Low-Dose Aspirin Acetylates Cyclooxygenase-1 in Human Colorectal Mucosa: Implications for the Chemoprevention of Colorectal Cancer.

The mechanism of action of low-dose aspirin in the prevention of colorectal cancer (CRC) remains largely hypothetical. We aimed to compare the effects of low-dose aspirin (100 mg/day for 7 days) given to 40 individuals undergoing CRC screening on the extent of cyclooxygenase (COX)-1 acetylation at serine-529 (AceCOX-1), in blood platelets vs. colorectal mucosa, at 7 (group 1) and 24 h (group 2) after dosing. A significantly (P < 0.01) lower %AceCOX-1 was detected in colonic and rectal mucosa (average 64%) vs. platelets (average 75%) in both groups. This effect was associated with an average 46% (P < 0.01) and 35% (P < 0.05) reduction in prostaglandin (PG) E2 levels and phosphorylated S6 (p-S6) levels, respectively. Rectal mucosal levels of p-S6/S6 significantly (P < 0.01) correlated with PGE2 . These findings demonstrate that low-dose aspirin produces long-lasting acetylation of COX-1 and downregulation of p-S6 in human colorectal mucosa, an effect that may interfere with early colorectal carcinogenesis.

Reappraisal of the clinical pharmacology of low-dose aspirin by comparing novel direct and traditional indirect biomarkers of drug action.

BACKGROUND: Even though the acetylation of platelet cyclooxygenase (COX)-1 at serine-529 is the direct mechanism of action of low-dose aspirin, its antiplatelet effect has been characterized using indirect indexes of COX-1 activity. OBJECTIVES: We performed a clinical study with enteric-coated low-dose aspirin (EC-aspirin), in healthy subjects, to evaluate the effects on the extent and duration of platelet COX-1 acetylation, using a novel proteomic strategy for absolute protein quantification (termed AQUA), as compared with traditional pharmacokinetic and pharmacodynamic parameters. SUBJECTS AND METHODS: In a phase I, single-arm, open-label study of EC aspirin (100 mg day(-1) ) administered to 24 healthy subjects, we compared, over a 24 h-period on day 1 and 7, % platelet acetylated COX-1 (AceCOX-1) with traditional pharmacokinetic and pharmacodynamics [i.e. serum thromboxane (TX) B2 , platelet function by monitoring CEPI(collagen/epinephrine) closure time (CT) using whole-blood PFA-100 and urinary excretion of 11-dehydro-TXB2 ] parameters. RESULTS: Acetylation of platelet COX-1 was measurable before detection of aspirin levels in the systemic circulation and increased in a cumulative fashion upon repeated dosing. After the last dose of EC-aspirin, %AceCOX-1, serum TXB2 and CEPI-CT values were maximally and persistently modified throughout 24 h; they averaged 76 ± 2%, 99.0 ± 0.4% and 271 ± 5 s, respectively. EC-aspirin caused 75% reduction in urinary 11-dehydro-TXB2 excretion. After chronic dosing with aspirin, the pharmacokinetics of acetylsalicylic acid was completely dissociated from pharmacodynamics. CONCLUSIONS: The demonstrated feasibility of quantifying the extent and duration of platelet COX-1 acetylation will allow characterizing the genetic, pharmacokinetic and pharmacodynamic determinants of the inter-individual variability in the antiplatelet response to low-dose aspirin as well as identifying extra-platelet sites of drug action.

The novel anti-inflammatory agent VA694, endowed with both NO-releasing and COX2-selective inhibiting properties, exhibits NO-mediated positive effects on blood pressure, coronary flow and endothelium in an experimental model of hypertension and endothelial dysfunction.

