LTA(4)-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB(4) receptor of human polymorphonuclear leukocytes.

5-oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A(4) (LTA(4)) in addition to 5,12-dihydroxy-(6E,8E,10E, 14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E, 11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA(4) was found to be pH-dependent. After incubation of LTA(4) in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA(4) in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC(50) of 250 nM, as compared to values of 3.5 nM for leukotriene B(4) (LTB(4)500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB(4) totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB(4). The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB(4) receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB(4) receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB(4).

Characterization of rofecoxib as a cyclooxygenase-2 isoform inhibitor and demonstration of analgesia in the dental pain model.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, and indomethacin (INN, indometacin) inhibit both the constitutive (COX-1) and inducible (COX-2) isoforms of cyclooxygenase. The induction of COX-2 after inflammatory stimuli has led to the hypothesis that COX-2 inhibition primarily accounts for the therapeutic properties of NSAIDs. METHODS: Chinese hamster ovary (CHO) cell lines that express each COX isoform were used to characterize the in vitro selectivity of rofecoxib. Single oral doses of rofecoxib and indomethacin were then assessed in subjects with use of ex vivo COX-isoform specific assays (serum thromboxane B2 [TXB2] and lipopolysaccharide [LPS]-stimulated whole blood prostaglandin E2 and assays of COX-1 and COX-2 activity, respectively). A double-blind, parallel-group study compared the analgesic efficacy of rofecoxib to placebo and ibuprofen in 102 patients with dental pain. RESULTS: Rofecoxib showed a >800-fold COX-2 selectivity with use of CHO cells that express human COX-1 and COX-2. In subjects, dose- and concentration-dependent inhibition of LPS-stimulated prostaglandin E2 was observed with both rofecoxib (IC50 [the concentration estimated to produce 50% inhibition], 0.77 micromol/L) and indomethacin (IC50, 0.33 micromol/L). Whereas indomethacin inhibited TXB2, (IC50, 0.14 micromol/L), no inhibition was observed with rofecoxib even at doses of up to 1000 mg. In the dental pain study, total pain relief (TOTPAR) over the 6 hours after dosing was similar between 50 mg and 500 mg rofecoxib and 400 mg ibuprofen (P > .20). All active treatments showed greater improvement than placebo (P < .001) CONCLUSIONS: Rofecoxib inhibited COX-2 without evidence of COX-1 inhibition, even at oral doses of up to 1000 mg. Nonetheless, rofecoxib showed analgesic activity indistinguishable from that observed with ibuprofen, a nonisoform-selective COX inhibitor. These results support the hypothesis that the analgesic effects of NSAIDs primarily derive from inhibition of COX-2.

Stereoselective carbonyl reductases from rat skin and leukocyte microsomes converting 12-ketoeicosatetraenoic acid to 12(S)-HETE.

Cell-free preparations from rat polymorphonuclear leukocytes and skin were found to catalyze the reduction of 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE) to 12-hydroxyeicosatetraenoic acid (12-HETE). The reductase activity was associated with the microsomal fraction and showed a marked preference for NADH over NADPH as reducing cofactor. Characterization of the reaction product by chiral phase HPLC of the methyl ester derivative indicated that 12-KETE reduction generated almost exclusively 12(S)-HETE. The results demonstrate that rat skin and leukocyte microsomes possess an NADH-dependent 12-KETE reductase activity that forms 12(S)-HETE as a major product. The identification of stereoselective 12-KETE reductases provides a basis for further defining the role these enzymes may play in the regulation of 12-KETE levels and in the protection against degradation of 12-KETE to the pro-inflammatory 12(R)-HETE by selectively generating 12-HETE of the S configuration.

Cloning and expression of rhesus monkey cathepsin K.

