Medical Hypotheses 84 (2015) 262–267

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Medical Hypotheses

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NOSH aspirin may have a protective role in Alzheimer’s disease

http://dx.doi.org/10.1016/j.mehy.2015.01.0080306-9877/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: manuelam@icmpp.ro (M. Murariu).

Gabi Drochioiu a, Lucia Tudorachi a, Manuela Murariu b,⇑a Faculty of Chemistry, Al. I. Cuza University of Iasi, 11 Carol I, Iasi 700506, Romaniab Petru Poni Institute of Macromolecular Chemistry, Romanian Academy, 41A Grigore Ghica Voda Alee, Iasi 700487, Romania

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 August 2014Accepted 6 January 2015

Evidence indicates that inflammation, oxidative stress, and the disruption of normal conformation of pro-teins might be directly linked to Alzheimer’s disease (AD). The present study was undertaken using lit-erature data to find a possible drug to address the multiple disorders involved in AD-associated Abaccumulation and plaque formation. We consider NOSH-aspirin a drug of choice for reducing the inflam-matory areas in the brain (aspirin moiety), removing the noxious heavy metals from plaques (hydrogensulfide), and increasing the oxygen supply to neurons since nitrogen oxide is a potent vasodilator and ananti-inflammatory agent. Several confirmatory data in literature and possible mechanisms for cellulardefence as well as novel therapeutical pathways are discussed.

� 2015 Elsevier Ltd. All rights reserved.

Introduction

Alzheimer’s disease (AD) is a major cause of disability and mor-tality, being characterized by insidious decline in memory and byits unique pathology, which affect language, visuospatial percep-tion, calculations, and executive functioning. Behavioral changesare common in AD and include psychosis, agitation, depression,anxiety, personality alterations, and neurovegetative changes [1].The cardinal feature of AD is the extracellular deposition of pro-teinaceous amyloid-b fibrils as senile plaques [2,3]. In fact, proteinaggregation into amyloid fibrils is involved both in AD and otherneurodegenerative diseases such as Parkinson’s, various dementiasand prion diseases. Nevertheless, the accumulation and depositionof Ab proteins in the brain to form neuritic plaques are the keypathological features of AD, even though its mechanism has notbeen yet completely understood [4]. Gradual accumulation ofaggregated Ab initiates a complex, multistep cascade that includesgliosis, inflammatory changes, neuritic/synaptic change, tanglesand transmitter loss [2]. Oxidative stress and amyloid fibril forma-tion are consistent major themes among processes thought to beinvolved in the pathogenesis of AD [5,6]. Oxidative stress has beenproposed as a molecular mechanism linking c-secretase to b-secre-tase activity [7]. For example, the total amount of iron ions in ADbrain tissues is significantly higher compared to control samples[9–11], iron accumulation in Alzheimer disease being a source ofredox-generated free radicals. Consequently, antioxidant protec-tion in the brain is largely provided by vitamin E, glutathione,

ascorbate and carnosine [8]. Besides, metal ions and acid pH inducethe formation of b-amyloid protein oligomers. However, they aredistinct from those generated by slow spontaneous ageing at neu-tral pH [12].

Clinicopathological and neuroradiological data show that Abdeposition in the neuroparenchyma is closely associated with alocally-induced, non-immune-mediated chronic inflammatoryresponse [13]. After activation by amyloid-beta deposits, glial cellsmay secrete inflammatory mediators and reactive oxygen species,which, in turn, may aggravate the aggregation of amyloid-b[14,15]. In fact, amyloid plaques are co-localized with a variety ofinflammation-related proteins, like complement factors, acute-phase proteins, pro-inflammatory cytokines, and clusters ofactivated microglia [16]. Some authors consider that AD may bea consequence of inflammation, whereas homocysteic acid wasshown to induce amyloid-b accumulation in neuronal cells [17,18].

