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The G protein-coupled cysteinyl leukotriene receptor CysLT 1 R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT 1 R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT 1 R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue-coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The role of inflammation and oxidative stress is critical during onset of metabolic disorders and this has been sufficiently established in literature. In the present study, we evaluated the effects of sesamol and sesamin, two important bioactive molecules present in sesame oil, on the generation of inflammatory and oxidative stress factors in LPS injected rats. Sesamol and sesamin lowered LPS induced expression of cPLA 2 (61 and 56%), 5-LOX (44 and 51%), BLT-1(32 and 35%) and LTC 4 synthase (49 and 50%), respectively, in liver homogenate. The diminished serum LTB 4 (53 and 64%) and LTC 4 (67 and 44%) levels in sesamol and sesamin administered groups, respectively, were found to be concurrent with the observed decrease in the expression of cPLA 2 and 5-LOX. The serum levels of TNF-α (29 and 19%), MCP-1 (44 and 57%) and IL-1β (43 and 42%) were found to be reduced in sesamol and sesamin group, respectively, as given in parentheses, compared to LPS group. Sesamol and sesamin offered protection against LPS induced lipid peroxidation in both serum and liver. Sesamol, but not sesamin, significantly restored the loss of catalase and glutathione reductase activity due to LPS (P<.05). However, both sesamol and sesamin reverted SOD activities by 92 and 98%, respectively. Thus, oral supplementation of sesamol and sesamin beneficially modulated the inflammatory and oxidative stress markers, as observed in the present study, in LPS injected rats. Our report further advocates the potential use of sesamol and sesamin as an adjunct therapy wherein, inflammatory and oxidative stress is of major concern., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The pro-inflammatory leukotrienes (LTs) are formed from arachidonic acid (AA) in activated leukocytes, where 5-lipoxygenase (5-LO) translocates to the nuclear envelope to assemble a functional complex with the integral nuclear membrane protein 5-LO-activating protein (FLAP). FLAP, a MAPEG family member, facilitates AA transfer to 5-LO for efficient conversion, and LT biosynthesis critically depends on FLAP. Here we show that the novel LT biosynthesis inhibitor BRP-187 prevents the 5-LO/FLAP interaction at the nuclear envelope of human leukocytes without blocking 5-LO nuclear redistribution. BRP-187 inhibited 5-LO product formation in human monocytes and polymorphonuclear leukocytes stimulated by lipopolysaccharide plus N-formyl-methionyl-leucyl-phenylalanine (IC 50 =7-10nM), and upon activation by ionophore A23187 (IC 50 =10-60nM). Excess of exogenous AA markedly impaired the potency of BRP-187. Direct 5-LO inhibition in cell-free assays was evident only at >35-fold higher concentrations, which was reversible and not improved under reducing conditions. BRP-187 prevented A23187-induced 5-LO/FLAP complex assembly in leukocytes but failed to block 5-LO nuclear translocation, features that were shared with the FLAP inhibitor MK886. Whereas AA release, cyclooxygenases and related LOs were unaffected, BRP-187 also potently inhibited microsomal prostaglandin E 2 synthase-1 (IC 50 =0.2μM), another MAPEG member. In vivo, BRP-187 (10mg/kg) exhibited significant effectiveness in zymosan-induced murine peritonitis, suppressing LT levels in peritoneal exudates as well as vascular permeability and neutrophil infiltration. Together, BRP-187 potently inhibits LT biosynthesis in vitro and in vivo, which seemingly is caused by preventing the 5-LO/FLAP complex assembly and warrants further preclinical evaluation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Inhibition of drug metabolizing enzymes is a major mechanism in drug-drug interactions (DDIs). A number of cases of DDIs via inhibition of UDP-glucuronosyltranseferases (UGTs) have been reported, although the changes in pharmacokinetics are relatively small in comparison with drugs that are metabolized by cytochrome P450s. Most of the past studies have investigated hepatic UGTs, although recent studies have revealed a significant contribution of UGTs in the small intestine to drug clearance. To evaluate potential DDIs caused by inhibition of intestinal UGTs, we assessed inhibitory effects of 578 compounds, including drugs, xenobiotics, and endobiotics, on human UGT1A8 and UGT1A10, which are major contributors to intestinal glucuronidation. We identified 29 inhibitors by monitoring raloxifene glucuronidation with recombinant UGTs. All of the inhibitors potently inhibited UGT1A1 activity, as well. We found that zafirlukast is a potent general inhibitor of UGT1As and a moderate inhibitor of UGT2Bs because it monitors 4-methylumbelliferone glucuronidation by recombinant UGTs. However, zafirlukast did not potently inhibit diclofenac glucuronidation, suggesting that the inhibitory effects might be substrate specific. Inhibitory effects of zafirlukast on some UGT substrates were further investigated in human liver and human small intestine microsomes in order to evaluate potential DDIs. The R values (the ratios of intrinsic clearance with and without an inhibitor) revealed that zafirlukast has potential to cause clinical DDIs in the small intestine. Although we could not identify specific UGT1A8 and UGT1A10 inhibitors, zafirlukast was identified as a general inhibitor for UGTs in vitro. The present study suggests that the inhibition of UGT in the small intestine would be an underlying mechanism for DDIs., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

