antioxidants Article Characterization of a CholesteroNitrone (ISQ-201), a Novel Drug Candidate for the Treatment of Ischemic Stroke Emma Martínez-Alonso 1 , Alejandro Escobar-Peso 1 , Maria I. Ayuso 2 , Rafael Gonzalo-Gobernado 2 , Mourad Chioua 3 , Juan J. Montoya 4 , Joan Montaner 2,5 , Israel Fernández 6, * , José Marco-Contelles 3 and Alberto Alcázar 1, * 1 Department of Research, IRYCIS, Hospital Ramón y Cajal, Ctra. Colmenar km 9.1, 28034 Madrid, Spain 2 Neurovascular Research Group, Institute of Biomedicine of Seville, IBiS, Hospital Universitario Virgen del Rocío, Av. Manuel Siurot s/n, 41013 Seville, Spain 3 Laboratory of Medicinal Chemistry (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain 4 Isquaemia Biotech SL, Scientific Technological Park, C/Astrónoma Cecilia Payne s/n, 14014 Córdoba, Spain 5 Neurovascular Research Laboratory, Institut de Recerca Vall d’Hebron, Autònoma University of Barcelona, 08035 Barcelona, Spain 6 Departamento de Química Orgánica I y Centro de Innovación en Química Avanzada (ORFEO-CINQA), Complutense University of Madrid, Av. Complutense s/n, 28040 Madrid, Spain * Correspondence: israel@quim.ucm.es (I.F.); alberto.alcazar@hrc.es (A.A.); Tel.: +34-913945155 (I.F.); +34-913369016 (A.A.)  Received: 28 February 2020; Accepted: 28 March 2020; Published: 31 March 2020  Abstract: Nitrones have a well-recognized capacity as spin-traps and are considered powerful free radical scavengers, which are two important issues in hypoxia-induced oxidative stress and cell death in brain ischemia. Consequently, nitrones have been proposed as therapeutic agents in acute ischemic stroke (AIS). In this paper, we update the biological and pharmacological characterization of ISQ-201, a previously identified cholesteronitrone hybrid with antioxidant and neuroprotective activity. This study characterizes ISQ-201 as a neuroprotective agent against the hypoxia-induced ischemic injury. Transitory four-vessel occlusion and middle cerebral artery occlusion (tMCAO) were used to induce cerebral ischemia. Functional outcomes were determined using neurofunctional tests. Infarct area, neuronal death, and apoptosis induction were evaluated. In addition, ISQ-201 reactivity towards free radicals was studied in a theoretical model. ISQ-201 significantly decreased the ischemia-induced neuronal death and apoptosis, in a dose-dependent manner, showing its therapeutic effect when administered up until 6 h after post-ischemic reperfusion onset, effects that remained after 3 months from the ischemic episode. Furthermore, ISQ-201 significantly reduced infarct volume, leading to recovery of the motor function in the tMCAO model. Finally, the theoretical study confirmed the reactivity of ISQ-201 towards hydroxyl radicals. The results reported here prompted us to suggest ISQ-201 as a promising candidate for the treatment of AIS. Keywords: antioxidant; brain ischemia; hydroxyl radical; ischemic stroke; neuroprotection; nitrones; steroids; reactive oxygen species 1. Introduction Strokes represent the second largest cause of death worldwide. The ischemic subtype accounts for approximately 80% of registered cases [1], which is caused by a decrease in cerebral blood flow that affects the whole or part of the brain (global or focal cerebral ischemia, respectively). Reduction of blood flow usually originates from a thrombus or embolus occluding a blood vessel or by systemic Antioxidants 2020, 9, 291; doi:10.3390/antiox9040291 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 291 2 of 21 hypoperfusion in the brain. As a consequence of the decrease in blood supply, which is modulated by its severity, duration, and area of the brain that is affected, diverse effects are triggered within the components of the neurovascular unit. This set of processes, also known as the ischemic cascade, leads to the loss of cell function and, eventually, to the loss of cell integrity and death [2]. In the last decades, a plethora of strategies has been directed against different components of the ischemic cascade to find an adequate approach for the treatment of ischemic disease, overcoming the limitations of recombinant tissue plasminogen activator (rtPA) for thrombolysis, the only approved agent in the treatment of ischemic stroke by the US Food and Drug Administration (FDA). Either alone or in combination with other strategies, antioxidant therapy has long been considered an interesting field of research regarding the relevance of oxidative stress in an ischemic episode [3]. Briefly, during the duration of occlusion, but especially after recanalization, when a massive flow of oxygen reaches the compromised tissue, an overproduction of highly reactive oxygen and nitrogen species (ROS and RNS) takes place [4]. If uncontrolled by the endogenous antioxidant systems, which are compromised due to energy deprivation, radical species might further contribute to exacerbating the damage of the ischemia/reperfusion period. Antioxidant drugs are more often aimed at scavenging radical species (i.e., radical traps), to inhibit the ROS/RNS enzyme producers, or to recover or increase the activity of endogenous antioxidant systems (glutathione, SOD, Nrf2, etc.) [5]. Nitrones were initially developed as detection tools of radical species due to the formation of stable nitrone–radical adducts that can be detected by electron paramagnetic resonance methods [6]. In the following years, after the establishment of the pathological role of free radicals in several diseases, the trapping behavior of nitrone–radicals was suggested to be a relevant feature in the fight against oxidative stress [7]. N-tert-Butyl-α-phenylnitrone (PBN), which is extensively studied in spin-trapping experiments, was reported to exert a protective effect in rats, after traumatic brain injury [8]. After some development, the PBN-derived nitrone NXY-059 (which has a greater solubility than the parent compound) was described to be a good neuroprotective agent in preclinical models of ischemic stroke. This led to it being considered as a candidate for clinical trials [9,10]. Phase I and II studies revealed adequate pharmacokinetic characteristics and tolerability [11–13]. Phase III studies, however, were not able to show a conclusive beneficial effect when compared to placebo, a fact that led to NXY-059 withdrawal from the clinical trial path [14]. From this point, and despite the lack of benefit of NXY-059 in clinical phases, nitrone development has remained an appealing field of research, in the search for new candidates for the treatment of ischemic stroke and other diseases. It is the nature, position, and connectivity of the substituents on the nitrone group (the N-oxide of an imine), which are the main features modulating the chemical and pharmacological activity of