Stephen M. Coutts

Arylmethyl Phenyl Ethers

Oxidative metabolites of arachidonic acid produced by the 5-lipoxygenase pathway are potent mediators of hypersensitivity and inflammation (see O’Flaherty, 1982; Lewis and Austen, 1984 for reviews). Their proposed roles in the pathophysiology of asthma, chronic inflammation, and psoriasis have prompted an intense search for specific inhibitors of their biosynthesis (Bach, 1984). Three years ago a program was initiated at Revlon Health Care Group to find compounds that would modulate the biosynthesis of leukotrienes or antagonize their spasmogenic actions on smooth muscle. In particular, we sought specific and stable inhibitors of the 5-lipoxygenase (5-LOX) enzyme, utilizing a cellular assay as our primary screening tool.

Antiallergic Activity of Tiaramide (RHC 2592-A)

Tiaramide (RHC 2592-A) is an analgesic agent with antiallergic activity in vivo. We have investigated the antianaphylactic properties of tiaramide and its metabolites in three in vitro models of anaphylaxis; namely, IgE-induced release of histamine from rat mast cells and human basophils, and IgG1-induced release of histamine from guinea pig lung slices. Tiaramide and one of the metabolites, desethanol tiaramide (DETR), were found to inhibit immunologic release of histamine in all three of these in vitro models. Although tiaramide and DETR were less potent than disodium cromoglycate (DSCG) and/or proxicromil as inhibitors of mediator release, they were not cross-tachyphylactic to DSCG in the rat mast cell model. These data indicate that tiaramide is a unique inhibitor of histamine release whose mechanism of action differs from that of DSCG, and which in vivo is converted to a more potent metabolite.

RHC 3024: Antiallergic Activity in vitro and Comparison with Disodium Cromoglycate and Other Antiallergic Agents

RHC 3024 has been investigated for its antiallergic activity in three in vitro models of anaphylaxis. We have also compared its activity profile in these models with that of disodium cromoglycate (DSCG) and other antiallergic agents. As an inhibitor of antigen-induced release of histamine from rat mast cells RHC 3024 was 4 times more potent than DSCG. In the same model the activity profile of RHC 3024 was identical to that of DSCG in the following respects: loss of inhibitory activity with increasing preincubation time, reversibility of the inhibition, tachyphylaxis and cross-tachyphylaxis to each other and inability to inhibit histamine release stimulated by Ca++ ionophore, dextran/phosphatidyl serine and compound 48/80. Both drugs had no effect in the other two models, IgG1-mediated histamine release from guinea pig lung and anti-IgE-induced histamine release from human basophils. We conclude: (1) RHC 3024 is a potent inhibitor of mediator release with a mechanism of action similar to that of DSCG, M&B 22,948, PRD-92-Ea and AH-7725 and (2) the in vitro activity profiles of proxicromil, doxantrazole, ICI 74,917 and WY-16,922 are different from DSCG and RHC 3024.

REV 2871 (CHBZ): A novel inhibitor of histamine release

CHBZ is a novel, non-disodium cromoglycate (DSCG)-like, antiallergic drugin vitro. Whereas DSCG loses anti-secretory activity with increasing time of preincubation with rat mast cells (RMC) before antigen challenge, and does not inhibit non-immunologically mediated release from human leukocytes, CHBZ exerts long-lived inhibition of both types of release from both types of cells. CHBZ is taken up by RMC in a saturable and time-dependent manner. It is enzymatically converted inside RMC to a DSCG-like metabolite, REV 3579, which, because of its ionic character, accumulates inside the cell. CHBZ, presumably through the action of the REV 3579 generated intracellularly, may mediate long-lived phosphorylation of the same 78 K protein which is transiently phosphorylated upon exposure of RMC to DSCG.

Antiallergic Activity of Nylidrin Hydrochloride (RHC 3432-A)

Nylidrin hydrochloride has weak beta-adrenergic agonist properties with rat mast cells, and shows synergy with 3-isobutyl-1-methylxanthine (IBMX) in raising intracellular cAMP in purified mast cells; both of these properties can be blocked by dl-propranolol. However, the action of nylidrin hydrochloride as an inhibitor of histamine secretion from purified rat mast cells is not antagonized by dl-propranolol, nor is it potentiated by IBMX. Nylidrin-induced elevation of cellular cAMP in purified rat mast cells shows no correlation with nylidrin-induced inhibition of histamine release. Thus, nylidrin hydrochloride inhibits release of histamine from mast cells by a novel, non-adrenergic mechanism, which is not dependent on increased levels of cAMP.

RHC 3288 [1-methyl-2(1,3,4-oxadiazol-2(3H)-one-5-yl) benzimidazole] and related compounds: Novel inhibitors of histamine release from rat mast cells and human basophils

RHC 3288 [1-methyl-2(1,3,4-oxadiazol-2(3H)-one-5-yl) benzimidazole] and twenty-five related 5-substituted oxadiazolones have been investigated for their antiallergic activities in three in vitro models of anaphylaxis. Sixteen compounds were potent (I50 less than or equal to 50 microM) inhibitors of antigen-induced release of histamine (AIR) from rat mast cells (RMC), and seven compounds inhibited anti-IgE-induced release of histamine from human basophils (I50 less than or equal to 100 microM). The antiallergic activity profiles of RHC 3288 and three other compounds in these models have been compared with that of disodium cromoglycate (DSCG). As inhibitors of AIR from RMC, RHC 3288, 3334, 3354 and 3380 were 3 to 10 times more potent than DSCG. In the same model (AIR from RMC), activity profiles of all four RHC compounds were identical to that of DSCG in the following respects: loss of inhibitory activity with increasing preincubation time, tachyphylaxis and cross-tachyphylaxis to each other, and inability to inhibit histamine release stimulated by Ca2+ ionophore, dextran + phosphatidyl serine and compound 48/80. RHC 3288, 3334, 3354 and DSCG had no effect in the other two models, histamine release from guinea pig lung mediated predominantly by IgG1 class of antibodies and anti-IgE-induced histamine release from human basophils. We conclude that RHC 3288 is a potent inhibitor of mediator release with a mechanism of action similar to that of DSCG.

