**4.7 Retinal degenerative diseases**

In recent years, others and we have investigated the potential of MMF in the treatment of degenerative retinal diseases. In an early study, we showed MMF to be protective against reactive gliosis, a characteristic response of Muller glial cells to an environment rich in pro-oxidant and inflammatory factors in retinal disease. Folate uptake by Muller cells is considered a key event in this process [82]. MMF treatment significantly reduced folate uptake by Muller cells by decreasing the expression and

**209**

*Repurposing Fumaric Acid Esters to Treat Conditions of Oxidative Stress and Inflammation…*

activity of proton-coupled folate transporter (PCFT), a transporter integral to the uptake of folate. This was the first report demonstrating that MMF could regulate folate transport in retinal glial cells and therefore, be potentially useful in the treatment of degenerative retinal diseases. To determine whether, in addition to downregulating pro-inflammatory mechanisms, MMF affects counteractive or protective signaling, in a subsequent study we evaluated also the effect of the compound on the expression and activity of the cysteine/glutamate exchanger SLC7A11 (system

<sup>−</sup>), a transport system critical for the intracellular entry of the amino acid cysteine which is required for glutathione synthesis [28]. Glutathione is the most abundant endogenous antioxidant in the retina and is therefore essential for the protection of retinal cells against oxidative stress. Further, retinal pigment epithelial (RPE) cells are one of the highest producers of glutathione of any cell type in the body. As such, we exposed human retinal pigment epithelial (ARPE-19) cells to MMF in the presence or absence of pro-oxidant stimuli and evaluated the dose- and time-dependent

regulate each of these parameters and additionally, up-regulate hypoxia-inducible factor 1-alpha (Hif-1α), nuclear factor erythroid 2-related factor 2 (Nrf2) expression and increase total reduced glutathione (GSH) content. Collectively, our early *in vitro* studies demonstrated that MMF affects multiple pathways in multiple retinal cell types in a manner that is overall protective against oxidative damage. We sought next to determine whether our findings extrapolate to the *in vivo* condition, therefore, we evaluated the efficacy of MMF in a living animal model of retinal disease. Retinopathy is a major cause of vision loss in sickle cell disease (SCD) and therapies to prevent and treat sickle retinopathy (SR) are very limited. Therapeutic induction of γ-globin expression and subsequent induction of fetal hemoglobin (HbF) production can alleviate some SCD-associated complications. Interestingly, Nrf2 inducers have been demonstrated to be effective γ-globin inducers [83]. The robust inductive properties of MMF on Nrf2 translocation and activity have been long recognized therefore, it was logical to explore the effects of MMF in SCD. Not only did we confirm that RPE cells, cells integral to retinal health and function, produce HbF but that MMF treatment of Townes humanized SCD mice of SCD resulted in reductions in the expression of pro-oxidant and inflammatory factors and turn, preserved retinal morphology [35]. Shortly after this study, Cho et al. [51] too reported on the potential benefit of MMF in the treatment of retinal disease in a mouse model of retinal ischemia-reperfusion. Specifically, they showed that MMF promotes Nrf2-neuroprotection in this model. MMF treatment was associated with significant increases in the expression of Nrf2-responsive antioxidant genes and a suppression of inflammatory responses as evidenced by increased expression of NAD (P) H quinone dehydrogenase 1, thioredoxin reductase 1 and heme oxygenase-1 along with decrease in interleukin-1β, chemokine (C-C motif) ligands (2, 7 and 12), expressions. Collectively, these molecular improvements interpreted to improved retinal function as evidenced by electroretinogram recordings performed on live mice and were heavily dependent upon the expression and activity of Nrf2.