nitrones. Thus, radical trapping activity can be easily modified with subtle chemical changes on the main scaffold, leading to more potent drugs. Nevertheless, this confers upon nitrones a great versatility, which makes it possible to find compounds that are not only able to act as radical traps but which also serve other functions, as examples of pleiotropic drugs. Drugs aimed for the treatment of ischemic stroke must feature precise characteristics, regarding stroke physiopathology, in order to increase the chances of success. Even in the best situations, patient arrival to the hospital, diagnosis, and start of treatment span several hours since the production of the occlusion. Treatments must, therefore, be effective and safe in an extended time window, covering the period required before treatment and that for recanalization—if not spontaneous. Recent studies have also pointed out the relevance of long-term recovery in patients who have suffered a stroke. The average age of stroke incidence has dramatically decreased in the last years, mainly because of poorer lifestyles and accumulated risk factors in younger individuals [15]. Apart from the sequelae common to older patients, these young adults (i.e., younger than 50) might eventually develop compromised functional, neuropsychiatric, or cardiovascular outcomes in the long-term yet earlier in their life, conditioning their extended life expectancy [16]. Consequently, the long-term effect should also be a priority in the drug development process and not only during the observation phase Antioxidants 2020, 9, 291 3 of 21 after drug approval, i.e., it should also be considered during the preclinical development by taking the limitations of experimental models into account. Previously, our group reported the first approach into the biological activity of a cholesterol-derived nitrone (i.e., cholesteronitrone) as a potential treatment for ischemic stroke [17]. ISQ-201, previously named as ChN 2, was identified as a promising candidate after it was administered to primary neuronal cultures subjected to oxygen and glucose deprivation (OGD) as a model of experimental ischemia [17]. Cell viability studies revealed a good neuroprotective effect in the micromolar range, which prompted us to explore the ISQ-201 effect on an in vivo model of cerebral ischemia. Intraperitoneal administration of the candidate at the onset of reperfusion after cerebral ischemia afforded a neuroprotective effect, when compared to the vehicle group [17]. Following these promising results, we decided to continue exploring the biological and pharmacological properties of ISQ-201. 2. Materials and Methods 2.1. Computational Methods All calculations reported in this paper were performed with the Gaussian 09 suite of programs [18]. Electron correlation was partially taken into account by using the hybrid function that is usually denoted as B3LYP [19–21], in conjunction with the D3 dispersion correction suggested by Grimme et al. [22], using the standard double-ζ quality def2-SVP [23] basis set for all atoms. Geometries were fully optimized in solution without any geometry or symmetry constraints. All species were characterized by frequency calculations [24], and had definite positive Hessian matrices. Frequency calculations were also used to determine the difference between the potential (E) and Gibbs (G) energies, G − E, which contained the zero-point, thermal, and entropy energies. Potential energies were refined in solution, Esol , by means of single point (SP) calculations at the same level, with a larger basis set, def2-TZVPP [23], where all elements were described with a triple-ζ + a polarization quality basis set. Solvent effects (solvent = water) were considered in the single-point calculations, using the polarizable continuum method (PCM) [25–27]. This level was denoted by PCM (water)-B3LYP-D3/def2-TZVPP// B3LYP-D3/def2-SVP. The ∆GR , a given in the text, were obtained from the Gibbs energy in solution, Gsol , which was calculated by adding the thermochemistry corrections, G − E, to the refined SP energies, Esol , i.e., Gsol = Esol + G − E. 2.2. Synthesis Cholesteronitrone ISQ-201 was prepared and purified as described in Section A of the Supplementary Material and as reported in [17]. 2.3. Pharmacokinetics Study For the study of the variation of the concentration of ISQ-201 in plasma, male Wistar rats (10–12 weeks, Charles River) were randomly divided into three groups (n = 6 per group) and treated intravenously, as a bolus with different concentrations of ISQ-201 (0.07, 0.25, and 1.0 mg/kg, respectively). Plasma samples were taken 0.25, 0.5, 1, 2, 4, and 8 h after the administration and the ISQ-201 concentration was determined by LC–MS/MS analysis. A noncompartmental analysis was performed for the determination of the toxicokinetic parameters, using the Phoenix 64 WinNonlin program (version 8.0). This study was carried out by Swiss BioQuant AG (Reinach, Switzerland). 2.4. Animal Model of Global Cerebral Ischemia, Experimental Design, and Treatment Transient forebrain ischemia was induced in adult male Wistar rats (10–12 weeks, Charles River) by the standard four-vessel occlusion model (4VO) described previously [28,29]. Briefly, both vertebral arteries were irreversibly occluded by electrocoagulation under anesthesia, with a mixture of atropine, ketamine, and diazepam (0.25, 62.5, and 5 mg/kg, respectively) delivered through intraperitoneal injection. After 24 h, ischemia was induced by carotid occlusion with atraumatic clips, for 15 min, and Antioxidants 2020, 9, 291 4 of 21 then the clips were removed from the carotid arteries to allow reperfusion. Body temperature was maintained at 37 ◦ C during the surgical procedure. The animals were studied after 5 days of reperfusion (R5d) after which they were sacrificed. Sham control animals were prepared in the same way, without carotid occlusion. We performed a power analysis (http://www.biomath.info/power/ttest.htm) to determine the sample size. Significance level and statistical power were set at 0.05 and 0.8 (80%), respectively, which afforded a sample size of <6 subjects per group. The treatments were administered with concealed allocation, and in randomized order, according to a computer-generated randomization program. Ischemic animals were treated with a vehicle solution (saline/ethanol, 90:10 vol/vol) or nitrone ISQ-201, dissolved in a vehicle solution through a single intravenous tail injection, when the carotid arteries were unclamped for reperfusion. In therapeutic window experiments, the animals were treated once at the reperfusion onset or at the set reperfusion time points, after ischemia. For the administration, at 6 h and 48 h of reperfusion after ischemia, the animals were anesthetized with isoflurane for 2–3 min and 5 min, respectively, prior to the treatment administration. An independent researcher prepared the treatments for each animal, according to the randomization schedule. Data of each experimental group were normalized to each vehicle control group. All procedures associated with animal experiments were approved by the Animal Care Ethics Committee of the Hospital Universitario Ramón y Cajal (Madrid, Spain, code no. 06/2015) and were performed according to the ARRIVE guidelines. 