NSAIDs induce both necrosis and apoptosis in guinea pig gastric mucosal cells in primary culture

A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is gastropathy. In this study, we examined, using guinea pig gastric mucosal cells in primary culture, how NSAIDs damage gastric mucosal cells. The short-term treatment of cells with high concentrations of indomethacin decreased cell viability in the absence of apoptotic DNA fragmentation, chromatin condensation, or caspase activation. Cells lost membrane integrity with this short-term indomethacin treatment, suggesting that indomethacin induced necrosis under these conditions. In contrast, the long-term treatment of cells with low concentrations of indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation, chromatin condensation, and caspase activation. Pretreatment of cells with inhibitors of caspases or protein synthesis suppressed cell death caused by long-term indomethacin treatment, suggesting that apoptosis was induced when the inhibitors were not present. These results imply that NSAIDs cause gastric mucosal damage through both necrosis and apoptosis of gastric mucosal cells.     

Effect of licofelone against NSAIDs-induced gastrointestinal ulceration and inflammation

The present study was aimed to evaluate the effect of licofelone, a dual inhibitor of cycloxygenase1/2-5-lipoxygenase against indomethacin-induced gastric damage in rats and mice in order to assess the role of leukotrienes if any, in non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastrointestinal inflammation. Acute pretreatment with licofelone reversed the indomethacin-induced gastric ulceration, neutrophil adhesion in mesentery venules, neutrophil count in blood, lipid peroxides and vascularity in the stomachs of mice and rats. Further, chronic pretreatment of licofelone also prevented indomethacin-induced gastric morphological changes and cellular infiltration in mesentery venules. Moreover, acute administration of indomethacin elevated leukotriene B4 levels in gastric mucosa, which was reversed by pretreatment with licofelone The results suggest that licofelone offered gastroprotection against NSAIDs-induced gastropathy through its effect on leukotrienes and by inhibiting extravasation of neutrophils.     

Nonsteroidal anti-inflammatory drugs inhibit gastric peroxidase activity

The peroxidase activity of the mitochondrial fraction of rat gastric mucosa was inhibited with various nonsteroidal anti-inflammatory drugs (NSAIDs) in vitro. Indomethacin was found to be more effective than phenylbutazone (PB) or acetylsalicylic acid (ASA). Mouse gastric peroxidase was also very sensitive to indomethacin inhibition. Indomethacin has no significant effect on submaxillary gland peroxidase activity of either of the species studied. Purified rat gastric peroxidase activity was inhibited 75% with 0.15 mM indomethacin showing half-maximal inhibition at 0.04 mM. The inhibition could be withdrawn by increasing the concentration of iodide but not by H2O2. NSAIDs inhibit gastric peroxidase activity more effectively at acid pH (pH 5.2) than at neutral pH. Spectral studies showed a bathochromic shift of the Soret band of the enzyme with indomethacin indicating its interaction at or near the heme part of the enzyme.     

Gastric mucosal cell model for estimating relative gastrointestinal toxicity of non-steroidal anti-inflammatory drugs

The study objective was to characterize the AGS human gastric mucosal cell line as a model for estimating gastrointestinal toxicity of COX-inhibiting compounds. Rofecoxib, celecoxib, nimesulide, ibuprofen, indomethacin, aspirin, salicylic acid, naproxen and acetaminophen were tested for inhibition of COX-2-mediated prostaglandin E2 synthesis in A549 and AGS cells. The IC50 ratio AGS/A549 was calculated as an estimate of the therapeutic index (TI) for gastrointestinal toxicity. Calculated IC50 values of non-steroidal anti-inflammatory drugs (NSAIDs) in A549 cells were in excellent agreement with published values (r = 0.996; P < 0.005). Calcium ionophore induction of arachidonic acid release in AGS cells provided TI similar to those using platelets and A549 cells (r = 0.918; P < 0.01). The AGS/A549 model exhibited lower TI than the platelet/A549 model. Spearman ranking correlated clinical NSAID gastropathy with lower AGS TI values. The AGS cell line has excellent potential to serve as a model for assessing the gastrointestinal effects of COX-inhibiting compounds.     

