an ioxidan s
A icle
Ta ge ing P o-Oxidan I on wi h De e oxamine as a T ea men
o Ischemic S oke: Sa e y and Op imal Dose Selec ion in a
Randomized Clinical T ial
Mònica Millán1,*, Nú ia DeG ego io-Rocasolano 1,2 , Na àlia Pé ez de la Ossa 1, Síl ia Re e é1, Joan Cos a 3,
Pila Gine 4, Yolanda Sil a 5, Tomás Sob ino 6, Manuel Rod íguez-Yáñez 7, Flo en ino Nombela 8,
F ancisco Campos 6, Joaquín Se ena 5, JoséVi ancos 8, Oc a i Ma í-Sis ac 2,9, Jo di Co és10 ,
An oni Dá alos 1and Te esa Gasull 1,2,*
Ci a ion: Millán, M.;
DeG ego io-Rocasolano, N.; Pé ez de
la Ossa, N.; Re e é, S.; Cos a, J.;
Gine , P.; Sil a, Y.; Sob ino, T.;
Rod íguez-Yáñez, M.;
Nombela, F.; e al. Ta ge ing
P o-Oxidan I on wi h De e oxamine
as a T ea men o Ischemic S oke:
Sa e y and Op imal Dose Selec ion in
a Randomized Clinical T ial.
An ioxidan s 2021,10, 1270.
h ps://doi.o g/10.3390/
an iox10081270
Academic Edi o s: Alessand a
Napoli ano and Rosa M.
Lamuela-Ra en os
Recei ed: 14 June 2021
Accep ed: 5 Augus 2021
Published: 10 Augus 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Depa men o Neu osciences, Hospi al Ge mans T ias i Pujol, 08916 Badalona, Ba celona, Spain;
[email p o ec ed] (N.D.-R.); npe [email p o ec ed] (N.P.d.l.O.); sil ia. e e e@u .ca (S.R.);
[email p o ec ed] (A.D.)
2
Cellula and Molecula Neu obiology Resea ch G oup, Depa men o Neu osciences, Ge mans T ias i Pujol
Resea ch Ins i u e (IGTP), 08916 Badalona, Ba celona, Spain; [email p o ec ed] o [email p o ec ed]
3Depa men o Clinical Pha macology, Hospi al Ge mans T ias i Pujol, 08916 Badalona, Ba celona, Spain;
[email p o ec ed]
4Depa men o Pha macy, Hospi al Ge mans T ias i Pujol, 08916 Badalona, Ba celona, Spain;
[email p o ec ed]
5
Depa men o Neu ology, Hospi al D . Josep T ue a, 17007 Gi ona, Spain; [email p o ec ed] (Y.S.);
jse ena.gi [email p o ec ed] (J.S.)
6Clinical Neu osciences Resea ch Labo a o y, Heal h Resea ch Ins i u e o San iago de Compos ela, Hospi al
Clínico Uni e si a io, Uni e sidade de San iago de Compos ela, 15706 San iago de Compos ela, Spain;
omas.sob ino.mo ei as@se gas.es (T.S.); F ancisco.Campos.Pe ez@se gas.es (F.C.)
7Depa men o Neu ology, Hospi al Clínico Uni e si a io, 15706 San iago de Compos ela, Spain;
manuel. od iguez.yanez@se gas.es
8Depa men o Neu ology, Hospi al La P incesa, 28006 Mad id, Spain;
[email p o ec ed] (F.N.); [email p o ec ed]g (J.V.)
9Depa men o Cellula Biology, Physiology and Immunology, Uni e si a Au ònoma de Ba celona,
08193 Bella e a, Spain
10 Depa men o S a is ics and Ope a ions Resea ch, Uni e si a Poli ècnica de Ca alunya (UPC),
08028 Ba celona, Spain; jo [email p o ec ed]
*Co espondence: [email p o ec ed] (M.M.); [email p o ec ed] o
[email p o ec ed] (T.G.)
Abs ac :
A ole o i on as a a ge o p e en s oke-induced neu odegene a ion has been ecen ly
e isi ed due o new e idence showing ha e op osis inhibi o s a e p o ec i e in expe imen al
ischemic s oke and migh be he apeu ic in o he neu odegene a i e b ain pa hologies. Fe op osis
is a new o m o p og ammed cell dea h a ibu ed o an o e whelming lipidic pe oxida ion due o
excessi e ee i on and eac i e oxygen species (ROS). This s udy aims o e alua e he sa e y and
ole abili y and o explo e he he apeu ic e icacy o he i on chela o and an ioxidan de e oxamine
mesyla e (DFO) in ischemic s oke pa ien s. Adminis a ion o placebo o a single DFO bolus ollowed
by a 72 h con inuous in usion o h ee escala ing doses was ini ia ed du ing he PA in usion, and he
impac on blood ans e in i on was de e mined. P ima y endpoin was sa e y and ole abili y, and
seconda y endpoin was good clinical ou come (clinicalT ials.go NCT00777140). DFO was ound
sa e as ad e se e ec s we e no di e en be ween placebo and DFO a ms. DFO (40–60 mg/Kg/day)
educed he i on sa u a ion o blood ans e in. A end o e icacy was obse ed in pa ien s wi h
mode a e-se e e ischemic s oke (NIHSS > 7) ea ed wi h DFO 40–60 mg/Kg/day. A good ou come
was obse ed a day 90 in 31% o placebo s. 50–58% o he 40–60 mg/Kg/day DFO- ea ed pa ien s.
