in patients with acquired 
methemoglobinemia. 
7.1.3. 
Exploratory Objective 
• To collect sparse pharmacokinetic (PK) data in patients. 
7.2. 
STUDY ENDPOINTS 
7.2.1. 
Primary Efficacy Endpoint 
• A 50% reduction in metHb level within 1 hour of the first dose of ProvayBlueTM for 
treatment of acquired methemoglobinemia. 
7.2.2. 
Secondary Efficacy Endpoints 
• Concomitant normalization of the respiratory rate, heart rate and blood pressure (see 
standard values in Section 13.1) within 2 hours of the first dose of ProvayBlueTM; 
• Evaluation of the patients who achieve a 50% reduction in metHb level after a single dose 
of ProvayBlue™, in addition to normalization of the respiratory rate, heart rate and blood 
pressure; 
• Evaluation of the number of patients who achieve a 50% reduction in metHb level within 
one hour after treatment with ProvayBlue™ in addition to normalization of the respiratory 
rate, heart rate and blood pressure within 2 hours of receiving the first dose of 
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• Evaluation of the patients who achieve a 50% reduction in metHb level after a second dose 
of ProvayBlueTM, in addition to normalization of the respiratory rate, heart rate and blood 
pressure, in cases where a single dose did not completely normalize metHb level; 
7.2.3. 
Safety Endpoints 
The following safety parameters will be assessed: 
• Physical examination; 
• Vital signs; 
• Hematology and biochemistry (see tests in Section 15.2); 
• ECG; 
• AEs. 
7.2.4. 
Pharmacokinetic (PK) Endpoints 
• PK samples will be analyzed for methylene blue and azure B. 


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8. 
INVESTIGATIONAL PLAN 
8.1. 
SUMMARY OF STUDY DESIGN 
This is an open label, uncontrolled Phase 4 study of 10 patients who present in hospital/urgent care 
setting with acquired methemoglobinemia. The population may include pediatric and adult patients 
(males and females of all ages are included).  
The aim of the study is to confirm safety and efficacy of ProvayBlueTM for the treatment of 
acquired methemoglobinemia.  
This study comprises of an analysis of data for patients presenting with acquired 
methemoglobinemia and requiring ProvayBlueTM. The analysis itself will focus on clinical 
outcome of treatment with ProvayBlueTM alone. If possible, depending on the number of patients, 
the data will be summarized by age, sex and body weight.  
The study will include sites which will be based in France, Germany, UK and the US. 
ProvayBlueTM will be administered IV at a dose of 1 mg/kg over 5-30 minutes. If the metHb level 
remains greater than 30% or if clinical signs and symptoms persist, a repeat dose of 1 mg/kg may 
be given 1 hour after the first dose. 
Depending on the clinical status of the patient, additional doses may be administered at the 
discretion of the Investigator. The data on these additional doses will be recorded in the study Case 
Report Form (CRF). 
Due to the nature of the disease and indication, the patient’s treatment with ProvayBlue™ may 
take place per the Investigator’s standard of care prior to or after Patient Informed Consent 
Document (PICD) signature. Data collection (retrospective and prospective) for this study and 
study specific assessments will only take place after PICD signature.  
Safety will be monitored throughout the study by repeated clinical and laboratory evaluations 
according to protocol requirements. 
The expected duration of the study will vary for each patient depending on the clinical situation 
and outcome of treatment. Patients will be followed-up 5 to 10 days (follow-up visit), and 10 to 
15 days (follow-up call) after the last administration of ProvayBlueTM. End of study is defined as 
the Last Patient Last Visit (LPLV). 
The study design is summarized in Figure 8-1. 
 
 
 
 
 


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Figure 8-1: 
Study Design 
 
A schedule for the tests and evaluations to be conducted in this study is found in the flow chart in 
APPENDIX 2: Study Flow Chart. 
The study duration of the trial will vary for each patient depending on the clinical situation and 
outcome of treatment.  
8.2. 
DISCUSSION OF TRIAL DESIGN 
As a post-marketing requirement imposed by the US-FDA, Provepharm is required to conduct a 
clinical study to evaluate the safety and efficacy of ProvayBlueTM for the treatment of acquired 
methemoglobinemia. The minimal efficacy endpoints should include achieving a 50% reduction 
in metHb within 1 hour of the first dose of ProvayBlueTM in addition to normalization of the 
respiratory rate, heart rate and blood pressure within 2 hours of the first dose of ProvayBlueTM 
(refer to Section 6.1). 
This is a prospective, interventional, open label, uncontrolled Phase 4 study to confirm that 
ProvayBlueTM is efficacious and safe in patients with acquired methemoglobinemia. 
The open-label conduct of the trial allows for rapid assessment of the drug-relatedness status of 
any toxicity. 
 
 


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8.3. 
TRIAL TIMETABLE (STUDY PERIODS) 
The trial includes the following visits:  
• Screening and enrolment 
• Study Treatment 
- 
H0: ProvayBlueTM Dose 1 
o H1: 1 hour after the end of ProvayBlue™ infusion. Administration of 
ProvayBlueTM Dose 2 (if applicable)  
- 
H2: 2 hours after the end of ProvayBlue™ Dose 1 infusion 
- 
H3: 2 hours after the end of ProvayBlue™ Dose 2 infusion (if applicable) 
• H24 (24 hours after the end of ProvayBlue™ Dose 1 infusion)  
• Hospital Discharge (if earlier than H24) 
• Follow up visit (5 -10 days following the last ProvayBlue™ administration) 
• Follow up call (10 -15 days following the last ProvayBlue™ administration) 
 


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9. 
SELECTION AND WITHDRAWAL OF PATIENTS 
10 patients (adults and pediatric population of all ages) diagnosed with acquired 
methemoglobinemia and treated with ProvayBlueTM will be included in the study. 
9.1. 
INFORMED CONSENT 
Each potentially eligible patient (and their parent[s] or legally authorized representative[s] as 
applicable) will be informed of the study's objectives and overall requirements.  
ProvayBlueTM will be administered as per the treating physician’s diagnosis and hospital standard 
of care. It may be possible that the patient will be administered their first dose of ProvayBlueTM as 
per standard of care prior to obtaining the consent for this study. However, patient consent (and 
their parent[s] or legally authorized representative[s] as applicable) MUST be obtained prior to 
any data collection (retrospective and prospective) for this study and study specific assessments. 
The Investigator will explain the study fully to the patient (as relevant) and his/her parent(s)/ 
legally authorized representative(s) (as required) using the PICD approved by the institution’s 
Independent Ethics Committee (IEC)/ Institutional Review Board (IRB). If a parent/legally 
authorized representative is willing for the patient to participate in the study, (s)he will be requested 
to give written informed consent. The PICD will be signed and personally dated by both a 
parent/legally authorized representative and the Investigator.  
If capable, the patient should provide assent/consent to participate in the study and also sign and 
personally date the Informed Consent if possible (according to local laws and regulations). 
A copy of the signed form will be provided to the parent(s)/ legally authorized representative(s) 
and the original retained with the source documents.  
Although nursing staff may be involved in describing the trial to a patient and his/her 
parent(s)/legally authorized representative(s), the Investigator must participate in discussions with 
the patient and his/her parent(s)/legally authorized representative(s) and sign and personally date 
the PICD. 


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9.2. 
INCLUSION CRITERIA 
To be eligible for inclusion into this study, each patient must fulfil the following inclusion criteria: 
1. Pediatric or adult patients (males and females of all ages are included) diagnosed with 
acquired methemoglobinemia and receiving treatment with ProvayBlue™ as per the 
treating physician’s diagnosis and hospital standard of care. 
Acquired methemoglobinemia is defined as a level of methemoglobinemia >30% or ≤30% 
with associated clinical symptoms (e.g. sleepiness, cyanosis, dizziness, etc.). 
2. Written informed consent obtained prior to any data collection (retrospective and 
prospective) for this study and study specific assessments. 
9.3. 
EXCLUSION CRITERIA 
To be eligible for inclusion into this study, each patient must violate none of the following 
exclusion criteria: 
1. Known severe hypersensitivity reactions to methylene blue or any other thiazine dye. 
2. Known deficiency in glucose-6-phosphate dehydrogenase (G6PD) due to the risk of 
hemolytic anemia as well as lack of therapeutic effect.  
3.  Known deficiency in NADPH reductase. 
4. Known use of selective serotonin reuptake inhibitors (SSRIs), serotonin and 
norepinephrine reuptake inhibitors (SNRIs), MonoAmine Oxidase (MAO) inhibitors or 
drugs metabolised via CYP isoenzymes anticipated during the treatment phase of the study 
(see Section 11.2). 
5. Women who refuse to stop breastfeeding for up to 8 days after receiving the last dose of 
ProvayBlueTM (see Section 11.2). 
9.4. 
ASSIGNMENT OF PATIENT NUMBER 
All patients who sign the PICD will be allocated a patient number sequentially at each center. The 
number of any patient who is withdrawn from the study will not be reallocated. 
9.5. 
PREMATURE WITHDRAWAL OF PATIENTS FROM STUDY AND 
REPLACEMENT POLICY 
9.5.1. 
Discontinuation Criteria 
Patients (and their parent[s] or legally authorized representative[s] as applicable) will be informed 
that they have the right to withdraw from the study at any time, without prejudice to their medical 
care, and are not obliged to state their reasons. Any withdrawals must be fully documented in the 
CRF and should be followed up by the Investigator. 
Additionally, the Investigator may withdraw a patient from the study or from ProvayBlue™ 
administration at any time if he/she considers this to be in the patient’s best interest.  


