Acidic NSAIDs Research Articles

Determination of non-steroidal anti-inflammatory drugs and their metabolites in milk by liquid chromatography–tandem mass spectrometry

Non-steroidal anti-inflammatory drugs are widely used for treatment of animals. According to Council Directive 96/23/EC, residues of these drugs must be monitored because of the potential risk they pose to the consumers' health. For this reason an LC-MS-MS method was developed for detection of wide range of NSAIDs, including both "acidic" NSAIDs (carprofen, diclofenac, flunixin, meloxicam, phenylbutazone, oxyphenbutazone, tolfenamic acid, mefenamic acid, naproxen, ketoprofen, ibuprofen, firocoxib, rofecoxib, and celecoxib) and "basic" NSAIDs (four metamizole metabolites). Analytes were extracted from milk samples with acetonitrile in the presence of ammonium acetate. One portion of the extract was directly analyzed for the presence of metamizole metabolites; a second portion was cleaned with an amino cartridge. All NSAIDs were separated on a Phenomenex Luna C8(2) column and analyzed by LC-MS-MS in negative (acidic NSAIDs) and positive (metamizole metabolites) ion modes. The method was validated in accordance with the requirements of Commission Decision 2002/657/EC. Within-laboratory reproducibility was in the range 7-28%, and accuracy was in the range 71-116%. The method enabled detection of all the analytes with the expected sensitivity, below the recommended concentrations. The method fulfills the criteria for confirmatory methods and, because of its efficiency, may also be used for screening purposes. The procedure was also successfully verified in the proficiency test organized by EU-RL in 2010. As far as the authors are aware, this is one of the first methods capable of detecting diclofenac residues below the MRL in milk (0.1 μg kg(-1)). An additional advantage is the possibility of simultaneous determination of "acidic" NSAIDs and metamizole metabolites.

Stable ester and amide conjugates of some NSAIDs as analgesic and antiinflammatory compounds with improved biological activity

A set of ester and amide derivatives of some acidic NSAIDs, including ibuprofen, ketoprofen, and mefenamic acid (1-3), were synthesized and evaluated for their in vivo analgesic and antiinflammatory activity using the p-benzoquinone-induced writhing test and the carrageenan-induced paw edema model, respectively. Among the synthesized compounds, ester derivatives of ketoprofen showed especially potent analgesic and antiinflammatory activity as compared to the parent drug. In vitro chemical stability studies revealed that ester and amide derivatives were chemically stable in simulated gastric (pH 1.2) and intestinal fluids (pH 6.8). In 80\% human plasma, the ester derivatives were found to be relatively stable against plasma esterases over periods of 24 h, indicating that the observed activity was not due to the parent NSAIDs. Most of the compounds were found to be nonulcerogenic under the tested conditions.

Development of Quantitative Structure-Property Relationship Models for Self-Emulsifying Drug Delivery System of 2-Aryl Propionic Acid NSAIDs

We developed the quantative structure-property relationships (QSPRs) models to correlate the molecular structures of surfactant, cosurfactant, oil, and drug with the solubility of poorly water-soluble 2-aryl propionic acid nonsteroidal anti-inflammatory drugs (2-APA-NSAIDs) in self-emulsifying drug delivery systems (SEDDSs). The compositions were encoded with electronic, geometrical, topological, and quantum chemical descriptors. To obtain reliable predictions, we used multiple linear regression (MLR) and artificial neural network (ANN) methods for model development. The obtained equations were validated using a test set of 42 formulations and showed a great predictive power, and linear models were found to be better than nonlinear ones. The obtained QSPR models would greatly facilitate fast screening for the optimal formulations of SEDDS at the early stage of drug development and minimize experimental effort.

Inclusion complexation of weakly acidic NSAID with β-cyclodextrin: selection of arginine, an amino acid, as a novel ternary component

The purpose of the present study was to investigate the influence of an amino acid, arginine, as a ternary component on the complexation of Lornoxicam, a poorly water-soluble and weakly acidic anti-inflammatory agent, with β-cyclodextrin (β-CD). The molecular inclusion of Lornoxicam with βCD alone and in combination with ternary component was aimed at improvement in solubility and, subsequently, dissolution rate limited oral absorption. The solid complexes of Lornoxicam and β-CD with or without arginine (binary and ternary systems, respectively) were prepared as Freeze dried product in different stoichiometric ratios. The formation of inclusion complexes in solid state was confirmed by using classical instrumental techniques like IR, DSC, XRD, and in liquid state by phase solubility analysis, UV spectroscopy, HPLC and 1H NMR. The in vitro dissolution and the saturation solubility of complex are determined analyzing by UV spectrophotometer. Assay and level of related substance was monitored by developed RP-HPLC method. Inclusion ternary complex of Lornoxicam with β-CD and arginine showed significant improvement in dissolution compared with uncomplexed drug and binary system. This improved physicochemical behavior of ternary complex with the novel inclusion of an arginine translated into enhanced in vitro dissolution of Lornoxicam compared with standard rapid marketed formulation.

