WY-50,295 tromethamine, a novel, orally active 5-lipoxygenase inhibitor: biochemical characterization and antiallergic activity.

WY-50,295 tromethamine demonstrates significant 5-lipoxygenase inhibitory activity with IC50 values ranging from 0.055 microM in rat peritoneal exudate cells, to 0.16 microM in mouse macrophages, 1.2 microM in human peripheral neutrophils and 8.1 microM in rat blood leukocytes. This activity appeared selective for 5-lipoxygenase as concentrations up to 10 microM in rat peritoneal exudate cells, and 1 microM in mouse macrophages did not effect prostaglandin generation. In non-cellular enzyme assays, WY-50,295 tromethamine displayed inhibitory activity against a soluble 5-lipoxygenase from guinea pig peritoneal exudate cells (IC50 = 5.7 microM), while it was essentially inactive against 12-lipoxygenase, 15-lipoxygenase, or prostaglandin H synthetase at concentrations up to 500 microM, or against human phospholipase A2 at concentrations up to 50 microM. In purified human blood neutrophils the inhibitory activity was reversible but did not appear dependent upon substrate concentration. IN contrast, in the guinea pig cell-free 5-lipoxygenase assay changing the arachidonic acid substrate concentration from 5 to 500 microM produced a concentration-dependent reduction in inhibitory activity. WY-50,295 tromethamine inhibited the release of peptidoleukotrienes from fragmented guinea pig lung with an IC50 of 0.63 microM. When administered p.o. with a 4 h pretreatment time, WY-50,295 tromethamine inhibited ex vivo leukotriene B4 production in rat blood leukocytes with an ED50 of 19.6 mg/kg. Against an ovalbumin-induced leukotriene dependent bronchoconstriction in anesthetized sensitized guinea pigs, WY-50,295 tromethamine inhibited the ovalbumin-induced bronchoconstriction with an i.v. ED50 of 2.5 mg/kg (5 min pretreatment) and a p.o. ED50 of 7.3 mg/kg (4 h pretreatment). Significant activity was also evident with an 18 h pretreatment. Thus WY-50,295 tromethamine is an potent and selective 5-lipoxygenase inhibitor in a number of in vitro systems. Additionally the compound is orally efficacious and has a long duration of action in an allergic bronchoconstriction model. This data suggests that WY-50,295 tromethamine may have utility in the treatment of asthma and other leukotriene-dependent pathologies.

Anti-leukotriene effects of WY-50,295 tromethamine in isolated guinea pig pulmonary tissues.

The abilities of WY-50,295 tromethamine, a 5-lipoxygenase inhibitor, to inhibit antigen-induced leukotriene (LT) release from guinea pig lung fragments, and to prevent LTD4 or antigen-induced contraction of isolated guinea pig tracheal muscle were compared with the activities of zileuton and MK-886 (two selective 5-lipoxygenase inhibitors), and LY171883 (a LTD4 receptor antagonist). In fragmented guinea pig lung, WY-50,295 tromethamine inhibited antigen-induced LT release with an IC50 of 0.63 microM, and was 4.6- and 5.2-fold more potent than zileuton and MK-886, respectively. WY-50,295 tromethamine differed from these 5-lipoxygenase inhibitors, however, in that WY-50,295 tromethamine competitively antagonized LTD4-induced tracheal contractions (pA2 = 6.06) at concentrations that inhibited LT release. LY171883 was an effective LTD4 receptor antagonist (pA2 = 6.96), that only inhibited antigen-induced LT release at higher concentrations (IC50 = 7.9 microM). WY-50,295 tromethamine almost completely inhibited antigen-induced leukotriene-dependent tracheal contractions, whereas high concentrations of zileuton, MK-886, or LY171883 produced only partial inhibition. This partial inhibition was likely to result from 'breakthrough' 5-lipoxygenase activity, because combinations of zileuton plus MK-886 or zileuton plus LY171883, were more effective than zileuton, MK-886, or LY171883 alone. The greater efficacy of WY-50,295 tromethamine in the antigen-challenged guinea pig trachea is likely to result from its combined abilities to prevent LT biosynthesis and block LTD4 receptors.

Phospholipase A2 acyl-hydrolytic activity in rat RPAR-induced pleurisy.

