Pharmacokinetics and intraocular pressure-lowering activity of TAK-639, a novel C-type natriuretic peptide analog, in rabbit, dog, and monkey.

TAK-639 is a topical, 9-amino acid, synthetic, C-type natriuretic peptide analog in development for the treatment of primary open-angle glaucoma and ocular hypertension. This study investigated the impact of TAK-639 on intraocular pressure (IOP), the levels of TAK-639 in aqueous humor, and the pharmacokinetic/pharmacodynamic relationship of TAK-639 following topical ocular administration to normotensive female Dutch belted rabbits, beagle dogs, and cynomolgus monkeys. In the IOP studies, rabbits (n = 6/group) and dogs (n = 8/group) received a single topical ocular dose of TAK-639 0.03%, 0.1%, 0.3%, or 0.6% in the right eye and vehicle in the left eye; monkeys (n = 8/group) received TAK-639 0.1%, 0.3%, 0.6%, 0.9%, or 1.2% in the right eye only. IOP was measured pre dose and at various time points from 0.5 to 24 h post dose for rabbits, and 1-48 h post dose for dogs and monkeys. To assess exposure in aqueous humor, another set of animals received a single ocular dose of TAK-639 0.03%, 0.1%, 0.3%, or 0.6% (rabbits, n = 20/group; dogs, n = 14/group) or TAK-639 0.3%, 0.6%, or 1.2% (monkeys, n = 10/group) in both eyes. Aqueous humor and plasma were collected at the same post dose time points at which IOP was measured. Aqueous humor and plasma TAK-639 concentrations were measured by liquid chromatography-mass spectrometry, and pharmacokinetic parameters were estimated with non-compartmental analysis. Topical ocular administration of TAK-639 resulted in a dose-dependent decrease in IOP, with maximum mean decreases in IOP ranging from -8.90% to -34.4% in the rabbit, from -16.5% to -26.4% in the dog, and from -3.43% to -13.5% in the monkey. The duration of the IOP-lowering effect was 12 h in the rabbit and monkey and 48 h in the dog. TAK-639 exposure in aqueous humor (both maximum concentration and area under the curve) was also dose dependent, with maximum concentration ranging from 0.152 to 93.6 ng/mL (0.03% and 0.6% doses, respectively) in rabbits, 0.490-13.8 ng/mL (0.03% and 0.3% doses, respectively) in dogs, and 1.16-18.1 ng/mL (0.3% and 1.2% doses, respectively) in monkeys. The pharmacokinetic/pharmacodynamic profile, when fitted to an inhibitory sigmoidal model, demonstrated that TAK-639 exposure in aqueous humor correlated well with IOP reduction in these species. The TAK-639 exposure in aqueous humor at half maximal IOP reduction (EC50) was lower in monkey and dog than in rabbit (0.2 and 0.4 vs. 2.0 ng/mL, respectively). In plasma, quantifiable concentrations of TAK-639 were low and detectable predominantly at early time points. In conclusion, in rabbit, dog, and monkey, a single topical ocular drop of TAK-639 had a significant IOP-lowering effect that correlated well with increases in TAK-639 levels in aqueous humor and resulted in minimal systemic exposure of TAK-639.

Multiple Natural and Experimental Inflammatory Rabbit Lacrimal Gland Phenotypes.

