**4.3 Nephrotoxicity**

Very little information exists on the protective effect of FAEs on renal function. A study by Takasu et al. [42] evaluated the effect of DMF treatment on CsA (calcineurin inhibitor)-induced nephrotoxicity. Male *Sprague–Dawley* rats were treated with 20 mg/kg CsA or CsA + 50 mg/kg DMF (i.g.) for 28 days. At the end of the treatment schedule, renal function, histopathology, malondialdehyde (MDA), myeloperoxidase levels, and antioxidant enzyme expression were determined. DMF co-treatment ameliorated CsA-induced renal dysfunction as evidenced by a significant decrease in serum creatinine and urea levels, as well as improvement of creatinine clearance. DMF also significantly decreased serum and renal MDA

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

every 24 h, starting 3 h after the administration of DNBS and continuing over the course of 4 days. DMF treatment to DNBS treated mice significantly improved colon injury and histological score. Further DMF also reduced lipid peroxidation by regulating the expression of SOD2 (superoxide dismutase 2, mitochondrial) and Nrf2. The anti-inflammatory effect of DMF was evident by a reduction in the expression of TNF-α (tumor necrosis factor- α), IL-1β (Interleukin 1 beta) and ICAM-1 (intercellular adhesion molecule 1) and P- selectin. This effect was thought to be a result of reduced IκB-α degradation to prevent nuclear translocation of p65 NF-κB (Nuclear factor-κB). Moreover, *in vitro* DMF treatment improved hydrogen peroxide-induced barrier dysfunction of human intestinal epithelial cells. The authors also confirmed the protective effect of DMF on experimental colitis using another model (9-weekold IL-10KO mice). Collectively, this study demonstrated that DMF could reduce experimental colitis by regulating inflammation and oxidative stress [38]. In another study, Liu et al., 2016 evaluated the efficacy of DMF in reducing DSS-induced murine colitis. Wild-type and *Nrf2−/−* mice received either vehicle or 3% (w/v) DSS in drinking water for 7 days and thereafter provided with only drinking water for another 3 days. Groups of mice were also given 30 or 60 mg/kg DMF (i.g.) from day 1 to 10. DMF treatment significantly reduced oxidative stress and inflammation and thereby improved signs/symptoms of colitis in DSS-treated mice. However, these

mechanism of action of the drug. This was supported by additional *in vitro* studies in which the authors showed that DMF-mediated Nrf2 activation reduces NLRP3 (NLR family pyrin domain containing 3) inflammasome activation to control intestinal

Consistent with the above gastrointestinal benefits derived from DMF/MMF treatment, the efficacy of MMF treatment in improving stomach ulcers in rats has also been described. Although the detailed mechanism of action was not evaluated, the authors attributed the protective effect of the compound in this condition to be due primarily to the anti-inflammatory activity of MMF [39]. Collectively, these studies indicate that DMF/MMF therapy may be of benefit the clinical management of inflammatory gastrointestinal disorders. This is interesting given that gastrointestinal (GI) side effects (e.g., nausea, vomiting, diarrhea, and upper abdominal pain) are one of the most commonly reported complaints in patients receiving DMF therapy [62, 63]. Indeed, during phase 3 clinical trials for multiple sclerosis, adverse events (AEs) involving the GI system were reported in 40% of patients treated with DMF compared with 30% of patients treated with placebo [64, 65]. Though the adverse GI events are generally mild in severity and typically resolve within the first 2 months of treatment, these issues may impact patient quality of life and ultimately medication adherence. Thus, while a number of experimental studies have reported gastroprotective effects of DMF, there is some concern as to whether such therapy could reliably be extrapolated to clinical management of gastrointestinal disorders in human patients. However, the increasing number of additional reports of DMF/MMF benefit in the digestive system that continue to arise in the scientific literature suggests that perhaps efforts to implement DMF/MMF therapy for use in this regard should not be dismissed completely. For example, Rao and Mishra [66] performed a preliminary study demonstrating the hepatoprotective effects of MMF isolated from *Fumaria Indica* extract in various models of hepatotoxicity. Although the study was preliminary and had some limitations, it does introduce a possible hepatoprotective effect of MMF. This is supported also by a recent study by Abdelrahman et al. [67] that reported the protective effects of DMF treatment on acetaminophen-induced hepatic injury in mice. Acetaminophen-treated mice receiving a single or double dose of DMF (100 mg/kg) showed reduced oxidative stress, inflammation, and associated liver damage compared to non-DMF treated

