antioxidants Article Targeting Pro-Oxidant Iron with Deferoxamine as a Treatment for Ischemic Stroke: Safety and Optimal Dose Selection in a Randomized Clinical Trial Mònica Millán 1, *, Núria DeGregorio-Rocasolano 1,2 , Natàlia Pérez de la Ossa 1 , Sílvia Reverté 1 , Joan Costa 3 , Pilar Giner 4 , Yolanda Silva 5 , Tomás Sobrino 6 , Manuel Rodríguez-Yáñez 7 , Florentino Nombela 8 , Francisco Campos 6 , Joaquín Serena 5 , José Vivancos 8 , Octavi Martí-Sistac 2,9 , Jordi Cortés 10 , Antoni Dávalos 1 and Teresa Gasull 1,2, * 1 Department of Neurosciences, Hospital Germans Trias i Pujol, 08916 Badalona, Barcelona, Spain; ndgregorio@igtp.cat (N.D.-R.); nperez.germanstrias@gencat.cat (N.P.d.l.O.); silvia.reverte@urv.cat (S.R.); adavalos.germanstrias@gencat.cat (A.D.) 2 Cellular and Molecular Neurobiology Research Group, Department of Neurosciences, Germans Trias i Pujol Research Institute (IGTP), 08916 Badalona, Barcelona, Spain; omarti@igtp.cat or octavi.marti@uab.cat  3 Department of Clinical Pharmacology, Hospital Germans Trias i Pujol, 08916 Badalona, Barcelona, Spain;  joan.costa.pages@gmail.com Citation: Millán, M.; 4 Department of Pharmacy, Hospital Germans Trias i Pujol, 08916 Badalona, Barcelona, Spain; DeGregorio-Rocasolano, N.; Pérez de pginerb@gmail.com 5 Department of Neurology, Hospital Dr. Josep Trueta, 17007 Girona, Spain; ysilva.girona.ics@gencat.cat (Y.S.); la Ossa, N.; Reverté, S.; Costa, J.; Giner, P.; Silva, Y.; Sobrino, T.; jserena.girona.ics@gencat.cat (J.S.) 6 Clinical Neurosciences Research Laboratory, Health Research Institute of Santiago de Compostela, Hospital Rodríguez-Yáñez, M.; Clínico Universitario, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain; Nombela, F.; et al. Targeting tomas.sobrino.moreiras@sergas.es (T.S.); Francisco.Campos.Perez@sergas.es (F.C.) Pro-Oxidant Iron with Deferoxamine 7 Department of Neurology, Hospital Clínico Universitario, 15706 Santiago de Compostela, Spain; as a Treatment for Ischemic Stroke: manuel.rodriguez.yanez@sergas.es Safety and Optimal Dose Selection in 8 Department of Neurology, Hospital La Princesa, 28006 Madrid, Spain; a Randomized Clinical Trial. fnombela.hlpr@salud.madrid.org (F.N.); joseaurelio.vivancos@salud.madrid.org (J.V.) 9 Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Antioxidants 2021, 10, 1270. https://doi.org/10.3390/ 08193 Bellaterra, Spain 10 Department of Statistics and Operations Research, Universitat Politècnica de Catalunya (UPC), antiox10081270 08028 Barcelona, Spain; jordicortes40@gmail.com * Correspondence: mmillan.germanstrias@gencat.cat (M.M.); tgasull@igtp.cat or Academic Editors: Alessandra teresagasull@yahoo.com (T.G.) Napolitano and Rosa M. Lamuela-Raventos Abstract: A role of iron as a target to prevent stroke-induced neurodegeneration has been recently Received: 14 June 2021 revisited due to new evidence showing that ferroptosis inhibitors are protective in experimental Accepted: 5 August 2021 ischemic stroke and might be therapeutic in other neurodegenerative brain pathologies. Ferroptosis Published: 10 August 2021 is a new form of programmed cell death attributed to an overwhelming lipidic peroxidation due to excessive free iron and reactive oxygen species (ROS). This study aims to evaluate the safety and Publisher’s Note: MDPI stays neutral tolerability and to explore the therapeutic efficacy of the iron chelator and antioxidant deferoxamine with regard to jurisdictional claims in mesylate (DFO) in ischemic stroke patients. Administration of placebo or a single DFO bolus followed published maps and institutional affil- by a 72 h continuous infusion of three escalating doses was initiated during the tPA infusion, and the iations. impact on blood transferrin iron was determined. Primary endpoint was safety and tolerability, and secondary endpoint was good clinical outcome (clinicalTrials.gov NCT00777140). DFO was found safe as adverse effects were not different between placebo and DFO arms. DFO (40–60 mg/Kg/day) reduced the iron saturation of blood transferrin. A trend to efficacy was observed in patients with Copyright: © 2021 by the authors. moderate-severe ischemic stroke (NIHSS > 7) treated with DFO 40–60 mg/Kg/day. A good outcome Licensee MDPI, Basel, Switzerland. was observed at day 90 in 31% of placebo vs. 50–58% of the 40–60 mg/Kg/day DFO-treated patients. This article is an open access article distributed under the terms and Keywords: iron; deferoxamine; antioxidant; ferroptosis; neuroprotection; outcome conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Antioxidants 2021, 10, 1270. https://doi.org/10.3390/antiox10081270 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 1270 2 of 15 1. Introduction A growing body of novel evidence points to a newly described type of programmed cell death having a pivotal role in neurodegeneration. This cell death is known as ferropto- sis, which is iron- and lipid peroxidation-dependent, and associated with reduced cellular antioxidant activity. In support of this concept, ferroptosis has been recently reported to drive the acute neurodegeneration observed in ischemic and hemorrhagic stroke [1–6]; in traumatic brain injury [7]; or in long-term neurodegeneration observed in pathologies such as Alzheimer’s disease, Parkinson’s disease, or Huntington’s disease [8]. Preclinical investigations indicate that iron, either resident in brain cells, released from hemolyzed red blood cells that reach the brain parenchyma during hemorrhage, imported from blood to brain as free-labile iron or by iron-carrying molecules, or acting at the cerebral vasculature, is a potent pro-oxidant that contributes to the neurodegeneration and brain damage observed in acute ischemic stroke (AIS) and in intracerebral hemorrhage (ICH) [4,6,8–12]. Iron exacerbates excitotoxicity and induces neurodegeneration through the production of highly reactive and cytotoxic hydroxyl radicals which foster oxidative DNA damage and lipid peroxidation [12–14]. In the clinical arena, iron overload conditions at admission, measured as high ferritin levels in blood, have