PURPOSE: To investigate lacrimal gland (LG) immunophysiological and immune-mediated inflammatory process (IMIP) phenotype diversity. METHODS: Ex vivo matured dendritic cells (mDC) were loaded with acinar cell microparticles (MP). Peripheral blood lymphocytes (PBL) were activated in mixed cell reactions with mDC and injected directly into autologous, unilateral LG (1°ATD-LG) of two rabbit cohorts, one naïve, one immunized with a LG lysate membrane fraction (Pi). Autoimmune IgG titers were assayed by ELISA, MCR PBL stimulation indices (SI) by [3H]-thymidine incorporation. Schirmer tests without and with topical anesthetic (STT-I, STT-IA) and rose Bengal (RB) staining tests were performed. H&E and immunohistochemically stained sections were examined. RNA yields and selected transcript abundances were measured. Immune cell number and transcript abundance data were submitted to Principal Component Analysis (PCA). RESULTS: Immunizing Pi dose influenced SI but not IgG titers. STT scores were decreased, and rose Bengal scores increased, by day 118 after immunization. Previous immunization exacerbated scores in 1°ATD-eyes and exacerbated 1°ATD-LG atrophy. IMIP were evident in 2°ATD-LG as well as 1°ATD-LG. PCA described diverse immunophysiological phenotypes in control LG and diverse IMIP phenotypes in ATD-LG. IgG titers and SI pre-adoptive transfer were significantly associated with certain post-adoptive transfer IMIP phenotype features, and certain LG IMIP features were significantly associated with RB and STT IA scores. CONCLUSIONS: The underlying variability of normal states may contribute to the diversity of experimental IMIP phenotypes. The ability to generate and characterize diverse phenotypes may lead to phenotype-specific diagnostic and therapeutic paradigms.

Conservative Secondary Shell Substitution In Cyclooxygenase-2 Reduces Inhibition by Indomethacin Amides and Esters via Altered Enzyme Dynamics.

The cyclooxygenase enzymes (COX-1 and COX-2) are the therapeutic targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Neutralization of the carboxylic acid moiety of the NSAID indomethacin to an ester or amide functionality confers COX-2 selectivity, but the molecular basis for this selectivity has not been completely revealed through mutagenesis studies and/or X-ray crystallographic attempts. We expressed and assayed a number of divergent secondary shell COX-2 active site mutants and found that a COX-2 to COX-1 change at position 472 (Leu in COX-2, Met in COX-1) reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings.

High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker.

Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H4]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H4]EA) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H4]EA was linear from 10 nM to 10 µM, and the analysis time was less than 6 min/sample. Results using the [²H4]AEA and HPLC-MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC-MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials.

(R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2.

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid and the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. Evaluation of a series of COX-2 inhibitors revealed that many weak competitive inhibitors of arachidonic acid oxygenation are potent inhibitors of endocannabinoid oxygenation. (R) enantiomers of ibuprofen, naproxen and flurbiprofen, which are considered to be inactive as COX-2 inhibitors, are potent 'substrate-selective inhibitors' of endocannabinoid oxygenation. Crystal structures of the COX-2­(R)-naproxen and COX-2­(R)-flurbiprofen complexes verified this unexpected binding and defined the orientation of the (R) enantiomers relative to (S) enantiomers. (R)-Profens selectively inhibited endocannabinoid oxygenation by lipopolysaccharide-stimulated dorsal root ganglion (DRG) cells. Substrate-selective inhibition provides new tools for investigating the role of COX-2 in endocannabinoid oxygenation and a possible explanation for the ability of (R)-profens to maintain endocannabinoid tone in models of neuropathic pain.

Differential sensitivity and mechanism of inhibition of COX-2 oxygenation of arachidonic acid and 2-arachidonoylglycerol by ibuprofen and mefenamic acid.

Ibuprofen and mefenamic acid are weak, competitive inhibitors of cyclooxygenase-2 (COX-2) oxygenation of arachidonic acid (AA) but potent, noncompetitive inhibitors of 2-arachidonoylglycerol (2-AG) oxygenation. The slow, tight-binding inhibitor, indomethacin, is a potent inhibitor of 2-AG and AA oxygenation whereas the rapidly reversible inhibitor, 2'-des-methylindomethacin, is a potent inhibitor of 2-AG oxygenation but a poor inhibitor of AA oxygenation. These observations are consistent with a model in which inhibitors bind in one subunit of COX-2 and inhibit 2-AG binding in the other subunit of the homodimeric protein. In contrast, ibuprofen and mefenamate must bind in both subunits to inhibit AA binding.