Endogenous phospholipase A2 (PLA2) specific activity (SA) (% hydrolysis/min/mg protein) in the rat pleural cavity, measured as the acyl-hydrolysis of [3H]-arachidonic acid E. coli substrate, was quantitated after induction of a reverse passive Arthus reaction (RPAR). PLA2 SA, derived when the rate of hydrolysis was linear (1-5 min), was 1.9, 1.4, 3.8 and 4.1% h/min/mg at 10 min, 2 h, 4 h and 24 h, respectively, after induction of the RPAR. This time course appeared to correlate with the influx of mononuclear inflammatory cells, although the effect of plasma leakage on changes in exudate PLA2 SA could not be determined. Oral administration of antiinflammatory drugs significantly inhibited pleural fluid exudation and inflammatory cell influx to varying degrees. However, whereas these drugs additionally reduced total exudate protein and PLA2, they had no effect on the concentration of either parameter, implying that pleural PLA2 may be passively linked to fluid and cell movement.

Characterization of phospholipase A2 activity in rat RPAR-induced pleurisy.

At 10 min, 2 h and 4 h after induction of a reverse passive Arthus reaction (RPAR) in the pleural cavity of rats, pleural exudate was collected and analyzed in vitro for phospholipase A2 (PLA2) acyl hydrolytic activity. Inasmuch as exudate PLA2 was inhibited by addition of EDTA, the acyl hydrolytic activity appeared calcium-dependent. However, addition of exogenous calcium to the exudate had only a minor effect on the activity. Maximum hydrolysis in all exudates was observed over a pH range of 6-9 with a neutral optimum. The 4 h exudate PLA2 activity was almost fully inhibited by para-bromophenacyl bromide (pBPB), whereas the acyl hydrolytic activity in the 10 min and 2 h exudates was reduced by 44% and 46%, respectively, by 200 microM pBPB. Although the PLA2 in the 4 h exudate displayed an increased sensitivity to high exogenous calcium concentrations and to pBPB inactivation, the basic properties of the exudate PLA2 at the three time points appeared similar.

Comparison of several new 5-lipoxygenase inhibitors in a rat Arthus pleurisy model.

The 5-lipoxygenase inhibitors WY-50,295 tromethamine, A-64,077, L-663,536 and ICI-207,968 were compared in a reverse passive Arthus reaction-induced pleurisy model of eicosanoid biosynthesis in the rat. When a 1 h pretreatment schedule was employed, all four inhibitors equivalently blocked leukotriene B4 (LTB4) production with ED50 values of 2.0-2.9 mg/kg p.o. Conversely, WY-50,295 tromethamine (225 mg/kg p.o.) and L-663,536 (100 mg/kg p.o.) did not significantly alter thromboxane B2 (TxB2) levels, whereas A-64,077 (50 mg/kg p.o.) and ICI-207,968 (100 mg/kg p.o.) significantly reduced TxB2 by 50 and 72%, respectively. When 3 and 18 h pretreatment schedules were employed, WY-50,295 tromethamine demonstrated a longer duration of action than the other 5-lipoxygenase inhibitors with ED50 values of 1.7 and 6.3 mg/kg p.o., respectively. At doses of 50 and 100 mg/kg p.o., all drugs tested significantly inhibited inflammatory cell influx by 15-27%, albeit in a non-dose-related manner. However, only A-64,077 significantly lowered fluid extravasation by 35%, presumably due to inhibition of cyclooxygenase product formation. These results demonstrate that in this rat reverse passive Arthus pleurisy model, WY-50,295 tromethamine potently and selectively inhibits 5-lipoxygenase in vivo, and possesses a longer duration of action than the other 5-lipoxygenase inhibitors employed.

WY-50, 295 tromethamine: an orally active 5-lipoxygenase inhibitor with anti-allergic activity.

WY-50,295 tromethamine inhibited antigen-induced peptidoleukotriene (pLT) release from fragmented sensitized guinea pig lung (IC50 = 0.63 microM), antagonized LTD4-induced contractions of isolated guinea pig trachea (pA2 = 6.06), and suppressed antigen-induced contraction of sensitized guinea pig trachea over the 0.1-10 microM concentration range. In vivo, WY-50,295 tromethamine inhibited LTD4-induced bronchoconstriction (ED50 = 1.3 mg/kg i.v. and 6.6 mg/kg p.o.) and antigen-induced bronchoconstriction (ED50 = 2.5 mg/kg i.v. and 7.3 mg/kg p.o.) in anesthetized guinea pigs. Peak activity vs antigen was noted at 4-6 h after oral dosing and remained significant through 18 h. These studies demonstrate that WY-50,295 tromethamine possesses the complimentary actions of 5-LO inhibition and LTD4 receptor antagonism.

