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The discovery of a second isoform of cyclooxygenase (COX) led to the search for compounds that could selectively inhibit COX-2 in humans while sparing prostaglandin formation from COX-1. Celecoxib and rofecoxib were among the molecules developed from these efforts. We report here the pharmacological properties of a third selective COX-2 inhibitor, valdecoxib, which is the most potent and in vitro selective of the marketed COX-2 inhibitors that we have studied. Recombinant human COX-1 and COX-2 were used to screen for new highly potent and in vitro selective COX-2 inhibitors and compare kinetic mechanisms of binding and enzyme inhibition with other COX inhibitors. Valdecoxib potently inhibits recombinant COX-2, with an IC50of 0.005 μM; this compares with IC values of 0.05 μM for celecoxib, 0.5 μM for rofecoxib, and 5 μM for etoricoxib. Unique binding interactions of valdecoxib with COX-2 translate into a fast rate of inactivation of COX-2 (110,000 M/s compared with 7000 M/s for rofecoxib and 80 M/s for etoricoxib). The overall saturation binding affinity for COX-2 of valdecoxib is 2.6 nM (compared with 1.6 nM for celecoxib, 51 nM for rofecoxib, and 260 nM for etoricoxib), with a slow off-rate (t1/2∼98 min). Valdecoxib inhibits COX-1 in a competitive fashion only at very high concentrations (IC50= 150 μM). Collectively, these data provide a mechanistic basis for the potency and in vitro selectivity of valdecoxib for COX-2. Valdecoxib showed similar activity in the human whole-blood COX assay (COX-2 IC50= 0.24 μM; COX-1 IC50= 21.9 μM). We also determined whether this in vitro potency and selectivity translated to significant potency in vivo. In rats, valdecoxib demonstrated marked potency in acute and chronic models of inflammation (air pouch ED50= 0.06 mg/kg; paw edema ED50= 5.9 mg/kg; adjuvant arthritis ED50= 0.03 mg/kg). In these same animals, COX-1 was spared at doses greater than 200 mg/kg. These data provide a basis for the observed potent anti-inflammatory activity of valdecoxib in humans.

The discovery of a second isoform of cyclooxygenase (COX) led to the search for compounds that could selectively inhibit COX-2 in humans while sparing prostaglandin formation from COX-1. Celecoxib and rofecoxib were among the molecules developed from these efforts. We report here the pharmacological properties of a third selective COX-2 inhibitor, valdecoxib, which is the most potent and in vitro selective of the marketed COX-2 inhibitors that we have studied. Recombinant human COX-1 and COX-2 were used to screen for new highly potent and in vitro selective COX-2 inhibitors and compare kinetic mechanisms of binding and enzyme inhibition with other COX inhibitors. Valdecoxib potently inhibits recombinant COX-2, with an IC(50) of 0.005 microM; this compares with IC values of 0.05 microM for celecoxib, 0.5 microM for rofecoxib, and 5 microM for etoricoxib. Unique binding interactions of valdecoxib with COX-2 translate into a fast rate of inactivation of COX-2 (110,000 M/s compared with 7000 M/s for rofecoxib and 80 M/s for etoricoxib). The overall saturation binding affinity for COX-2 of valdecoxib is 2.6 nM (compared with 1.6 nM for celecoxib, 51 nM for rofecoxib, and 260 nM for etoricoxib), with a slow off-rate (t(1/2) approximately 98 min). Valdecoxib inhibits COX-1 in a competitive fashion only at very high concentrations (IC(50) = 150 microM). Collectively, these data provide a mechanistic basis for the potency and in vitro selectivity of valdecoxib for COX-2. Valdecoxib showed similar activity in the human whole-blood COX assay (COX-2 IC(50) = 0.24 microM; COX-1 IC(50) = 21.9 microM). We also determined whether this in vitro potency and selectivity translated to significant potency in vivo. In rats, valdecoxib demonstrated marked potency in acute and chronic models of inflammation (air pouch ED(50) = 0.06 mg/kg; paw edema ED(50) = 5.9 mg/kg; adjuvant arthritis ED(50) = 0.03 mg/kg). In these same animals, COX-1 was spared at doses greater than 200 mg/kg. These data provide a basis for the observed potent anti-inflammatory activity of valdecoxib in humans.

Background: Subjects with aspirin-intolerant asthma (AIA) respond with bronchoconstriction and extrapulmonary adverse reactions to conventional nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit the cyclooxygenase (COX) step in the biosynthesis of prostaglandins. Recently, 2 isotypes of COX have been identified, and COX-2-selective NSAIDs have been developed for treatment of inflammatory disorders. Objective: We investigated whether 33 subjects with a typical history of AIA tolerated the new COX-2-selective NSAID celecoxib. Methods: All subjects displayed current aspirin sensitivity in oral or inhalation challenge tests. The subjects first underwent a double-blind, randomized, cross-over, increasing-dose challenge with placebo or celecoxib (10, 30, or 100 mg in suspension) on 2 occasions 7 days apart. Thereafter, all subjects were exposed to 400 mg of celecoxib administered during an open challenge session as two 200-mg doses 2 hours apart. Lung function, clinical symptoms, and urinary excretion of leukotriene E4(LTE4) were monitored, with the latter being a sensitive biochemical marker of aspirin intolerance. Results: There were no changes in lung function or extrapulmonary symptoms during the double-blind sessions or in urinary excretion of LTE4. Also, the highest recommended daily dose of celecoxib was well tolerated, with no symptoms, lung function changes, or alterations in urinary LTE4levels. Conclusions: A group of subjects with clinically well-documented AIA tolerated acute challenge with the selective COX-2 inhibitor celecoxib. The findings indicate that the intolerance reaction in AIA is due to inhibition of COX-1. Large long-term studies of COX-2 inhibitors in AIA should be undertaken. (J Allergy Clin Immunol 2003;111:1116-21.)

