Characterization of the leukotriene D4 receptor in guinea-pig lung

The effects of monovalent, divalent cations, buffere species and pH dependency on [3H]leukotriene D4 binding to the receptor have been characterized in vitro by using a radioligand binding assay. It was found that Ca2+, Mg2+, Co2+ and Mn2+ enhanced the specific binding. High concentrations of NaCl (150-300 mM) inhibited the specific binding to the receptor. The specific binding was also found to be higher in Pipes buffer (pH 6.5) than in Tris, Hepes and phosphate buffer at pH 7.0-8.0. Conversion of [3H]leukotriene D4 was minimized by inclusion of 1 mM cysteine, glycine in the incubation buffer and maintaining the temperature at 22 degrees C. Under the conditions employed, the dissociation constant (KD) and the receptor density (Bmax) were calculated to be 1.8 +/- 0.9 nM and 2100 +/- 375 fmol/mg protein respectively. The leukotriene antagonist FPL 55712, agonist 5R, 6S-LTD4 and LTE4 competed with the [3H]LTD4 binding to the receptor. Prostaglandins, alpha-, beta-adrenergic and dopaminergic receptor agonists and antagonists did not compete significantly.     

Characterization of the leukotriene D4 receptor in dimethylsulphoxide-differentiated U937 cells: comparison with the leukotriene D4 receptor in human lung and guinea-pig lung

The leukotriene D₄ receptor has been fully characterized by radioligand binding in membrane preparations from dimethyl sulphoxide-differentiated U937 cells, a human monocyte leukemia cell line, and, in parallel experiments, compared with leukotriene D₄ receptor found in human lung and guinea-pig lung preparations. [³H]Leukotriene D₄ specific binding in differentiated U937 cell membranes is of high affinity (K_D = 0.35 nM), saturable (B_max = 287 fmol/mg protein), with differentiation resulting in a 3–5-fold increase in the number of detectable binding sites. [³H]Leukotriene D₄-specific binding in differentiated U937 cell membranes displays several features of G-protein-cupled receptors, being inhibited by GTP analogues and sodium ions, but increased by divalent cations. These characteristics are shared with [³H]leukotriene D₄-specific binding in human and guinea-pig lung preparations. However, differences between these leukotriene D₄ receptor types were observed. [³H]Leukotriene D₄ equilibrium binding to differentiated U937 cell membranes could be dissociated to non-specific binding levels by 1000-fold excess of competing ligand, whereas binding to guinea-pig lung membranes was only partially dissociated under these conditions. In addition, differences in potency were demonstrated in competition studies with leukotriene E₄ and leukotriene C₄, although leukotriene D₄ and the leukotriene D₄-receptor antagonists MK-571 and ICI 204,219 were equipotent in competing for [³H]leukotriene D₄-specific binding in all three membranes preparations. In conclusion, the leukotriene D₄ receptor in differentiated U937 cell membranes resembles that in human lung, validating the use of this cell line as a suitable source of receptor in the development of potent specific antagonists.     

Inhibition of allergen-induced bronchoconstriction in hyperreactive rats as a model for testing 5-lipoxygenase inhibitors and leukotriene D4 receptor antagonists

Sensitized inbred hyperreactive rats showed reproducible episodes of dyspnea when exposed to aerosols of antigen. Following inhibition of the serotonin component of the response by pretreatment with methysergide, the model was shown to be useful for studying the oral activity of compounds that affect the production or action of leukotrienes. This was shown through inhibition of the duration of dyspnea by two selective 5-lipoxygenase inhibitors, L-651,392 and L-615,919, and two selective leukotriene D4 receptor antagonists, L-647,438 and L-649,923. Selectivity of the compounds could be demonstrated by reducing inhibition of the antigen response in the absence of methysergide and failure to inhibit serotonin-induced dyspnea. It is concluded that the model provides a reproducible method for screening large numbers of leukotriene inhibitors and antagonists and gives a measurement of their duration of biological activity.     