Synthesis and biological evaluation of loxoprofen derivatives

Non-steroidal anti-inflammatory drugs (NSAIDs) achieve their anti-inflammatory actions through an inhibitory effect on cyclooxygenase (COX). Two COX subtypes, COX-1 and COX-2, are responsible for the majority of COX activity at the gastrointestinal mucosa and in tissues with inflammation, respectively. We previously suggested that both gastric mucosal cell death due to the membrane permeabilization activity of NSAIDs and COX-inhibition at the gastric mucosa are involved in NSAID-induced gastric lesions. We have also reported that loxoprofen has the lowest membrane permeabilization activity among the NSAIDs we tested. In this study, we synthesized a series of loxoprofen derivatives and examined their membrane permeabilization activities and inhibitory effects on COX-1 and COX-2. Among these derivatives, 2-{4'-hydroxy-5-[(2-oxocyclopentyl)methyl]biphenyl-2-yl}propanoate 31 has a specificity for COX-2 over COX-1. Compared to loxoprofen, oral administration of 31 to rats produced fewer gastric lesions but showed an equivalent anti-inflammatory effect. These results suggest that 31 is likely to be a therapeutically beneficial and safer NSAID.     

Cyclooxygenase-2 selective and nitric oxide-releasing nonsteroidal anti-inflammatory drugs and gastric mucosal responses.

Occurrence of gastrointestinal damage and delayed healing of pre-existing ulcer are commonly observed in association with clinical use of nonsteroidal antiinflammatory drugs (NSAIDs). We examined the effects of NS-398, the cyclooxygenase (COX)-2 selective inhibitor, and nitric oxide (NO)- releasing aspirin (NCX-4016) on gastric mucosal ulcerogenic and healing responses in experimental animals, in comparison with those of nonselective COX inhibitors such as indomethacin and aspirin. Indomethacin and aspirin given orally were ulcerogenic by themselves in rat stomachs, while either NS-398 or NCX-4016 was not ulcerogenic at the doses which exert the equipotent antiinflammatory action with indomethacin or aspirin. Among these NSAIDs, only NCX-4016 showed a dose-dependent protection against gastric lesions induced by HCl/ethanol in rats. On the other hand, the healing of gastric ulcers induced in mice by thermal-cauterization was significantly delayed by repeated administration of these NSAIDs for more than 7 days, except NCX-4016. Gastric mucosal prostaglandin contents were reduced by indomethacin, aspirin and NCX-4016 in both normal and ulcerated mucosa, while NS-398 significantly decreased prostaglandin generation only in the ulcerated mucosa. Oral administration of NCX-4016 in pylorus-ligated rats and mice increased the levels of NO metabolites in the gastric contents. In addition, both NS-398 and NCX-4016 showed an equipotent anti-inflammatory effect against carrageenan-induced paw edema in rats as compared with indomethacin and aspirin. These results suggest that both indomethacin and aspirin are ulcerogenic by themselves and impair the healing of pre-existing gastric ulcers as well. The former action is due to inhibition of COX-1, while the latter effect may be accounted for by inhibition of COX-2 and mimicked by NS-398, the COX-2 selective NSAID. NCX-4016, despite inhibiting both COX-1 and COX-2, protects the stomach against damage and preserves the healing response of gastric ulcers, probably because of the beneficial action of NO.     

Effect of the antiinflammatory prodrug, nabumetone and its principal active metabolite on rat gastric mucosal, aortic and platelet eicosanoid synthesis, in vitro and ex vivo

