Grafting a NO-donating moiety into the structure of flurbiprofen, one of the most potent non-selective anti-inflammatory agents, yielded a drug devoided of side toxicity and endowed with a remarkable neuroprotective activity against MPTP-induced DAergic neurotoxicity, motor impairment and microglia activation in ageing mice. HCT1026 was effective in both the acute and subchronic models of MPTP, and its neuroprotective activity lasted up to 40 d, as opposed to age-matched controls indicating the longterm safety and efficacy of the HCT1026, as opposed to flurbiprofen. The different outcome of mice treated with HCT1026 was not due to differences in daily food consumption, or to poor MPP+ metabolism. HCT1026-induced neuroprotection was associated with a marked down-regulation of activated microglial cell number and a signiof ficant decrease of MPTP-induced pro-inflammatory mediators, including iNOS, Mac-1 and PHOX expression, as well reduced 3-NT formationin SNpc DAergic neurons, suggesting that a switch in microglia pro-inflammatory phenotype might contribute to nigrostriatal neuroprotection.
The experimental study was designed to compare the oral activity of HCT1026 with that of flurbiprofen. However, due to the significant gastric toxicity of flurbiprofen observed in the short-term study, only HCT1026-medicated diet was further studied. In long-term experimental protocols, HCT1026 proved to have a safe profile and a significant efficacy in counteracting MPTP-induced striatal DAergic toxicity.
Nitric oxide (•NO) is a biological molecule known to play a major role in a wide variety of physiological and pathological conditions . Some of its functions include vasodilation of blood vessels, GI mucosal healing and defense. Therefore NSAIDs containing NO-donor groups have been developed to obtain effective treatment of inflammation with reduced GI side effects [57–61]. Indeed, grafting an organic nitrate moiety onto the NSAID scaffold has been shown to result in the release of NO through slow kinetics (in comparison with others NO donors, i.e. sodium nitroprusside, S-nitroso-N-acetyl-D,L, penicillamine), possibly mimicking the physiological levels of NO produced by constitutive NO synthases. Thanks to NO release, and the combination of a balanced inhibition of the two main COX isoforms, NO-NSAIDs are endowed with little gastrointestinal and renal toxicity compared to their parents compounds [56–61, 65, 85]. It is believed that the NO, which is released by the metabolism of nitrate as the compounds are broken down, may counteract the consequences of the NSAID-induced decrease in gastric mucosal PGs . It seems important to mention that a clear identification of the metabolic steps by which NO-NSAIDs produce NO has not been established. Experimental findings obtained, in vivo, show that HCT1026 is metabolized into flurbiprofen and NO species, i.e. nitrates and nitrites, [60, 61, 70], which are detected in plasma and in brain at 2-4 h  and 3 h , respectively from drug administration. In vitro, HCT1026 is converted into flurbiprofen with different kinetics depending on the cell assay. In rat plasma, 30 min of incubation is required to fully convert HCT1026 to flurbiprofen [see ]. By contrast, approximately 35% of HCT1026 is converted into flurbiprofen within 1 h of incubation in human blood, and 24 h of incubation was necessary to reach the almost complete dissociation of HCT1026 [62, 63]. Given the rapid action of HCT1026 demonstrated in vitro, Bernardo et al.  suggested the possibility that HCT1026 might reach the brain parenchyma and act on brain cells before being cleaved to the nitrate moiety and flurbiprofen . Alternatively, the metabolites flurbiprofen and NO might act concomitantly by activating parallel pathways that ultimately determine the unique effects of HCT1026 . Other studies have suggested a potential action of the HCT-1026 metabolite, 4-hydroxybutyl nitrate, since animal studies have shown that the level of inorganic nitrite in the brain increases after oral administration of HCT-1026 . Plasma nitrite itself has been shown to provide a source of NO under certain conditions [see ].