Post soluble binding to the leukotriene D4 receptor from guinea pig lung membranes

Guinea pig lung membranes were extracted with 1% digitonin and yielded a preparation that contained soluble leukotriene D4 (LTD4) receptor. Specific binding of the high affinity radiolabeled receptor antagonist [3H]ICI-198615 to the soluble LTD4 receptor was time dependent and reversible. The dissociation constant (Kd) and the density (Bmax) of [3H]ICI-198615 binding to the soluble LTD4 receptor was 0.2 +/- 0.08 nM and 380 +/- 40 fmol/mg of protein, respectively. Radioligand competition studies showed several classes of structurally diverse, functionally defined, receptor antagonists competed with [3H]ICI-198615 binding to the soluble receptor. The rank order of potency and specificity of these antagonists in binding to the soluble receptor were equivalent to those determined from the membrane-bound receptor binding assay and from the smooth muscle contraction assay. Binding of LTD4 to the soluble receptor was observed, in the competition assay, only in the low affinity state (Ki = 2 microM). Size-exclusion chromatography of the soluble LTD4 receptor showed that the apparent molecular weight of the LTD4 receptor in digitonin micelle was approximately 300,000.     

SK&F S-106203 inhibits leukotriene C4, leukotriene D4 and leukotriene E4 vasopressor responses in the conscious rat.

1. The purpose of these experiments was to investigate the effects of the selective peptidoleukotriene receptor antagonist, SK&F S-106203, on leukotriene C4 (LTC4), LTD4 and LTE4 vasopressor responses in the conscious, normotensive rat. SK&F S-106203 was administered as a bolus followed by a continuous infusion in order to provide information on the relationship between antagonism of leukotriene responses and steady-state plasma concentrations. 2. Infusion of SK&F S-106203 at doses of 0.2 mgkg-1 + 1 mgkg-1 h-1, 1 mgkg-1 + 3 mgkg- h-1 or 2 mgkg-1 + 10 mgkg-1 h-1 produced dose-dependent steady-state plasma drug concentrations of 1.0, 3.2 and 23.8 micrograms ml-1, respectively. Plasma SK&F S-106203 concentrations appeared to increase in a linear fashion at doses of 1 and 3 mgkg-1 h-1; at the highest dose the increment in plasma drug concentrations (i.e., 7-8 fold) was greater than the increment in dose (i.e., 3 fold), suggesting saturation of the primary clearance mechanism(s) at this dose. 3. SK&F S-106203 (2 mgkg-1 + 10 mgkg-1 h-1) had no effect on noradrenaline-, vasopressin-, isoprenaline-, or U 46619-induced responses. 4. SK&F S-106203 produced dose-dependent rightward shifts in the LTC4 and LTE4 dose-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)     

The signal transduction system of the leukotriene D4 receptor

During the past several years, substantial progress in understanding the receptors and signal transduction processes for peptidyl leukotrienes has been reported. Receptors have been identified and characterized, the major steps in the signal transduction pathway have been described, and the genetic and epigenetic regulatory processes have been characterized. Very recent studies have defined the mechanisms by which LTE4 acts as a partial agonist at the LTD4 receptor. The cloning of the genes for the proteins involved in the major steps of the signalling process has also been initiated. Stanley Crooke and co-authors summarize this recent progress and present their current notions about the LTD4 receptor signalling process.     

ICI 198615 is an antagonist of leukotriene C4, leukotriene D4 and leukotriene E4 vasopressor responses in the conscious rat