Nabumetone is a novel non-steroidal antiinflammatory drug which although a weak cyclooxygenase inhibitor is converted by the liver to metabolites that are more potent inhibitors of cyclooxygenase. Nabumetone may thus avoid the occurrence of prostanoid-mediated gastropathy while maintaining its efficacy as an antiinflammatory agent. We compared the effect of nabumetone and 6-methoxy-2-naphthylacetic acid (6-MNA; the principal active metabolite of nabumetone) with that of naproxen and indomethacin on the synthesis of rat gastric prostaglandins I2 and E2, in vitro and ex vivo. Ex vivo platelet TXA2 and aortic PGI2 synthesis was also investigated in order to assess peripheral activity of nabumetone metabolites. In vitro, nabumetone was completely without effect on gastric mucosal prostanoid synthesis, whereas indomethacin, naproxen and 6-MNA (in this order of potency) inhibited prostanoid synthesis. Ex vivo, low dose naproxen and indomethacin (less than 5mg.kg-1) markedly inhibited gastric mucosal prostanoid synthesis at 30 min and 2 h post gavage, whereas nabumetone was without significant effect. Nabumetone administration also resulted in the inhibition of platelet TXA2 synthesis, whereas aortic PGI2 synthesis was unaltered. These data indicate that the administration of nabumetone may avoid NSAID gastropathy by leaving gastric mucosal prostanoid synthesis intact and also that the active metabolite(s) of nabumetone are effective inhibitors of cyclooxygenase in an NSAID-target tissue (platelet). The lack of effect of nabumetone administration on vascular PGI2 synthesis may confer an additional advantage over other NSAIDs, since the inhibition of peripheral PGI2 has been implicated in hypertensive and nephrotoxic side effects of NSAIDs.     

Non-steroid anti-inflammatory drugs: Combined assay for anti-edemic potency and gastric ulcerogenesis in the same animal

Simultaneous assessments of the anti-inflammatory and ulcerogenic activities of a test compound or new formulations of known anti-inflammatory drugs are conducted rapidly in starved rats, subjected to mild cold stress (?15°, 45 min) then challenged in a rear paw with carrageenan. Data are provided showing that some anti-ulcerant adjuncts may negate the anti-inflammatory activity of drugs such as aspirin, while other adjuncts do not and are therefore therapeutically beneficial (e.g. licorice extract, glucose with aspartic acid). Problems arising from drug esterification, use of suspending agents and alternative assays for gastrotoxicity, are clearly demonstrated by data generated in this bi-modal assay.     

Role of nitric oxide in the gastrointestinal tract

Worldwide osteoarthritis (OA) affects more than 9.6% of men and 18% of women older that 60 years. Treatment for OA often requires chronic use of selective or nonselective nonsteroidal anti-inflammatory drugs (NSAIDs), which have been associated with gastrointestinal and cardiovascular complications. An increased risk for upper gastrointestinal bleeding with NSAIDs alone and when combined with low-dose aspirin has been described in numerous studies. Although cyclo-oxygenase-2 inhibitors have been shown to carry a lower risk for gastrointestinal injury than nonselective NSAIDs, research continues to identify new treatments that not only are effective but also provide an improved benefit/risk profile, including better gastrointestinal tolerability. Nitric oxide (NO) is known to have a protective effect on the gastrointestinal tract. In preclinical studies NO was shown to help maintain gastric mucosal integrity, to inhibit leukocyte adherence to the endothelium, and to repair NSAID-induced damage. In addition, epidemiologic studies have shown that the use of NO-donating agents with NSAIDs or aspirin resulted in reduced risk for gastrointestinal bleeding. Recent studies have shown that cyclo-oxygenase inhibiting NO-donating drugs (CINODs), in which a NO molecule is chemically linked to an NSAID, are effective anti-inflammatory agents and may result in less gastrointestinal damage than is associated with NSAID use. Therefore, these agents provide a potential therapeutic option for patients with arthritis who require long-term NSAID therapy.     

NSAIDs counteract H. pylori VacA toxin-induced cell vacuolation in MKN 28 gastric mucosal cells

The relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori-induced gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells. Whether and how NSAIDs affect VacA-induced cytotoxicity is unclear. This study was designed to evaluate the effect of NSAIDs on H. pylori VacA toxin-induced cell vacuolation in human gastric mucosal cells in culture (MKN 28 cell line). Our data show that 1) NSAIDs (indomethacin, aspirin, and NS-398) inhibit VacA-induced cell vacuolation independently of inhibition of cell proliferation and prostaglandin synthesis; 2) NSAIDs impair vacuole development/maintenance without affecting cell binding and internalization of VacA; and 3) NSAIDs, as well as the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid, also inhibit cell vacuolation induced by ammonia. We thus hypothesize that NSAIDs might protect MKN 28 cells against VacA-induced cytotoxicity by inhibiting VacA channel activity required for vacuole genesis.     

