Since many years, NSAID such as aspirin (ASA) are widely prescribed and also used over the counter mainly for their anti-inflammatory, antipyretic and analgesic effects (1). The major limitation of their clinical application are serious side-effects such as induction of acute hemorrhagic erosions, potentiation of gastric ulcerogenic response to various stimuli, exaggeration of colitis and impairment of healing of pre-existing ulcers (2-5). More recently, the ability of NSAIDs to adversely affect renal function has become recognized. As presented in previous studies, NSAID exhibit potent anti-inflammatory and analgesic actions but their use in humans is limited due to untoward effects such as formation of gastric mucosal bleeding erosions. Aspirin is recommended for the anti-thrombotic prophylactics, particularly in patients for prevention at recurrence of myocardial infarction and angina pectoris, but also in healthy individuals in an attempt to prevent myocardial infarction (5). The beneficial effects of aspirin is attributable to its ability to irreversibly inhibit the enzyme cyclooxygenase (COX), thereby preventing the formation of the pro-aggregatory, vasoconstrictor substance, thromboxane A2 (6).

Many attempts have been made in last decade to develop more effective and safer NSAIDs. One of this attempt was the introduction of new NSAIDs that have been launched into the market on the basis of lower gastrointestinal toxicity. These approaches have included chiral NSAIDs and enteric coating of the drug to prevent absorption in the stomach, formulation as a pro-drug, to prevent contact between the active drug and the gastric mucosa, and formulation of the drugs for parenteral administration (7). The basis for each of these approaches was the observation that some NSAIDs, particularly those which are soluble in acidic milieu of the stomach, can directly damage gastric epithelial cells. Reducing the topical irritancy, it was postulated, should prevent the epithelial damage and, in turn, prevent ulceration and bleeding. Interestingly, topical irritant properties of NSAID do not appear to make a major contribution to their ability to produce acute gastric erosions and chronic ulcerations because untoward effects such as mucosal damage associated with fall in gastroduodenal microcirculation can be observed after parenteral administration of these drugs (7). Apparently, each of these approaches has failed in terms of substantially reducing the clinically significant adverse effects of NSAIDs in the gastrointestinal tract.

As mentioned above gastrointestinal ulceration associated with the use of all NSAID is directly related to the ability of these drugs to inhibit cyclooxygenase (COX) activity, thereby suppressing the formation of prostaglandins and shifting the arachidonate cascade into overproduction of vasoconstrictive leukotrienes (LT) such as LTC4 and LTD4 (8 - 10). However, besides an suppressive action on the generation of arachidonate cascade products, NSAID cause the GI damage through a variety of different mechanisms unrelated to simple inhibition of COX enzymes. This includes the non-ionic diffusion of NSAID with low pKa such as aspirin into the surface epithelial cells resulting in the suppression of oxidative phosphorylation and disturbance of cell viability. Following NSAID, there is an enhanced expression and release of TNF-, that promotes cell apoptosis and triggers activation of adhesion molecules and leukocyte recruitment leading to microvascular perturbations (11-14). Furthermore, the inhibition of nuclear transcription factor (NFB) and suppression of extracellular regulated kinase (ERK) pathway were proposed to act as an important component of the gastropathy associated with the use of NSAID (14).


