R. Fruchtmann

Mode of action of the new selective leukotriene synthesis inhibitor BAY X 1005 {(R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid} and structurally related compounds

BAY X 1005 ((R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) has been demonstrated to be a potent inhibitor of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) synthesis in various in vitro systems. Using mainly human polymorphonuclear leukocytes (PMNL) this study elucidates the mechanism of inhibition of 5-lipoxygenase (5-LOX, EC 1.13.11.34)-derived arachidonic acid metabolites by BAY X 1005. At concentrations of BAY X 1005 which almost totally inhibited the formation of 5-LOX-derived metabolites, both arachidonic acid release and platelet-activating factor synthesis were only modestly affected. This suggests that the inhibitory effect of BAY X 1005 is not due to a limitation of substrate availability for 5-LOX. Compared to the inhibition of leukotriene synthesis in intact human PMNL about 800-fold higher concentrations of BAY X 1005 were required to inhibit leukotriene formation in a cell-free system suggesting that the inhibitory effect of BAY X 1005 cannot be explained by a direct effect on 5-LOX. In an attempt to identify possible target proteins of BAY X 1005, [14C]BAY X 1005 was used in binding studies under equilibrium conditions. The quantitative analysis of specific binding in intact human PMNL revealed two binding sites for BAY X 1005. Upon subcellular fractionation of these cells the BAY X 1005 high affinity binding site was localized in the microsomal fraction whereas the low affinity binding site was localized in the granule fraction. The Kd for BAY X 1005 binding to the high affinity binding site (0.165 mumol/L) was almost identical to the IC50 value for inhibition of LTB4 synthesis (0.22 mumol/L). Furthermore, the IC50 values for competition of BAY X 1005 binding at the high affinity binding site were almost identical to the IC50 values for inhibition of LTB4 synthesis in the case of BAY X 1005, 12 other structurally related quinoline derivatives and the reference compounds REV-5901, WY-50,295 and MK-886, but not in the case of the direct 5-LOX inhibitors A-64077 and AA-861. The analysis of BAY X 1005 binding in rat PMNL also revealed two binding sites. Whereas the low affinity binding site in rat PMNL exhibited a Kd similar to the human, the rat high affinity binding site showed a 5.5-fold higher affinity for BAY X 1005 compared to the human. This correlates well with the 8.5-fold higher sensitivity of rat versus human PMNL concerning inhibition of LTB4 synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro pharmacology of BAY X1005, a new inhibitor of leukotriene synthesis

BAY X1005, (R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid, is an enantioselective inhibitor of leukotriene biosynthesis. It effectively inhibits the synthesis of LTB4 in A23187-stimulated leukocytes from rats, mice and humans (IC50 0.026, 0.039 and 0.22 μmol/l, respectively) as well as the formation of LTC4 (IC50 0.021 μmol/l) in mouse peritoneal macrophages stimulated with opsonized zymosan. The compound is, however, less active in inhibiting LTB4 synthesis in human whole blood (IC50 17.0 and 11.6 μmol/l, as measured by RIA or HPLC, respectively). BAY X1005 exhibits a high enantioselectivity in human whole blood (31 times over the (S)-enantiomer). BAY X1005 is shown to be a selective inhibitor of the formation of 5-lipoxygenase-derived metabolitesin vitro, without effects on other routes of arachidonic acid metabolism such as 12-lipoxygenase in human whole blood and cyclooxygenase in both mouse macrophages and human whole blood. BAY X1005 is devoid of any antioxidant activity (methemoglobin induction and xanthine-xanthine oxidase assay), without effects on granule release and with only weak effects on reactive oxygen species generation in human PMNL.

Inversely-correlated inhibition of human 5-lipoxygenase activity by BAY X1005 and other quinoline derivatives in intact cells and a cell-free system--implications for the function of 5-lipoxygenase activating protein.

