Subset Of Nonsteroidal Anti-inflammatory Drugs Research Articles

The Structural Basis for Nonsteroidal Anti-Inflammatory Drug Inhibition of Human Dihydrofolate Reductase.

Although nonsteroidal anti-inflammatory drugs (NSAIDs) target primarily cyclooxygenase enzymes, a subset of NSAIDs containing carboxylate groups also has been reported to competitively inhibit dihydrofolate reductase (DHFR). In this study, we have characterized NSAID interactions with human DHFR based on kinetic, NMR, and X-ray crystallographic methods. The NSAIDs target a region of the folate binding site that interacts with the p-aminobenzoyl-l-glutamate (pABG) moiety of folate and inhibit cooperatively with ligands that target the adjacent pteridine-recognition subsite. NSAIDs containing benzoate or salicylate groups were identified as having the highest potency. Among those tested, diflunisal, a salicylate derivative not previously identified to have anti-folate activity, was found to have a Ki of 34 μM, well below peak plasma diflunisal levels reached at typical dosage levels. The potential of these drugs to interfere with the inflammatory process by multiple pathways introduces the possibility of further optimization to design dual-targeted analogs.

Administration of meloxicam to cows experiencing dystocia does not increase the incidence of retained placenta

The post partum use of analgesics in cows has increased in consequence to society's increased awareness of animal welfare. However, it is known that the post partum administration of a subset of nonsteroidal anti-inflammatory drugs to cows may lead to an increased rate of retained placenta. Therefore, the aim of this study was clarify, whether a single administration of meloxicam to cows that experienced dystocia results in an increased rate of this disease. The study was conducted in 63 Holstein-Friesian cows in 4 farms with random allocation into 2 groups: In animals of the treatment group (n = 27) a single subcutaneous injection of meloxicam (0.5 mg/kg) was administered within 2-8 hours after calving. The animals of the control group (n = 36) received the same volume of a bovine infusion solution (Amynin®, bovine infusion solution, Boehringer Ingelheim) subcutaneously. A clinical examination of the cows was performed on the day of parturition and on the 21st day post partum. Findings on the days in between were obtained by questioning of the farmers. The exact Fisher-test was calculated in order to test for statistical differences between the treatment and control groups. In cows of the treatment group, retained placenta was not observed more frequently than in cows of the control group. A single administration of meloxicam to cows that experienced dystocia does not increase the incidence of retained placenta.

Substrate Sequence Influences γ-Secretase Modulator Activity, Role of the Transmembrane Domain of the Amyloid Precursor Protein

A subset of non-steroidal anti-inflammatory drugs modulates the γ cleavage site in the amyloid precursor protein (APP) to selectively reduce production of Aβ42. It is unclear precisely how these γ-secretase modulators (GSMs) act to preferentially spare Aβ40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on γ-cleavage of artificial constructs containing several γ-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. γ-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to γ-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on γ-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the γ-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.

Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.

ChemInform Abstract: Halogen Bonding in Halocarbon—Protein Complexes: A Structural Survey

AbstractReview: 42 refs.

Attenuated Aβ42 Responses to Low Potency γ-Secretase Modulators Can Be Overcome for Many Pathogenic Presenilin Mutants by Second-generation Compounds