FUNCTIONAL SIGNIFICANCE OF COX-2 AND THERAPEUTIC EFFICACY OF COXIBS
In the early 1990s two structurally related isoforms of cyclooxygenase (COX) have been identified, namely COX-1, constitutively expressed in most mammalian tissues, and COX-2, which is usually undetectable under resting conditions and is rapidly induced at sites of inflammation in response to noxious stimuli (15). This led to the hypothesis that COX-1 izoform produced PG which exert house-keeping function but COX-2 isoenzyme produced detrimental PG responsible for the pain and inflammation. A considerable resources of pharmaceutical industry were invested to develop selective COX-2 inhibitors, unfortunately with the unfulfilled promise that these drugs would improve tolerability profile. These drugs named coxibs were introduced into the market and gained an impressive success (16). Although COX-1 is the predominant isoenzyme in the gastric mucosa, there is increasing evidence that COX-2 mRNA and protein are either constitutive or inducible in specific areas of the GI-tract in animals and humans (17,18). Preliminary observation on the physiological role of COX-2 in gastric mucosal defense was obtained in COX-1 knockout animals (19). Moreover, neither inhibition of COX-1 nor COX-2 activity alone at the doses capable of inhibiting both enzymes in vivo induced gastric lesions but the combined administration of both COX-1 and COX-2 inhibitors caused gastric mucosal injury of the rodent stomach (20). Furthermore, we and others have shown that COX-2 is upregulated in the gastric mucosa infected by Helicobacter pylori, in that pretreated with mild irritants or exposed to ischemia-reperfusion and other noxious stimuli such as ASA (21,22). Enhanced expression of COX-2 was demonstrated in the proliferating zone of gastric mucosa undergoing mucosal repair or regeneration during ulcer healing suggesting that this enzyme acts as the second line in the process of mucosal repair and ulcer healing. Corroborative with this notion were the observations that selective COX-2 inhibitors delayed ulcer healing in similar fashion as conventional NSAIDs (17,23). Apparently, COX-2 enzyme seems to be of lesser importance for the proper mucosal lining and mucosal defense under resting conditions but is of crucial importance in face of pending injury or preexisting ulcerations, possibly cooperating with COX-1 in promotion of gastric mucosal recovery from mucosal damage and ulcer healing. Consequently, gastric damaging effect of coxibs is absent in the healthy stomach but becomes evident when gastric mucosal defense is impaired. Thus, the selective COX-2 inhibitors (coxibs), appeared as the newer class of anti-inflammatory agents, that spare COX-1 but produced gastrointestinal ulcer complication at about half the rate of conventional NSAIDs (24). However, in contrast to classic NSAIDs, coxibs not only are devoid of antiplatelet activity, but their use has been linked to an increased risk of nonfatal myocardial infarction (25) raising the question whether cardioprotection should be recommended to patients with cardiovascular risk factors that take a coxib. Although the use of low doses of ASA has been recommended (25), human studies also suggest that co-administration of a coxib with ASA increase the risk of gastrointestinal injury (26). Further evidence confirmed better safety profile of coxibs in humans, however, under certain circumstances such as therapy with low dose of ASA, the selective COX-2 inhibitors loose their beneficial effect showing the same risk of bleedings as conventional NSAIDs. Therefore, in these group of patients or in patients subjected to ASA therapy the recommendation to use proton pump inhibitors is mandatory.


NITRIC OXIDE, CINODS AND GASTRIC MUCOSA DEFENSE
NO is a putative signaling molecule recognized for its ability to enhance gastric mucus/alkaline secretion, inhibit gastric acid secretion, and prevent neutrophil activation and adherence to vascular endothelium thus affording gastroprotection (27). Earlier studies revealed that endogenous NO released from vascular endothelium, sensory nerves or gastric epithelium cooperates with endogenous prostaglandins in the maintenance of gastric mucosa integrity and microcirculation (28, 34). Therefore, a new strategy in limiting adverse effects associated with the use of NSAID was developed that NO once linked to NSAID could be released from these derivatives and exert beneficial influence on gastric mucosa by enhancing the mucosal defensive ability and preventing pathogenic events resulting from suppression of prostanoids synthesis mainly the reduction in gastric microcirculation and the leukocyte-endothelial adherence (29). Indeed, a novel series of compounds was introduced into preclinical trials by pioneering group of investigators (John Wallace and Piero del Soldato) that consist of an non-selective NSAID (e.g. aspirin, flurbiprofen, diclofenac, ketoprofen) linked to a nitric-oxide (NO)-releasing moiety (30). This new class of anti-inflammatory drugs, named the COX-inhibiting NO donating drugs (CINODs) was exploiting the concept that gaseous mediators play a functional role in gastroprotection in vivo and in vitro as well as in ulcer healing (31, 32). Numerous studies including our own, have shown that such NSAID constructed by adding of nitroxybuthyl or a nitrosothiol moiety containing NO to the native NSAID resulted in gastroprotection, stimulation of mucosal repair and enhancement of the gastric ulcer healing (33 - 35). While these NO-NSAID retain their anti-inflammatory properties comparable to those of parent NSAID, they significantly reduced typical NSAID-induced gastropathy which definitely accounted for their greater tolerability in the gastrointestinal tract.