Neurochemical, morphological and behavioral changes clearly indicate that with the ageing process, increased vulnerability of the nigrostriatal DAergic system and limited recovery from MPTP injury are observed [4, 5, 17, 18, 77, 78]. Indeed, the nigrostriatal DAergic neurons exhibit compensatory mechanisms in response to MPTP injury, but the degree of plasticity becomes reduced with age [18, 77, 78]. Accordingly, a diminished compensatory capacity of nigrostriatal DAergic neurons "as a prelude" to PD is recognized to accompain the process of aging [see [88, 89]]. Among the mechanisms at play, increased neuronal vulnerability to degenerative conditions, dysfunction of glia-neuron crosstalk, reduced repair capacity of injured DAergic neurons and/or limited neurogenesis may contribute to the poor recovery observed with age [see  for review]. Given the role of the ageing process as a critical risk factor for developing PD, we addressed the efficacy of HCT1026 preventive administration schedule in 9 to 11 month-old mice and found that longterm administration of HCT1026 resulted in a significant DAergic neuroprotection following MPTP insult, at both and SNpc levels. These effects lasted up to 40 dpt, supporting HCT1026 as promising approach towards the development of effective pharmacological neuroprotective strategies against PD.
Variable neuroprotective effects have been, so far, reported for both steroidal and non-sterodal, mixed and COX-2-selective inhibitors in different MPTP-mouse models of PD [37–48]. The mixed COX-1/COX-2 inhibitor indomethacine  and a COX-2 selective NSAID, rofecoxib  treatment rescued DAergic neurons from MPTP injury. Indomethacine, however, appeared toxic at high doses, and rofecoxib, failed to keep its protective properties when used in the prolonged treatment . Studies on mice deficient with COX-2 showed that COX-2 plays a critical role in animal models of DAergic degeneration [38, 32, 43]. In particular, the role of increased levels of COX-2 in generating a toxic dopamine-quinone species which was responsible for DAergic neuronal degeneration, was demonstrated, whereas the selective COX-2 inhibitor, rofecoxib, exhibited a neuroprotective effect (38). In the study of Sanchez-Pernaute , the COX-2 antagonist, celecoxib, was capable to prevent or slow down DAergic degeneration induced by intrastriatal administration of 6-OHDA. Accordingly, Vijitruth et al.  showed that pharmacological or genetic inhibition of COX-2 was capable to reduce motor impairment and to protect DAergic neuronal cell bodies in the SNpc as well as the striatal TH-stained fibres against MPTP-induced neurotoxicity .
On the other hand, Ibuprofen, a non selective blocker, was shown to diminish the decline of dopamine content in striatum in the MPTP mouse model of PD, in a dose-dependent manner, and was not toxic to the DAergic system . In accord with these experimental results, ibuprofen, but not other non-selective NSAIDs, was shown to diminish the risk/incidence of PD in men [5, 6]. The present results showing the longterm DAergic neuroprotection in ageing mice and the safety profile of HCT1026 are of special interest, given that non -selective NSAIDs long-term therapies are hampered by their significant gastrointestinal, renal and cardiovascular side-effects [56, 85].
HCT1026-induced neuroprotection observed in the present study was accompanied by a sharp downregulation of all studied markers of microglial activation including ameboid-like microglial cell number, pro-inflammatory mediators as well as two key harmfull elements, MAC-1 and PHOX, likely suggesting that a shift from microglial pro-inflammatory ("harmfull") phenotype might be a major contributing factor.
Indeed, under inflammatory conditions, PHOX is the major source of peroxides in the brain. Activation of microglial PHOX is synergistic with glial iNOS expression in inducing DAergic neuron death [27–36]. Accordingly, PHOX/Mac-1-deficiency mitigates MPTP-induced DAergic neurotoxicity both in vivo and in vitro [30–33]. Inflammatory stimuli together with ROS and RNS activate nuclear factor-kB (NF-kB) in microglial cells, oligodendrocytes and neurons to promote the transcription of inflammatory cytokines, COX-2, iNOS, and apoptosis-promoting factor including p53/Bax [see [45, 46]]. Various studies (reported in Backgroud section) have clearly underlined a "dual key mechanism" whereby simultaneous activation glial iNOS and PHOX synergistically act in killing DAergic neurons . This mechanism may mediate inflammatory degeneration in response to cytokines, bacteria, ATP, arachidonate, whereas neuroprotection was observed by NO and peroxynitrite scavengers . Indeed, nitrative stress is among the factors potentially underlying DAergic neurodegeneration [8, 27, 28, 33–36]. Interestingly, the number of DAergic neurons containing 3-NT increased significantly in rhesus monkey midbrain DAergic neurons with age, suggesting a role for aging-related increase of nitrative damage in the selective vulnerability of SN neurons to degeneration in PD . Here, ageing mice fed with a control diet exhibited a dramatic increase of 3-NT colocalization with TH+ neurons by 3 d after MPTP, corresponding to the active DAergic degeneration phase [27, 28, 32, 33, 36], while by 7 d a certain decrease in 3-NT accumulation within TH+ neurons was observed, possibly indicating the end of the degeneration phase. In HCT1026-fed mice, the generation of PHOX and iNOS-derived cytotoxic mediators, including 3-NT accumulation within DAergic neurons, were markedly abated, supporting that a significant proportion of SNpc neurons survived MPTP insult.