Sequential processing of the β-amyloid precursor protein by β- and γ-secretase generates the amyloid β-peptide (Aβ), which is widely believed to play a causative role in Alzheimer disease. Selective lowering of the pathogenic 42-amino acid variant of Aβ by γ-secretase modulators (GSMs) is a promising therapeutic strategy. Here we report that mutations in presenilin (PS), the catalytic subunit of γ-secretase, display differential responses to non-steroidal anti-inflammatory drug (NSAID)-type GSMs and more potent second-generation compounds. Although many pathogenic PS mutations resisted lowering of Aβ(42) generation by the NSAID sulindac sulfide, the potent NSAID-like second-generation compound GSM-1 was capable of lowering Aβ(42) for many but not all mutants. We further found that mutations at homologous positions in PS1 and PS2 can elicit differential Aβ(42) responses to GSM-1, suggesting that a positive GSM-1 response depends on the spatial environment in γ-secretase. The aggressive pathogenic PS1 L166P mutation was one of the few pathogenic mutations that resisted GSM-1, and Leu-166 was identified as a critical residue with respect to the Aβ(42)-lowering response of GSM-1. Finally, we found that GSM-1-responsive and -resistant PS mutants behave very similarly toward other potent second-generation compounds of different structural classes than GSM-1. Taken together, our data show that a positive Aβ(42) response for PS mutants depends both on the particular mutation and the GSM used and that attenuated Aβ(42) responses to low potency GSMs can be overcome for many PS mutants by second generation GSMs.

Inflammation and Parkinson's Disease

After more than two decades of research, the latest published studies regarding the protective effects of anti-inflammatory drugs in Parkinson's disease (PD) indicate that only a subset of nonsteroidal anti-inflammatory drugs (NSAIDs) may be efficient in decreasing the risk of PD. In particular, recent epidemiology studies and meta-analysis have shown that, among these NSAIDS, the inhibitors of the enzyme cyclooxygenase (COX) were the most potent compounds reaching the highest rate of PD prevention. These data clearly support a COX-specific mechanism of neuroprotection and reinforce the idea that neuroinflammation in PD comprises specific features that should be unraveled. Such that knowledge should help the development of specific drugs targeting inflammatory mediators. Several clinical trials currently ongoing have focused their goals in evaluating in vivo potential imaging biomarkers for inflammatory changes in neurodegeneration. [18F] FEPPA and [(11) C] PBR28 are being evaluated for their capacity in detecting neuroinflammation by single photon emission computed tomography (SPECT). This is an important step regarding the safe monitoring of neuroinflammation in patients. If successful, these in vivo imaging methods will be valuable not only to determine precisely the right therapeutic window but also to accurately measure the biological outcomes of neuroprotective treatments. Most importantly, this also means that the inflammatory component of PD is having significant attention among researchers and it will probably be assumed in the clinical scenario in the coming years. In addition, there are numerous preclinical trials testing the protective effects of anti-inflammatory drugs in animal models of PD and hopefully some of them will soon be brought into Phase I trials. However, further research and new perspectives are needed to understand the specific aspects of inflammation in PD. In the present special issue, we present 9 review articles that explore new insights into the inflammatory reaction associated with PD. D. Litteljohn et al. show an excellent review of how the toxin-based models of PD have contributed significantly to the study of the mechanisms underlying neuroinflammatory processes in Parkinsonism and outline the role of TNF-α and IFN-γ, two cytokines critically involved in glial cell activation and dopaminergic degeneration. Then, T. Farooqui and A. A. Farooqui describe in a comprehensive review how lipid-derived factors are able to induce cellular stress and inflammation, which may be involved in PD pathogenesis. In line with this, M. Liu and G. Bing show in their revision how lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria and prototypical inflammogen, induces dopaminergic cell death indicating that the inflammatory response is by itself detrimental. Importantly, inflammation-induced toxicity seems to be highly specific for dopaminergic cells and with very special distinctiveness in some dopaminergic areas. In fact, V. Roca et al. nicely described, in their review, the unique susceptibility to inflammation of the Substantia Nigra, the prime locus of dopaminergic cell death in PD. On the other hand, C. C. Ferrari and R. Tarelli describe in their complete study how systemic inflammation may impact central inflammation and dopaminergic cell death. Then, they explore the possibility that peripheral inflammation could be a contributing factor in PD development. This view is further complemented by the review from A. Machado et al., which gives a different perspective of this particular topic but focused on studies performed in animal models of PD. Finally, in a last series of review, authors consider new perspectives and therapeutic targets to avert toxic inflammation in PD. First, A. R. Carta et al. discuss an emerging strategy to block neuroinflammation in PD using PPAR-γ agonists. This is particularly interesting in light of the above discussion since ibuprofen but not aspirin or acetaminophen has been shown to display PPAR-γ agonistic activity. P. M. Flood et al. proposed new ways for targeting NF-kB through IKK complex inhibition, a promising route that may be a useful approach in a close future in the treatment of PD. Finally, in a last review article, A. Zinger et al. describe how the kynurenine pathway, a metabolic pathway involved in the production of nicotinamide adenine dinucleotide, may be critically involved in the neuroinflammatory process in PD and how this pathway could be controlled for therapeutic purposes. Carlos Barcia Stephane Hunot Gilles J. Guillemin Fernando Pitossi