The ABC protein P-glycoprotein (Pgp or ABCB1) is a multidrug efflux pump capable of transporting many structurally diverse substrates from within the lipid bilayer. Previous studies have demonstrated the importance of the membrane in modulating Pgp function, but few have quantified these effects. We employed purified Pgp reconstituted into phospholipid bilayers with defined gel to liquid-crystalline melting transitions to investigate the effect of membrane environment on the transporter and three of its substrates. Equilibrium dialysis measurements indicated that Hoechst 33342, LDS-751, and MK-571 partitioned much more readily into liquid-crystalline phase bilayers than into gel phase bilayers. However, drug binding affinities revealed that Pgp bound the three substrates more tightly when the lipid bilayer was in the gel phase. The binding affinity of the transporter for substrates within the bilayer was low, in the millimolar range, suggesting that it interacts with them weakly. Thermodynamic analysis revealed that both drug-Pgp and drug-lipid interactions contribute to binding affinity. The kinetics of LDS-751 and Hoechst 33342 transport by reconstituted Pgp was monitored using a real-time fluorescence-based assay to obtain apparent turnover frequencies. Transport rates were found to be sensitive to both drug structure and lipid environment. Arrhenius and transition state analysis of transport rates suggested that the rate of drug transport depends on both the affinity of Pgp for substrate and protein conformational changes. Transport rates did not appear to be limited exclusively by the rate of ATP hydrolysis and may be partially controlled by the rate of drug dissociation.

Multidrug resistance-associated protein 4 (MRP4, also known as Abcc4) regulates intracellular levels of cAMP and cGMP in arterial SMCs. Here, we report our studies of the role of MRP4 in the development and progression of pulmonary arterial hypertension (PAH), a severe vascular disease characterized by chronically elevated pulmonary artery pressure and accompanied by remodeling of the small pulmonary arteries as a prelude to right heart failure and premature death. MRP4 expression was increased in pulmonary arteries from patients with idiopathic PAH as well as in WT mice exposed to hypoxic conditions. Consistent with a pathogenic role for MRP4 in PAH, WT mice exposed to hypoxia for 3 weeks showed reversal of hypoxic pulmonary hypertension (PH) following oral administration of the MRP4 inhibitor MK571, and Mrp4-/- mice were protected from hypoxic PH. Inhibition of MRP4 in vitro was accompanied by increased intracellular cAMP and cGMP levels and PKA and PKG activities, implicating cyclic nucleotide-related signaling pathways in the mechanism underlying the protective effects of MRP4 inhibition. Our data suggest that MRP4 could represent a potential target for therapeutic intervention in PAH.