been consistently associ- ated with poor functional outcome in patients with either intracranial hemorrhage [15,16] or ischemic stroke [17,18]. Moreover, increased systemic iron stores are associated with severe edema and with symptomatic hemorrhagic transformation in ischemic stroke pa- tients treated with thrombolytic reperfusion therapy with intravenous recombinant tissue plasminogen activator (tPA) [18]. Importantly, the biological iron chelator and powerful antioxidant deferoxamine mesylate (DFO), that has long been used in clinic as a first line treatment to remove excess iron in iron-overload diseases such as thalassemia [19], has demonstrated its effectiveness as a neuroprotective agent in experimental stroke models of AIS and ICH [4,20–23] and prevents the excess of mitochondrial free radicals induced in transient ischemic stroke models [24]. Given the pivotal role of iron in reactive oxygen species production, ferroptosis, and ischemia/reperfusion damage, and since iron chelation with DFO is neuroprotective in experimental stroke models and well-tolerated in ICH patients [25], the TANDEM 1 study aimed to evaluate safety and tolerability, and to explore potential efficacy of intravenous DFO administered to ischemic stroke patients. 2. Materials and Methods Study design and participants: TANDEM-1 (Thrombolysis And Deferoxamine in Middle Cerebral Artery Occlusion study) was a multicenter, randomized, double-blind, placebo-controlled, dose-finding phase II clinical trial approved by the Spanish Drug Agency (eudraCT 2007-0006731-31) and local Ethics Committee, and registered in clinicalTrials.gov as NCT00777140. Consecutive patients with acute ischemic stroke af- fecting the middle cerebral artery (MCA) territory, with baseline National Institute of Health Stroke Scale (NIHSS) ≥ 4, treated with IV tPA within 3 h of symptoms onset, and with written informed consent were enrolled at four Spanish centers: Hospital Germans Trias i Pujol, Hospital de La Princesa, Hospital Dr. Josep Trueta, and Hospital Clínico de Santiago de Compostela. CONSORT work flow diagram and inclusion and exclusion criteria are depicted in Figure 1. Hypertension, diabetes or dyslipidemia conditions were diagnosed as explained in the legend in Table 1. Alcohol consumption, current smoking habit, or iron supplementation were assessed at inclusion. Vital signs, laboratory param- eters and other parameters of interest for the stroke evolution such as pre-stroke Rankin Scale, NIHSS neurological scale at baseline, previous stroke, inflammatory conditions, or time to recanalization treatment (tPA or endovascular) were recorded. Study interventions and procedures: Previous reports on pharmacokinetics in healthy humans administered at the maximum recommended DFO dose (10 mg/Kg as a bolus) demonstrate an extremely short half-life of DFO [26]. To quickly reach sustained meaning- Antioxidants 2021, 10, 1270 3 of 15 Antioxidants 2021, 10, x FOR PEER REVIEW ful concentrations in blood, we administered DFO as a bolus followed 4 of 16 by a continuous three-day IV infusion of three DFO doses up to a maximum of 60 mg/Kg/day. Figure Figure 1. Inclusion and1.exclusion Inclusioncriteria and exclusion criteria and and CONSORT CONSORT flow diagramflow diagram of the of thetier three-dose three-dose tier sub- sub-studies (DTS). In each DTS studies (DTS). In each DTS early termination cases due to mortality, discontinuation due to serious early termination cases due to mortality, discontinuation due to serious adverse events (SAE) possibly related to treatment, adverse events (SAE) possibly related to treatment, and patients excluded are indicated. and patients excluded are indicated. Study interventions and procedures: Previous reports on pharmacokinetics in healthy humans administeredThe basis at thefor analyzing maximum the safetyDFO recommended of updoseto (10 60 mg/Kg mg/Kg/day DFO in the preferred as a bolus) demonstrateintravenous an extremelyadministration short half-life ofroute DFOin terms [26]. of pharmacometabolism To quickly reach sustained mean- and pharmacokinetics were as follows. ingful concentrations Firstly, in blood, we to neverDFO administered reach as athe maximal bolus followedrecommended by a continuousdose in infusion of three-day IV 15infusion mg/Kg/hourof three (Available DFO doses online: https://www.medicines.org.uk/emc/product/5/smpc up to a maximum of 60 mg/Kg/day. (accessed The basis on 12the for analyzing June 2021)). safety The of up to 60 continuous mg/Kg/day DFO infusion of 60 mg/Kg/day in the preferred intra- results in the venous administration administration routeofin2.5 terms mg/Kgof pharmacometabolism of DFO each hourand pharmacokinetics of infusion. As 10 mg/Kg DFO was were as follows. Firstly, to never administered reach the as a bolus maximal recommended immediately preceding dose in infusionduring the infusion, of 15 the first hour of mg/Kg/hourtreatment (Availablepatients online: https://www.medicines.org.uk/emc/product/5/smpc receive 10 mg in the bolus + 2.5 mg in infusion (ac- = 12.5 mg/Kg DFO, cessed on 12this June 2021)). The continuous infusion of 60 mg/Kg/day results in the admin- being close to, but below, the maximum recommended dose. Secondly, according to istration of 2.5 mg/Kg of DFO each hour of infusion. As 10 mg/Kg DFO was administered the recommendations, DFO dosage should be reduced as soon as possible and should as a bolus immediately preceding the infusion, during the first hour of treatment patients not exceed 80 mg/Kg/day. The dose of 60 mg/Kg/day used added to the 10 mg/Kg receive 10 mg in the bolus + 2.5 mg in infusion = 12.5 mg/Kg DFO, this being close to, but DFO bolus results in 70 mg/Kg/day during the first 24 h, close to, but again below, the maximum dose allowed. In addition, the large individual differences in pharmacokinetics Antioxidants 2021, 10, 1270 4 of 15 and pharmacometabolism of DFO reported in the literature further recommend not to use the higher doses allowed. Table 1. Description of the demographic and baseline clinical characteristics in the placebo