Two compounds (celecoxib and valdecoxib) from the diarylheterocycle class of cyclooxygenase inhibitors were radiolabeled and used to characterize their binding to cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), several single-point variants of COX-2 (Val523Ile, Tyr355Ala, Arg120Ala, Arg120Gln, Arg120Asn) and one triple-point variant of COX-2 [Val523Ile, Arg513His, Val434Ile (IHI)]. We demonstrate highly specific and saturable binding of these inhibitors to COX-2. Under the same assay conditions, little or no specific binding to COX-1 could be detected. The affinity of [3H]celecoxib for COX-2 (KD= 2.3 nM) was similar to the affinity of [3H]valdecoxib (KD= 3.2 nM). The binding to COX-2 seems to be both rapid and slowly reversible with association rates of 5.8 × 106/M/min and 4.5 × 106/M/min and dissociation rates of 14 × 10−3/min (t1/2= 50 min) and 7.0 × 10−3/min (t1/2= 98 min) for [3H]celecoxib and [3H]valdecoxib, respectively. These association rates increased (4- to 11-fold) when the charged arginine residue located at the entrance to the main hydrophobic channel was mutated to smaller uncharged amino acids (Arg120Ala, Arg120Gln, and Arg120Asn). Mutation of residues located within the active site of COX-2 that define a ‘side pocket’ (Tyr355Ala, Val523Ile, IHI) of the main channel had a greater effect on the dissociation rate than the association rate. These mutations, which modified the shape of and access to the ‘side pocket’, affected the binding affinity of [3H]valdecoxib more than that of [3H]celecoxib. These binding studies provide direct insight into the properties and binding constants of celecoxib and valdecoxib to COX-2.

Prostaglandin E2(PGE2) is the major prostaglandin produced both centrally and in the periphery in models of acute and chronic inflammation, and its formation in both locations is blocked by cyclooxygenase-2 (COX-2) inhibitors such as celecoxib. In animal models of inflammation, PGE2inhibition in the brain may occur secondarily to a peripheral action by inhibiting local PG formation that elicits increased firing of pain fibers and consequent activation of PG synthesis in the central nervous system (CNS). Celecoxib was studied in the kainate-induced seizure model in the rat, a model of direct central prostaglandin induction, to determine whether it can act directly in the CNS. In the kainate-treated rat brain there was increased PGE2, PGF2α, and PGD2production, with COX activity and PGE2formation increased about 7-fold over normal. We quantitated mRNA levels for enzymes involved in the prostaglandin biosynthetic pathways and found that both COX-2 and PGE synthase (PGEs) mRNA levels were increased in the brain; no changes were found for expression of COX-1 or PGD synthase mRNA. By Western blot analysis, COX-2 and PGEs were induced in total brain, hippocampus, and cortex, but not in the spinal cord. Immunohistological studies showed that COX-2 protein expression was enhanced in neurons. Dexamethasone treatment reduced the expression of both COX-2 and PGEs in kainate-treated animals. Celecoxib reduced the elevated PGE2levels in brain of kainate-treated rats and inhibited induced COX activity, demonstrating the ability of this compound to act on COX-2 in CNS. Doses of celecoxib that inhibited brain COX-2 were lower than those needed for anti-inflammatory activity in adjuvant arthritis, demonstrating a potent direct central action of the compound.

Cyclo-oxygenase (COX) enzymes are the targets for non-steroidal anti-inflammatory drugs (NSAIDs). These drugs demonstrate a variety of inhibitory mechanisms, which include simple competitive, as well as slow binding and irreversible inhibition. In general, most NSAIDs inhibit COX-1 and −2 by similar mechanisms. A unique class of diarylheterocyclic inhibitors has been developed that is highly selective for COX-2 by virtue of distinct inhibitory mechanisms for each isoenzyme. Several of these inhibitors, with varying selectivity, have been utilized to probe the mechanisms of COX inhibition. Results from analysis of both steady-state and time-dependent inhibition were compared. A generalized mechanism for inhibition, consisting of three sequential reversible steps, can account for the various types of kinetic behaviour observed with these inhibitors.

A series of heteroaryl modified 1,2-diarylimidazoles has been synthesized and found to be potent and highly selective (1000−9000-fold) inhibitors of the human COX-2. 3-Pyridyl derived COX-2 selective inhibitor (25) exhibited excellent activity in acute (carrageenan induced paw edema, ED50 = 5.4 mg/kg) and chronic (adjuvant induced arthritis, ED50 = 0.25 mg/kg) models of inflammation. The relatively long half-life of 25 in rat and dog prompted investigation of the pyridyl and other heteroaromatic systems containing potential metabolic functionalities. A number of substituted pyridyl and thiazole containing compounds (e.g., 44, 46, 54, 76, and 78) demonstrated excellent oral activity in every efficacy model evaluated. Several orally active diarylimidazoles exhibited desirable pharmacokinetics profiles and showed no GI toxicity in the rat up to 100 mg/kg in both acute and chronic models. The paper describes facile and practical syntheses of the targeted diarylimidazoles. The structure−activity relationships and antiinflammatory properties of a series of diarylimidazoles are discussed.

Conventional nonsteroidal anti‐inflammatory drugs (NSAIDs) nonspecifically inhibit cyclooxygenase‐1 (COX‐1), an enzyme critical to normal platelet function, and COX‐2, which mediates inflammatory response mechanisms. Celecoxib, an antiarthritic agent that inhibits COX‐2 but spares COX‐1 at therapeutic doses, is expected to have minimal effects on platelet function. A double‐blind, randomized, placebo‐controlled study of 10 days' duration was conducted in 24 healthy adults to compare the effects on platelet function of a supratherapeutic dose of celecoxib (600 mg bid) with a standard dose of naproxen (500 mg bid), a conventional NSAID. Ex vivo platelet aggregation in response to standard agonists (collagen, arachidonate, or U46619 [a thromboxane A2receptor agonist]), bleeding time, and serum thromboxane B2(TxB2level were measured. Unlike celecoxib or placebo, naproxen produced statistically significant reductions in platelet aggregation and serum TxB2levels and increased bleeding time. The results indicate that even at supratherapeutic doses, celecoxib will not interfere with normal mechanisms of platelet aggregation and hemostasis, supporting the premise that celecoxib is COX‐1 sparing relative to conventional NSAIDs.

