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While free radicals and inflammation constitute major routes of neuronal injury occurring in neurodegenerative diseases, neither antioxidants nor nonsteroidal anti-inflammatory drugs (NSAIDs) have shown significant efficacy in human clinical trials. To explore the possibility that concurrent blockade of free radicals and PGE2-mediated inflammation might constitute a safe and effective therapeutic approach to certain neurodegenerative diseases, we have developed 2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobezoic acid (AAD-2004) as a derivative of aspirin. AAD-2004 completely removed free radicals at 50 nM as a potent spin trapping molecule and inhibited microsomal prostaglandin E synthase-1 (mPGES-1) with an IC50 of 230 nM. Oral administration of AAD-2004 blocked free radical formation, PGE2 formation, and microglial activation in the spinal motor neurons of SOD1 mice. As a consequence, AAD-2004 reduced autophagosome formation, axonopathy, and motor neuron degeneration, improving motor function and increasing life span. In these assays, AAD-2004 was superior to ibuprofen or riluzole. Gastric bleeding was not induced by AAD-2004 even at a dose 400-fold higher than that required to obtain maximal therapeutic efficacy in SOD1 mice. Targeting both mPGES-1 and free radicals may be a promising approach to reduce neurodegeneration in ALS and possibly other neurodegenerative diseases. Introduction Extensive evidence supports the central role of free radicals in the pathogenesis of amyotrophic lateral sclerosis (ALS) as well as other neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Elevated oxidative products of protein, DNA, and lipid have been reported in the brain, spinal cord, and cerebrospinal fluid (CSF) in subjects with ALS . Transgenic (SOD1) mice that overexpress mutant SOD1 in familial ALS show motor neuron degeneration, movement deficit, and decreased survival rates . In SOD1 mice, oxidative stress is induced in spinal cord regions known to undergo the pathological changes in ALS . Excess accumulation of pro-oxidants such as iron is also observed and expected to cause neurodegeneration in familial as well as sporadic forms of ALS . A causative role for oxidative stress in the neurodegenerative pathology is supported by experimental findings that administration of antioxidants reduces neurological deficits apparent in SOD1 mice . However, two clinical trials using oral supplementation of vitamin E at either 500 mg/d for 12 months or 1500 mg/d for 18 months produced no beneficial effect on mortality in ALS patients . The therapeutic potential of anti-oxidants such as vitamin E, N-acetylcysteine and Coenzyme Q10 that were advanced to clinical trial for ALS was limited by side effects and poor BBB permeability . Antioxidants that are capable of effectively and safely removing free radicals in the nervous system are needed to conduct a proof of concept study for ALS patients. Inflammation constitutes an additional contributor to the neurodegenerative events observed in ALS. Inflammatory responses such as PGE2, tumor necrosis factor-alpha (TNF-α), and C-reactive protein are significantly elevated in serum and CSF of patients with ALS . In particular, COX-2, the inducible isoform of cyclooxygenase (COX), is induced in neurons, microglia, astrocytes, and endothelial cells in both SOD1 mice and patients with ALS . COX-2 is thought to mediate inflammation and neuronal injury in the spinal cord of SOD1 mice through the generation of PGE2 . Celecoxib, a selective COX-2 inhibitor, reduced levels of PGE2 and neuronal death in the spinal cord and prolonged survival in SOD1 mice . However, chronic treatment with the maximum dosage of celecoxib (800 mg/d) for 12 months improved neither motor function nor survival in ALS patients . As levels of PGE2, a surrogate marker for the pharmacological action of celecoxib, were not reduced in the cerebrospinal fluid of ALS patients treated with celecoxib, it remained to be resolved if selective COX-2 inhibitors would attenuate PGE2, neurodegeneration, and neurological deficits in ALS patients in the absence of adverse gastric and cardiovascular effects. While antioxidants or anti-inflammatory drugs (e.g. COX inhibitors) have reduced disease progression in SOD1 mice, a combination approach targeting both free radicals and inflammation may synergistically improve motor function and survival. In the SOD1 mice, combined treatment of the selective COX-2 inhibitors, celecoxib or rofecoxib, with creatine, a mitochondrial transition pore stabilizer shown to reduce oxidative stress in SOD1 mice , resulted in additive neuroproection and survival compared to the COX-2 inhibitors alone . A phase II clinical trial of combination therapy for ALS patients revealed that the combination of celecoxib and creatine produced slower deterioration in the ALS Functional Rating ScaleRevised than the historical controls or the combination of minocycline and creatine . Thus, concurrent blockade of free radicals and PGE2-mediated