and DFO group in each DTS. KERRYPNX DTS 1 DTS 2 DTS 3 Placebo DFO 20 Placebo DFO 40 Placebo DFO 60 (n = 5) (n = 15) (n = 5) (n = 16) (n = 5) (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 history, % patients Hypertension 80 53 80 50 80 63 Diabetes 60 20 20 19 40 19 Current smoking habit 20 20 20 13 20 6 Dislipemia 40 40 40 31 40 44 Alcohol consumption 20 0 60 19 20 31 Atrial fibrillation 20 40 20 13 20 13 Prior stroke 0 7 0 13 0 19 Vital signs and laboratory parameters Systolic BP, mmHg 166 ± 41 148 ±21 143 ± 10 140 ± 16 147 ± 16 150 ± 21 Diastolic BP, mmHg 79 ± 9 78 ± 18 80 ± 15 77 ± 11 85 ± 24 80 ± 14 Body temperature, ◦ 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 Heart rate, bpm 62 ± 12 82 ± 22 81 ± 19 71 ± 19 94 ± 9.4 70 ± 10 Serum glucose, mg/dL 142 ± 19 107 ± 33 155 ± 71 160 ± 94 212 ± 197 147 ± 72 Platelet count (×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 Hematocrit, % 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 Creatinin, 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 14 11 16 17 21.5 12 NIHSS at baseline [12, 15] [9, 18.5] [13, 21] [8, 21] [17.5, 25] [7.5, 16.5] Stroke subtype, % patients Atherothrombotic 40 7 0 13 40 0 Cardioembolic 20 60 20 44 60 50 Undetermined 40 33 80 31 0 50 Other 0 0 0 6 0 0 10 10 10 10 9.5 10 ASPECTS score on baseline CT scan [9, 10] [10, 10] [10, 10] [9, 10] [7.5, 10] [9, 10] 110 136 100 140 132 140 Time from onset to tPA, min [90, 110] [102, 168] [90, 130] [95, 155] [88, 174] [115, 157] Time from onset to trial 140 163 125 170 163 155 treatment, min [135, 143] [150, 213] [125, 150] [140, 190] [128, 192] [141, 195] Rescue endovascular treatment, 0 7 20 19 80 31 % patients Values are presented as mean ± SD, % percentage, or median [quartiles]. DTS: dose tier sub-study; DFO: deferoxamine (in mg/Kg/day for 3 days); NIHSS: National Institutes of Health Stroke Scale; BP: blood pressure; tPA: tissue plasminogen activator; aPTT: activated partial thromboplastin time. Hypertension is diagnosed if, when it is measured on two different days, the systolic blood pressure on both days is ≥140 mmHg and/or the diastolic blood pressure on both days is ≥90 mmHg. Diabetes, a condition where the body can0 t control the amount of glucose in blood, is diagnosed when fasting plasma glycemia is ≥126 mg/dL. Alcohol consumption is here considered as an actual average daily alcohol consumption of less than 40 g/day. Dyslipidemia is diagnosed as an elevation of plasma cholesterol, triglycerides, or both, or a low HDL cholesterol level. The clinical trial was performed as three sequential dose tier sub-studies (DTS), DTS1 to DTS3, from lowest to highest DFO dose. Patients received IV tPA and were randomized using a computer-generated number sheet in a 3:1 ratio to receive either IV DFO or IV placebo treatment starting within the one-hour tPA infusion with no stratification tech- niques since primary outcome was safety. Some patients received rescue endovascular Antioxidants 2021, 10, 1270 5 of 15 treatment according to the local protocols. Dose tier protocol was: for DTS1, 0.9% saline so- lution as placebo (n = 5) or a 10 mg/Kg DFO bolus + continuous infusion of 20 mg/Kg/day DFO (n = 15); for DTS2, saline as placebo (n = 5) or a 10 mg/Kg DFO bolus + continuous infusion of 40 mg/Kg/day DFO (n = 16), and for DTS 3, saline as placebo (n = 5) or a 10 mg/Kg DFO bolus + continuous infusion of 60 mg/Kg/day DFO (n = 16). At the end of the study, 15 placebo patients had been included. The maximum dose per hour never reached the maximal recommended dose in infusion of 15 mg/Kg/hour (Available online: https://www.medicines.org.uk/emc/product/5/smpc (accessed on 12 June 2021)). Trial drug was prepared according to Good Manufacturing and Clinical Practices and masking process and drug randomization was held centralized in the Pharmacy Service of one of the participating sites. Drug trial preparation was done by local non-blinded nurses who signed a confidentiality agreement. Containers and venous and urinary catheters were opaque to prevent viewing of the particular color of the drug and urine in order to maintain the blinding plan. Safety stopping rules were prespecified as either the presence of 15% of patients (n = 3) with symptomatic intracranial hemorrhage (sICH) or 25% mortality (n = 5) or the presence of unexpected serious adverse events (SAE) in the DFO arm in one DTS. After each DTS an independent data safety monitoring board (DSMB) reviewed all SAE. The next DTS only started after a positive DSMB evaluation, otherwise termination of the study was mandatory. Patients were continuously monitored in the stroke units of participating centers. Neurological examination was assessed using the NIHSS by certified neurologists at admission and during hospitalization at 24, 48, and 72 h, and also at 7 and 90 days. Stroke worsening was considered at least a four-point increase in the widely used and reported scale NIHSS score. Follow-up CT scans were performed at 24–36 h after tPA administration to assess intracranial hemorrhage (ICH) and hypodensity volume. Functional outcome was evaluated using the modified Rankin Scale (mRS) at 7 days and 90 days. Serum was obtained before, and at several time points after DFO administration and was stored at −80 ◦ C. Samples were taken longitudinally from each patient before (baseline), right after the bolus administration, and at different times after the infusion treatment with placebo or DFO, including sampling at 24 and 72 h after the baseline pre- sampling at exactly the same time of day to avoid circadian interferences in the variables under study. At completion of the study, we determined serum levels of DFO (time course along 72 h; n = 5–7 patients in each DTS), % of iron saturation of blood transferrin (TSAT) (in patients with serum available pre-, 24 and 72 h post treatment) and ferritin (at admission and at 24 and 72 h) respectively, using an HPLC-based modification of a previously described protocol [27], a method that allows a direct and accurate measure of TSAT in serum samples [12], or an