Recently, new hybrids known as NOSH-aspirin, which are NO-and H2S-releasing agents, with anti-inflammatory activity, havebeen reported [19–21]. They proved to have both anti-inflamma-tory and cytoprotective actions probably due to the slowly-liber-ated hydrogen sulfide into cell environment [22,23]. Since thereis a link between inflammation and cancer, NOSH compounds havebeen recommended as anti-cancer agents. Experiments showedthat NOSH-aspirin inhibited HT-29 colon cancer growth [20]. Theauthors claimed that NOSH-aspirin is the first nonsteroidal anti-inflammatory drug (NSAID) based agent with such high degree ofpotency. NOSH-aspirin inhibited cell proliferation, induced apopto-sis, and caused G0/G1 cell cycle block.

Therefore, we were interested in exploring the most significantevents in the etiology of AD and the molecular mechanisms of Abaccumulation and aggregation in order to find new therapeutically

G. Drochioiu et al. / Medical Hypotheses 84 (2015) 262–267 263

solutions. Consequently, we are searching for a possible drug toaddress the multiple disorders involved in Ab accumulation andplaque formation. Since both inflammatory events and the effectof heavy metal or ROS are associated with AD, we consider the fea-sibility of using new derivatives of aspirin, like NOSH compounds,in order to address both the prevention and improvement of thispathology.

Premises of the hypothesis

Inflammatory processes and pH

Since a mildly acidic environment together with increased Zn2+

and Cu2+ are common features of inflammation, it was proposedthat Ab aggregation by these factors may be a response to localinjury [24]. There is evidence that inflammatory processes mayplay a very important role in the mechanism of neuronal damagein AD [25]. During normal aging, a progressive neuroinflammatorystate builds in the brain, involving astrocytes and microglia, theprimary cellular components of neuroinflammation [26]. Underinflammatory conditions, low pH has a dual effect by activatingb-secretase (BACE-1) and inhibiting a-secretase (ADAM10).

Cleavage of amyloid beta precursor protein (APP) is predomi-nantly catalyzed by a-secretases, which have an optimum pHrange of 7.4–8.0. Some of the cleavage is catalyzed by b secre-tase(s), which leave 28 amino acids on the outside of the mem-brane. The substrate for c-secretase is the C-terminal fragment ofAPP resulted from the b-secretase activity. Then, as a result ofthe c-secretase cleavage, different Ab types are rendered, butmostly Ab40 and Ab42, both species involved in AD [27,28]. Theoptimum pH of b secretases is approximately 5.0 [29], the samewith that in acidic intracellular compartments, such as endosomesand the trans-Golgi network [30]. c-Secretase is an intramem-brane-cleaving aspartyl protease complex that mediates the finalcleavage of b-amyloid precursor protein to liberate the neurotoxicamyloid-b peptide [31]. The aspartyl proteases generally have anoptimum pH range of 4.0–5.0.

Although low pH values (pH 4.0–5.0) might be associated withthe complete degradation of Ab peptides, a slight decrease in pHresults in partial APP degradation and Ab accumulation. Onceformed, the deposits (plaques), which contain b-amyloid aggre-gates and heavy metal ions [32], can hardly be decomposed viaproteolytic degradation (active a-secretase are found within neu-ronal membrane, limited access of enzymes to each Ab backbone,inappropriate pH) or by solubilization.

Anti-inflammatory effect of aspirin

Cyclo-oxygenase-2 (COX-2) is induced in sites of inflammation,whereas cyclo-oxygenase-1 (COX-1) is believed to produce prosta-glandins, vital to stomach mucosal defense [33]. Aspirin causes adramatic increase in COX-2 mRNA expression and a high increasein COX-2 immunoreactivity. Besides, the involvement of nitricoxide (NO) in antioxidant cellular protection induced by aspirinwas demonstrated [34]. It was concluded that endothelial NO syn-thase is a site of action for aspirin, and that the NO/cyclic GMP sys-tem takes on a crucial function in mediating the cytoprotectiveaction of aspirin.

From aspirin to NOSH compounds

Recently, several derivatives of aspirin [20,35], which incorpo-rate in their molecules both nitric oxide (NO) and hydrogen sulfide(H2S)-releasing moieties have been synthesized (they are known asNOSH compounds or NOSH-aspirin). NOSH-aspirin proved to

inhibit the cyclooxygenase enzyme activity, being thus a potentanti-inflammatory agent.