Keywo ds: i on; de e oxamine; an ioxidan ; e op osis; neu op o ec ion; ou come
An ioxidan s 2021,10, 1270. h ps://doi.o g/10.3390/an iox10081270 h ps://www.mdpi.com/jou nal/an ioxidan s
An ioxidan s 2021,10, 1270 2 o 15
1. In oduc ion
A g owing body o no el e idence poin s o a newly desc ibed ype o p og ammed
cell dea h ha ing a pi o al ole in neu odegene a ion. This cell dea h is known as e op o-
sis, which is i on- and lipid pe oxida ion-dependen , and associa ed wi h educed cellula
an ioxidan ac i i y. In suppo o his concep , e op osis has been ecen ly epo ed o
d i e he acu e neu odegene a ion obse ed in ischemic and hemo hagic s oke [
1
–
6
]; in
auma ic b ain inju y [
7
]; o in long- e m neu odegene a ion obse ed in pa hologies such
as Alzheime ’s disease, Pa kinson’s disease, o Hun ing on’s disease [8].
P eclinical in es iga ions indica e ha i on, ei he esiden in b ain cells, eleased
om hemolyzed ed blood cells ha each he b ain pa enchyma du ing hemo hage,
impo ed om blood o b ain as ee-labile i on o by i on-ca ying molecules, o ac ing a
he ce eb al ascula u e, is a po en p o-oxidan ha con ibu es o he neu odegene a ion
and b ain damage obse ed in acu e ischemic s oke (AIS) and in in ace eb al hemo hage
(ICH) [
4
,
6
,
8
–
12
]. I on exace ba es exci o oxici y and induces neu odegene a ion h ough
he p oduc ion o highly eac i e and cy o oxic hyd oxyl adicals which os e oxida i e
DNA damage and lipid pe oxida ion [
12
–
14
]. In he clinical a ena, i on o e load condi ions
a admission, measu ed as high e i in le els in blood, ha e been consis en ly associ-
a ed wi h poo unc ional ou come in pa ien s wi h ei he in ac anial hemo hage [
15
,
16
]
o ischemic s oke [
17
,
18
]. Mo eo e , inc eased sys emic i on s o es a e associa ed wi h
se e e edema and wi h symp oma ic hemo hagic ans o ma ion in ischemic s oke pa-
ien s ea ed wi h h omboly ic epe usion he apy wi h in a enous ecombinan issue
plasminogen ac i a o ( PA) [
18
]. Impo an ly, he biological i on chela o and powe ul
an ioxidan de e oxamine mesyla e (DFO), ha has long been used in clinic as a i s line
ea men o emo e excess i on in i on-o e load diseases such as halassemia [
19
], has
demons a ed i s e ec i eness as a neu op o ec i e agen in expe imen al s oke models o
AIS and ICH [
4
,
20
–
23
] and p e en s he excess o mi ochond ial ee adicals induced in
ansien ischemic s oke models [24].
Gi en he pi o al ole o i on in eac i e oxygen species p oduc ion, e op osis, and
ischemia/ epe usion damage, and since i on chela ion wi h DFO is neu op o ec i e in
expe imen al s oke models and well- ole a ed in ICH pa ien s [
25
], he TANDEM 1 s udy
aimed o e alua e sa e y and ole abili y, and o explo e po en ial e icacy o in a enous
DFO adminis e ed o ischemic s oke pa ien s.
2. Ma e ials and Me hods
S udy design and pa icipan s:
TANDEM-1 (Th ombolysis And De e oxamine in
Middle Ce eb al A e y Occlusion s udy) was a mul icen e , andomized, double-blind,
placebo-con olled, dose- inding phase II clinical ial app o ed by he Spanish D ug
Agency (eud aCT 2007-0006731-31) and local E hics Commi ee, and egis e ed in
clinicalT ials.go as NCT00777140. Consecu i e pa ien s wi h acu e ischemic s oke a -
ec ing he middle ce eb al a e y (MCA) e i o y, wi h baseline Na ional Ins i u e o
Heal h S oke Scale (NIHSS)
≥
4, ea ed wi h IV PA wi hin 3 h o symp oms onse , and
wi h w i en in o med consen we e en olled a ou Spanish cen e s: Hospi al Ge mans
T ias i Pujol, Hospi al de La P incesa, Hospi al D . Josep T ue a, and Hospi al Clínico
de San iago de Compos ela. CONSORT wo k low diag am and inclusion and exclusion
c i e ia a e depic ed in Figu e 1. Hype ension, diabe es o dyslipidemia condi ions we e
diagnosed as explained in he legend in Table 1. Alcohol consump ion, cu en smoking
habi , o i on supplemen a ion we e assessed a inclusion. Vi al signs, labo a o y pa am-
e e s and o he pa ame e s o in e es o he s oke e olu ion such as p e-s oke Rankin
Scale, NIHSS neu ological scale a baseline, p e ious s oke, in lamma o y condi ions, o
ime o ecanaliza ion ea men ( PA o endo ascula ) we e eco ded.
S udy in e en ions and p ocedu es: P e ious epo s on pha macokine ics in heal hy
humans adminis e ed a he maximum ecommended DFO dose (10 mg/Kg as a bolus)
demons a e an ex emely sho hal -li e o DFO [
26
]. To quickly each sus ained meaning-
An ioxidan s 2021,10, 1270 3 o 15
ul concen a ions in blood, we adminis e ed DFO as a bolus ollowed by a con inuous
h ee-day IV in usion o h ee DFO doses up o a maximum o 60 mg/Kg/day.
An ioxidan s 2021, 10, x FOR PEER REVIEW 4 o 16
Figu e 1. Inclusion and exclusion c i e ia and CONSORT low diag am o he h ee-dose ie sub-
s udies (DTS). In each DTS ea ly e mina ion cases due o mo ali y, discon inua ion due o se ious
ad e se e en s (SAE) possibly ela ed o ea men , and pa ien s excluded a e indica ed.
