**Pathophysiology of NSAIDs peptic ulceration: Defense and injury mechanisms**

#### *Defense mechanisms*

150 New Advances in the Basic and Clinical Gastroenterology

100.000 patients were admitted every year for NSAIDs related adverse events, resulting in

Non-selective NSAIDs (nsNSAID) inhibit both cyclooxigenase-1 (COX1) and cyclooxigenase-2 (COX2). These two enzyme have different roles in the cell and, in particular, COX1 mediates prostaglandin (PG) secretion which is one of the upper GI protective mechanisms. That is why, with the aim of reducing NSAIDs related upper GI toxicity, selective COX2 inhibitors (coxibs) were developed in the last decade. Coxibs weakly inhibit COX1 and a reduced relative risk of developing upper GI injury was

The incidence of peptic ulcer disease is tightly related to epidemiological changes in environmental factors, reflecting aging, prevalence of *Helicobacter pylori* infection and use of

Non-steroidal anti-inflammatory drugs (NSAIDs) are estimated to be the most prescribed therapy worldwide (Clinard, 2001); unfortunately, chronic NSAIDs therapy may induce upper gastrointestinal injury, leading to symptoms such as dyspepsia, chest pain or heartburn or severe complications (i.e. gastroduodenal ulcers bleeding or perforation).

The incidence of GI injuries is significantly higher (about four fold) in patients receiving NSAIDs chronic therapy and 1-2.5 clinically significant adverse events were recorded for 100 patients treated/year; it was estimated that 20-40% of patients receiving chronic NSAIDs therapy present endoscopic finding of gastroduodenal mucosal injury (MacDonald, 1997; Ramey, 2005; Targownik 2006; Taha, 1996). All these evidences, lead to an increased

Moreover, these adverse event rates, resulting from observational studies, refer to general population receiving NSAIDs; when clinical studies evaluate high-risk categories, the relative risk for upper GI events significantly increase. Therefore, the available guidelines identify these high-risk categories of patients and try to outline possible specific management strategies

Different guidelines identify various risk factors for the development of upper GI injury under NSAIDs therapy: age, previous history of an upper GI event, the need of high-dose NSAIDs, Helicobacter pylori infection, use of antiplatelet agents, use of warfarin or other anticoagulant agents, corticosteroids, selective serotonin re-uptake inhibitors (SSRI), and alendronate (Langman 1994; Garcia Rodriguez 1994; Papatheodoridis, 2006; Huang, 2002). On the other hand, GI risk factors in patients receiving coxibs are not well defined, with a significant lack of data: only a previous history of peptic disease or ulcer bleeding, presence of *Helicobacter pylori* infection and concomitant assumption of antiplatelet agents are

In order to minimize NSAID-related events, evidence-based guidelines suggest to prescribe coxibs or a gastroprotective agent combined to a nsNSAID to high risk patients (Lanza, 2009). The first drug registered as a gastroprotective agent, in patients receiving NSAID, was misoprostol, a PG analogue. Clinical studies assessed the efficacy of misoprostol in reducing

for each category (Anon, 2000; Lanza, 2009; Moens, 2004; MacLean 2001).

about 15000 deaths (Weil et al., 2000; Ofman et al., 2002).

demonstrated in clinical trials in patients receiving coxibs.

**2. Epidemiology** 

mortality of patients receiving NSAIDs.

considered independent risk factors (Lanas, 2005).

NSAIDs.

The gastroduodenal mucosa is continuously exposed to endogenous (HCl, pepsin and bile acids) and exogenous (drugs, alcohol and bacteria) noxious agents; therefore, upper GI tract is characterized by a complex biological defense system, in order to prevent and heal any injury.