A series of quinoline derivatives were analysed for the influence on leukotriene synthesis as a parameter for 5-LOX (EC 1.13.11.34) activity in a cell-free system of the 10,000 g supernatant of human PMNL (polymorphonuclear leukocytes). The ratios of the IC50 values for leukotriene synthesis inhibition in this cell-free system and in A23187-stimulated intact PMNL ranged from 1-1100. Consequently, plotting of the two values resulted in a random distribution (r = -0.281, N = 18) suggesting that no relationship between the inhibition of leukotriene synthesis in the cell-free system and in intact cells exists. At first sight this finding was not surprising since we have shown earlier that in intact cells this class of quinoline derivatives shares the same mode of action as MK-886, i.e. an indirect inhibition of 5-LOX activity by binding to FLAP. However, we found that the potency of these compounds in intact cells is strongly influenced by the K value (partition coefficient) which is a parameter for the ability of a substance to accumulate in a lipid (membrane) phase compared to the water phase. Therefore, the IC50 values for leukotriene synthesis inhibition in intact PMNL were corrected for the corresponding K value of the compounds and the resulting values again plotted against the IC50 values for inhibition of leukotriene synthesis in the cell-free system. As a result, a significant correlation (r = -0.878, N = 18) was obtained. In order to simplify this relationship the influence of the partition coefficient was eliminated by comparing compounds with about the same K value (K = 7243 +/- 1646, N = 7). As a result, the IC50 values for inhibition of leukotriene synthesis in the 10,000 g supernatant fraction (indicative for the affinity of the compounds to 5-LOX) and in intact cells (indicative for the affinity of the compounds to FLAP) were highly, but inversely correlated (r = -0.992). That means that a compound with a high affinity to 5-LOX will have a low affinity to FLAP and vice versa. We hypothesized that this pharmacologically obtained relationship could be indicative of a physiologically occurring equivalent. We therefore propose a model in which FLAP binds arachidonic acid as its physiological substrate with low affinity and allows 5-LOX to get access to its substrate (assuming a higher affinity of 5-LOX to arachidonic acid) after 5-LOX translocation from the cytosol to the membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Mode of action of the leukotriene synthesis (FLAP) inhibitor BAY X 1005: implications for biological regulation of 5-lipoxygenase.

Five-lipoxygenase (5-LOX) inhibition is gaining increasing importance as a novel approach to therapy of allergic asthma and other inflammatory diseases. Presently, two types of inhibitors are known, direct 5-LOX inhibitors (LOI) and the FLAP (five lipoxygenase activating protein) binding leukotriene synthesis inhibitors (LSI). The 5-LOX selective and orally active quinoline LSI, BAY X 1005, shares many mechanistic features with the indole LSI, MK-886. The binding of BAY X 1005 to FLAP correlates with LTB4 synthesis inhibition. BAY X 1005 has been shown to bind to the 18 kD protein FLAP. BAY X 1005 inhibits 5-LOX translocation from the cytosol to membranes and reverses 5-LOX translocation. The use of BAY X 1005 has helped to elucidate part of the complex FLAP/5-LOX interaction by showing that FLAP appears to represent a 5-LOX substrate transfer protein channelling endogenous and exogenous arachidonic acid to the leukotriene synthetizing 5-LOX. This notion presented by our group in 1992 has stimulated further mechanistic studies. These findings have additionally led to the hypothesis that substrate competition is not confined to the LSI/FLAP interaction but may also be true for the LOI/5-LOX interaction and that even mixed LSI/LOI 5-LOX inhibitors are feasible, yet have not been described. Further mechanistic work on LSI will be orientated not only to further elucidate the complex FLAP/5-LOX interaction, but also to identify FLAP-related eicosanoid binding proteins.