S udy in e en ions and p ocedu es: P e ious epo s on pha macokine ics in heal hy
humans adminis e ed a he maximum ecommended DFO dose (10 mg/Kg as a bolus)
demons a e an ex emely sho hal -li e o DFO [26]. To quickly each sus ained mean-
ing ul concen a ions in blood, we adminis e ed DFO as a bolus ollowed by a con inuous
h ee-day IV in usion o h ee DFO doses up o a maximum o 60 mg/Kg/day.
The basis o analyzing he sa e y o up o 60 mg/Kg/day DFO in he p e e ed in a-
enous adminis a ion ou e in e ms o pha macome abolism and pha macokine ics
we e as ollows. Fi s ly, o ne e each he maximal ecommended dose in in usion o 15
mg/Kg/hou (A ailable online: h ps://www.medicines.o g.uk/emc/p oduc /5/smpc (ac-
cessed on 12 June 2021)). The con inuous in usion o 60 mg/Kg/day esul s in he admin-
is a ion o 2.5 mg/Kg o DFO each hou o in usion. As 10 mg/Kg DFO was adminis e ed
as a bolus immedia ely p eceding he in usion, du ing he i s hou o ea men pa ien s
ecei e 10 mg in he bolus + 2.5 mg in in usion = 12.5 mg/Kg DFO, his being close o, bu
Figu e 1.
Inclusion and exclusion c i e ia and CONSORT low diag am o he h ee-dose ie sub-s udies (DTS). In each DTS
ea ly e mina ion cases due o mo ali y, discon inua ion due o se ious ad e se e en s (SAE) possibly ela ed o ea men ,
and pa ien s excluded a e indica ed.
The basis o analyzing he sa e y o up o 60 mg/Kg/day DFO in he p e e ed
in a enous adminis a ion ou e in e ms o pha macome abolism and pha macokine ics
we e as ollows. Fi s ly, o ne e each he maximal ecommended dose in in usion o
15 mg/Kg/hou (A ailable online: h ps://www.medicines.o g.uk/emc/p oduc /5/smpc
(accessed on 12 June 2021)). The con inuous in usion o 60 mg/Kg/day esul s in he
adminis a ion o 2.5 mg/Kg o DFO each hou o in usion. As 10 mg/Kg DFO was
adminis e ed as a bolus immedia ely p eceding he in usion, du ing he i s hou o
ea men pa ien s ecei e 10 mg in he bolus + 2.5 mg in in usion = 12.5 mg/Kg DFO,
his being close o, bu below, he maximum ecommended dose. Secondly, acco ding o
he ecommenda ions, DFO dosage should be educed as soon as possible and should
no exceed 80 mg/Kg/day. The dose o 60 mg/Kg/day used added o he 10 mg/Kg
DFO bolus esul s in 70 mg/Kg/day du ing he i s 24 h, close o, bu again below, he
maximum dose allowed. In addi ion, he la ge indi idual di e ences in pha macokine ics
An ioxidan s 2021,10, 1270 4 o 15
and pha macome abolism o DFO epo ed in he li e a u e u he ecommend no o use
he highe doses allowed.
Table 1. Desc ip ion o he demog aphic and baseline clinical cha ac e is ics in he placebo and DFO g oup in each DTS.
KERRYPNX DTS 1 DTS 2 DTS 3
Placebo
(n= 5)
DFO 20
(n= 15)
Placebo
(n= 5)
DFO 40
(n= 16)
Placebo
(n= 5)
DFO 60
(n= 16)
Age 64.4 ±8 67.8 ±13 67.6 ±8 64.1 ±10 60.0 ±16 70.0 ±11
Sex, % male 40 60 80 75 100 81
Medical his o y, % pa ien s
Hype ension 80 53 80 50 80 63
Diabe es 60 20 20 19 40 19
Cu en smoking habi 20 20 20 13 20 6
Dislipemia 40 40 40 31 40 44
Alcohol consump ion 20 0 60 19 20 31
A ial ib illa ion 20 40 20 13 20 13
P io s oke 0 7 0 13 0 19
Vi al signs and labo a o y
pa ame e s
Sys olic BP, mmHg 166 ±41 148 ±21 143 ±10 140 ±16 147 ±16 150 ±21
Dias olic BP, mmHg 79 ±9 78 ±18 80 ±15 77 ±11 85 ±24 80 ±14
Body empe a u e, ◦C 35.8 ±0.5 36.0 ±0.3 35.5 ±0.3 36.0 ±0.4 35.9 ±0.6 35.9 ±0.5
Hea a e, bpm 62 ±12 82 ±22 81 ±19 71 ±19 94 ±9.4 70 ±10
Se um glucose, mg/dL 142 ±19 107 ±33 155 ±71 160 ±94 212 ±197 147 ±72
Pla ele coun (×1000) 226 ±61 218 ±61 277 ±54 237 ±75 285 ±102 234 ±63
aPTT, s 28 ±5 26 ±3 26 ±6 25 ±4 28 ±5 27 ±3
Hema oc i , % 40.4 ±3.8 41.4 ±3.1 45.8 ±3.4 41.0 ±3.6 45.1 ±1.1 41.8 ±4.6
Hemoglobin, g/dL 13.5 ±1.5 14.0 ±1.1 15.3 ±1.0 13.8 ±1.3 15.3 ±0.4 14.2 ±1.5
C ea inin, mg/dL 0.9 ±0.1 0.9 ±0.1 0.9 ±0.5 0.9 ±0.2 1.1 ±0.3 0.9 ±0.2
NIHSS a baseline 14 11 16 17 21.5 12
[12, 15] [9, 18.5] [13, 21] [8, 21] [17.5, 25] [7.5, 16.5]
S oke sub ype, % pa ien s
A he o h ombo ic 40 7 0 13 40 0
Ca dioembolic 20 60 20 44 60 50
Unde e mined 40 33 80 31 0 50
O he 0 0 0 6 0 0
ASPECTS sco e on baseline CT scan 10 10 10 10 9.5 10
[9, 10] [10, 10] [10, 10] [9, 10] [7.5, 10] [9, 10]
Time om onse o PA, min 110 136 100 140 132 140
[90, 110] [102, 168] [90, 130] [95, 155] [88, 174] [115, 157]
Time om onse o ial
ea men , min
140 163 125 170 163 155
[135, 143] [150, 213] [125, 150] [140, 190] [128, 192] [141, 195]
Rescue endo ascula ea men ,
% pa ien s 0 7 20 19 80 31
Values a e p esen ed as mean
±
SD, % pe cen age, o median [qua iles]. DTS: dose ie sub-s udy; DFO: de e oxamine (in mg/Kg/day
o 3 days); NIHSS: Na ional Ins i u es o Heal h S oke Scale; BP: blood p essu e; PA: issue plasminogen ac i a o ; aPTT: ac i a ed
pa ial h omboplas in ime. Hype ension is diagnosed i , when i is measu ed on wo di e en days, he sys olic blood p essu e on bo h
days is
≥
140 mmHg and/o he dias olic blood p essu e on bo h days is
≥
90 mmHg. Diabe es, a condi ion whe e he body can
0
con ol
he amoun o glucose in blood, is diagnosed when as ing plasma glycemia is
≥
126 mg/dL. Alcohol consump ion is he e conside ed
as an ac ual a e age daily alcohol consump ion o less han 40 g/day. Dyslipidemia is diagnosed as an ele a ion o plasma choles e ol,
iglyce ides, o bo h, o a low HDL choles e ol le el.