immunodiagnosis ELECSYS 2010 System. To determine TSAT, 0.26 µL of human serum were loaded in Precast 6% TBE urea gels (U-PAGE) (Life Technologies). Human ATf (hATf) and human holotrasferrin (hHTf) from Sigma-Aldrich were used as electrophoretic standards in U-PAGE, ATf, monoferric and diferric hHTf molecules show different electrophoretic mobility in those gels, allowing to detect the amount of each form. Gels were electroblotted onto PVDF-LF membranes (Millipore) which were incubated overnight at 4 ◦ C with the specific anti-transferrin primary antibody and thereafter with the NIR-conjugated secondary antibody. Bands were measured using an Odyssey imaging system and its dedicated software. The anti-transferrin antibody used equally recognizes ATf and iron-containing Tf forms in WB. In contrast with the usual indirect methods, we calculate TSAT (%) directly by combining electrophoretic U-PAGE (that separates Tf into ATf (devoid of iron), two monoferric Tf, and the diferric Tf bands) with immunodetecttion and individual band quantification. We calculated % TSAT in serum samples of stroke patients using our U-PAGE/WB results, according to the following formula: TSAT (%) = (0.5 *mFe·Tf + diFe·Tf)*100/(ATf + mFe·Tf + diFe·Tf. Outcomes: Primary end points were any SAE that occurred within 90 days, the presence of sICH in the 24–36 h CT scan according to the ECASS II criteria [28] (neurological Antioxidants 2021, 10, 1270 6 of 15 worsening of NIHSS ≥ 4 in any bleeding), early neurological worsening (worsening of NIHSS ≥ 4 within 24 h after stroke onset), and mortality at 90 days. Secondary end points were good clinical outcomes defined as a dichotomized score (mRS ≤ 2) at 7 and 90 days, the percent reduction of NIHSS at 90 days, pharmacokinetics of IV DFO and the effect of DFO on iron saturation of blood transferrin. Statistical analysis: The stopping safety rules were calculated according to the higher confidence interval of the accepted risk of sICH and mortality in the SITS-MOST study [29] assuming that all drug-related SAE will occur in the DFO arm. All patients enrolled in the trial, including those who had treatment discontinuation at any time point, were included in the safety analysis. Proportions between two groups were compared by using the X2 test or Fisher’s exact test, as appropriate. Data are expressed as the mean and SD or the median and quartiles. For vital signs, slope (resulting from a linear regression) and maximum increase were used, whereas for most routine laboratory parameters, the change from baseline was used. Groups were compared using the Student’s t-test, the Mann–Whitney U test, or independent or repeated measures ANOVA as appropriate. Statistical analyses were performed using SPSS (version 24) or GraphPad Prism (version 8.3); statistical significance was considered at p ≤ 0.05. 3. Results 3.1. Subject Clinical Characteristics A total of 62 subjects were enrolled, 45 men and 17 women. The percentage of males is high in all the groups and similar among them: 73%, 60%, 75%, and 81% males were present respectively in the placebo pool and in the treatment groups of DTS1, DTS2, and DTS3. Mean ± SD age of the sample was 66 ± 11 years, median (quartiles) of NIHSS score was 14 [9, 20] and time from stroke onset to IV thrombolysis was 130 [100, 160] minutes and to investigational trial initiation 157 [140, 190] minutes. All patients were treated with the full dose of IV tPA and received at least one dose of the investigational product according to the estimated weight; 58 (93%) subjects completed the 72 h drug infusion. Forty-seven patients received DFO and 15 placebo. All patients completed the follow up. The treatment was kept blind for all patients during the 90 days of the study, except for one patient displaying an allergic reaction. Table 1 summarizes the demographic and baseline clinical characteristics in both groups of treatment in each DTS, which were similar across treatment subgroups, except in the DTS3 subgroups that showed higher NIHSS (21.5 versus 12) in the placebo sub- group. By pooling all the placebo groups, the basal NIHSS differences among treatment groups disappear. 3.2. Safety Data Intracranial hemorrhage, neurological worsening and mortality are events that as- sociate to the normal evolution of the stroke pathology. No significant differences were found between placebo and DFO groups in any DTS in the total number of reported adverse events (AE) or SAE in the clinical trial (Table 2), indicating no safety concerns of the treatment with DFO. Each DTS terminated without crossing the safety stopping rules. SAE was reported in 4 patients in the placebo arm pooled from the 3 DTS (26% of placebo pool-treated patients) and in 5 (33.4%), 4 (25%), and 4 (25%) of DFO patients in DTS1, DTS2, and DTS3, respectively. Five events were deemed possibly or definitely drug-related by the local investigator, consisting in a sICH, an asymptomatic bradycardia, a symptomatic hypotension, a patient who suffered an early neurological worsening, and an anaphylaxis considered to be an allergic reaction to DFO during the bolus; in the last four events the drug was discontinued. Only 2 of 62 patients, both treated with the lower dose (IV 20 mg/Kg/day DFO), presented sICH during the study. Nine (14.5%) patients died during the 90-day follow-up post-stroke (causes of mortality are shown in Table S1). No differences were found in the early neurological worsening, early and late mortality Antioxidants 2021, 10, 1270 7 of 15 (Table 2), or hypodensity volume during the 36 h (Figure S1) post stroke-onset between placebo pool and DFO groups. Table 2. Safety and outcome results in the placebo and DFO group in each DTS. DTS 1 DTS 2 DTS 3 Placebo DFO 20 Placebo DFO 40 Placebo DFO 60 p p p (n = 5) (n = 15) (n = 5) (n = 16) (n = 5) (n = 16) Patients with AE, % 100 73.3 0.197 100 75 0.214 100 87.5 0.406 n = 13 n = 28 n = 12 n = 36 n = 18 n = 44 AE 0.349 0.603 0.603 2.6 ± 1.1 1.9 ± 1.8 2.4 ± 1.1 2.1 ± 1.8 3.5 ± 3.1 2.7 ± 1.8 Patients with SAE, % 40 33.4 0.787 0 25 0.214 40 25 0.517 n=2 n=6 n=6 n=4 n=6 SAE 0.933 n=0 0.445 0.548 0.4 ± 0.5 0.4 ± 0.6 0.4 ± 0.8 0.5 ± 1 0.4 ± 0.8 ENW, % 20 20 1 0 6 0.567 0 6 0.567 sICH, % 0 13.3 0.389 0 0 - 0 0 - Mortality 7 days, % 20 6.7 0.389 0 6.3 0.567 20 12.5 0.676 Mortality 90 days, % 20 13.3 0.718 0 18.8 0.296 20 12.5 0.676 Most stroke patients show adverse events (AE), and a significant number of patients show serious adverse events (SAE) associated to the normal evolution of the stroke pathology. These include symptomatic intracranial hemorrhage (sICH), early neurological worsening (ENW) and mortality. Values are presented as percentage %, n and mean ± SD. DTS: dose tier sub-study. DFO: deferoxamine (in mg/Kg/day for three days). The AE and SAE recorded within each DTS allowed to proceed with the next DTS, since each DTS terminated without crossing the safety stopping rules. 