Identification of the TPO1 gene in yeast, and its human orthologue TETRAN, which cause resistance to NSAIDs

Non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, have serious gastrointestinal side effects. Since their direct cytotoxicity was suggested to be involved in this side effect, we here tried to identify NSAID-resistant genes. We screened for Saccharomyces cerevisiae genes whose overexpression causes indomethacin resistance and identified the TPO1 gene, which encodes a major facilitator superfamily transporter. Its overexpression or deletion made yeast cells resistant or sensitive, respectively, to some NSAIDs. A BLAST search identified the possible human orthologue of Tpo1p, tetracycline transporter-like protein (TETRAN), whose overexpression in cultured human cells caused resistance to some NSAIDs, suggesting that TETRAN is an efflux pump for some NSAIDs.     

Modulations in the intestinal disaccharide hydrolases and membrane dynamics: Effect of non-steroidal anti-inflammatory drugs aspirin and nimesulide

The present study was designed to evaluate the influence of two commonly prescribed non-steroidal anti-inflammatory drugs (NSAIDs), aspirin and nimesulide on the biochemical composition and membrane dynamics of rat intestine. Female Wistar rats were divided into three different groups viz: Group I (Control), Group II (aspirin-treated, 50 mg/kg body weight) and Group III (nimesulide-treated, 10 mg/kg body weight). After 28 days, biochemical estimations in both drug treated groups showed an increase in sucrase, lactase, maltase and alkaline phosphatase as compared to the control. Alterations in the intestinal membrane dynamics by fluidity studies and Fourier Transform Infra Red (FTIR) spectroscopy also showed considerable changes. The alterations in the histoarchitecture of the intestine were also seen, which correlated well with the changes in structure and composition of the intestine. The use of NSAIDs like aspirin and nimesulide may cause the gastrointestinal side effects due to initial changes in the enzyme activities and membrane dynamics.     

Current state of therapy for pain and inflammation

Nonsteroidal anti-inflammatory drugs (NSAIDs), including both traditional nonselective NSAIDs and the selective cyclo-oxygenase (COX)-2 inhibitors, are among the most widely used medications in the USA. Traditional NSAIDs, although effective at relieving pain and inflammation, are associated with a significant increase in the risk for gastrointestinal adverse events. Throughout the 1990s these events were estimated to result in approximately 100,000 hospitalizations and 16,500 deaths each year nationally. Recent studies have indicated that the risk for serious NSAID gastropathy has declined substantially during the past decade as a result of a number of factors, including lower doses of NSAIDs, the use of gastroprotective agents such as proton pump inhibitors and misoprostol, and the introduction of the selective COX-2 inhibitors. One therapeutic approach that may reduce the risk for gastrointestinal side effects associated with traditional NSAIDs while retaining their efficacy is the inclusion of co-therapy with a proton pump inhibitor; these agents inhibit acid secretion and have been demonstrated to promote ulcer healing in patients with NSAID-related gastric ulcers. Alternatively, COX-2 selective agents have been used to treat patients at high risk for such events. Both nonselective and selective COX-2 inhibitors have now been shown to be associated with an increased risk for cardiovascular events. These studies, together with the outcomes of the recent US Food and Drug Administration decision to require 'black box' warnings regarding potential cardiovascular risks associated with NSAIDs, suggest that the use of COX-2 inhibitors as the sole strategy for gastroprotection in patients with arthritis and other pain syndromes must be reconsidered, particularly among those at risk for cardiovascular events.     