The clinical ial was pe o med as h ee sequen ial dose ie sub-s udies (DTS), DTS1
o DTS3, om lowes o highes DFO dose. Pa ien s ecei ed IV PA and we e andomized
using a compu e -gene a ed numbe shee in a 3:1 a io o ecei e ei he IV DFO o IV
placebo ea men s a ing wi hin he one-hou PA in usion wi h no s a i ica ion ech-
niques since p ima y ou come was sa e y. Some pa ien s ecei ed escue endo ascula
An ioxidan s 2021,10, 1270 5 o 15
ea men acco ding o he local p o ocols. Dose ie p o ocol was: o DTS1, 0.9% saline so-
lu ion as placebo (n= 5) o a 10 mg/Kg DFO bolus + con inuous in usion o 20 mg/Kg/day
DFO (n= 15); o DTS2, saline as placebo (n= 5) o a 10 mg/Kg DFO bolus + con inuous
in usion o 40 mg/Kg/day DFO (n= 16), and o DTS 3, saline as placebo (n= 5) o a
10 mg/Kg DFO bolus + con inuous in usion o 60 mg/Kg/day DFO (n= 16). A he end
o he s udy, 15 placebo pa ien s had been included. The maximum dose pe hou ne e
eached he maximal ecommended dose in in usion o 15 mg/Kg/hou (A ailable online:
h ps://www.medicines.o g.uk/emc/p oduc /5/smpc (accessed on 12 June 2021)). T ial
d ug was p epa ed acco ding o Good Manu ac u ing and Clinical P ac ices and masking
p ocess and d ug andomiza ion was held cen alized in he Pha macy Se ice o one o
he pa icipa ing si es. D ug ial p epa a ion was done by local non-blinded nu ses who
signed a con iden iali y ag eemen . Con aine s and enous and u ina y ca he e s we e
opaque o p e en iewing o he pa icula colo o he d ug and u ine in o de o main ain
he blinding plan.
Sa e y s opping ules we e p especi ied as ei he he p esence o 15% o pa ien s
(
n= 3
) wi h symp oma ic in ac anial hemo hage (sICH) o 25% mo ali y (n= 5) o he
p esence o unexpec ed se ious ad e se e en s (SAE) in he DFO a m in one DTS. A e
each DTS an independen da a sa e y moni o ing boa d (DSMB) e iewed all SAE. The
nex DTS only s a ed a e a posi i e DSMB e alua ion, o he wise e mina ion o he s udy
was manda o y.
Pa ien s we e con inuously moni o ed in he s oke uni s o pa icipa ing cen e s.
Neu ological examina ion was assessed using he NIHSS by ce i ied neu ologis s a
admission and du ing hospi aliza ion a 24, 48, and 72 h, and also a 7 and 90 days. S oke
wo sening was conside ed a leas a ou -poin inc ease in he widely used and epo ed
scale NIHSS sco e. Follow-up CT scans we e pe o med a 24–36 h a e PA adminis a ion
o assess in ac anial hemo hage (ICH) and hypodensi y olume. Func ional ou come
was e alua ed using he modi ied Rankin Scale (mRS) a 7 days and 90 days.