Comparative bioavailability and pharmacokinetics of investigational enteric- and film-coated formulations of flurbiprofen 100-mg tablets: A single-dose, randomized, open-label, two-period, two-way crossover study in healthy Pakistani male volunteers

Background: Flurbiprofen, a chiral, 2-arylpropionic acid NSAID with analgesic and antipyretic properties, has been associated with important gastrointestinal adverse events, including peptic ulcer and gastrointestinal perforation. An investigational enteric-coated tablet formulation of flurbiprofen was produced to evaluate whether it would improve the gastric tolerability of flurbiprofen. Objective: This study compared the pharmacokinetic parameters and bioavailability of flurbiprofen from the investigational enteric-coated tablet (test) and from a film-coated immediate-release tablet compounded for the purposes of this study (reference). Methods: This was a randomized, open-label, 2-period, 2-way crossover study conducted in healthy male volunteers at a single center in Pakistan. Small batches of the test and reference tablets were manufactured and evaluated according to US Pharmacopoeia criteria. Each volunteer received a single 100-mg tablet of the test and reference formulations, separated by a 14-day washout period. Tablets were administered after an overnight fast. Blood samples were obtained before dosing (0) and at 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 12, and 24 hours after drug administration. Safety monitoring was performed by an unblinded physician and included adverse events, biochemistry and hematology tests, urinalysis, and ECGs. Plasma concentrations of the 2 formulations were determined, and pharmacokinetic parameters were compared using noncompartmental analysis. The 2 formulations were considered bioequivalent if the 90% CI for the ratios (test:reference) of log-transformed C max, AUC 0−t, and AUC 0–∞ were between 0.80 and 1.25. Results: Of the 23 healthy male subjects originally recruited, 2 withdrew before commencement of the study. Twenty-one subjects (mean [SD] age, 25.4 [2.7] years [range, 20–30 years]; weight, 63.4 [7.2] kg [range, 56–78 kg]) were enrolled in and completed the study. There were significant differences in C max, AUC 0−t, and AUC 0–∞ between the test and reference formulations (all, P < 0.001). The 90% CIs for the geometric mean ratios of log-transformed C max, AUC 0−t, and AUC 0–∞ (49.78–55.22, 57.51–64.42, and 58.48–65.30, respectively) were not within the predetermined bioequivalence range. There were no clinically meaningful changes in hematology, biochemistry, urinalysis, or physical variables with either formulation over the course of the study. Mild headache was reported in 2 volunteers who received the reference formulation during the first study period; this was not considered related to study drug. Conclusions: In this small study in healthy Pakistani male subjects, there were significant differences in the bioavailability and pharmacokinetic parameters of the enteric- and film-coated tablet formulations of flurbiprofen. Thus, the 2 formulations could not be considered bioequivalent. Both formulations were well tolerated.

Synthesis and biological evaluation of N-difluoromethyl-1,2-dihydropyrid-2-one acetic acid regioisomers: Dual Inhibitors of cyclooxygenases and 5-lipoxygenase

A new group of acetic acid ( 7a– c, R 1 = H), and propionic acid ( 7d– f, R 1 = Me), regioisomers wherein a N-difluoromethyl-1,2-dihydropyrid-2-one moiety is attached via its C-3, C-4, and C-5 position was synthesized. This group of compounds exhibited a more potent inhibition, and hence selectivity, for the cyclooxygenase-2 (COX-2) relative to the COX-1 isozyme. Attachment of the N-difluoromethyl-1,2-dihydropyrid-2-one ring system to an acetic acid, or propionic acid, moiety confers potent 5-LOX inhibitory activity, that is, absent in traditional arylacetic acid NSAIDs. 2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetic acid ( 7c) exhibited the best combination of dual COX-2 and 5-LOX inhibitory activities. Molecular modeling (docking) studies showed that the highly electronegative CHF 2 substituent present in 7c, that showed a modest selectivity for the COX-2 isozyme, is oriented within the secondary pocket (Val523) present in COX-2 similar to the sulfonamide (SO 2NH 2) COX-2 pharmacophore present in celecoxib, and that the N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore is oriented close to the region containing the LOX enzyme catalytic iron (His361, His366, and His545). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-one moiety possesses properties suitable for the design of dual COX-2/5-LOX inhibitory drugs.