To compare the efficacy and safety of celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, with those of naproxen, a nonsteroidal anti-inflammatory drug (NSAID), and placebo in the treatment of osteoarthritis of the knee.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the formation of prostaglandins by cyclo-oxygenases (COX). The discovery of a second COX isoform (COX-2) associated with inflammation led to agents that selectively inhibit COX-2, e.g. celecoxib. We evaluated the kinetics of inhibition of celecoxib and several NSAIDs. Celecoxib displays classic competitive kinetics on COX-1 (Ki = 10-16 μM). An initial competitive interaction with COX-2 can also be discerned with celecoxib (Ki = 11-15 μM), followed by a time-dependent interaction leading to potent inhibition, characterized as inactivation (Kinact = 0.03-0.5 s-1). Half-maximal inhibition (IC50) using end-point assays reflects the competitive component on COX-1 (IC50 = 4-19 μM) and the inactivation component on COX-2 (IC50 = 0.003-0.006 μM). NSAIDs exhibit four distinct modes of COX inhibition based on kinetic behaviour: (1) competitive, e.g. ibuprofen; (2) weak binding, time-dependent, e.g. naproxen, oxicams; (3) tight binding, time-dependent, e.g. indomethacin; (4) covalent, e.g. aspirin. In addition, most NSAIDs display different kinetic behaviour for each isoform. Weakly binding inhibitors show variable behaviour in enzyme assays, with apparent inhibitory activity being markedly influenced by experimental conditions; determination of kinetic constants with this class is unreliable and IC50 values are strongly dependent on assay conditions. Although IC50 determinations are useful for structure/activity analyses, the complex and distinct mechanisms of enzyme inhibition of each COX isoform by the NSAIDs renders comparison of inhibitory activity on COX-1 and COX-2 using IC50 ratios of questionable validity.

PGs derived from cyclooxygenase-2 (COX-2), in particular PGE2, play important roles in the initiation of inflammation and pain. In the present study, we evaluated the role of COX-2-derived PGE2in an animal model of established hyperalgesia. Inflammation and hyperalgesia were first induced by injection of carrageenan into rat footpads. Then we investigated the effects of subsequent therapeutic treatment with a selective COX inhibitor, with a nonsteroidal anti-inflammatory drug and with anti-PGE2antibody. Test compounds were administered 1 to 3 hr after carrageenan challenge, and inhibition of pain (hyperalgesia, measured by withdrawal from a thermal stimulus), and changes in paw edema and PG levels were evaluated. The i.v. administration of a nonselective COX inhibitor, ketorolac, caused a rapid reduction in hyperalgesia in the inflamed footpad, returning it to near-normal values within 1 hr. Normal (control) paw response times were not affected. Therapeutic administration of ketorolac prevented most further swelling caused by carrageenan but did not reverse edema already present at the time of dosing. Administered p.o., a selective COX-2 inhibitor (SC-58635) was as efficacious as ketorolac in reducing inflammatory hyperalgesia. Footpad PG levels returned to base line or below within 5 min of dosing with ketorolac, which suggests rapid turnover of PG in the inflamed tissue. Therapeutic treatment with a monoclonal anti-PGE2antibody also fully reversed the hyperalgesia response. These studies suggest that continuous production of PGE2by the COX-2 enzyme is a critical element in sustaining the hyperalgesic response at sites of tissue inflammation.

Intrathecal administration of nonsteroidal anti-inflammatory drugs in the rat blocks the thermal hyperalgesia induced by tissue injury, which suggests a role for spinal cyclooxygenase (COX) products in this facilitated state. Two isozymes of the COX enzyme have been reported, COX-1 and COX-2, but the agents thus far examined are not isozyme selective. We examined the effects of intrathecally (i.t.) or systemically (i.p.) administered S(+)-ibuprofen (a nonselective COX inhibitor) or 1-[(4-methysulfonyl)phenyl]-3-tri-fluoromethyl-5-(4-fluorophenyl) pyrazole (SC58125; a COX-2 selective inhibitor) on carrageenan-induced thermal hyperalgesia (reduced hindpaw-withdrawal latency). The following observations were made: 1) Thermal hyperalgesia otherwise observed during the first 170 min was blocked in a dose-dependent manner by S(+)-ibuprofen or SC58125 administered i.t. or i.p. before carrageenan treatment. 2) Intraperitoneal, but not i.t., administration of either inhibitor after the establishment of hyperalgesia (170 min after carrageenan injection) reversed thermal hyperalgesia in a dose-dependent manner. Thus, the initial component of thermal hyperalgesia after tissue injury was blocked by systemic or spinal administration of both COX inhibitors, whereas established hyperalgesia was reversed only by systemic inhibitors. This study demonstrates that at least spinal COX-2, if not both COX-1 and COX-2, are necessary for the initiation of thermal hyperalgesia, whereas nonspinal sources of prostanoids (synthesized by COX-2 and perhaps also COX-1) are important for the maintenance of thermal hyperalgesia associated with tissue injury and inflammation.

Adherent murine stromal cells support long-term in vitro lymphopoiesis or myelopoiesis dependent on the culture conditions used. A cell line, TC-1, isolated from long-term liquid murine marrow cultures under conditions approaching those permissive for lymphoid growth, has been found to produce an activity that acts synergistically with interleukin-3 (IL-3) or colony-stimulating factor-1 (CSF-1) to stimulate in vitro myeloid colonies, but which has no intrinsic colony-stimulating activity. We report here the presence of multiple growth factors in conditioned medium (CM) from the TC-1 line, including granulocyte-macrophage colony-stimulating factor (GM-CSF) (bioassay with antibody blocking and messenger RNA [mRNA] analysis), granulocyte CSF (G-CSF) and IL-4 (factor-dependent cell line bioassay), and CSF-1 (radioimmunoassay, mRNA) along with a pre-B cell inducing activity, which appears separate from these CSFs and segregates with the myeloid synergizing activity through anion exchange, sizing, and Conconavalin A chro-matography. Because these activities are not yet purified to homogeneity, their identity or lack of identity remains an open question. Assays of TC-1 CM or cellular mRNA analysis have given negative results for IL-1, IL-2, IL-3, IL-6, and IL-7, and IL-6 does not stimulate pre-B cells in this assay. However, IL-4 and G-CSF do stimulate in vitro induction of pre-B cells from pre-B and B-cell-depleted Balb/C marrow and are present in CM by selective cell line assay. A monoclonal antibody to IL-4 that inhibited its pre-B inducing activity did not inhibit pre-B inducing activity of TC-1 CM. These data suggest the existence of a unique synergizing and pre-B inducing factor(s) in TC-1 CM. Given the known capacity of subliminal levels of growth factors to act synergistically, an alternate possibility is that these biologic phenomena represent the actions of low concentrations of growth factors acting synergistically and possibly associated with some core protein.