2.5. Evaluation of Neurological Deficits Neurological deficits in rats subjected to global cerebral ischemia were blindly evaluated after 5 days of reperfusion (R5d). General deficit and deficit in movement and sensory states were quantified, according to a scale ranging from 0 (best) to 10 (rats had a depressed level of consciousness). This scale was validated in an entire cohort of R5d animals (n = 20), as described previously [17]. 2.6. Behavioral Tests For the long-term studies, animals were subjected to behavioral tests, in order to assess their spontaneous spatial alternation and spatial memory skills, as markers of neurological damage present in longer periods, after the surgical procedure. According to previous literature [30], we selected the Y maze, in which both the exploration activity (spatial recognition) and spatial memory were evaluated. For the exploration activity and spatial recognition, control animals and ischemic animals treated with the vehicle or ISQ-201 were placed in the center of the maze and let to explore for 8 min. The total number of arms that the animal entered indistinctly (number of arm entries) and the times that the animal explored consecutively—the three different arms of the maze (number of alternations or triads)—were registered by an observer blind to the treatment. For the assessment of spatial memory, the control animals and ischemic animals treated with the vehicle or ISQ-201 were placed into one of the arms of the Y maze (start arm) and were allowed to explore the maze with one of the arms closed for 10 min (training trial). After 1 h, the rats were returned to the Y maze by placing them in the start arm and allowing them to freely explore all three arms of the maze for 3 min. The arm of the first choice of entry, the new or old, was registered manually by an observer blind to the treatment. 2.7. Brain Sections After 5 days of reperfusion (R5d), the animals were euthanized by transcardiac perfusion, under deep anesthesia. A washout step with 200 mL of saline via the left ventricle was followed by a fixation with 4% (w/v) paraformaldehyde solution in PBS. Brains were then removed and post-fixed in the same solution, overnight at 4 ◦ C, sequentially washed with 10%, 20%, and 30% (w/v) sucrose in PBS, and embedded in Tissue-Tek O.C.T. (Sakura Finetek), before freezing at −80 ◦ C. Brain coronal sections containing the hippocampus were obtained by cryostat sectioning at the level of interaural line +5.7 ± 0.2 mm on real capillary gap microscope slides (Dako). Antioxidants 2020, 9, 291 5 of 21 2.8. Neuronal Death Evaluation Brain cryosections (10 µm thick) from ischemic animals reperfused for 5 days were used after fixation to detect neuronal death by Fluoro-Jade B staining [28], and visualized by fluorescence microscopy. Labeled (dead) neuronal cells (in green) were counted, as in TUNEL assay (see below). Data from different animals of each experimental group were independently analyzed by two observers, and treatment information was kept concealed throughout the study. 2.9. TUNEL Assay Apoptotic neurons within the brain sections were detected by using the Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) assay (Promega). For this test, 5-µm-thick coronal cryostat brain sections containing the dorsal hippocampal formation were obtained, as described above, and post-fixed with 4% formaldehyde in PBS for 5 min, at room temperature. After washing in PBS for three times, the sections were permeabilized with 0.1 M sodium citrate, pH 6.0, for 1 min at 95 ◦ C, then quickly cooled in ice and washed in PBS for 5 min at room temperature. Next, the brain sections were incubated with a blocking solution (0.1 M Tris-HCl, pH 7.5, 3% bovine serum albumin, and 20% fetal bovine serum), washed in PBS for 5 min, and post-fixed with 4% formaldehyde in PBS, for 5 min. After washing in PBS for three times, the sections were incubated with a buffer kit for 10 min, and then incubated with terminal deoxynucleotidyl transferase (TdT) and fluorescein-12-dUTP for 1.5 h at 37 ◦ C, as described by the supplier. The reaction was stopped by extensive washing in saline-sodium citrate buffer (30 mM sodium citrate in 0.3 M NaCl) at room temperature. After washing in PBS for three times, the sections were then mounted with coverslips in an antifade solution, with a glycerol-buffer containing p-phenylenediamine and 30 µM bisbenzimide (Hoechst 33342), for nuclear staining. The hippocampal CA1 subfield and cerebral and lateral cortex fields from a given section were analyzed with fluorescence microscopy (40× objective), to count the number of apoptotic nuclei (green). A grid of 330 × 220 µm was used to count the cells in the regions of interest and digitized with a color CCD camera (1280 × 960 pixel resolution). TUNEL-positive cells were counted by two independent observers with a total area of 1.017 mm2 analyzed per section. Four sections per brain sample were averaged per experiment and the treatment information was kept concealed throughout the study. 2.10. Animal Model of Focal Cerebral Ischemia Transient occlusion of the distal branch of the middle cerebral artery (tMCAO) was induced in male C57BL/6 mice (9–11 weeks old, Charles River), as described previously [31]. Mice were anesthetized with 4% isoflurane for induction and 1.5–2% isoflurane for maintenance (in 79% N2 /21% O2 ). After drilling a small hole on the temporal bone at the level of the distal portion of the middle cerebral artery (MCA), the artery was compressed for 60 min, with a 30-G needle by using a micromanipulator. Body temperature was maintained between 36.5–37.5 ◦ C and cerebral blood flow was monitored using laser-Doppler flowmetry to confirm MCA occlusion. Buprenorphine (0.05–0.1 mg/kg) was administered subcutaneously immediately before the procedure. A total of 33 mice were subjected to tMCAO, according to the study design. A power analysis was performed to determine the sample size (significance level set at 0.05, and the power set at 0.8 (80%)), which identified the need for 8 mice to be included in each experimental group. Additional mice were allocated by taking into account mortality and technical issues that arise during the experiments, based on experience. The surgical inclusion criteria were—a reduction in blood flow to <25% of baseline value during the ischemia period, and a recovery of 75% of the baseline value in the reperfusion period. Mice that met the following criteria were excluded—(i) mice that failed to meet the inclusion criteria explained above (4 mice); and (ii) mice that died during the induction of MCA occlusion (2 mice). Mice were randomly assigned by using a randomization software to the following experimental groups (n = 9 per group)—vehicle (saline-ethanol 90:10 vol/vol), ISQ-201 0.05 mg/kg, and ISQ-201 0.1 mg/kg in vehicle. The treatments were administered intraperitoneally at the onset of the reperfusion period, by a blinded investigator. Antioxidants 2020, 9, 291 6 of 21 All procedures were approved by the local Animal Care Committee and were conducted in compliance with the ARRIVE guidelines, the Spanish legislations, and EU directives. 