Hydrolysis of a series of parabens by skin microsomes and cytosol from human and minipigs and in whole skin in short-term culture.

Parabens are esters of 4-hydroxybenzoic acid and used as anti-microbial agents in a wide variety of toiletries, cosmetics and pharmaceuticals. It is of interest to understand the dermal absorption and hydrolysis of parabens, and to evaluate their disposition after dermal exposure and their potential to illicit localised toxicity. The use of minipig as a surrogate model for human dermal metabolism and toxicity studies, justifies the comparison of paraben metabolism in human and minipig skin. Parabens are hydrolysed by carboxylesterases to 4-hydroxybenzoic acid. The effects of the carboxylesterase inhibitors paraoxon and bis-nitrophenylphosphate provided evidence of the involvement of dermal carboxylesterases in paraben hydrolysis. Loperamide, a specific inhibitor of human carboxylesterase-2 inhibited butyl- and benzylparaben hydrolysis in human skin but not methylparaben or ethylparaben. These results show that butyl- and benzylparaben are more selective substrates for human carboxylesterase-2 in skin than the other parabens examined. Parabens applied to the surface of human or minipig skin were absorbed to a similar amount and metabolised to 4-hydroxybenzoic acid during dermal absorption. These results demonstrate that the minipig is a suitable model for man for assessing dermal absorption and hydrolysis of parabens, although the carboxylesterase profile in skin differs between human and minipig.

The distribution of esterases in the skin of the minipig.

Skin esterases serve an important pharmacological function as they can be utilised for activation of topically applied ester prodrugs. Understanding the nature of these enzymes, with respect to their role and local activity, is essential to defining the efficacy of ester prodrugs. Minipigs are used as models to study the kinetics of absorption of topically applied drugs. Their skin has structural properties very similar to human skin. However, regional distribution differences in esterase activity from site-to-site could influence cross-species extrapolation. Investigation of the regional site variation of minipig skin esterase activity will facilitate standardization of topically applied drug studies. Furthermore, the characterization of regional skin variation, will aid in translation of minipig results to better predictions of human esterase activity. Here we report the variation in rates of hydrolysis by minipig skin taken from different regional sites, using the esterase-selective substrates: phenyl valerate (carboxylesterase), phenyl acetate (arylesterase) and p-nitrophenyl acetate (general esterase). Skin from ears and back of male minipig showed higher activity than female. Skin from minipig ears and the back showed the highest level of esterase activity and was similar to human breast skin used in vitro absorption studies. These results suggest that skin from the minipig back is an appropriate model for preclinical human skin studies, particularly breast skin. This study supports the use of the minipig, with topical application to the back, as a model for the investigation of pharmacokinetics and metabolism of ester prodrugs.

Oxidative metabolism of lipoamino acids and vanilloids by lipoxygenases and cyclooxygenases.

The lipoamino acids and endovanilloids have multiple roles in nociception, pain, and inflammation, yet their biological reactivity has not been fully characterized. Cyclooxygenases (COXs) and lipoxygenases (LOs) oxygenate polyunsaturated fatty acids to generate signaling molecules. The ability of COXs and LOs to oxygenate arachidonyl-derived lipoamino acids and vanilloids was investigated. COX-1 and COX-2 were able to minimally metabolize many of these species. However, the lipoamino acids were efficiently oxygenated by 12S- and 15S-LOs. The kinetics and products of oxygenation by LOs were characterized. Whereas 15S-LOs retained positional specificity of oxygenation with these novel substrates, platelet-type 12S-LO acted as a 12/15-LO. Fatty acid oxygenases may play an important role in the metabolic inactivation of lipoamino acids or vanilloids or may convert them to bioactive derivatives.

Parabens inhibit human skin estrogen sulfotransferase activity: possible link to paraben estrogenic effects.