According to published in vitro studies, cytochrome P450 3A4 catalyzes montelukast 21-hydroxylation (M5 formation), whereas CYP2C9 catalyzes 36-hydroxylation (M6), the primary step in the main metabolic pathway of montelukast. However, montelukast is a selective competitive CYP2C8 inhibitor, and our recent in vivo studies suggest that CYP2C8 is involved in its metabolism. We therefore reevaluated the contributions of different cytochrome P450 (P450) enzymes, particularly that of CYP2C8, to the hepatic microsomal metabolism of montelukast using clinically relevant substrate concentrations in vitro. The effects of P450 isoform inhibitors on montelukast metabolism were examined using pooled human liver microsomes, and montelukast oxidations by human recombinant CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were investigated. The results verified the central role of CYP3A4 in M5 formation. The CYP2C8 inhibitors gemfibrozil 1-O-β glucuronide and trimethoprim inhibited the depletion of 0.02 μM montelukast and formation of M6 from 0.05 μM montelukast more potently than did the CYP2C9 inhibitor sulfaphenazole. Likewise, recombinant CYP2C8 catalyzed montelukast depletion and M6 formation at a 6 times higher intrinsic clearance than did CYP2C9, whereas other P450 isoforms produced no M6. On the basis of depletion of 0.02 μM montelukast, CYP2C8 was estimated to account for 72% of the oxidative metabolism of montelukast in vivo, with a 16% contribution for CYP3A4 and 12% for CYP2C9. Moreover, CYP2C8 catalyzed the further metabolism of M6 more actively than did any other P450. In conclusion, CYP2C8 plays a major role in the main metabolic pathway of montelukast at clinically relevant montelukast concentrations in vitro.

Leukotrienes generated by 5-lipoxygenase (5-LOX)-catalyzed reaction are key regulators of inflammation. In ionophore-stimulated (A23187; 1-2.5 μM) human blood neutrophils or differentiated HL-60 cells, vitamin E forms differentially inhibited leukotriene B(4) (LTB(4)) with an IC(50) of 5-20 μM for γ-tocopherol, δ-tocopherol (δT), and γ-tocotrienol, but a much higher IC(50) for α-tocopherol. 13'-Carboxychromanol, a long-chain metabolite of δT, suppressed neutrophil- and HL-60 cell-generated LTB(4) with an IC(50) of 4-7 μM and potently inhibited human recombinant 5-LOX activity with an IC(50) of 0.5-1 μM. In contrast, vitamin E forms had no effect on human 5-LOX activity but impaired ionophore-induced intracellular calcium increase and calcium influx as well as the subsequent signaling including ERK1/2 phosphorylation and 5-LOX translocation from cytosol to the nucleus, a key event for 5-LOX activation. Further investigation showed that δT suppressed cytosolic Ca(2+) increase and/or LTB(4) formation triggered by ionophores, sphingosine 1-phosphate, and lysophosphatidic acid but not by fMLP or thapsigargin, whereas 13'-carboxychromanol decreased cellular production of LTB(4) regardless of different stimuli, consistent with its strong inhibition of the 5-LOX activity. These observations suggest that δT does not likely affect fMLP receptor-mediated signaling or store depletion-induced calcium entry. Instead, we found that δT prevented ionophore-caused cytoplasmic membrane disruption, which may account for its blocking of calcium influx. These activities by vitamin E forms and long-chain carboxychromanol provide potential molecular bases for the differential anti-inflammatory effects of vitamin E forms in vivo.

Objective and Design: To investigate the sensitizing effects of the cysteinyl leukotrienes (CysLTs) C(4) and D(4) on the proinflammatory responses of chemoattractant-activated human neutrophils in vitro., Materials: Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers., Treatment: Cells were exposed to LTC(4) and LTD(4) (50-300 nM) prior to activation with 1 microM of N-formyl-L-methionyl- L-leucyl-L-phenylalanine (fMLF)., Methods: A fura-2/AM-based spectrofluorimetric procedure, lucigenin-enhanced chemiluminescence (LECL), a colourimetric method and an ELISA procedure, were used to measure Ca(2+) mobilization, superoxide production, elastase and MMP-8 release respectively following activation of LTC(4)/ D(4)-primed neutrophils with fMLF. Superoxide generation was also measured in the presence and absence of the CysLT receptor 1 antagonist, montelukast (100 nM)., Results: Exposure of neutrophils to either LTC(4) or LTD(4) alone had modest effects on Ca(2+) mobilization, while superoxide generation and elastase release were unaffected. However, relative to the responses of neutrophils activated with fMLF in the absence of the CysLTs, pre-treatment of the cells with either LTC(4)or LTD(4) resulted in significant, augmentation of fMLF-activated elastase and MMP-8 release and superoxide generation, which was attenuated by montelukast., Conclusion: These previously undocumented sensitizing interactions of LTs C(4) and D(4) with neutrophils may contribute to the activation of these cells in acute and chronic inflammation of both atopic and non-atopic aetiology, while identifying a role for montelukast in regulating neutrophil reactivity.