Halogen bonding in halocarbon–protein complexes: a structural survey

Halogen bonding has been extensively described in the context of a variety of self-assembled supramolecular systems and efficiently utilized in the rational design of materials with specific structural properties. However, it has so far received only little recognition for its possible role in the stabilization of small molecule-protein complexes. In this tutorial review, we provide a few examples of halogen bonds occurring between small halogen-substituted ligands and their biological substrates. Examples were drawn from a diverse set of compounds, ranging from chemical additives and possible environmental agents such as triclosan to pharmacologically active principles such as the volatile anesthetic halothane or HIV-1 reverse transcriptase inhibitors or a subset of non-steroidal anti-inflammatory drugs (NSAIDs) that are halogen-substituted. The crystal structures presented here, where iodine, bromine, or chlorine atoms function as halogen bonding donors and a variety of electron rich sites, such as oxygen, nitrogen and sulfur atoms, as well as aromatic π-electron systems, function as halogen bonding acceptors, prove how halogen bonds can occur in biological systems and provide a class of highly directional stabilizing contacts that can be exploited in the process of rational drug design.

A randomized controlled study on effects of ibuprofen on cognitive progression of Alzheimer’s disease

Epidemiological studies have examined the association between the use of non-steroidal anti-inflammatory drugs (NSAIDs) and the risk of Alzheimer's disease (AD). Recently, a variety of experimental studies indicates that a subset of NSAIDs, such as ibuprofen or flurbiprofen, also have Abeta-lowering properties in both AD transgenic mice and cell cultures of peripheral, glial and neuronal origin. In this trial, we evaluated whether the non-selective NSAID ibuprofen slows disease progression in patients with mild to moderate AD. This was a 12-month multicenter, randomized, double-blind, placebo-controlled, parallel group trial. Participants with mild-moderate AD (Mini-Mental State Examination score >15, <26; Clinical Dementia Rating= 0.5-1), 65 years or older, with reliable caregivers, were recruited between April 2003 and September 2004. Seven AD Outpatient Treatment Centers screened 530 patients, 132 of whom were enrolled. Intervention consisted of 400 mg ibuprofen twice a day or placebo, together with 20 mg once a day of esomeprazol, or placebo. The primary measure was any one-year change in the Alzheimer Disease Assessment Scale- Cognitive (ADAS-Cog) subscale score. Secondary measures included changes in MMSE, CDR, Basic and Instrumental Activities of Daily Living scales, and Neuropsychiatric Inventory (NPI). Fifty-one patients (77%) in the ibuprofen vs 46 (70%) in the placebo group completed the protocol (p>0.20). In intention-to- treat analysis, ADAS-Cog score worsening was similar in the two groups (p=0.951, treatment difference= 0.1, CI -2.7; 2.9). No differences were found for any secondary outcomes. In a subsample of genotyped patients, ApoE epsilon4 carriers treated with ibuprofen (n=27) were the only group without significant cognitive decline. Ibuprofen, if used for relatively short periods of time and although well tolerated thanks to gastroprotection, does not seem to be effective in tertiary prevention of mild-moderate AD. Our results suggest the need to examine whether differences in the response to NSAIDs exist, based on ApoE epsilon4 carrier status.