It is of interest that the major importance of NO included into the ASA structure is its slow release from NO-ASA in the gastric mucosa and direct prevention of mucosal damage and acceleration of ulcer healing ability as expected based on previous studies showing that both endogenous NO released by capsaicin or NO originating from L-arginine, a substrate for NO-synthase (NOS), and finally that released from glyceryl trinitrate exert gastroprotective activity and accelerate ulcer healing (28, 32, 36). In contrast to native NSAID, their NO-releasing derivatives such as NO-aspirin (NO-ASA) were found to exhibit lower gastric toxicity exerting a potent anti-thrombotic, anti-inflammatory and analgesic action, showing equal ability to native NSAID in terms of inhibiting both COX-1 and COX-2 activity in the gastric mucosa (26, 29, 30). Studies by Fiorucci and colleagues have demonstrated that NO-NSAIDs are able to modulate inflammation through actions other than just suppression of PG biosynthesis, including modulation of caspase activity (37). The NSAID examined in our previous study (33), such as aspirin was ulcerogenic by itself in the stomach and its damaging action was strongly aggravated by addition of exogenous acid that was employed to mimic the natural fate of this NSAID occurring in highly acidic conditions in the stomach. In contrast to conventional NSAID, NO-NSAID failed to induce gastric lesions upon acidification and remained without deleterious influence on gastric blood flow. Moreover, both non-specific COX-inhibitors inhibited PGE2 generation confirming previous observations that the suppression of COX and subsequent deficiency of endogenous PG in the gastric mucosa could account, at least in part, for their damaging effect. In contrast, NO-releasing ASA and NO-releasing naproxen which by themselves were devoid of ulcerogenic properties, failed to augment the gastric damage after acidification of these drugs, despite the sustained ability of these agents to suppress of PGE2 generation similar to that observed after application of their parent drugs. Furthermore, pretreatment with NO-ASA and NO-naproxen resulted in the attenuation of ethanol-induced damage and raised significantly the GBF and these effects were counteracted by ODQ, an inhibitor of NO-dependent guanylyl cyclase but remained unaffected by inhibition of NO-synthase with L-NNA (33).

The mechanism of protection and accompanying hyperemia induced by NO-releasing NSAID still remains to be elucidated but could be attributable to the local release of NO from NO-NSAID. This is supported by the fact that a considerable amount of NO metabolites were detected in luminal content of the stomach of rats pretreated with NO-releasing NSAID. Moreover, Snitroso-N-acetyl penicillamine (SNAP), a potent NO donor, which by itself reduced significantly the gastric damage provoked by ethanol, added to native ASA or naproxen resulted in protection and hyperemia comparable to those exhibited by NO-releasing NSAID suggesting that, indeed, NO released from these compounds plays a crucial role in this protection and accompanying hyperemia (31,33). The mechanism of this release could be attributed to the activity of not yet defined esterases present in the biological fluids such as blood, cell body fluids and cell homogenates. This hypothesis was supported by the observations that this release of NO from CINODs which requires the activity of cell esterases has the different kinetics as compared to that of NO donors. Moreover, the gastric sparing effect of CINODs in the stomach was also preserved when these compounds were given systemically indicating that reduction of topical irritant effect due to different route of administration does not impair the gastroprotective efficacy of CINODs in the stomach. The question still remained how fast this NO should be released from NO-NSAID to exert therapeutic activity in the stomach? Previous studies revealed that abundance of NO due to administration of NO donors (glyceryl trinitrate, sodium nitroprusside) exerts beneficial influence against injury induced in the gastric mucosa by various strong irritants. The critical issue was, however, addressed to the rate of delivery of NO because these NO donors at higher doses used were capable of attenuating the extent of gastric damage induced by an NSAID or ethanol while significantly reduced systemic blood pressure possibly due to excessive amount of NO (16,31). Thus, it become apparent that slow and more discrete release of NO from NO-NSAID without affecting systemic arterial pressure could be of interest once the sparing effect of the gastric mucosa exerted by these drugs is concerned. Unfortunately the site of the NO formation released from CINODs are still unknown and detailed pharmacokinetics studies as to how the CINODs are absorbed and metabolized are lacking.