The purpose of these studies was to evaluate the effects of the peptidoleukotriene (LT) receptor antagonist, ICI 198615, on the vasopressor responses produced by LTC4, LTD4 and LTE4. Conscious, normotensive rats were prepared with arterial and venous catheters for measurement of changes in arterial blood pressure and administration of drugs, respectively. Complete dose-response curves were first generated to LTC4, LTD4 and LTE4: those agents produced dose-dependent increases in arterial blood pressure, with ED20 values (i.e. dose to increase blood pressure 20 mm Hg) of 1.7 +/- 0.2, 2.1 +/- 0.2 and 19.8 +/- 3.7 nmol/kg i.v., respectively. ICI 198615 (intravenous bolus followed by a continuous infusion) produced dose-dependent, parallel shifts to the right in the LTC4 dose-response curve. At doses of 0.2 mg/kg + 1 mg/kg/h, 1 mg/kg + 3 mg/kg/h or 2 mg/kg + 10 mg/kg/h, ICI 198615 produced dose ratios of 4.5, 17.1 and 50.0, respectively. Against LTD4 responses, ICI 198615 at a dose of 0.1 mg/kg + 0.3 mg/kg/h produced a dose ratio of 3.4, whereas at doses of 0.2 mg/kg + 1 mg/kg/h, 1 mg/kg + 3 mg/kg/h or 2 mg/kg + 10 mg/kg/h ICI 198615 produced dose ratios of 16.3, 24.9 and 16.2, respectively. The difference in the dose ratios between these three groups was not statistically significant (p greater than 0.05). However, a dose of 10 mg/kg + 30 mg/kg/h produced a dose ratio of greater than 100. Against LTE4 responses, ICI 198615 at doses of 0.2 mg/kg + 1 mg/kg/h or 1 mg/kg + 3 mg/kg/h produced dose ratios of 4.1 and 11.3, respectively. The similarity in the LTD4 dose ratios despite a 3- or 10-fold increase in the dose of ICI 198615 suggests the existence of high- and low-affinity LTD4 receptor sites, whereas the responses to LTC4 and LTE4 appeared to be mediated via a single receptor population. These results indicate that ICI 198615 is a potent and competitive antagonist of LTC4, LTD4 and LTE4 vascular responses in the rat.     

Leukotriene receptor antagonists. III. Pharmacological and chemical studies with LY137617, a leukotriene D4 and leukotriene E4 receptor antagonist

A series of chlorophenoxyalkyl acids were prepared and evaluated as pharmacological antagonists of leukotriene D4. Structure-activity relationship studies pointed to LY137617 as a compound with possible therapeutic value. In experiments on isolated smooth muscles from the guinea-pig, this agent was a selective and moderately potent antagonist of leukotriene D4 and also leukotriene E4. Other in vitro experiments demonstrated that LY137617 had a high affinity for protein molecules. This was reflected in vivo as a weaker than expected efficacy against leukotriene-mediated events, limiting the compound's potential as a clinical candidate. Nevertheless, agents of this type will prove useful in the laboratory to increase knowledge of leukotriene receptor-antagonist interactions.     

Solubilization of [3H]leukotriene D4 receptor complex from guinea pig lung membranes

Guinea pig lung membrane leukotriene D4 (LTD4) receptors were prelabeled with [3H]LTD4 and solubilized using digitonin, 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulfonate, and other non-ionic, zwitterionic, and ionic detergents. [3H]LTD4 remains tightly associated with the receptor complex in the digitonin solubilized state. The dissociation rate of [3]LTD4 from the soluble receptor complex was increased in the presence of guanine nucleotides and sodium ions in a manner similar to that observed for the receptors in the membrane-bound state. The soluble [3H]LTD4 receptor complex was retained on wheat germ lectin affinity columns and destabilized by heat (40 +/- 4 degrees), trypsin, and chymotrypsin treatment, suggesting that the receptor is a glycoprotein. Size exclusion high pressure liquid chromatography of the soluble receptor complex showed that an apparent molecular weight of the soluble receptor complex, in the presence of digitonin, is in the range of 240,000-500,000. An approximately 20-fold enrichment of receptor-radioligand complex was achieved by passing the solubilized LTD4 receptor preparation successively through size exclusion and wheat germ lectin chromatography columns. These data provide the first step toward the purification and chemical characterization of LTD4 receptors.     

Development of a novel series of (2-quinolinylmethoxy)phenyl-containing compounds as high-affinity leukotriene D4 receptor antagonists. 4. Addition of chromone moiety enhances leukotriene D4 receptor binding affinity.

The combination of the benzopyran-4-one ring, a moiety found in the prototype leukotriene antagonist, FPL 55,712, with the (2-quinolinylmethoxy)phenyl group led to a significant increase in leukotriene receptor binding affinity. This modification resulted in a 10,000-fold improvement in binding affinity compared to FPL 55,712. Compound 7 (RG 12553), with a Ki value of 0.1 nM, has higher affinity than the natural agonist LTD4 and is one of the most potent LTD4 antagonists reported. The structure-activity relationships of this series of potent leukotriene antagonists are discussed.     