Distinct Pharmacological Effects of Inhibitors of Signal Peptide Peptidase and γ-Secretase

Signal peptide peptidase (SPP) and gamma-secretase are intramembrane aspartyl proteases that bear similar active site motifs but with opposite membrane topologies. Both proteases are inhibited by the same aspartyl protease transition-state analogue inhibitors, further evidence that these two enzymes have the same basic cleavage mechanism. Here we report that helical peptide inhibitors designed to mimic SPP substrates and interact with the SPP initial substrate-binding site (the "docking site") inhibit both SPP and gamma-secretase, but with submicromolar potency for SPP. SPP was labeled by helical peptide and transition-state analogue affinity probes but at distinct sites. Nonsteroidal anti-inflammatory drugs, which shift the site of proteolysis by SPP and gamma-secretase, did not affect the labeling of SPP or gamma-secretase by the helical peptide or transition-state analogue probes. On the other hand, another class of previously reported gamma-secretase modulators, naphthyl ketones, inhibited SPP activity as well as selective proteolysis by gamma-secretase. These naphthyl ketones significantly disrupted labeling of SPP by the helical peptide probe but did not block labeling of SPP by the transition-state analogue probe. With respect to gamma-secretase, the naphthyl ketone modulators allowed labeling by the transition-state analogue probe but not the helical peptide probe. Thus, the naphthyl ketones appear to alter the docking sites of both SPP and gamma-secretase. These results indicate that pharmacological effects of the four different classes of inhibitors (transition-state analogues, helical peptides, nonsteroidal anti-inflammatory drugs, and naphthyl ketones) are distinct from each other, and they reveal similarities and differences with how they affect SPP and gamma-secretase.

Processes of β-Amyloid and Intracellular Cytoplasmic Domain Generation by Presenilin/γ-Secretase

Background/Aims: Following extracellular shedding, transmembrane domains (TMs) of β-amyloid precursor protein (βAPP) and Notch-1 undergo proteolysis by presenilin (PS)/γ-secretase at least at two sites, near the middle of the TM (γ-/S4 cleavage) and at the interface between cytosol and the TM (Ε-/S3 cleavage), releasing Alzheimer disease (AD)-associated β-amyloid (Aβ)/Notch-1β (Nβ) and βAPP intracellular cytoplasmic domain (AICD)/Notch-1 intracellular cytoplasmic domain (NICD). Inhibiting PS/γ-secretase activity is an essential approach to AD treatment, but it also decreases NICD production, which may cause severe side effects. Therefore, it is important to investigate the differences between the cleavages at the two sites. γ-/S4 and Ε-cleavages have diversity, and produce a number of Aβ/Nβ and AICD species. S3 cleavage diversity has been recently identified. It is significant that each cleavage occurs with strict precision, not randomly. Methods: Biochemical analysis of cultured cells was performed to explore the processing mechanisms. Results: Familial AD-associated PS1 mutations as well as a subset of nonsteroidal anti-inflammatory drugs cause similar changes in γ-/S4 cleavage precision, suggesting a common process for these cleavages near the middle of the TM. While the precision of the Ε-cleavage is drastically affected by physiological factors, that of Ε-/S3 cleavage is not. Conclusion: The processes of the two cleavages occurring in different portions of TMs may be diverse, thus representing possible targets for anti-AD therapeutics to selectively reduce Aβ.

Generation of Aβ38 and Aβ42 Is Independently and Differentially Affected by Familial Alzheimer Disease-associated Presenilin Mutations and γ-Secretase Modulation

Alzheimer disease amyloid beta-peptide (Abeta) is generated via proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretase. Gamma-secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Abeta(42) and concomitantly increase the levels of the rather benign Abeta(38). Here we show that Abeta(42) and Abeta(38) generation occur independently from each other. The amount of Abeta(42) produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of gamma-secretase, appeared to correlate with the respective age of onset in patients. However, Abeta(38) levels did not show a negative correlation with the age of onset. Modulation of gamma-secretase activity by sulindac sulfide reduced Abeta(42) in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Abeta(42) or only rather subtle effects. Strikingly, even the mutations that showed no effect on Abeta(42) levels allowed a robust increase of Abeta(38) upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Abeta(42) and to decrease Abeta(38). For mutants that predominantly produce Abeta(42), the ability of fenofibrate to further increase Abeta(42) levels became diminished, whereas Abeta(38) levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Abeta(38) and Abeta(42) production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Abeta(42)-lowering capacity of gamma-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to gamma-secretase modulators.