Cathepsin K is a cysteine protease involved in degradation of human type I collagen and plays a primary role in bone resorption. We have cloned rhesus monkey cathepsin K by reverse transcriptase-polymerase chain reaction (RT-PCR) from rhesus ovary poly A+ RNA. The sequence for the rhesus enzyme is 98% identical to that of the human with 100% identity within the mature active form of cathepsin K. Rhesus monkey cathepsin K was transiently expressed in Chinese hamster ovary (CHO) cells and found to be secreted as the proenzyme in the culture media and 50% activated to the mature form intracellularly. The substrate specificity preference of aminomethylcoumarin and rhodamine peptide substrates was Leu > Phe > Pro in the P2 position when tested with constant arginine at P1. The enzyme activity expressed in CHO cell extracts was sensitive to inhibition by E-64 and cystatin with IC50s of 3.5 nmol/L and 13 ng/mL, respectively. The apparent second order rate constants of inactivation by E-64 were 66,000 M(-1) s(-1) and 130,000 M(-1) s(-1) for the recombinantly expressed rhesus monkey and human cathepsin K, respectively. The high similarity between the sequences and the kinetic properties of rhesus monkey and human cathepsin K establishes this monkey species as a suitable animal model for development of novel cathepsin K inhibitors as antiresorptive agents.

Effects of COX-2 inhibitors on aortic prostacyclin production in cholesterol-fed rabbits.

Prostacyclin (PGI(2)) is a potent vasodilator and inhibitor of platelet aggregation that is produced by prostacyclin synthase via the cyclooxygenase (COX) pathway of arachidonic acid metabolism. We investigated the potential role of COX-2 in the production of vasoactive prostanoids by aortic tissue in a rabbit model of dietary cholesterol-induced atherosclerosis. COX-1 was detected as the major isoform by immunoblot analysis in extracts from aortas of normal and 8 week cholesterol-fed animals with COX-2 being induced in atherosclerotic plaques from cholesterol-fed animals. Aortic tissue from cholesterol-fed animals showed decreased levels of basal 6-keto-PGF(1 alpha) and PGE(2) production as compared to the normal controls but showed no difference with respect to their ability to synthesize these prostanoids in response to exogenous arachidonic acid. The highly selective COX-2 inhibitors rofecoxib and the furanone DFP at concentrations of up to 10 micromol/l had no effect on the arachidonic acid-dependent production of 6-keto-PGF(1 alpha), in contrast to indomethacin, which caused a complete inhibition at 0.5 micromol/l. Celecoxib caused a significant inhibition of 6-keto-PGF(1 alpha) at 10 micromol/l but had little effect when the dose was lowered to 1 micromol/l. Similar effects of these inhibitors were observed with respect to the production of PGE(2) and no major difference was observed between aortic tissues from normal and cholesterol-fed animals with regard to inhibitor sensitivity. These results indicate that in a rabbit model of early stage cardiovascular disease, the basal production of 6-keto-PGF(1 alpha) and PGE(2) by aortic tissue is decreased. Furthermore, COX-2 expression is induced in atherosclerotic plaques and may play a role in altering localized synthesis of prostanoids in these lesions but does not appear to significantly impact the arachidonic acid-dependent prostacylin production of aortic tissues, which is largely mediated by COX-1.

Development of 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (L-670,630) as a potent and orally active inhibitor of 5-lipoxygenase.

Leukotrienes are potent biological mediators of allergic and inflammatory diseases and are derived from arachidonic acid through the action of the 5-lipoxygenase. In this study, the syntheses and comparative biological activities of three series of 2,3-dihydro-2,6-disubstituted-5-benzofuranols with various substituents on position 3 are described. Compounds from each series were evaluated for their ability to inhibit the production of leukotriene B4 (LTB4) in human peripheral blood polymorphonuclear (PMN) leukocytes and the 5-lipoxygenase reaction in cell-free preparations from rat PMN leukocytes. The structure-activity relationships of each series in vitro and in vivo are presented. The bioavailability, metabolism, and toxicity profile of each series are discussed. The series with no substituent at position 3 was the most potent and among the compounds in that series 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (46, L-670,630) was chosen for further development.

Thiopyrano[2,3,4-cd]indoles as 5-lipoxygenase inhibitors: synthesis, biological profile, and resolution of 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]-4,5 -dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]butanoic acid.