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Patients MUST be discontinued from the study for any of the following reasons:  
• Severe or life-threatening hypersensitivity/allergic reaction (refer also to Section 11.4.1); 
• Hemolytic anemia (refer also to Section 11.4.2); 
• Changes in medical status of the patient such that the Investigator believes that patient 
safety will be compromised; 
• Death; 
• Withdrawal by patient or at the request of their legally authorized representative;  
• Physician decision;  
• At the specific request of the Sponsor;  
• Study terminated by Sponsor. 
Patients MAY be discontinued from the study for any of the following reasons: 
• Adverse events including serious intercurrent illness or significant worsening of 
intercurrent illness; 
• Protocol violations, including non-compliance with study assessment and patient refusal; 
• Other study-specific reasons; 
• If a patient fails to return for a scheduled visit/follow-up, 3 attempts should be made to 
contact the patient to ensure that the reason for not returning is not an AE. Likewise if a 
patient declares his/her wish to discontinue from the study, e.g. for personal reasons. 
If the ProvayBlue™ therapy is prematurely discontinued, the primary reason for discontinuation 
must be recorded in the appropriate section of the CRF and all efforts will be made to complete 
and report the observations as thoroughly as possible. Discontinuing of ProvayBlue™ 
administration is not a reason to discontinue from the study. Every effort should be made to collect 
complete information on patients whether ot not they complete the planned treatment.  
A complete final evaluation following withdrawal from the study should be made, as described in 
Section 12.5, and any AEs will be followed 10 to 15 days from the last dose of ProvayBlue™.  
The study will be terminated if, in the opinion of the Investigator and the Sponsor, significant 
safety concerns arise during the conduct of the study. 
9.5.2. 
Replacement Policy 
Should a patient drop out or be withdrawn from the study, his/her patient number will not be 
reallocated. During the study, recruitment may be increased in order to include 10 evaluable 
patients. A patient will be considered evaluable if he/she is diagnosed with acquired 
methemoglobinemia (refer to Section 6.3), received a first dose of ProvayBlue™ of 1 mg/kg and 
had adequate available data to measure the primary efficacy analysis. 


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10. 
STUDY MEDICATION 
Patients will receive treatment with ProvayBlue™ as per the treating physician’s diagnosis and 
hospital standard of care. 
ProvayBlue™ has been approved by the US FDA for patients to be administered IV at a dose of 
1 mg/kg over 5-30 minutes and if the metHb level remains above 30% or if clinical symptoms 
persist, to give a repeat dose of up to 1 mg/kg 1 hour after the first dose.  
Depending on the clinical status of the patient, additional doses may be administered at the 
discretion of the Investigator. The data on these additional doses will be recorded in the study CRF. 
If ProvayBlue™ is administered as an infusion, it MUST be diluted in 5% dextrose in water (D5W) 
only. Dilution in saline will result in precipitation of the product and must not be administered. 
For further information regarding ProvayBlue™, including packaging, labelling, presentation, 
storage and preparation prior to patient administration, please refer to the prescribing information 
for ProvayBlue™ in APPENDIX 1: US Package Insert of ProvayBlue™. 


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11. 
CONCOMITANT THERAPY/MEDICAL MANAGEMENT OF 
ADVERSE EVENTS (AEs) 
Medications or clinical interventions, which are considered necessary for the patient welfare or to 
assist in the management of AEs, may be given at the discretion of the Investigator. 
Refer to APPENDIX 1: 
US Package Insert of ProvayBlue™ for further information on 
potential drug interactions with ProvayBlueTM. 
Safety measures are to be considered for certain concomitant medications. These medications are 
included in Section 11.2. Medications that are prohibited during the course of this study are listed 
in Section 11.3. 
11.1. 
RECORDING OF PREVIOUS CONCOMITANT THERAPY 
All concomitant therapy received during the study, from PICD signature or the start of 
ProvayBlue™ treatment (whichever occurs sooner) until the follow up visit, will be recorded using 
the generic terms. The use of herbal/natural products, as well as diagnostic, therapeutic or surgical 
procedures performed during the study period will be recorded as concomitant therapy. 
All prescription, non-prescription, or over-the-counter medications including herbal remedies 
given to or taken by the patient before inclusion in the study at study entry and within 4 weeks 
prior to study entry, must be clearly documented on the prior medications CRF. 
Investigational ProvayBlue™ should be used for this study/ However, commercial ProvayBlueTM 
that could be administered for treatment of acquired methemoglobinemia prior to study entry as 
per hospital standard of care must be retained at the site until the monitoring visit of the Orion 
Clinical Research Associate (CRA) and all information need to be recorded in the CRF. In 
particular, batch number, expiration date and reason for not using investigational product should 
be captured on the drug accountability log. 
11.2. 
PRECAUTIONS FOR USE, INTERACTIONS WITH OTHER MEDICINAL 
PRODUCTS AND OTHER FORMS OF INTERACTION OF THE IMP 
Agents Metabolized by Cytochrome P450 Enzymes 
Methylene blue inhibits a range of Cytochrome (CYP) isozymes in vitro, including 1A2, 2B6, 
2C8, 2C9, 2C19, 2D6 and 3A4/5. This interaction could be more pronounced with narrow 
therapeutic index drugs that are metabolized by one of these enzymes (e.g., digoxin, warfarin, 
phenytoin, alfentanil, cyclosporine, dihydroergotamine, ergotamine, fentanyl, pimozide, 
quinidine, sirolimus and tacrolimus). However, the clinical relevance of these in vitro interactions 
is unknown. 
Interaction with other medicinal products and other forms of interaction 
Methylthioninium chloride is a potent inhibitor of the transporters OCT2, MATE1 and MATE2-
K. The clinical consequences of the inhibition are not known. The administration of 
methylthioninium chloride Proveblue has the potential to transiently increase the exposure of drugs 


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primarily cleared by renal transport involving the OCT2/MATE pathway, including cimetidine, 
metformin and acyclovir. 
Methylthioninium chloride is a substrate of P-glycoprotein (P-gp). The clinical consequences are 
considered likely to be minimal due to the transient and single dose use that normally occurs in 
the emergency setting. 
Contraindications 
In Europe patients with chlorate poisoning, nitrite-induced methemoglobinemia during treatment 
of cyanide poisoning and patients with a deficiency in NADPH (nicotinamide adenine dinucleotide 
phosphate) reductase are contraindicated for receiving ProvayBlue™ treatment for 
methemoglobinemia. Although this is not the case in the United States and other countries, special 
care should be given to ensure the efficacy benefit of administering ProvayBlue™ in patients 
known to meet these restrictions. 
Pregnancy 
There are no adequate data from the use of ProvayBlue™ in pregnant women. Studies in animals 
have shown reproductive toxicity (see section 5.3). The potential risk for humans is unknown. 
ProvayBlue™ should not be used during pregnancy unless clearly necessary, e.g. in life-
threatening methemoglobinemia. 
Breastfeeding 
It is unknown whether ProvayBlue™ is excreted in human breast milk. The excretion of 
ProvayBlue™ in milk has not been studied in animals. A risk to the suckling child cannot be 
excluded. Based on kinetic data, breastfeeding should be discontinued upon receiving 
ProvayBlue™ and for a period of 8 days after the administration of the last dose of ProvayBlue™. 
Otherwise, breastfeeding patients must be excluded for safety reasons (see also Section 9.3). In 
case a breastfeeding woman needs specific therapy that is urgently indicated, drugs shall be 
administered as current standard therapy. 
Fertility 
In vitro, ProvayBlue™ has been shown to reduce motility of human sperm in a dose dependent 
manner.  
Contraception  
Both patients and their partners with child-bearing potential must employ a reliable method of 
contraception, for example,  
• Oral Contraceptive + Condom with Spermicide; 
• Inter Uterine Device (IUD) + Condom with Spermicide; 
• Diaphragm with Spermicide + Condom with Spermicide; 
• Injectable progesterone + Condom with Spermicide; 
• Subdermal Implant + Condom with Spermicide; 
• Sexual abstinence. 


