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

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. As previously reported , SOD1 mice revealed marked oxidative stress in motor neurons of the lumbar spinal cord at 10 weeks of age as evident by increased immunoreactivity to nitrotyrosine and 8-OHdG (Fig. 3a). Administration of AAD-2004 (i.p., b.i.d) from 8 weeks of age alleviated motor function deficits and increased survival in SOD1 mice. Maximal effects were observed from doses of 2.5 mg/kg (supplementary fig. 1). In mice, the oral administration of 2.5 mg/kg AAD-2004 showed an area under the curve (AUC) of 7.7 μg.h/mL which was 2-fold higher than the intraperitoneal administration of 2.5 mg/kg AAD-2004 (Unpublished data). Thus, the pharmacological effects of AAD-2004 were examined by the oral administration of 2.5 mg/kg (b.i.d.) in SOD1 mice. The administration of AAD-2004 from 8 weeks of age significantly blocked elevated levels of nitrotyrosine and 8-OHdG in the lumbar spinal cord of SOD1 mice at 10 weeks of age (Fig. 3a,b). The number of microglia immunoreactive to Iba-1 (ionized calcium-binding adaptor molecule-1), a marker of activated microglia/macrophage, and Iba-1 expression were increased in the ventral horn of the lumber spinal cord of SOD1 mice at 16 weeks of age compared to the wild type (Fig. 4a). The presence of Iba-1 positive microglia was prevented by AAD-2004 as evidenced by a decrease in immunoreactivity in spinal cord sections as well as Western blots (Fig. 4a-c). In addition, immunohistofluorescence studies revealed that the expression of mPGES-1 was increased throughout the lumbar ventral horn of SOD1 mice at 16 weeks of age (Fig. 4d-f). PGE2 levels were significantly increased in the lumbar spinal cord and also in plasma of SOD1 mice, which was significantly reduced following administration of AAD-2004 (Fig. 4g). As the maximum plasma concentration of AAD-2004 is approximately 8.1 μM following the oral administration of 2.5 mg/kg in SOD1 mice (unpublished data), AAD-2004 is expected to prevent inflammation in the lumbar spinal cord of SOD1 mice through blockade of mPGES-1. AAD-2004 prevents motor neuron degeneration, axonal damage, and autophagosome formation in the lumbar spinal cord of SOD1 mice. Widespread motor neuron degeneration was observed in the ventral horn of the lumbar spinal cord in 16-week-old SOD1 mice. The administration of 2.5 mg/kg AAD-2004 beginning 8 weeks of age significantly prevented the loss of spinal motor neurons in the SOD1 mice compared to vehicle treatment (Fig. 5a, b). Immunohistochemistry with the tau-5 antibody further demonstrated degradation of cell bodies and axons originating from the motor neurons (Fig. 5c). Such degenerative changes were significantly ameliorated by the administration of AAD-2004 (Fig. 5c, d). However, the axonopathy was not prevented by ibuprofen or riluzole, a disease-modifying neuroprotectant known to reduce glutamate neurotoxicity and used as the only approved treatment for ALS. The conversion of LC3-I to LC3-II, microtubule-associated protein 1 light chain 3-II, known as a marker for autophagosome formation, was induced in the lumbar spinal cord of 16-week-old SOD1 mice as previously reported . The conversion to LC3-II was not observed in SOD1 mice treated with AAD-2004 (Fig. 5e, f). In addition, administration of AAD-2004 also blocked the abnormal aggregation of mutant SOD1 observed in the lumbar spinal cord of SOD1 mice (Fig. 5g). AAD-2004 shows better beneficial effects than ibuprofen or riluzole in SOD1 mice. Finally, we carried out a study comparing the functional efficacy of AAD-2004 with that of riluzole or ibuprofen, a nonselective COX inhibitor that inhibited microglial activation and PGE2 production in the lumbar spinal cord of SOD1 mice (unpublished data). As reported , SOD1 mice that orally received a maximally effective dose of riluzole revealed significant improvement in motor function and survival (Fig. 6). Administration of 25 mg/kg ibuprofen improved motor function and extended life span in SOD1 mice comparable to riluzole. SOD1 mice treated with 2.5 mg/kg AAD-2004 showed significantly better motor function and survival relative to riluzole or ibuprofen. The onset of Rotarod deficits was significantly delayed by 12% and 15.6% in the riluzole and ibuprofen -treated groups, respectively, as compared with the control group. The disease onset was further delayed by 36% in SOD1 mice treated with AAD-2004 (Fig. 6e). Survival was extended by 8.2%, 9.4%, and 21% in the riluzole, ibuprofen, or AAD-2004-treated groups. While there was no difference in the disease onset and survival between the riluzole and ibuprofen groups, the AAD-2004 group significantly improved motor performance and survival compared to the riluzole or ibuprofen