Because these initial reports of MMF's potential efficacy in protecting against retinal degeneration were conducted acutely, we decided to evaluate the effect of long-term administration of the compound (5 months administration of 15mg/ml MMF in drinking water) in the humanized SCD model [34]. Importantly we found via high-pressure liquid chromatography (HPLC) and hematological analyses of peripheral blood that MMF treatment reduced sickle hemoglobin (HbS) content and white blood cell counts, and improved hematocrit, red blood cell number, and hemoglobin concentrations significantly in SCD mice. In retina specifically, the mRNA and protein expression of well-established markers of inflammation and oxidative stress (i.e., vascular endothelial growth factor, intercellular adhesion molecule-1,

<sup>−</sup> mRNA, protein, and activity levels. MMF was found to up-

*DOI: http://dx.doi.org/10.5772/intechopen.91915*

xc

effects on system xc

#### *Repurposing Fumaric Acid Esters to Treat Conditions of Oxidative Stress and Inflammation… DOI: http://dx.doi.org/10.5772/intechopen.91915*

activity of proton-coupled folate transporter (PCFT), a transporter integral to the uptake of folate. This was the first report demonstrating that MMF could regulate folate transport in retinal glial cells and therefore, be potentially useful in the treatment of degenerative retinal diseases. To determine whether, in addition to downregulating pro-inflammatory mechanisms, MMF affects counteractive or protective signaling, in a subsequent study we evaluated also the effect of the compound on the expression and activity of the cysteine/glutamate exchanger SLC7A11 (system xc <sup>−</sup>), a transport system critical for the intracellular entry of the amino acid cysteine which is required for glutathione synthesis [28]. Glutathione is the most abundant endogenous antioxidant in the retina and is therefore essential for the protection of retinal cells against oxidative stress. Further, retinal pigment epithelial (RPE) cells are one of the highest producers of glutathione of any cell type in the body. As such, we exposed human retinal pigment epithelial (ARPE-19) cells to MMF in the presence or absence of pro-oxidant stimuli and evaluated the dose- and time-dependent effects on system xc <sup>−</sup> mRNA, protein, and activity levels. MMF was found to upregulate each of these parameters and additionally, up-regulate hypoxia-inducible factor 1-alpha (Hif-1α), nuclear factor erythroid 2-related factor 2 (Nrf2) expression and increase total reduced glutathione (GSH) content. Collectively, our early *in vitro* studies demonstrated that MMF affects multiple pathways in multiple retinal cell types in a manner that is overall protective against oxidative damage.

We sought next to determine whether our findings extrapolate to the *in vivo* condition, therefore, we evaluated the efficacy of MMF in a living animal model of retinal disease. Retinopathy is a major cause of vision loss in sickle cell disease (SCD) and therapies to prevent and treat sickle retinopathy (SR) are very limited. Therapeutic induction of γ-globin expression and subsequent induction of fetal hemoglobin (HbF) production can alleviate some SCD-associated complications. Interestingly, Nrf2 inducers have been demonstrated to be effective γ-globin inducers [83]. The robust inductive properties of MMF on Nrf2 translocation and activity have been long recognized therefore, it was logical to explore the effects of MMF in SCD. Not only did we confirm that RPE cells, cells integral to retinal health and function, produce HbF but that MMF treatment of Townes humanized SCD mice of SCD resulted in reductions in the expression of pro-oxidant and inflammatory factors and turn, preserved retinal morphology [35]. Shortly after this study, Cho et al. [51] too reported on the potential benefit of MMF in the treatment of retinal disease in a mouse model of retinal ischemia-reperfusion. Specifically, they showed that MMF promotes Nrf2-neuroprotection in this model. MMF treatment was associated with significant increases in the expression of Nrf2-responsive antioxidant genes and a suppression of inflammatory responses as evidenced by increased expression of NAD (P) H quinone dehydrogenase 1, thioredoxin reductase 1 and heme oxygenase-1 along with decrease in interleukin-1β, chemokine (C-C motif) ligands (2, 7 and 12), expressions. Collectively, these molecular improvements interpreted to improved retinal function as evidenced by electroretinogram recordings performed on live mice and were heavily dependent upon the expression and activity of Nrf2.

Because these initial reports of MMF's potential efficacy in protecting against retinal degeneration were conducted acutely, we decided to evaluate the effect of long-term administration of the compound (5 months administration of 15mg/ml MMF in drinking water) in the humanized SCD model [34]. Importantly we found via high-pressure liquid chromatography (HPLC) and hematological analyses of peripheral blood that MMF treatment reduced sickle hemoglobin (HbS) content and white blood cell counts, and improved hematocrit, red blood cell number, and hemoglobin concentrations significantly in SCD mice. In retina specifically, the mRNA and protein expression of well-established markers of inflammation and oxidative stress (i.e., vascular endothelial growth factor, intercellular adhesion molecule-1,

*Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

HO-1 and elevated levels of glutathione, and increased NeuN<sup>+</sup>

the potential viability of this candidate therapy for PD is enhanced.