Hydrogen sulfide, H2S, was demonstrated to play an importantrole in many biological systems [21]. The mechanisms of action ofNOSH aspirin assumes hydrolyzing the hybrid molecules of H2S-releasing NSAIDs, which results in the parent NSAID and an organicmolecule from which H2S is slowly released. The NSAID compo-nent inhibits COX-1 and COX-2 resulting in compromised mucosaldefense mechanisms. However, the released H2S counteracts manyof the damaging effects of NSAIDs, by activation of KATP channels.Moreover, H2S causes vasodilatation, thus leading to cardioprotec-tive effects. Both NSAID moiety and the released H2S have anti-inflammatory effects.

Heavy metal ions

Both the degenerative diseases like cancer or diabetes and theneurodegenerative ones are associated with metal ion imbalance.Heavy metal ions are involved in the pathogenesis of several neu-rodegenerative and vascular diseases [9,12,32]. Treating thepatients with calcium disodium edetate may result in removingthe intoxicating traces of heavy metals [36]. The neurotoxicity ofAb peptides has been linked to peroxide generation [37]. Ab pro-duces hydrogen peroxide by the reduction of metal ions, Fe(III)or Cu(II), setting up conditions for Fenton-type chemistry. AD isclosely related to the aggregation of Ab within the neocortex dueto metal-ion–protein interactions [38–40]. Ab precipitation andtoxicity in AD are provoked by abnormal interactions with neocor-tical metal ions, such as Zn, Cu and Fe [41–44]. Since heavy metalsare implicated in the aggregation of proteins, a combination of ana-lytical methods among them intrinsic fluorescence, circular dichro-ism, and high-resolution fourth-derivative absorbance analysis hasbeen developed to prove the metal-induced conformationalchanges [45].

Hypothesis on AD prevention by NOSH-aspirin

Putting together the main AD-related findings, one can get apicture of the etiology and course of this neurodegenerative dis-ease, in which is highlighted the role of inflammatory agents(Fig. 1). Pro-inflammatory mediators play key roles in the etiologyof neurodegenerative diseases including AD, and neuroinflamma-tion is associated with a decrease in pH, which induce an increasein amyloid-b peptide production by enhanced proteolysis of amy-loid precursor protein. Depending on age, genetic or metabolic fac-tors and life style, and other risk factors such as the inflammatoryagents, including Ab species and their association with heavy met-als, a decrease in intracellular pH may occur. Decreasing pHenhances BACE-1 activity responsible for Ab formation and inhibitsADAM10. Aspirin and other anti-inflammatory agents may coun-teract the inflammatory reactions and also inhibit the ROS produc-tion. However, the anti-inflammatory action of NOSH-aspirin maybe more elevated than that of simple aspirin due to H2O and NOrelease, which have also potent anti-inflammatory properties.Moreover, heavy metal ions are known to induce b-sheet confor-mation of Ab peptides, binding then to the resulted Ab oligomersto form fibrils and plaques. The last ones are ROS-generatingagents. H2S may protect against increasing concentrations of freeheavy metal ions, since it is able to form insoluble sulfides. Besides,H2S may react with heavy metal ions enclosed in the plaques, andliberate Ab oligomers, which are easier proteolytically degraded.

In brief, since the NSAIDs proved to be anti-AD agents,whereas H2S and NO may attenuate the neurodegenerative symp-toms, we consider that NOSH-aspirin, chemically named 4-(3-thi-oxo-3H-1,2-dithiol-5-yl) phenyl 2-((4-(nitrooxy)butanoyl)oxy)

Aging, Genetic& RiskFactors

Aβaccumulation

Metal binding ROS

HeavyMetalions

Cross-linkage &polymerization of Aβ peptides

Neuriticplaque

formation

Activatedmicroglia

InflammationBACE-1activation

Aβaccumulation

Inflammation

NOSHaspirin

NOSHaspirin

NOSHaspirin

H2S

NOSH

aspirinH2S

NO

Inflammation

H2S

H2S

NO

Inflammatoryagents

H2S

Aspirin

NOSHaspirin

Aspirin

Fig. 1. Proposed mechanism for multi-leveled anti-Alzheimer action of NOSH-aspirin. NOSH-aspirin provides by hydrolysis a NSAID compound with both anti-inflammatoryaction and ROS scavenging one. NO and H2S may potentate the NSAID action. H2S may react with heavy metal ions and reduce the ROS production.