Selective cyclooxygenase 2 (COX2) inhibitors (COXIBs) are effective anti-inflammatory and analgesic drugs with improved gastrointestinal (GI) safety compared to nonselective nonsteroidal anti-inflammatory drugs known as traditional (tNSAIDs). However, their use is associated with a cardiovascular (CV) hazard (i.e. increased incidence of thrombotic events and hypertension) due to the inhibition of COX2-dependent vascular prostacyclin. Aiming to design COX2-selective inhibitors with improved CV safety, new NO-releasing COXIBs (NO-COXIBs) have been developed. In these hybrid drugs, the NO-mediated CV effects are expected to compensate for the COXIB-mediated inhibition of prostacyclin. This study evaluates the potential CV beneficial effects of VA694, a promising NO-COXIB, the anti-inflammatory effects of which have been previously characterized in several in vitro and in vivo experimental models. When incubated in hepatic homogenate, VA694 acted as a slow NO-donor. Moreover, it caused NO-mediated relaxant effects in the vascular smooth muscle. The chronic oral administration of VA694 to young spontaneously hypertensive rats (SHRs) significantly slowed down the age-related development of hypertension and was associated with increased plasma levels of nitrates, stable end-metabolites of NO. Furthermore, a significant improvement of coronary flow and a significant reduction of endothelial dysfunction were observed in SHRs submitted to chronic administration of VA694. In conclusion, VA694 is a promising COX2-inhibiting hybrid drug, showing NO releasing properties which may mitigate the CV deleterious effects associated with the COX2-inhibition.

Clinical pharmacology of cyclooxygenase inhibition and pharmacodynamic interaction with aspirin by floctafenine in Thai healthy subjects.

Floctafenine, a hydroxyquinoline derivative with analgesic properties, is widely used in Thailand and many other countries. The objectives of this study were to evaluate in Thai healthy volunteers: i) the inhibition of whole blood cyclooxygenase(COX)-2 and COX-1 activity by floctafenine and its metabolite floctafenic acid in vitro and ex vivo after dosing with floctafenine; ii) the possible interference of floctafenine administration with aspirin antiplatelet effects. We performed an open-label, cross-over, 3-period study, on 11 healthy Thai volunteers, who received consecutively floctafenine(200mg/TID), low-dose aspirin(81mg/daily) or their combination for 4 days, separated by washout periods. Floctafenine and floctafenic acid resulted potent inhibitors of COX-1 and COX-2 in vitro (floctafenic acid was more potent than floctafenine) showing a slight preference for COX-1. After dosing with floctafenine alone, whole blood COX-1 and COX-2 activities were inhibited ex vivo in a time-dependent fashion which paralleled floctafenic acid plasma concentrations. Aspirin alone inhibited profoundly and persistently platelet COX-1 activity and AA-induced platelet aggregation throughout 24-h dosing interval which was affected by the co-administration of floctafenine. At 24 h after dosing with aspirin and floctafenine, the inhibition of platelet thromboxane(TX)B2 generation and aggregation were significantly(P less than 0.05) lower than that caused by aspirin alone. Therapeutic dosing with floctafenine profoundly inhibited prostanoid biosynthesis through the rapid conversion to floctafenic acid. Floctafenine interfered with the antiplatelet effect of aspirin. Our results suggest that floctafenine should be avoided in patients with cardiovascular disease under treatment with low-dose aspirin.

Enlarging the NSAIDs family: ether, ester and acid derivatives of the 1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents.

The development of the coxib family has represented a stimulating approach in the treatment of inflammatory disorders, such as arthritis, and for the management of acute pains, in relation to the well-known traditional Non-Steroidal Anti-inflammatory Drugs (t-NSAIDs). Prompted by the pursuit for new cyclooxygenase-2 (COX-2) inhibitors, endowed with fine tuned selectivity and high potency, in the past years we have identified novel classes of ether, ester and acid molecules characterized by the 1,5-diarylpyrrole scaffold as potentially powerful anti-inflammatory molecules (12-66). All compounds proved to exert an in vitro inhibition profile as good as that shown by reference compounds. Compounds bearing a p-methylsulfonylphenyl substituent at C5 displayed the best issues. In particular, ester derivatives proved to perform the best in vitro profile in terms of selectivity and activity toward COX-2. The cell-based assay data showed that an increase of hindrance at the C3 side chain of compounds could translate to activity enhancement. The human whole blood (HWB) test let to highlight that submitted compounds displayed 5-10 fold higher selectivity for COX-2 vs COX-1 which should translate clinically to an acceptable gastrointestinal safety and mitigate the cardiovascular effects highlighted by highly selective COX-2 inhibitors. Finally, to assess in vivo anti-inflammatory and analgesic activity three different tests (rat paw pressure, rat paw oedema and abdominal constriction) were performed. Results showed good in vivo anti-inflammatory and analgesic activities. The issues gained with these classes of compounds represent, nowadays, a potent stimulus for a further enlargement of the NSAIDs family. In this review we describe the results obtained by our research group on this topic.