PHENOMENON OF GASTRIC ADAPTATION TO PROLONGED NSAID INSULT
As documented in numerous studies before NSAIDs such as ASA can damage gastric mucosa when given for the first time but with more prolonged administration of ASA to the stomach gastric mucosa the adaptation to ulcerogenic action of these drugs develops in the gastric mucosa of experimental animals and humans resulting in attenuation of gastric damage despite administration of NSAID in ulcerogenic dose is continued (38-41). The mechanism of this adaptation still should be elucidated but increase in gastric blood flow, enhancement in gastric cell proliferation mediated by the rise in plasma growth factors such epidermal growth factor (EGF) and its mucosa content as well as the fall in neutrophil infiltration of the gastric mucosa were observed after continued ASA administration (40,41). Inhibition of PG by NSAID seems to not play a major role in the gastric adaptation since, for instance, a better tolerance to repeated ASA insult occurred regardless complete suppression of endogenous PG in the gastric mucosa. Vascular etiology for NSAID gastropathy has been proposed by the demonstrable ability of these drugs to cause vascular endothelial damage, neutrophil activation within the mucosa, production of free radical oxygen metabolites and decrease in the gastric blood flow. All these changes observed with initial ASA insult that was applied to the rodent gastric mucosa, were markedly diminished in the stomach subjected to 5 repetitive ASA treatments, even ASA was applied in a dose which produced hemorrhagic lesions (42). Recent experimental evidence indicates that nitric oxide (NO) released from sensory nerves or produced by gastric epithelium cooperates with cytoprotective prostaglandins (PG) in the mucosal integrity and gastroprotection (43) but the hypothesis that sensory neuropeptides and NO could mediate gastric adaptation to ASA should be further investigated.

As mentioned above, NO-releasing derivatives of flurbiprofen, ketoprofen or diclofenac have been shown to exhibit low gastrointestinal toxicity and, therefore, to spare the gastrointestinal tract even when administered repeatedly for several weeks (29,33). We have demonstrated that repeated administration to ASA results in gastric adaptation (42) but it is unknown, whether NO-NSAID such as NO-aspirin or NO-naproxen can induce adaptation similar to that achieved with their native ASA or naproxen. We have assessed this hypothesis in rats treated once or repeatedly with NO-ASA and NO-naproxen to check whether these compounds induce gastric adaptation similar to that observed with classic NSAIDs. Finally, we examined whether adaptation induced by repeated exposures to NO-ASA or NO-naproxen would enhance the resistance to the subsequent mucosal damage induced by ASA applied in ulcerogenic dose or to topical application of strong irritant such as absolute ethanol. Since the adaptation to ASA is PG-independent but involves enhancement in cell proliferation and an increase in gastric blood flow (GBF), possibly mediated by NO (43), it was of interest to study whether these protective mechanisms contribute to adaptation induced by NO-aspirin or NO-naproxen. The NO-NSAIDs which were shown to inhibit activity of both COX-1 and COX-2 enzymes with comparable potency to parent NSAIDs, failed to induce initial gastric mucosal injury and did not interfere with gastric adaptation developed in response to these NO-derivatives of NSAID. These results could be interpreted that NO-NSAID which were reported as much safer for the gastric mucosa than classic NSAIDs, do not cause initial injury which was believed earlier as prerequisite for the adaptation to occur (44). This is in keeping with our previous finding that ASA applied intragastrically in the doses of lower than 50 mg/kg which does not produce gastric lesions in rat gastric mucosa failed to exhibit gastric adaptation and did not show the enhanced mucosal resistance to mucosa challenged with supramaximal dose of 250 mg/kg (42). We demonstrated that repetitive treatment with NO-ASA similarly to ASA applied in a dose which by itself is not ulcerogenic one, caused only moderate initial injury and consequently failed to develop gastric adaptation. NO-ASA increased the GBF when given for the first time and this increase in GBF was sustained during 5 subsequent exposures to NO-ASA suggesting that enhanced NO can contribute to the enhanced resistance of NO-ASA-treated gastric mucosa to damage induced by strong irritants.