inflammation may provide better therapeutic outcome than monotherapy for intervention of neurodegenerative process and neurological deficits in ALS patients. We have investigated the premise that a single agent combining the anti-inflammatory attributes of aspirin with powerful anti-oxidant efficacy would constitute an effective disease modifying therapeutic for ALS, based on the additive/synergistic neuroprotective effects of these two actions. We took a structural lead from sulfasalazine, and developed synthetic derivatives conjugated to 5-aminosalicylate that prevent free radical formation, as well as inflammation without causing gastric damage. AAD-2004 was chosen as a final drug candidate, based upon safety and efficacy profile through multiple in vitro and in vivo screening processes. Result AAD-2004 blocks free radical neurotoxicity as a potent spin trapping molecule. Mixed cortical cell cultures containing neurons and glia produced reactive oxygen species (ROS) within 4 h and widespread neuronal death over 24 h after continuous exposure to 50 μM Fe, a transition metal ion catalyzing hydroxyl radicals from H2O2. Concurrent addition of 1 μM AAD-2004 blocked Fe-induced ROS production and neuronal death (Fig. 1a). The efficacy and potency of AAD-2004 were compared to those of antioxidants that were included in clinical trials for treatment of neurodegenerative diseases. Vitamin E, a free radical scavenger, attenuated Fe neurotoxicity in a dose-dependent manner (IC50 = 22.03 μM). Estrogen and melatonin revealed an IC50 of 2.41 μM and 311.6 μM in reducing Fe neurotoxicity, respectively. However, administration of acetyl-L-carnitine up to 1 mM slightly reduced Fe-induced neuronal death. In contrast, AAD-2004 showed IC50 of 0.097 μM and all but completely blocked Fe neurotoxicity even at 0.3 μM (Fig. 1b), suggesting that AAD2004 has better efficacy and potency against free radical neurotoxicity than the other antioxidants examined. AAD-2004 also protected against free radical injury by DLbuthionine-[S,R]-sulfoximine, a glutathione-depleting agent, and sodium nitroprusside, a nitric oxide donor (Unpublished data). As salicylate and acetyl salicylate (aspirin) can scavenge free radicals at millimolar concentrations , the antioxidant action of AAD-2004 may be attributable to direct scavenging of free radicals. Salicylate, aspirin, and sulfasalazine slightly reduced levels of 2,2-diphenyl-1-picrylhydrazyl (DPPH), a stable free radical that is widely used to analyze radical scavenging activity. Compared to the limited scavenging action of the salicylates, AAD-2004 rapidly reacted with DPPH with potency higher than vitamin E, suggesting that AAD-2004 is a potent free radical scavenger (Fig. 1c). The free radical scavenging action of AAD-2004 was further examined using the spectroscopic technique of electron spin resonance (ESR). 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a spin trapping agent, reacted with hydroxyl radicals, producing the ESR spectra of DMPO-OH (Fig. 1d). The addition of AAD-2004 reduced levels of DMPO-OH in a dose-dependent manner. The ESR spectra of DMPO-OH were almost completely blocked in the presence of AAD-2004 as low as 50 nM, demonstrating that AAD-2004 is a potent spin trapping molecule. AAD-2004 is an mPGES-1 inhibitor and does not cause gastric damage. AAD-2004 was derived from aspirin and thus expected to prevent inflammation as a COX inhibitor. The IC50 values of AAD-2004 for ovine COX-1 and COX-2 were 334.75 μM and 21.47 μM, respectively (Fig. 2a). However, AAD-2004 prevented PGE2 production following exposure of BV2 cells to lipopolysaccharide (LPS) with IC50 of 1.4 μM (Fig. 2b). This implies that AAD-2004 inhibits PGE2 production through another target besides cyclooxygenases. We examined the possibility that AAD-2004 would inhibit mPGES-1, an isomerase converting COX-derived PGH2 to PGE2 . Addition of PGH2 in extracts of LPStreated BV2 cells resulted in increased PGE2 production in the presence of excess indomethacin, a dual COX-1/COX-2 inhibitor, compared to those of control BV2 cells, suggesting that the bacterial endotoxin induces mPGES-1-mediated PGE2 production as previously reported . AAD-2004 inhibited the conversion of PGH2 to PGE2 by mPGES-1 with an IC50 of 0.23 μM (Fig. 2c). Oral administration of 200 mg/kg ibuprofen and 300 mg/kg aspirin caused severe gastric damage 24 h later (Fig. 2d). Celecoxib, a selective COX-2 inhibitor, also produced mild gastric damage at an extremely high dose of 1000 mg/kg. However, oral administration of 1000 mg/kg AAD-2004 did not damage gastric mucosal membrane. Interestingly, aspirin-induced gastric damage was prevented by co-administration of trolox, a vitamin E analogue. This implies that AAD-2004 prevents inflammation with reduced gastric risk possibly due to selectivity to mPGES-1 and spin-trapping property. AAD-2004 blocks oxidative stress and inflammation in SOD1 mice

AAD-2004, a potent spin trapping molecule and microsomal prostaglandin E synthase-1 inhibitor, shows safety and efficacy in a mouse model of ALS