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11.3. 
MEDICATIONS THAT MUST NOT BE ADMINISTERED 
Methemoglobinemia may not resolve or may rebound after response to treatment with 
ProvayBlue™ in patients with methemoglobinemia due to aryl amines such as aniline or sulfa 
drugs such as dapsone. 
Serotonergic Drugs 
Avoid concomitant use of ProvayBlue™ with medicinal products that enhance serotonergic 
transmission including SSRIs, SNRIs, MAO inhibitors, bupropion, buspirone, clomipramine, 
mirtazapine and venlafaxine; because of the potential for serious CNS reactions, including 
potentially fatal serotonin syndrome. Although the mechanism is not clearly, understood, literature 
reports suggest inhibition of MAO by methylene blue may be involved. In addition, in vitro studies 
cannot rule out the potential involvement of CYP 2D6 inhibition by methylene blue. If the 
intravenous use of ProvayBlue™ cannot be avoided in patients treated with serotonergic medicinal 
products, choose the lowest possible dose and observe closely the patient for CNS effects for up 
to 4 hours after administration [see Warning and Precautions (5.1), Clinical Pharmacology (12.3) 
in APPENDIX 1: 
US Package Insert of ProvayBlue™]. 
11.4. 
GUIDANCE ON MEDICATIONS TO BE ADMINISTERED/PATIENT 
MANAGEMENT FOR EXPECTED ADVERSE EVENTS (AEs) 
11.4.1. 
Hypersensitivity 
Anaphylactic reactions to methylene blue class products have been reported. Patients treated with 
ProvayBlue™ should be monitored for anaphylaxis. If severe or life threatening allergic reaction 
(e.g., angioedema, urticaria, bronchospasm) should occur, discontinue ProvayBlue™ and initiate 
supportive treatment. Refer also to APPENDIX 1: US Package Insert of ProvayBlue™ for 
further information. 
11.4.2. 
Hemolytic Anemia 
Hemolysis can occur during treatment of methemoglobinemia with ProvayBlue™. Laboratory 
testing may show Heinz bodies, elevated indirect bilirubin and low haptoglobin, but the Coombs 
test is negative. The onset of anemia may be delayed 1 or more days after treatment with 
ProvayBlue™. In the event that a patient has hemolytic anemia, discontinue ProvayBlue™ and 
initiate appropriate treatment (e.g. red blood cell transfusion). Refer also to APPENDIX 1:
 
US Package Insert of ProvayBlue™ for further information. 


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12. 
SCHEDULE OF ASSESSMENTS 
12.1. 
SCREENING AND ENROLMENT 
Refer to APPENDIX 2: 
Study Flow Chart for data collection time points and schedule of 
assessments. 
Once the patient (and/or the patient’s parent[s]/legal guardian[s]) has signed the PICD, the formal 
screening assessments to determine the patient’s study eligibility must be completed. These 
assessments and relevant collection of data (as per standard of care) include the following: 
• Date of signed informed consent(s); 
• Demographics – date of birth, sex and race (as allowed by local regulations); 
• Medical history (including all concomitant disease[s] and previous medications); 
• Date, time and initial reason for hospitalization; 
• Methemoglobinemia diagnosis (including name of toxic agents responsible for 
methemoglobinemia if available, clinical symptoms related to methemoglobinemia [e.g. 
sleepiness, cyanosis, dizziness, etc.], methemoglobinemia level at presentation in hospital); 
• Confirmation of study eligibility (inclusion and exclusion criteria); 
• Physical examination including weight (refer to Section 15.2.1); 
• Vital signs (refer to Section 15.2.2 for the list of parameters) If the patient is treated prior 
to consent, all available pre-treatment vital sign information should be retrieved where 
possible; 
• Pregnancy test: All females of childbearing potential will be submitted to a pregnancy test 
(both urine and serum unless one is not possible); 
• Blood sample for:  
o Biochemistry: 
 Sodium 
 Potassium 
 Chloride 
 Urea 
 Creatinine 
 Calcium 
 Inorganic phosphorous 
 Total Protein 
 Albumin 
 Gamma glutamyl transferase 
 Bilirubin (Total) 
 Alkaline phosphatase 
 Aspartate transaminase 


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 Alanine aminotransferase 
 Glucose 
Additional tests (for example blood gas) to be done at admission according to hospital standard of 
care (Refer to Section 15.2.3). 
o Hematology: 
 Haemoglobin 
 Red blood cell count 
 White blood cells 
 Platelets 
 Differential white cell count, neutrophils, lymphocytes, monocytes, 
basophils and eosinophils 
Additional tests to be done at admission according to hospital standard of care (Refer 
 
to Section 15.2.4); 
o metHb (Refer to Section 15.2.5);  
• 12-lead ECG (refer to Section 15.2.6); 
• Review of concomitant therapy since PICD signature or the start of ProvayBlue™ 
treatment (whichever occurs sooner) (refer to Section 11); 
• Collect of AEs that may have occurred since the start of ProvayBlue™ treatment (refer to 
Section 15.3). 
Results of all the screening evaluations and procedures must be reviewed by the Investigator to 
ensure that all eligibility criteria have been satisfied prior to patient enrolment. 
12.2. 
TREATMENT 
Due to the nature of the disease indication, the patient’s treatment with ProvayBlue™ as per 
standard of care can take place prior to or after PICD signature. Data collection (retrospective and 
prospective) for this study and study specific assessments will only take place after PICD 
signature. 
Refer to APPENDIX 2: 
Study Flow Chart for data collection time points and schedule of 
assessments. 
Assessments during treatment and the relevant collection of data (as per standard of care) include 
the following: 
• ProvayBlue™ administration (including dose, lot number, date, time and route of 
administration, and if any dilution took place); 
• Clinical symptoms related to methemoglobinemia and severity: 
o Within 1 hour after the end of each ProvayBlue™ infusion (i.e. both Dose 1, Dose 
2 and any additional dose if applicable); 


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o Within 2 hours after the end of each ProvayBlue™ infusion (i.e. both Dose 1, Dose 
2 and any additional dose if applicable); 
o Approximately 24 hours post the end of the last ProvayBlue™ infusion, or just prior 
to discharge from the clinic if earlier than 24 hours. 
• Vital signs (refer to Section 15.2.2 for the list of parameters): 
o Within 1 hour after the end of each ProvayBlue™ infusion (i.e. both Dose 1, Dose 
2 and any additional dose if applicable); 
o Within 2 hours after the end of each ProvayBlue™ infusion (i.e. both Dose 1, Dose 
2 and any additional dose if applicable); 
o Approximately 24 hours post the end of the last ProvayBlue™ infusion, or just prior 
to discharge from the clinic if earlier than 24 hours. 
• Blood sample for:  
o Biochemistry, hematology and metHb (refer to Section 15.2): 
 Within 1 hour after the end of each ProvayBlue™ Dose 1 infusion (i.e. Dose 
1,  Dose 2 and any additional dose if applicable); 
 Prior to ProvayBlue™ Dose 2 if applicable (i.e., if the metHb level is 
acceptable within 1 hour after the end of Dose 1 ProvayBlue™ infusion, the 
repeat dose of ProvayBlue™ will not be needed); 
 Approximately 24 hours post the end of the last ProvayBlue™ infusion, or 
just prior to discharge from the clinic if earlier than 24 hours. 
o PK analysis (refer to Section 14): 
 Approximately 1 hour after the end of each ProvayBlue™ infusion (i.e. 
Dose 1, Dose 2 and any additional dose if applicable); 
 Prior to Dose 2 (or any additional dose) ProvayBlue™ infusion (if 
applicable);  
 Approximately 24 hours post the end of the last ProvayBlue™ infusion, or 
just prior to discharge from the clinic if earlier than 24 hours. 
Additional tests to be done according to hospital standard of care. 
• 12-lead ECG (refer to Section 15.2.6): 
o Approximately 24 hours post the end of the last ProvayBlue™ infusion, or just prior 
to discharge from the clinic if earlier than 24 hours. 
• Review of concomitant therapy until follow-up (refer to Section 11); 
• Review of AEs (refer to Section 15.3). 
Should any of the blood samples collected for the purposes of the study be compromised or 
destroyed in error, a repeat sample should be taken during the visit. Care should be taken to ensure 
that the maximum amount of blood drawn does not exceed what is stated in the PICD. 