Leukotriene biosynthesis inhibitors have potential as new therapies for asthma and inflammatory diseases. The recently disclosed thiopyrano[2,3,4-cd]indole class of 5-lipoxygenase (5-LO) inhibitors has been investigated with particular emphasis on the side chain bearing the acidic functionality. The SAR studies have shown that the inclusion of a heteroatom (O or S) in conjunction with an alpha-ethyl substituted acid leads to inhibitors of improved potency. The most potent inhibitor prepared contains a 2-ethoxybutanoic acid side chain. This compound, 14d (2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methox y]- 4,5-dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]-butanoic acid, L-699,333), inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50s of 22 nM, 7 nM and 3.8 microM, respectively). The racemic acid 14d has been shown to be functionally active in a rat pleurisy model (inhibition of LTB4, ED50 = 0.65 mg/kg, 6 h pretreatment) and in the hyperreactive rat model of antigen-induced dyspnea (50% inhibition at 2 and 4 h pretreatment; 0.5 mg/kg po). In addition, 14d shows excellent functional activity against antigen-induced bronchoconstriction in the conscious squirrel monkey [89% inhibition of the increase in RL and 68% inhibition in the decrease in Cdyn (0.1 mg/kg, n = 3)] and in the conscious sheep models of asthma (iv infusion at 2.5 micrograms/kg/min). Acid 14d is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of human 15-LO, porcine 12-LO and ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or competition in a FLAP binding assay (IC50 > 10 microM). Resolution of 14d affords 14g, the most potent diastereomer, which inhibits the 5-HPETE production of human 5-LO and LTB4 biosynthesis of human PMN leukocytes and human whole blood with IC50s of 8 nM, 4 nM, and 1 microM respectively. The in vitro and in vivo profile of 14d is comparable to that of MK-0591, which has showed biochemical efficacy in inhibiting ex vivo LTB4 biosynthesis and urinary LTE4 excretion in clinical trials.

Naphthalenic lignan lactones as selective, nonredox 5-lipoxygenase inhibitors. Synthesis and biological activity of (methoxyalkyl)thiazole and methoxytetrahydropyran hybrids.

Combinations of structural elements found in (methoxyalkyl)thiazole 1a and methoxytetrahydropyran 2a with a naphthalenic lignan lactone produce the potent 5-lipoxygenase (5-LO) inhibitors 3 and 4. While the nature of link Y-Z has a major effect on the in vitro activity of compounds 1 and 2, inhibitors 3 and 4 retain their potencies with either an oxymethylene (Y = O, Z = CH2) or a methyleneoxy (Y = CH2, Z = O) link. Compound 4b inhibits the oxidation of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid by 5-LO (IC50 = 14 nM) and the formation of leukotriene B4 in human polymorphonuclear leukocytes (IC50 = 1.5 nM) as well as in human whole blood (IC50 = 50 nM). Compound 4b is a selective 5-LO inhibitor showing no significant inhibition of human 15-lipoxygenase or porcine 12-lipoxygenase or binding to human 5-lipoxygenase-activating protein up to 10 microM and inhibits leukotriene biosynthesis by a direct, nonredox interaction with 5-LO. Compound 15, the open form of lactone 4b, is well absorbed in the rat and is transformed into the active species 4b. In addition, 15 is orally active in the rat pleurisy model (ED50 = 0.6 mg/kg) and in the functional model of antigen-induced bronchoconstriction in allergic squirrel monkeys (95% inhibition at 0.3 mg/kg).

Novel, nonpeptidic cyanamides as potent and reversible inhibitors of human cathepsins K and L.