Development of a series of phenyltetrazole leukotriene D4 (LTD4) receptor antagonists.

A hypothetical model for receptor binding of leukotriene D4 (LTD4) was deduced from conformational analysis of LTD4 and from the structure-activity relationships (SAR) of known LTD4 receptor antagonists. A new structural series of LTD4 receptor antagonists exemplified by 5-[4-(4-phenylbutoxy)phenyl]-2-[4-(tetrazol-5-yl)butyl]-2H-t etrazole was designed in which a phenyltetrazole moiety was incorporated as a receptor binding equivalent of the triene unit of LTD4. A number of these phenyltetrazoles were prepared and found to possess LTD4 receptor antagonist activity. The structure-activity relationship (SAR) of this series is described.     

SKF 104353, a high affinity antagonist for human and guinea pig lung leukotriene D4 receptor, blocked phosphatidylinositol metabolism and thromboxane synthesis induced by leukotriene D4

SKF 104353 (2(S)-hydroxyl-3(R)-carboxyethylthio)-3-[2-(8-phenyloctyl) phenyl] propanoic acid) is a synthetic structural analog of leukotrienes D4 and E4 (LTD4, LTE4). This compound binds to guinea pig and human lung LTD4 receptors with affinities (Ki) of 5 +/- 2 and 10 +/- 3 nM, respectively. The Ki values of a reference compound, FPL 55712, were 2200 and 4500 nM, respectively, approximately 400- and 500-fold less effective than SKF 104353. LTD4- and LTE4-induced biosynthesis of thromboxane B2 has been shown to be mediated by LTD4 receptors in guinea pig lung in vitro. SKF 104353 did not induce synthesis of TxB2 in this system at concentrations of 1-20 microM. When SKF 104353 and increasing concentrations of LTD4 were incubated with guinea pig lung, the dose response curve of LTD4-induced TxB2 biosynthesis was shifted to the right with a -log[KB] = 8.4 +/- 0.2. LTD4-induced phosphatidylinositol (PI) hydrolysis in guinea pig lung has been shown to be the major signal transduction mechanism. In this system, SKF 104353 (1-20 microM) did not promote PI hydrolysis. Pretreatment of the [3H]myo-inositol-labeled guinea pig lung with SKF 104353 shifted the LTD4-induced PI hydrolysis dose response curve to the right, indicating that SKF 104353 inhibited LTD4 receptor-mediated intracellular second messenger formation. These results demonstrate that SKF 104353 is a high affinity, specific LTD4 receptor antagonist. It inhibited LTD4-induced PI hydrolysis and TxB2 biosynthesis in guinea pig lung. SKF 104353 may prove to be an important research tool for research on the activities of leukotrienes and of value therapeutically in the treatment of leukotriene-mediated diseases.     

Inhibition by ibudilast of leukotriene D4-induced formation of inositol phosphates in guinea-pig lung.

1. The effects of a novel anti-asthmatic drug, 3-isobutyryl-2-isopropylpyrazolo [1,5-a]pyridine (ibudilast, KC-404) on leukotriene D4 (LTD4)-induced formation of inositol phosphates were studied in slices of guinea-pig lung and hippocampus. 2. In guinea-pig lung, ibudilast inhibited LTD4 (0.01-1 microM)-induced formation of inositol monophosphate (IP1) in a concentration-dependent manner (IC50 = 10 microM) without affecting LTD4 receptor binding. 3. Ibudilast (10 microM) inhibited histamine (0.1-1 mM)-induced formation of IP1 in guinea-pig lung slices but not in hippocampal slices. 4. Inhibition of agonist-induced formation of IP1 by ibudilast was non-competitive. 5. Ibudilast had no effect on either GTP- or calcium-stimulated phosphatidylinositol specific-phospholipase C activity of lung membranes. 6. These results suggest that ibudilast has no direct effect on LTD4 receptors, GTP binding proteins (G proteins) or phospholipase C, but inhibits inositol phosphate formation, possibly by interfering with the coupling between receptors and G proteins.     

