**6. Conclusions**

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

casein kinase 2 and Nrf2 signaling pathways in mice.

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

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

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.

By and large, the short-term safety profile for DMF in patients with RMS is highly favorable [64, 65] and long-term safety analyses from the ENDORSE study sustains a favorable benefit: risk ratio [94]. The most common adverse events observed in patients receiving DMF include flushing, gastrointestinal (GI) events (e.g., diarrhea, nausea, abdominal pain, and vomiting), proteinuria, and pruritus [64, 65]. Aspirin pretreatment has been shown to reduce DMF induced adverse GI events [95]. Additionally, the leukotriene-receptor antagonist montelukast has been shown to help as well [96]. Further, it has been observed that consuming a high fat

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*Nrf2<sup>−</sup>/<sup>−</sup>*

**5. Safety profile**

Drug repurposing is a very viable therapeutic strategy [18]. Many agents approved for other uses already have been tested in humans, so detailed information is available on their pharmacology, formulation and potential side effects. Since repurposing expands upon past innovative endeavors, hopeful new treatments could be prepared for clinical trials rapidly. Historically, pharmaceutical companies have achieved a number of successes via drug repositioning (e.g., for Viagra, thalidomide, metformin, etc.). Based on the literature available, DMF/MMF has been shown to protect against a variety of diseases other than MS and psoriasis.

## **7. Future perspectives**

FAE are perhaps most noted for the robust antioxidant effects that they elicit via Nrf2 induction. A number of additional (non-FAE based) Nrf2 inducing drugs have been developed and tested in experimental and clinical systems in recent years (e.g., resveratrol, sulforaphane, etc.) and several have been with considerable success with regard to potential for clinical development [111]. However, the multimodal actions of FAE make this emerging drug stand out among the rest. It is commonly said that oxidative stress and inflammation go hand-in-hand, meaning that one potentiates the other in somewhat of a cyclic manner. Thus, it can only be hoped that in turn, if one is suppressed then the other similarly complies. However, things are usually not that simple. In the case of FAE, there are two arms of action: one induces Nrf2 and the other interacts with the anti-inflammatory hydroxycarboxylic acid receptor (HCAR2 or HCA2; **Figure 2**). Thus, the compound has a direct impact on inflammation independent of its actions on oxidative stress. The fascinating thing about these two mechanistic arms, is that they appear to act simultaneously in many cell and tissue systems. This may explain why FAE has exceled in so many variable pathologic conditions. MMF through its interaction with HCAR2, which is expressed by primary immune cells and a multitude of accessory immune cells (i.e., those that initiate the immune response and those cells like retinal pigment

epithelial cells, for example, that aren't truly "immune" cells but are capable just the same of secreting pro- and anti-inflammatory factors depending upon the stimulus), elicits a tremendous anti-inflammatory response. The combined Nrf2 inducing and immune-modulatory properties of FAE have enabled this drug to be efficacious in a broad range of body systems. The evidence provided in this chapter alone demonstrates convincingly that the benefits of FAE have been realized in the central nervous system (brain and retina), the cardiovascular system, the digestive and/or gastrointestinal system, the immune system, the integumentary system and the renal system; this list continues to grow. Thus, the potential clinical impact of FAE therapy use is high and importantly extremely broad. It is acknowledged that as with virtually all pharmacologic agents, FAE therapy is not without adverse effects. Importantly, however, the effects are relatively mild and the benefit(s) indisputably outweigh the risks. As such, there is a prompt need for additional experimental and clinical studies to translate the information gleaned from exploratory trials of FAE therapy in various cell, tissue, and disease types into clinical use.