Pre-epithelial, epithelial and post-epithelial defenses were together involved in this complex mechanism preventing mucosal injury and maintaining integrity. The *pre-epithelial defense* level consists of mucus and a bicarbonate barrier, secreted by upper GI epithelial cells. Mucus is composed by water (95%), lipids (fatty acids and phospholipids) and glycoproteins (mucin), and constitutes an hydrophobic layer preventing ions and molecules (eg. pepsine) passage. Bicarbonate, directly secreted into the mucus layer, forms a high pH gradient (6-7) able to neutralize lumen acidity even when pH falls below 2. The *epithelial defense* layer is constituted by a continuous layer of GI epithelial surface cells linked to each other by tight junctions, these complexes constitute an hydrophobic barrier limiting the diffusion of hydrogen ions and water-soluble agents through the mucosa; moreover, hydrogen ions that enter into the epithelial cells can be removed by basolateral ion pumps (i.e. Na+/H+ and a Cl- /HCO3 exchanger). Minimal mucosal injury can be rapidly recovered thanks to the migration of the nearest healthy cells able to close the mucosal gap, a phenomenon known as *rapid restitution*. This event involves several growth factors such as *epidermal growth factor (EGF), transforming growth factor alpha (TGFα) and fibroblast growth factor (FGF)*. Rapid restitution involves only cell migration not cell division so that only minor mucosal defects can be healed; large peptic lesions requires cellular proliferation and neoangiogenesis *(regeneration)*. The rich vascular system that underlies the mucosa represents the *post-epithelial defense* mechanism. Blood flow continuously provides bicarbonate to neutralize the acids released and supplies nutrients and oxygen essential for cells metabolism while taking away all the toxic catabolites produced. (Malfertheiner, 2009; Laine, 2008).

GI injury occur when the caustic acid-peptic factors on gastrointestinal lumen overwhelm all three components of epithelial defense or when those mechanisms are impaired.

Chronic NSAIDs Therapy and Upper Gastrointestinal Tract – Mechanism of Injury,

nitronaproxen) that is in phase III trials for the treatment of osteoarthritis.

(Brzozowski, 2008).

*Aspirin* 

dose and GI toxic dose.

Mucosal Defense, Risk Factors for Complication Development and Clinical Management 153

coxibs significantly reduce, but do not completely abolish, the risk of gastrointestinal events. Moreover, as observed with nsNSAIDs other than naproxen, coxibs increase CV risk because of their pro-aggregating action; the selective inhibition of COX-2 create a disequilibrium between endothelial synthesis of PGs (mostly COX-2 dependent) and the platelets TxA2 synthesis (COX-1 dependent), with relative increased activity of the latter (Antman, 2005). Coxibs are now strongly contraindicated in patients with CV disease (Abraham, 2010; Bhatt, 2008). Finally, the evidences that COX-2 is considerably expressed in the proliferating zone of gastric mucosa undergoing mucosal repair or regeneration during ulcer healing, suggest that COX-2, although being of lesser significance in resting conditions, possess a crucial role in processes of mucosal repair and ulcer healing

The impairment of mucosal microcirculation should be considered one of the most important mechanism of damage that results from NSAIDs consumption. It originate both from the PGs inhibited biosynthesis and, at the same time, from the phlogosis that brings to leukocytes recruitment, activation and endothelial-adherence. An answer to this key source of mucosal injury has been found in nitric oxide (NO). Thanks to NO vasodilatatory activity mucosal defense mechanisms, including mucus/alkaline secretion and inhibition of leukocytes activation, result enhanced. CINODs (COX-inhibiting NO-donating drugs), a new class of anti-inflammatory compounds putting its conceptual basis on the protective action of NO, appear to preserve their anti-inflammatory proprieties with a greater gastrointestinal safety (Brzozowski, 2008). Several CINODs are currently being tested in clinical trials, the most advanced of which regards naproxcinod (NO-naproxen,

Acetylsalicylic acid (ASA), the first molecule studied for its anti-inflammatory properties, presents various effects; the mechanisms underlying these effects appear to be related to the doses: low doses (< 80 mg/day) induce an acetylation of cyclooxygenase-1 in an irreversible way, leading to antithrombotic effect; medium doses (650 mg - 4 g/day) block prostaglandin production through an inhibition of both COX-1 and COX-2; higher doses ( > 4g/day) induce an anti-inflammatory effect through both a cyclooxygenase-dependent and a COXindependent way (Lauer, 2002). Most of aspirin effects, like non-salicylate NSAIDs, are mediated by inhibition of cyclooxigenase active site of PGH2 (prostaglandin synthase H2). Aspirin acts through an irreversible inhibition of both COX isoenzymes, impairing PG production. Inhibition of COX-1 is about 10-fold greater than COX-2 ones; on this basis, the dose necessary to achieve an anti-inflammatory effect is significantly higher than antiplatelet

Moreover, aspirin inhibits (although not completely) the expression of iNOS (inducible Nitric Oxide Synthase) independently from COX-inhibition; this effect leads to an impaired production of nitric oxide, a molecule responsible for inflammatory response, host defenses and tissue healing process. This partial COX-independent suppression of NO production lead to a synergistic anti-inflammatory effect (coupled with COX inhibition) induced by

Moreover, aspirin GI toxicity, is worsened by its topic injury due to the rapid absorption of this drug from the stomach (low PKa) that result in an enhanced local gastric toxicity.