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12.3. 
DISCHARGE 
Assessments prior to discharge from the clinic and the relevant collection of data (as per standard 
of care) include the following: 
• Clinical symptoms related to methemoglobinemia; 
• Physical examination; 
• Vital signs (refer to Section 15.2.2 for the list of parameters); 
• Blood sample for:  
o Biochemistry 
o Hematology  
o metHb 
o PK analysis (refer to Section 14) 
Additional tests to be done according to hospital standard of care 
• 12-lead ECG (refer to Section 15.2.6); 
• Review of concomitant therapy (refer to Section 11); 
• Review of AEs (refer to Section 15.3). 
12.4. 
FOLLOW-UP 
Patients will be followed up with: 
 
• A visit with blood sample for hematology and biochemistry, and assessment of clinical 
symptoms, physical examination and vital signs 5 to 10 days following the last 
ProvayBlue™ administration. 
• A telephone call to take place 10 to 15 days following the last ProvayBlue™ 
administration. 
Refer to APPENDIX 2: 
Study Flow Chart for data collection time points and schedule of 
assessments. 
Additional follow-up assessments include the following: 
• Review of concomitant therapy (refer to Section 11); 
• Review of AEs (refer to Section 15.3). 
12.5. 
WITHDRAWAL/PREMATURE STUDY DISCONTINUATION 
Should the patient discontinue prematurely and choose to withdraw from the study, the following 
procedures should be performed: 
• Clinical symptoms related to methemoglobinemia; 


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• Vital signs (refer to Section 15.2.2 for the list of parameters); 
• Blood sample for:  
o Biochemistry  
o Hematology 
o MetHb  
Additional tests to be done according to hospital standard of care. 
• Review of concomitant therapy (refer to Section 11); 
• Review of AEs (refer to Section 15.3). 
The Investigator should document the reason for study discontinuation in the CRF. 
In the case of an ongoing AE, appropriate safety evaluations should be repeated more frequently 
and/or additional tests performed at any time when clinically indicated or at the discretion of the 
Investigator. All ongoing AEs and SAEs will be followed up until resolution, until the condition 
stabilizes, until the event is otherwise explained or until the patient is lost to follow up. If the 
patient is lost to follow up, then this should be noted in the CRF. 




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14. 
ASSESSMENTS OF PHARMACOKINETIC (PK) 
Whenever feasible and ONLY if the total blood drawn for any patient does not exceed the 
recommended maximum blood sampling volume (refer also to Section 6.6 for regulation and 
guidance), PK samples are to be taken at the discretion of the Investigator based on the condition 
of the patient. The time blood sample(s) are taken should be recorded, but there is no absolute time 
window for the PK samples. Suggested PK sampling time points are as follows: 
• Approximately 1 hour after the end of each ProvayBlue™ infusion (i.e. Dose 1,  Dose 2 
and any additional dose if applicable); 
• Prior to Dose 2 (or any additional dose) ProvayBlue™ infusion (if applicable);  
• Approximately 24 hours post the end of the last ProvayBlue™ infusion, or just prior to 
discharge from the clinic if earlier than 24 hours. 
PK blood samples (venous) must be drawn from the arm opposite to the ProvayBlue™ dosing arm. 
Whole blood samples will be analyzed for methylene blue and azure B using a validated 
bioanalytical method. Arterial blood taken should be submitted for analysis with a note that it is 
an arterial sample. 
Further instruction on PK sampling will be provided in a separate laboratory manual. 
The volume of whole blood taken per venipuncture sample is no more than 2 ml. The number of 
samples drawn from any patient up to 2 years of age should be limited (refer also to Section 6.6 
for regulation and guidance). Care should be taken to ensure that the maximum amount of blood 
drawn does not exceed what is stated in Section 6.6 (refer also to APPENDIX 3: estimates 
of 
1% of the patient’s blood volume) and in the PICD. 
 
 


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15. 
ASSESSMENTS OF SAFETY 
15.1. 
SPECIFICATION OF SAFETY PARAMETERS 
The following safety parameters will be assessed: 
• Physical examination; 
• Vital signs; 
• Hematology and biochemistry; 
• ECG; 
• AEs. 
15.2. 
METHODS AND TIMING FOR ASSESSING, RECORDING AND 
ANALYSING SAFETY PARAMETERS 
Refer to the Study Flow Chart (APPENDIX 2: 
Study Flow Chart) for data collection time 
points for the safety parameters. 
15.2.1. 
Physical Examination 
Physical examinations during the study will be based on patient standard of care as appropriate to 
determine general condition. 
Weight will be recorded at screening. 
New or worsening clinically significant abnormalities following PICD signature will be reported 
as an AE. 
15.2.2. 
Vital Signs 
Vital signs will include: 
• SBP and DBP (mmHg) (controlled posture if possible); 
• Heart rate (beats per minute [bmp]); 
• Body temperature (°C or °F); 
• Respiration rate (breaths per minute); 
• Oxygen saturation by co-oximetry. 
New or worsening clinically significant abnormalities following PICD signature will be reported 
as an AE. 


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15.2.3. 
Biochemistry  
Biochemistry samples will be analyzed by the hospital laboratory. 
The following biochemistry parameters will be collected: 
• Sodium 
• Potassium 
• Chloride 
• Urea 
• Creatinine 
• Calcium 
• Inorganic phosphorous 
• Total Protein 
• Albumin 
• Gamma glutamyl transferase 
• Bilirubin (Total) 
• Alkaline phosphatase 
• Aspartate transaminase 
• Alanine aminotransferase 
• Glucose 
Additional (optional) biochemistry parameters (for example blood gas) will be collected based on 
available data from patient standard of care. 
15.2.4. 
Hematology 
Hematology samples will be analyzed by the hospital laboratory. 
The following hematology parameters will be collected: 
• Haemoglobin 
• Red blood cell count 
• White blood cells 
• Platelets 
• Differential white cell count, neutrophils, lymphocytes, monocytes, basophils and 
eosinophils 


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Additional (optional) hematology parameters will be collected based on available data from patient 
standard of care. 
15.2.5. 
Methemoglobin 
Methemoglobin samples will be analyzed by the hospital laboratory.  
15.2.6. 
12-lead Electrocardiogram (ECG) 
Collection of ECG data is mandatory at admission and at the time points defined in Section 8.3. 
The collection of additional data will be based on available 12-lead ECG recordings performed as 
per patient standard of care as appropriate to determine general condition. 
Any new or worsening clinically significant abnormalities following PICD signature will be 
reported as an AE. 
15.2.7. 
Adverse Events (AEs) 
AEs (disease related and/or treatment related) reported by the patients (and/or their parent[s]/legal 
guardian[s]) will be recorded in the CRF (refer to Section 15.3 for more information). 
15.3. 
ADVERSE EVENT (AE) REPORTING 
It is of the utmost importance that all staff involved in the study are familiar with the content of 
this section. It is the Investigator’s responsibility to ensure compliance with reporting of AEs from 
his/her site.  
Adverse events will be collected from the start of ProvayBlue™ treatment up to follow up 10 to 
15 days after the last administration of ProvayBlue™. 
Any AE that occurs after the 10 to 15 days following the last administration of ProvayBlue™ 
which the Investigator assesses as related to a study procedure and/or ProvayBlue™, should also 
be reported as an AE or a SAE. 
All AEs should be followed until they are resolved or a clinically-stable endpoint is reached if they 
are considered chronic or until the patient is lost to follow-up.  
15.3.1. 
Definition of an Adverse Event (AE) 
An AE is defined as: Any untoward medical occurrence in a patient or clinical investigation subject 
administered a pharmaceutical product and which does not necessarily have to have a causal 
relationship with this treatment.  
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding, for example), symptom, or disease temporally associated with the use of a medicinal 
product, whether or not considered related to the medicinal product. 


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An AE can also refer to an untoward response to the administration of ProvayBlue™, but can also 
occur as a result of the protocol-required procedures or be unrelated to both and include worsening 
of pre-existing conditions, for example. 
AEs include the following: 
• Suspected adverse medication reactions; 
• Reactions from medication overdose, abuse, sensitivity, or toxicity; 
• Apparently unrelated illnesses, including the worsening of a pre-existing illness; 
• Injury or accidents; 
Note: If a documented medical condition is known to have caused the injury or accident, only the 
accident should be reported as an AE; 
• New or aggravated clinically relevant abnormal medical finding at a physical examination 
as compared with previous assessments; 
• Laboratory abnormalities or other abnormal assessments (e.g. physical examination, vital 
signs, ECG) that require clinical intervention or further investigation (beyond ordering a 
repeat [confirmatory] test) unless they are associated with an already reported clinical 
event. 
A suspected adverse drug reaction (ADR) means any AE for which there is a reasonable possibility 
that the medicinal product caused the AE. Reasonable possibility means there is evidence to 
suggest a causal relationship between the medicinal product and the AE. 
15.3.2. 
Definition of a Serious Adverse Events (SAE) 
An SAE is defined as any untoward medical occurrence that: 
• Results in death; 
Note: Death is an outcome of an AE, and not an AE in itself. Event which led to death should be recorded 
with fatal outcome. 
All deaths occurring on the study or up to 10 to 15 days following the last administration of ProvayBlue™ 
or patient withdrawal must be reported. 
• Is life-threatening; 
A life-threatening event places the patient 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). 
• Requires inpatient hospitalization or prolongation of existing hospitalization; 
Note: In general, hospitalization signifies that the patient has been detained (usually involving at least an 
overnight stay) at the hospital or emergency ward for observation and/or treatment that would not have been 
appropriate in the physician’s office or outpatient setting. Complications that occur during hospitalization are 
AEs. If a complication prolongs hospitalization or fulfils any other serious criteria, the event is serious. When 
in doubt as to whether other events meet the serious criteria, the event is to be considered serious. When in 
doubt as to whether “hospitalization” occurred or was necessary, the AE should be considered serious. 
Hospitalization for elective treatment of a pre-existing condition that did not worsen from baseline is not 
considered an AE. 


