Discovery of CP-199,330 and CP-199,331: two potent and orally efficacious cysteinyl LT1 receptor antagonists devoid of liver toxicity.

CP-199,330 (3) and CP-199,331 (4) are cysLT1 receptor antagonists that are equipotent to marketed cysLT1 receptor antagonists zafirlukast and pranlukast, show good pharmacokinetics in rats and monkeys, and are devoid of liver toxicity in monkeys as seen in CP-85,958 (1).

Development of new chromanol antagonists of leukotriene D4.

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).

N-carbamoyl analogs of Zafirlukast: potent receptor antagonists of leukotriene D4.

Exploration of the indole nitrogen region of Zafirlukast (1) has uncovered a potent series of cysteinyl leukotriene D4 (LTD4) antagonists. These studies showed that a variety of functionality could be incorporated in this region of the molecule without sacrificing potency. Efforts to exploit this site in order to improve oral efficacy are discussed.

Development of 2,2-dimethylchromanol cysteinyl LT1 receptor antagonists.

A new series of cysLT1 receptor antagonists represented by CP-288,886 (7) and CP-265,298 (8) were developed which are equipotent to clinical cysLT1 receptor antagonists Zafirlukast (1) and Pranlukast (2).

Biarylcarboxylic acids and -amides: inhibition of phosphodiesterase type IV versus [3H]rolipram binding activity and their relationship to emetic behavior in the ferret.

In addition to having desirable inhibitory effects on inflammation, anaphylaxis, and smooth muscle contraction, PDE-IV inhibitors also produce undesirable side effects including nausea and vomiting. In general, compounds that inhibit PDE-IV also potently displace [3H]rolipram from a high-affinity binding site in rat cortex. While this binding site has not been identified, it has been proposed to be an allosteric binding site on the PDE-IV enzyme. Preliminary studies have suggested that the emetic potency of PDE-IV inhibitors is correlated with affinity for the brain rolipram binding site rather than potency at inhibiting PDE-IV enzyme activity. Efforts to eliminate the emetic potential of PDE-IV inhibitors were directed toward developing compounds with decreased [3H]rolipram binding affinity while retaining PDE-IV potency. Thus, a novel series of 4-(3-alkoxy-4-methoxyphenyl)benzoic acids and their corresponding carboxamides were prepared and evaluated for their PDE-IV inhibitory and rolipram binding site properties. Modification of the catechol ether moiety led to phenylbutoxy and phenylpentoxy analogues that provided the desired activity profile. Specifically, 4-[3-(5-phenylpentoxy)-4-methoxyphenyl]-2-methylbenzoic acid, 18, was found to exhibit potent PDE-IV inhibitory activity (IC50 0.41 microM) and possessed 400 times weaker activity than rolipram for the [3H]rolipram binding site. In vivo, compound 18 was efficacious in the guinea pig aerosolized antigen induced airway obstruction assay (ED50 8.8 mg/kg, po) and demonstrated a significant reduction in emetic side effects (ferret, 20% emesis at 30 mg/kg, po).

Antigen-mediated pulmonary eosinophilia in immunoglobulin G1-sensitized guinea pigs: eosinophil peroxidase as a simple specific marker for detecting eosinophils in bronchoalveolar lavage fluid.

Eosinophil peroxidase (EPO) has been used previously to detect the number of eosinophils in the peritoneal exudate and bone marrow of mice. The present study was undertaken to determine 1) whether EPO activity may provide a measure of a change in eosinophils in bronchoalveolar lavage fluid (BALF) of guinea pigs, 2) whether immunoglobulin (Ig)G1 could play a role in pulmonary eosinophilia and 3) effects of pharmacological agents on the EPO response in an IgG1 passively sensitized animal model. The activity of EPO was assessed by the ability of cell lysates (0.1% Triton-100 treatment) to oxidize 1 mM o-phenylenediamine in the presence of 1 mM H2O2 for 5 min at 22 degrees C. The enzyme activity was found to be eosinophil dependent, inhibited by the EPO inhibitor 3-amino-1,2,4-triazole (IC50 = approximately 0.1 mM) and relatively resistant to heat treatment (no loss of activity after 2-hr preincubation at 56 degrees C). To determine antigen-dependent eosinophil and EPO responses, guinea pigs were passively sensitized i.p. with 0.5 mg/kg of an affinity-purified antiovalbumin (OA) IgG1. Two to 3 days later, the sensitized animals were injected with pyrilamine (5 mg/kg, i.p.) before OA aerosol challenge. Aerosolized OA (0.1%) caused a significant increase in both eosinophil number and EPO activity in BALF of sensitized guinea pigs at 18 to 24 hr post-challenge. At a given concentration of aerosolized OA, the enzyme activity increased as a function of the antibody dose and time post-OA challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Competition of leukotrienes and ICI-198,615 for [3H]LTD4 binding sites in guinea pig lung membranes suggests the involvement of two LTD4 receptor subtypes.