**4.7 Retinal degenerative diseases**

in the striatum. Moreover, DMF reduced IL-1β levels, cyclooxygenase 2 activities, and neuronal nitrite oxide synthase expression. This treatment also modulated microglial activation (**Figure 3**), restored nerve growth factor levels, and preserved microtubule-associated protein 2 alterations. Using the Nrf2 inhibitor trigonelline, the authors were able to confirm the Nrf2 dependency of the protective mechanism. Collectively, these results demonstrated that DMF protects against experimental PD via NF-κB/Nrf2 pathway [50]. Several other antioxidants have shown potential as therapeutic options for PD, however, because DMF/MMF is already FDA-approved,

In recent years, others and we have investigated the potential of MMF in the treatment of degenerative retinal diseases. In an early study, we showed MMF to be protective against reactive gliosis, a characteristic response of Muller glial cells to an environment rich in pro-oxidant and inflammatory factors in retinal disease. Folate uptake by Muller cells is considered a key event in this process [82]. MMF treatment significantly reduced folate uptake by Muller cells by decreasing the expression and

/Nrf2+

cell number

rates in a dose-dependent manner. Contrary to this, MMF increased these activities *in vitro*. However, both DMF and MMF activated the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 which promoted the nuclear exit of the Nrf2 repressor Bach1 to improve mitochondrial biogenesis. Despite the *in vitro* differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild type but not in *Nrf2−/−* mice. It was concluded that DMF and MMF exhibit neuroprotective effects because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Therefore, the authors advocated for the possible development of MMF rather than DMF as a novel therapy for PD. Synucleinopathies (also called α-synucleinopathies; α-SYN) are neurodegenerative diseases characterized by the abnormal accumulation of aggregates of alpha-synuclein protein in neurons, nerve fibers or glial cells [81]. Lastres-Becker et al. [49] conducted a study in which they focused primarily on the role of DMF in regulating synucleinopathies associated with oxidative stress and inflammation. In brief, an adeno-associated pseudotype 6 (rAAV6) viral vector was used to express human α-SYN under the neuron-specific human synapsin 1 promoter to create conditions of PD and animals were treated daily with DMF (100–300 mg/kg) via oral gavage. DMF protected nigral dopaminergic neurons against α-SYN toxicity and decreased astrocytosis and microgliosis. However, this protective effect was not observed in *Nrf2−/−* mice. Additionally, *in vitro* studies indicated that the neuroprotective effect was correlated with altered regulation of autophagy markers and with a shift in microglial dynamics toward a less pro-inflammatory and a more wound-healing phenotype (**Figure 3**). These experiments provide a compelling rationale for targeting Nrf2 with DMF as a therapeutic strategy to reinforce endogenous brain defense mechanisms against PD-associated synucleinopathy. These findings are supported by another study in which daily oral administration of DMF (10, 30, and 100mg/kg) significantly reduced neuronal cell degeneration of the dopaminergic tract and behavioral impairments induced by four injections of the dopaminergic neurotoxin MPTP. Moreover, treatment with DMF prevented dopamine depletion, increased tyrosine hydroxylase, and dopamine transporter activities, and also reduced the number of α-synuclein-positive neurons. Furthermore, DMF treatment up-regulated Nrf2 as evidenced by the increased activation of SOD2 and