Se um was ob ained be o e, and a se e al ime poin s a e DFO adminis a ion
and was s o ed a
−
80
◦
C. Samples we e aken longi udinally om each pa ien be o e
(baseline), igh a e he bolus adminis a ion, and a di e en imes a e he in usion
ea men wi h placebo o DFO, including sampling a 24 and 72 h a e he baseline p e-
sampling a exac ly he same ime o day o a oid ci cadian in e e ences in he a iables
unde s udy. A comple ion o he s udy, we de e mined se um le els o DFO ( ime
cou se along 72 h; n= 5–7 pa ien s in each DTS), % o i on sa u a ion o blood ans e in
(TSAT) (in pa ien s wi h se um a ailable p e-, 24 and 72 h pos ea men ) and e i in
(a admission and a 24 and 72 h) espec i ely, using an HPLC-based modi ica ion o a
p e iously desc ibed p o ocol [
27
], a me hod ha allows a di ec and accu a e measu e o
TSAT in se um samples [
12
], o an immunodiagnosis ELECSYS 2010 Sys em. To de e mine
TSAT, 0.26
µ
L o human se um we e loaded in P ecas 6% TBE u ea gels (U-PAGE) (Li e
Technologies). Human AT (hAT ) and human holo as e in (hHT ) om Sigma-Ald ich
we e used as elec opho e ic s anda ds in U-PAGE, AT , mono e ic and di e ic hHT
molecules show di e en elec opho e ic mobili y in hose gels, allowing o de ec he
amoun o each o m. Gels we e elec oblo ed on o PVDF-LF memb anes (Millipo e)
which we e incuba ed o e nigh a 4
◦
C wi h he speci ic an i- ans e in p ima y an ibody
and he ea e wi h he NIR-conjuga ed seconda y an ibody. Bands we e measu ed using
an Odyssey imaging sys em and i s dedica ed so wa e. The an i- ans e in an ibody used
equally ecognizes AT and i on-con aining T o ms in WB. In con as wi h he usual
indi ec me hods, we calcula e TSAT (%) di ec ly by combining elec opho e ic U-PAGE
( ha sepa a es T in o AT (de oid o i on), wo mono e ic T , and he di e ic T bands)
wi h immunode ec ion and indi idual band quan i ica ion. We calcula ed % TSAT in
se um samples o s oke pa ien s using ou U-PAGE/WB esul s, acco ding o he ollowing
o mula: TSAT (%) = (0.5 *mFe·T + diFe·T )*100/(AT + mFe·T + diFe·T .
Ou comes
: P ima y end poin s we e any SAE ha occu ed wi hin 90 days, he
p esence o sICH in he 24–36 h CT scan acco ding o he ECASS II c i e ia [
28
] (neu ological
An ioxidan s 2021,10, 1270 6 o 15
wo sening o NIHSS
≥
4 in any bleeding), ea ly neu ological wo sening (wo sening o
NIHSS ≥4 wi hin 24 h a e s oke onse ), and mo ali y a 90 days.
Seconda y end poin s we e good clinical ou comes de ined as a dicho omized sco e
(mRS
≤
2) a 7 and 90 days, he pe cen educ ion o NIHSS a 90 days, pha macokine ics
o IV DFO and he e ec o DFO on i on sa u a ion o blood ans e in.
S a is ical analysis
: The s opping sa e y ules we e calcula ed acco ding o he highe
con idence in e al o he accep ed isk o sICH and mo ali y in he SITS-MOST s udy [
29
]
assuming ha all d ug- ela ed SAE will occu in he DFO a m. All pa ien s en olled in he
ial, including hose who had ea men discon inua ion a any ime poin , we e included
in he sa e y analysis. P opo ions be ween wo g oups we e compa ed by using he X
2
es
o Fishe ’s exac es , as app op ia e. Da a a e exp essed as he mean and SD o he median
and qua iles. Fo i al signs, slope ( esul ing om a linea eg ession) and maximum
inc ease we e used, whe eas o mos ou ine labo a o y pa ame e s, he change om
baseline was used. G oups we e compa ed using he S uden ’s - es , he Mann–Whi ney U
es , o independen o epea ed measu es ANOVA as app op ia e. S a is ical analyses we e
pe o med using SPSS ( e sion 24) o G aphPad P ism ( e sion 8.3); s a is ical signi icance
was conside ed a p≤0.05.
3. Resul s
3.1. Subjec Clinical Cha ac e is ics
A o al o 62 subjec s we e en olled, 45 men and 17 women. The pe cen age o males
is high in all he g oups and simila among hem: 73%, 60%, 75%, and 81% males we e
p esen espec i ely in he placebo pool and in he ea men g oups o DTS1, DTS2, and
DTS3. Mean
±
SD age o he sample was 66
±
11 yea s, median (qua iles) o NIHSS sco e
was 14 [9, 20] and ime om s oke onse o IV h ombolysis was 130 [100, 160] minu es
and o in es iga ional ial ini ia ion 157 [140, 190] minu es.
All pa ien s we e ea ed wi h he ull dose o IV PA and ecei ed a leas one dose o
he in es iga ional p oduc acco ding o he es ima ed weigh ; 58 (93%) subjec s comple ed
he 72 h d ug in usion. Fo y-se en pa ien s ecei ed DFO and 15 placebo. All pa ien s
comple ed he ollow up. The ea men was kep blind o all pa ien s du ing he 90 days
o he s udy, excep o one pa ien displaying an alle gic eac ion.
Table 1summa izes he demog aphic and baseline clinical cha ac e is ics in bo h
g oups o ea men in each DTS, which we e simila ac oss ea men subg oups, excep
in he DTS3 subg oups ha showed highe NIHSS (21.5 e sus 12) in he placebo sub-
g oup. By pooling all he placebo g oups, he basal NIHSS di e ences among ea men
g oups disappea .
3.2. Sa e y Da a
In ac anial hemo hage, neu ological wo sening and mo ali y a e e en s ha as-
socia e o he no mal e olu ion o he s oke pa hology. No signi ican di e ences we e
ound be ween placebo and DFO g oups in any DTS in he o al numbe o epo ed
ad e se e en s (AE) o SAE in he clinical ial (Table 2), indica ing no sa e y conce ns
o he ea men wi h DFO. Each DTS e mina ed wi hou c ossing he sa e y s opping
ules. SAE was epo ed in 4 pa ien s in he placebo a m pooled om he 3 DTS (26%
o placebo pool- ea ed pa ien s) and in 5 (33.4%), 4 (25%), and 4 (25%) o DFO pa ien s
in DTS1, DTS2, and DTS3, espec i ely. Fi e e en s we e deemed possibly o de ini ely
d ug- ela ed by he local in es iga o , consis ing in a sICH, an asymp oma ic b adyca dia,
a symp oma ic hypo ension, a pa ien who su e ed an ea ly neu ological wo sening, and
an anaphylaxis conside ed o be an alle gic eac ion o DFO du ing he bolus; in he las
ou e en s he d ug was discon inued. Only 2 o 62 pa ien s, bo h ea ed wi h he lowe
dose (IV 20 mg/Kg/day DFO), p esen ed sICH du ing he s udy. Nine (14.5%) pa ien s
died du ing he 90-day ollow-up pos -s oke (causes o mo ali y a e shown in Table S1).