Chlorzoxazone esters of some non-steroidal anti-inflammatory (NSAI) carboxylic acids as mutual prodrugs: Design, synthesis, pharmacological investigations and docking studies

The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-inflammatory agents devoid of the undesirable effects associated with classical NSAIDs. New chlorzoxazone ester prodrugs ( 6– 8) of some acidic NSAIDs ( 1– 3) were designed, synthesized and evaluated as mutual prodrugs with the aim of improving the therapeutic potency and retard the adverse effects of gastrointestinal origin. The structure of the synthesized mutual ester prodrugs ( 6– 8) were confirmed by IR, 1H NMR, mass spectroscopy (MS) and their purity was ascertained by TLC and elemental analyses. In vitro chemical stability revealed that the synthesized ester prodrugs ( 6– 8) are chemically stable in hydrochloric acid buffer pH 1.2 as a non-enzymatic simulated gastric fluid (SGF) and in phosphate buffer pH 7.4 as non-enzymatic simulated intestinal fluid (SIF). In 80% human plasma, the mutual prodrugs were found to be susceptible to enzymatic hydrolysis at relatively faster rate ( t 1/2 ≈ 37 and 34 min for prodrugs 6 and 7, respectively). Mutual ester prodrugs ( 6– 8) were evaluated for their anti-inflammatory and muscle relaxation activities. Scanning electromicrographs of the stomach showed that the ester prodrugs induced very little irritancy in the gastric mucosa of rats after oral administration for 4 days. In addition, docking of the mutual ester prodrugs ( 6– 8) into COX-2 active site was conducted in order to predict the affinity and orientation of these prodrugs at the enzyme active site.

Preferential uptake of the non steroid anti-inflammatory drug diclofenac into inflamed tissues after a single oral dose in rats

BackgroundDiclofenac is a nonsteroidal anti-inflammatory drug which is available as prescription (RX) and over-the-counter (OTC) medication for the systemic and topical treatment of painful and inflammatory conditions such as arthritis and back pain. This study was undertaken to investigate the distribution and retention of diclofenac and/or its metabolites in inflamed tissues, using the carrageenan-induced inflammation model and quantitative whole body autoradiography in rats.Methods[14C]diclofenac sodium was administrated as a single 2 mg/kg oral dose 1 h after injection of carrageenan into one front and one hind footpads and subcutaneously into the dorsum of the neck of rats. A control animal received saline injections. Three carrageenan-treated rats and one control rat were sacrificed at 1, 4, 8, and 24 h after [14C]diclofenac sodium administration (total of 4 rats/time point). The carcasses were immediately snap-frozen and prepared for cryosectioning. Lengthwise whole-body sections (40 μm thick), including all major tissues, were obtained from different levels across the body. The tissue concentrations of total radiolabeled components were determined using quantitative autoradioluminography.ResultsThe radioactivity patterns demonstrated that diclofenac and/or its metabolites preferentially distributed into the inflamed tissues. In unharmed tissues the distribution was similar in control and treated animals. The exposure, based on the areas under the tissue concentration versus time (AUC0-tlast), was 26 and 53 fold higher in the inflamed neck and inflamed footpads of treated animals than in control rats; the exposures in unharmed tissues were similar in the treated and control rats, and the AUC0-tlast was 17 fold higher in the inflamed paws than in the non inflamed footpads of the carrageenan-treated rats. The higher exposure in the inflamed tissues may be explained partly to the fact that the elimination of total radiolabeled components from inflamed tissues (t1/2 = 6 h) appeared lower than from the corresponding unharmed tissues (t1/2 = 2 h).ConclusionThis animal study demonstrated that diclofenac and/or its metabolites were rapidly and preferentially taken up and retained in inflamed tissues. Although there were theoretical considerations that mildly acidic NSAID may show some preferential distribution in inflamed tissues there was no clear experimental proof for diclofenac until the present study.