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• Results in persistent or significant disability/incapacity; 
Note: The term “significant disability” means a substantial disruption of a person’s ability to conduct normal 
life functions. This definition is not intended to include experiences of relatively minor medical significance 
such as uncomplicated headache, nausea, vomiting, hospital, influenza, and accidental trauma (e.g., sprained 
ankle) which may interfere or prevent everyday life functions, but do not constitute a substantial disruption. 
• Is a congenital anomaly/birth defect; 
• Is an important medical event(s) that may not be immediately life threatening or result in 
death or hospitalization but that may jeopardize the patient or require intervention to 
prevent one of the above outcomes. Examples of such events are intensive treatment in an 
emergency room or at home for allergic bronchospasm; blood dyscrasias or convulsions 
that do not result in hospitalization; or development of drug dependency or drug abuse. 
Medical and scientific judgement should be exercised in deciding whether expedited 
reporting is appropriate in such instances. 
Refer to Section 15.3.3.9 for reporting SAEs to IEC(s), IRB(s) and regulatory authorities (RAs). 
15.3.3. 
Recording and Reporting of Adverse Events (AEs) 
All AEs, as defined above, encountered during the clinical study as well as any SAEs (see 
Section 15.3.2) will be reported in the appropriate section of the CRF. Information will include the 
following: 
• Duration of the AE (onset date and time and resolution date); 
• Relationship to ProvayBlue™ (refer to Section 15.3.3.5); 
• Time relationship to ProvayBlue™ dosing time; 
• Assessment of whether AE is an infusion site reaction; 
• Severity (refer to Section 15.3.3.6); 
• Outcome of the AE; 
• Concomitant treatment dispensed (or other action taken); 
• Action taken with respect to the ProvayBlue™. 
If an AE increases in severity it will be recorded as a new record with the same AE identifier. 
AE data should be obtained through observation of the patient, from any information volunteered 
by the patient (and the patient’s parent[s]/legal guardian[s]) and through patient (and the patient’s 
parent[s]/legal guardian[s]) questioning. The patient may be asked “Do you have any health 
problems?” or “Have you had any health problems since your last site visit?” 
Refer to Section 15.3.3.9 for guidance relating to reporting of suspected, unexpected serious 
adverse reactions (SUSARs) to regulatory agencies. 


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15.3.3.1. 
Adverse Event (AE) Reporting Period 
ALL AEs or the worsening of any ongoing events which occur from PICD signature or the start 
of ProvayBlue™ treatment (whichever occurs sooner) up to 10 to 15 days after the last 
administration of ProvayBlue™ will be recorded in the CRF. All ongoing AEs and SAEs will be 
followed up until resolution, until the condition stabilizes, until the event is otherwise explained 
or until the patient is lost to follow up. 
All AEs experienced by a patient, irrespective of the suspected causality, will be monitored until 
the event has resolved, any abnormal laboratory values have returned to baseline or stabilized at a 
level acceptable to the Investigator and Medical Monitor, until there is a satisfactory explanation 
for the changes observed, or until the patient is lost to follow-up.  
Any untoward event that occurs after the AE reporting period but which the Investigator assesses 
as possibly related to ProvayBlue™ should also be reported as an AE or SAE. 
15.3.3.2. 
Reporting of Signs and Symptoms versus a Diagnosis 
Recording a diagnosis (when possible) is preferred to recording a list of associated signs and 
symptoms. However, if a diagnosis is known but there are associated signs or symptoms not 
generally attributed to the diagnosis, the diagnosis and each sign or symptom must be recorded 
separately. 
15.3.3.3. 
Death 
All deaths that occur during the AE Reporting period (refer to Section 15.3.3.1) must be reported 
as follows: 
• Will be reported as a SAE within 24 hours. The report should detail the main and 
contributory causes of death. This information should also be accompanied by a death 
certificate or autopsy report (if available). 
15.3.3.4. 
Pregnancy 
Should a pregnancy occur in a female patient or the partner of a male patient, it must be reported 
in accordance with the procedures described in Section 15.3.3.7. Pregnancy in itself is not regarded 
as an AE unless there is a suspicion that ProvayBlue™ may have interfered with the effectiveness 
of a contraceptive medication. 
15.3.3.5. 
Definition of Relationship of Adverse Events (AEs) to Study Medication 
The Investigator must assess the possible relationship between the AE and ProvayBlue™ and 
record that assessment in the CRF. The Investigator is to make his/her own assessment of each 
SAE to be recorded on the CRF and on the SAE form. 
The Investigator should provide a Yes or No assessment as to whether there is a reasonable 
possibility that the event may have been caused by ProvayBlue™. 


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The relationship should be assessed according to the criteria in Table 15-1 below: 
Table 15-1: Relationship of the Adverse Event (AE) to Study Medication 
Unrelated 
• 
The AE must clearly be caused by the participants clinical state, or 
the study procedure/conditions; 
• 
Definitely not related to ProvayBlue™; 
• 
Temporal sequence of an AE onset relative to administration of 
ProvayBlue™ not reasonable; 
• 
Another obvious cause of an AE. 
Unlikely 
• 
Time sequence is unreasonable; 
• 
There is another more likely cause for an AE. 
Possibly 
• 
Corresponds to what is known about ProvayBlue™; 
• 
Time sequence is reasonable; 
• 
Could have been due to another equally, likely cause. 
Probably 
• 
Is a known effect of ProvayBlue™; 
• 
Time sequence from taking ProvayBlue™ is reasonable; 
• 
Ceases on stopping ProvayBlue™; 
• 
Cannot be reasonably explained by the known characteristics of 
the participant’s clinical state. 
Likely 
• 
Is a known effect of ProvayBlue™ (e.g. listed in APPENDIX 1:
 US Package Insert of ProvayBlue™); 
• 
Time sequence from taking ProvayBlue™ is reasonable; 
• 
Event stops upon stopping ProvayBlue™, event returns upon 
restarting ProvayBlue™; 
 
15.3.3.6. 
Definition of Severity of Adverse Events (AEs) 
Severity of any AE will be recorded in the CRF. 
Severity of any AE will be graded according to the National Cancer Institute - Common 
Terminology Criteria for Adverse Event (NCI-CTCAE) (Version 4.03) as reported in Table 15-2. 


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Table 15-2: Definition of Severity of Adverse Events (AEs) 
Grade 1 
Mild; asymptomatic or mild symptoms; 
clinical or diagnostic observations only; intervention not indicated. 
Grade 2 
 
Moderate; minimal, local or non-invasive intervention indicated;  
limiting age-appropriate instrumental ADL*. 
Grade 3  
 
Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; 
limiting self-care ADL**. 
Grade 4 
Life-threatening consequences; urgent intervention indicated. 
Grade 5 
Death related to AE. 
National Cancer Institute - Common Terminology Criteria for Adverse Event (NCI-CTCAE) Version 4.03 
A Semi-colon indicates ‘or’ within the description of the grade. 
ADL: Activities of daily living 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the telephone, managing money, 
etc. 
**Self-care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking medications, and not 
bedridden. 
 
 
Note the distinction between serious and severe AEs: Severe is a measure of intensity whereas an 
event must meet one of the criteria for serious events listed in Section 15.3.3.6 to be considered 
serious; thus, a severe reaction is not necessarily a serious reaction. For example, a headache may 
be severe in intensity, but would not be classified as serious unless it met one of the criteria for 
serious events listed in Section 15.3.3.6. An AE that is assessed as Grade 3 (severe) or Grade 4 
(potentially life-threatening) should not be confused with a SAE.  
15.3.3.7. 
Serious Adverse Event (SAE) Reporting Procedure for Investigators to Regulis 
The Investigator must report (by fax, telephone or email) all initial and follow-up SAE and 
pregnancy reports to Regulis’ Pharmacovigilance Department within 24 hours of awareness of an 
SAE. 
 