Modulation of NSAID-induced antinociceptive and anti-inflammatory effects by alpha2-adrenoceptor agonists with gastroprotective effects

Tizanidine, an alpha2-adrenergic receptor agonist with myospasmolytic action, is indicated for the treatment of back pain either as monotherapy or in combination with nonsteridal anti-inflammatory drugs (NSAIDs). Tizanidine (0.25-1.0 mg/kg) significantly produced analgesic and anti-inflammatory effect in acetic acid induced writhing in mice and carrageenan-induced paw edema in rats, respectively. The effects were comparable with clonidine (0.25 and 0.50 mg/kg), another alpha2-agonist. Yohimbine (1 mg/kg), alpha2-adrenergic antagonist reversed the effect of tizanidine. Tizanidine (0.25 mg/kg) and clonidine (0.25 mg/kg) significantly potentiated the antinociceptive and anti-inflammatory effect of NSAIDs (nimesulide, meloxicam and naproxen). Tizanidine (1 mg/kg) did not alter basal pH, acidity (free and total) of gastric content and did not produce any mucosal injury in fasted rats. Tizanidine (1 mg/kg) significantly reduced meloxicam (UD50 3.21 mg/kg), nimesulide (UD50 24.52 mg/kg) and naproxen (UD50 14.10 mg/kg)-induced ulcerogenic effect (ulcer index, pH and free/total acidity). It is expected that tizanidine exerted gastrotprotection through stimulation of gastric and central alpha2-adrenergic receptors. Present investigation suggested that tizanidine not only enhance the analgesic and anti-inflammatory effect of NSAIDs but also improved gatstrointestinal tolerability of NSAIDs through modulation of central alpha-2-receptors. From this study, it can be speculated that tizanidine and NSAID combination therapy would prove to be a novel approach to treat nociceptive/inflammatory conditions with improved gastric tolerability of NSAIDs.     

Nonsteroidal anti-inflammatory drugs impair oral mucosal repair by eliciting disturbances in endothelin-converting enzyme-1 and constitutive nitric oxide synthase.

Although the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is known to cause the impairment in mucosal defenses that are well recognized and clinically emphasized with respect to the gastrointestinal tract, less apparent is the extent of their interference with the repair of soft oral tissue. As the disturbances in nitric oxide generation and the release of endothelin-1 (ET-1) are the early signs of injury by NSAIDs, we investigated oral mucosal ulcer healing in the presence of NSAID administration by analyzing the expression of endothelin-converting enzyme-1(ECE-1), responsible for ET-1 generation, and the mucosal activity of inducible (NOS-2) and constitutive (cNOS) nitric oxide synthase responsible for nitric oxide production. Groups of rats with acetic-induced buccal mucosal ulcers were subjected twice daily for up to 10 days to intragastric administration of either indomethacin (5 mg/kg), aspirin (20 mg/kg), or the vehicle and their mucosal tissue subjected to macroacopic assessment of ulcer healing rate and biochemical measurements. While in the control group the ulcer healed by the tenth day, only a 57.2% reduction in the ulcer crater area was attained in the animals subjected to indomethacin and a 54.8% reduction in ulcer occurred in the presence of aspirin administration. Futhermore, by the tenth day, the delay in healing in the presence of indomethacin was manifested by a 4.9-fold higher rate of apoptosis, a 2.7-fold higher expression of ECE-1 activity, a 3.9-fold higher expression of NOS-2 activity and a 2.2-fold decline in cNOS activity, while the interference in ulcer healing by aspirin was characterized by a 5.6-fold higher rate of apoptosis, a 2.8-fold expressiom of ECE-1 activity, a 3.7-fold higher expression of NOS-2 activity and a 2.3-fold lower expression of cNOS activity. Our findings demonstrate that NSAIDs not only pose a well-known risk of gastrointestinal injury, but also interfere with soft oral tissue repair. The impairment in buccal mucosal ulcer healing by NSAID ingestion is manifested in up-regulation in the expression of ECE-1 responsible for ET-1 generation, suppression in cNOS, and amplification of apoptotic events that delay the healing process.     