Ca2+ ionophore A23187-stimulated secretion of azurophil granules in human polymorphonuclear leukocytes is largely mediated by endogenously formed leukotriene B4

The mode of action of the new leukotriene synthesis inhibitor BAY X1005 ((R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) and structurally-related quinoline derivatives is reflected by the binding to a high-affinity binding site presumably identical to FLAP (five lipoxygenase activating protein). In addition to FLAP, we have identified a second BAY X1005 (low-affinity) binding site localized in the granule fraction of human PMNL (polymorphonuclear leukocytes). Based on the hypothesis that the corresponding target protein might be involved in the regulation of granule release, the influence of the leukotriene synthesis inhibitors BAY X1005 and MK-886 and the direct 5-LOX (5-lipoxygenase, EC 1.13.11.34) inhibitor A-64077 on the A23187- and fMLP (N-formyl-methionyl-leucyl-phenylalanine)-stimulated release of beta-glucuronidase (as a marker for azurophil granules) and vitamin B12-binding protein (as a marker for specific granules) was investigated. In contrast to MK-886, neither BAY X1005 nor A-64077 significantly affected fMLP-stimulated granule release. This was also true for the A23187-stimulated release of specific granules; however, under the same conditions the A23187-stimulated release of azurophil granules was almost totally inhibited by all three compounds. No obvious relationship between the corresponding IC50 values and the ability of these compounds to compete for BAY X1005 binding at the low-affinity binding site existed. Instead, by extending these studies to additional inhibitors, a correlation between the IC50 values for inhibition of A23187-stimulated (i) beta-glucuronidase release and (ii) LTB4 (leukotriene B4) synthesis was found (r = 0.969, N = 7). This relationship was independent of the mode of action of the compounds, namely direct 5-LOX inhibition or indirect 5-LOX inhibition mediated via binding to FLAP. These results suggest that 5-LOX metabolites may be involved in A23187-stimulated azurophil granule release. Of the two main biologically active 5-LOX metabolites synthesized under these conditions (LTB4 and 5-hydroxyeicosatetraenoic acid), only LTB4 stimulated beta-glucuronidase release to nearly the same extent as A23187. In addition, this metabolite significantly enhanced A23187-stimulated beta-glucuronidase release, but only at A23187 concentrations (> or = 0.25 mumol/L) which by themselves were not sufficient to trigger LTB4 formation. Moreover, the inhibition of A23187-stimulated beta-glucuronidase release by BAY X1005 or A-64077 was totally reversed by the addition of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)

ROLE OF 5-LIPOXYGENASE-ACTIVATING PROTEIN IN THE REGULATION OF 5-LIPOXYGENASE ACTIVITY IN HUMAN NEUTROPHILS

Using intact and fractionated human polymorphonuclear leukocytes (PMNL), we provide evidence that the enantioselective leukotriene synthesis inhibitor (LSI) BAY X 1005, (R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid binds specifically to a high-affinity binding site, which is most likely identical to FLAP.BAY X 1005 blocks the translocation of 5-lipoxygenase (5-LOX) in PMNL stimulated by the calcium ionophore A23187 or chemotactic stimuli such as PAF, C5a or fMLP as does MK-886. In contrast to the direct 5-LOX inhibitors (LOI) A-64077 and AA-861, the degree of leukotriene synthesis inhibition declines with increasing duration of A23187-induced leukocyte activation in the presence of BAY X 1005 and MK-886. Kinetic studies performed with BAY X 1005 showed that this effect was not accompanied by a significant translocation of 5-LOX from the cytosol to the microsomal fraction. Because FLAP has been implicated in the transfer of arachidonic acid to 5-LOX and A23187 is a potent activator of leukocyte phospholipase A2, we hypothesized that the observed loss of leukotriene synthesis inhibition may be due to competition of BAY X 1005 binding by endogenously released arachidonic acid. Accordingly, binding of BAY X 1005 to FLAP in intact and fractionated cells is dose-dependently inhibited by arachidonic acid and other unsaturated long-chain fatty acids, but not by saturated fatty acids. Therefore, we conclude that BAY X 1005 or MK-886 inhibit leukotriene biosynthesis by binding to FLAP, thereby preventing 5-LOX translocation and substrate transfer to the enzyme.