Although NSAIDs pharmacological actions are related to their ability to inhibit PG biosynthesis, some of their beneficial therapeutical effects are thought to be mediated by a panel of COX-independent mechanisms. NSAIDs are able to inactivate the transcription NF-kB and activator protein-1 (AP-1), critically involved in the induction of multiple inflammatory gene products involved in the inflammatory response (i.e. iNOS, TNF-α). In addition, NSAIDs in neuronal cells might directly and dose-dependently scavenge ROS and RNS, thereby blocking their detrimental effects [45, 46]. On the other hand, high concentrations of NSAIDs such as ibuprofen and indomethacin, activate PPARγ. PPARγ is a ligand activated inhibitory transcription factor that antagonizes the activity of NFkB, AP1, signal transducer and activator of transcription-1 (STAT-1) and nuclear factor of activated T cells (NFAT). PPARγ activation is then associated with a reduction in the expression of several inflammatory genes and the production of inflammatory cytokines and iNOS [45, 46]. In particular, in vitro studies have reported that selective PPARγ agonists such as pioglitazone, ibuprofen, or indomethacin, can activate PPARγ in microglia, decreasing the number of activated glial cells [see [45, 46]].
The mechanisms that differentiate HCT-1026 from flurbiprofen remains a matter of debate. In vitro studies demonstrated that a low concentration of (1 μM) of HCT1026, but not flurbiprofen, activated PPARγ in primary cultures of rat microglia, with kinetics similar to those of the synthetic agonist, ciglitazone , supporting additional anti-inflammatory action through PPARγ . In addition PPARγ agonists were reported to mitigate MPTP-induced DAergic neurotoxicity in different PD models [see [13–15, 45, 46]]. In the recent studies of Abdul-Hay et al. , flurbiprofen was 10-fold less potent than HCT-1026 in inhibiting iNOS induction in RAW 264.7 cell cultures. In LPS/IFNγ-induced primary astroglial cultures, HCT-1026 showed anti-inflammatory potency towards inhibition of cytokine and iNOS elevation, providing similar observations to those in microglial cultures . That the anti-inflammatory activity of HCT-1026 could translate into neuroprotection was further demonstrated in a co-culture experiment with LPS-stimulated RAW cells and a neuroblastoma cell culture, where HCT-1026 was highly efficacious neuroprotectant .
Grafting a NO-donating moiety to flurbiprofen was reported to confer additional anti-inflammatory properties [51, 52, 60–66, 87, 92, 93]. It has been suggested that this effect may depend on the negative feedback regulation exerted by low physiologic concentration of NO (nanomolar range) on different inflammatory mediators such as iNOS and COX-2, as well as on their associated functions [94–97]. Indeed, there is evidence that at low concentrations, NO has anti-inflammatory properties as it inhibits the expression of pro-inflammatory proteins (i.e. COX-2 and iNOS), and it counteracts the release of pro-inflammatory cytokines, such as TNF-α, in activated macrophages [94–97].