The anti-inflammatory effects of pranlukast, an antagonist of cysteinyl leukotriene receptor 1, may be rendered not only by antileukotriene activity but also by other pharmacological activities. Previous studies indicate that pranlukast reduces ischemic tissue injury partially through decreasing vascular permeability, but its effect on ischemic injury in endothelial cells is not known. Thus, in this study, we investigated the effect of pranlukast on ischemia-like injury induced by oxygen-glucose deprivation (OGD) in EA.hy926 cells, a human endothelial cell line, and the possible mechanisms. We found that cell viability was reduced, lactate dehydrogenase release was increased 4-8 hours after OGD, and necrosis was induced 8 hours after OGD. Production of reactive oxygen species (ROS) increased by 211%, 176%, and 128%, respectively, 0.5, 1, and 2 hours after OGD. Nuclear factor-kappaB (NF-kappaB) was translocated to the nuclei 4-8 hours after OGD. Pranlukast ameliorated the reduced viability, the increased lactate dehydrogenase release, and necrosis after OGD. It also reduced ROS production and inhibited NF-kappaB nuclear translocation after OGD. The ROS scavenger, edaravone, inhibited OGD-induced nuclear translocation of NF-kappaB as well. Edaravone and pyrrolidine dithiocarbamate (a specific NF-kappaB inhibitor) protected endothelial cells from the OGD-induced injury. However, zileuton, a 5-lipoxygenase inhibitor, did not affect the cell injury, ROS production, and NF-kappaB nuclear translocation after OGD. The exogenous leukotriene D4 did not induce cell injury, ROS production, and NF-kappaB translocation. Thus, we conclude that pranlukast protects endothelial cells from ischemia-like injury via decreasing ROS production and inhibiting NF-kappaB activation, which is leukotriene independent.

Background and Purpose: KP-496 is a novel dual antagonist for cysteinyl leukotriene receptor 1 (CysLT(1)) and thromboxane A(2) (TXA(2)) receptor (TP). The aim of this study was to evaluate the pharmacological profile of inhaled KP-496 and its effects on airway obstruction., Experimental Approach: Antagonist activities of inhaled KP-496 were investigated using bronchoconstriction induced in guinea pigs by LTD(4) or U46619, a stable TXA(2) mimetic. Guinea pigs sensitized with injections of ovalbumin were used to assess the effects of inhaled KP-496 on bronchoconstriction induced by antigen (i.v.). Another set of guinea pigs were sensitized and challenged with ovalbumin by inhalation and the effects of inhaled KP-496 on immediate and late airway responses and airway hyperresponsiveness were investigated., Key Results: KP-496 significantly inhibited LTD(4)- and U46619-induced bronchoconstriction in a dose-dependent manner. The inhibitory effects of KP-496 (1%) were comparable to those of montelukast (a CysLT(1) antagonist, p.o., 0.3 mg kg(-1)) or seratrodast (a TP antagonist, p.o., 3 mg kg(-1)). KP-496 (1%) and oral co-administration of montelukast (10 mg kg(-1)) and seratrodast (20 mg kg(-1)) significantly inhibited antigen-induced bronchoconstriction, whereas administration of montelukast or seratrodast separately did not inhibit antigen-induced bronchoconstriction. KP-496 exhibited dose-dependent and significant inhibitory effects on the immediate and late airway responses and airway hyperresponsiveness following antigen challenge., Conclusions and Implications: KP-496 exerts effects in guinea pigs which could be beneficial in asthma. These effects of KP-496 were greater than those of a CysLT(1) antagonist or a TP antagonist, in preventing antigen-induced airway obstruction.