ASA, but also to a synergistic GI toxic effect with both nsNSAIDs and coxibs.

#### *Injury mechanisms*

NSAID-induced upper GI injury result from both topical damage and systemic effects mainly related to COX inhibition. *Topical injury* is a direct consequence of the chemical proprieties of these drugs. NSAIDs are weak acids that remain in non-ionized lipophilic form in the highly acid gastric environment. This condition promote the NSAIDs migration through the hydrophobic cell membrane into the cell where, because of the neutral pH, they get trapped inside in an ionized form (ion trapping). The resulting hydrogen ions are responsible of cellular toxicity; oxidative phosphorylation is compromised with impaired mithocondrial energy production, reduced cellular integrity and increased permeability. All these changes, lead to retrodiffusion of H+ and pepsin with consequent amplification of cellular toxicity (Sostres C., 2010).

Topical injury was once thought to be the main mechanism of NSAID-induced damage, but it is now clear that most of the NSAID-related gastrointestinal injuries come from their systemic effects; NSAID-related inhibition of GI mucosal cyclooxygenase, regardless of the drug administration modality, could lead to clinically significant GI toxicity.

Cyclooxigenase converts arachidonic acid into active prostaglandins (PGs); in humans (at least) two isozymes of COX were described, COX-1 and COX-2 (Wallace et al., 2000). These two isoforms present different characteristics of expression in human cells and substrates: COX-1 is almost ubiquitary and necessary for cellular homeostasis (gastric protection, vascular regulation, platelet aggregating effect and kidney function), while COX-2 is expressed in cells exposed to inflammatory signals (cytokines or chemokines) or growth factors.

Gastric cells COX-1 is the rate-limiting enzyme in PGs biosynthesis; these molecules guarantee the mucosal coating protection from the caustic action of acid and pepsin in many ways. First of all, PGs reduce gastric acid secretion and stimulate the production of glycoprotein (mucin), bicarbonate and phospholipid by epithelial cells. Moreover, PGs guarantee mucosal blood flow and oxygen delivery through vasodilatation, promote epithelial cells migration towards the luminal surface during restitution and finally enhance cells proliferation (Brzozowski, 2008; Sostres, 2010).

Most of nsNSAIDs inhibit both COX-1 and COX-2, leading to a strong impairment of gastric PG biosynthesis; therefore, in the last decades, research interest was focused on the development of new molecules with a COX-2 selective inhibitory effect, in order to obtain an effective anti-inflammatory effect and preserve PG-mediated gastrointestinal mucosal protection. (Malfertheiner, 2009; Laine, 2008).

First trials evaluating coxibs GI safety profile (Laine, 1999) were very promising; rofecoxib appeared to be safer than ibuprofen with a reported GI event rate similar to that observed in the placebo group.

However, the initial enthusiasm secondary to coxibs' GI safety was put in perspective because of the evidence of serious CV side effects (hypertension, edema, hearth failure and acute coronary syndrome) that, in some cases, brought to their withdrawal from the market (rofecoxib, precoxib and valdecoxib). Coxibs, when given at clinically effective doses, present a significantly reduced but still effective COX-1 inhibitory effect leading to a blockade of gastrointestinal mucosal COX-1-dependent PGs production: therefore, coxibs significantly reduce, but do not completely abolish, the risk of gastrointestinal events. Moreover, as observed with nsNSAIDs other than naproxen, coxibs increase CV risk because of their pro-aggregating action; the selective inhibition of COX-2 create a disequilibrium between endothelial synthesis of PGs (mostly COX-2 dependent) and the platelets TxA2 synthesis (COX-1 dependent), with relative increased activity of the latter (Antman, 2005). Coxibs are now strongly contraindicated in patients with CV disease (Abraham, 2010; Bhatt, 2008). Finally, the evidences that COX-2 is considerably expressed in the proliferating zone of gastric mucosa undergoing mucosal repair or regeneration during ulcer healing, suggest that COX-2, although being of lesser significance in resting conditions, possess a crucial role in processes of mucosal repair and ulcer healing (Brzozowski, 2008).