Intraperitoneal administration of zymosan to mice resulted in marked biosynthesis of eicosanoids and influx of neutrophils with distinct time course profiles. 6-Keto-prostaglandin-F1 alpha (6-KPA) increased between 30 and 60 min and rapidly decreased thereafter. Leukotriene (LT)C4 levels showed similar patterns, but were sustained for several hours. LTB4 increased in a biphasic manner with peak increases between 2 to 3 hr. Repeated injections with zymosan suggested that incoming neutrophils generate most of the LTB4. Myeloperoxidase (MPO), an enzyme marker for neutrophils, continued to increase throughout the time course. Mast cells regulate LTB4 biosynthesis and neutrophil trafficking, whereas resident macrophages contribute to 6-KPA and LTC4 biosynthesis. The complement fragment C5a has a minimal role in zymosan-induced inflammation. Selective 5-lipoxygenase (5-LO) inhibitors, zileuton [N-(1-benzo[b]thienyl-2yl-ethyl)-N-hydroxyurea], TZI-41127 [2-(4-hydroxy-3,5-dimethylphenyl)-5-methoxy-3-methylindole] and cyclooxygenase (CO) inhibitors selectively modulated eicosanoid biosynthesis. Both 5-LO and CO inhibitors attenuated influx of neutrophils to varying degrees. A LTB4 receptor antagonist, SC-41930 [7-(3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid) and an LTD4 receptor antagonist, LY-171883 [1-(2-hydroxy-3-propyl-4-(4-1H-tetrazol-5-yl)butoxy-phenyl) ethanone)] (i.v.) attenuated influx of neutrophils and associated LTB4 biosynthesis. These results suggest that both 5-LO and CO metabolites regulate neutrophil influx in this model. Marked eicosanoid biosynthesis and cellular influx in response to zymosan provides an attractive experimental paradigm to evaluate anti-inflammatory effects of inhibitors of arachidonate CO or 5-LO pathways.

We have determined whether B cells previously activated by anti-Ig (anti-Ig blasts) are responsive to lymphokines that induce isotype switching. Culture of anti-Ig blasts with a mixture of lymphokines, including BSF-1, resulted in marked secretion of IgM and IgG1, but not other IgG isotypes. The IgG1 response of anti-Ig blasts to lymphokines was 13-fold greater than was observed with splenic B cells. B cell blasts induced by 8-mercaptoguanosine or dextran sulfate did not secrete high levels of any IgG isotype in response to lymphokines alone. An mAb against BSF-1 suppressed the IgG1 response of anti-Ig blasts, but not the IgM response to lymphokines. These data suggest that anti-Ig-treated B cells respond to at least one of the effects of BSF-1.

A novel series of terphenyl methyl sulfones and sulfonamides have been shown to be highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. The sulfonamide analogs 17 and 21 were found to be much more potent COX-2 inhibitors and orally active anti-inflammatory agents than the corresponding methyl sulfone analogs 16 and 20, respectively, albeit with some decrease in COX-2 selectivity. Structure−activity relationship studies have determined that incorporation of two fluorine atoms in the central phenyl group, as in 20 and 21, is extremely advantageous for both in vitro COX-2 potency and selectivity as well as in vivo activity. Several noticeable examples in the 1,2-diaryl-4,5-difluorobenzenesulfonamide series are 21a−c,k,l,n (COX-2, IC50 = 0.002−0.004 μM), in which all have in vitro COX-1/COX-2 selectivity >1000. In addition, sulfonamides 21a,b,d,g,j,m,n,q were shown to have greatly enhanced oral activity with more than 90% inhibition of prostaglandin E2 production in the air pouch model of inflammation. Furthermore, sulfonamide 21b was found to be very active in the rat adjuvant-induced arthritis model (ED50 = 0.05 mg/kg) and carrageenan-induced hyperalgesia assay (ED50 = 38.7 mg/kg) with no indication of gastrointestinal toxicity in rats at doses as high as 200 mg/kg.

The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.

Series of 1,2-diarylpyrroles has been synthesized and found to contain very potent and selective inhibitors of the human cyclooxygenase-2 (COX-2) enzyme. The paper describes short and practical syntheses of the target molecules utilizing the Paal−Knorr reaction. Electrophilic substitution on 1 proceeds in a regioselective fashion, and the method was used to generate a number of tetrasubstituted pyrroles. Detailed SAR on the series has been studied by modifications of the aryl rings and the substituents in the pyrrole ring. Diarylpyrrole 1 is a very potent (COX-2, IC50 = 60 nm) and selective (COX-1/COX-2 = >1700) inhibitor whereas the isomeric 2 is completely inactive against COX-2. Modifications of the substituents on the fluorophenyl ring in 1 yields very potent inhibitors of COX-2 (IC50 = 40−80 nm) with excellent selectivity (1200 to >2500) vs COX-1. Analog 20 containing a sulfonamide group is an excellent inhibitor of COX-2 with an IC50 of 14 nm. Tetrasubstituted pyrroles containing groups such as COCF3, SO2CF3, or CH2OAr at position 3 in the pyrrole ring give excellent inhibitors (COX-2, IC50 = 30−120 nm). In vivo testing in the carrageenan-induced paw edema model in the rat establishes that the 1,2-diarylpyrroles are orally active antiinflammatory agents. Compound 3 is the most potent inhibitor of edema with an ED50 of 4.7 mpk.

Series of 1,2-diarylimidazoles has been synthesized and found to contain highly potent and selective inhibitors of the human COX-2 enzyme. The paper describes a short synthesis of the target 1,2-diarylimidazoles starting with aryl nitriles. Different portions of the diarylimidazole (I) were modified to establish SAR. Systematic variations of the substituents in the aryl ring B have yielded very potent (IC50 = 10−100 nm) and selective (1000−12500) inhibitors of the COX-2 enzyme. The study on the influence of substituents in the imidazole ring established that a CF3 group at position 4 gives the optimum oral activity. A number of the diarylimidazoles showed excellent inhibition in the adjuvant induced arthritis model (e.g., ED50 = 0.02 mpk for 22 and 34). The diarylimidazoles are also potent inhibitors of carrageenan-induced edema (ED50 = 9−30 mpk) and hyperalgesia (ED50 = 11−40 mpk). Several orally active diarylimidazoles show no GI toxicity in the rat and mouse up to 200 mpk.