OH

COOH

Salicylic acid(2-hydroxybenzoic acid)

OCOCH3

COOH

Aspirin(2-acetoxybenzoic acid)

O

CH2

O

O2NO

O

OS

S

S

NOSH-aspirin

Fig. 2. The chemical structure of NOSH-aspirin (in bold, aspirin moiety); it is the first dual acting NO and H2S releasing hybrid containing a 3H-1,2-dithiole-3-thione structureable to release hydrogen sulfide and another one, 4-(nitrooxy)butanoic acid, releasing NO (H2S ‘‘donating’’ compounds).

264 G. Drochioiu et al. / Medical Hypotheses 84 (2015) 262–267

benzoate (Fig. 2), could be a drug of choice for AD prevention andtreatment with multiple therapeutic indications, since it mayreduce the inflammatory areas in the brain [25], remove the nox-ious heavy metals in the plaques [12,32], and increase the oxygenadmission to the neurons due to NO chemical groups it contains[46].

Hydrogen sulfide may play an antioxidant function and pre-vents free radical-induced impairment, being beneficial in treatingage-associated diseases [47]. H2S reduces cystine to cysteine in theextracellular space, increasing the intracellular concentrations ofcysteine to increase the production of intracellular GSH. Thus,H2S enhances the redistribution of GSH into mitochondria in Neu-ro2a cells [48]. Consequently, H2S suppresses oxidative stress inthe mitochondria. H2S can prevent cytokine- or oxidant-inducedoxidative damage through its antioxidative effects [36]. Besides,it can inhibit the expression of proinflammatory factors by down-regulating NF-jB activation or by upregulating heme oxygenase 1expression.

These findings suggest that NOSH-aspirin has significant anti-inflammatory properties and may be a new candidate for treatingneurodegenerative disorders that have a prominent neuroinflam-matory component such as AD. Moreover, NO and H2S releasedfrom the molecules of NOSH-aspirin may enhance the effect ofAspirin, which has both an anti-inflammatory action and a ROSscavenging one. H2S may react with heavy metal ions and reducethe ROS production.

Hypothesis-supporting literature data

Intracerebral injection of dilute, Ab-containing brain extractsfrom humans with AD or APP transgenic mice induced cerebralb-amyloidosis and associated pathology in APP transgenic micein a time- and concentration-dependent manner [49,50]. The seed-ing activity of brain extracts is reduced or abolished by Ab immun-odepletion, protein unfolding, or by Ab immunization of the host.Studies on the long-term treatment with non-steroidal anti-inflammatory drugs (NSAIDs) demonstrated a decrease in the riskof AD; besides, such treatment delays its onset or slows down itsprogression [51]. The administration of COX-2 inhibitors preventsboth the inflammatory reaction and the cholinergic hypofunction.The involvement of the local inflammatory reaction is confirmedmainly by studies dealing with activated microglia, cytokines, reac-tive astrocytes, complement system and reactive oxygen species(ROS) [52]. The inflammatory events occur in close proximity ofbeta-amyloid and tau protein deposits. The most appropriate pro-phylactic effect seems to be achieved by specific inhibitors of COX-2. COX-2 is expressed in higher concentrations in the degeneratingcells of the brain. The NSAIDs are selective inhibitors of COX-2, andcan thereby have an anti-inflammatory effect. Moreover, the NSA-IDs decrease the excessive activation of some transcription factors,like PPARgama and the nuclear factor kapa-B, which are responsi-ble for the transcription initiation of a number of pro-inflammatorygenes.