[Italian consensus on EULAR recommendations 2005 for the management of hip osteoarthritis]. / Consensus italiana sulle raccomandazioni EULAR 2005 per il trattamento dell'artrosi dell'anca.

The recommendations for the management of osteoarthritis (OA) of the hip were proposed by EULAR in 2005. Among the most important objectives of the expert charged to provide these recommendations were their wide dissemination and implementation. Thus, the information generated can be used by each individual country to produce their own set of management guidelines and algorithms for treatment in primary care. According with that previously executed for the EU-LAR recommendation 2003 for the knee, the Italian Society of Rheumatology (SIR) has organised a Consensus on the EULAR recommendations 2005 for the management of hip OA. To obtain an acceptability as large as possible, the group of experts was composed by many physicians interested in the management of hip OA, including Orthopaedics, Rheumatologists, Physiatrists, and General Practitioners. Main aim of the Consensus was to analyse the acceptability and applicability of the recommendations according to own experience and local situations in the Italy. The results of this Consensus have demonstrated that a large majority of the EULAR recommendations are endorsed by the Italian experts. Furthermore, the final document of the Italian Consensus clearly indicated the need that the specialists involved in the management of hip OA strongly encourage the dissemination of the EULAR 2005 recommendations also in Italy.

Platelet activation in patients with colorectal cancer.

Aspirin may reduce the risk of colorectal neoplasia at doses similar to those recommended for the prevention of cardiovascular disease. Thus, we aimed to address whether enhanced platelet activation, as assessed by the measurement of the urinary excretion of 11-dehydro-TXB(2) (a major enzymatic metabolite of TXB(2)), occurs in patients with colorectal cancer. In 10 patients with colorectal cancer, the urinary excretion of 11-dehydro-TXB(2) was significantly higher than in 10 controls, matched for sex, age and cardiovascular risk factors [1001(205-5571) versus 409(113-984) pg/mg creatinine, respectively, median (range), P<0.05]. The administration of aspirin 50 mg daily for 5 consecutive days to colorectal cancer patients caused a cumulative inhibition of platelet cyclooxygenase (COX)-1 activity either ex vivo, as assessed by the measurement of serum TXB(2) levels, or in vivo, as assessed by urinary 11-dehydro-TXB(2) excretion. In conclusion, enhanced platelet activation occurs in colorectal cancer patients. Permanent inactivation of platelet COX-1 by low-dose aspirin might restore anti-tumor reactivity.

[Italian consensus on Eular 2003 recommendations for the treatment of knee osteoarthritis]. / Consensus italiana sulle raccomandazioni dell'EULAR 2003 per il trattamento dell'artrosi del ginocchio.

The recommendations for the management of osteoarthritis (OA) of the knee firstly proposed by the EULAR in 2000, have been updated in 2003. One of the most important objectives of the expert charged to provide these recommendations was their dissemination. Thus, the information generated may be used by each individual country to produce their own set of management guidelines and algorithms for treatment in primary care. The Italian Society of Rheumatology (SIR) and the Italian League against Rheumatism (LIMAR) have organised a Consensus on the EULAR recommendations 2003 with the aim to analyse their acceptability and applicability according to our own experience and local situations in the Italy. The results of this Consensus have demonstrated that a large majority of the EULAR recommendations are endorsed by the Italian experts. Furthermore, the final document of the Italian Consensus clearly indicated the need that specialists involved in the management of knee OA strongly encourage the dissemination of the EULAR 2003 recommendations also in Italy.

Clinical pharmacology of novel selective COX-2 inhibitors.