Compounds containing a 1-cyanopyrrolidinyl ring were identified as potent and reversible inhibitors of cathepsins K and L. The original lead compound 1 inhibits cathepsins K and L with IC(50) values of 0. 37 and 0.45 M, respectively. Modification of compound 1 by replacement of the quinoline moiety led to the synthesis of N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide (2). Compound 2 was found to be a potent inhibitor of cathepsins K and L with a K(i) value of 50 nM for cathepsin K. Replacement of the 1-cyanopyrrolidine of compound 2 by a 1-cyanoazetidine increased the potency of the inhibitor by 10-fold. This increase in potency is probably due to an enhanced chemical reactivity of the compound toward the thiolate of the active site of the enzyme. This is demonstrated when the assay is performed in the presence of glutathione at pH 7.0 which favors the formation of a GSH thiolate anion. Under these assay conditions, there is a loss of potency in the 1-cyanoazetidine series due to the formation of an inactive complex between the GSH thiolate and the 1-cyanoazetidine inhibitors. 1-Cyanopyrrolidinyl inhibitors exhibited time-dependent inhibition which allowed us to determine the association and dissociation rate constants with human cathepsin K. The kinetic data obtained showed that the increase of potency observed between different 1-cyanopyrrolidinyl inhibitors is due to an increase of k(on) values and that the association of the compound with the enzyme fits an apparent one-step mechanism. (13)C NMR experiments performed with the enzyme papain showed that compound 2 forms a covalent isothiourea ester adduct with the enzyme. As predicted by the kinetic analysis, the addition of the irreversible inhibitor E64 to the enzyme-cyanopyrrolidinyl complex totally abolished the signal of the isothiourea bond as observed by (13)C NMR, thereby demonstrating that the formation of the covalent bond with the active site cysteine residue is reversible. Finally, compound 2 inhibits bone resorption in an in vitro assay involving rabbit osteoclasts and bovine bone with an IC(50) value of 0.7 M. 1-Cyanopyrrolidine represents a new class of nonpeptidic compounds that inhibit cathepsin K and L activity and proteolysis of bone collagen.

Substituted thiopyrano[2,3,4-c,d]indoles as potent, selective, and orally active inhibitors of 5-lipoxygenase. Synthesis and biological evaluation of L-691,816.

Thiopyrano[2,3,4-c,d]indoles are a new class of 5-lipoxygenase (5-LO) inhibitors. SAR studies have demonstrated that the thiopyran ring, the 5-phenylpyridine substituent, and an acidic functional group on a four-carbon C-2 side chain are all required for optimal inhibitor potency. In contrast, the indolic nitrogen may be substituted with a variety of lipophilic groups. As a result of the SAR investigation, 44 (L-691,816; 5-[3-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy ]- 4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]-2,2-dimethylpro pyl]-1H- tetrazole) has been identified as a potent inhibitor of the 5-LO reaction both in vitro and in a range of in vivo models. Compound 44 inhibits 5-HPETE production by both rat and human 5-LO and LTB4 synthesis in human PMN leukocytes (IC50s 16, 75, and 10 nM, respectively). The mechanism of inhibition of 5-LO activity by compound 44 appears to involve the formation of a reversible deadend complex with the enzyme and does not involve reduction of the nonheme iron of 5-LO. Compound 44 is highly selective for 5-LO when compared to the inhibition of human FLAP, porcine 12-LO, and also ram seminal vesicle cyclooxygenase. In addition, 44 is orally active in a rat pleurisy model (inhibition of LTB4, ED50 = 1.9 mg/kg; 8 h pretreatment) as well as in the hyperreactive rat model of antigen-induced dyspnea (ED50 = 0.1 mg/kg; 2-h pretreatment). Excellent functional activity was also observed in both the conscious allergic monkey and sheep models of asthma. In the latter case, the functional activity observed correlated with the inhibition of urinary LTE4 excretion.

2,3-Diarylcyclopentenones as orally active, highly selective cyclooxygenase-2 inhibitors.