3.3. Hemodynamic Vital Signs and Routine Clinical Laboratory Results Continuous monitoring of vital signs and also time-course measures of several lab- oratory parameters were collected during the 72 h drug infusion. DFO administration at the doses of 20 mg/Kg/day and 40 mg/Kg/day did not modify systolic and diastolic blood pressure (Figure S2), although there was a patient in the 40 mg/Kg/day DFO arm who suffered a symptomatic hypotension requiring medical treatment with complete recovery 10 min later without sequelae. The 60 mg/Kg/day DFO infusion was associ- ated with mild systolic blood pressure-lowering effects that were not clinically significant (Figure S2). A trend to increased heart rate which is not clinically relevant was observed in the 60 mg/Kg/day DFO, with a maximum increase of 13 bpm (95% CI, 4.1 to 22.6). Administration of DFO at any dose did not change body temperature or serum glucose levels (Figure S2). No clinically significant safety concerns or differences across treatment groups were observed for clinical biochemistry or hematological parameters. 3.4. Ferritin, DFO Pharmacokinetics, and Effects on TSAT Large individual differences in blood DFO levels were observed among patients within a given dose tier group. DFO levels in serum were found higher in most subjects within the first 30 min following the initial 10 mg/Kg DFO bolus administration (average values were 21 µM ± 31). No differences were observed in blood DFO levels when comparing patients in the DFO arms DTS1 (n = 7), DTS2 (n = 5) and DTS3 (n = 5) (Figure 2A). Baseline serum TSAT in ischemic stroke patients was 31.2 ± 11.0, this being consistent with the TSAT levels and variability reported for healthy non-hemochromatosis individ- uals [30]. TSAT levels remained steady along the 3 days following ischemia onset in the placebo group as stated using a direct method using longitudinal measures performed in each individual pre- and post- treatment (Figure 2C). Repeated measures analysis show that DFO 20 mg/Kg/day did not change TSAT (Figure 2D). DFO 40 and 60 mg/Kg/day reduced by 30% and 40%, respectively, the TSAT when measured 72 h post-treatment onset (Figure 2E,F). At the 60 mg/Kg/day dose, DFO showed a trend to reduce TSAT (p = 0.101) after only 24 h of treatment. Ferritin levels in serum showed a 32% increase 72 h post-stroke onset as compared with baseline (p = 0.0046), in agreement with a previous study [18], and DFO treatment did not alter ferritin levels. Antioxidants 2021, 10, Antioxidants 10, 1270 x FOR PEER REVIEW 9 8of of 16 15 A B 100 DFO 20 DFO 40 80 DFO 60 DFO (µM) 60 40 20 0 0 5 0 2 4 6 24 48 72 0. 0. 1. time (h) C 70 D 70 60 60 50 50 % TSAT % TSAT 40 40 30 30 20 20 10 10 0 0 0 24 h 72 h 0 24 h 72 h placebo DFO 20 E 70 F ** 70 60 p = 0.101 50 * 60 50 % TSAT 40 % TSAT 40 30 30 20 20 10 10 0 0 24 h 72 h 0 0 24 h 72 h DFO 40 DFO 60 Figure 2. Deferoxamine (DFO) reduces TSAT time- and dose-dependently. (A) Time-course of serum Figure 2. Deferoxamine (DFO) reduces TSAT time- and dose-dependently. (A) Time-course of se- DFODFO rum levels along levels the the along infusion in AIS infusion patients in AIS patients treated with treated witha 10 a 10mg/Kg mg/Kgbolusbolusof ofDFO DFO IVIV followed followed by a 72 h continuous IV infusion of DFO in escalating dose tiers of 20, 40, or by a 72 h continuous IV infusion of DFO in escalating dose tiers of 20, 40, or 60 mg/Kg/day (blood 60 mg/Kg/day (blood DFO levels DFO levels at time == 00 of of infusion infusion in this graph are the result of the the initial initial previous previous 1010 mg/Kg mg/Kg bolus of DFO of DFO IV). Mean ±±SD IV). Mean SDareareshown; shown;no nosignificant significanteffects effectswere werefound found(repeated (repeatedmeasures measuresANOVA). ANOVA). (B) (B) U-PAGE/WB U-PAGE/WBdepicting depictingthe thebands bandsof of the the iron-devoid iron-devoid formform ofof human human transferrin transferrin standard standard (Std) (Std) (apotransferrin, (apotransferrin, ATf) and human diferric transferrin (diFe-Tf) standard (holotransferrin, HTf). The ATf) and human diferric transferrin (diFe-Tf) standard (holotransferrin, HTf). The iron iron load load ofof transferrin transferrin determines determines the the electrophoretic electrophoretic mobility mobility of of the the different different Tf Tf forms forms in these in these urea gels. Arrows indicate the different electrophoretic pattern of ATf, the two monoferric forms of urea gels. Arrows indicate the different electrophoretic pattern of ATf, the two monoferric forms of transferrin (mFe-Tf), and the diferric transferrin (diFe-Tf) form in serum samples of stroke patients. transferrin (mFe-Tf), and the diferric transferrin (diFe-Tf) form in serum samples of stroke patients. Placebo and DFO depict bands of transferrin of two representative patients (one of the placebo Placebo group andone and DFO depict of the DFO bands of transferrin 60 group) before theof two representative onset of treatmentpatients (0), and(one of the 24 and 72 hplacebo group after admin- and one of istration. Inthe eachDFO 60 optical lane, group) density before the of onset of treatment the bands (0), and 24ofand allow calculation the72%hTSAT after administration. for a given pa- In each tient at alane, givenoptical density time point of the formula: using bands allow TSAT calculation (%) = (0.5 of the % +TSAT *mFe·Tf for a given patient diFe·Tf)*100/(ATf + mFe·Tf at +a diFe·Tf). given time point (C–F) usingbefore % TSAT the formula: the onsetTSAT (%) = (0.5 of treatment *mFe (0 h), and ·Tf24+and diFe72·Tf)*100/(ATf + mFe·Tfof+ h after administration placebo diFe·Tf).