LIPOXINS IN THE MECHANISM OF GASTRIC MUCOSAL DEFENSE
AND GASTRIC ADAPTATION TO REPEATED ASA TREATMENTS
Lipoxin A4 (LXA4) and lipoxin B4 (LXB4) were first identified by Serhan and colleagues (45) as 5- and 15-lipoxygenase interaction products of activated leukocytes. But later on lipoxins were found also in tissue cells of patients with various immuno-inflammatory states such as rheumatoid arthritis, asthma, pneumonia to contribute to resolution of inflammatory state (7). Aspirin is considerably involved in lipoxin biosynthesis because it can acetylate a key serine residue in both COX-1 and COX-2, leading to inhibition of the production of prostanoids from arachidonic acid. While COX-1 activity appears to be completely inhibited by ASA, COX-2 can still convert arachidonic acid to 5-HETE (46). 5-HETE can be further metabolized via 5-lipoxygenase to 15(R)-epi-lipoxin A4, an isomer of LXA4, which is also referred to as aspirin-triggered lipoxin (ATL) (35) (Fig. 1). As mentioned above, the identification of two isoforms of cyclooxygenase (COX) in the early 1990s led to the hypothesis that selective suppression of COX-2 would produce many of the beneficial anti-inflammatory effects of NSAIDs (which inhibit both COX-1 and COX-2), but would spare gastric prostaglandin synthesis and therefore cause less stomach irritation (47). It is now clear, however, that COX-2 makes an important contribution to gastric mucosal defense. This is supported by the fact that selective inhibition of only one izoform does not cause gastric damage and NSAID-induced gastric injury in rats and humans requires the combined inhibition of both COX-1 and COX-2 (20,48). This finding prompted further investigation into the potential role of COX-2-derived factors in gastric mucosal defence by demonstration that lipoxin A4 and its 15-epimeric counterpart (ATL) exhibited a potent protective actions in the stomach. Intraperitoneal administration of synthetic lipoxin A4 to rats prior to oral administration of aspirin resulted in a dose-dependent reduction in the extent of hemorrhagic damage to the lining of the stomach (35). Lipoxin A4 and ATL not only inhibit ASA-induced gastric lesions but also the associated increase in neutrophil adherence and neutrophil chemotaxis, adherence, transmigration and superoxide anion production (49,50). The mechanism of this action should be still elucidated but the common pathway could be the activation of NO system by these lipid mediators. Moreover, the administration of a lipoxin receptor antagonist prior to oral administration of aspirin was found to significantly increase the extent of hemorrhagic damage. Interestingly, an increase in ATL generation is not observed following administration of non-aspirin NSAIDs such as indomethacin or ibuprofen. While the amounts of ATL produced by the normal rat stomach are negligible, ASA administration results in a marked increase in its synthesis. ATL production induced by ASA could be completely inhibited by a selective COX-2 inhibitor (celecoxib), and its inhibition by this COX-2 inhibitor led to a significant increase in the extent of gastric damage. This indicates that ASA-induced acetylation of COX-2 results in the generation of ATL, compensating for fall in PG biosynthesis, thereby reducing the severity of gastric damage. Most important is that lipoxin A4 can also be synthesized independently of aspirin administration, and has the capacity to make important contributions to mucosal defence (35). Besides inhibition of neutrophil activation, ATL could reduce the severity of ASA-induced damage is through elevation of mucosal NO production (Fig. 2). We tested this possibility by examining the effects of a NO-synthase inhibitor on the gastroprotective effects of lipoxin, which completely reversed the protective effects of lipoxin A4 against ASA-induced gastric damage in the rat stomach. Lipoxins may play an enhanced role in gastric mucosal defence with the inflamed stomach due to not only ASA ingestion but also by Helicobacter pylori (H. pylori) infection. The interaction between ASA and H. pylori is controversial suggesting that enhanced COX-2 during H. pylori infection can render gastric mucosa more resistant to damage induced by ASA (51-54). However, gastric adaptation developed by continues ASA administration is severely impaired in H. pylori-infected individuals (55). There is a lack of studies on the link between the persisted colonization of the stomach by Helicobacter pylori accompanied by gastritis and lipoxins. The increased levels of COX-2 expression in the inflamed stomach or that treated with ASA may provide a greater capacity for the production of aspirin-triggered lipoxins. This hypothesis was tested in a rat model of gastritis, however, not strictly related to Helicobacter pylori infection (56). Rats in which gastric inflammation was induced by oral administration of iodoacetamide exhibited enhanced resistance to aspirin-induced gastric damage as compared to controls (56). Aspirin administration to the rats with gastritis triggered significantly more ATL synthesis than that detected in controls without gastritis. The increased ATL formation and the increased resistance to damage were completely inhibited by a selective COX-2 inhibitor (56). This resulted in blockade of ATL synthesis, and a reversal of this gastric adaptive response because the susceptibility to aspirin-induced damage returned to normal. In addition, the rise in the mucosal generation of LTB4 was observed. These findings indicate that ATL are essential mediators of ASA-induced gastric adaptation and increased LTB4/ATL ratio in the gastric mucosa is associated with reversal of gastric adaptation to ASA and an increase in the index of gastric damage. Whether or not lipoxin A4 or stable analogues of this substance would influence healing of pre-existing gastric ulcers has not yet been examined.