In vitro and in vivo profiling of CHF5022 and CHF5074: Two β-amyloid 1–42 lowering agents

Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may delay or prevent the onset of Alzheimer's disease (AD). A subset of NSAIDs, including flurbiprofen, has been shown to selectively inhibit the production of β-amyloid 1–42 (Aβ42), independently from their cyclooxygenase (COX) inhibiting activity. We evaluated the in vitro and in vivo profiles of CHF5022 and CHF5074, two flurbiprofen analogues. The in vitro Aβ inhibiting activity was evaluated in a human neuroglioma cell line (H4) carrying the double Swedish mutation (K595N/M596L) of the human amyloid precursor protein (APPsw). The in vitro anti-COX activity was evaluated using human recombinant enzymes isolated from transfected Sf-9 cells. The in vivo pharmacokinetic and pharmacodynamic profiles of the two compounds were evaluated in young APPsw transgenic mice (Tg2576) after oral gavage (100 or 300 mg kg −1 day −1 for 4–5 days) and after medicated diet (375 ppm for 4 weeks). R-Flurbiprofen was used as comparator. In vitro, CHF5022 and CHF5074 were found to be 3- and 7-fold more potent than R-flurbiprofen in inhibiting Aβ42 secretion (IC 50s of 92, 40 and 268 μM, respectively). Differently from R-flurbiprofen, CHF5022 and CHF5074 did not affect COX-1 (at 100 μM) and COX-2 (at 300 μM) activity. Similarly to R-flurbiprofen, no significant alteration in the expression profile of a subset of Notch intracellular domain-responsive genes was observed with either CHF5022 or CHF5074. In Tg2576 mice, CHF5022 was well tolerated when administered by oral gavage (100 mg kg −1 day −1 for 5 days) or by medicated diet (56 mg kg −1 day −1 for 4 weeks). R-Flurbiprofen was poorly tolerated in the diet (32 mg kg −1 day −1) with 55% of the animals dying during the first week of treatment. After 4–5 days of oral gavage, CHF5022 and CHF5074 plasma and brain levels at 3 h were found to increase with the dose, leading to brain concentrations of about 10% and 5% of the corresponding plasma concentrations, respectively. In animals fed for 4 weeks with compound-supplemented diet, mean plasma (580 μM) and brain (20 μМ) concentrations of CHF5022 were 8 and 15 times higher than those of R-flurbiprofen. Plasma Aβ42 concentration was dose-dependently decreased by CHF5022 and CHF5074. Brain Aβ levels (formic acid-extractable) were not significantly affected by either compound, although Aβ42 levels tended to inversely correlate ( P = 0.105) with CHF5022 concentration in the brain. CHF5022 and CHF5074 thus appear to have a promising in vitro and in vivo profile. This warrants further evaluation of their long-term effects on Aβ brain pathology.

Are the benefits of NSAIDs to decrease the risk of Alzheimer's disease a MIRAGE?

Evaluation of: Yip AG, Green RC, Huyck M et al. Nonsteroidal anti-inflammatory drug use and Alzheimer's disease risk: the MIRAGE Study. BMC Geriatr. 5(1), 2 (2005). The Multi-Institutional Research in Alzheimer’s Genetic Epidemiology study data reveal an inverse association between nonsteroidal anti-inflammatory drugs (NSAIDs) and Alzheimer’s disease that is more robust among subjects who carried the apolipoprotein E e4 allele. Randomized trials of NSAIDs for the primary prevention of Alzheimer’s disease are unlikely to show effects with treatment until participants have been followed for several years. Inhibition of γ-secretase, as identififed in three Aβ-lowering NSAIDs, is an avenue of research likely to demonstrate preventive efficacy in prospective clinical trials. A subset of NSAIDs, or their analogs, will probably contribute to a combination-therapy approach targeted to different mechanisms of the neuropathology associated with the amyloid cascade.