N-Pyridinyl(methyl)-indole-1- or 3-propanamides and propenamides acting as topical and systemic inflammation inhibitors

In this study, the synthetic way to new N-pyridinyl(methyl)indolylpropanamides acting as non acidic NSAIDs has been described. Pharmacomodulation was carried out at N1 and C5 of the indole ring and at the level of the propanamide chain. N3-pyridinylmethyl-[1(4-chlorobenzyl-5-chloroindol-3-yl)propanamide represents one of the most potent compounds in the TPA-induced mouse ear swelling assay, with a level of activity higher than that of ibuprofen and comparable to that of dexamethasone.

Acute NSAID-related transmural duodenitis and extensive duodenal ulceration

Background: A 40-year-old previously healthy white man presented to the emergency department at American University of Beirut Medical Center, Beirut, Lebanon, with severe upper abdominal pain of 36-hour duration. The pain started a few hours after the intake of a single tablet of tiaprofenic acid and became more intense after the intake of another tablet 24 hours later. He had no other symptoms. He had no prior upper gastrointestinal (GI) symptoms, ulcer disease, steroidal or nonsteroidal anti-inflammatory drug use, or ethanol intake. Physical examination revealed mild upper abdominal tenderness. Complete blood count, amylase, lipase, and liver function tests were unremarkable. Computed tomography of the abdomen showed marked thickening of the duodenal wall with surrounding mesenteric streaking. Upper GI endoscopy revealed extensive ulceration involving the duodenal bulb, apex, and proximal D2, as well as a few gastric erosions. Histopathologic examination of duodenal biopsy samples showed extensive epithelial cell necrosis and infiltration of the lamina propria with neutrophils and eosinophils. The patient responded well to rabeprazole 20 mg BID and remains well 5 months later. Methods: We performed a literature search of PubMed for all English-language articles published between January 1970 and present (June 2007) using the key words tiaprofenic acid, nonsteroidal anti-inflammatory drugs, NSAID, duodenitis, duodenal erosion, duodenal ulcer, gastritis, gastric erosion, gastric ulcer, or peptic ulcer. We reviewed all randomized controlled trials involving NSAIDs found using PubMed, with a focus on their GI adverse effects. Results: Based on the PubMed search, there were no published reports of acute transmural duodenitis and complicated duodenal ulcers associated with short-term exposure to tiaprofenic acid or other NSAIDs. The Naranjo adverse drug reaction (ADR) probability scale was used and a score of 6 was obtained, indicating a probable ADR from tiaprofenic acid use. Conclusion: We report a patient who developed symptomatic severe transmural duodenitis and periduodenal mesenteric streaking, consistent with a complicated ulcer, probably associated with very short-term exposure to tiaprofenic acid.

Assessment and prevention of gastrointestinal toxicity of non-steroidal anti-inflammatory drugs

Abstract Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for analgesic, anti-inflammatory and, in the case of aspirin, for anti-thrombotic actions. The serious gastrointestinal side-effects associated with these drugs are of concern and pose a significant obstacle to their use. This review discusses the pathogenic mechanisms by which the conventional acidic NSAIDs induce gastrointestinal toxicity, with particular emphasis on non-prostaglandin effects. Methods of assessment of NSAID-induced enteropathy are reviewed, with particular emphasis on the use of functional measurement of NSAID-induced changes in the gastrointestinal tract. The advances in our knowledge of the pathogenesis of these effects have resulted in the development of a range of novel NSAIDs. Where functional assessment of the effects of NSAIDs has been employed, it appears to be more useful as an indicator of early-stage changes rather than a predictor of the effects of long-term NSAID exposure. Successful pharmaceutical strategies now offer considerable promise for reducing the severity of NSAID damage to the gastrointestinal tract. The utility of intestinal permeability measurements for selection and assessment of these strategies is discussed.