Churg-Strauss syndrome (CSS) is a rare vasculitic disorder that generally occurs in patients with bronchial asthma. CSS is being increasingly recognized in asthmatic patients treated with leukotriene receptor antagonists. However, the nature of this relationship remains to be elucidated. The present report describes three asthmatic patients who developed clinical manifestations highly suggestive of CSS, although one patient lacked the presence of eosinophilia. The patient, however, exhibited biopsy-proven cutaneous necrotizing vasculitis, which improved after withdrawal of montelukast. The second patient presented with systemic constitutional signs including fever, malaise, arthralgias, clinical jaundice, peripheral blood eosinophilia, and biopsy-proven eosinophilic hepatitis. The third patient also had circulating eosinophilia, scleritis, and arthritis. All patients improved after discontinuation of the leukotriene receptor antagonist (montelukast).

Leukotriene (LT) B4 is a powerful chemotactic and immune modulating agent that signals via two receptors denoted BLT1 and BLT2. Here we report that BLT1 and BLT2 are expressed at low levels in an apparently silent state in human umbilical vein endothelial cells (HUVEC). However, treatment with LPS leads to a >10 fold increase in the levels of BLT1 mRNA without any significant effects on BLT2 mRNA. In parallel, LPS also increases the amounts of BLT1 protein. Tumor necrosis factor-alpha (TNF-alpha) increases the expression of BLT2 mRNA approximately 6 times above basal levels with only a modest increase in BLT1 mRNA. Interleukin-1beta causes variable and parallel increases of both BLT1 and BLT2 mRNA. The natural ligand LTB4 also increases BLT1, but not BLT2, mRNA and protein expression. Along with the induction of BLT1 and/or BLT2, HUVEC acquire the capacity to respond to LTB4 with increased levels of intracellular calcium and these signals can be blocked by isotype selective BLT antagonists, CP-105696 and LY-255283. In addition, treatment of HUVEC with LTB4 causes increased release of both nitrite, presumably reflecting nitric oxide (NO), and monocyte chemoattractant protein-1. Our data indicate that expression of functional BLT receptors may occur at the surface of endothelial cells in response to LPS, cytokines, and ligand, which in turn may have functional consequences during the early vascular responses to inflammation. Moreover, the results point to BLT receptors as potential targets for pharmacological intervention in LT-dependent inflammatory diseases such as asthma, rheumatoid arthritis, and arteriosclerosis.

Background: Evidence suggests that small airways contribute to clinically significant processes in asthma. Cysteinyl leukotrienes (CysLTs) are considered to be pivotal mediators in the pathogenesis of asthma. Montelukast (MK), a specific CysLT1 receptor antagonist, is metabolized in two main hydroxylated metabolites (termed M5 and M6, respectively)., Objectives: The aims of this study were to compare the responsiveness of small and large human bronchi to the three CysLTs, to evaluate the antagonist activity of MK, M5 and M6 in these preparations of human bronchi, and to characterize the CysLT receptors involved in the contractile response., Methods and Results: In isolated small bronchus (i.d. 0.5-2 mm), the potencies (-log molar EC50) of LTC4, LTD4 and LTE4 were 9.3 (n=11), 9.1 (n=30) and 8.4 (n=14), respectively. The three CysLTs were about 30-fold more potent in small bronchi than in larger bronchi (i.d. 4-6 mm). In small bronchi, MK significantly shifted to the right the CysLT concentration-effect curves with pA2 values against LTC4, LTD4 and LTE4 of 9.1 (n=3), 9.0 (n=11) and 8.7 (n=5), respectively. The antagonist potencies of M6 and M5 were similar to MK and fivefold lower, respectively. A similar activity of MK against the three CysLTs suggested that CysLT1 receptors are involved in the contraction of human bronchus. Analysis by RT-PCR also indicated that human bronchus mainly expressed CysLT1 receptors., Conclusion: MK exerts a potent antagonist activity against the particularly potent constricting effects of CysLTs in isolated human small bronchi, which only expressed the CysLT1 receptor subtype. The metabolites of MK are also potent in vitro antagonists, but may not participate in the therapeutic activity of MK due to their low plasma concentrations in patients treated with the recommended dose of MK.