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Secretase activity and pH

The active a-secretase is present in the neuronal membraneand has a limited access to the Ab backbone. In fact, chronicinflammation is associated with low pH values which increasethe b-secretase activity in the cerebral cortex and enhance theb-amyloid aggregation and metal-associated oxidative stress.ADAM10 is the most widely expressed in neurons and seems tobe the most active a-secretase [50]. Some a-secretases have aconsensus HEXXH zinc-binding motif (X represents any aminoacid) which is involved in the proteolytic activity. The putativea-secretase cleaves the amyloid precursor protein (APP) ofAlzheimer’s disease in the middle of the amyloid b peptide (Ab)domain [53]. ADAM9, ADAM 10, and ADAM 17 catalyze a-secre-tory cleavage and therefore act as a-secretases in A172 cells [54].ADAM8 may play a role in soluble CD23-mediated inflammationand cell migration.

AD patients show an increase of b-secretase activity in the cere-bral cortex [55]. It was shown that insulin-degrading enzyme is themain soluble beta-amyloid degrading enzyme at neutral pH inhuman brain [56]. Nevertheless, the highest beta-amyloid proteindegrading activity of this aspartyl protease in the soluble fractionsoccurs between pH 4 and 5. Synthetic beta-amyloid protein (1–40)is rapidly degraded by a human brain soluble fraction, optimumactivity occurring at around pH = 4 [57]. Besides, BACE-1 isexpressed in activated astrocytes around senile plaques [58], sug-gesting that various inflammatory cytokines and forms of oxidativestress could provoke BACE-1 expression in astrocytes. The AD brainshows chronic inflammation characterized by an abundance ofreactive astrocytes and activated microglia, which are near senileplaques and secrete a variety of cytokines [25]. A large body of evi-dence suggests that by transforming from a basal to a reactivestate, astrocytes neglect their neurosupportive functions, thus ren-dering neurons vulnerable to neurotoxins, including proinflamma-tory cytokines and ROS [59].

The complex dynamics of Ab may also contribute to the causa-tive role of Ab in the pathogenesis of AD [60].

NOSH-aspirin is a NO and H2S releasing hybrid, which provokescell growth inhibition in the low nano-molar range [20]. NOSH-aspirin inhibits COX-1 more than COX-2. H2S, in turn, is an impor-tant endogenous signaling molecule, with therapeutic potential inage-associated diseases, being involved in aging by inhibiting free-radical reactions and activating SIRT1 [17,47]. Moreover, H2S canprevent cytokine- or oxidant-induced oxidative damage, andinhibits the expression of proinflammatory factors either by down-regulating NF-jB activation or by upregulating heme oxygenase 1expression. Endogenous levels of 50–160 lM H2S are detected innormal human brains [61]. However, lower levels of H2S as wellas accumulation of homocysteine, a strong risk factor for the devel-opment of AD, are observed in the brains of AD patients [62].Besides, neurotoxicity of homocysteine is associated with inhibi-tion of endogenous H2S generation and downregulation of expres-sion and activity of CBS in PC12 cells, being mediated byextracellular signal-regulated kinase 1 and 2 (ERK1/2) activation.Moreover, it is believed that H2S could reduce neurotoxicityinduced by Hcy and that H2S may be a useful therapeutic strategyagainst homocysteine-induced AD [63]. H2S may have protectiveeffects against Ab-induced cell injury by inhibiting inflammation,promoting cell growth, and preserving mitochondrial function ina p38- and Jun N-terminal protein kinase (JNK)-mitogen-activatedprotein kinase (MAPK)-dependent manner [64]. Moreover, H2S canprotect neurons from oxidative stress, which is characteristic forAD. Besides, H2S protects neurons against glutamate-mediated oxi-dative stress by enhancing the activities of c-GCS and cystinetransport, which results in incremental changes of glutathione lev-els [65]. These findings suggest that H2S, alone or released from

NOSH-aspirin, is a promising therapeutic target for treating neuro-degenerative diseases.