Cyclopentenones containing a 4-(methylsulfonyl)phenyl group in the 3-position and a phenyl ring in the 2-position are selective inhibitors of cyclooxygenase-2 (COX-2). The selectivity for COX-2 over COX-1 is dramatically improved by substituting the 2-phenyl group with halogens in the meta position or by replacing the phenyl ring with a 2- or 3-pyridyl ring. Thus the 3,5-difluorophenyl derivative 7 (L-776,967) and the 3-pyridyl derivative 13 (L-784,506) are particularly interesting as potential antiinflammatory agents with reduced side-effect profiles. Both exhibit good oral bioavailability and are potent in standard models of pain, fever, and inflammation yet have a much reduced effect on the GI integrity of rats compared to standard nonsteroidal antiflammatory drugs.

Dioxabicyclooctanyl naphthalenenitriles as nonredox 5-lipoxygenase inhibitors: structure-activity relationship study directed toward the improvement of metabolic stability.

Naphthalenic lignan lactone 3a (L-702,539), a potent and selective 5-lipoxygenase (5-LO) inhibitor, is extensively metabolized at two different sites: the tetrahydropyran and the lactone rings. Early knowledge of the metabolic pathways triggered and directed a structure-activity relationship study aimed toward the improvement of metabolic stability in this series. The best modifications discovered, i.e., replacement of the lactone ring by a nitrile group, replacement of the tetrahydropyran ring by a 6,8-dioxabicyclo[3.2.1]octanyl moiety, and replacement of the pendant phenyl ring by a 3-furyl ring, were incorporated in a single molecule to produce inhibitor 9ac (L-708,780). Compound 9ac inhibits the oxidation of arachidonic acid to 5-hydroperoxy-eicosatetraenoic acid by 5-LO (IC50 = 190 nM) and the formation of leukotriene B4 in human polymorphonuclear leukocytes (IC50 = 3 nM) as well as in human whole blood (IC50 = 150 nM). The good inhibitory profile shown by naphthalenenitrile 9ac is accompanied by an improved resistance to oxidative metabolism. In addition, 9ac is orally active in the functional model of antigen-induced bronchoconstriction in allergic squirrel monkeys (95% inhibition at 0.1 mg/kg).

Substituted (pyridylmethoxy)naphthalenes as potent and orally active 5-lipoxygenase inhibitors; synthesis, biological profile, and pharmacokinetics of L-739,010.

Dioxabicyclooctanyl naphthalenenitriles have been reported as a class of potent and nonredox 5-lipoxygenase (5-LO) inhibitors. These bicyclo derivatives were shown to be metabolically more stable than their tetrahydropyranyl counterparts but were not well orally absorbed. Replacement of the phenyl ring in the naphthalenenitrile 1 by a pyridine ring leads to the potent and orally absorbed inhibitor 3g (L-739,010, 2-cyano-4-(3-furyl)-7-[[6-[3-(3-hydroxy-6,8-dioxabicyclo[3.2.1] octanyl)]-2-pyridyl]methoxy]naphthalene). Compound 3g inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50S of 20, 1.6, and 42 nM, respectively). Derivative 3g is orally active in the rat pleurisy model (inhibition of LTB4, ED50 = 0.3 mg/kg) and in the anesthetized dog model (inhibition of ex vivo whole blood LTB4 and urinary LTE4, ED50 = 0.45 and 0.23 microgram/kg/min, respectively, i.v. infusion). In addition, 3g shows excellent functional activity against ovalbumin-induced dyspnea in rats (60% inhibition at 0.5 mg/kg, 4 h pretreatment) and Ascaris-induced bronchoconstriction in conscious sheep (50% and > 85% inhibition in early and late phases, respectively at 2.5 micrograms/kg/min, i.v. infusion) and, more particularly in the conscious antigen sensitive squirrel monkey model (53% inhibition of the increase in RL and 76% in the decrease of Cdyn, at 0.1 mg/kg, po). In rats and dogs, 3g presents excellent pharmacokinetics (estimated half-lives of 5 and 16 h, respectively) and bioavailabilities (26% and 73% when dosed as its hydrochloride salt at doses of 20 and 10 mg/kg, respectively, in methocel suspension). Based on its overall biological profile, compound 3g has been selected for preclinical animal toxicity studies.

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor.