Fig. 1. Biosynthetic pathways of proinflammatory (cysteine-leukotrienes) and anti-inflammatory lipoxins (lipoxin A4) from arachidonic acid. Irreversible acetylation of COX-2 enzyme by NSAID such as aspirin leads to the formation of epi-lipoxin A4, an isomer of lipoxinA4, also known as aspirin-triggered lipoxin (ATL).

Interestingly, the single administration of NO-aspirin failed to influence lipoxin generation suggesting that acute administration of NCX-4016 (NO-ASA) did not trigger ATL formation, in contrast to the significant production of ATL by the stomach of rats given an equimolar dose of aspirin (34, 35). This was confirmed in arthritic rats by demonstration that HCT-3012 (NO releasing naproxen), which inhibited COX-1 and COX-2 activity, also suppressed ATL formation when co-administered in combination with ASA (34). Although these data indicate that HCT-3012 inhibits the acetylated and non-acetylated form of COX-2, it appears that NO released by its NO-donating moiety compensates for ATL and PGE2 deficiency in these experimental settings.

Fig. 2. Aspirin (ASA) applied once damages gastric mucosa but when administered 5 times it results in the mucosal resistance to damage (gastric adaptation) manifested by a decrease in gastric damage possibly due to upregulation of COX-2, acetylation of this enzyme and subsequent production of 15-epi-lipoxin A4 (15-epi-LXA4). Aspirin-triggered lipoxins (ATL) exhibit gastroprotective action against ASA-induced gastric damage and inhibit associated increase in neutrophil activation and adherence, thus contributing to gastric adaptation. ATL may also trigger NO-synthase (eNOS and/or iNOS or both) to release NO, which causes vasodilatation and inhibits neutrophil activation.

It is of interest, however, that the prolonged administration of NCX-4016 which was administered each day for 5 days, significantly increased the gastric ATL formation. This increase was inhibited by co-administration of a selective COX-2 inhibitors celecoxib and rofecoxib. This findings were in part, confirmed in clinical trials where the urinary excretion of ATL was monitored after 8 and 15 days treatment with aspirin (100 mg once a day) or NCX-4016 (800 mg bid) (57). It was found that ATL formation was significantly elevated with both drugs being suppressed by co-administration of a selective COX-2 inhibitor (celecoxib). While co-administration of celecoxib and aspirin resulted in a significant increase in the severity of gastric damage as compared to aspirin alone, celecoxib and NCX-4016 (NO releasing ASA) co-administration did not result in significant injury to the stomach. This strongly suggests that the ATL formed in response to NCX-4016 administration did not make a significant contribution to protection of the stomach but the NO released from NCX-4016 appears more likely to act as the major factor accounting for the gastric safety of this compound and protects the rodent and human gastric mucosa even in the presence of suppression of COX-1 and COX-2.

Acknowledgments: This work was supported by the grant # 2P05A 11830 from Ministry of Education & Science, Warsaw, Poland.

Conflict of interest statement: None declared.


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