Leukotriene antagonists block the proinflammatory actions of leukotrienes (LT) and have been introduced as new treatments for asthma. Conventional therapy with glucocorticosteroids does not inhibit the biosynthesis of leukotrienes. We therefore tested whether addition of the leukotriene receptor antagonist montelukast was of therapeutic benefit in a group of aspirin-intolerant patients with asthma of whom 90% already were treated with moderate to high doses of glucocorticosteroids. Under double-blind conditions, 80 aspirin-intolerant patients with asthma were randomized to receive 4 wk oral treatment of either 10 mg of montelukast or placebo once daily at bedtime. Pulmonary function was measured as forced expiratory volume in 1 s (FEV(1)) once a week in the clinic and daily as morning and evening peak expiratory flow rate (PEFR). Asthma symptoms and use of rescue bronchodilator were also recorded daily. Asthma specific quality of life (QoL) was assessed before and after the treatments. The group receiving montelukast showed a remarkable improvement of their asthma, whereas the group given placebo showed no change. Thus, from equal baseline values, the mean difference between the groups over the 4-wk treatment period was 10.2% for FEV(1) and 28.0 L for morning PEFR (p for both < 0.001). The improved pulmonary function in the group receiving montelukast occurred at the same time as 27% less bronchodilator was used (p < 0.05), and it was associated with fewer asthma symptoms than in the group given placebo, including 1.3 nights more of sleep per week and 54% fewer asthma exacerbations (p < 0.05). There was also an improvement in asthma-specific QoL (p < 0.05). The therapeutic response to montelukast was consistent across patients with different baseline characteristics and did not correlate with baseline urinary LTE(4). Addition of a leukotriene receptor antagonist such as montelukast improves asthma in aspirin-intolerant patients over and above what can be achieved by glucocorticosteroids.

The leukotriene antagonists have represented the first novelty in the treatment of asthma over the last 20 years. Their easy oral administration that favours a suitable completion of the treatment, especially on children, together with the proven efficiency and their apparent safety, gave rise to the fact in 1999 the asthma committee of the Spanish Society of Clinical Immunology and Paediatric Allergology (SSCIPA) in the bronchial asthma treatment guide that was presented a year ago during the XXIII National Congress of our Society, considered the antileukotrienes, together with the chromones, as important medicines for stage 2 asthma (frequent episodes) and as useful medicines to reduce the need to take corticoides in cases of persistent asthma. A year later, in our study we proposed to value too suitability or non suitability of this positioning, in the light of the new investigations published and from our own experience. We believe that the bibliography available to date shows that the agonist cysteinyl-leukotrienes are anti-inflammatory medicines, that have an efficiency similar to that of low doses of inhaled corticoids, equally efficient in controlling bronchospasm-induced exercise and they have a good safety profile. We present two studies that are the result of our own experience, one carried out on children with persistent asthma and the other on children with frequent episodic asthma, from which we can apparently deduce that both nedocromil (2 inhalations every 8 hours) and montelukast (5 mg every 24 hours) are medicines that have similar efficacy in the control of asthma. We conclude that the antileukotrienes, together with the chromones, should be seen as important medicines when treating infant asthma.

Background: The ATP-dependent drug transporter proteins, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) are known to be involved in drug efflux that reduces drug accumulation and so renders tumor cells resistant to the cytotoxic effects of a number of anticancer agents. The ways in which these transporters bring about drug expulsion are not fully explained and may involve intracellular factors as well. Thus detailed evidence may be difficult to obtain from studies on intact cells., Material and Methods: Inside-out plasma membrane vesicles prepared from multidrug-resistant cells expressing high amounts of Pgp or of MRP provide a simpler system for investigating the interactions of putative substrates and resistance modifiers with the transport process. We consider here some aspects of the accumulation of radiolabelled vincristine and of dinitrophenol glutathione conjugate by these vesicles and demonstrate the usefulness of this approach for determining whether potential inhibitors have their effects on transport at the cell membrane or by more indirect means., Conclusions: We show that information gained from analysis of the ATP-dependence, time course and osmotic sensitivity of accumulation is helpful in distinguishing between transport and changes in binding. We have also used the technique to demonstrate the effects of the resistance modifier, XR-9051 on Pgp-mediated transport and to explore interactions of MK571, indomethacin and ethacrynic acid with MRP.