Nonsteroidal anti-inflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho.

A subset of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to preferentially reduce the secretion of the highly amyloidogenic, 42-residue amyloid-beta peptide Abeta42. We found that Rho and its effector, Rho-associated kinase, preferentially regulated the amount of Abeta42 produced in vitro and that only those NSAIDs effective as Rho inhibitors lowered Abeta42. Administration of Y-27632, a selective Rock inhibitor, also preferentially lowered brain levels of Abeta42 in a transgenic mouse model of Alzheimer's disease. Thus, the Rho-Rock pathway may regulate amyloid precursor protein processing, and a subset of NSAIDs can reduce Abeta42 through inhibition of Rho activity.

Enhancement of chemotherapeutic drug toxicity to human tumour cells in vitro by a subset of non-steroidal anti-inflammatory drugs (NSAIDs)

The effect on cytotoxicity of combining a range of clinically important non-steroidal anti-inflammatory drugs (NSAIDs) with a variety of chemotherapeutic drugs was examined in the human lung cancer cell lines DLKP, A549, COR L23P and COR L23R and in a human leukaemia line HL60/ADR. A specific group of NSAIDs (indomethacin, sulindac, tolmetin, acemetacin, zomepirac and mefenamic acid) all at non-toxic levels, significantly increased the cytotoxicity of the anthracyclines (doxorubicin, daunorubicin and epirubicin), as well as teniposide, VP-16 and vincristine, but not the other vinca alkaloids vinblastine and vinorelbine. A substantial number of other anticancer drugs, including methotrexate, 5-fluorouracil, cytarabine, hydroxyurea, chlorambucil, cyclophosphamide, cisplatin, carboplatin, mitoxantrone, actinomycin D, bleomycin, paclitaxel and camptothecin, were also tested, but displayed no synergy in combination with the NSAIDs. The synergistic effect was concentration dependent. The effect appears to be independent of the cyclo-oxygenase inhibitory ability of the NSAIDs, as (i) the synergistic combination could not be reversed by the addition of prostaglandins D 2 or E 2; (ii) sulindac sulphone, a metabolite of sulindac that does not inhibit the cyclooxygenase enzyme, was positive in the combination assay: and (iii) many NSAIDs known to be cyclo-oxygenase inhibitors, e.g. meclofenamic acid, diclofenac, naproxen, fenoprofen, phenylbutazone, flufenamic acid, flurbiprofen, ibuprofen and ketoprofen, were inactive in the combination assay. The enhancement of cytotoxicity was observed in a range of drug sensitive tumour cell lines, but did not occur in P-170-overexpressing multidrug resistant cell lines. However, in the HL60/ADR and COR L23R cell lines, in which multidrug resistance is due to overexpression of the multidrug resistance-associated protein MRP, a significant increase in cytotoxicity was observed in the presence of the active NSAIDs. Subsequent Western blot analysis of the drug sensitive parental cell lines, DLKP and A549, revealed that they also expressed MRP and reverse-transcription–polymerase chain reaction studies demonstrated that mRNA for MRP was present in both cell lines. It was found that the positive NSAIDs were among the more potent inhibitors of [ 3H]-LTC 4 transport into inside-out plasma membrane vesicles prepared from MRP-expressing cells, of doxorubicin efflux from preloaded cells and of glutathione-S-transferase activity. The NSAIDs did not enhance cellular sensitivity to radiation. The combination of specific NSAIDs with anticancer drugs reported here may have potential clinical applications, especially in the circumvention of MRP-mediated multidrug resistance.