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furan one) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX-2 (IC50 = 41 +/- 14 nM) over COX-1 (IC50 > 50 microM). Indomethacin was a potent inhibitor of both COX-1 (IC50 = 18 +/- 3 nM) and COX-2 (IC50 = 26 +/- 6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50 > 50 microM and 4.1 +/- 1.7 nM, respectively). 3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a Ki, value of 140 +/- 68 microM for the initial reversible binding to enzyme and a kappa 2 value of 0.11 +/- 0.06 s-1 for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62 +/- 26 microM and 0.06 +/- 0.01 s-1, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1:1 stoichiometry and to dissociate only very slowly (t1/2 = 1-3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50 = 63 +/- 5 microM at 0.1 microM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5. DFU inhibited lipopolysaccharide (LPS)-induced PGE2 production (COX-2) in a human whole blood assay with a potency (IC50 = 0.28 +/- 0.04 microM) similar to indomethacin (IC50 = 0.68 +/- 0.17 microM). In contrast, DFU was at least 500 times less potent (IC50 > 97 microM) than indomethacin at inhibiting coagulation-induced TXB2 production (COX-1) (IC50 = 0.19 +/- 0.02 microM). 6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 microM), DFU inhibited COX-1 with an IC50 value of 13 +/- 2 microM as compared to 20 +/- 1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac > indomethacin approximately naproxen > nimesulide approximately meloxicam approximately piroxicam > NS-398 approximately SC-57666 > SC-58125 > CGP 28238 approximately etodolac > L-745,337 > DFU. 7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED50 of 1.1 mg kg-1 vs 2.0 mg kg-1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg-1 vs 1.5 mg kg-1 for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED50 = 0.76 mg kg-1 vs 1.1 mg kg-1 for indomethacin). 8. In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg-1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg-1), meloxicam (3 mg kg-1) or etodolac (10-30 mg kg-1). A 5 day administration of DFU in squirrel monkeys (100 mg kg-1) did not affect chromium leakage in contrast to diclofenac (1 mg kg-1) or naproxen (5 mg kg-1). 9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.

Expression, maturation, and rhodamine-based fluorescence assay of human cathepsin K expressed in CHO cells.

Cathepsin K is a cysteine protease that degrades type I human collagen during bone resorption. We have expressed the recombinant human cathepsin K in Chinese hamster ovary (CHO) cells as a pre-proenzyme and demonstrated that it is processed intracellularly to an active enzyme form and that only the proenzyme form is secreted. Immunofluorescence detection of cathepsin K in CHO cells resulted in discrete punctate distribution consistent with a lysosomal localization of the enzyme. With both extract and cell preparations of CHO cells expressing cathepsin K, [Z-Leu-Arg](2)-rhodamine was the best substrate for analyzing cathepsin K activity over background proteases. We have established a cellular-based assay to analyze cell-permeable inhibitors of cathepsin K and validated the assay with detection of intracellular versus extracellular activity, fluorescence-assisted cell sorter (FACS) analysis, and a selective cathepsin K inhibitor. The intracellular activity of cathepsin K was monitored by FACS analysis using the rhodamine substrate, which demonstrated an increased fluorescence over mock-transfected cells that was also inhibitable by (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E64d). A selective cathepsin K inhibitor, 1, 3-bis(CBZ-Leu-NH)-2-propanone, had an IC(50) of 134 nM in the CHO/Cat K cells, which is the same potency as that measured against a purified enzyme preparation of cathepsin K. Therefore, we have established a system to evaluate intracellular cathepsin K activity and inhibition by cell-permeable inhibitors of this thiol protease.

Criteria for the identification of non-redox inhibitors of 5-lipoxygenase.