2.11. Motor-Deficit Evaluation, Grip Strength Test Neurobehavioral studies were performed to evaluate ischemic outcomes and monitor motor function after focal ischemia. A grip strength test was performed 24 h before the surgery and 24 and 48 h after tMCAO, by a researcher blinded to the experimental conditions. This test was designed to assess the maximum force displayed by mouse forelimbs (in grams), using a metallic grid connected to a force sensor (Bioseb). A total of six trials were conducted for each test and the strength value (expressed in grams) was calculated as the mean of all six trials. 2.12. Infarct Volume Evaluation The size of infarction was evaluated 48 h after tMCAO, using 2,3,5-triphenyltetrazolium chloride (TTC) stain [32]. Mice were sacrificed by transcardiac perfusion with ice-cold saline, under deep anesthesia. Brains were removed and cut into 1-mm-thick coronal sections and stained with 2.5% TTC in saline, at room temperature for 20 min. All analyses were performed in a blinded manner. The infarct areas were analyzed using the Image-J software and the infarct volume was determined through linear integration of the quantified lesion areas and the distances over the sections. To avoid the brain edema effects, the infarct area was corrected by the ratio of the area of the ipsilateral and the contralateral hemisphere. 2.13. Statistical Analysis Data from each treatment and the different animals from each experimental condition or group were independently analyzed and their averaged values were used for statistical analysis. The treatment information was kept concealed throughout the study. Data were represented as mean ± SE. Analysis of variance (ANOVA) or the non-parametric Kruskal–Wallis test (for scoring variables) were performed to compare the data between multiple concentrations or groups, followed by post-hoc tests, to compare pairs of group means when analysis of variance was significant. Statistical significance was set at p < 0.05, using Prism statistical software (GraphPad Software). 3. Results 3.1. In Silico DFT-Study Based on previous biological data on nitrone ISQ-201 [17] and as superoxide (O2 •– ) and hydroxyl • (HO ) radicals play a major role [33,34] among toxic radical oxygenated species involved in an ischemic event, we investigated the reactivity of these species with nitrone ISQ-201 using density functional theory (DFT) methods, to investigate the exceptional neuroprotective profile of this compound. To this end, and by using a model system of ISQ-201 where the long lateral chain was replaced by an iso-propyl group (ISQ-201’), we explored the thermodynamics of the addition reaction of the oxygen-centered radicals HO• and O2 •– to ISQ-201’, as well as the most favorable hydrogen abstraction reactions promoted by these species. Four different processes, shown in Scheme 1, are therefore considered. These include the addition of the oxygen-radicals directly to the nitrone (1,2-addition, path A) and, alternatively, to the conjugated double-bond (i.e., 1,4-Michael type addition, path B), and the hydrogen abstractions involving either H2 (path C) or H5 (path D). reaction of the oxygen-centered radicals HO• and O2•– to ISQ-201’, as well as the most favorable hydrogen abstraction reactions promoted by these species. Four different processes, shown in Scheme 1, are therefore considered. These include the addition of the oxygen-radicals directly to the nitrone (1,2-addition, path A) and, alternatively, to the conjugated double-bond (i.e., 1,4-Michael type addition, path B), and the hydrogen abstractions Antioxidants 2020, 9, 291 7 of 21 involving either H2 (path C) or H5 (path D). Scheme1.1.Possible Scheme Possiblereaction reactionpaths pathsfor forthe thereaction reactionofofnitrone nitroneISQ-201 ISQ-201and andoxygen-centered oxygen-centeredradicals radicals HO ••and O •– . HO and O22 . •– Table 1 shows the computed free reaction energies in water (∆GR , at 298 K) for the above- Table 1 shows the computed free reaction energies in water (∆G R, at 298 K) for the above- commented processes. From the data in this Table, it becomes obvious that the reactions involving the commented processes. From the data in this Table, it becomes obvious that the reactions involving superoxide radical are unfeasible, given the high endergonicities computed for these processes. In the superoxide radical are unfeasible, given the high endergonicities computed for these processes. sharp contrast, all reactions involving the hydroxyl radical are highly exergonic, therefore, suggesting In sharp contrast, all reactions involving the hydroxyl radical are highly exergonic, therefore, a favorable reaction of nitrone ISQ-201 and this radical. Moreover, the abstraction reaction of H5 suggesting a favorable reaction of nitrone ISQ-201 and this radical. Moreover, the abstraction (PATH D), which leads to radical species 4 (see Scheme 1) and a molecule of water, becomes the most reaction of H5 (PATH D), which leads to radical species 4 (see Scheme 1) and a molecule of water, thermodynamically preferred pathway. The remarkable stability of the latter species can be ascribed becomes the most thermodynamically preferred pathway. The remarkable stability of the latter to the delocalization of the unpaired electron into the entire conjugated system, which also involves species can be ascribed to the delocalization of the unpaired electron into the entire conjugated the nitrone moiety. Indeed, the spin density computed for species 4 (see Scheme 1) confirms that the system, which also involves the nitrone moiety. Indeed, the spin density computed for species 4 (see unpaired electron is fully delocalized into this conjugated fragment (Figure 1). Scheme 1) confirms that the unpaired electron is fully delocalized into this conjugated fragment (Figure 1). Table 1. Computed free reaction energies (∆GR , at 298 K, in kcal/mol) for the reactions involving ISQ-201’ and radicals HO• and O2 •– . All data were computed at the polarizable continuum method Table 1. Computed free reaction energies (∆GR, at 298 K, in kcal/mol) for the reactions involving (PCM)(water)–B3LYP-D3/def2-TZVPP//B3LYP-D3/def2-SVP level. ISQ-201’ and radicals HO• and O2•–. All data were computed at the polarizable continuum method Reaction Path HO• (PCM)(water)–B3LYP-D3/def2-TZVPP//B3LYP-D3/def2-SVP O2 •−level. PATH A −36.5 +14.5 Reaction Path HO• O2•– PATH B −37.1 +6.9 PATH A –36.5 +14.5 PATH C −41.1 +15.6 PATH B –37.1 +6.9 PATH D −48.7 +18.5 Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 21 PATH C –41.1 +15.6 PATH D –48.7 +18.5 Computed Figure 1.Figure spinspin 1. Computed density density (isovalue ==0.01 (isovalue 0.01au)au) for for compound compound 4 of Scheme 4 of Scheme 1. 