groups. Discussion AAD-2004, a dual function drug derived from aspirin and sulfasalazine, has been developed to protect against both free radicals and PGE2-mediated inflammation associated with certain forms of neurodegeneration in the central nervous system (CNS). AAD-2004 is a potent spin trapping molecule and mPGES-1 inhibitor effective at nanomolar concentrations. Administration of AAD-2004 improves motor function and survival in SOD1 mice with a maximally effective dose of 2.5 mg/kg while no gastric damage was observed following oral administration of doses as high as 1000 mg/kg. AAD-2004 blocks oxidative stress and inflammation through inhibition of mPGES-1-mediated PGE2 production in SOD1 mice, which results in blockade of neuronal death, axonopathy, and autophagosome formation normally observed in the lumbar spinal cord of these mice. As a consequence, blockade of oxidative stress and mPGES-1-mediated inflammation significantly extended disease onset and survival compared to riluzole and ibuprofen. Salicylate (2-hydroxybenzoate) can react with hydroxyl radical to produce catecol, 2,3dihydroxybenzoate, and 2,5-duhydroxybenzoate that act as a free radical trap . However, salicylate weakly reacts with DDPH and does not reduce Fe-induced free radical injury up to 1 mM, suggesting that salicylate is a poor anti-oxidant. Interestingly, sulfasalazine and 5aminosalicylate prevented Fe-induced free radical neurotoxicity at ~ 30 μM . The antioxidant effects of sulfasalazine and 5-aminosalicylate appear to be related with p-amine relative to the hydroxyl group of salicylate that increases stability of the peroxyl radical . Furthermore, the anti-oxidant potency and efficacy of AAD-2004 were remarkably increased with the electron-rich moiety (4-trifluoromethylpheny group) linked to p-amine that favors reaction with hydroxyl radical. In light of anti-inflammatory actions of salicylates as inhibitors of cyclooxygenases, we reasoned that AAD-2004 would inhibit COX-2. AAD-2004 was indeed a direct COX-2 selective inhibitor with IC50 of 21.47 μM, but reduced LPS-induced PGE2 production with IC50 of 1.4 μM in BV2 cells. This led us to examine mPGES-1, an inducible terminal isomerase catalyzing PGE2 biosynthesis, as a potential target of AAD-2004. AAD-2004 reduced activity of mPGES-1 with IC50 of 0.23 μM in extracts of LPS-treated BV2 cells. Thus, AAD-2004 is expected to selectively reduce PGE2 production as an mPGES-1 inhibitor at nanomolar concentrations while it prevents production of PGI2 as well as PGE2 as a moderate COX-2 selective inhibitor at high doses (≥20 μM). mPGES-1 mediates inflammatory responses in the CNS as well as peripheral inflammation . Expression of mPGES-1 was sparsely detectable in normal brain but markedly increased in brain endothelial cells and the paraventricular nucleus of the hypothalamus during fever, arthritis, and burn injury in rodents . Genetic deletion of mPGES-1 was shown to reduce levels of PGE2 in the CSF and fever following exposure to peripheral LPS injection , suggesting that mPGES-1-dependent PGE2 production is a mediator of CNS inflammation. Increased expression of mPGES-1 was also observed in neurons, astrocytes, and microglia as well as endothelial cells in postmortem brain of AD . We found that levels of PGE2 and mPGES-1 were significantly increased in the lumbar spinal cord of SOD1 mice. The latter was observed in neurons, astrocytes, microglia, and endothelial cells in the ventral horn undergoing widespread neuronal death and inflammation in SOD1 mice (Unpublished data). The plasma concentration profiles of AAD-2004 after single or 4-week oral administration of 2.5 mg/kg show a maximal concentration of ~ 2.7 μg/ml (~ 8 μM) within 30 min after the final dosing and blood-brain barrier (BBB) permeability of AAD-2004 is 3 – 5 % in mouse and rat. Therefore, it can be suggested that AAD-2004 prevents inflammation in SOD1 mice primarily by inhibiting mPGES-1mdiated PGE2 production in the spinal cord. As an mPGES-1 inhibitor selectively lowering PGE2 production, AAD-2004 appears to show better safety than conventional NSAIDs including selective COX-2 inhibitors that cause the risk of cardiovascular infarction and thrombosis by preventing production of vascular prostacyclin (PGI2) as well as adverse gastrointestinal events. The pharmacological property of AAD-2004 as a spin trapping molecule provides an additional safety profile. This is supported by recent reports demonstrating that anti-oxidants or free radical scavengers such as vitamin E, melatonin, DL-alpha-tocopherol, and L carnitine protect against NSAIDs-induced gastric injuries in rats 36, . In line with this, oral administration of trolox dramatically attenuates aspirin-induced gastric bleeding in rat. Thus, the dual pharmacological properties of AAD-2004 are