New N‐Pyridinyl(methyl)‐indolalkanamides Acting as Topical Inflammation Inhibitors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Application of quantitative structure–toxicity relationships for acute NSAID cytotoxicity in rat hepatocytes

Non-steroidal anti-inflammatory agents (NSAIDs) are widely used for pain relief. However, they have been associated with harmful and sometimes fatal side effects. Usually, the target organs are the GI tract and liver. In this study, we have investigated the physicochemical requirements of 21 NSAIDs for glucuronidation and cytotoxicity by quantitative structure–toxicity relationships (QSTRs) in isolated rat hepatocytes. Furthermore, we have investigated the contrast in physicochemical variables that correlated with NSAID-induced hepatocyte cytotoxicity when glucuronidation was inhibited with borneol. The competitive inhibition of hepatocyte p-nitrophenol glucuronidation by NSAIDs was determined by HPLC. Glucuronidation-inhibited hepatocytes were more susceptible to NSAID-induced cytotoxicity. Also, we found a parabolic correlation between lipophilicity and the inhibition of glucuronidation for a subset of NSAIDs. For NSAIDs with a benzoic acid moiety, cytotoxicity also correlated parabolically with lipophilicity, but correlated linearly with the HOMO-LUMO gap, and the first-order valence connectivity index. The cytotoxicity of NSAIDs with a phenylacetic acid (or propionic acid) substructure also correlated with lipophilicity, but not with the HOMO-LUMO gap. Our findings indicated that the inhibition of glucuronidation resulted in increased NSAID cytotoxicity, suggesting that acyl-glucuronide metabolites were acutely less cytotoxic. Also, comparative QSTRs revealed that benzoic acid NSAIDs may form cytotoxic radical metabolites (parameterized by the HOMO-LUMO gap) or alter mitochondrial respiration (parameterized by the connectivity index), whereas phenylacetic acid derived NSAIDs may form different cytotoxic metabolites, since they did not correlate with these parameters. In summary, we have used QSTRs as a tool to distinguish the cytotoxic mechanism of two groups of NSAIDs, which, if analyzed together as one group, did not reveal such mechanism-based differences.

R-Flurbiprofen, a Non-Steroidal Anti-Inflammatory Agent (NSAID) with Anti-Tumor Activity, Inhibits the Stromal-Induced Upregulation of Interleukin-8 in Chronic Lymphocytic Leukemia Cells.

NSAIDs have documented activity in the prevention or treatment of human epithelial cancers. Little information exists, however, to determine if similar anti-tumor effects can occur with hematologic malignancies. We have evaluated R-flurbiprofen (RFB), an enantiomer of an arylpropionic acid NSAID with known anti-tumor effects, as a potential treatment for CLL. PBMNCs from 7 CLL patients (WBC 30,000 –104,000/mcL; all CD5+/CD19+/CD38−) were exposed in short term culture to RFB. All samples showed caspase-dependent cell death following addition of RFB, with a median LD50 of approximately 90mcg/mL, a clinically achievable concentration. Addition of PGE2 or caffeic acid did not prevent RFB-induced cell death, demonstrating that the effect was not due to cyclooxygenase or lipooxygenase modulation. To identify molecular events resulting from RFB treatment, we performed microarray anaylsis (with Affymetrix U74v2A chips) on drug-treated CLL cells. PBMNCs were initially cultured in medium alone, or co-cultured with allogeneic primary marrow stromal cells (MSC). MSC co-culture increased or decreased (by >2-fold) approximately 13% of the 12,000 genes/probe sets interrogated by these microarrays (2 independent experiments), but had little effect on RFB cell kill. Approximately 35 genes (fusion of 2 independent experiments) were reproducibly up- or down-regulated by RFB in the non-adherent CLL cells cultured with MSC, but not in CLL cultured alone. Among the down-regulated genes were several chemokines and adhesion molecules. These included the gene for interleukin-8 (IL8). The expression of IL8 was markedly increased in CLL cells by co-cultivation with MSC. Furthermore, IL8 expression showed reproducible and dose-dependent down-regulation in CLL cells by RFB treatment. In contrast RFB did not affect expression of IL8 in CLL cells cultured without MSC. Intracellular IL8 was measured in CLL cells by flow cytometry; RFB markedly decreased expression of IL8 protein in addition to its effects on IL8 mRNA. IL8 has been identified as a growth/survival factor for CLL cells, and is a potent tumor-derived stimulator of angiogenesis. Furthermore, increased blood levels of IL8 have been identified as a poor prognostic sign in patients with CLL. RFB may modulate leukemia cell/MSC interactions by blocking the expression of chemokines and other pro-angiogenic mediators. RFB may play a supporting role as part of a multi-modality therapy regimen for CLL.