Parabens (p-hydroxybenzoate esters) are a group of widely used preservatives in topically applied cosmetic and pharmaceutical products. Parabens display weak associations with the estrogen receptors in vitro or in cell based models, but do exhibit estrogenic effects in animal models. It is our hypothesis that parabens exert their estrogenic effects, in part, by elevating levels of estrogens through inhibition of estrogen sulfotransferases (SULTs) in skin. We report here the results of a structure-activity-relationship of parabens as inhibitors of estrogen sulfation in human skin cytosolic fractions and normal human epidermal keratinocytes. Similar to reports of paraben estrogenicity and estrogen receptor affinity, the potency of SULT inhibition increased as the paraben ester chain length increased. Butylparaben was found to be the most potent of the parabens in skin cytosol, yielding an IC(50) value of 37+/-5 microM. Butylparaben blocked the skin cytosol sulfation of estradiol and estrone, but not the androgen dehydroepiandrosterone. The parabens were also tested as inhibitors of SULT activity in a cellular system, with normal human epidermal keratinocytes. The potency of butylparaben increased three-fold in these cells relative to the IC(50) value from skin cytosol. Overall, these results suggest chronic topical application of parabens may lead to prolonged estrogenic effects in skin as a result of inhibition of estrogen sulfotransferase activity. Accordingly, the skin anti-aging benefits of many topical cosmetics and pharmaceuticals could be derived, in part, from the estrogenicity of parabens.

Comparison of paraben stability in human and rat skin.

Parabens are widely used preservatives in topical products, and are estrogenic in numerous experimental models. The typical cutaneous metabolism model, rat skin, hydrolyzes parabens much faster than human skin. Chronic application and absorption of parabens, combined with low metabolism rates, may lead to prolonged estrogenic effects in the skin.

Comparison of skin esterase activities from different species.

PURPOSE: Many topically applied drugs contain esters that are hydrolyzed in the skin. Minipigs have emerged as potential models of human dermatology and, in some aspects, may be superior to commonly used rat skin. The aims of this study were to evaluate the suitability of minipig and rat skin as in vitro models of human epidermal esterase activity. METHODS: Naphthyl acetate and para-nitrophenyl acetate were tested as prototypical substrates of carboxylesterases from skin, plasma, and liver. Reaction products were monitored by high-performance liquid chromatography/ultraviolet analysis. RESULTS: Hydrolysis efficiency in skin was higher than plasma, but lower than liver. The esterase efficiency of rat skin microsomes (580-1100 min(-1) mg(-1)) was two to three orders of magnitude higher than human (1.3-4.2 min(-1) mg(-1)) and minipig microsomes (1.2-4.2 min(-1) mg(-1)). Rat skin cytosol (80-100 min(-1) mg(-1)) was 2- to 10-fold more efficient than human (2.4-67 min(-1) mg(-1)) or minipig cytosol (18-61 min(-1) mg(-1)). Most importantly, human skin fractions displayed kinetics of hydrolysis very similar to minipig skin. CONCLUSIONS: These studies show minipig skin as an appropriate, potentially valuable model for human epidermal ester metabolism and support the use of minipig skin in preclinical development of topically applied compounds.

Molecular basis of the time-dependent inhibition of cyclooxygenases by indomethacin.

Cyclooxygenases (COXs) are the therapeutic targets of nonsteroidal antiinflammatory drugs. Indomethacin (INDO) was one of the first nonsteroidal antiinflammatory drugs to be characterized as a time-dependent, functionally irreversible inhibitor, but the molecular basis of this phenomenon is uncertain. In the crystal structure of INDO bound to COX-2, a small hydrophobic pocket was identified that surrounds the 2'-methyl group of INDO. The pocket is formed by the residues Ala-527, Val-349, Ser-530, and Leu-531. The contribution of this pocket to inhibition was evaluated by altering its volume by mutagenesis of Val-349. The V349A mutation expanded the pocket and increased the potency of INDO, whereas the V349L mutation reduced the size of the pocket and decreased the potency of INDO. Particularly striking was the reversibility of INDO inhibition of the V349L mutant. The 2'-des-methyl analogue of INDO (DM-INDO) was synthesized and tested against wild-type COX-1 and COX-2, as well as the Val-349 mutants. DM-INDO bound to all enzymes tested, but only inhibited wt mCOX-2 and the V349I enzyme. Without the 2'-methyl group anchoring DM-INDO in the active site, the compound was readily competed off of the enzyme by arachidonic acid. The kinetics of inhibition were comparable to the kinetics of binding as evaluated by fluorescence quenching. These results highlight binding of the 2'-methyl of INDO in the hydrophobic pocket as an important determinant of its time-dependent inhibition of COX enzymes.