Study Contact for Reporting Serious Adverse Events 
 
 
 
  


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If, for any reason, it is not possible to complete all sections of the SAE form within 24 hours, 
transmission of the form must not be delayed and the outstanding information should be sent on a 
follow-up SAE form. 
If the SAE is reported by telephone, all points on the SAE form should be covered in the initial 
telephone report and followed by a completed and signed SAE form to verify the verbal 
information given previously. In addition, the event must be documented in the CRF. 
Blank copies of the SAE forms are included in the Investigator Site File. 
The SAE form must be completed as fully as possible with information relevant to the SAE(s) 
being reported. All fields should be populated or marked accordingly if no information is available.  
Copies of relevant Case Report Form (CRF) pages, such as concomitant medications and medical 
history may be sent as attachments to the SAE forms. 
Death and life threatening SAEs must be reported by the Investigator to the Medical Monitor 
immediately by phone on +44 7590893277 or +44 1753 578080 (out of hours the main reception 
will contact ‘first call’ who will take a message and contact the medical monitor if they are 
unavailable by mobile). The SAE Form must then be e-mailed or faxed to Regulis Safety 
Department as above. 
All incidents of pregnancy (of the patient or patient’s partner) during the study must be reported 
and follow-up of the pregnancy outcome must be made. Specific pregnancy forms should be 
completed. 
15.3.3.8. 
Follow-up SAE Reports 
For all SAEs where important or relevant information is missing, active follow-up should be 
undertaken. Investigators or other site personnel should inform Regulis of any follow-up 
information on a previously reported SAE immediately but no later than 24 hours after they 
become aware of the SAE. The follow-up information must be presented on an SAE form marked 
as follow-up. It is necessary only to provide the new information, with the SAE form signed by an 
Investigator. 
Investigators or other site personnel should send relevant or requested anonymized supporting 
documentation (e.g. ECG, laboratory results, autopsy report) to Regulis. 
The Investigator will ensure that all the necessary information is provided within the timelines 
stipulated by Regulis when the request for information is made. 
Follow-up reports (as many as required) should be completed and faxed following the same 
procedure above. 


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15.3.3.9. 
Reporting Serious Adverse Events (SAEs) to Independent Ethics Committee(s) 
(IEC[s])/ Institutional Review Board(s) (IRB[s])/ Regulatory Authorities (RA[s]) 
The Sponsor is responsible for informing local IEC(s)/IRB(s)/RA(s) of the applicable safety 
reports in compliance with local regulations. Copies of all correspondence relating to reporting of 
any safety reports to the IEC/IRB/RA[s] should be maintained in the Investigator Site File and 
provided to Orion. 
With respect to SUSARs, the reference document for definition of expectedness is the Package 
Insert of ProvayBlue™ (refer to APPENDIX 1: 
US Package Insert of ProvayBlue™). 
The Sponsor, or its designee, Orion, will inform Investigators, central IEC(s)/IRB(s) and RAs of 
applicable safety reports, as required. 


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16. 
EVALUATION OF RESULTS 
16.1. 
SAMPLE SIZE AND STUDY POWER 
Because this is an ultra-orphan indication, the enrollment will be 10 patients, in adult and pediatric 
population. 
Based on evaluation of historical data, the potential enrollment for this study is estimated to be 1/3 
patient per site per year (i.e., one patient per site over the study period of approximately 32 
months). Thus it is expected that 10 patients may be enrolled during the 3-year study. 
A sample size of 10 will provide at least 80% power to detect a 90% response rate and exclude a 
response rate less than 50%. 
As this is an open label study with only one treatment there will be no randomization and no 
blinding of subjects. Study numbers will be allocated to subjects in the order in which they are 
enrolled into the study. 
16.2. 
STATISTICAL METHODS 
Statistical analyses will be performed by Orion under the authority of the Sponsor. Statistical 
analyses will be carried out using SAS®, Version 9.3 or later, SAS Institute, Cary, North Carolina, 
USA. 
The statistical methods for this study, summarized below, will be described in a detailed statistical 
analysis plan (SAP), which will be finalized prior to database lock. Any deviations from the 
planned analysis as described in the SAP will be justified and recorded in the final clinical study 
report. 
16.2.1. 
General Considerations 
Continuous variables will be summarized by descriptive statistics (number of patients, mean, 
standard deviation [SD], minimum, median and maximum). Geometric mean and coefficient of 
variation (CV) will be presented for PK parameters when appropriate. Categorical data will be 
summarized by absolute and relative frequencies. 
A 95% confidence interval (CI) for the responder rate will be presented for each of the primary 
and secondary endpoints. 
All variables will be listed on a patient level. 
Unless indicated otherwise, summary statistics will be reported for observed data only. Missing 
data will not be imputed. If a baseline value is missing, no change from baseline will be calculated. 
Baseline for all assessments will be defined as the last measurement taken prior to dosing with 
study drug. 
 
 


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16.2.2. 
Analysis Sets 
The following analysis sets will be analyzed: 
Full Analysis Set (FAS) 
The Full Analysis Set (FAS) is defined as all patients who are enrolled. The FAS will be used for 
all efficacy analyses. 
Safety Set 
The Safety Set is defined as all patients who receive at least one dose of ProvayBlue™. The Safety 
Set will be used for all safety analyses. 
PK Analysis Set 
The PK Analysis Set is defined as those patients in the FAS who have any PK results. 
16.3. 
EVALUATION OF BASELINE DATA  
All data will be listed and summary tables will be provided by means of summary statistics and 
frequency tables as appropriate. 
16.4. 
EVALUATION OF EFFICACY 
Refer to Sections 7.2.1 and 7.2.2 for the primary and secondary efficacy endpoints, respectively. 
Efficacy evaluations will be performed using the FAS.  
Efficacy will be assessed based on the metHb level and on the normalization of respiratory rate, 
heart rate and blood pressure after administration of ProvayBlue™ (single- or repeat dose). 
The results will be descriptive only. A 95% CI for the responder rate will be presented for each of 
the primary and secondary endpoints. The results will be compared with historical methylene blue 
results to confirm that ProvayBlue™’s results are not unexpected. 
16.5. 
EVALUATION OF SAFETY 
Refer to Section 7.2.3 for the safety endpoints. 
All AEs will be displayed in summary tables, by Medical Dictionary for Regulatory 
Activities (MedDRA) system organ class and preferred term. Other safety data (including 
laboratory data) data will be summarized using appropriate descriptive statistics. 
16.6. 
EVALUATION OF PHARMACOKINETICS (PK) 
Refer to Section 7.2.4 for further details. 
Results from PK analyses will be summarized and presented in the clinical study report. 
16.7. 
INTERIM ANALYSIS 
No interim analysis is planned. 


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17. 
OBLIGATIONS OF THE PRINCIPAL INVESTIGATOR (PI) 
The study will be performed in accordance with  
• The protocol; 
• The Declaration of Helsinki (Version 1996); 
• The International Council on Harmonization (ICH) Harmonized Tripartite Guideline for 
Good Clinical Practice (GCP)1;  
• All local regulations and IEC/IRB guidance. 
17.1. 
INDEPENDENT ETHICS COMMITTEE/ INSTITUTIONAL REVIEW 
BOARD (IEC/IRB) 
It is the responsibility of the Investigator to obtain approval of the trial protocol/amendments from 
the IEC/IRB of each participating Institution. Prior to the initiation of the study, the 
Investigator/Orion will submit the following documents to the appropriate IECs/IRBs for approval: 
• Study protocol and any amendments; 
• PICD and any other written documents to be provided to the patient; 
• Package Insert of ProvayBlue™ (refer to APPENDIX 1: 
US 
Package 
Insert 
of 
ProvayBlue™); 
• Details of any compensation to patients; 
• Current Curriculum Vitae of the PI; 
• Any other requested document(s). 
A copy of the approval will be sent to Orion along with all other correspondence with the IEC/IRB, 
including the submission documents. The Investigator should file all IEC/IRB correspondence in 
the Investigator Site File. 
The study will not start until approval of the protocol and the PICD has been obtained from the 
appropriate IECs/IRBs. The letter of approval should be dated, and should specify the protocol 
number and date of the protocol or amendment that was reviewed and approved. It should also 
specify the date of the PICD that was reviewed and approved. 
For sites in the United States, the Federal Wide Assurance (FWA) number and appropriate 
documentation that study staff was not involved in the voting should be provided by the 
Investigator to Orion prior to study initiation. 
A dated list of the voting members of the IEC/IRB who were present when the protocol was 
reviewed and approved, including their titles/occupations and institutional affiliations should be 
provided by the Investigator to Orion prior to study initiation. The Investigator will make all 
 
 
1 ICH Harmonized Tripartite Guideline for Good Clinical Practice (June 1996), as recommended for adoption to the three regulatory parties to ICH. 
Transmitted to the CPMP in July 1996 and approved on 17th July 1996 (CPMP/ICH/135/95). The proposed date for coming into operation is (for 
studies commencing after) 17th January 1997. Approved by FDA on 9th May 1997 and effective 9th May 1997 (62 FR 25692). 