A series of sulfonamide-containing 1,5-diarylpyrazole derivatives were prepared and evaluated for their ability to block cyclooxygenase-2 (COX-2) in vitro and in vivo. Extensive structure−activity relationship (SAR) work was carried out within this series, and a number of potent and selective inhibitors of COX-2 were identified. Since an early structural lead (1f, SC-236) exhibited an unacceptably long plasma half-life, a number of pyrazole analogs containing potential metabolic sites were evaluated further in vivo in an effort to identify compounds with acceptable pharmacokinetic profiles. This work led to the identification of 1i (4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide, SC-58635, celecoxib), which is currently in phase III clinical trials for the treatment of rheumatoid arthritis and osteoarthritis.

A novel series of 5,6-diarylspiro[2.4]hept-5-enes was shown to provide highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. A study of structure−activity relationships in this series suggests that 3,4-disubstituted phenyl analogs are generally more selective than 4-substituted phenyl analogs and that replacement of the methyl sulfone group on the 6-phenyl ring with a sulfonamide moiety results in compounds with superior in vivo pharmacological properties, although with lower COX-2 selectivity. Several compounds have been shown to possess promising pharmacological properties in adjuvant-induced arthritis and edema analgesia models. The absence of gastrointestinal (GI) toxicity at 200 mpk of several selected compounds in rats and mice corresponds well with the weak potency for inhibition of COX-1 observed in the enzyme assay. Methyl sulfone 55 and sulfonamide 24 were shown to have superior in vivo pharmacological profiles, low GI toxicity, and good oral bioavailability and duration of action.

Assessment of eicosanoid levels in biological systems is important for understanding their role in cell function and pathophysiological events. Current methods of eicosanoid quantitation are limited by sensitivity, scope, or throughput. The development of a new method for eicosanoid assessment in biological samples by electrospray and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring mode is described here. In this study, 14 biologically significant eicosanoids were quantitated in a single sample. Complete sample analysis required two repeated injections of 5 μl with an analysis time of 1.5 min/injection. Limits of detection ranged from 0.5 pg for thromboxane B2(TxB2) to 10 pg for 6-keto prostaglandin F1α(6-keto PGF1α). The reliability, reproducibility, sensitivity, and cross-detection of the method is also described. The MS/MS method was used to explore eicosanoid production in two inflammation models: lipopolysaccharide (LPS)-stimulated human whole blood and carrageenan-challenged rat air pouch. The most abundant metabolites in LPS-stimulated whole blood were prostaglandin E2(PGE2), TxB2, and 6-keto PGF1α; prostaglandins E1, D2, and F2αand leukotrienes B4and C4were detected in lower amounts. Eicosanoid levels determined by MS/MS were similar to those obtained by immunoassay and GC-MS. The most abundant metabolites detected in carrageenan-challenged rat air pouch were PGE2, 6-keto PGF1α, and TxB2. The method described in this work is accurate and rapid and should greatly aid in evaluating the role of multiple eicosanoids in future biological studies.

Murine BCL1 tumor cells bear large amounts of surface IgM and trace amounts of surface IgD. In the present studies we have shown that cultivation of these cells, in the absence of lipopolysaccharide, results in the acquisition of IgD by virtually all the cells. These results suggest that BCL1 cells can differentiate in vitro into more mature B cells and offer an attractive model for analyzing the factors controlling appearance of IgD on a monoclonal cell line.

Cyclooxygenase or prostaglandin endoperoxide H synthase-2 (PGHS-2) is the first enzyme in the prostanoid biosynthetic pathways and, in brain, it is regulated as an immediate-early gene (IEG). PGHS-2 mRNA and protein are rapidly induced by physiological synaptic activity, and high basal expression in cerebral cortex appears to be maintained by the natural synaptic activity. In contrast to other IEGs, PGHS-2 is a dendritic protein that is enriched in dendritic spines and is, therefore, likely to play a direct role in synaptic physiology. Consistent wish a signaling function in mature dendritic spines, PGHS-2 expression is strongly regulated during normal postnatal development in the rat, with peak expression during the third and fourth weeks. Here we use immunocytochemical approaches to compare the developmental expression of PGHS-2 in rat neocortex with that of other well characterized markers of dendritic maturation. PGHS-2 immunoreactivity (ir) follows histogenetic gradients and expression in secondary or more distal dendrites postdates that of even the most delayed dendritic proteins. This developmental pattern parallels the critical period for somatosensory and visual cortex development. Accordingly, PGHS-2-ir may be a useful marker of the final activity-dependent stages of cortical development. Consistent with this potential histochemical utility, we demonstrate that the normal laminar pattern of PGHS-2-ir in human cortex is altered in patients with Rett syndrome, a form of mental retardation with known alterations of dendritic maturation. Further studies of the developmental expression of PGHS-2 in human cortical development may permit analyses of dendritic abnormalities, in syndromes associated with disturbances of activity-dependent development, as well as provide an anatomic basis for understanding the role of prostaglandin signaling in cortical development.

To investigate the efficacy and safety of SC‐58635 (celecoxib), an antiinflammatory and analgesic agent that acts by selective cyclooxygenase 2 (COX‐2) inhibition and is not expected to cause the typical gastrointestinal (GI), renal, and platelet‐related side effects associated with inhibition of the COX‐1 enzyme. Four phase II trials were performed: a 2‐week osteoarthritis efficacy trial, a 4‐week rheumatoid arthritis efficacy trial, a 1‐week endoscopic study of GI mucosal effects, and a 1‐week study of effects on platelet function. The 2 arthritis trials identified SC‐58635 dosage levels that were consistently effective in treating the signs and symptoms of arthritis and were distinguished from placebo on standard arthritis scales. In the upper GI endoscopy study, 19% of subjects receiving naproxen (6 of 32) developed gastric ulcers, whereas no ulcers occurred in subjects receiving SC‐58635 or placebo. The study of platelet effects revealed no meaningful effect of SC‐58635 on platelet aggregation or thromboxane B2levels, whereas aspirin caused significant decreases in 2 of 3 platelet aggregation measures and thromboxane B2levels. In all 4 trials, SC‐58635 was well tolerated, with a safety profile similar to that of placebo. SC‐58635 achieves analgesic and antiinflammatory efficacy in arthritis through selective COX‐2 inhibition, without showing any evidence of 2 of the toxic effects of COX‐1 inhibition associated with nonsteroidal antiinflammatory drugs.