In vitro and in vivo biotransformations of the potent leukotriene D4 antagonist verlukast in the rat.

Verlukast, (S)3-((((3-(2-(7-chloroquinolin-2-yl)-(E)-ethenyl)phenyl)- 3-dimethylamino-3-oxopropylthio)methyl)thio)propionic acid, formerly known as MK-679, is a potent leukotriene D4 antagonist. Verlukast was incubated with rat liver microsomes under oxidative conditions to generate five metabolites, which were identified as the four possible isomeric monosulfoxides (M1-M4), and the N-hydroxymethyl amide (M5). This latter metabolite loses the elements of formaldehyde to yield the N-monomethyl amide (M6). These metabolites were isolated from a large microsomal incubation and were characterized by UV, 1H-NMR, and fast atom bombardment-MS. These data were identical to those obtained from synthetically prepared standards. Microsomal incubations of verlukast supplemented with UDP-glucuronic acid yielded the acyl glucuronide metabolite (M7), which was isolated and characterized by UV, 1H-NMR, and fast atom bombardment-M5. Verlukast was regenerated from M7 upon treatment with either beta-glucuronidase or strong aqueous base (pH greater than 11). The metabolites described above were all detected in bile collected from a rat dosed with verlukast.     

Oral pharmacokinetics and food interaction of the leukotriene D4 receptor antagonist verlukast

The influence of dose and food on the pharmacokinetic profile of orally administered verlukast, a leukotriene D4 receptor antagonist, was investigated in 12 healthy male volunteers. This was an open, four-period, single dose, randomised, crossover design including the following doses: one 75 mg tablet, one 250 mg tablet, 500 mg (2 x 250 mg) and 500 mg immediately following a standard meal. There were dose-related increases in the AUC, although after 500 mg verlukast this was disproportionately greater than with 75 mg (P = 0.04). Similarly, there were dose-related increases in C(max). No differences were observed in the t(max) between treatments. With respect to food, there was a 22% decrease (P = 0.02) in C(max) after 500 mg, and the AUC was 13% less (P = 0.052). The differences in the plasma concentration profiles betweeen fasted and fed states are not considered to be of clinical importance.     

Leukotriene receptor antagonists. 4. Synthesis and leukotriene D4/E4 receptor antagonist activity of 4-(alkyl)acetophenone derivatives

Analogues of the leukotriene D4/E4 receptor antagonist LY171883 (1a) were synthesized in which the tetrazole was linked to the hydroxyacetophenone moiety by an all-methylene carbon chain. A key step in the synthesis involved a Wittig olefin-forming reaction between 3-methoxy-2-propylbenzaldehyde and the ylide derived from (4-carboxybutyl)triphenylphosphonium bromide to form the desired carbon chain. A regioselective Fries rearrangement was employed to form the o-hydroxyacetophenone. Compounds in which the tetrazole was separated from the acetophenone by four and five methylene groups were compared to the corresponding derivatives in which an oxygen atom linked the tetrazole chain to the aromatic ring for their ability to antagonize LTD4- or LTE4-induced contractions of the isolated guinea pig ileum. When compared to 1a, the "carba" analogue, 7a, showed nearly identical LTD4 antagonist activity. The LTE4 antagonist activity for these two compounds was also identical. In the shorter chain series, the "carba" analogue, 7b, showed enhanced LTD4 antagonist activity and approximately 10-fold greater LTE4 antagonist activity. These results suggest that the oxygen atom para to the acetyl group of 1a and 1b is not of major importance for association with the LTD4 or LTE4 receptor sites in the guinea pig ileum.     

Characterization and autoradiographic distribution of the beta-adrenergic receptor in the rat lung

An unexpected distribution of the beta-adrenergic receptor in the rat lung was revealed by an autoradiographic technique. [3H]-Dihydroalprenolol binding was stereoselective. L-propranolol was 300 times more potent than D-propranolol in competition experiments. Scatchard analysis revealed a Kd of 1.1 nM and Bmax of 14 fmol/mg wet weight. Relative potencies of beta-adrenergic ligands were: propranolol greater than alprenolol greater than timolol greater than pindolol greater than isoproterenol greater than epinephrine greater than soterenol greater than metoprolol greater than terbutaline greater than norepinephrine. The autoradiograms generated revealed a diffuse pattern with binding always associated with tissue structures. We conclude that beta-adrenergic receptors are extensively distributed in the lung, being present in large and small airways and blood vessels.     