Phospholipase A2-induced pleural inflammation in rats.

Injection of Naja mocambique mocambique phospholipase A2 [PLA2] into the rat pleural cavity induced dose- and time-dependent fluid accumulation and cellular infiltration. The time course of the cell influx was initially neutrophilic [2-6 h] and later mononuclear [6-24 h]. During reverse passive Arthus reaction [RPAR] induced pleurisy, endogenously produced PLA2 activity, quantitated by the hydrolysis of [3H]-arachidonic acid E. coli substrate, was detected in the pleural exudate. However, the biosynthesis of eicosanoids and plasma extravasation in the pleural cavity preceded the 9-fold elevation in PLA2 activity which was obtained at 4 h. Whereas the exact role of PLA2 in the inflammatory response remains to be determined, these results demonstrate that exogenous PLA2 can induce pleural inflammation in the rat, and that this enzyme is released endogenously during experimental pleurisy.

Production of prostacyclin in mice following intraperitoneal injection of acetic acid, phenylbenzoquinone and zymosan: its role in the writhing response.

The magnitude and temporal production of PGI2, PGE2 and LTB4 were measured in the mouse peritoneal cavity for a 15 min period following the intraperitoneal injection of either acetic acid, phenyl-p-benzoquinone (PBQ) or zymosan. For each algogenic substance, PGI2 (assayed as the stable metabolite, 6-keto-PGF1 alpha) represented the major eicosanoid with lower levels of PGE2 also detected. Zymosan induced the greatest 6-keto-PGF1 alpha production among the three algogenic agents, but only a weak writhing response was observed. LTB4 was detected in the peritoneal lavage only after zymosan. The magnitude of eicosanoid production did not correlate with the writhing response induced by the algogenic agents, even though the inhibition of both 6-keto-PGF1 alpha and writhing by several peripheral analgesics was positively correlated. PGI2, (100 ng), 6-keto-PGF1 alpha (1 microgram) and PGE2 (100 ng) did not induce writhing. However, only PGI2 acted synergistically with acetic acid to produce writhing. Presumably due to the short biological lifetime of PGI2, this synergism was noted only when PGI2 was administered after the acetic acid. These results suggest that PGI2 acts to sensitize the animal for the writhing response.

17-Heteroaroyl esters of corticosteroids. 2. 11 beta-Hydroxy series.

The preparation and topical antiinflammatory potencies of a series of 17-furoyl and -thenoyl esters of 9 alpha-fluoro-11 beta-hydroxy-16 methyl and 9 alpha-chloro-11 beta-hydroxy-16-methyl corticosteroids are described. The 17 alpha-esters were introduced to the 9 alpha-fluoro 11-ketones or to the appropriate delta 9(11) compounds by direct acylation with the appropriate heteroaryl carbonyl chloride in the presence of 4-(dimethylamino)pyridine. Functionalization of the C ring was completed by standard methods. The most extensively studied heterocyclic acyl group was 2-furoyl, but 3-furoyl and 2- and 3-thenoyl derivatives were also investigated. Antiinflammatory potencies were measured in mice by a 5-day modification of the Tonelli croton oil ear assay. The most potent topical antiinflammatory agents were 1e, dexamethasone 17-(2'-furoate) 21-propionate, and 2c, the 21-chloro 17-(2'-furoate) in the 9 alpha-chloro series, both being 6 times as potent as betamethasone 17-valerate. Several other 9 alpha-chloro-11 beta-hydroxy-17-heteroaryl carboxylates (2a, 2b, 2d, and 2g) were at least 4 times as potent as betamethasone 17-valerate. Evaluation of 2c in the clinic confirmed that the compound is a potent topical antiinflammatory agent in humans.

Leukotriene B4 production and pharmacologic regulation of reverse passive Arthus pleurisy: importance of antigen dose.