mice, highlighting the importance of the Nrf2-mediated

**204**

effects were lost in *Nrf2<sup>−</sup>/<sup>−</sup>*

inflammation.

and myeloperoxidase contents whereas, protein expression of NQO-1 (NAD (P) H quinone oxidoreductase-1), a major cellular antioxidant and the detoxifying enzyme, was significantly enhanced by DMF administration. Although evidence is limited, the above study supports the protective potential of DMF/MMF therapy in a clinically relevant model of nephrotoxicity, an effect that is afforded in part via DMF's robust ability to enhance the cellular antioxidant capacity and thereby, inhibit oxidative stress and inflammation [42] as described in other cell and tissue systems. Thus, while much remains to be learned about the possible use of DMF/ MMF in the treatment of renal diseases, initial results are encouraging.

## **4.4 Non-HIV related neurotoxicity**

Prior discussion (subSection 4.2) of neurotoxicity in this chapter was related specifically to that occurring in HIV. Irrespective, however, of the mitigating disease or pathologic process, the brain is indisputably sensitive to pro-inflammatory and/or oxidative insult. Hence, neurotoxicity can emanate from multiple variable causes. Kume et al. [43] evaluated the ability of DMF to protect against *in vitro* and *in vivo* oxidative stress in the central nervous system induced via pro-oxidant agents like sodium nitroprusside and hydrogen peroxide (H2O2). DMF pretreatment (60–200 mg/kg) for 24 h dose-dependently protected against 10 nM sodium nitroprusside-induced brain damage and in rat primary striatal cell cultures, 10 μM DMF markedly prevented cytotoxicity stemming from exposure of cells to H2O2 (1 mM). Interestingly, the protective effects of DMF against *in vitro* oxidative stress were countered by the HO-1 inhibitor zinc protoporphyrin IX however, buthionine sulfoximine, an inhibitor of glutathione synthesis, did not interfere with the protection afforded by DMF. Collectively, these results support the potential of DMF/ MMF therapy in conditions of neurotoxicity and suggest that its ability to activate HO-1 may be critical. Neural stem/progenitor cells (NPCs) are a heterogeneous population of self-renewing and multi-potent cells that can differentiate into neurons, astrocytes, or oligodendrocytes (post-mitotic daughter cells) [73, 74]. Hence, the survival of these cells could greatly impact various forms of neurodegenerative diseases. Wang et al. [75] reported on the neuroprotective effects of DMF on mouse and rat neural stem/progenitor cells (NPCs) and neurons. DMF treatment reduced reactive oxygen species (ROS) production, increased the frequency of the multipotent neurospheres and enhanced the survival of NPCs following H2O2-mediated oxidative stress. DMF also decreased oxidative stress-induced apoptosis and promoted the survival of motor neurons, effects that this group demonstrated to be mediated via the Nrf2-ERK1/2 MAPK pathway. These studies provide additional support of the overwhelmingly protective effects of FAE in multiple brain cell types and therefore, of the potential feasibility of this therapy in the prevention and treatment of neurodegenerative diseases.