1. 3.2. Dose-Response Study Study 3.2. Dose-Response Previous Previous studiesstudies of ISQ-201 of ISQ-201 effective effective concentration determined concentration determined an optimal concentration an optimal range concentration range between 1.0 and 10 µ M in primary neuronal cultures subjected to an oxygen and glucose deprivation between 1.0 and 10 µM in primary neuronal cultures subjected to an oxygen and glucose deprivation (OGD) experimental ischemia to achieve significant neuroprotection values [17]. Considering the (OGD) experimental ischemia observed solubility and thetopenetration achieve significant neuroprotection profile predicted values for ISQ-201, doses of 0.05 [17]. and 0.1Considering mg/kg the observed were solubility and the penetration profile predicted for ISQ-201, doses of 0.05 and administered through intraperitoneal injection at the onset of reperfusion, in animals subjected 0.1 mg/kg to cerebral ischemia were administered through[17]. As was previously intraperitoneal reported, injection at significant the onsetdecreases in neuronal of reperfusion, in death and subjected animals apoptosis, as well as in neurodeficit score, were obtained for both concentrations [17]. In our attempt to improve the applicability of the treatment with ISQ-201, we decided to explore the suitability of an intravenous administration. According to the doses studied previously, we selected a concentration range of 0.01–0.1 mg/kg. Administration of ISQ-201 or a vehicle solution was carried out by an intravenous tail injection at the reperfusion onset, after the global ischemia, according to the procedure described in the Materials and Methods section. Animals were let to Antioxidants 2020, 9, 291 8 of 21 to cerebral ischemia [17]. As was previously reported, significant decreases in neuronal death and apoptosis, as well as in neurodeficit score, were obtained for both concentrations [17]. In our attempt to improve the applicability of the treatment with ISQ-201, we decided to explore the suitability of an intravenous administration. According to the doses studied previously, we selected a concentration range of 0.01–0.1 mg/kg. Administration of ISQ-201 or a vehicle solution was carried out by an intravenous tail injection at the reperfusion onset, after the global ischemia, according to the procedure described in the Materials and Methods section. Animals were let to recover for 5 days (R5d), after which neuronal death, apoptosis, and the neurodeficit score were assessed. Results are shown in Figure 2. 3.2.1. Analysis of Neuronal Death Blood supply cessation in the global ischemia model, during a 15-min-induced neuronal death, especially in the hippocampal CA1 and cortical regions of the brain. This neuronal damage could be observed after 5 days of reperfusion through Fluoro-Jade B staining, as a marker of dead neurons. In these experiments, a decrease in the dead neuron numbers could be observed with ISQ-201 treatment in the whole concentration range tested in the hippocampal CA1 region and in the cerebral cortex, when compared to the group treated with a vehicle (Figure 2a). Remarkably, a significant reduction of the dead neuron number observed in the vehicle group could be observed in those groups treated with 0.05 and 0.1 mg/kg of ISQ-201. 3.2.2. Analysis of Neuronal Apoptosis Neuronal apoptosis could be observed as a consequence of the 15-min-long global ischemia, when brain slices were subjected to a TUNEL assay, after 5 days of reperfusion, as described in the Materials and Methods section. Cortical and, especially, hippocampal CA1 regions were the most vulnerable to the blood flow impairment in this experimental model, therefore, being adequate for the observation of potential neuroprotective effects. Results shown in Figure 2b describe the reduction in cell apoptosis observed when animals were treated with ISQ-201 (ranging 0.01–0.1 mg/kg), compared to the vehicle group. Consistent with the Fluoro-Jade B experiments, a significant reduction of apoptotic cells is obtained with ISQ-201 at 0.05 and 0.1 mg/kg, in both CA1 and cortical regions. 3.2.3. Dose-Response Curve Both, a decrease in neuronal death and the apoptosis results are indicative of the neuroprotective effect of ISQ-201 in the experimental model used in this study. In Figure 2c, 0% neuroprotection is assigned to the cell death or apoptosis obtained for the vehicle group through the Fluoro-Jade B or TUNEL assays, respectively, whereas a complete neuroprotection (100%) stands for the complete absence of cell death or apoptosis. At the most potent concentration (0.05 µM), ISQ-201 exerts a maximum neuroprotection (Emax ) of 80% and 31% in neuronal death assays—in the cortex and hippocampal CA1, respectively—and 81% and 35% in TUNEL assays—again in the cortex and CA1, respectively. Considering the dose-response curve, an EC50 could be determined, affording a value of 0.015 and 0.025 mg/kg for the graphical representations in cortex and CA1, respectively (Figure 2c). These results point out the use of the concentrations of 0.05 mg/kg of ISQ-201 as the most adequate among the set of doses evaluated, due to its higher value than EC50 which is closer to Emax , in both, the cerebral regions and the efficacy studies tested. and hippocampal CA1, respectively—and 81% and 35% in TUNEL assays—again in the cortex and CA1, respectively. Considering the dose-response curve, an EC50 could be determined, affording a value of 0.015 and 0.025 mg/kg for the graphical representations in cortex and CA1, respectively (Figure 2c). These results point out the use of the concentrations of 0.05 mg/kg of ISQ-201 as the most adequate among the set of doses evaluated, due to its higher value than EC50 which is closer to Emax, Antioxidants 2020, 9, in both, the291 cerebral regions and the efficacy studies tested. 