Oxidative metabolism of endocannabinoids by COX-2.

The last decade has witnessed a rapid expansion in our understanding of the mammalian endogenous cannabinoid system. In just a few short years since the discovery of endogenous lipids that serve as cannabinoids in vivo, these molecules have been shown to participate in a broad array of physiological and pathological processes. Consequently, attention has been directed at defining the proteins responsible for endocannabinoid synthesis, transport, and metabolism. Recently, multiple fatty acid oxygenases including, most notably, cyclooxygenase-2 (COX-2), have been implicated in endocannabinoid metabolism. This review will highlight connections between COX-2 and the endogenous cannabinoid system. The available biochemical evidence supporting a role for COX-2 in endocannabinoid metabolism will be presented. Finally, the potential biological consequences of COX-2-mediated endocannabinoid oxygenation will be discussed.

A novel mechanism of cyclooxygenase-2 inhibition involving interactions with Ser-530 and Tyr-385.

A variety of drugs inhibit the conversion of arachidonic acid to prostaglandin G2 by the cyclooxygenase (COX) activity of prostaglandin endoperoxide synthases. Several modes of inhibitor binding in the COX active site have been described including ion pairing of carboxylic acid containing inhibitors with Arg-120 of COX-1 and COX-2 and insertion of arylsulfonamides and sulfones into the COX-2 side pocket. Recent crystallographic evidence suggests that Tyr-385 and Ser-530 chelate polar or negatively charged groups in arachidonic acid and aspirin. We tested the generality of this binding mode by analyzing the action of a series of COX inhibitors against site-directed mutants of COX-2 bearing changes in Arg-120, Tyr-355, Tyr-348, and Ser-530. Interestingly, diclofenac inhibition was unaffected by the mutation of Arg-120 to alanine but was dramatically attenuated by the S530A mutation. Determination of the crystal structure of a complex of diclofenac with murine COX-2 demonstrates that diclofenac binds to COX-2 in an inverted conformation with its carboxylate group hydrogen-bonded to Tyr-385 and Ser-530. This finding represents the first experimental demonstration that the carboxylate group of an acidic non-steroidal anti-inflammatory drug can bind to a COX enzyme in an orientation that precludes the formation of a salt bridge with Arg-120. Mutagenesis experiments suggest Ser-530 is also important in time-dependent inhibition by nimesulide and piroxicam.

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide.

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.

Peroxisome proliferator-activated receptor gamma-mediated differentiation: a mutation in colon cancer cells reveals divergent and cell type-specific mechanisms.

Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and induces differentiation in both adipocyte and epithelial cell lineages, although it is unclear whether this occurs through common or cell-type specific mechanisms. We have identified four human colon cancer cell lines that do no undergo growth inhibition or induce markers of differentiation after exposure to PPARgamma agonists. Sequence analysis of the PPARgamma gene revealed that all four cell lines contain a previously unidentified point mutation in the ninth alpha-helix of the ligand binding domain at codon 422 (K422Q). The mutant receptor did not exhibit any defects in DNA binding or retinoid X receptor heterodimerization and was transcriptionally active in an artificial reporter assay. However, only retroviral transduction of the wild-type (WT), but not mutant, receptor could restore PPARgamma ligand-induced growth inhibition and differentiation in resistant colon cancer cell lines. In contrast, there was no difference in the ability of fibroblast cells expressing WT or K422Q mutant receptor to undergo growth inhibition, express adipocyte differentiation markers, or uptake lipid after treatment with a PPARgamma agonist. Finally, analysis of direct PPARgamma target genes in colon cancer cells expressing the WT or K422Q mutant allele suggests that the mutation may disrupt the ability of PPARgamma to repress the basal expression of a subset of genes in the absence of exogenous ligand. Collectively, these data argue that codon 422 may be a part of a co-factor(s) interaction domain necessary for PPARgamma to induce terminal differentiation in epithelial, but not adipocyte, cell lineages and argues that the receptor induces growth inhibition and differentiation via cell lineage-specific mechanisms.