It should be recalled, that NO signaling plays an important role in the functioning of the CNS, and activation of soluble guanylate cuclase (sGC) represent one important effect of NO. Of note, physiological release of low concentrations of NO by constitutive neuronal NOS is recognized to modulate extracellular levels of dopamine in the striatum and to critically participate in striatal DAergic homeostasis . The NO/sGC/cGMP signal transduction system is also considered to be important for modulating synaptic transmission and plasticity in brain regions such as the hippocampus, cerebral cortex, and cerebellum, and further studies are required to unravel potential involvement of these pathways in DAergic neuroprotection afforded by HCT1026. Besides the NO-mediated effects, most recently proposed are NO-independent and NSAID-independent actions on NFκB and MAPK/ERK signaling pathways [see ]. In their study, Idris and co  reported the ability of HCT1026 to inhibit receptor activator of NFkB (RANKL), as well as RANKL-induced activation of NFkB and ERK pathway in LPS-stimulated macrophage cultures. In addition, HCT1026 also inhibited TNF-α, IL-1 and LPS-induced signaling. Interestingly enough, the pathways inhibited by HCT1026 all share a similar kinase complex upstream of the NFkB and ERK pathways and this is the most likely target for the action of HCT1026 .
It seems important to underline that inflammatory pathways may become hyperactivated with age and/or become more sensitive to immune/neurotoxic challenge, thereby promoting degeneration [99–102]. Given that with age, dysfunctional microglia and altered glia-neuron crosstalk may contribute to the progression of neuronal degeneration , HCT1026 preventive and long-term treatment might thus reduce age-dependent and MPTP-induced increase in oxidative and inflammatory attacks to nigrostriatal DAergic neurons. Of special interest, in view of the role of both systemic and central inflammation in modulating the severity of neuronal insult, including DAergic injury [see [13–15, 18, 24, 81–83]], a potential effect of HCT1026 in influencing systemic inflammation cannot be excluded. In addition, the mitigation of the nitrosative/oxidative status of the nigral microenvironment as revealed by downregulation of Mac-1, PHOX and 3-NT in the VM, likely have beneficial consequences for glial expression of critical neuroprotective/neurotrophic factors , thereby supporting TH+ neuron survival/neurorescue, possibly through an amelioration/mitigation of SNpc nicroenvironment.
After brain injury, the inflammatory environment is recognized to have both detrimental and beneficial effects on neuronal outcome, depending on mouse strain, age and sex of the host, the severity of the lesion, the degree and timing glial activation, the hormonal background, the specific cellular context and intrinsic region-specific neuronal characteristics [see [8–18, 21–26, 40, 77, 78, 100, 103, 104]]. In degenerative conditions, glia serve neuroprotective functions including the removal of dead cells by phagocytic activity and the production of neurotrophic factors. By contrast, overactivation of microglia or dysfunctional microglial cells as a consequence of ageing and age-related events within the SN microenviroment, [18, 25, 100–102] likely increase DAergic neuron vulnerability and/or may limit DAergic self-repair abilities. In vivo experiments have recently shown that intranigral administration of prostaglandin J2 (PJ2) induces microglia activation, selective degeneration of DAergic neurons in the SNpc, formation of ubiquitin- and α-synuclein-immunoreactive aggregates in the spared DAergic neurons, and locomotor deficit . These and other findings have underlined the role of a transient initiation factor, triggering an active self-perpetuating cycle of chronic neuroinflammation, contributing to DAergic neuronal dysfunction [13, 83]. By reducing exacerbation of inflammation, HCT1026 may then improve mitochondrial performance, increase glial-mediated neurotrophic support, thus creating a more favorable milieu for nigrostriatal DAergic neuron survival/rescue.
The present results are in line with data obtained in different animal models of brain inflammation, where HCT1026 significantly reduced neuronal loss and decreased the number of reactive microglial cells to a greater extent than the parent compound, flurbiprofen [66, 86, 87, 92, 93]. In experimental allergic encephalomyelitis (EAE), oral treatment with HCT1026 which delayed disease onset and decreased the severity of clinical signs in mice immunized with myelin oligodendrocyte peptide (MOG35-55) . In addition, HCT1026 fed mice exhibited significantly reduced mRNA levels of pro-inflammatory cytokines, caspase-1, and iNOS in blood cells, with reduced number of CNS-infiltrating T cells . Recently, HCT1026 was reported to mitigate amyloid-β-induced toxicity, in cell culture, in vitro, while enhancing cognition in response to cholinergic blockade, in vivo . Other studies reported the ability of HCT1026 to reduce microglia activation and to prevent muscular dystrophy pathology in two murine models .