**208**

interleukin-1β, dihydroethidium labeling) was reduced and the development and progression of SCD-like retinal pathology in these mice were ameliorated. Additional related *in vitro* studies performed toward elucidating the molecular mechanisms responsible for the MMF-induced improvements that were observed implicate Nrf2 and Bcl11A (B-cell lymphoma/leukemia 11A) as key players. This study was of extreme significance because not only did it support strongly the notion that fumaric acid ester therapy may be of benefit for the treatment of retinal pathology, especially in SCD, but for SCD in general, a concept that we have since patented [84]. Perhaps equally as astounding is the fact that MMF delivered systemically induced such robust effects in retina, meaning that MMF must be capable of crossing in significant quantities or otherwise inducing signaling across the blood-retinal barriers. Given the known difficulties with non-invasive yet efficacious drug delivery to the posterior segment of the eye (retina) and the commonality of oxidative stress and inflammation as key causative factors in the development and progression of numerous retinal diseases, the clinical relevance and therefore potential impact of the above findings is extremely high. Indeed, new reports of potential benefit derived from MMF in animal models of the degenerative retinal disease continue to surface, such as the recent study by Jiang et al. [52] demonstrating that MMF treatment protects against light-induced retinal damage on BALB/C mice and effect due potentially to HCAR2-dependent signaling in retinal microglia cells (**Figure 3**). Eventually, data emanating from these preclinical reports may spur increased interest in moving toward clinical testing and implementation of FAE therapy in the near future.

### **4.8 Sepsis**

Sepsis is a potentially fatal illness that can lead to the damage of multiple organs [85]. The condition is deeply associated with oxidative stress and inflammation. Firstly, a study by Giustina et al. [53] reported the protective effects of DMF against multi-organ sepsis by modulating oxidative stress and inflammation. It was reported that oral administration of 15 mg/kg of DMF provides significant protection against sepsis-induced multi-organ (heart, liver, and lung) damage in rats. Later, the same research group reported the protective effects of DMF treatment on sepsis-associated inflammation and oxidative stress and cognitive impairment in the brain [54]. Although both these studies were descriptive in nature as neither evaluated in detail the underlying mode of action, they provide evidence that DMF might be used successfully for the clinical management of sepsis. This is supported by a study by Shalmani et al. [55] in which it was reported that 50 mg/kg (i.p.) MMF treatment improved sepsis-induced liver dysfunction by regulating the TLR-4/NF-κB signaling pathway. Collectively, these preclinical studies provide a great foundation for future clinical evaluations of the utility of FAE in the management of organ damage in sepsis.

#### **4.9 Sickle cell disease-associated oxidative stress and inflammation**

Uncontrolled hemolysis and subsequent release of hemoglobin (Hb) and heme into the vasculature is a hallmark of sickle cell disease (SCD) [86, 87]. Heme, a damage-associated molecular pattern, is highly pro-oxidative and proinflammatory and induces vaso-occlusion in murine models of sickle cell disease (SCD) [88]. A study by Belcher et al. evaluated the protective effect of DMF treatment on SCD associated oxidative stress and inflammation in the liver and kidneys [56]. DMF (30 mg/kg/day) or vehicle (0.08% methylcellulose) was administered for 3–7 days to NY1DD and HbSS-Townes SCD mice. DMF had a significant reductive impact on vaso-occlusion in SCD mice. It increased the nuclear translocation

**211**

**4.11 Stroke**

*Repurposing Fumaric Acid Esters to Treat Conditions of Oxidative Stress and Inflammation…*

of Nrf2 and cellular mRNA of Nrf2-responsive genes in livers and kidneys, and increased heme defenses, including HO-1, haptoglobin, hemopexin, and ferritin heavy chain, without altering plasma Hb and heme levels. Markers of inflammation were also reduced. Interestingly, much of the DMF-induced benefit was blunted by the HO-1 inhibitor, protoporphyrin. Chronic treatment (24 weeks) of SCD with DMF decreased hepatic necrosis, inflammatory cytokines, and irregularly shaped erythrocytes, and increased HbF but did not alter hematocrit, reticulocyte counts, lactate dehydrogenase or plasma heme levels or, spleen weights. These results [56] together with our previously highlighted findings in SCD (subSection 4.7) [34, 35], are supportive of the multiple beneficial effects of DMF/MMF on the pathogenesis of SCD and the need for further clinical evaluation of the drug for this indication.