Novel coxibs (i.e. etoricoxib, valdecoxib, parecoxib and lumiracoxib) with enhanced biochemical cyclooxygenase (COX)-2 selectivity over that of rofecoxib and celecoxib have been recently developed. They have the potential advantage to spare COX-1 activity, thus reducing gastrointestinal toxicity, even when administered at high doses to improve efficacy. They are characterized by different pharmacodynamic and pharmacokinetics features. The higher biochemical selectivity of valdecoxib than celecoxib, evidenced in vitro, may be clinically relevant leading to an improved gastrointestinal safety. Interestingly, parecoxib, a pro-drug of valdecoxib, is the only injectable coxib. Etoricoxib shows only a slightly improved COX-2 selectivity than rofecoxib, a highly selective COX-2 inhibitor that has been reported to halve the incidence of serious gastrointestinal toxicity compared to nonselective nonsteroidal antiinflammatory drugs (NSAIDs). Lumiracoxib, the most selective COX-2 inhibitor in vitro, is the only acidic coxib. The hypothesis that this chemical property may lead to an increased and persistent drug accumulation in inflammatory sites and consequently to an improved clinical efficacy, however, remains to be verified. Several randomized clinical studies suggest that the novel coxibs have comparable efficacy to nonselective NSAIDs in the treatment of osteoarthritis, rheumatoid arthritis and acute pain, but they share similar renal side-effects. The apparent dose-dependence of renal toxicity may limit the use of higher doses of the novel coxibs for improved efficacy. Large-size randomized clinical trials are ongoing to define the gastrointestinal and cardiovascular safety of the novel coxibs.

[Rationale of the use of COX-2 inhibitors in ENT pathologies]. / Razionale d'uso dei cox-2 inibitori nelle affezioni ORL.

Upper airways inflammations (rhinitis, rhinosinusitis, polyposis, otitis, pharyngitis, etc) the pathologies most commonly encountered in the daily clinical practice and they represent, because of the high sanitary costs, an important social problem. The Literature suggests that almost all the symptoms, which characterize upper airways inflammations, are induced by the production of prostaglandins by cyclooxigenase (COX); it is obvious the need of a therapeutic action at this level. The non steroidal anti-inflammatory drugs (NSAID) block the activity of both COX-1 and COX-2, whereas the selective inhibitors of COX-2 (the coxibs) act only on this isoform. Actually, the therapeutic effects of both NSAIDs and coxibs are due to their actions on COX-2, while the system toxicity of NSAIDs (gastrointestinal perforation or ulcer, reduction of glomerular filtration rate, prolongation of bleeding time) is ascribable to the inference of these drugs with the COX-1. In conclusion, a correct approach to ENT inflammations must implies the use of drugs efficacious against the typical symptoms of the inflammatory process (and specifically the symptom: pain), eventually joined with an appropriate antibiotic treatment; in this context, a selective inhibitor of COX-2 short course treatment offers the double advantage of managing the inflammation and avoiding damages to the gastric mucosa.

Clinical pharmacology of selective COX-2 inhibitors.

The discovery of cyclooxygenase (COX)-2 has provided the rationale for the development of a new class of nonsteroidal antiinflammatory drugs (NSAIDs), the selective COX-2 inhibitors (denominated coxibs), with the aim of reducing the gastrointestinal (GI) toxicity associated with the administration of NSAIDs by virtue of COX-1 sparing. Rofecoxib and celecoxib are the first selective COX-2 inhibitors approved by the FDA and EMEA for the treatment of rheumatoid arthritis (RA), osteoarthritis (OA) and for relief of acute pain. Rofecoxib has been shown to spare COX-1 activity ex vivo, in platelets and gastric mucosa, when administered at therapeutic doses or above. In a large clinical trial, COX-2 inhibitors have been demonstrated to halve the incidence of serious upper GI events vs a nonselective NSAID. Recently, other selective COX-2 inhibitors with different COX-1/COX-2 selectivity and pharmacokinetic features have been developed, i.e. valdecoxib, parecoxib, etoricoxib and lumiracoxib. The improved biochemical selectivity of valdecoxib vs celecoxib in vitro (COX-1/COX-2 ratio: 60 vs 30, respectively) may be clinically relevant leading to an improved GI safety. Interestingly, parecoxib, a pro-drug of valdecoxib, is the only injectable coxib. Etoricoxib, showing only a slightly higher COX-2 selectivity than rofecoxib in vitro (COX-1/COX-2 ratio: 344 vs 272, respectively), has been reported to cause a similar specific COX-2 inhibition ex vivo that should translate into comparable GI safety. Lumiracoxib, the most selective COX-2 inhibitor in vitro (COX-1/COX-2 ratio: 400), is the only acidic coxib. It has been hypothesized that this pecular chemical feature may lead to an enhanced concentration in inflammatory sites that may translate into an improved clinical efficacy. The results of clinical trials have shown that coxibs have a comparable clinical efficacy and renal toxicity and an improved GI safety vs nonselective NSAIDs. Whether the different pharmacodynamic and pharmacokinetics features of the various coxibs will produce detectable differences in efficacy and toxicity remains to be evaluated in appropriate comparative randomized clinical studies.