9 of 21 Figure 2. Dose-response Figure curve 2. Dose-response of ISQ-201. curve of ISQ-201.Ischemic animals Ischemic animals were were treated treated withwith a vehicle a vehicle or ISQ-201 or ISQ-201 (range 0.01–0.1 mg/kg) (range 0.01–0.1 by an mg/kg) by intravenous an intravenousinjection, atthe injection, at theonset onset of of reperfusion reperfusion after after globalglobal cerebralcerebral ischemia, ischemia, and ISQ-201 and ISQ-201 effect effect waswas evaluatedby evaluated byassessing assessing the theneuronal neuronaldeath at 5 days death of reperfusion at 5 days of reperfusion (R5d). (a) Brain sections from vehicle- and ISQ-201-treated animals (R5d+vehicle and R5d+ISQ-201, respectively), were fixed and stained with Fluoro-Jade B to detect neuronal death. Fluoro-Jade-positive dead neurons were visualized by fluorescence microscopy (in green) in the hippocampal CA1 (CA1) and cortical (C) regions (images), and counted, as described in Materials and Methods (bar graph). (b) Brain sections as in (a) were used for apoptosis detection by TUNEL assay after fixation, and were visualized by fluorescence microscopy (in green) in the CA1 and C regions (images). TUNEL-positive cells were counted in the CA1 and C regions, as described in the Materials and Methods (bar graph). In (a) and (b), images are representative results of CA1 or C regions from vehicle- and ISQ-201-treated (0.05 mg/kg) animals. In bar graphs, data represent the mean of 6−11 independent animals per group, and the error bars indicate the SE. * p < 0.05 and ** p < 0.01, when compared with their respective R5d+vehicle (Vh), through Dunnett’s post-test, and # p < 0.05, when compared by Student’s t-test, after ANOVA. In (c), data from (a) and (b) are represented as neuroprotective activity of the different concentrations of ISQ-201. Neuroprotection was defined as the percentage achieved from the neuronal death or apoptosis value in the R5d+vehicle (Vh) (0% value), to the absence of neuronal death or apoptosis (100% value). Data are represented as mean ± SE. Antioxidants 2020, 9, 291 10 of 21 3.3. Therapeutic Window In the following study, delayed administration of ISQ-201, ranging from 0 h to 48 h after reperfusion onset was evaluated in the animal model of cerebral ischemia, to determine its therapeutic window. ISQ-201 was administered as a single intravenous injection of 0.05 mg/kg, the most effective dose was found in the dose-response study (see above). Neuronal death and apoptosis were evaluated after 5 days Antioxidants of reperfusion and a neurodeficit score was determined. In these experiments, each 11 2020, 9, x FOR PEER REVIEW time of 21 point of treatment was conducted with a respective vehicle group. No significant differences were found between afterthe vehicle 48 h groups (p of reperfusion, > 0.158, ISQ-201 by ANOVA treatment couldtest) and datathe not improve were represented neurological asofnormalized, state the animal, with compared respect with thegroup to the vehicle vehicleadministered treatment. at the onset of the reperfusion (Figure 3a,b). FigureFigure 3. The 3. The therapeutic therapeutic windowofofISQ-201. window ISQ-201. Ischemic Ischemicanimals animals were weretreated treatedwithwith vehicle (Vh) (Vh) vehicle or or ISQ-201 (0.05 mg/kg), by an intravenous injection at the onset of reperfusion, or at ISQ-201 (0.05 mg/kg), by an intravenous injection at the onset of reperfusion, or at 1, 3, 6, or 48 h of1, 3, 6, or 48 hours of reperfusion after global cerebral ischemia; and the neuroprotection of ISQ-201 was evaluated after reperfusion after global cerebral ischemia; and the neuroprotection of ISQ-201 was evaluated after 5 days of reperfusion, following ischemia. (a) The bar graph represents the neuronal death quantified 5 days of reperfusion, following ischemia. (a) The bar graph represents the neuronal death quantified in in brain sections from vehicle- and ISQ-201-treated animals, after fixation and staining with brainFluoro-Jade sections from vehicle- and ISQ-201-treated animals, after fixation and staining with Fluoro-Jade B. B. Neuroprotection was determined by quantification of the decrease of neuronal death Neuroprotection was in the hippocampal determined CA1 (CA1, byblack quantification bars) andofcortical the decrease of neuronal (C, white deathas bars) regions, in described the hippocampal in CA1 Materials (CA1, black bars) and cortical (C, white bars) regions, as described in Materials and Methods. (b) The bar graph represents the apoptosis quantified in brain sections and Methods. from (b) The bar graph vehicle- andrepresents the apoptosis ISQ-201-treated animals,quantified through TUNELin brain sections assay. from vehicle- Neuroprotection wasand ISQ-201-treated determined by animals, through TUNEL assay. Neuroprotection was determined by quantification of the decrease quantification of the decrease of neuronal apoptosis in the CA1 and C regions, as described in of Materials and Methods. (c) Images of the representative results of CA1 or neuronal apoptosis in the CA1 and C regions, as described in Materials and Methods. (c) Images ofC regions from vehicle- and ISQ-201-treated the representative animals results of CA1at 3orh,C6 regions h, or 48 h of reperfusion, from vehicle- and after apoptosis detection ISQ-201-treated by TUNEL animals at 3 h, 6 h, assay and was visualized by fluorescence microscopy. TUNEL-positive cells were or 48 h of reperfusion, after apoptosis detection by TUNEL assay and was visualized by fluorescence visualized in CA1 and C fields (in green). In (a) and (b), the data represent the mean of 6−7 independent animals, and microscopy. TUNEL-positive cells were visualized in CA1 and C fields (in green). In (a) and (b), the the error bars indicate the SE, * p < 0.05 and ** p < 0.01, compared to their respective vehicle (Vh), data represent the mean of 6−7 independent animals, and the error bars indicate the SE, * p < 0.05 and through Dunnett’s post-test after ANOVA. ** p < 0.01, compared to their respective vehicle (Vh), through Dunnett’s post-test after ANOVA. Antioxidants 2020, 9, 291 11 of 21 3.3.1. Analysis of Neuronal Death As observed with the Fluoro-Jade B staining, the administration of ISQ-201 (0.05 mg/kg) significantly decreased neuronal death at the time points evaluated between 0 and 6 h after reperfusion, when compared to the vehicle group. This effect was observed in both the hippocampal CA1 and the cortical regions. In the cortex, a significant reduction in neuronal death could be observed when ISQ-201 was administered later than 6 h after the reperfusion onset (Figure 3a). 