We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammation in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by high levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-directed antiinflammatory drugs.

Leukotriene-C4 synthase (LTC4S; EC 2.5.1.37) catalyzes the committed step in the biosynthesis of the peptidoleukotrienes, which are important in the pathogenesis of asthma. Antibodies were generated to a synthetic peptide based on the partial amino acid sequence previously reported for human LTC4S [Nicholson, D.W., Ali, A., Vaillancourt, J.P., Calaycay, J.R., Mumford, R.A., Zamboni, R.J. & Ford-Hutchinson, A. W. (1993) Proc. Natl. Acad. Sci. USA 90, 2015-2019] and specifically bound detergent-solubilized LTC4S obtained from THP-1 cells, confirming that the published sequence is associated with enzyme activity. Inosine-containing oligonucleotides based on the partial protein sequence were used to isolate a 679-bp cDNA for LTC4S from THP-1 cells. The cDNA contains an open reading frame that encodes a 150-amino acid protein (M(r) = 16,568) that has a calculated pI value of 11.1. The deduced protein sequence is composed predominantly of hydrophobic amino acids; hydropathy analysis predicts three transmembrane domains connected by two hydrophilic loops. Analysis of the deduced sequence identified two potential protein kinase C phosphorylation sites and a potential N-linked glycosylation site. The amino acid sequence for human LTC4S is unique and shows no homology to other glutathione S-transferases. LTC4S was found to be most similar to 5-lipoxygenase activating protein (31% identity, 53% similarity), another protein involved in leukotriene biosynthesis. Active enzyme was expressed in bacterial, insect, and mammalian cells as shown by the biosynthesis of LTC4 in incubation mixtures containing LTA4 and reduced glutathione. The cloning and expression of human LTC4S provide the basis for a better understanding of this key enzyme in peptidoleukotriene biosynthesis.

The prostaglandins (PGs) released from the heart have generally been characterized as resembling PGE by bioassay techniques. The major PG formed from [14C]arachidonate (C20:4) by the isolated perfused rabbit heart has chromatographic mobility similar to that of PGE2 in most solvent systems. However, additional analysis of this radioactive "PGE" peak suggests that two substances are formed by the heart and migrate like PGE2: one has chemical properties similar to those of authentic PGE2 and the other is a novel PG. The unknown compound is the major PG formed by the heart from either exogenous arachidonate or hormonal stimulation of PG biosynthesis. The novel PG produced by the heart may be identical with either 6(9)-oxy-PGF or 6-keto-PGF1 alpha.

In isolated perfused rabbit hearts, bradykinin produced a concentration-dependent decrease in coronary resistance directly associated with biosynthesis and release of prostaglandin-E-like substance. An inhibitor of bradykinin destruction (the nonapeptide SQ-20881) markedly enhanced both the coronary vasodilation and release of prostaglandin-E-like substance produced by cardiac injection of bradykinin. Indomethacin inhibited both the myocardial prostaglandin biosynthesis and the decrease in coronary resistance induced by bradykinin. The demonstration that bradykinin is a potent stimulator of prostaglandin biosynthesis in the heart has implications as to the cause of the afferent cardiovascular reflexes and pain in myocardial infarction and angina pectoris.

Induction of switch recombination to the gamma 1 and epsilon immunoglobulin (Ig) heavy chain loci was examined in B cells preactivated with anti-Ig (B lymphoblasts). In B lymphoblasts cultured with interleukin 4 (IL-4), IL-5 induced the accumulation of S micro-S gamma 1 rearrangements, but not epsilon recombination. Thus, IL-5 facilitates switch recombination directed to the gamma 1 heavy chain locus by IL-4, but additional signals are required to drive rearrangements to epsilon. Lipopolysaccharide (LPS), in the presence of IL-4, induced the accumulation of both S micro-S gamma 1 and S micro-S epsilon rearrangements, and cells treated with LPS exhibited 40-50-fold more S micro-S gamma 1 rearrangements than cells cultured with IL-5. Induction of switch recombination was not always associated with secretion of the respective Ig isotype, since concentrations of IL-4 that were sufficient to direct switch recombination to gamma 1 and epsilon in blasts treated with LPS failed to elicit secretion of IgG1 and IgE. These results demonstrate differential requirements for switch recombination to the gamma 1 and epsilon loci, as well as independent regulation of Ig gene rearrangement and secretion of each isotype.

The role of prostaglandin E2 (PGE2) in the development of inflammatory symptoms and cytokine production was evaluated in vivo using a neutralizing anti-PGE2 monoclonal antibody 2B5. In carrageenan-induced paw inflammation, pretreatment of rats with 2B5 substantially prevented the development of tissue edema and hyperalgesia in affected paws. The antibody was shown to bind the majority of PGE2 produced at the inflammatory site. In adjuvant-induced arthritis, the therapeutic administration of 2B5 to arthritic rats substantially reversed edema in affected paws. Anti-PGE2 treatment also reduced paw levels of IL-6 RNA and serum IL-6 protein without modifying tumor necrosis factor RNA levels in the same tissue. In each model, the antiinflammatory efficacy of 2B5 was indistinguishable from that of the nonsteroidal antiinflammatory drug indomethacin, which blocked the production of all PGs. These results indicate that PGE2 plays a major role in tissue edema, hyperalgesia, and IL-6 production at sites of inflammation, and they suggest that selective pharmacologic modulation of PGE2 synthesis or activity may provide a useful means of mitigating the symptoms of inflammatory disease.