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attempts to ensure that the IEC/IRB is constituted and operates in accordance with the ICH GCP 
and any local regulations.  
The Investigator will submit any protocol amendments to the IEC/IRB (and other local authorities, 
according to local regulations) prior to implementation. 
The Investigator will submit required progress reports to the IEC/IRB that approved the protocol 
at least annually, as well as report any SAEs, life-threatening problems or deaths, to comply with 
ICH GCP. The Investigator will also inform the IEC/IRB of reports of SAEs (provided to him/her 
by the Sponsor/Orion) that occurred in other clinical studies conducted with ProvayBlue™.  
The Investigator must inform the IEC/IRB of the termination of the study. 
17.2. 
REGULATORY BODY APPROVAL 
The study will not start until receipt by the Sponsor/Orion of approval from the relevant regulatory 
authorities. 
17.3. 
INFORMED CONSENT 
PICDs will be based on a master document provided by Orion who must approve it prior to 
submission to the IEC/IRB. The content of the PICD should reflect the information described in 
Section 4.8.10 of the ICH Guidelines and any local requirements. Any change requested by the 
IEC/IRB must be approved by the Sponsor/Orion prior to the documents being used. A copy of 
the final, IEC/IRB-approved PICD must be submitted to the Sponsor/Orion prior to initiation and 
activation (green light approval) of this study. 
Written informed consent will be obtained from each patient prior to inclusion in the trial, and 
prior to any study-related assessments are performed, as described in Section 12. 
17.4. 
CASE REPORT FORMS AND SOURCE DOCUMENT VERIFICATION 
CRFs of a design mutually agreed upon by the Sponsor and Orion will be supplied by Orion. CRFs 
are the sole property of the Sponsor and should not be made available in any form to third parties, 
except for authorized representatives of appropriate Health/Regulatory Authorities, without 
written permission from the Sponsor. 
A CRF is required and should be completed for each included (consented) patient. The Investigator 
will be responsible for the accuracy of the data entered into the CRFs. All data must be entered in 
English in black ballpoint pen. Corrections of data should be made using one single line, leaving 
the corrected data clearly visible. The accurate data should be entered next to the inaccurate data. 
All changes must be initialled and dated. Correction fluids are not allowed. The Investigator will 
sign the relevant CRF pages as clearly indicated in the CRF. If a clinically significant change is 
made on any of the CRF pages after the Investigator has signed it, the Investigator must re-sign 
the relevant page to document that he agrees with the change. 
The relevant completed CRF pages must be available for review/collection to designated Orion 
representatives at each scheduled monitoring visit. 


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The Investigator will allow designated Orion representatives and regulatory bodies to have direct 
access to the source documents to verify the data reported in the CRFs. 
Source documents (e.g., medical records, raw data collection forms, pharmacy dispensing records, 
recorded data from automated instruments, laboratory data) are the originals of any documents 
used by the Investigator or hospital/institution that allow verification of the existence of the patient 
and substantiate the integrity of the data collected during the trial. Source documents should be 
available to support all the data recorded in the CRF. The Investigator will prepare and maintain 
adequate and accurate source documents. These documents are designed to record all observations 
and other pertinent data for each patient included in this clinical trial. 
17.5. 
CONFIDENTIALITY 
The Investigator must ensure that the patients’ anonymity will be maintained. On the CRFs or 
other documents submitted to the Sponsor/Orion, patients should NOT be identified by their 
names, but by the assigned patient number. 
If patient names are included on copies of documents submitted to Orion, the names will be 
obliterated and the assigned patient number added to the document. 
The Investigator should keep a separate log (Patient Master List) of patient’s codes (assigned 
patient number), names, addresses, telephone numbers and hospital numbers (if applicable). 
Documents not for submission to Orion (e.g. signed PICDs) should be maintained by the 
Investigator in strict confidence. 
17.6. 
STAFF INFORMATION AND RESPONSIBILITIES 
It is the responsibility of the Investigator to ensure that all personnel involved in the study are fully 
informed of all relevant aspects of the study, including detailed knowledge of and training in all 
procedures to be followed to allow collection of accurate, consistent, complete and reliable data.  
The Investigator will provide a list of delegated responsibility to Orion, detailing the various study 
tasks to be performed by each member of his/her study staff. Each staff member should sign in 
agreement to their performing each of the tasks delegated to them on the list. Orion should ensure 
that all staff members have the required knowledge and training for the tasks delegated to them. 
17.7. 
ESSENTIAL DOCUMENT RETENTION 
All clinical information shall be recorded, handled and stored in such a way that it can be accurately 
reported, interpreted and verified, while ensuring confidentiality of the trial patients’ personal data. 
Documents that enable both the conduct of the clinical trial and the quality of the data produced to 
be evaluated; and show whether the trial is, or has been, conducted in accordance with ICH-GCP 
and applicable regulatory requirements are considered essential documents. 
The Investigator will retain copies of all the essential documents (as defined by ICH-GCP) until 
at least 2 years after the last approval of a marketing application in an ICH region, and until there 
are no pending or contemplated marketing applications in an ICH region, or at least 2 years have 
elapsed since the formal discontinuation of clinical development of ProvayBlue™. These 


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documents should be retained for a longer period, however, if required by the applicable regulatory 
requirements (e.g. EEC Directive 91/507 requires retention of patient codes for at least 15 years 
after the completion or discontinuation of a trial and retention of hospital records and other source 
data for the maximum time permitted by the institution where the study takes place). The 
Investigator should take measures to prevent accidental or premature destruction of these 
documents. 
The essential documents include, but are not limited to: the signed protocol, copies of the 
completed CRFs, signed PICDs from all patients (and/or the patient’s parent[s]/legal guardian[s]) 
who consented, hospital records, and other source documents, IRB/IEC approval and all related 
correspondence, including approved documents, and all other documentation included in the 
Investigator Site File. 
The Investigator will inform the Sponsor of the storage location of these essential documents and 
must contact the Sponsor before disposing of any. If the Investigator wishes to assign the files to 
someone else (e.g. if he/she retires) or to remove them to another location, the Sponsor should be 
consulted about this change. 
The Sponsor will inform the Investigator in writing when these documents no longer need to be 
retained. 


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18. 
STUDY MANAGEMENT 
18.1. 
MONITORING 
Prior to study commencement, the Investigator will be informed of the anticipated frequency of 
the monitoring visits. He/she will also receive a notification prior to each monitoring visit during 
the course of the study. It is expected that the Investigator and/or his/her Sub-investigator(s) and 
other appropriate staff will be available on the day of the visit to discuss study conduct. Orion is 
responsible for ensuring the proper conduct of the clinical trial with regards protocol adherence 
and validity of the data recorded in the CRFs. 
A site initiation visit must be conducted by Orion and the site must be activated prior to the 
commencement of any study activities requiring informed consent. 
18.2. 
QUALITY ASSURANCE (QA) AND QUALITY CONTROL (QC) 
An independent audit of the study may be conducted during the study or after completion. The 
audit may be conducted by either Orion or the Sponsor’s Quality Assurance (QA) department or 
an independent auditor or a regulatory authority. 
18.2.1. 
Quality Control (QC) 
Quality control (QC) is defined as the operational techniques and activities undertaken within the 
QA system to verify that the requirements for quality of the trial-related activities have been 
fulfilled. 
QC should be applied to each stage of data handling to ensure that all data are reliable and have 
been processed correctly. 
18.2.2. 
Quality Assurance (QA) 
Quality assurance (QA) is defined as the planned and systematic actions that are established to 
ensure that the trial is performed and the data are generated, documented (recorded) and reported 
in compliance with GCP and the applicable regulatory requirements. 
18.2.3. 
Audit 
The Investigator will permit an audit mandated by the Sponsor after reasonable notice.  The 
purpose of an audit is to confirm that the study is conducted as per protocol, GCP and applicable 
regulatory requirements, that the rights and well-being of patients enrolled have been protected 
and that all data relevant for the evaluation of ProvayBlue™ have been captured, processed and 
reported in compliance with the planned arrangements. The Investigator will permit direct access 
to all study documents, ProvayBlue™ accountability records, medical records and source data. 
The Investigator and his/her study team will also be available for discussion regarding study 
progress and procedures during the audit (both during the audit and at the end of the audit for an 
“exit” discussion). 
 