Patients with spinal cord injury (SCI) usually have permanent and often devastating neurologic deficits and disabilities. The currently available therapeutic options include surgical decompression, methylprednisolone and hemodynamic control [89, 90]. Hence, the development of a new therapy for SCI holds great merits. Recent work by Cordaro et al. [57] evaluated the beneficial effects of DMF and MMF in a mouse model of traumatic SCI. Using an aneurysm clip, SCI was induced by extradural compression of the spinal cord at T6-T7 for 1 min. Mice were then treated with 30 mg/kg (i.g) DMF or MMF one and 6 h post-SCI. To evaluate the locomotor activity, study mice were treated with DMF/MMF once daily for 10 days. It was observed that mice treated with DMF exhibited a significant and sustained recovery of motor function. DMF/MMF significantly reduced the severity of inflammation by modulation of pro-inflammatory cytokines and apoptosis factors and increased neurotrophic factors. The authors concluded that the observed results were attributable to reduced secondary inflammation and tissue injury and therefore, DMF may constitute a promising target for future SCI therapies [57]. This study provided the first scientific evidence for the protective role of DMF in the treatment of SCI, however, additional detailed experimental and preclinical studies are needed to identify the potential mechanism(s) of action and enhance the likelihood that this therapy could be advanced to clinical testing and implementation.

Over the past 2 years, researchers worldwide have published several articles on the role of FAE in the treatment of stroke. In one of the early studies on intracerebral hemorrhage (ICH), male rats and mice (including *Nrf2*-deficient animals) were subjected to intracerebral injection of blood and then treated with DMF [40]. In rats, 5 mg/kg DMF was administered at 2 h post-ICH and again orally twice a day on days 1–3, whereas in mice, the same dose of DMF was injected (i.p.) 24 h post-ICH and then at days 2 and 3. Treatment with DMF induced Nrf2-target genes, improved hematoma resolution, reduced brain edema and eventually enhanced neurological recovery in rats and wild type mice, but not in *Nrf2−/−* mice. Based on these findings, the authors proposed that DMF may offer an impressive 24 h therapeutic window of opportunity in which to treat ICH, a concept certainly worthy of further evaluation. The potential of DMF/MMF therapy in ICH is supported further by work by Iniaghe et al. [41] in which male CD-1 mice were subjected to intrastriatal infusion of bacterial collagenase, autologous blood or sham surgery. After ICH, animals either received vehicle, DMF (10 mg or 100 mg/kg) or casein kinase 2 inhibitor (E)-3-(2,3,4,5-tetrabromophenyl) acrylic acid (TBCA). Some mice also received scrambled siRNA or MAFG siRNA 24 h before ICH. DMF

*DOI: http://dx.doi.org/10.5772/intechopen.91915*

**4.10 Spinal cord injury**

*Repurposing Fumaric Acid Esters to Treat Conditions of Oxidative Stress and Inflammation… DOI: http://dx.doi.org/10.5772/intechopen.91915*

of Nrf2 and cellular mRNA of Nrf2-responsive genes in livers and kidneys, and increased heme defenses, including HO-1, haptoglobin, hemopexin, and ferritin heavy chain, without altering plasma Hb and heme levels. Markers of inflammation were also reduced. Interestingly, much of the DMF-induced benefit was blunted by the HO-1 inhibitor, protoporphyrin. Chronic treatment (24 weeks) of SCD with DMF decreased hepatic necrosis, inflammatory cytokines, and irregularly shaped erythrocytes, and increased HbF but did not alter hematocrit, reticulocyte counts, lactate dehydrogenase or plasma heme levels or, spleen weights. These results [56] together with our previously highlighted findings in SCD (subSection 4.7) [34, 35], are supportive of the multiple beneficial effects of DMF/MMF on the pathogenesis of SCD and the need for further clinical evaluation of the drug for this indication.

#### **4.10 Spinal cord injury**

*Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

interleukin-1β, dihydroethidium labeling) was reduced and the development and progression of SCD-like retinal pathology in these mice were ameliorated. Additional related *in vitro* studies performed toward elucidating the molecular mechanisms responsible for the MMF-induced improvements that were observed implicate Nrf2 and Bcl11A (B-cell lymphoma/leukemia 11A) as key players. This study was of extreme significance because not only did it support strongly the notion that fumaric acid ester therapy may be of benefit for the treatment of retinal pathology, especially in SCD, but for SCD in general, a concept that we have since patented [84]. Perhaps equally as astounding is the fact that MMF delivered systemically induced such robust effects in retina, meaning that MMF must be capable of crossing in significant quantities or otherwise inducing signaling across the blood-retinal barriers. Given the known difficulties with non-invasive yet efficacious drug delivery to the posterior segment of the eye (retina) and the commonality of oxidative stress and inflammation as key causative factors in the development and progression of numerous retinal diseases, the clinical relevance and therefore potential impact of the above findings is extremely high. Indeed, new reports of potential benefit derived from MMF in animal models of the degenerative retinal disease continue to surface, such as the recent study by Jiang et al. [52] demonstrating that MMF treatment protects against light-induced retinal damage on BALB/C mice and effect due potentially to HCAR2-dependent signaling in retinal microglia cells (**Figure 3**). Eventually, data emanating from these preclinical reports may spur increased interest in moving toward clinical testing and implementation of FAE therapy in the near future.