#### **4.5 Pancreatitis**

Chronic pancreatitis (CP) is a progressive inflammatory disorder that results in the destruction and fibrosis of the pancreatic parenchyma and its endocrine and exocrine dysfunctions [76]. Various research groups have evaluated the effect of DMF treatment on acute and chronic pancreatitis. In one of the studies, chronic pancreatitis in rats was induced by five injections of 250 mg/100 g L-Arginine and sacrificed 7 weeks later. In another group 25 mg/kg DMF was given orally 24 before L-arginine treatment and continued thereafter until the end of the study. DMF treatment significantly improved glucose tolerance, pancreas histology, biochemical parameters (MDA and MPO; myeloperoxidase), and induced HO-1 expression [44].

**207**

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

However, this study did not evaluate the mechanism of action for DMF-induced protection. Another study by Robles et al. [45] evaluated the efficacy of DMF in an acute model of pancreatitis. Acute pancreatitis was induced by two injections of 3 g/kg L-Arginine (1 hr. apart) to rats and sacrificed later at 24 and 72 hr. DMF (25 mg/kg) was orally administered to rats 24 h before L-arginine and continued until sacrifice. The histology of the pancreas was significantly improved in DMFtreated animals possibly due to decreased cleaved caspase-3 (apoptosis) and MDA levels. This group additionally stimulated splenocytes with 1 μg/ml for 24 h with or without DMF 20 μM. *In vitro* DMF treatment significantly reduced proinflammatory cytokine secretion in rat splenocytes, although a definitive mechanism for this DMF-mediated action was not put forward. Recently, however, Zhang and colleagues [46] too evaluated the effect of DMF on L-arginine induced chronic pancreatitis. In brief, this group treated *Wistar* rats intraperitoneally with L-arginine 5 times (250 mg/100 kg, twice per time, each interval of 1 h) to induce chronic pancreatitis (CP). One group of rats was treated with 20 mg/kg DMF. Compared with control (untreated) group, the weight of rats in CP group was significantly reduced at weeks 2, 4 and 6; blood glucose levels were significantly increased, the histopathological scores of pancreatic atrophy, acinar injury, edema, and cellular infiltration increased, levels of MDA and MPO increased, and the islet equivalent and islet activity decreased at 0, 30, 60, 120 and 180 min., parameters that were all prevented or reversed in the DMF-treated CP group. Thus, DMF treatment can protect against CP induced by L-arginine and islet function in rats. Although these three studies support the potential of DMF/MMF therapy in pancreatitis, the exact mechanism (s) to explain the benefits attained remains unknown. Because therapies to impact pancreatitis are extremely limited at present, additional detailed studies to test the efficacy of FAE in this condition would certainly be worthwhile in hopes that findings emanating therefrom could be carried forward to use in a clinical setting.

Again, the brain is especially sensitive to perturbations caused by oxidative and/or inflammatory stress. In fact, these factors, particularly oxidative stress, are central to the pathology of several neurodegenerative diseases, including Parkinson's disease (PD) [77, 78] therefore, therapies designed to enhance antioxidant potential and counter this stress may be of clinical value [79, 80]. Scientific studies published within the last couple of years highlight the high clinical potential the repurposing of DMF/MMF for the treatment of PD holds. Using various *in vitro* and *in vivo* studies it has been demonstrated that DMF/MMF induced Nrf2 signaling can protect against oxidative stress and inflammatory conditions related to PD. In an initial study by Jing et al. [47], DMF (2–4 μM) pre-treatment significantly reduced hydroxydopamine (6-OHDA) induced generation of ROS and subsequent cytotoxicity in SH-SY5Y cells. The increase in ROS production caused by 6-OHDA treatment was also attenuated by DMF. Further, siNrf2 treatment blocked DMF's protection against 6-OHDA-induced neurotoxicity. *In vivo*, oral administration of DMF (50 mg/kg) to C57BL/6 mice up-regulated expression of Nrf2 and Nrf2-dependent cytoprotective genes. Taken together, this study provided initial evidence for the protective role of DMF in PD. This was followed by three different studies focusing on the mechanism of action for DMF and its metabolite, MMF in mediated protection against PD. Ahuja et al. [48] compared the effects of DMF and MMF on Nrf2 signaling by evaluating its ability to block 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-induced experimental PD. Their results showed that Nrf2 activation by DMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis

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

**4.6 Parkinson's disease**

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

However, this study did not evaluate the mechanism of action for DMF-induced protection. Another study by Robles et al. [45] evaluated the efficacy of DMF in an acute model of pancreatitis. Acute pancreatitis was induced by two injections of 3 g/kg L-Arginine (1 hr. apart) to rats and sacrificed later at 24 and 72 hr. DMF (25 mg/kg) was orally administered to rats 24 h before L-arginine and continued until sacrifice. The histology of the pancreas was significantly improved in DMFtreated animals possibly due to decreased cleaved caspase-3 (apoptosis) and MDA levels. This group additionally stimulated splenocytes with 1 μg/ml for 24 h with or without DMF 20 μM. *In vitro* DMF treatment significantly reduced proinflammatory cytokine secretion in rat splenocytes, although a definitive mechanism for this DMF-mediated action was not put forward. Recently, however, Zhang and colleagues [46] too evaluated the effect of DMF on L-arginine induced chronic pancreatitis. In brief, this group treated *Wistar* rats intraperitoneally with L-arginine 5 times (250 mg/100 kg, twice per time, each interval of 1 h) to induce chronic pancreatitis (CP). One group of rats was treated with 20 mg/kg DMF. Compared with control (untreated) group, the weight of rats in CP group was significantly reduced at weeks 2, 4 and 6; blood glucose levels were significantly increased, the histopathological scores of pancreatic atrophy, acinar injury, edema, and cellular infiltration increased, levels of MDA and MPO increased, and the islet equivalent and islet activity decreased at 0, 30, 60, 120 and 180 min., parameters that were all prevented or reversed in the DMF-treated CP group. Thus, DMF treatment can protect against CP induced by L-arginine and islet function in rats. Although these three studies support the potential of DMF/MMF therapy in pancreatitis, the exact mechanism (s) to explain the benefits attained remains unknown. Because therapies to impact pancreatitis are extremely limited at present, additional detailed studies to test the efficacy of FAE in this condition would certainly be worthwhile in hopes that findings emanating therefrom could be carried forward to use in a clinical setting.

#### **4.6 Parkinson's disease**

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

MMF in the treatment of renal diseases, initial results are encouraging.

**4.4 Non-HIV related neurotoxicity**

ment of neurodegenerative diseases.

**4.5 Pancreatitis**

and myeloperoxidase contents whereas, protein expression of NQO-1 (NAD (P) H quinone oxidoreductase-1), a major cellular antioxidant and the detoxifying enzyme, was significantly enhanced by DMF administration. Although evidence is limited, the above study supports the protective potential of DMF/MMF therapy in a clinically relevant model of nephrotoxicity, an effect that is afforded in part via DMF's robust ability to enhance the cellular antioxidant capacity and thereby, inhibit oxidative stress and inflammation [42] as described in other cell and tissue systems. Thus, while much remains to be learned about the possible use of DMF/