(C), (C–F)20 mg/Kg/day % TSAT before DFOthe (D), 40 mg/Kg/day onset of treatment DFO (E), (0 h), or 24 and 60 and mg/Kg/day 72 h afterDFO (F). Values are administration of presented placebo (C),as 20 mean ± SD. * p ≤DFO mg/Kg/day 0.05,(D), ** p 40 ≤ 0.005 (repeatedDFO mg/Kg/day measures (E), or one-way 60 mg/Kg/dayANOVA DFOplus theValues (F). post- hoc Benjamini–Krieger–Yekutieli are presented as mean ± SD. * p ≤test). 0.05, ** p ≤ 0.005 (repeated measures one-way ANOVA plus the post-hoc Benjamini–Krieger–Yekutieli test). 3.5. Clinical Outcome No differences were observed in baseline parameters between five of the experimental groups of the study. The lack of patient stratification in the randomization process resulted in a difference in the baseline NIHSS of placebo/DFO arms within the DTS3 (Table 1), this distorting the correct assessment of the effect of treatment within the DTS3 sub-study but not affecting the study when placebo patients were considered as a pool. A post-hoc Antioxidants 2021, 10, 1270 9 of 15 exploratory analysis was performed to evaluate the trend to outcome improvement of DFO in those patients with moderate-severe ischemic stroke (NIHSS > 7) (n = 47) of the whole cohort. Placebo patients in each of the three sub-studies were compiled within a single placebo group, which we term “placebo pool”; NIHSS at admission was not different between placebo and DFO groups in this patient population (Figure 3A). Interestingly, in this patient subpopulation, a trend of reduction of neurological impairment as expressed in percentage of the initial score: (NIHSS baseline-NIHSS 90 days) * 100/NIHSS baseline) was observed in the patient groups administered with the higher DFO doses (DFO 40 and DFO 60, Figure 3B). In addition, a higher proportion of patients having a good outcome (mRS ≤ 2) was observed in the higher DFO dose groups when assessed early (at day 7) or at 90 days after the stroke event (Figure 3C). At day 7, 40% of patients treated with DFO ≥ 40 mg/Kg/day showed a good outcome vs. 23% of patients in the placebo arm. Antioxidants 2021, 10, x FOR PEER REVIEW 10 of 16 Similarly, at 90 days 50–60% of patients treated with DFO ≥ 40 mg/Kg/day showed a good outcome vs. only 30% in the placebo arm. Figure 3. Exploratory analysis to examine whether the TSAT-modifier deferoxamine (DFO) doses Figure 3. Exploratory analysis to examine whether the TSAT-modifier deferoxamine (DFO) doses favors favors aa good goodoutcome. outcome.(A) (A) Median Median and and quartiles quartiles of baseline of baseline NIHSS NIHSS in a subpopulation in a subpopulation of theof the TAN- TANDEM-1 study with NIHSS at admission > 7. Baseline NIHSS were found DEM-1 study with NIHSS at admission > 7. Baseline NIHSS were found balanced between the four balanced between the four treatment treatment groups groups when considering when considering this patient this patient subpopulation subpopulation (NIHSS (NIHSS > 7) > 7) (p (p = 0.24, = 0.24, one- one-way ANOVA). way ANOVA). (B) In (B)this subpopulation In this subpopulation(NIHSS > 7) >we (NIHSS 7) calculated the the we calculated percentage of neurological percentage im- of neurological provement with improvement thethe with formula: formula:% reduction of NIHSS % reduction of NIHSS at 90 at days = ((NIHSS 90 days = ((NIHSS at admission-NIHSS at admission-NIHSS at a given at time)*100/NIHSS a given time)*100/NIHSS at admission). As DFO at admission). 40 and As DFO 40DFO and 60, DFO but 60,not butDFO not 20, DFOreduce TSAT,TSAT, 20, reduce these two groups were pooled and compared to the placebo group. We observed that these two groups were pooled and compared to the placebo group. We observed that half of the half of the patients in the 40 + 60 DFO group showed a 100% reduction of their initial neurological impairment, in con- patients in the 40 + 60 DFO group showed a 100% reduction of their initial neurological impairment, trast to those in the placebo group (p = 0.0546, Mann–Whitney U test). (C) Graph depicting the pro- in contrast to those in the placebo group (p = 0.0546, Mann–Whitney U test). (C) Graph depicting portion of AIS patients showing good functional outcome (in black) in the placebo and DFO groups. the DFO proportion dose tiersofofAIS patients 40 and showing good 60 mg/Kg/day havefunctional outcome of higher proportion (inAIS black) in the placebo classified as goodand DFO outcome groups. DFO dose tiers of 40 and 60 mg/Kg/day have higher proportion of AIS patients (mRS ≤ 2) at 7 and 90 days. Values are presented as median and quartiles and compared classified as good outcome patients with one-way ANOVA(mRS (A)≤ 2)oratMann–Whitney 7 and 90 days. UValues are presented as median and quartiles and test (B). compared with one-way ANOVA (A) or Mann–Whitney U test (B). 