No di e ences we e ound in he ea ly neu ological wo sening, ea ly and la e mo ali y
An ioxidan s 2021,10, 1270 7 o 15
(Table 2), o hypodensi y olume du ing he 36 h (Figu e S1) pos s oke-onse be ween
placebo pool and DFO g oups.
Table 2. Sa e y and ou come esul s in he placebo and DFO g oup in each DTS.
DTS 1 DTS 2 DTS 3
Placebo
(n= 5)
DFO 20
(n= 15) pPlacebo
(n= 5)
DFO 40
(n= 16) pPlacebo
(n= 5)
DFO 60
(n= 16) p
Pa ien s wi h AE, % 100 73.3 0.197 100 75 0.214 100 87.5 0.406
AE n= 13
2.6 ±1.1
n= 28
1.9 ±1.8 0.349 n= 12
2.4 ±1.1
n= 36
2.1 ±1.8 0.603 n= 18
3.5 ±3.1
n= 44
2.7 ±1.8 0.603
Pa ien s wi h SAE, % 40 33.4 0.787 0 25 0.214 40 25 0.517
SAE n= 2
0.4 ±0.5
n= 6
0.4 ±0.6 0.933 n= 0 n= 6
0.4 ±0.8 0.445 n= 4
0.5 ±1
n= 6
0.4 ±0.8 0.548
ENW, % 20 20 1 0 6 0.567 0 6 0.567
sICH, % 0 13.3 0.389 0 0 - 0 0 -
Mo ali y 7 days, % 20 6.7 0.389 0 6.3 0.567 20 12.5 0.676
Mo ali y 90 days, % 20 13.3 0.718 0 18.8 0.296 20 12.5 0.676
Mos s oke pa ien s show ad e se e en s (AE), and a signi ican numbe o pa ien s show se ious ad e se e en s (SAE) associa ed o he
no mal e olu ion o he s oke pa hology. These include symp oma ic in ac anial hemo hage (sICH), ea ly neu ological wo sening (ENW)
and mo ali y. Values a e p esen ed as pe cen age %, nand mean
±
SD. DTS: dose ie sub-s udy. DFO: de e oxamine (in mg/Kg/day o
h ee days). The AE and SAE eco ded wi hin each DTS allowed o p oceed wi h he nex DTS, since each DTS e mina ed wi hou c ossing
he sa e y s opping ules.
3.3. Hemodynamic Vi al Signs and Rou ine Clinical Labo a o y Resul s
Con inuous moni o ing o i al signs and also ime-cou se measu es o se e al lab-
o a o y pa ame e s we e collec ed du ing he 72 h d ug in usion. DFO adminis a ion
a he doses o 20 mg/Kg/day and 40 mg/Kg/day did no modi y sys olic and dias olic
blood p essu e (Figu e S2), al hough he e was a pa ien in he 40 mg/Kg/day DFO a m
who su e ed a symp oma ic hypo ension equi ing medical ea men wi h comple e
eco e y 10 min la e wi hou sequelae. The 60 mg/Kg/day DFO in usion was associ-
a ed wi h mild sys olic blood p essu e-lowe ing e ec s ha we e no clinically signi ican
(Figu e S2). A end o inc eased hea a e which is no clinically ele an was obse ed
in he 60 mg/Kg/day DFO, wi h a maximum inc ease o 13 bpm (95% CI, 4.1 o 22.6).
Adminis a ion o DFO a any dose did no change body empe a u e o se um glucose
le els (Figu e S2). No clinically signi ican sa e y conce ns o di e ences ac oss ea men
g oups we e obse ed o clinical biochemis y o hema ological pa ame e s.
3.4. Fe i in, DFO Pha macokine ics, and E ec s on TSAT
La ge indi idual di e ences in blood DFO le els we e obse ed among pa ien s wi hin
a gi en dose ie g oup. DFO le els in se um we e ound highe in mos subjec s wi hin
he i s 30 min ollowing he ini ial 10 mg/Kg DFO bolus adminis a ion (a e age alues
we e 21
µ
M
±
31). No di e ences we e obse ed in blood DFO le els when compa ing
pa ien s in he DFO a ms DTS1 (n= 7), DTS2 (n= 5) and DTS3 (n= 5) (Figu e 2A).
Baseline se um TSAT in ischemic s oke pa ien s was 31.2
±
11.0, his being consis en
wi h he TSAT le els and a iabili y epo ed o heal hy non-hemoch oma osis indi id-
uals [
30
]. TSAT le els emained s eady along he 3 days ollowing ischemia onse in he
placebo g oup as s a ed using a di ec me hod using longi udinal measu es pe o med in
each indi idual p e- and pos - ea men (Figu e 2C). Repea ed measu es analysis show
ha DFO 20 mg/Kg/day did no change TSAT (Figu e 2D). DFO 40 and 60 mg/Kg/day
educed by 30% and 40%, espec i ely, he TSAT when measu ed 72 h pos - ea men onse
(Figu e 2E,F). A he 60 mg/Kg/day dose, DFO showed a end o educe TSAT (p= 0.101)
a e only 24 h o ea men . Fe i in le els in se um showed a 32% inc ease 72 h pos -s oke
onse as compa ed wi h baseline (p= 0.0046), in ag eemen wi h a p e ious s udy [
18
], and
DFO ea men did no al e e i in le els.