3.3.2. Analysis of Neuronal Apoptosis Results obtained for the evaluation of neuronal apoptosis were similar to those obtained in the neuronal death analysis. Administration of ISQ-201 at extended periods after reperfusion, significantly reduced apoptosis-positive cells, including at 6 h postreperfusion, both in the hippocampal and the cortical regions, when compared to the vehicle group. Administration at 48 h postreperfusion did not show significant differences in cell apoptosis (Figure 3b). Representative images of TUNEL images are shown in Figure 3c. 3.3.3. Functional Test In order to quantify potential functional impairments, the general state of the animals was assessed. In this test, each variable related to the general state of the animal (breathing, coordination, aspect, etc.) was assigned a grade corresponding to the degree of severity by which that variable was affected. The overall grade accounted for the neurological deficit score (NDS), which represented the general consciousness and neurological activity of the animal (0, healthy animal; 10, the animal had a depressed level of consciousness). As shown in Figure 4, an improvement was observed when the animal was treated with ISQ-201 at any of time points between 0 and 6 h of post-reperfusion, showing significant differences when compared to the vehicle group. When administration occurred after 48 h of reperfusion, ISQ-201 treatment could not improve the neurological state of the animal, compared with the vehicle Antioxidants 2020, 9,treatment. x FOR PEER REVIEW 12 of 21 Neurological deficit score (NDS) 6 5 4 + * * * 3 2 1 0 Vh 0 1 3 6 48 Time (h) of the ISQ-201 injection after reperfusion onset Figure 4.4. Neurodeficit Figure Neurodeficit score score outcomes outcomes after after cerebral cerebral ischemia ischemia in in the the therapeutic therapeutic window window study study of of ISQ-201. The bar graph shows the neurological deficit score (NDS) in ischemic animals ISQ-201. The bar graph shows the neurological deficit score (NDS) in ischemic animals at 5 days of at 5 days of reperfusion reperfusionafter afterischemia. ischemia.Animals Animalswere weretreated treatedwith withthethevehicle vehicle(Vh) (Vh)or orISQ-201 ISQ-201(0.05 (0.05mg/kg), mg/kg),by byan an intravenous injection at the onset of reperfusion, or at 1, 3, 6, or 48 h of reperfusion after global intravenous injection at the onset of reperfusion, or at 1, 3, 6, or 48 hours of reperfusion after global cerebral ischemia. The values cerebral ischemia. Therepresent the meanthe values represent of 6−7 mean independent animals, animals, of 6−7 independent and the error barserror and the indicate bars the SE, * p < 0.05 or + p < 0.05, compared with the vehicle by Dunn’s or Mann–Whitney indicate the SE, * p < 0.05 or + p < 0.05, compared with the vehicle by Dunn’s or Mann–Whitney post-test, respectively, after the Kruskal−Wallis post-test, respectively, test. after the Kruskal−Wallis test. 3.4. Long-Term Efficacy Assessment As described in the previous sections, a short-term (5 days) neuroprotective effect was achieved when administrating 0.05 mg/kg of ISQ-201 at 0–3 h, after the reperfusion onset. Being aware of the relevance of a long-term effect in the stroke therapeutics, we wanted to assess whether the optimal concentration and administration time had any effect at longer times, after the ischemic episode. In previous experiments, neuronal damage and death produced by the ischemic episode were Antioxidants 2020, 9, 291 12 of 21 3.4. Long-Term Efficacy Assessment As described in the previous sections, a short-term (5 days) neuroprotective effect was achieved when administrating 0.05 mg/kg of ISQ-201 at 0–3 h, after the reperfusion onset. Being aware of the relevance of a long-term effect in the stroke therapeutics, we wanted to assess whether the optimal concentration and administration time had any effect at longer times, after the ischemic episode. In previous experiments, neuronal damage and death produced by the ischemic episode were determined by Fluoro-Jade B and TUNEL assays, 5 days after post-ischemic reperfusion. The study of the effect at longer periods demands a different experimental approach, since the damaged or dead cells are naturally reabsorbed by the brain parenchyma and are not observed through Fluoro-Jade B or TUNEL assays. In the following experiments, we assessed the neuronal death at 11 weeks of reperfusion through immunostaining and quantification of the ribosomal protein S6, a constitutive protein that is present in neuron cytoplasm and is a marker of cell viability [35]. S6 protein staining was observed in the hippocampal CA1 region, one of the most vulnerable brain regions to the ischemic damage. Within this approach, we were able to observe whole neurons, indicating proper cell viability in the brain sections of sham control animals (Figure 5a, control, S6-positive (S6+) cells). Ischemic animals were treated with vehicle or ISQ-201, through intravenous injections, at the onset of reperfusion, Antioxidants 2020, 9, x FOR PEER REVIEW 13 of 21 after cerebral ischemia and neuronal loss was evaluated in the brain sections, at 11 weeks after treatment (Figure 5a, out IR, by S6+counting cells). Additionally, animal S6-positive neurons behavior (viable neurons)was also in the evaluated CA1 for thethat region, neurons neurofunctional were outcome assessment. preserved with the administration of ISQ-201 (Figure 5b). Figure 5. Long-term efficacy Figure 5. Long-term studystudy efficacy of ISQ-201. of ISQ-201. (a) (a) Brain sections Brain sections fromfrom the sham the sham control control animals animals (control), or from vehicle (Vh)- and ISQ-201-treated (ISQ-201, 0.05 mg/kg) (control), or from vehicle (Vh)- and ISQ-201-treated (ISQ-201, 0.05 mg/kg) ischemic animals ischemic animals were were evaluated for cell detection at 11 weeks after ischemic reperfusion (IR). IR animals were treated by an evaluated for cell detection at 11 weeks after ischemic reperfusion (IR). IR animals were treated by intravenous injection at the onset of reperfusion, after global cerebral ischemia. Neurons were an intravenous injection labelled at thewith after fixation onset of reperfusion, an anti-protein after global S6 antibody cerebral ischemia. and X red-secondary antibody,Neurons were were labelled after fixation visualized bywith an anti-protein fluorescence microscopyS6(in antibody and red), and the Xwere cells red-secondary antibody, also stained with were the Hoechst visualized dye (in blue). by fluorescence The images (in microscopy are representative red), and the results cellsofwere the hippocampal also stained CA1with region. the(b)Hoechst The bar graph dye (in blue). represents the number of positive-cells for S6 labeling (S6+ cells), per field of the hippocampal CA1 The images are representative results of the hippocampal CA1 region. (b) The bar graph represents region from the control animals and the vehicle (Vh)- and ISQ-201-treated ischemic reperfused the numberanimals. of positive-cells for S6 labeling (S6+ cells), per field of the hippocampal CA1 region from Values represent the mean of six independent animals, and the