Methoxyalkyl thiazoles have been identified as a novel series of selective 5-lipoxygenase inhibitors with anti-inflammatory properties (Bird et al., J Med Chem 34: 2176-2186, 1991). Based on their structure, it was proposed that the potency of these compounds is not due to redox or iron-chelating properties. In the studies reported here, it was found that the model compounds 1-[3-(naphth-2-ylmethoxy)phenyl]-1-(thiazol-2-yl)propy l methyl ether (ICI 211965) and 3-[1-(4-chlorobenzyl)-4-methyl-6-(5- phenylpyridin-2-ylmethoxy)-4,5-dihydro-1H-thiopyrano[2 ,3,4-c,d]indol-2- yl]-2,2-dimethylpropanoic acid (L-689,065) (1) are inactive as reducing substrates in the 5-lipoxygenase-catalyzed decomposition of lipid hydroperoxides, (2) inhibit the 5-lipoxygenase-catalyzed reaction of reducing agents with lipid hydroperoxides, and (3) strongly inhibit the turnover-dependent inactivation of 5-lipoxygenase. These three observations with ICI 211965 and L-689,065 are in contrast to the behavior of other potent 5-lipoxygenase inhibitors from other structural classes, such as L-670,630, BW A4C, and zileuton, which all function as reducing substrates for 5-lipoxygenase. The data indicate that methoxyalkyl thiazoles and thiopyranoindoles are reversible dead-end inhibitors of 5-lipoxygenase and that the effects of inhibitors on the pseudoperoxidase activity and rate of enzyme inactivation provide simple tests to distinguish between redox and non-redox inhibitors of 5-lipoxygenase.

Inhibition of human leukocyte 5-lipoxygenase by a 4-hydroxybenzofuran, L-656,224. Evidence for enzyme reduction and inhibitor degradation.

Detailed studies of the interaction of L-656,224 (2-[(4'-methoxyphenyl)methyl]-3-methyl-4-hydroxy-5-propyl-7- chlorobenzofuran) with 5-lipoxygenase were conducted using the enzymes from human and pig leukocytes. L-656,224 was a potent inhibitor of these 5-lipoxygenases although its efficiency varied with enzyme concentration. L-656,224 also stimulated the pseudoperoxidase activity of 5-lipoxygenase as measured by the consumption of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD), indicating that this compound can reduce the enzyme. Furthermore the inhibitor was degraded rapidly by both cell-free leukocyte extracts and purified 5-lipoxygenase after incubation with 13-HPOD, ATP and calcium ions. The degradation of L-656,224 was also observed during inhibition of the lipoxygenase reaction and occurred mainly after the initial lag phase of the reaction when hydroperoxides begin to accumulate. A single major radioactive product was formed after incubation of [3H]L-656,224 with purified 5-lipoxygenase in the presence of 13-HPOD. This product was unstable and could not be isolated. During the course of the pseudoperoxidase reaction, [3H]L-656,224 covalently labelled the enzyme, suggesting that a chemically reactive species had been formed. These data are consistent with the hypothesis that L-656,224 reduces the oxidized form of the 5-lipoxygenase to an inactive form, with degradation of the inhibitor and regeneration of the active enzyme with hydroperoxides.

Sensitivity of immunoaffinity-purified porcine 5-lipoxygenase to inhibitors and activating lipid hydroperoxides.

The requirement for hydroperoxide activation and the effect of inhibitors from different structural classes on 5-lipoxygenase activity were determined on the immunoaffinity-purified enzyme from porcine leukocytes. The 5-lipoxygenase activity was measured using a continuous spectrophotometric assay monitoring the increase in conjugated diene formation (A235) upon incubation of the enzyme with arachidonic acid. Under standard assay conditions, the reaction progress curves showed little or no lag phase, with a rapid first-order decay in enzyme activity (T1/2 = 0.7 to 1.1 min). Both the initial rate of the reaction and total product formation were stimulated by the addition of ATP, Ca2+ and phosphatidylcholine (PC). PC (24 micrograms/ml) was also found to increase the recovery of radiolabeled arachidonic acid from the assay mixture and thus part of the stimulation may be due to an increase in substrate availability and reduction of surface adsorption effects. The requirement of hydroperoxides for the initiation of the reaction was shown by the induction of 0.1 to 1-min lag phases using NaBH4 or glutathione peroxidase and by the reduction in lag times by 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 13-hydroperoxyoctadecadienoic acid (13-HPOD). The following compounds were evaluated as inhibitors of the 5-lipoxygenase reaction and caused a 50% decrease in product accumulation (IC50) at the indicated concentrations: quercetin, L-651,896, L-656,224, MTPPH and L-651,392 (0.3-0.5 microM); diphenyldisulfide (2-5 microM); phenidone (5-10 microM); AA861 (4-10 microM) and BW755C (4-15 microM). In addition, the presence of inhibitors extended the initial lag phase of the reaction and increased the dependence of the initiation of the reaction on exogenous lipid hydroperoxides. The inhibition by phenidone was accompanied by a 2-fold increase in the rate of enzyme inactivation, whereas other compounds such as AA861 and L-656,224 did not show this effect. The results indicate that the presence of inhibitors can modify the kinetics of 5-lipoxygenase at the levels of the initiation of the reaction and the rate of enzyme inactivation, with variations depending on the structural class of the inhibitor and the concentration of lipid hydroperoxides.

Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells.

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.

Phosphodiesterase 4-dependent regulation of cyclic AMP levels and leukotriene B4 biosynthesis in human polymorphonuclear leukocytes.

Several selective phosphodiesterase 4 inhibitors were found to be potent inhibitors of the N-formyl-Met-Leu-Phe (fMLP)-induced leukotriene B4 biosynthesis by human polymorphonuclear leukocytes with IC50s in the nanomolar range (0.09-26 nM). The rank order of potency was 6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline (RS-14203) > 3-benzyl-5-phenyl-3H-imidazo[4,5-c][1,8]naphthyridin-4(5H)-one (KF18280) > 8-aza-1-(3-nitrophenyl)-3-(4-pyridylmethyl)-2,4-quinazoline dione (RS-25344) > 3-cyclo-pentyloxy-N-[3,5-dichloro-4-pyridyl]-4-methoxybenzamide (RP-73401) > R-rolipram > R-4-[2-(3-cyclopentyloxy-4-methoxyphenyl)-2-phenylethyl] pyridine (CDP840)> S-rolipram. Isoproterenol (IC50 = 350 nM) and prostaglandin E2 (IC50 = 59 nM) also suppressed leukotriene B4 biosynthesis. Inhibitors of the phosphodiesterase 1 (8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine (8-MeOMe-IBMX)), phosphodiesterase 2 (erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA)), phosphodiesterase 3 (quazinone and milrinone) and phosphodiesterase 5 (zaprinast and dipyridamole) had no inhibitory effects on the fMLP-induced leukotriene B4 biosynthesis (IC50s > 20 microM). All phosphodiesterase 4 inhibitors caused an accumulation of cellular cyclic AMP to 140-185% over the basal level of fMLP-treated control cells, comparable to that observed with high concentrations of isoproterenol and prostaglandin E2. In contrast, the complete inhibition of leukotriene B4 production by 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) inhibitors had no effect on cyclic AMP levels. Phosphodiesterase 1, 2, 3 and 5 inhibitors had little effect on the level of cellular cyclic AMP (89-126% of the basal cyclic AMP level). Dose-dependencies for R-rolipram, RS-14203 and CDP840 indicated that the maximal accumulation of cyclic AMP occurred at concentrations of phosphodiesterase 4 inhibitors higher than those required for the inhibition of leukotriene B4 production. The presence of a mixture of 8-MeOMe-IBMX, EHNA, milrinone and zaprinast to inhibit phosphodiesterase 1, 2, 3 and 5 had little effect on the dose-dependence of R-rolipram for the inhibition of leukotriene B4 biosynthesis or cyclic AMP accumulation. These data demonstrate that selective phosphodiesterase 4 inhibitors can inhibit the fMLP-induced leukotriene B4 biosynthesis in human polymorphonuclear leukocytes with a potency similar or greater than that of potent 5-lipoxygenase or FLAP inhibitors. This inhibition is accompanied by small variations in the levels of cellular cyclic AMP and appears to proceed independently of the other phosphodiesterases.