 


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18.3. 
DATA QUERY PROCESS 
Data management of the CRF will be performed by Orion on behalf of the Sponsor. 
Sites will enter the data from source documents into the CRF and the data will be verified for 
missing data, inconsistencies, and for any necessary medical clarifications. Queries arising from 
these checks will be flagged within the CRF. The site staff will correct, confirm or clarify the data 
as appropriate. All possible attempts should be made by the site staff to resolve the queries within 
the requested timeframes. If the site staff are unsure about the meaning of a query, or what data is 
required, then they should seek clarification from the Orion CRA assigned to their site. 
Once all data queries have been resolved, the study will be declared to be “clean”, and the CRF 
will be locked ready for statistical analysis. After clean-file status has been achieved, Orion will 
provide copies of each patient’s CRF to the Investigator for archiving. Copies of each patient’s 
CRF will also be archived by the Sponsor/Orion. 
The data management, data handling, and analysis will be conducted in accordance with good 
clinical, scientific and data management principles and in compliance with Orion’s Standard 
Operating Procedures. 
18.4. 
PROTOCOL DEVIATIONS/AMENDMENTS 
The trial must be conducted in accordance with: 
• The protocol; 
• Applicable regulatory requirement(s) or conditions linked to the approval(s) of the study; 
• Applicable IEC/IRB requirement or conditions linked to the approval(s) of the study; 
• Any particulars or documents, other than the protocol, accompanying the regulatory or 
IEC/IRB request or that application. 
Any deviation from the protocol that has not been approved by the Sponsor and the relevant 
regulatory authority/IRB/IEC could result in a discontinuation from the study at the site involved. 
Any amendment(s) to the protocol must be approved by both the Sponsor and the relevant 
regulatory authority/IRB/IEC which granted the original approval of the study prior to their 
implementation (unless only logistical or administrative aspects of the trial are involved). All 
substantial amendments to the protocol must be approved by the applicable regulatory bodies prior 
to their implementation. 
However, in the event of any medical emergency, the Investigator is free to institute any medical 
procedure he/she deems appropriate. Such events and procedures must be promptly reported to 
representatives of the Sponsor and Orion. 


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18.5. 
DISCONTINUATION OF THE STUDY 
The Sponsor reserves the right to stop the study at any time on the basis of new information 
regarding safety or efficacy, or if study progress is unsatisfactory, or for other valid administrative 
reasons. After such a decision is made, the Investigator must inform all patients being screened or 
in the study as soon as possible. All delivered study materials must be collected and all CRFs 
completed to the extent possible. 
18.6. 
PUBLICATIONS 
Any manuscript, abstract or other publication or presentation of results or information arising in 
connection with the study (including any ancillary studies involving trial patients) must be 
prepared in conjunction with the Sponsor and must be submitted to the Sponsor for review and 
comment at least 8 weeks prior to submission for publication or presentation. 


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19. 
REFERENCES 
Hersh EV. Appendix IV. Methemoglobinemia and Topical Benzocaine Use. Review and Expert 
Opinion 
by: 
Elliot 
V. 
Wersh, 
DMD, 
MS, 
PhD. 
2004. 
Cited: 
http://www.fda.gov/ohrms/dockets/dailys/04/June04/060304/81n-0033-sup0007-13-Appendix-
04-vol17.pdf. 
Lunenfeld E, Kane GC. Methemoglobinemia: sudden dyspnea and oxyhemoglobin desaturation 
after esophagoduodenoscopy. Respir Care. 2004 Aug;49(8):940-2. 
Beutler E. Methemoglobinemia and Other Causes of Cyanosis: overview. Williams Hematology. 
5th ed. New York: McGraw-Hill. AccessMedicine. 2005. 
Kane GC, Hoehn SM, Behrenbeck TR, et al. Benzocaine-Induced Methemoglobinemia Based on 
the Mayo Clinic Experience From 28 478 Transesophageal Echocardiograms: Incidence, 
Outcomes, 
and 
Predisposing 
Factors. 
Arch 
Intern 
Med. 
2007;167(18):1977-1982. 
doi:10.1001/archinte.167.18.1977. 
Umbreit J. Methemoglobin--it's not just blue: a concise review. Am J Hematol. 2007 
Feb;82(2):134-44. 
Do Nascimento TS, Pereira RO, de Mello HL, Costa J. Methemoglobinemia: from Diagnosis to 
Treatment. Rev Bras Anestesiol. 2008; 58: 6: 651-664. 
Martindale. The complete drug reference. Methylene Blue. In: Sweetman S, editor. London: 
Pharmaceutical Press. 2007; 984-5. 
Therapeutic Drugs. Methylene blue. In: Dollery C, editor. Edinburgh: Churchill Livingston. 1999; 
117-8. 
European Medicines Agency (EMA) guideline on ethical considerations for clinical trials on 
medicinal products with the paediatric population, Final Version 2008. 
Study PVP-2013001: The collection of retrospective clinical data on the use of Proveblue® 
Injection, 0.5%, methylene blue, for treatment of acquired methemoglobinemia. Submitted in 
SN0008 of NDA204630. 
Study PVP-2014001: Provepharm Phase I clinical study report: A single-dose, parallel, 
bioequivalence study of methylene blue intravenous injection in healthy adult volunteers, 
September 2015. 
National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) 
Version 4.03 
NCI, 
NIH, 
DHHS. 
14 June 2010: 
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE 4.03 2010-06-14 QuickReference 8.5x11.pdf 
Chameides, Leon, Ricardo A. Samson, Stephen M. Schexnayder, and Mary Fran Hazinski, eds. 
Pediatric Advanced Life Support Provider Manual: Professional Edition. United States of 
America: American Heart Association, 2011. 
Kliegman, R.M., et al. Nelson Textbook of Pediatrics, 20th Edition. Philadelphia, PA: Elsevier, 
2015. 


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Usher, R., Shephard, M., and Lind, J. (1963). The blood volume of the newborn infant and 
placental transfusion. Acta paediat. (Uppsala), 52, 497. 


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20. 
APPENDICES 


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APPENDIX 1: 
US PACKAGE INSERT OF PROVAYBLUE™ 
 
 
 
 


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c. Due to the nature of the disease indication, the patient’s treatment with ProvayBlue™ as per standard of care can take place prior to or after PICD signature. 
Data collection (retrospective and prospective) for this study and study specific assessments will only take place after PICD signature. 
d. Date, time and initial reason for hospitalization. 
e. Methemoglobinemia diagnosis (including name of toxic agents responsible for methemoglobinemia if available, clinical symptoms related to 
methemoglobinemia [e.g. sleepiness, cyanosis, dizziness, etc.], methemoglobinemia level at presentation in hospital). 
f. Clinical symptoms related to methemoglobinemia and severity (e.g. sleepiness, cyanosis, dizziness, etc.). 
g. Physical examinations during the study will be based on patient standard of care as appropriate to determine general condition. Weight will be recorded at 
screening. 
h. Vital signs will include: Systolic (SBP) and diastolic (DBP) blood pressure (mmHg), heart rate (beats per minute [bmp]), body temperature (ºC or ºF), respiration 
rate (breaths per minute), oxygen saturation by co-oximetry. 
i. If the patient is treated prior to consent, all available pre-treatment vital sign information should be retrieved where possible; 
j. Within 1 and 2 hours after the end of each ProvayBlue™ infusion (i.e. both Dose 1, Dose 2 and any additional dose if applicable). 
k. Additional (optional) parameters (for example blood gas) will be collected based on available data from patient standard of care. 
l. Within 1 hour after the end of ProvayBlue™ infusion.  
m. If applicable (if the metHb level is acceptable within 1 hour after the end of Dose 1 ProvayBlue™ infusion, the repeat dose of ProvayBlue™ will not be needed). 
n. If applicable, in case of administration of additional doses of ProvayBlue at the discretion of the Investigator  
o. Collection of ECG data is mandatory at admission and approximately at 24 hours after the end of the last ProvayBlue™ infusion or just prior to discharge from 
the clinic if earlier than 24 hours. The collection of additional data will be based on available 12-lead ECG recordings performed as per patient standard of care 
as appropriate to determine general condition. 
p. ProvayBlueTM will be administered IV at a dose of 1 mg/kg over 5-30 minutes. If the methemoglobin level remains greater than 30% or if clinical signs and 
symptoms persist, a repeat dose of 1 mg/kg may be given 1 hour after the first dose. Depending on the clinical status of the patient, additional doses may be 
administered at the discretion of the Investigator. The data on these additional doses will be recorded in the study CRF. 
q. PK blood samples are to be drawn at the discretion of the Investigator. The suggested schedule of PK blood sampling is:  
• 
Approximately 1 hour after the end of each ProvayBlue™ infusion (i.e. Dose 1, Dose 2 and any additional dose if applicable); 
• 
Prior to Dose 2 (or any additional dose) ProvayBlue™ infusion (if applicable);  
• 
Approximately 24 hours post the end of the last ProvayBlue™ infusion, or just prior to discharge from the clinic if earlier than 24 hours. 
r. Collection of concomitant medications/medical procedures from PICD signature or the start of ProvayBlue™ treatment (whichever occurs sooner) until 10 to 
15 days after the last administration of ProvayBlueTM; 
s. Collection of AEs that may have occurred since the start of ProvayBlue™ treatment until 10 to 15 days after the last ProvayBlue™ dosing. 
t. All females of childbearing potential will be submitted to a pregnancy test (both urine and serum unless one is not possible) at Screening. 
 
 


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APPENDIX 3: 
ESTIMATES OF 1% OF THE PATIENT’S BLOOD VOLUME  
 
 
 
Source: Usher et al, 1963