appropriate for intervention of chronic PGE2-mediated inflammation and free radical production in the CNS with reduced adverse effects. Although either anti-oxidants or NSAIDs improve motor function and prolonged life span in SOD1 mice, none of them have shown significant benefits in the translational clinical studies for ALS patients . Such unsatisfactory outcomes may be attributable to low numbers of patients and short duration of the trials, but may also be associated with low permeability of anti-oxidants through the BBB and adverse effects of NSAIDs that limit pharmacological action of anti-oxidants or NSAIDs in the CNS . It is of note that combined treatment of celecoxib and creatine improves motor function in a randomized clinical trial phase II of ALS patients as well as SOD1 mice , suggesting better efficacy of combined antioxidant and NSAID therapy than monotherapy. AAD-2004 blocked free radical production and PGE2-mediated inflammatory responses induced in the spinal cord of SOD1 mice. Compared to beneficial effects of ibuprofen or riluzole in SOD1 mice, AAD-2004 significantly improved survival and onset of motor function deficit approximately up to 2 to 3-fold. Thus, concurrent blockade of free radicals and mPGES-1-mediated PGE2 production with AAD-2004 has potential to improve neurological function and survival in ALS patients with a better safety profile compared to NSAIDs. AAD-2004 and ibuprofen attenuate motor neuron death in the ventral horn of the lumbar spinal cord as riluzole or other COX-2 inhibitors do in SOD1 mice . The loss of ventral root axons correlates well with motor function deficit and appears before motor neuron death in SOD1 mice and ALS patients . While neither riluzole nor ibuprofen attenuated degeneration of ventral root axons, AAD-2004 significantly protected the axons in SOD1 mice. Impaired autophagy has been proposed as a cause of progressive dystrophy and degeneration of axons 37-38, . Administration of AAD-2004 prevented levels of LC3-II and SOD1 aggregates that were increased in the ventral horn of lumbar spinal cord in SOD1 mice. AAD-2004 prevents abnormal protein aggregates and autophagosome formation possibly by blocking free radicals and PGE2-mediated inflammation. In support of this, iron or mitochondrial reactive oxygen species are shown to induce autophagy and autophagic cell death . By inhibiting abnormal protein aggregation and axonopathy, AAD2004 produces better motor function and survival in SOD1 mice than COX-2 inhibitors or riluzole. Undoubtedly, free radicals and PGE2-dependant inflammation contribute to progression of neuronal damage and neurological deficit in ALS. Neither antioxidants nor NSAIDs, however, showed significant efficacy in ALS patients due to poor BBB permeability and drug-related adverse effects at therapeutic doses. AAD-2004 blocks free radical production and inflammation in vitro and in SOD1 mice by scavenging free radicals as a spin trapping molecule and preventing PGE2 production as an mPGES-1 inhibitor. With the dual pharmacological actions, AAD-2004 did not cause gastric damage at a dose 400-fold higher than efficacy doses in SOD1 mice, prevented protein aggregation and axonopathy, and improved neurological function and survival better than NSAIDs or riluzole. A phase I study of AAD-2004 in healthy human volunteers has demonstrated a safety profile that AAD-2004 does not produce serious adverse events at doses higher than the therapeutic target dose determined in SOD1 mice (unpublished data). The present findings support the need for a novel medication that exhibits concurrent blockade of free radicals and mPGES-1 as a means to combat devastating neuronal cell loss in ALS and also has implications for the treatment of other neurodegenerative diseases including AD and PD. Methods Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturemedicine/. Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS This work was supported by grants from the 21C Frontier R&D Program in Neuroscience from Ministry of Education, Science, and Technology, a National Drug Discovery Program from Ministry of Health and Welfare, the Brain Korea 21 project at Ajou University School of Medicine, and Neurotech Pharmaceuticals. AUTHOR CONTRIBUTIONS B.J.G designed and directed the study.; J.H.S. and J.K.L. performed the in vivo experiments.; Y.A.L. performed free radical neurotoxicity experiments in vitro.; Y.B.L, W.C., and J.H.S. designed and performed mPGES-1 activity assay in vitro.; Y.A.L. and J.H.S. performed COX activity assay in vitro.; D.S.I., J.H.L., and S.Y.B. performed gastric damage experiments.; S.J.S., S.M.P., and S.Y.B prepared for experiments.; J.H.S., S.J.S., S.M.P., and S.Y.B. discussed the experimental data.; J.E.S. discussed the manuscript; J.H.S. and B.J.G. wrote the manuscript.