New N-pyridinyl(methyl)-indolalkanamides acting as topical inflammation inhibitors

The authors have described the synthetic way to new N-pyridinyl(methyl)indolylpropanamides acting as non acidic NSAIDs. Pharmacomodulation was carried out at N-1 and C-5 of the indole ring and at the level of the propanamide chain. N-(pyridin-3-ylmethyl)-3-[5-chloro-1-(4-chlorobenzyl)-indol-3-yl]propanamide 32 represents one of the most potent compounds evaluated in the TPA-induced mouse ear swelling assay, with a level of activity higher than that of ibuprofen and comparable to that of dexamethasone.

Options for Treatment of Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is a chronic progressive cholestatic disease where there is progressive, granulomatous destruction of the middle-sized bile ducts. The disease affects mainly middle-aged women. The association with other autoimmune diseases and the widespread disturbance of the humoral and cellular immune systems has led to the inclusion of PBC as an autoimmune disease. However, there are several lines of evidence that suggest that both host and environmental factors are implicated in triggering the disease. Without a clear aetiology, it is difficult to find a logical approach to treatment. Well constructed clinical trials are difficult to run because of the variable and long natural history of the disease; and suitable endpoints are difficult to define and validated surrogate endpoints have not been defined. The only drug licensed for use is the bile acid, ursodeoxycholic acid. This drug is associated with significant biochemical improvement and improvement in the immunological disturbances (including a reduction in the titre of the diagnostic autoantibody, antimitochondrial antibody), but the effect on survival and histological progression is still controversial. There is little effect on symptoms. Nonetheless, its safety and lack of toxicity have meant that it has become the drug of choice and most studies now assess the effect of additional treatments. Many other agents have been studied. There is some evidence, from prospective, controlled studies, for a beneficial effect of azathioprine and ciclosporin (cyclosporine); evidence for a beneficial effect of corticosteroids and of mycophenolate is limited and there is little firm evidence for a beneficial effect of methotrexate, penicillamine, thalidomide or colchicine. Other treatments being evaluated include fibric acid derivatives (fibrates), NSAIDs and leukotriene antagonists. Liver transplantation remains the only option for end-stage disease but recurrence of disease may be found in the graft. Experimental therapies include antiretroviral therapy. Symptomatic treatment is required for pruritus and the mainstays are the bile acid binding agents such as colestyramine. For those who are intolerant of the drug or where it is ineffective, rifampicin and naltrexone may be effective. There is no effective treatment for the associated lethargy.

COX-1, COX-2 and the topical effect in NSAID-induced enteropathy.

The side effects of NSAIDs are equally evident in the stomach and the small bowel. The latter is increasingly seen as being clinically significant, contributing substantially to the iron-deficiency anaemia that is so common in patients with rheumatoid arthritis. Furthermore, NSAID-enteropathy may be associated with life-threatening events. The pathogenesis of NSAID-enteropathy is uncertain but inhibition of COX-1 is believed to be of pivotal importance. However there is increasing evidence that COX-2 inhibition and the topical effect may have a synergistic detrimental action. We examined the role of COX-1, COX-2 and the so called topical effect of acidic NSAIDs. We found that COX-1 or COX-2 inhibition and the topical effect alone do not damage the GI tract. Dual inhibition of COX-1 and COX-2 results in intestinal inflammation similar to that caused by Indomethacin. The topical effect may act synergistically in this damage. The conventional view that the mechanism of gastrointestinal damage is principally caused by COX-1 inhibition needs to be revised in view of recent studies using selective inhibitors of the COX enzymes and COX knockout animals.

Anandamide metabolism by fatty acid amide hydrolase in intact C6 glioma cells. Increased sensitivity to inhibition by ibuprofen and flurbiprofen upon reduction of extra- but not intracellular pH