An ioxidan s 2021,10, 1270 8 o 15
An ioxidan s 2021, 10, x FOR PEER REVIEW 9 o 16
Figu e 2. De e oxamine (DFO) educes TSAT ime- and dose-dependen ly. (A) Time-cou se o se-
um DFO le els along he in usion in AIS pa ien s ea ed wi h a 10 mg/Kg bolus o DFO IV ollowed
by a 72 h con inuous IV in usion o DFO in escala ing dose ie s o 20, 40, o 60 mg/Kg/day (blood
DFO le els a ime = 0 o in usion in his g aph a e he esul o he ini ial p e ious 10 mg/Kg bolus
o DFO IV). Mean ± SD a e shown; no signi ican e ec s we e ound ( epea ed measu es ANOVA).
(B) U-PAGE/WB depic ing he bands o he i on-de oid o m o human ans e in s anda d (S d)
(apo ans e in, AT ) and human di e ic ans e in (diFe-T ) s anda d (holo ans e in, HT ). The
i on load o ans e in de e mines he elec opho e ic mobili y o he di e en T o ms in hese
u ea gels. A ows indica e he di e en elec opho e ic pa e n o AT , he wo mono e ic o ms o
ans e in (mFe-T ), and he di e ic ans e in (diFe-T ) o m in se um samples o s oke pa ien s.
Placebo and DFO depic bands o ans e in o wo ep esen a i e pa ien s (one o he placebo
g oup and one o he DFO 60 g oup) be o e he onse o ea men (0), and 24 and 72 h a e admin-
is a ion. In each lane, op ical densi y o he bands allow calcula ion o he % TSAT o a gi en pa-
ien a a gi en ime poin using he o mula: TSAT (%) = (0.5 *mFe·T + diFe·T )*100/(AT + mFe·T
+ diFe·T ). (C–F) % TSAT be o e he onse o ea men (0 h), and 24 and 72 h a e adminis a ion o
placebo (C), 20 mg/Kg/day DFO (D), 40 mg/Kg/day DFO (E), o 60 mg/Kg/day DFO (F). Values a e
p esen ed as mean ± SD. * p ≤ 0.05, ** p ≤ 0.005 ( epea ed measu es one-way ANOVA plus he pos -
hoc Benjamini–K iege –Yeku ieli es ).
0.0
0.5
1.0 2
4
6
24
48
72
0
20
40
60
80
100
ime (h)
DFO (µM)
DFO 20
DFO 40
DFO 60
0 24 h 72 h
0
10
20
30
40
50
60
70
% TSAT
placebo
0 24 h 72 h
0
10
20
30
40
50
60
70
% TSAT
*
DFO 40
0 24 h 72 h
0
10
20
30
40
50
60
70
% TSAT
DFO 20
0 24 h 72 h
0
10
20
30
40
50
60
70
% TSAT
**
p = 0.101
DFO 60
A
CD
EF
B
Figu e 2.
De e oxamine (DFO) educes TSAT ime- and dose-dependen ly. (
A
) Time-cou se o se um
DFO le els along he in usion in AIS pa ien s ea ed wi h a 10 mg/Kg bolus o DFO IV ollowed
by a 72 h con inuous IV in usion o DFO in escala ing dose ie s o 20, 40, o 60 mg/Kg/day (blood
DFO le els a ime = 0 o in usion in his g aph a e he esul o he ini ial p e ious 10 mg/Kg bolus
o DFO IV). Mean
±
SD a e shown; no signi ican e ec s we e ound ( epea ed measu es ANOVA).
(
B
) U-PAGE/WB depic ing he bands o he i on-de oid o m o human ans e in s anda d (S d)
(apo ans e in, AT ) and human di e ic ans e in (diFe-T ) s anda d (holo ans e in, HT ). The
i on load o ans e in de e mines he elec opho e ic mobili y o he di e en T o ms in hese
u ea gels. A ows indica e he di e en elec opho e ic pa e n o AT , he wo mono e ic o ms o
ans e in (mFe-T ), and he di e ic ans e in (diFe-T ) o m in se um samples o s oke pa ien s.
Placebo and DFO depic bands o ans e in o wo ep esen a i e pa ien s (one o he placebo g oup
and one o he DFO 60 g oup) be o e he onse o ea men (0), and 24 and 72 h a e adminis a ion.
In each lane, op ical densi y o he bands allow calcula ion o he % TSAT o a gi en pa ien a
a gi en ime poin using he o mula: TSAT (%) = (0.5 *mFe
·
T + diFe
·
T )*100/(AT + mFe
·
T +
diFe
·
T ). (
C
–
F
) % TSAT be o e he onse o ea men (0 h), and 24 and 72 h a e adminis a ion o
placebo (
C
), 20 mg/Kg/day DFO (
D
), 40 mg/Kg/day DFO (
E
), o 60 mg/Kg/day DFO (
F
). Values
a e p esen ed as mean
±
SD. * p
≤
0.05, ** p
≤
0.005 ( epea ed measu es one-way ANOVA plus he
pos -hoc Benjamini–K iege –Yeku ieli es ).