The impairment of mucosal microcirculation should be considered one of the most important mechanism of damage that results from NSAIDs consumption. It originate both from the PGs inhibited biosynthesis and, at the same time, from the phlogosis that brings to leukocytes recruitment, activation and endothelial-adherence. An answer to this key source of mucosal injury has been found in nitric oxide (NO). Thanks to NO vasodilatatory activity mucosal defense mechanisms, including mucus/alkaline secretion and inhibition of leukocytes activation, result enhanced. CINODs (COX-inhibiting NO-donating drugs), a new class of anti-inflammatory compounds putting its conceptual basis on the protective action of NO, appear to preserve their anti-inflammatory proprieties with a greater gastrointestinal safety (Brzozowski, 2008). Several CINODs are currently being tested in clinical trials, the most advanced of which regards naproxcinod (NO-naproxen, nitronaproxen) that is in phase III trials for the treatment of osteoarthritis.

#### *Aspirin*

152 New Advances in the Basic and Clinical Gastroenterology

NSAID-induced upper GI injury result from both topical damage and systemic effects mainly related to COX inhibition. *Topical injury* is a direct consequence of the chemical proprieties of these drugs. NSAIDs are weak acids that remain in non-ionized lipophilic form in the highly acid gastric environment. This condition promote the NSAIDs migration through the hydrophobic cell membrane into the cell where, because of the neutral pH, they get trapped inside in an ionized form (ion trapping). The resulting hydrogen ions are responsible of cellular toxicity; oxidative phosphorylation is compromised with impaired mithocondrial energy production, reduced cellular integrity and increased permeability. All these changes, lead to retrodiffusion of H+ and pepsin with consequent amplification of

Topical injury was once thought to be the main mechanism of NSAID-induced damage, but it is now clear that most of the NSAID-related gastrointestinal injuries come from their systemic effects; NSAID-related inhibition of GI mucosal cyclooxygenase, regardless of the

Cyclooxigenase converts arachidonic acid into active prostaglandins (PGs); in humans (at least) two isozymes of COX were described, COX-1 and COX-2 (Wallace et al., 2000). These two isoforms present different characteristics of expression in human cells and substrates: COX-1 is almost ubiquitary and necessary for cellular homeostasis (gastric protection, vascular regulation, platelet aggregating effect and kidney function), while COX-2 is expressed in cells exposed to inflammatory signals (cytokines or chemokines) or growth

Gastric cells COX-1 is the rate-limiting enzyme in PGs biosynthesis; these molecules guarantee the mucosal coating protection from the caustic action of acid and pepsin in many ways. First of all, PGs reduce gastric acid secretion and stimulate the production of glycoprotein (mucin), bicarbonate and phospholipid by epithelial cells. Moreover, PGs guarantee mucosal blood flow and oxygen delivery through vasodilatation, promote epithelial cells migration towards the luminal surface during restitution and finally enhance

Most of nsNSAIDs inhibit both COX-1 and COX-2, leading to a strong impairment of gastric PG biosynthesis; therefore, in the last decades, research interest was focused on the development of new molecules with a COX-2 selective inhibitory effect, in order to obtain an effective anti-inflammatory effect and preserve PG-mediated gastrointestinal mucosal

First trials evaluating coxibs GI safety profile (Laine, 1999) were very promising; rofecoxib appeared to be safer than ibuprofen with a reported GI event rate similar to that observed in

However, the initial enthusiasm secondary to coxibs' GI safety was put in perspective because of the evidence of serious CV side effects (hypertension, edema, hearth failure and acute coronary syndrome) that, in some cases, brought to their withdrawal from the market (rofecoxib, precoxib and valdecoxib). Coxibs, when given at clinically effective doses, present a significantly reduced but still effective COX-1 inhibitory effect leading to a blockade of gastrointestinal mucosal COX-1-dependent PGs production: therefore,

drug administration modality, could lead to clinically significant GI toxicity.

*Injury mechanisms* 

factors.

the placebo group.

cellular toxicity (Sostres C., 2010).

cells proliferation (Brzozowski, 2008; Sostres, 2010).

protection. (Malfertheiner, 2009; Laine, 2008).

Acetylsalicylic acid (ASA), the first molecule studied for its anti-inflammatory properties, presents various effects; the mechanisms underlying these effects appear to be related to the doses: low doses (< 80 mg/day) induce an acetylation of cyclooxygenase-1 in an irreversible way, leading to antithrombotic effect; medium doses (650 mg - 4 g/day) block prostaglandin production through an inhibition of both COX-1 and COX-2; higher doses ( > 4g/day) induce an anti-inflammatory effect through both a cyclooxygenase-dependent and a COXindependent way (Lauer, 2002). Most of aspirin effects, like non-salicylate NSAIDs, are mediated by inhibition of cyclooxigenase active site of PGH2 (prostaglandin synthase H2).