Prior discussion (subSection 4.2) of neurotoxicity in this chapter was related specifically to that occurring in HIV. Irrespective, however, of the mitigating disease or pathologic process, the brain is indisputably sensitive to pro-inflammatory and/or oxidative insult. Hence, neurotoxicity can emanate from multiple variable causes. Kume et al. [43] evaluated the ability of DMF to protect against *in vitro* and *in vivo* oxidative stress in the central nervous system induced via pro-oxidant agents like sodium nitroprusside and hydrogen peroxide (H2O2). DMF pretreatment (60–200 mg/kg) for 24 h dose-dependently protected against 10 nM sodium nitroprusside-induced brain damage and in rat primary striatal cell cultures, 10 μM DMF markedly prevented cytotoxicity stemming from exposure of cells to H2O2 (1 mM). Interestingly, the protective effects of DMF against *in vitro* oxidative stress were countered by the HO-1 inhibitor zinc protoporphyrin IX however, buthionine sulfoximine, an inhibitor of glutathione synthesis, did not interfere with the protection afforded by DMF. Collectively, these results support the potential of DMF/ MMF therapy in conditions of neurotoxicity and suggest that its ability to activate HO-1 may be critical. Neural stem/progenitor cells (NPCs) are a heterogeneous population of self-renewing and multi-potent cells that can differentiate into neurons, astrocytes, or oligodendrocytes (post-mitotic daughter cells) [73, 74]. Hence, the survival of these cells could greatly impact various forms of neurodegenerative diseases. Wang et al. [75] reported on the neuroprotective effects of DMF on mouse and rat neural stem/progenitor cells (NPCs) and neurons. DMF treatment reduced reactive oxygen species (ROS) production, increased the frequency of the multipotent neurospheres and enhanced the survival of NPCs following H2O2-mediated oxidative stress. DMF also decreased oxidative stress-induced apoptosis and promoted the survival of motor neurons, effects that this group demonstrated to be mediated via the Nrf2-ERK1/2 MAPK pathway. These studies provide additional support of the overwhelmingly protective effects of FAE in multiple brain cell types and therefore, of the potential feasibility of this therapy in the prevention and treat-

Chronic pancreatitis (CP) is a progressive inflammatory disorder that results in the destruction and fibrosis of the pancreatic parenchyma and its endocrine and exocrine dysfunctions [76]. Various research groups have evaluated the effect of DMF treatment on acute and chronic pancreatitis. In one of the studies, chronic pancreatitis in rats was induced by five injections of 250 mg/100 g L-Arginine and sacrificed 7 weeks later. In another group 25 mg/kg DMF was given orally 24 before L-arginine treatment and continued thereafter until the end of the study. DMF treatment significantly improved glucose tolerance, pancreas histology, biochemical parameters (MDA and MPO; myeloperoxidase), and induced HO-1 expression [44].

**206**

Again, the brain is especially sensitive to perturbations caused by oxidative and/or inflammatory stress. In fact, these factors, particularly oxidative stress, are central to the pathology of several neurodegenerative diseases, including Parkinson's disease (PD) [77, 78] therefore, therapies designed to enhance antioxidant potential and counter this stress may be of clinical value [79, 80]. Scientific studies published within the last couple of years highlight the high clinical potential the repurposing of DMF/MMF for the treatment of PD holds. Using various *in vitro* and *in vivo* studies it has been demonstrated that DMF/MMF induced Nrf2 signaling can protect against oxidative stress and inflammatory conditions related to PD. In an initial study by Jing et al. [47], DMF (2–4 μM) pre-treatment significantly reduced hydroxydopamine (6-OHDA) induced generation of ROS and subsequent cytotoxicity in SH-SY5Y cells. The increase in ROS production caused by 6-OHDA treatment was also attenuated by DMF. Further, siNrf2 treatment blocked DMF's protection against 6-OHDA-induced neurotoxicity. *In vivo*, oral administration of DMF (50 mg/kg) to C57BL/6 mice up-regulated expression of Nrf2 and Nrf2-dependent cytoprotective genes. Taken together, this study provided initial evidence for the protective role of DMF in PD. This was followed by three different studies focusing on the mechanism of action for DMF and its metabolite, MMF in mediated protection against PD. Ahuja et al. [48] compared the effects of DMF and MMF on Nrf2 signaling by evaluating its ability to block 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-induced experimental PD. Their results showed that Nrf2 activation by DMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis

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 HO-1 and elevated levels of glutathione, and increased NeuN<sup>+</sup> /Nrf2+ cell number 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, the potential viability of this candidate therapy for PD is enhanced.