REV 2871 (CHBZ): A potent antiallergic agent with a novel mechanism of action: I. Activity profile as an inhibitor of mediator release☆☆☆

REV 2871 (CHBZ) and its putative metabolite REV 3579-Z (also designated in the literature as RHC 3579-Z) were shown to be potent and orally effective inhibitors of passive cutaneous anaphylaxis (PCA) in the rat (ED50 = 12 mg/kg). The activity profiles of CHBZ, REV 3579-Z and disodium cromoglycate (DSCG) were compared as inhibitors of histamine release (HR) in vitro from rat mast cells, human basophils, and guinea pig lung slices. CHBZ was a potent inhibitor of both immunologic and non-immunologic HR (I50 2-20 microM from rat mast cells). The activity profile of CHBZ as an inhibitor of HR from rat mast cells differed from that of DSCG and REV 3579-Z in the following respects: increasing inhibition of HR with increasing preincubation time; irreversibility of the inhibition; lack of tachyphylaxis and cross-tachyphylaxis to DSCG; potentiation of the inhibition of antigen-induced release of histamine (AIR) by DSCG; and inhibition of HR induced by dextran + phosphatidyl serine, compound 48/80, ionophore A23187 and platelet activating factor (PAF). In the human basophil model, CHBZ was: a potent inhibitor (I50 = 25 microM) of anti-IgE-induced release (AbIR), whereas DSCG and REV 3579-Z had no effect on AbIR; more potent as an inhibitor of AbIR than ionophore-induced release, whereas the reverse was true for proxicromil; an inhibitor of PAF-induced release, whereas proximcromil stimulated it; and potentiative with proxicromil for inhibition of AbIR. In the guinea pig lung slice model, CHBZ inhibited AIR (I50 = 800 microM) whereas DSCG and REV 3579-Z did not (I50 greater than 300 microM). We conclude that CHBZ is an orally effective antiallergic agent whose mechanism of action as an inhibitor of mediator release is different from DSCG and proxicromil.

Antiallergic activity profiles in vitro of RHC 3164 and related compounds. I: A lack of correlation between inhibition of cyclic nucleotide phosphodiesterases and antigen-induced release of histamine from rat mast cells

A series of 26 compounds belonging to the chemical class of (1,2,4)triazolo(4,3-a)-quinoxaline-1,4-diones have been investigated for their antiallergic activities in 3 in vitro models of anaphylaxis. Effects of these and other known antiallergic agents on cyclic nucleotide phosphodiesterases (cNUC-PDE) from purified rat mast cells have also been investigated. 18 compounds were potent (I50 less than or equal to 45 microM) inhibitors of antigen-induced release of histamine (AIR) from rat mast cells (RMC), 3 compounds inhibited anti-IgE-induced release of histamine from human basophils (I50 less than or equal to 25 microM) and none of the compounds significantly affected AIR from guinea pig lung slices. 13 of the compounds were more potent than theophylline as inhibitors of cyclic AMP-PDE and/or cyclic GMP-PDE from RMC. Parallel concentration-response curves for the inhibition of cyclic AMP-PDE and cyclic GMP-PDE indicated that these compounds probably interact with enzyme in the same manner. Paired regression analysis of the I50 values for inhibition of AIR and cNUC-PDE from RMC by these compounds revealed no statistically significant correlation between the inhibition of AIR and inhibition of cyclic AMP-PDE or cyclic GMP-PDE. We conclude: (1) some of these compounds are potent inhibitors of immunologic release of histamine from RMC with an in vitro activity profile similar to that of DSCG, and (2) inhibition of cyclic AMP or cyclic GMP hydrolysis by cNUD-PDE by these compounds, DSCG, and 6 known antiallergic agents is not the biochemical mechanism by which they inhibit AIR from RMC.

REV 2871 (CHBZ): A potent antiallergic agent with a novel mechanism of action: II. Studies on the mechanism of action☆

REV 2871 (CHBZ) was taken up by rat mast cells and human leukocytes in a specific and saturable manner. The compound can be hydrolyzed by a granule-associated enzyme in the mast cell to an ionic metabolite (REV 3579) whose in vitro profile is identical to that of disodium cromoglycate (DSCG). REV 3579, although achieving millimolar concentrations inside cells incubated with CHBZ, was not itself taken up by rat mast cells or human leukocytes. The unusual in vitro activity of CHBZ is postulated to arise from the fact that it is a prodrug for delivering a DSCG-like drug to the interior of a secretory cell. The internalized drug apparently exerts a more general and longer-lived inhibition of the secretory process than it can by acting on exterior membrane receptors. CHBZ thus represents a novel drug for studying anaphylactic responses in vitro.

Purification of small peptides labeled with Bolton-Hunter reagent

A method utilizing high-voltage electrophoresis on paper is described whereby a pentapeptide (Asp-Ser-Asp-Pro-Arg) labeled with Bolton-Hunter reagent is separated from hydrolyzed reagent and unreacted peptide and is recovered from the electrophoretogram in high yield. The general applicability to other peptides is discussed.