Sepsis is a potentially fatal illness that can lead to the damage of multiple organs [85]. The condition is deeply associated with oxidative stress and inflammation. Firstly, a study by Giustina et al. [53] reported the protective effects of DMF against multi-organ sepsis by modulating oxidative stress and inflammation. It was reported that oral administration of 15 mg/kg of DMF provides significant protection against sepsis-induced multi-organ (heart, liver, and lung) damage in rats. Later, the same research group reported the protective effects of DMF treatment on sepsis-associated inflammation and oxidative stress and cognitive impairment in the brain [54]. Although both these studies were descriptive in nature as neither evaluated in detail the underlying mode of action, they provide evidence that DMF might be used successfully for the clinical management of sepsis. This is supported by a study by Shalmani et al. [55] in which it was reported that 50 mg/kg (i.p.) MMF treatment improved sepsis-induced liver dysfunction by regulating the TLR-4/NF-κB signaling pathway. Collectively, these preclinical studies provide a great foundation for future clinical evaluations of the utility of FAE in the manage-

**4.9 Sickle cell disease-associated oxidative stress and inflammation**

Uncontrolled hemolysis and subsequent release of hemoglobin (Hb) and heme into the vasculature is a hallmark of sickle cell disease (SCD) [86, 87]. Heme, a damage-associated molecular pattern, is highly pro-oxidative and proinflammatory and induces vaso-occlusion in murine models of sickle cell disease (SCD) [88]. A study by Belcher et al. evaluated the protective effect of DMF treatment on SCD associated oxidative stress and inflammation in the liver and kidneys [56]. DMF (30 mg/kg/day) or vehicle (0.08% methylcellulose) was administered for 3–7 days to NY1DD and HbSS-Townes SCD mice. DMF had a significant reductive impact on vaso-occlusion in SCD mice. It increased the nuclear translocation

**210**

**4.8 Sepsis**

ment of organ damage in sepsis.

Patients with spinal cord injury (SCI) usually have permanent and often devastating neurologic deficits and disabilities. The currently available therapeutic options include surgical decompression, methylprednisolone and hemodynamic control [89, 90]. Hence, the development of a new therapy for SCI holds great merits. Recent work by Cordaro et al. [57] evaluated the beneficial effects of DMF and MMF in a mouse model of traumatic SCI. Using an aneurysm clip, SCI was induced by extradural compression of the spinal cord at T6-T7 for 1 min. Mice were then treated with 30 mg/kg (i.g) DMF or MMF one and 6 h post-SCI. To evaluate the locomotor activity, study mice were treated with DMF/MMF once daily for 10 days. It was observed that mice treated with DMF exhibited a significant and sustained recovery of motor function. DMF/MMF significantly reduced the severity of inflammation by modulation of pro-inflammatory cytokines and apoptosis factors and increased neurotrophic factors. The authors concluded that the observed results were attributable to reduced secondary inflammation and tissue injury and therefore, DMF may constitute a promising target for future SCI therapies [57]. This study provided the first scientific evidence for the protective role of DMF in the treatment of SCI, however, additional detailed experimental and preclinical studies are needed to identify the potential mechanism(s) of action and enhance the likelihood that this therapy could be advanced to clinical testing and implementation.