Aspirin acts through an irreversible inhibition of both COX isoenzymes, impairing PG production. Inhibition of COX-1 is about 10-fold greater than COX-2 ones; on this basis, the dose necessary to achieve an anti-inflammatory effect is significantly higher than antiplatelet dose and GI toxic dose.

Moreover, aspirin inhibits (although not completely) the expression of iNOS (inducible Nitric Oxide Synthase) independently from COX-inhibition; this effect leads to an impaired production of nitric oxide, a molecule responsible for inflammatory response, host defenses and tissue healing process. This partial COX-independent suppression of NO production lead to a synergistic anti-inflammatory effect (coupled with COX inhibition) induced by ASA, but also to a synergistic GI toxic effect with both nsNSAIDs and coxibs.

Moreover, aspirin GI toxicity, is worsened by its topic injury due to the rapid absorption of this drug from the stomach (low PKa) that result in an enhanced local gastric toxicity.

Chronic NSAIDs Therapy and Upper Gastrointestinal Tract – Mechanism of Injury,

Mucosal Defense, Risk Factors for Complication Development and Clinical Management 155

2000 – 3900 mg 1.2 > 4000 mg 1.0

1200 – 1799 mg 1.8 > 1800 mg 4.6

75 – 149 mg 3.2 > 150 mg 12.2

10 – 19 mg 12.0 > 20 mg 79.0

**Drug Dose RR**  *Acetaminophen* < 2000 mg 1.2

*Ibuprofen* < 1200 mg 1.1

*Diclofenac* < 75 mg 2.2

*Piroxicam* < 10 mg 9.0

Table 1. Dose-dependent risk for Upper GI bleeding (Acetaminophen and ns-NSAIDs)

subsequently valdecoxib) reported an alarming increase of CV events (congestive heart failure, polmunary edema and myocardial infarction), leading to withdrawal from market of

Currently, this new drug generation accounts for about 33% prescription (60% of the relative healthcare expenditure). Initially, a completely safe profile of coxibs was speculated based on preclinical and clinical trial, even for high risk patients (Skelly and Hawkey, 2002). However, these benefit effects were initially demonstrated only in patients without GI risk factors. The incidence of GI events in patients with one or more risk factors was similar in those receiving coxibs or nsNSAIDs (Silverstein et al., 2000; Bombardier et al., 2000; Skelly

Clinical trials demonstrated that coxibs have a reduced relative risk of development of peptic ulcers and other GI complications (Hooper et al.,2004); in fact, a significant reduction in ulcers found on endoscopy studies (about 4-fold reduction) was observed (FitzGerald and Patrono, 2001),. High doses of coxibs (rofecocix or celecoxib) allow an approximately 50% reduction in the incidence of GI injury when compared to nsNSAIDs (Bombardier et al., 2000). Coxibs present a reduced but not abolished GI toxicity when compared to nsNSAIDs. For example, patients receiving Rofecoxib present an increased risk for peptic ulcer bleeding when compared to patients receiving placebo (0.88 vs. 0.18 clinically significant

Moreover, coxibs do not show advantages over nsNSAIDs in healing ulcers in patients with recent bleeding, because they inhibit the natural healing process of peptic ulcers (Perini et

In addition, clinical evidences showed that all GI benefits of coxibs disappear in patients receiving also low-dose aspirin (i.e. for CV primary prevention) (Schnitzer et al., 2004; Farkouh et al., 2004). Moreover, when coxibs are used in combination to antiplatelet agents

Dose-dependent risk for Upper GI bleeding (Acetaminophen and ns-NSAIDs)

both drugs (Juni et al., 2004; Abraham et al., 2007).

events registered/year; relative risk 4.9) (Lanas et al., 2007).

and Hawkey, 2002; Farkouh et al., 2004).

al., 2003).

Finally, the use of acetylsalicylic acid, even if prescribed at low doses, seems to abolish the GI safety profile of coxibs. Although the use of a COX-2 selective inhibitors could lead to a significant decrease in GI adverse events, when coxibs are prescribed together with aspirin the overall GI toxicity appear to be similar to that observed with standard NSAIDs (Silverstein, 2000).