4. Discussion This is the first report testing DFO, that targets pro-oxidant iron, in patients with ischemic stroke. DFO as a 10 mg/Kg IV bolus during the tPA infusion followed by a three- day IV continuous infusion of up to 60 mg/Kg/day is safe and well-tolerated, reduces TSAT, and shows a promising trend to better outcome. We did not find thrombolysis complications due to DFO or any other DFO-induced change in the number and type of adverse events (see Table 2). This safety DFO data in Antioxidants 2021, 10, 1270 10 of 15 4. Discussion This is the first report testing DFO, that targets pro-oxidant iron, in patients with ischemic stroke. DFO as a 10 mg/Kg IV bolus during the tPA infusion followed by a three- day IV continuous infusion of up to 60 mg/Kg/day is safe and well-tolerated, reduces TSAT, and shows a promising trend to better outcome. We did not find thrombolysis complications due to DFO or any other DFO-induced change in the number and type of adverse events (see Table 2). This safety DFO data in ischemic stroke patients set the groundwork for a larger clinical trial given the recently reported role of iron-induced ferroptosis as a main contributor of brain damage in exper- imental stroke models [1,4–6,31–33] and of previous reports in which DFO treatment is associated with lower infarct volume, less mitochondrial free radicals [24], less hemor- rhagic transformation, and improved neurological status in experimental ischemic stroke models [4,20,21,24,34–37]. Of note, either treatment with DFO or prevention of cellular iron uptake have been reported to protect neuronal phenotype cells from excitotoxic cell death through a reduction of oxidative stress [12,38,39]. In iron intoxication/overload, DFO administration for several days as a subcutaneous or intravenous slow infusion is indicated [40]. Although DFO administered systemically does not penetrate very well into the brain through an intact blood–brain barrier (BBB), DFO has been found in the experimentally-induced ischemic brain within the first hour post-stroke [34]. Thus, DFO would preferentially reach the ischemic brain areas, with a leaky BBB, while precluding iron chelation by DFO affecting the activity of cellular metalloenzymes in the healthy brain areas. Our study provides information about the pharmacokinetics of DFO in ischemic stroke patients, these data being of interest given that in all the species tested so far DFO demonstrate an extremely short half-life in serum/plasma. In healthy humans, IV or i.m. bolus injection of 10 mg/Kg is safe and serum concentrations of DFO range from 5 to 120 µM within minutes after the bolus injection but levels drop quickly [26,41]. To overcome DFO clearance from blood and to secure sustained therapeutic effects during the critical three days post-stroke-onset, the present study used a single 10 mg/Kg bolus of DFO IV, to reach high blood concentrations within minutes, followed by a 72 h continuous IV infusion of 20, 40, or 60 mg/Kg/day DFO. The higher serum DFO levels were observed within the first 30 min of treatment onset as a result of the initial DFO bolus administration (Figure 2A); thereafter, blood DFO levels dropped despite the three-day continuous infusion protocol. We did not find significant differences in DFO pharmacokinetics among different dose tiers along the 72 h follow-up (Figure 2A); this lack of differences could be anticipated in view of: (1) the limited stability and high individual variability of DFO pharmacokinetics reported in a previous clinical study [42], and (2) the rapid clearance of DFO from systemic circulation at clinically relevant doses (half-life of 5–17 min [26,42]). Of note, previous clinical studies using DFO as a continuous infusion in ICH stroke patients did not report pharmacokinetic data [25,43], and a recent study in rats concluded that the biodistribution and clearance from blood was too rapid to generate meaningful blood DFO pharmacokinetics [44]. A dose-dependent effect of the bolus + three-day sustained DFO infusion was ob- served on the reduction of an important systemic iron parameter, the % saturation of blood transferrin (Tf) with iron (TSAT), which gives the relative amount of iron-free and iron-loaded Tf in blood. Transferrin is the physiological carrier and provider of iron to neurons. We have previously reported the importance of blood Tf load with iron in ischemia events, as iron-loaded transferrin promotes ROS production at reperfusion in stroke models whereas iron-free transferrin prevents the production of 4-hydroxynonenal during ischemia/excitotoxicity in vitro [12]. Blood TSAT remained steady along 72 h in the stroke patients in the placebo group. DFO 40 and 60 mg/Kg/day significantly reduced TSAT by 30% and 40%, respectively, when measured at 72 h (Figure 2E,F) indicating an effective reduction of systemic iron, and the higher 60 mg/Kg/day DFO dose showed a trend to reduce TSAT after only 24 h of treatment (p = 0.107, repeated measures analysis). Although DFO is unable to directly remove iron from transferrin at physiological pH Antioxidants 2021, 10, 1270 11 of 15 (Available online: https://www.medicines.org.uk/emc/product/5/smpc (accessed on 12 June 2021)) [45], TSAT can be modulated by the iron available in blood to bind circulating Tf and/or the iron availability in the Tf-synthesizing cells. In this regard, previous reports have showed that DFO IV infusion rapidly clears around one third of the non-transferrin- bound iron in the blood [46,47] and reduces intracellular labile iron stores in breast cancer cells [48]. The reduction of TSAT by DFO might well be meaningful to the final outcome in the patients according to the fact that: (1) reduction of TSAT by IV administration of iron-free Tf (apotransferrin) at reperfusion has demonstrated neuroprotective and found associated to better outcome in experimental ischemic stroke models [12]; and (2) blood TSAT at reperfusion positively correlated with infarct volume and neurological impairment in experimental stroke; in rats, a 25% reduction in transferrin saturation decreases ischemic brain damage accordingly [12]. In addition, in the placebo group of the present TANDEM-1 study, the endogenous TSAT level seems to impact the outcome of stroke patients, as indicated by preliminary evidence obtained in the present study (Figure S3). Results in Figure 2F show that a bolus + three-day 60 mg/Kg/day DFO treatment reduces TSAT to half the baseline levels, and the time-effect suggests that administration of 60 mg/Kg/day exceeding three consecutive days would result in too low TSAT (below 16%). Given that TSAT < 16% is usually considered inadequate for erythropoiesis [49], this provides a rationale for the harmful side effects observed associated to the five-day treatment with 62 mg DFO/Kg/day that was initially proposed in the high dose HI- DEF trial protocol [50]. After a three-day high dose DFO treatment, other interventions known to have a mild effect on TSAT, such as