3.5. Clinical Ou come
No di e ences we e obse ed in baseline pa ame e s be ween i e o he expe imen al
g oups o he s udy. The lack o pa ien s a i ica ion in he andomiza ion p ocess esul ed
in a di e ence in he baseline NIHSS o placebo/DFO a ms wi hin he DTS3 (Table 1),
his dis o ing he co ec assessmen o he e ec o ea men wi hin he DTS3 sub-s udy
bu no a ec ing he s udy when placebo pa ien s we e conside ed as a pool. A pos -hoc
An ioxidan s 2021,10, 1270 9 o 15
explo a o y analysis was pe o med o e alua e he end o ou come imp o emen o
DFO in hose pa ien s wi h mode a e-se e e ischemic s oke (NIHSS > 7) (n= 47) o he
whole coho . Placebo pa ien s in each o he h ee sub-s udies we e compiled wi hin a
single placebo g oup, which we e m “placebo pool”; NIHSS a admission was no di e en
be ween placebo and DFO g oups in his pa ien popula ion (Figu e 3A). In e es ingly, in
his pa ien subpopula ion, a end o educ ion o neu ological impai men as exp essed
in pe cen age o he ini ial sco e: (NIHSS baseline-NIHSS 90 days) * 100/NIHSS baseline)
was obse ed in he pa ien g oups adminis e ed wi h he highe DFO doses (DFO 40 and
DFO 60, Figu e 3B). In addi ion, a highe p opo ion o pa ien s ha ing a good ou come
(mRS
≤
2) was obse ed in he highe DFO dose g oups when assessed ea ly (a day 7)
o a 90 days a e he s oke e en (Figu e 3C). A day 7, 40% o pa ien s ea ed wi h
DFO ≥40 mg/Kg/day
showed a good ou come s. 23% o pa ien s in he placebo a m.
Simila ly, a 90 days 50–60% o pa ien s ea ed wi h DFO
≥
40 mg/Kg/day showed a
good ou come s. only 30% in he placebo a m.
An ioxidan s 2021, 10, x FOR PEER REVIEW 10 o 16
Figu e 3. Explo a o y analysis o examine whe he he TSAT-modi ie de e oxamine (DFO) doses
a o s a good ou come. (A) Median and qua iles o baseline NIHSS in a subpopula ion o he TAN-
DEM-1 s udy wi h NIHSS a admission > 7. Baseline NIHSS we e ound balanced be ween he ou
ea men g oups when conside ing his pa ien subpopula ion (NIHSS > 7) (p = 0.24, one-way
ANOVA). (B) In his subpopula ion (NIHSS > 7) we calcula ed he pe cen age o neu ological im-
p o emen wi h he o mula: % educ ion o NIHSS a 90 days = ((NIHSS a admission-NIHSS a a
gi en ime)*100/NIHSS a admission). As DFO 40 and DFO 60, bu no DFO 20, educe TSAT, hese
wo g oups we e pooled and compa ed o he placebo g oup. We obse ed ha hal o he pa ien s
in he 40 + 60 DFO g oup showed a 100% educ ion o hei ini ial neu ological impai men , in con-
as o hose in he placebo g oup (p = 0.0546, Mann–Whi ney U es ). (C) G aph depic ing he p o-
po ion o AIS pa ien s showing good unc ional ou come (in black) in he placebo and DFO g oups.
DFO dose ie s o 40 and 60 mg/Kg/day ha e highe p opo ion o AIS classi ied as good ou come
pa ien s (mRS ≤ 2) a 7 and 90 days. Values a e p esen ed as median and qua iles and compa ed
wi h one-way ANOVA (A) o Mann–Whi ney U es (B).
4. Discussion
This is he i s epo es ing DFO, ha a ge s p o-oxidan i on, in pa ien s wi h
ischemic s oke. DFO as a 10 mg/Kg IV bolus du ing he PA in usion ollowed by a h ee-
day IV con inuous in usion o up o 60 mg/Kg/day is sa e and well- ole a ed, educes
TSAT, and shows a p omising end o be e ou come.
We did no ind h ombolysis complica ions due o DFO o any o he DFO-induced
change in he numbe and ype o ad e se e en s (see Table 2). This sa e y DFO da a in
ischemic s oke pa ien s se he g oundwo k o a la ge clinical ial gi en he ecen ly
epo ed ole o i on-induced e op osis as a main con ibu o o b ain damage in expe -
imen al s oke models [1,4–6,31–33] and o p e ious epo s in which DFO ea men is
associa ed wi h lowe in a c olume, less mi ochond ial ee adicals [24], less hemo -
hagic ans o ma ion, and imp o ed neu ological s a us in expe imen al ischemic s oke
models [4,20,21,24,34–37]. O no e, ei he ea men wi h DFO o p e en ion o cellula
i on up ake ha e been epo ed o p o ec neu onal pheno ype cells om exci o oxic cell
dea h h ough a educ ion o oxida i e s ess [12,38,39].
In i on in oxica ion/o e load, DFO adminis a ion o se e al days as a subcu aneous
o in a enous slow in usion is indica ed [40]. Al hough DFO adminis e ed sys emically
Figu e 3.
Explo a o y analysis o examine whe he he TSAT-modi ie de e oxamine (DFO) doses
a o s a good ou come. (
A
) Median and qua iles o baseline NIHSS in a subpopula ion o he
TANDEM-1 s udy wi h NIHSS a admission > 7. Baseline NIHSS we e ound balanced be ween
he ou ea men g oups when conside ing his pa ien subpopula ion (NIHSS > 7) (p= 0.24, one-
way ANOVA). (
B
) In his subpopula ion (NIHSS > 7) we calcula ed he pe cen age o neu ological
imp o emen wi h he o mula: % educ ion o NIHSS a 90 days = ((NIHSS a admission-NIHSS
a a gi en ime)*100/NIHSS a admission). As DFO 40 and DFO 60, bu no DFO 20, educe TSAT,
hese wo g oups we e pooled and compa ed o he placebo g oup. We obse ed ha hal o he
pa ien s in he 40 + 60 DFO g oup showed a 100% educ ion o hei ini ial neu ological impai men ,
in con as o hose in he placebo g oup (p= 0.0546, Mann–Whi ney U es ). (
C
) G aph depic ing
he p opo ion o AIS pa ien s showing good unc ional ou come (in black) in he placebo and DFO
g oups. DFO dose ie s o 40 and 60 mg/Kg/day ha e highe p opo ion o AIS classi ied as good
ou come pa ien s (mRS
≤
2) a 7 and 90 days. Values a e p esen ed as median and qua iles and
compa ed wi h one-way ANOVA (A) o Mann–Whi ney U es (B).