Sepharose-bound anti-immunoglobulins, which are potent mitogens for normal adult B cells, are not mitogenic for tumor cells freshly isolated from mice carrying the B-cell leukemia BCL1. However, after 4 or more days of in vitro cultivation, BCL1 cells can be stimulated to divide by either anti-mu or anti-delta antibodies. These results suggest that in vitro cultivation of BCL1 cells results in their differentiation into more mature cells which can be triggered to proliferate by their interaction with anti-Ig antibodies. Addition of T-cell helper factors to anti-Ia treated BCL1 cells results in their differentiation into Ig-secreting cells. These results indicate that surface Ig molecules on BCL1 cells are capable of delivering an activation signal to the cells but that the cells require a second signal from T cells for induction of Ig secretion.

In these studies we have determined the molecular weights of B cell growth factor (BCGF) (less than 20,000), and B cell differentiation factors (BCDF) that induce immunoglobulin M (IgM) secretion (BCDF mu) (30-60,000) and IgG secretion (BCDF gamma) (less than 20,000). Thus, the molecular weight of BCDF mu is distinct from that of BCGF and BCDF gamma; BCGF and BCDF gamma cannot be distinguished. In addition, BCGF, BCDF mu, and BCDF gamma are distinguishable by their presence or absence in different supernatants from a panel of mitogen-induced T cell clones. These results suggest that the three lymphokines are different. This conclusion is supported by their differential biological effect on B cells from adult and neonatal mice. Thus, treatment with anti-Ig induces B cells from adult mice to proliferate and this proliferation is sustained by BCGF. In contrast, even in the presence of BCGF, anti-Ig does not induce B cells from neonatal mice to proliferate. However, BCDF mu and BCDF gamma induce IgM and IgG secretion in B cells, respectively, from both adult and neonatal mice. Thus, mature B cells can both clonally expand and differentiate in response to anti-Ig, BCGF, and BCDF, whereas immature B cells can only differentiate. The poor response of neonatal B cells to anti-Ig and BCGF may partially explain the relative immunoincompetence of immature B cells.

We have analyzed requirements for IL-4-induced secretion of IgG1 from anti-Ig-activated B cells. Activated B cell blasts prepared by culture of high density B cells with anti-Ig failed to secrete IgG1 upon subsequent culture with LPS and IL-4. However, IL-4 markedly suppressed IgM secretion in the same cultures. Addition of a mixture of T cell-derived lymphokines or rIL-5 to LPS-stimulated anti-Ig blasts restored IL-4-stimulated IgG1 secretion; rIL-2 further enhanced the response to IL-4 + rIL-5. These results suggest that IL-4, IL-5, and IL-2 cooperate in the regulation of B lymphocyte Ig isotype expression.

Sepharose-anti-Ig and purified populations of small, high-density B cells have been used to study the formation and function of B lymphoblasts. Sepharose-anti-Ig converts small, Ia-poor B cells with a high-buoyant density to large, Ia-rich, B blasts with a low-buoyant density. We find that this response proceeds efficiently in the absence of IL-4 (BSF-1) as well as most T cells, macrophages, and dendritic cells. Further development of the blasts requires an additional stimulus, such as LPS or the conditioned medium of stimulated EL-4 thymoma cells. Within 6 h, blasts begin to enter S phase and within 24 h most divide. At later times (48-72 h) most of the blasts are actively secreting IgM. Recombinant IL-1, -2, -3, and -4 have little or no effect on the B blasts, and a neutralizing mAb to IL-4 does not block the response to EL-4 Sn. We conclude that Sepharose-anti-Ig induces B cell blastogenesis in a T-independent fashion and that these blasts represent a highly enriched population of cells that respond to distinct, T cell-derived lymphokines.

Several potent and selective inhibitors of 5-lipoxygenase (5-LO) have been recently developed with excellent activity in certain in vivo assays of leukotriene production. The efficacy of three such inhibitors that have been in clinical trials (zileuton, A-78773 and ZD2138) were evaluated in: 1) ex vivo whole blood assay, 2) dermal Arthus reaction, and 3) functional airway response. In addition, a model of eicosanoid production in rat lung was developed that provides a simple assay for evaluation of the biochemical efficacy of 5-LO inhibitors in the lung. Bronchoalveolar lavage of rat lung with calcium ionophore A23187 resulted in rapid and robust production of PGE2, 6-keto-PGF1 alpha, thromboxane (TxB2), and leukotriene B4 (LTB4). Supplementation of lavage fluid with archidonic acid markedly augmented production of all eicosanoids except LTB4. All three inhibitors were potent and selective blockers of LTB4 production in the ex vivo whole blood assay and in the dermal Arthus reaction. In contrast, higher doses of inhibitor were needed to block LTB4 production in the rat lung lavage model than were needed to block ex vivo whole blood LTB4 production when both end points were measured in the same animal. Similarly, zileuton and A-78733 were less effective in suppressing the functional airway response to antigen in sensitized guinea pigs, whereas both inhibitors were effective in suppressing LTB4 production in the ex vivo whole blood assay. These results demonstrate that different 5-LO inhibitors have markedly distinct efficacy for inhibition of leukotriene production, depending on the animal model.(ABSTRACT TRUNCATED AT 250 WORDS)

Adminstration of endotoxin to dogs caused a rapid initial decline in blood pressure followed by a transient recovery preceding death. Plasma renin activity was elevated 5 minutes after endotoxin administration and continued to rise throughout the course of shock. Indomethacin given 60 minutes after endotoxin caused an elevation of blood pressure and a 50% decrease in plasma renin activity. Pretreatment with indomethacin markedly attenuated both the hemodynamic changes and the rise in plasma renin activity caused by endotoxin administration. Prostaglandin (PG) E-like material was observed in renal venous blood 30 minutes after endotoxin administration and was abolished by indomethacin. In addtion, a non-PG substance was found in dialysate from both arterial and renal venous blood within 5 minutes of endotoxin administration, Renal and mesenteric angiograms were taken at various stages of shock. Endotoxin administration caused a substantial increase in the diameter of intrarenal arterial branches which was temporally associated with the appearance of PGE-like material in the renal venous effluent. The mesenteric arteries were initially and transiently constricted by endotoxin and then were markedly and chronically dilated. Indomethacin simultaneously abolished renal PG and decreased renal and mesenteric arterial diameter.