a daily intake of the functional compound polyphenol [51], might be worth being evaluated for their possible contribution to TSAT long-term effects and long-term recovery from stroke. Our findings explain both the safety issues of the HI-DEF trial protocol just mentioned and the lack of effect observed when using an alternative intermediate DFO dose (32 mg/Kg/day) and a shorter three-day infusion in the i-DEF [43] and the Chinese Clinical Trial Registry ChiCTR-TRC-14004979 studies [52]. A strength of our study is that we developed and used a direct measurement of TSAT as a reliable surrogate marker of iron status. The effect of each DFO dose was determined longitudinally in each individual patient, from pre-DFO to the end of treatment, each patient being its own control. Using this strategy, we observed that only 40 and 60 mg/Kg DFO reduced TSAT, and we next studied the effect of these TSAT-modifying doses on outcome. The present study focused on safety and it is not powered to statistically assess efficacy or to equally distribute covariates at baseline along experimental groups, this being a limitation of the study. However, an exploratory analysis of efficacy was performed introducing as a selection criteria that of patients with baseline NIHSS > 7, the less severe strokes not being included in this post-hoc analysis and, in addition, we used the percentage reduction of neurological deterioration as an index that considers the specific neurological score of each patient at admission. In this subpopulation in which baseline scores are similar, DFO induced a larger percent reduction of neurological impairment at 90 days (Figure 3B), and the proportion of good outcome patients (mRS ≤ 2) increased with the dose of DFO at 7 and at 90 days. The mRS, that has been used to assess neurological status mostly at 90 days to evaluate long-term outcomes, has been reported in recent papers to be informative in the evaluation of short-term outcomes as well [53,54]. Although this is a post hoc analysis based on a relatively small number of patients, our observations support that DFO, at doses capable of reducing systemic iron and TSAT, seem to be a promising therapy increasing the proportion of patients showing better functional outcome. The effects of DFO preventing neurological impairment induced by AIS does not seem to be due to a possible impact on circulating immune cells since no effect of DFO was observed in blood leucocyte counts in our study (Figure S4), but rather associated with its iron-chelation and antioxidant properties. Antioxidants 2021, 10, 1270 12 of 15 5. Conclusions This study demonstrates that DFO in a bolus followed by doses of 40 to 60 mg/Kg/day is safe and well-tolerated in AIS patients, reduces systemic iron over 1–3 days and might provide long term (three months) benefit to AIS patients. Our findings have important implications to select the appropriate DFO dosage for future trials and provide medical plausibility and the rationale to further explore DFO effect in a larger cohort of patients or to test other therapeutic interventions addressed to quickly reduce TSAT in stroke. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antiox10081270/s1, Figure S1: Effect of DFO on CT hypodensity; Figure S2: Effect of deferoxamine (DFO) on cardiovascular parameters, body temperature, and blood glucose; Figure S3: Relationship between % TSAT of each patient in the placebo arm at admission and % reduction of neurological impairment; Figure S4: Effect of 72 h treatment with DFO on blood leucocytes; Table S1: Reported serious adverse events in the placebo and DFO group in each DTS. Author Contributions: Conceptualization, M.M., A.D., and T.G.; Patient inclusion and clinical data collection, M.M., N.P.d.l.O., Y.S., F.N., P.G., M.R.-Y., J.C. (Joan Costa), J.S., J.V., and A.D.; Investigation and laboratory work, N.D.-R., F.C., T.S., and O.M.-S.; Data curation and statistics J.C. (Jordi Cortés); Writing—original draft preparation and writing-review and editing, M.M., A.D., and T.G.; Project administration, S.R.; Funding acquisition, A.D. All authors have read and agreed to the published version of the manuscript. Funding: This academic research was supported by grants from the Fondo de Investigaciones Sanitarias-Instituto de Salud Carlos III (2007-006731-31) to M.M., INVICTUS PLUS RD16/0019/0020 to A.D. that was susceptible to be co-financed by FEDER funds, and a grant from Agència de Gestió d’Ajuts Universitaris i de Recerca 2017 SGR 1520 to A.D. and 2019PROD00120 to T.G. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Spanish Drug Agency (eudraCT 2007-0006731-31) and the Institutional Ethics Committee of the Hospital Germans Trias i Pujol. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data is contained within the article and supplementary material. Acknowledgments: The authors acknowledge Karla Odendaal for her help in improving the stan- dard of English in the manuscript. Conflicts of Interest: The authors declare that there is no conflict of interest regarding the publication of this paper. Abbreviations AE adverse events AIS acute ischemic stroke aPTT activated partial thromboplastin time ASPECTS Alberta Stroke Program Early CT Score ATf apotransferrin BBB blood–brain barrier CT computed tomography DFO deferoxamine diFe-Tf diferric transferrin DSMB data safety monitoring board DTS dose tier sub-study ENW early neurological worsening HTf holotransferrin ICH intracerebral hemorrhage i.m. intramuscular IV intravenous Antioxidants 2021, 10, 1270 13 of 15 MCA middle cerebral artery mFe-Tf monoferric transferrin mRS modified Rankin Scale NIHSS National Institute of Health Stroke Scale ROS reactive oxygen species SAE serious adverse events sICH symptomatic intracranial hemorrhage; study TANDEM Thrombolysis And Deferoxamine in Middle Cerebral Artery Occlusion Tf transferrin tPA tissue plasminogen activator TSAT % of iron saturation of blood transferrin U-PAGE/WB urea-polyacrylamide gel electrophoresis/Western Blot References 1. 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