Immunoreactive leukotriene B4 (iLTB4), detected in the pleural cavity following induction of a reverse passive Arthus reaction (RPAR), was inhibited by the mixed lipoxygenase-cyclooxygenase inhibitors, phenidone and BW 755C, but not by cyclooxygenase inhibitors or by chlorpheniramine or methysergide. Both iLTB4 production and the subsequent pleural inflammation were dependent upon the dose of BSA antigen employed to elicit the RPAR pleurisy. However, inasmuch as BW 755C and phenidone were not distinguished from the cyclooxygenase inhibitors in their effects on fluid accumulation and cellular infiltration in RPAR pleurisy, it is doubtful that LTB4 plays a functional role in this inflammation model.

Synthesis and structure-activity studies of corticosteroid 17-heterocyclic aromatic esters. 1. 9 alpha, 11 beta-dichloro series.

The preparation and topical antiinflammatory potencies of a series of 9 alpha, 11 beta-dichloro-16-methyl corticosteroid 17-heteroaryl carboxylates are described. The 17-acyl group was introduced to the 9 alpha, 11 beta-dichloro 21-acetate by direct acylation with the appropriate heteroaryl carbonyl chloride in the presence of 4-(dimethylamino)pyridine. Alternatively, the 21-functionalized 17-hydroxy delta 9(11) compound was acylated at 17, followed by C-ring chlorination. The most extensively studied heterocyclic acyl functionality was the 2-furoyl, but the 3-furoyl, and 2- and 3-thenoyl derivatives were also investigated. Antiinflammatory potencies were measured in mice by a 5-day modification of the Tonelli croton oil ear assay. The most potent topical antiinflammatory compounds were 17-heteroaryl esters in the 16 alpha-methyl series where the 21-substituent was chloro or fluoro. Thus 2p [21-chloro 17-(2'-furoate)] was 8 times as potent as betamethasone valerate, while 2s [21-fluoro 17-(2'-furoate)], 2r [21-chloro 17-(2'-theonate)], and 2v [6 alpha-fluoro 21-chloro 17-(2'-furoate)] were 3 times as potent as betamethasone valerate.

Structure-activity relationships of a series of novel topical corticosteroids.

The effect of various heteroaroyl groups in the 17-position of topical corticosteroids has been studied. The corticosteroids esterified at C17 were of 9 alpha,11 beta-dichloro, 9 alpha-chloro 11 beta-hydroxy and 9 alpha-fluoro 11 beta-hydroxy series. Among the 17-acyl groups 2'-furoates were most extensively investigated, although 2'-thenoates, 3'-thenoates and 3'-furoates were also examined. Many of these esters exhibited enhanced topical anti-inflammatory potencies. The most potent compounds investigated were the 21-chloro 17(2'-furoates) either in the 9 alpha,11 beta-dichloro, or in the 9 alpha-chloro 11 beta-hydroxy series. These compounds were at least 6 times as potent as betamethasone 17-valerate. Among 16-substituents studied 16 alpha-methyl compounds had the highest potency. Topical anti-inflammatory potencies were determined by using a 5-day modification of the croton oil ear assay in mice. The more potent compounds were also evaluated in the P. ovale induced chronic psoriaform lesion in the guinea-pig.

Differential effects of anti-inflammatory drugs on fluid accumulation and cellular infiltration in reverse passive arthus pleurisy and carrageenan pleurisy in rats.

At 4 h following induction of pleural inflammation in rats using either an immune stimulus (reverse passive Arthus reaction, RPAR) or a chemical stimulus (carrageenan), the cellular infiltration and fluid accumulation responses were quantitated. The bell-shaped antigen (BSA) dose-response curve describing the fluid response was increased upward as the anti-BSA dose was increased from 0.25 to 1 mg, whereas the dose-response curve for cellular infiltration was both shifted upward and to the right. Both nonsteroidal anti-inflammatory drugs and a mixed lipoxygenase-cyclooxygenase inhibitor (BW 755C) preferentially inhibited fluid accumulation in RPAR pleurisy elicited with 5 mg BSA and 1 mg anti-BSA and in carrageenan pleurisy. In contrast, these drugs inhibited cellular infiltration preferentially in RPAR pleurisy elicited with 1 mg BSA and 1 mg anti-BSA. These results demonstrate that the fluid and cellular responses in rat pleural inflammation can be differentially regulated by anti-inflammatory drugs depending upon the doses of antigen and antibody employed in RPAR pleurisy and the identity of the inflammatory stimulus.