We have examined the contributions of Interleukin 4 (IL-4), IL-5, and other stimuli to the expression of Immunoglobulin G1 (IgG1) and IgE in murine B lymphoblasts activated with anti-Ig. The combination of IL-4 and -5 induced B lymphoblasts to proliferate and to secrete IgM and IgG1. However, an additional stimulus was required along with IL-4 and -5 for induction of IgE secretion. This stimulus was provided by lipopolysaccharides (LPS) or cytokines produced by TC-1 or EL4 cells. In the absence of IL-5, exceptionally high concentrations of IL-4 (greater than 1,000 U/ml) were required to elicit IgG1 and IgE secretion from B lymphoblasts cultured with either LPS or TC-1-conditioned media (CM). To investigate regulation of expression of gamma 1 and epsilon genes by IL-4, -5, and LPS, the requirements for induction of gamma 1 and epsilon germline and productive transcripts were examined. Germline gamma 1, but not epsilon, transcripts were detected in RNA from B lymphoblasts treated with IL-4 and -5 for 48 h. In contrast, both germline gamma 1 and epsilon transcripts could be detected in B lymphoblasts cultured with IL-4 and LPS, and steady state levels of germline gamma 1 transcripts were four- to sevenfold higher in blasts cultured with LPS and IL-4, compared with blasts cultured with IL-4 and -5. LPS enhanced steady state levels of germline transcripts induced by IL-4, but LPS did not promote substantial accumulation of productive gamma 1 and epsilon transcripts. In contrast, IL-5 did not affect steady state levels of germline transcripts stimulated by IL-4, but did markedly increase levels of productive gamma 1 and epsilon transcripts. Thus, lymphokines regulate two distinct events in isotype switching: induction of germline transcripts (IL-4), and production of VDJ-C gamma 1 and VDJ-C epsilon mRNA (IL-5), which leads to secretion of IgG1 and IgE.

Culturing BALB/c B cells for 6 d at low cell density in the presence of lipopolysaccharide (LPS) results in the appearance of a small number of IgG plaque-forming cells (PFC). The addition of supernatants from concanavalin A (Con A)-induced alloreactive (AKR anti-B6) long-term T cell lines (PK 7.1.1a and 7.1.2) or a T cell hybridoma (FS7-6.18) to LPS-treated B cells resulted in a marked increase in IgG PFC (3--10-fold higher than in cultures treated with LPS alone. The number of induced IgG PFC was not affected by removing IgG-bearing cells on the fluorescence-activated cell sorter, indicating that T cell-derived B cell differentiation factor enhances isotype switching of sIgG- cells, rather than selecting and expanding pre-existing subpopulations of sIgG+ cells. We also investigated the subclass of IgG produced in the absence or presence of T cell factors and found that PK 7.1.1a, PK 7.1.2, and FS7-6.18 supernatants selectively increased IgG1 production. Several other T cell supernatants containing a variety of lymphokines had no effect, suggesting that PK 7.1.1a, PK 7.1.2, and FS7-6.18 lines produce factor(s) that can specifically enhance the recovery of IgG secreting cells in culture in the presence of LPS. These factors, which we have termed B cell differentiation factors, are different from interleukin 1, interleukin 2, T cell-replacing factor, colony-stimulating factor, macrophage-activating factor, and immune interferon. Our results suggest that soluble factors produced by T cell lines and hybridomas can markedly influence both the class and subclass of Ig produced by B cells.

Several lines of evidence document a critical role for mast cells in immune complex-mediated inflammatory models. However, their role in nonimmune models of acute inflammation is largely unknown. In the present investigation, the role of mast cells was examined in calcium ionophore (A23187)-induced mouse peritoneal inflammation. Intraperitoneal injection of A23187 (20)µg/mouse) elicited marked and transient increases in immunoreactive levels of 6-ketoprostaglandin-F2a, leukotrienes B4, C4, D4, E4, and F4. There were no discernible differences in levels of these mediators in male Swiss Webster mice, mast cell-deficient mice (WBB6F1-W/W'), and age-matched controls (WBB6F1-+/+), suggesting a minimal role of mast cells in eicosanoid biosynthesis in this model. However W/W' mice showed smaller increases in levels of myeloperoxidase, a marker for neutrophils, compared to +/+ mice. Both W/W' and +/+ mice have lower constitutive levels of peritonealN-acetyl-ß-d-glucosaminidase (NAG), a marker for mononuclear cells. Similar to the changes seen in myeloperoxidase, W/W' mice exhibited a blunted NAG response compared to +/+ mice. These results suggest that mast cell products other than eicosanoids may contribute to the changes in cellular trafficking in response to intraperitoneal A23187. These results also suggest that mast cells are required for full expression of inflammatory responses.

The effects of topical application of arachidonic acid (AA) or phorbol ester, tetradecanoylphorbol 13-acetate (TPA), on edema response, vascular permeability, MPO, NAG, and generation of eicosanoids were studied in two murine models of cutaneous inflammation. AA produced a short-lived edema response with a rapid onset that was associated with marked increases in levels of prostaglandins (PGE2, 6-keto-PGF1a, PGF2a), thromboxane B2 (TxB2) and leukotriene B4 (LTB4), with smaller increases in levels of LTC4. TPA produced a longer-lasting edema that was associated with marked influx of neutrophils and predominant formation of LTB4 along with significant changes in levels of TxB2. Circulating T lymphocytes have no apparent role in the acute inflammatory responses induced by either agent. Arachidonic acid-induced vascular permeability preceded the edema response and neutrophil influx, whereas TPA-induced vascular permeability paralleled the edema response and influx of neutrophils. Mast cells appear to be important in the complete expression of inflammatory response, i.e., edema, cellular influx, and vascular permeability induced by either AA or TPA, as these responses were blunted in mast cell-deficient mice. Inhibitors of CO or 5-LO attenuated inflammatory responses in both models. The LTB4 receptor antagonist, SC-41930, inhibited the inflammatory response to TPA but had little effect on that initiated by AA. This suggests that LTB4 is an important mediator in the phorbol ester-induced inflammatory response, whereas peptidoleukotrienes and prostaglandins regulate vascular permeability responses in the arachidonate model.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.