Nitric oxide (NO) is a potent vasodilator and neurotransmitter,being also involved in inflammation and immunity [66]. AlthoughNO produced in infected and inflamed tissues could contribute tothe process of carcinogenesis by different mechanisms [67], someauthors demonstrated its potent anti-inflammatory properties[68]. Direct effects of NO occur under normal physiological condi-tions when the rates of NO production are low, as in NO-releasingreactions from NOSH-aspirin. Such reactions may serve regulatoryand/or anti-inflammatory functions. Indirect effects are mediatedby reactive nitrogen oxide species formed from the reaction ofNO either with oxygen or superoxide. Such species can mediateeither nitrosative or oxidative stress [69]. Activated astrocytes ormicroglia produce NO, which inhibits the cellular respiration ofneurons, also decreasing ATP levels and stimulating lactate pro-duction by these neurons. NO donors cause rapid release of gluta-mate from neuronal and neuronal–astrocytic cultures andsubsequent neuronal death [69]. High rate of oxygen may preventNO-induced neuronal death. Nevertheless, the activated glia killneurons via NO formation, which inhibits neuronal respirationresulting in glutamate release and subsequent excitotoxicity dur-ing times of active inflammation. Indeed, the treatment withNOSH-aspirin reduces the release of the TNFa and IL-6, and alsoattenuates the activation of P38 MAPK and NFjB proteins [70].

H2S releasing compounds

Substantial evidence showed that H2S attenuates cognitive dys-function and prevents neuronal impairment in the experimentalmodel of AD [71]. The mechanisms of the protective role of H2Sin AD may involve its antioxidant, anti-apoptotic, and anti-inflam-matory effects. Recent research also reveals that H2S afforded byNaHS treatment attenuates neuronal death in the hippocampusof rats injected with Ab peptide [72]. H2S dramatically suppressesthe release of TNF-a, IL-1b and IL-6, as well as inhibits the upreg-ulation of COX-2 and the activation of NF-jB in the hippocampus.H2S releasing compounds have effective anti-inflammatory andanti-tumor effects, as well as precise ion-channel regulation, car-diovascular protection and oxidation resistance [73]. However,the therapeutic effects of H2S are still controversial due to conflict-ing published results regarding the use of different H2S donors.Therefore, it is essential to select the best H2S releasing com-pounds, some of which being currently used in clinical trials alongwith their biological effects. Moreover, the actions of H2S are influ-enced by its concentration, reaction time, and cell/disease types,and is limited because of the instant release and short lifetime ofH2S [74]. Nevertheless, the H2S releasing compounds like NOSHaspirin suggest hope for future investigations [75].

Since NOSH-aspirin was successfully tested on colon cancergrowth [20], when concentrations between 7.7 and 45.5 nMproved to be effective, similar doses could be tested in AD. How-ever, further research is needed to establish therapeutic details,and to find out if NOSH-aspirin can be used as preventive drug,long lasting therapy or part of an overall treatment.

Concluding remarks

A close relationship between various concentrations of metalions, inflammatory processes, decreased pH and Ab accumulationand aggregation is thought to be of paramount significance forAD. The risk and severity of AD are reduced by antioxidant andanti-inflammatory agents or use of chelating drugs. Consequently,we discussed here the feasibility of using some new aspirin deriv-atives against many pathological features of AD. Taken together,

266 G. Drochioiu et al. / Medical Hypotheses 84 (2015) 262–267

the previous results reported in literature suggest that NOSH aspi-rin could have strong anti-AD potential and merits further evalua-tion. Even at nanomolar concentrations, NOSH-aspirin could be apotent anti-inflammatory agent, due to the three moieties its mol-ecule contains: NSAID, H2S, and NO. Each of them has demon-strated anti-inflammatory potential doubled by ROS scavengingproperties, regulatory functions, or metal binding capabilities.Finally, NOSH-aspirin may have potential for treating neurodegen-erative diseases, including AD.

Conflict of interest

All authors declare that there are not any financial and personalrelationships with other people or organizations that couldinappropriately influence (bias) their work.

Acknowledgments

Financial support by Romanian Government (UEFISCDI IDEI313/2011) is grateful acknowledged. LT gratefully acknowledgesthe strategic grant POSDRU/159/1.5/S/137750 from EU.

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