The human pharmacology of monocyte cyclooxygenase 2 inhibition by cortisol and synthetic glucocorticoids.

BACKGROUND: We studied the concentration dependence of the inhibitory effects of cortisol, 6-methylprednisolone, and dexamethasone on cyclooxygenase-2 (COX-2) expression and activity in human monocytes in response to lipopolysaccharide (LPS) in vitro. Moreover, we characterized the time and dose dependence of the inhibitory effects of 6-methylprednisolone, administered to healthy subjects, on LPS-inducible prostaglandin E2 (PGE2) biosynthesis in whole blood ex vivo. METHODS: Heparinized whole-blood samples obtained from healthy subjects and patients with rheumatoid arthritis were incubated with LPS (10 microg/ml) for 24 hours at 37 degrees C, and PGE2 was measured in plasma as an index of monocyte COX-2 activity. Comparative experiments were performed in LPS-stimulated isolated monocytes. The levels of COX-2-like immunoreactivity in monocyte lysates were measured by a specific Western blot technique. PGE2 was evaluated by radioimmunoassay. RESULTS: Nanomolar concentrations of cortisol, 6-methylprednisolone, and dexamethasone suppressed LPS-induced PGE2 biosynthesis both in whole blood and in isolated monocytes in vitro with relative potencies similar to those reported for their anti-inflammatory effects in vivo. The administration of single oral doses (4, 8, or 16 mg) of 6-methylprednisolone caused a dose- and time-dependent inhibition of whole-blood COX-2 activity. Whole-blood samples obtained from patients with rheumatoid arthritis treated with comparable maintenance doses of glucocorticoids produced significantly lower levels of LPS-inducible PGE2 than were found in untreated patients. CONCLUSIONS: Therapeutic plasma levels of synthetic glucocorticoids down-regulate inducible prostanoid biosynthesis in circulating monocytes. This effect may represent a readily measurable surrogate marker of their clinical efficacy for dose-finding studies.

Oxidized lipids.

The addition of oxygen to lipids, in response to inflammatory and mitogenic stimuli, is an important process developed by biological systems to generate a wide spectrum of compounds both by enzymatic and non-enzymatic mechanisms. These oxidized lipids may serve as messengers for communication both within and between cells or may induce structural and metabolic changes in the cell. Since chronic inflammation has been proposed as an important risk factor for coronary events by making atherosclerotic plaques prone to rupture, extensive studies have been conducted to probe the involvement of the different pathways of lipid oxidation in the pathogenesis of coronary heart disease. The oxidation of arachidonic acid and 2-arachidonylglycerol through the activity of cyclooxygenase-2 may play a role in plaque instability through dysregulation of vascular tone and induction of endothelial dysfunction. Moreover, two families of biologically active mediators formed by free-radical catalyzed oxidation of arachidonic acid and phosphatidylcholine, i.e. isoprostanes and platelet activating factor-like lipids, respectively, may be involved. Clarification of the metabolic steps of lipid oxidation altered in unstable coronary artery disease will be of valuable help in identifying new cardiovascular markers for the prediction of the long-term risk of death from cardiac causes. The integration of this information with the presence of mutations in the genes encoding the enzymatic machinery of lipid oxidation will be useful in the selection of patients with increased risk of myocardial infarction.

Oxidant stress and aspirin-insensitive thromboxane biosynthesis in severe unstable angina.