Identification and characterization of leukotriene D4 receptors in adult and fetal human lung

Leukotriene D4 (LTD4) receptors were identified and characterized in adult and fetal human lung membranes. Macroscopically normal adult lung tissue was selected from seventeen surgical biopsy specimens, and twenty-seven fetal lung samples were obtained from therapeutic abortions. Binding assays were performed using pooled adult or fetal human lung membranes at 30 degrees under conditions which prevented metabolism of [3H]LTD4. Specific binding reached equilibrium within 30 min, remained constant for 60 min, was enhanced by Mg2+, and was inhibited by Na+ and guanyl-5'-yl-imidodiphosphate. Computer-assisted analyses of saturation binding data showed a single class of binding sites with similar apparent Kd (0.15 +/- 0.09 and 0.12 +/- 0.003 nM) and Bmax (68 +/- 29 and 62 +/- 14 fmoles/mg protein) values for adult and fetal samples respectively. Competition binding studies with [3H]LTD4 showed the same rank order potency for adult and fetal lung receptors (5S, 6R-LTD4 greater than 5S, 6R-LTD1 greater than 5R, 6S-LTD4 greater than 5S,6R-LTE4 greater than FPL 55712). A comparison of the receptor binding affinities of these compounds with their smooth muscle contractile agonist (pD2) and antagonist (-log[KB]) activities in guinea pig lung and trachea showed a good correlation (r = 0.88), suggesting that the saturable, high-affinity, stereoselective [3H]LTD4 specific binding sites identified in human lung may be physiologically relevant receptor moieties.     

砒石对哮喘小鼠肺组织白三烯含量的影响

目的探讨砒石对哮喘小鼠的治疗作用及对肺组织LTB4、LTC4水平的影响。方法建立小鼠卵蛋白哮喘模型,灌胃给予砒石等药,对肺泡灌洗液进行白细胞分类计数,肺组织切片HE染色观察病理变化,用ELISA法检测肺组织匀浆LTB4、LTC4含量。结果哮喘组小鼠肺组织匀浆中LTB4、LTC4含量较生理盐水对照组升高。砒石1.25mg/kg能降低哮喘小鼠肺组织LTB4的含量;砒石0.625mg/kg可降低哮喘小鼠肺组织中LTC4水平至正常。砒石不能影响哮喘小鼠肺泡灌洗液中白细胞分类百分比。结论肺组织中LTB4、LTC4水平的增高可能在哮喘的发病中起作用。砒石能减轻哮喘小鼠肺的病理损害,降低哮喘小鼠肺组织LTB4、LTC4水平,从而表现出抗哮喘的活性。     

Optimization of the quinoline and substituted benzyl moieties of a series of phenyltetrazole leukotriene D4 receptor antagonists.

This report describes the development of a series of highly potent quinoline-based leukotriene D4 (LTD4) receptor antagonists containing an N-benzyl-substituted phenyltetrazole moiety. They were designed to provide both the correct positioning of the acidic function and secondary lipophilic domain required for strong receptor binding. Members of this series possess high activity in blocking LTD4-induced contractions of isolated guinea pig ileum. Compound 32, LY287192 (2-[[5-[3-[2-(7-chloroquinolin-2- yl)ethenyl]phenyl]-2H-tetrazol-2-yl]methyl]-5-fluorobenzoic acid sodium salt), blocked contraction with a pKB value of 9.1 +/- 0.3. Qualitative structure-activity studies have demonstrated specific requirements for the best activity. In particular, ortho substitution of the benzyl group with an acidic function was crucial for maximum potency. In cases similar to 32, where the benzyl group possesses an ortho carboxylate, the N-2-substituted tetrazole isomer showed 100-fold greater activity relative to the corresponding N-1 isomer. This pattern was reversed when the acid was substituted at the para position. The quinoline unit may be replaced by other nitrogen-containing heterocycles.