#### **4.11 Stroke**

Over the past 2 years, researchers worldwide have published several articles on the role of FAE in the treatment of stroke. In one of the early studies on intracerebral hemorrhage (ICH), male rats and mice (including *Nrf2*-deficient animals) were subjected to intracerebral injection of blood and then treated with DMF [40]. In rats, 5 mg/kg DMF was administered at 2 h post-ICH and again orally twice a day on days 1–3, whereas in mice, the same dose of DMF was injected (i.p.) 24 h post-ICH and then at days 2 and 3. Treatment with DMF induced Nrf2-target genes, improved hematoma resolution, reduced brain edema and eventually enhanced neurological recovery in rats and wild type mice, but not in *Nrf2−/−* mice. Based on these findings, the authors proposed that DMF may offer an impressive 24 h therapeutic window of opportunity in which to treat ICH, a concept certainly worthy of further evaluation. The potential of DMF/MMF therapy in ICH is supported further by work by Iniaghe et al. [41] in which male CD-1 mice were subjected to intrastriatal infusion of bacterial collagenase, autologous blood or sham surgery. After ICH, animals either received vehicle, DMF (10 mg or 100 mg/kg) or casein kinase 2 inhibitor (E)-3-(2,3,4,5-tetrabromophenyl) acrylic acid (TBCA). Some mice also received scrambled siRNA or MAFG siRNA 24 h before ICH. DMF

treatment reduced Evans blue dye extravasation, decreased brain water content, microglia activation (**Figure 3**), ICAM-1 expression and, improved neurological deficits and casein kinase 2 levels. Interestingly, TBCA and MAFG siRNA blunted protection afforded by DMF. Hence, it was concluded that DMF reduced inflammation, blood-brain barrier permeability, and improved neurological outcomes via casein kinase 2 and Nrf2 signaling pathways in mice.

Similar to other neurodegenerative disorders, oxidative stress is common also to the pathogenesis of ischemic stroke, potentiating the neuronal malfunction and cell death characteristic of this disease [91]. Given that the up-regulation of antioxidant genes through activation of the Nrf2 is one of the key mechanisms of cellular defense against oxidative stress [92], it is logical to explore the efficacy of FAE therapy in this condition. Congruent with this, three additional groups used experimental models of ischemic stroke to evaluate the efficacy of FAEs. In 2016, Lin et al. [36] observed that MMF (25–100 μM) rescued cultured cortical neurons from oxygen–glucose deprivation (OGD) and suppressed pro-inflammatory cytokines produced by primary mixed neuron/glia cultures subjected to OGD. In rats, DMF treatment (25 or 50 mg/kg twice daily) significantly decreased infarction volume by nearly 40% and significantly improved neurobehavioral deficits after middle cerebral artery occlusion (MCAO). In the acute early phase (72 h after MCAO), DMF induced Nrf2 expression and its downstream mediator HO-1. In addition to its antioxidant role, DMF also acted as a potent immunomodulator, reducing the infiltration of neutrophils and T-cells as well as the number of activated microglia/ macrophages in the infarct region. Concomitantly, levels of pro-inflammatory cytokines were greatly reduced in the plasma and brain and oxygen–glucose deprived neuron/glia cultures. Further, using a mouse model of transient focal brain ischemia, Yao et al. [37] showed that DMF and MMF (30 mg/kg i.p.) significantly reduced neurological deficits, infarct volume, brain edema, and cell death. Additionally, DMF and MMF suppress glial activation following brain ischemia. Importantly, the protection of DMF and MMF was most evident during the sub-acute stage and was abolished in *Nrf2<sup>−</sup>/<sup>−</sup>* mice, indicating that the Nrf2 pathway is required for the beneficial effects of DMF and MMF [37]. In another study, murine organotypic hippocampal slice cultures, and two neuronal cell lines were treated with DMF and MMF [93]. The ischemic condition was generated by exposing cells and slice cultures to oxygen-glucose deprivation. Treatment with both DMF and MMF (30–100 μM) immediately upon reoxygenation strongly reduced cell death in hippocampal cultures *ex vivo*. Both DMF and MMF promoted neuronal survival in HT-22 and SH-SY5Y cell lines exposed to ischemic stress. However, interestingly, DMF but not MMF activated the anti-oxidative Nrf2 pathway in neurons. Accordingly, the protective effect of DMF but not MMF was abrogated in the neurons of Nrf2-deficient mice. These results provide the basis for a new therapeutic approach to treat ischemic pathologies such as stroke using a drug that is already approved by US-FDA for clinical use.