BACKGROUND: Unstable angina is associated with enhanced lipid peroxidation and reduced antioxidant defenses. We have previously reported aspirin failure in the suppression of enhanced thromboxane (TX) biosynthesis in a subset of episodes of platelet activation in this setting. We tested the hypothesis that the in vivo formation of the F(2)-isoprostane 8-iso-prostaglandin (PG)F(2alpha), a bioactive product of arachidonic acid peroxidation, is enhanced in unstable angina and contributes to aspirin-insensitive TX biosynthesis. METHODS AND RESULTS: Urine samples were obtained from patients with unstable angina (n=32), stable angina (n=32), or variant angina (n=4) and from 40 healthy subjects for the measurement of immunoreactive 8-iso-PGF(2alpha) and 11-dehydro-TXB(2). 8-Iso-PGF(2alpha) excretion was significantly higher in patients with unstable angina (339+/-122 pg/mg creatinine) than in matched patients with stable angina (236+/-83 pg/mg creatinine, P:=0.001) and control subjects (192+/-71 pg/mg creatinine, P:<0.0001). In patients with unstable angina, 8-iso-PGF(2alpha) was linearly correlated with 11-dehydro-TXB(2) excretion (rho=0.721, P:<0.0001) and inversely correlated with plasma vitamin E (rho=-0.710, P:=0. 004). Spontaneous myocardial ischemia in patients with variant angina or ischemia elicited by a stress test in patients with stable angina was not accompanied by any change in 8-iso-PGF(2alpha) excretion, thus excluding a role of ischemia per se in the induction of increased F(2)-isoprostane production. CONCLUSIONS: These findings establish a putative biochemical link between increased oxidant stress and aspirin-insensitive TX biosynthesis in patients with unstable angina and provide a rationale for dose-finding studies of antioxidants in this setting.

Effects of vitamin E supplementation on F(2)-isoprostane and thromboxane biosynthesis in healthy cigarette smokers.

BACKGROUND: Increased formation of 8-iso-prostaglandin (PG) F(2alpha) and thromboxane (TX) A(2), potent agonists of platelet and vascular thromboxane (TH)/PGH(2) receptors, has been detected in cigarette smokers. We performed a randomized, double-blind, placebo-controlled study of the effects of vitamin E (300, 600, and 1200 mg/d, each dose for 3 consecutive weeks) on 8-iso-PGF(2alpha) and TXA(2) biosynthesis in 46 moderate cigarette smokers. METHODS AND RESULTS: Urinary immunoreactive 8-iso-PGF(2alpha) and 11-dehydro-TXB(2), plasma vitamin E, and serum TXB(2) were measured by previously validated techniques. Baseline urinary 8-iso-PGF(2alpha) and 11-dehydro-TXB(2) excretion averaged 241+/-78 and 430+/-293 pg/mg creatinine, respectively. Urinary 8-iso-PGF(2alpha) was significantly correlated with 11-dehydro-TXB(2) (r=0.360, n=138, P<0.0001). Baseline plasma vitamin E levels averaged 20.6+/-4.9 micromol/L and were inversely correlated with urinary 11-dehydro-TXB(2) (r=-0.304, P=0.039) but not with 8-iso-PGF(2alpha) (r=-0.227, P=0.129). Vitamin E supplementation caused a dose-dependent increase in its plasma levels that reached a plateau at 600 mg (42.3+/-11.2 micromol/L, P<0. 001). This was not associated with any statistically significant change in urinary 8-iso-PGF(2alpha) or 11-dehydro-TXB(2) excretion. CONCLUSIONS: Supplementation with pharmacological doses of vitamin E has no detectable effects on lipid peroxidation and thromboxane biosynthesis in vivo in healthy subjects with a mild degree of oxidant stress. These findings are consistent with the hypothesis that the basal rate of lipid peroxidation is a major determinant of the response to vitamin E supplementation and have implications for the use of vitamin E in healthy subjects as well as for the design and interpretation of clinical trials of antioxidant intervention.

Nonsteroidal anti-inflammatory drugs, COX-2 and colorectal cancer.

Use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) has been associated with reduced risk of colorectal cancer. Moreover, the NSAID sulindac reduces the number and size of polyps in patients with familial adenomatous polyposis. The mechanisms of these effects of NSAIDs are not known but several lines of evidence suggest the involvement of the inhibition of the inducible isoform of prostaglandin H synthase (known as COX-2). Specific COX-2 inhibitors, showing an improved profile of gastrointestinal safety vis-à-vis conventional NSAIDs, provide interesting tools to probe the COX-2 dependence of the apparent protection against colorectal cancer associated with the use of NSAIDs.