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Measurement of cyclooxygenase inhibition using liquid chromatography–tandem mass spectrometry

https://doi.org/10.1016/j.jpba.2010.08.001Get rights and content

Abstract

Because cyclooxygenases (COX) convert arachidonic acid into pro-inflammatory cyclic endoperoxides, inhibition of these enzymes and especially the inducible COX-2 form is an important therapeutic approach to manage inflammatory diseases and possibly prevent cancer. Due to side effects of existing non-selective and COX-2 selective non-steroidal anti-inflammatory agents, the discovery of new COX inhibitors continues to be an area of active investigation. Since existing assays are slow or lack specificity, a liquid chromatography–tandem mass spectrometry (LC–MS–MS) based COX inhibition assay was developed and validated for the rapid and accurate quantitative analysis of the COX product prostaglandin E2. The assay was validated using four COX inhibitors, celecoxib, indomethacin, resveratrol, and diclofenac that exhibit different selectivities towards COX-1 and COX-2. The IC50 values of celecoxib and resveratrol for ovine and human COX-2 were compared, and the Km values were determined. Since considerable inter-species variation was observed, human COX-2 should be used for the discovery of COX inhibitors intended for human use. This sensitive and accurate LC–MS–MS based assay is suitable for the rapid screening of ligands for COX-1 and COX-2 inhibition and for IC50 determinations.

Introduction

Cyclooxygenase (COX)-1 and/or COX-2 are the targets of widely used non-steroidal anti-inflammatory drugs (NSAIDs). Expressed constitutively in all tissues, COX-1 is essential for such physiological processes as maintenance of the gastrointestinal tract, renal function and fever [1]. COX-2 is normally undetectable in most tissues but is induced during inflammatory, degenerative, and neoplastic processes. COX-1 and COX-2 catalyze the conversion of arachidonic acid to the endoperoxide prostaglandin H2 (PGH2) (see Fig. 1) which is then metabolized to form prostaglandins, thromboxanes and prostacyclin by non-rate limiting enzymes [2]. In aqueous solution, unstable PGH2 can rearrange non-enzymatically to form PGD2 and PGE2. By inhibiting COX-1 and/or COX-2, NSAIDs prevent the enzymatic conversion of arachidonic acid to pro-inflammatory cyclic endoperoxides. Based on the assumption that selective inhibition of COX-2 might reduce the side effects of NSAIDs, the discovery of selective COX-2 inhibitors has become an important area of pharmaceutical research.

To facilitate the discovery of new COX inhibitors, in vitro screening assays have been developed that utilize cells in culture [3] or purified enzymes. By carrying out enzyme assays using COX-1 and COX-2, the selectivity of inhibitors may be determined based on the ratio of their IC50 values (IC50 is the concentration of a compound that inhibits enzyme activity by 50%) [4]. Since assays using purified enzymes are faster, more convenient and less expensive than cell-based assays, purified COX-1 and COX-2 were used in this investigation.

Several functional COX assays using purified or recombinant enzymes have been reported and include an oxygen consumption assay [5], a peroxidase co-substrate oxidation assay [5], a radiolabeled chemical inhibition assay [6], and an enzyme-linked immunosorbant assay (ELISA) [5]. The oxygen consumption assay uses a relatively insensitive O2 sensor and requires more COX than most other assays. The peroxidase co-substrate oxidation assay lacks accuracy since it can respond to various free radical intermediates formed during COX catalysis or might fail to respond when the test inhibitor is an antioxidant [7]. Alternatively, COX inhibition activity can been determined by assessing PGE2 production using an ELISA or using radiolabeled arachidonic acid and HPLC with radioactivity detection [8]. The ELISA requires almost 2 days per assay and lacks selectivity since it might respond to several prostaglandins, and the radioisotope method requires specialized licensing, training, handling, and disposal of wastes.

To overcome the limitations of previous assays, we developed an in vitro COX inhibition assay based on the selective and rapid LC–MS–MS quantitative analysis of PGE2. Although similar to existing ELISA and radioisotope methods and therefore not novel, this assay has technical advantages that include more selective measurement of PGE2 than either the ELISA or radioisotope assays due to the combination of HPLC separation and tandem mass spectrometric detection, faster analysis than ELISA (less than 1 h total including extraction, reaction and analysis), and no need for radioisotopes. The assay was validated using a range of potent and weak inhibitors of COX-1 and COX-2 inhibitors including celecoxib, indomethacin, resveratrol, and diclofenac.

Section snippets

Materials

Ovine COX-1 (oCOX-1), ovine COX-2 (oCOX-2), human recombinant COX-2 (hCOX-2), arachidonic acid, PGE2, d4-PGD2, and d2-PGE2 (labeled with deuterium atoms at positions 3 and 4, see Fig. 1) were purchased from Cayman Chemicals (Ann Arbor, MI). The co-factors (−)epinephrine and hematin, and the COX inhibitors indomethacin, resveratrol and diclofenac were purchased from Sigma–Aldrich (St. Louis, MO). Celecoxib was purchased from 3B PharmaChem International (Wuhan, China). All organic solvents were

Assay optimization

Beginning with the COX incubation conditions cited in the literature [10], multiple parameters were optimized including the enzyme level, the substrate concentration, the reaction time, and the post-reaction extraction time. A preincubation of at least 10 min was found to be necessary before adding the arachidonic acid substrate because potent COX-2 inhibitors such as celecoxib are often time-dependent and require several minutes of interaction with the enzyme to reach full inhibition potency

Conclusions

A COX inhibition assay based on the selective measurement of PGE2 using LC–MS–MS has been developed, optimized and validated by analyzing known COX inhibitors. This sensitive assay uses small amounts of enzyme that are comparable to ELISA-based assays. The assay is also fast and requires less than 5 min per sample for LC–MS–MS analysis. When comparing the IC50 values of well characterized COX inhibitors for ovine COX-2 vs. human COX-2, significant species differences were observed for some

Acknowledgements

This research was supported by grant P01 CA48112 from the National Cancer Institute and grant P50 AT00155 from the Office of Dietary Supplements, the National Center for Complementary and Alternative Medicine and the Office on Research in Women's Health.

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