The metabolism of anandamide by fatty acid amidohydrolase (FAAH) at different intra- and extracellular pH values has been investigated in intact C6 rat glioma cells. The cellular uptake of anandamide at 37 degrees C was found to decrease by 28% when the extracellular pH (pH(e)) was reduced from pH 7.4 to pH 6.2. In contrast, a selective decrease in intracellular pH (pH(i)), accomplished by acidifying the cells followed by incubation in sodium-free buffer at pH 7.4, did not affect the uptake. Anandamide uptake was inhibited by (R)-ibuprofen, with pI(50) values of 3.05+/-0.57, 3.66+/-0.23 and 3.94+/-0.88 at pH(e) values of 7.4, 6.8 and 6.2, respectively. In the presence of phenylmethylsulfonyl fluoride, however, (R)-ibuprofen failed to inhibit the uptake of anandamide. A reduction in pH(e) from 7.4 to 6.2 produced a 17% reduction in the FAAH-catalyzed metabolism of anandamide in the intact C6 cells. However, an increased sensitivity of FAAH activity to inhibition by (R)-ibuprofen as well as (R,S)-flurbiprofen and (S)-flurbiprofen was seen at a lower pH(e). For (R)-ibuprofen, pI(50) values of 3.57+/-0.08, 4.04+/-0.05 and 4.59+/-0.04 were found at pH(e) values of 7.4, 6.8 and 6.2, respectively. For (R,S)- and (S)-flurbiprofen, the pI(50) values at pH(e) 7.4 were 4.02+/-0.05 and 4.13+/-0.18, respectively at a pH(e) of 7.4, and 4.81+/-0.11 and 4.84+/-0.10, respectively, at a pH(e) of 6.2. In contrast, intracellular acidification did not affect either the rate of anandamide metabolism or its inhibition by (R)-ibuprofen or (S)-flurbiprofen. It is concluded that a reduction of extracellular pH produces an enhanced accumulation of the acidic NSAIDs ibuprofen and flurbiprofen into C6 glioma cells and thereby an inhibition of anandamide metabolism.

Occurrence and relapse of bleeding from duodenal ulcer: respective roles of acid secretion and Helicobacter pylori infection.

Helicobacter pylori infection, gastric acid hypersecretion and NSAID consumption may cause peptic ulcer. To investigate the respective roles of H. pylori and acid secretion in bleeding duodenal ulcer. A total of 99 duodenal ulcer patients were referred for evaluation of acid secretion: seven with Zollinger-Ellison Syndrome; 14 with hypersecretory duodenal ulcer, defined by the coexistence of elevated basal acid output and pentagastrin acid output; and 78 duodenal ulcer patients with normal acid output. All non-Zollinger-Ellison Syndrome patients were H. pylori-positive and cured of infection. All patients were followed-up for a 36-month period, to assess the occurrence of bleeding episodes. Twenty-nine patients had at least one bleeding episode in the 4 years before the study. Bleeding was more frequent in males and in patients on NSAIDs. The mean basal acid output was not higher among bleeders. In the 21 patients (14 hypersecretory duodenal ulcer, seven Zollinger-Ellison Syndrome) with basal acid output > 10 meg/h and pentagastrin acid output > 44.5 meg/h, the risk of bleeding was higher (OR 6.5; 95% CI: 2-21). In the follow-up period, three out of 83 (3.3%) non-Zollinger-Ellison Syndrome patients had a H. pylori-negative duodenal ulcer with bleeding. The risk of bleeding after H. pylori cure was not higher in hypersecretory duodenal ulcer patients (P > 0.3), nor among patients with previous bleeding episodes (P > 0.2). In H. pylori-positive duodenal ulcer patients, the coexistence of elevated basal acid output and pentagastrin acid output leads to a sixfold increase in the risk of bleeding. After H. pylori cure, gastric acid hypersecretion is not a risk factor for bleeding. However, duodenal ulcer recurrence with bleeding may occasionally occur in patients cured of H. pylori, even if acid output is normal.

Helix 6 of subdomain III A of human serum albumin is the region primarily photolabeled by ketoprofen, an arylpropionic acid NSAID containing a benzophenone moiety

It is well known that the subdomain III A (site II) of human serum albumin (HSA) binds a variety of endogenous and exogenous substances. However, the nature of the microenvironment of the binding site remains unclear. Ketoprofen (KP), an arylpropionic acid NSAID which contains a benzophenone moiety, was used as a photoaffinity labeling agent to label the binding region. Subsequent CNBr cleavage of the photolabeled HSA revealed that the 11.6 kDa and 9.4 kDa fragments contained most of the incorporated radioactivity. Competition experiments showed that the 11.6 kDa fragment contains the common binding region for site II ligands. This fragment was redigested with Achromobacter lyticus protease I (AP-I) and the amino acid sequence of the photolabeled peptide was determined to be XCTESLVNRR, which corresponds to the sequence 476C–485K of HSA. The complete amino acid sequence of the corresponding AP-I digested HSA peptide encompasses residues 476 to 499, which form helices 5 and 6 of subdomain III A. The HSA-Myr X-ray crystallography data showed that helix 5 is involved to the least extent in ligand binding. A docking model provided further support that helix 6 represents the photolabeled region of KP.