1. The fate of [14C]CP-195,543, a novel leukotriene B4 receptor antagonist, was studied following oral administration to the Long-Evans rat and Cynomolgus monkey. 2. Most of the radioactivity was primarily excreted in the faeces, and urine was a minor route of excretion. 3. CP-195,543 was extensively metabolized in the two species, primarily by two metabolic pathways: glucuronidation of unchanged CP-195,543 and oxidative metabolism, presumably by cytochrome P450. 4. The sites of glucuronidation were the carboxylic acid moiety and the hydroxy group. The ester glucuronide was the predominant glucuronide conjugate detected in the rat, whereas the monkey generated the ether as well as the ester glucuronide. 5. The structures of oxidative metabolites were elucidated using mass spectrometry (in the positive- and negative-ion mode) and 1H-NMR. The sites of hydroxylation were the benzylic group and the 3-position of the benzopyran ring. 6. This study has indicated that CP-195,543 was mainly eliminated by Phase II metabolism in both species.

The binding of iralukast to plasma (or serum) proteins and to erythrocytes was studied in vitro, at +37 degrees C, using the erythrocyte partitioning method (EPM) and/or ultrafiltration (UF) with 14C-labelled iralukast. Iralukast was highly bound in human and animal serum (>99%). Similar bound fraction values were obtained with the two methods: in whole human plasma (or serum) 99.8% (EPM) and 99.9% (UF), in albumin solution 99.8% (EPM and UF), in high density lipoprotein solution 97.3% (EPM) and 98.3% (UF), and in low density lipoprotein solution 97.2% (EPM) and 98.8% (UF). Moreover, the erythrocyte partitioning method allowed the evaluation of other binding parameters. The binding capacity (l/micromol) of proteins equalled 35 for low density lipoproteins, 3.6 for high density lipoproteins, 1.0 for albumin, 0.78 for alpha-1-acid glycoprotein, and 0.03 for gamma globulins. In whole blood, iralukast was distributed between plasma and erythrocytes in the proportion (%) 90/10. At physiological protein concentrations, iralukast was primarily bound to albumin (79%).

Arachidonic acid metabolites mediate inflammatory responses at a cellular level. The affinity of the newly synthesized compound YM158, 3-[(4-tert-butylthiazol-2-yl)methoxy]-5'-[3-(4-chlorobenzenesuf onyl)propyl-2'-(1H-tetrazol-5-ylmethoxy)benzanilide monosodium salt monohydrate, for leukotriene D4 and thromboxane A2 receptors was examined in radioligand binding assays. YM158 inhibited [3H]leukotriene D4 and [3H]U46619 (9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F2alpha) binding to guinea pig lung membrane preparations, with Ki values of 0.64+/-0.06 nM for leukotriene D4 and 5.0+/-0.88 nM for thromboxane A2 receptors. The Hill coefficients (nH) did not significantly differ from unity, indicating that this antagonism is competitive. In contrast, YM158 showed no affinity for several other receptors, including neurotransmitter-related (alpha1-, alpha2-, beta-adrenoceptors, histamine, 5-HT, muscarinic, sigma), C5a, opioid, Ca2+ channel, K+ channel, protein kinase C, bradykinin, endothelin, neurokinin and platelet activating factor receptors. These studies indicate that YM158 is a highly selective dual antagonist for leukotriene D4 and thromboxane A2 receptors, and this has potential clinical and research applications.

By addressing the issues of potency and metabolism in 3, a new series of LTD4 antagonists represented by (+)-26 was developed which is equipotent to clinical LTD4 antagonists Zafirlukast (1) and Pranlukast (2).