error bars indicate the SE, ** the control panimals and the < 0.01, compared vehicle to the control(Vh)- group,and ISQ-201-treated through ischemic Dunnett’s post-test, reperfused animals. Values after ANOVA. represent the mean of six independent animals, and the error bars indicate the SE, ** p < 0.01, compared 3.4.2. Behavioral Tests to the control group, through Dunnett’s post-test, after ANOVA. As for neuronal viability studies, previously described functional tests were not adequate for a long-term outcome assessment. Animals might overcome their initial functional deficits due to the nervous system’s inherent plasticity, recovering completely, several days after the ischemia. For this reason, exploration tests assessing spatial recognition, memory, orientation, and curiosity, whose deficits are maintained over a longer period after the insult were needed. In these experiments, we carried out two different behavioral tests, assessing the animal’s spatial recognition and spatial Antioxidants 2020, 9, 291 13 of 21 3.4.1. Analysis of Neuronal Viability Viable neurons in the hippocampal CA1 region, at 11 weeks after reperfusion were stained with the S6 protein in ISQ-201-treated animals and were compared with vehicle-treated animals (Figure 5a, IR, S6+ cells). In the pictures, a severe reduction in the S6-positive cells could be observed in vehicle-treated animals (Figure 5a, IR Vh). These results showed how 15 min of cerebral ischemia, followed by reperfusion, could devastate and extinguish neuronal cells, in the CA1 region of the brain hippocampus, the region where the short-term memory and spatial memory reside. Remarkably, it can be observed in the pictures that neurons in the CA1 region from the ISQ-201-treated animals were similar in number and morphology to hippocampal neurons from control animals (Figure 5a, control and IR ISQ-201). Quantification of S6 staining results was carried out by counting S6-positive neurons (viable neurons) in the CA1 region, neurons that were preserved with the administration of ISQ-201 (Figure 5b). 3.4.2. Behavioral Tests As forAntioxidants neuronal 2020,viability studies, 9, x FOR PEER REVIEW previously described functional tests were not 14 adequate of 21 for a long-term outcome assessment. Animals might overcome their initial functional deficits due to the 3.4.2.1. Exploration Activity and Spatial Recognition Test nervous system’s inherent plasticity, recovering completely, several days after the ischemia. For this For the tests reason, exploration exploration activityspatial assessing test, vehicle- or ISQ-201-treated recognition, memory,animals were compared orientation, to the and curiosity, whose control group. It was observed that the vehicle group had a significantly higher neurological deficit, deficits are maintained over a longer period after the insult were needed. In these experiments, compared to the control group (Figure 6a). In contrast, the animals treated with ISQ-201 showed no we carriedsignificant out twodifferences differentwithbehavioral the controltests, group.assessing In the samethe test,animal’s the numberspatial recognition of alternations or timesand spatial memory, to thatevaluate the animals long-term completedcognitive impairment a triad (i.e., they explored(see thethe Materials three and Methods different arms of the mazesection for consecutively) detailed procedure was also assessed as a parameter indicative of their spatial recognition (Figure 6b). As information). for the assessment of the number of arm entries, the vehicle group showed a significantly higher neurological Exploration Activity deficit, when compared and Spatial Recognitionto theTest control, as indicated by the smaller number of triads completed. The ISQ-201-treated group ameliorated their deficit, resulting in no significant differences, For the whenactivity exploration comparedtest, to the control. or ISQ-201-treated animals were compared to the vehicle- control group. It was observed that the vehicle group had a significantly higher neurological deficit, compared 3.4.2.2. Spatial Memory Test to the control group (Figure 6a). In contrast, the animals treated with ISQ-201 showed no significant To evaluate the animals’ long-term spatial memory, they were subjected to a novel arm differencesrecognition with thetest control group. In the same test, the number of alternations or times that the in the Y-maze. After the training period, with one of the arms closed, the animals animals completed a triad (i.e., were introduced to the maze they explored again, with two the three arms different available arms of the for exploration. The maze consecutively) exploration of the was also assessed newas arma parameter indicative as a first choice of their was indicative spatial of proper recognition spatial memory and,(Figure 6b). itAs therefore, for was the assessment assessed in of this test for control, vehicle-, or ISQ-201-treated animals. The results (Figure 6c) showed the number of arm entries, the vehicle group showed a significantly higher neurological deficit, when a decreased percentage of animals within the vehicle group, exploring the new arm as a first choice and compared indicating to the control, as indicated by the smaller number of triads completed. The ISQ-201-treated an impaired spatial memory. This was ameliorated with the ISQ-201-treatment, which group ameliorated allowed thetheir deficit, recovery resulting of values inthe similar to nocontrol significant group. differences, when compared to the control. (a) Arm Entries (b) Alternation Test (c) First Choice Novel Arm 20 100 Percentage of animals 30 No. of alternations No. of arm entries 15 75 * 20 10 ** *** 50 10 5 25 0 0 0 control Vh +ISQ-201 control Vh +ISQ-201 control Vh +ISQ-201 Figure 6. Behavioral Figure 6. Behavioral tests after tests after cerebral cerebral ischemia at ischemia at long-term. long-term.TheThebar graphs represent bar graphs the resultsthe results represent obtained after three spatial recognition and spatial memory test, to assess the long-term cognitive obtained after three spatial recognition and spatial memory test, to assess the long-term cognitive impairment of control animals and vehicle (Vh)- and the ISQ-201-treated ischemic animals, at 11 impairment of control animals and vehicle (Vh)- and the ISQ-201-treated ischemic animals, at 11 weeks after ischemic reperfusion. The values represent the mean of 6 independent tests, performed weeks afteronischemic reperfusion. different animals. In (a) andThe(b),values error barsrepresent the indicate the SE,mean of and * p < 0.05 6 independent tests, performed ** p < 0.01, as compared to on different animals. Ingroup the control (a) and (b),Dunn’s using error bars indicate post-test the SE, * p < 0.05 after Kruskal−Wallis test; and p