In vitro scavenging activity for reactive oxygen and nitrogen species by nonsteroidal anti-inflammatory indole, pyrrole, and oxazole derivative drugs

This study was undertaken to evaluate the scavenging activity for reactive oxygen species (ROS) and reactive nitrogen species (RNS) by several nonsteroidal anti-inflammatory drugs (NSAIDs), namely indole derivatives (indomethacin, acemetacin, etodolac), pyrrole derivatives (tolmetin and ketorolac), and an oxazole derivative (oxaprozin). The inhibition of prostaglandin synthesis constitutes the primary mechanism of the anti-inflammatory action of these drugs. Nevertheless, it has been suggested that the anti-inflammatory activity of NSAIDs may be also partly due to their ability to scavenge ROS and RNS and to inhibit the respiratory burst of neutrophils triggered by various activator agents. Thus, the scavenging activity of these NSAIDs was evaluated against an array of ROS (O(2)(-), HO, HOCl, and ROO) and RNS (NO and ONOO(-)) using noncellular in vitro systems. The results obtained demonstrated that tolmetin, ketorolac, and oxaprozin were not active against O(2)(-), while acemetacin, indomethacin, and etodolac exhibited concentration-dependent effects. Oxaprozin was also the least active scavenger for HO, among all the tested NSAIDs shown to be active. The scavenging effect for HOCl was not observed for any of the tested NSAIDs. The ROO was effectively scavenged by etodolac, with the other tested NSAIDs being much less active. NO and ONOO(-) were scavenged by all the tested NSAIDs. These effects may strongly contribute to the anti-inflammatory therapy benefits that may be attained with some of the studied NSAIDs.     

Synthesis and pharmacological evaluation of some dual-acting amino-alcohol ester derivatives of flurbiprofen and 2-[1,1'-biphenyl-4-yl]acetic acid: a potential approach to reduce local gastrointestinal toxicity

The search for safer non-steroidal anti-inflammatory drugs (NSAIDs) continues with the failure of anticipated 'ideal' anti-inflammatory agents, the coxibs, on long-term usage. Increased gastric motility and acidity due to the free carboxy group are involved in the etiology of gastric toxicity, common to conventional NSAIDs. Keeping this fact in mind, it was planned to modify some of the conventional NSAIDs to amino-alcohol ester derivatives, which satisfied the structural requirements for these compounds to possess anticholinergic activity in the intact form. Besides blocking the acidic carboxylic group, incorporation of anticholinergic acivity in these molecules was expected to reduce the gastric toxicity by decreasing gastric acid secretion and motility. Synthesis and pharmacological evaluation of six different N,N-disubstituted amino-ethyl ester derivatives, structurally resembling the amino-alcohol ester class of anticholinergic agents, each for [1,1'-biphenyl]-4-acetic acid (3) and flurbiprofen (10), have been reported as potential substitutes for these NSAIDs, with improved therapeutic profile. All the ester derivatives were found to have sufficient chemical stability in buffers (pH 2.0 and 7.4), ensuring them to be absorbed as intact moieties from the gastrointestinal tract. A significant reduction in ulcerogenic potency in comparison to the parent drugs with a slightly higher anti-inflammatory potency suggests that the majority of these candidates have an improved therapeutic profile over their parent drugs. Hence, a promising novel approach, different from the conventional prodrug concept, has been successfully worked out to overcome the local gastric toxicity, yielding therapeutically better compounds for long-term oral anti-inflammatory therapy.     

C-reactive protein (CRP) measurement in canine serum following experimentally-induced acute gastric mucosal injury

To establish the diagnostic significance of canine C-reactive protein (CRP) in gastrointestinal disorders, the serum canine CRP concentration was measured in dogs with experimentally-induced acute gastric mucosal injury. Gastric injury was induced in one male and one female beagle by a single dose oral administration of acetylsalicylic acid (200 mg/kg body weight) or indomethacin (60 mg/kg body weight), or sodium chloride (1,000 mg/kg body weight). CRP was measured prior to dose, and 1, 3, 7, and 14 days after the administration of the drugs, together with the total leucocyte counts and serum iron. Changes in the serum CRP in dogs with gastric injury were similar for the three test compounds, and reflected by the endoscopic findings. CRP values increased from 87 to 390 mg/l within 1 to 3 days after the compound administration but returned nearly to the predose levels within 14 days. Endoscopy revealed haemorrhagic erosion of the gastric mucosa in all dogs one day after dosing, with no evidence of the erosions observed after 7 days in many of the dogs. Changes of the total leucocyte and serum iron also occurred following gastric injury, but these changes were not as marked as those observed for CRP. The results of this study suggest that serum CRP level may be a useful indicator of a gastrointestinal mucosal injury in dogs.