Selective oxygenation of N-arachidonylglycine by cyclooxygenase-2.

Nonsteroidal anti-inflammatory drugs prevent hyperalgesia and inflammation by inhibiting the cyclooxygenase-2 (COX-2) catalyzed oxygenation of arachidonic acid to prostaglandin (PG) H(2). The lipoamino acid N-arachidonylglycine (NAGly) has also been shown to suppress tonic inflammatory pain and is naturally present at significant levels in many of the same mammalian tissues that express COX-2. Here, we report that COX-2 selectively metabolizes NAGly to PGH(2) glycine (PGH(2)-Gly) and hydroxyeicosatetraenoic glycine (HETE-Gly). Site-directed mutagenesis experiments identify the side pocket residues of COX-2, especially Arg-513, as critical determinants of the COX-2 selectivity towards NAGly. This is the first report of a charged arachidonyl derivative that is a selective substrate for COX-2. These results suggest a possible role for COX-2 in the regulation of NAGly levels and the formation of a novel class of eicosanoids from NAGly metabolism.

Isoform-selective interaction of cyclooxygenase-2 with indomethacin amides studied by real-time fluorescence, inhibition kinetics, and site-directed mutagenesis.

Conversion of carboxylate-containing nonsteroidal antiinflammatory drugs, such as indomethacin, to esters or amides provides potent and selective inhibitors of cyclooxygenase-2 (COX-2) [Kalgutkar et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 925-930]. Synthesis of cinnamyl- or coumarinyl-substituted ethanolamide derivatives of indomethacin produced fluorescent probes of inhibitor interaction with COX-2 and COX-1. Binding of either derivative to apoCOX-2 or apoCOX-1 resulted in a rapid, reversible enhancement of fluorescence. Following this rapid phase, a slow additional increase in fluorescence was observed with apoCOX-2 but not with apoCOX-1. The slow, COX-2-specific increase in fluorescence was prevented or reversed by addition of the nonfluorescent COX inhibitor (S)-flurbiprofen. Detailed kinetic studies of the interaction of the coumarinyl derivative with COX-2 showed that the observed changes in fluorescence could be described by two sequential equilibria, the first of which is rapid, reversible, and bimolecular in the forward direction. The second equilibrium is slower, reversible, and unimolecular in both directions. The forward rate constant for the slow equilibrium determined by fluorescence enhancement [(3.1 +/- 0.6) x 10(-3) s(-1)] corresponded closely to the forward rate constant for inhibition of COX-2 activity [(6.8 +/- 2.3) x 10(-3) s(-1)], suggesting that the slow fluorescence enhancement is associated with selective COX-2 inhibition. Site-directed mutagenesis indicated that residues in the carboxylate-binding region of the COX-2 active site (Arg-120, Tyr-355, and Glu-524) are critical for the binding of the indomethacin conjugates that leads to slow fluorescence enhancement and cyclooxygenase inhibition. The indomethacin conjugates described herein represent powerful tools for the investigation of a novel class of selective inhibitors of COX-2.

Enantiospecific, selective cyclooxygenase-2 inhibitors.

Cyclooxygenase inhibition studies with novel indomethacin alkanolamides demonstrate the potential for dramatic differences in inhibitor properties conferred by subtle structural modifications. The transformation of non-selective alpha-(S)-substituted indomethacin ethanolamides to potent, COX-2 selective inhibitors by simple stereocenter inversion highlights this property.