Synthesis and topical antiinflammatory potencies of a series of 6-keto- and delta 6-6-acyloxybetamethasone derivatives.

6-keto- and delta 6-6-acyloxybetamethasone esters were synthesized, and tested for topical antiinflammatory potency using a modification of the Tonelli croton ear assay. The introduction of a 6-keto group generally led to retention of topical antiinflammatory potencies when compared to the corresponding 6-desoxycorticoids. In contrast, introduction of the delta 6-6-acyloxy moiety into betamethasone 17,21-dipropionate reduced antiinflammatory potency.

The influence of esterification on the topical antiinflammatory activity of 7 alpha-chloro- and 7 alpha-bromo-16 alpha-methylprednisolones.

The effect on topical antiinflammatory potency of altering the ester functions of 7-halogenocorticosteroids was examined. Highest potencies for both 7 alpha-chloro- and 7 alpha-bromo-16 alpha-methylprednisolones were generally achieved through diesterification at the 17- and 21-positions. However, among these diesters, structure seems to be more important for topical potency than lipophilicity.

Vitamin E-induced chronic inflammation in rats.

Sub-plantar injection of vitamin E (VE) oil produces a highly localized, chronic inflammation which is sustained for at least 8 weeks. As a result of studies using mediator inhibitors and depletors, it appears that histamine, serotonin, kinins, and prostaglandins may be involved in at least the early response to VE. This early phase is dominated by a massive influx of neutrophils, with the reaction rapidly progressing to a sustained mononuclear cell pathology. Results of studies in leucopenic rats suggest that edema formation and accumulation of white cells may be dissociable, although at this time it is not possible to conclude that edema can occur in the absence of cells since profoundly leucopenic rats are still able to locally mobilize polymorphonuclear cells in response to VE. Vitamin E-induced inflammation, produced under the conditions described, can be effectively suppressed by corticosteroids but seems relatively insensitive to the action of most non-steroidal anti-inflammatory agents tested.

A novel class of potent topical antiinflammatory agents: 17-benzoylated, 7 alpha-halogeno substituted corticosteroids.

As part of continued efforts in the synthesis of structurally novel corticosteroids, a number of 17 alpha-benzoylated, 7 alpha-halogeno substituted prednisolones were tested for topical antiinflammatory activity. 7 alpha-Chloro, 7 alpha-bromo, and 7 alpha-iodo corticosteroids were synthesized by hydrogen halide addition to 1,4,6-triene-3-ones. The 7 alpha-fluoro substituted steroid was obtained by reaction of the appropriate 7 beta-hydroxy compound with N,N-diethyl(2-chloro-1,1,2-trifluoroethyl)amine. Antiinflammatory potencies were obtained using a croton oil-induced inflammation in the ears of mice. In this assay the greatest effect of a 7 alpha-halogen was observed in the 16 alpha-methylprednisolone series, where 7 alpha-fluoro and 7 alpha-bromo substitution yielded corticosteroids with topical potencies significantly higher than those of the corresponding 17,21-dipropionate analogs. Surprisingly, little potency enhancement due to 7 alpha-halogenation was discerned in the 16 beta-methylprednisolone series.

Selective effects of 7 alpha-halogeno substitution on corticosteroid activity: Sch 22219 and Sch 23409.

Two 7 alpha-halogeno substituted corticosteroids, 7 alpha-chloro-16 alpha-methylprednisolone-17,21 dipropionate (Sch 22219) and 7 alpha-bromo-16 alpha-methylprednisolone-17-benzoate-21-acetate (Sch 23409) were compared to other clinically utilized topical corticosteroids for local and parenteral antiinflammatory and glucocorticosteroid activity. Both compounds were at least equivalent to the most potent comparison corticosteroids in topical antiinflammatory activity, and exhibited favorable ratios of local to systemic effects. In mice, Sch 22219 showed a greater dissociation of antiinflammatory activity from side effects than Sch 23409, although the reverse was true in rats. On the basis of the data available, both compounds possess enhanced topical antiinflammatory potency, with the potential for reduced side effects in man.