Pharmacological analysis of the effects of leukotriene D4 (LTD4) antagonists on contraction of guinea pig airway smooth muscle to leukotrienes reveals the presence of two subtypes of the LTD4 receptor. This finding is, however, inconsistent with [3H]LTD4 equilibrium binding results, which show no evidence of a heterogeneity of pulmonary [3H]LTD4 binding sites. It is possible that LTD4 binds to the two receptor subsets with equal affinity, and the pharmacological difference between them lies in the relative ability of leukotriene (LT) agonists and antagonists to interact at the receptor sites. This study was, therefore, undertaken to determine the rank order of potency of LTs and ICI-198,615 in competing with [3H]LTD4 for their respective binding sites in guinea pig lung membranes. To determine precisely the inhibitory constant (Ki) of LTC4, we used the irreversible gamma-glutamyl transpeptidase inhibitor, acivicin (AT-125), to prevent LTC4 metabolism. Incubation of lung membranes with 5 mM AT-125 for 120 min at 25 degrees C resulted in greater than 98% recovery of LTC4. Unlike L-serine-borate complex, AT-125 failed to inhibit pulmonary [3H]LTD4 binding. These results suggest that AT-125 can be used in this study. Nonlinear least squares analysis of the results of LTD4/[3H]LTD4 or ICI-198,615/[3H]LTD4 competitive binding reveals that either LTD4 (Ki = 0.49 nM) or ICI-198,615 (Ki = 6.89 nM) interacts at a single homogenous population of [3H]LTD4 binding sites. However, the data of competitive binding results of LTC4 (in the presence of AT-125) or LTE4 are best fitted for its interaction with high- and low-affinity [3H]LTD4 binding sites, designated as LTD4 alpha and LTD4 beta sites, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that peptidoleukotriene is a prerequisite for antigen-dependent thromboxane synthesis in IgG1-passively sensitized guinea pig lungs.

Lungs from guinea pigs passively sensitized with an affinity-purified IgG1 antibody produce both leukotriene (LT)D4 and thromboxane (Tx)B2 upon ex vivo antigen challenge. This study was undertaken to determine the possibility of endogenously generated peptido-LTs being a prerequisite for Tx synthesis. In immunoglobulin G1-sensitized lungs, exogenous LTD4 induced TxB2 production with a median effective dose of 4.1 nM, whereas the response to LTE4, LTB4 or platelet-activating factor was relatively weak. Although LTC4 was as potent as LTD4 in stimulating TxB2 generation, LTC4's dose-response curve was shifted significantly to the right by AT-125, an irreversible gamma-glutamyl transpeptidase inhibitor, suggesting that at least a part of LTC4 sensitized lungs with antigen (0.01-30 micrograms/ml ovalbumin) for 20 min precipitated a significant amount of LTD4 production. The levels of LTD4 range from 8 to 26 nM (without taking LTD4 recovery into consideration). This level is 2- to 7-fold greater than the median effective dose value observed with exogenous LTD4. Moreover, pretreatment of sensitized lungs with ICI-198,615 a specific LTD4 antagonist, blocked equally both antigen (IC50 = 0.01 microM)- and LTD4 (IC50 = 0.017 microM)-induced TxB2 production. When sensitized lung fragments were treated with 5 mM AT-125, ICI-198,615 was effective in preventing not only antigen-but also LTC4-dependent production of TxB2 (IC50 = 0.018 and 0.021 microM, respectively). In contrast, neither WEB-2086, a platelet-activating factor antagonist, nor pyrilamine, a histamine antagonist, inhibited antigen and LTD4 responses (IC50 greater than 30 microM). Unlike its effect on antigen response, ICI-198,615 was unable to block Ca2+ ionophore-induced TxB2 production.2

Antigen-dependent leukotriene synthesis and histamine release from IgG1 passively-sensitized guinea pig lungs ex vivo: relationship between serum levels of antigen-specific IgG1 and mediator synthesis/release.

Naive guinea-pigs were passively sensitized with varying amounts of affinity column purified, homologous, anti-ovalbumin IgG1 (anti-OA IgG1) and then examined for a) the capacity of lung tissue to release mediators (histamine and LTB4/LTD4) in response to antigen-challenge ex vivo and b) the attendant circulating levels of anti-OA IgG1. Intraperitoneal administration of anti-OA IgG1 (0.125-0.75 mg/kg) to guinea-pigs facilitated the synthesis of LTB4 (8-25 ng/g lung) and LTD4 (18-80 ng/g) and the release of histamine (1-7 ug/g) from lung tissue after exposure to 10 micrograms/ml of ovalbumin for 20 min ex vivo. Peak levels of mediators were found using 0.5 mg/kg anti-OA IgG1 with an ED50 = 0.35 mg/kg. LTD4/LTB4 synthesis and histamine release were both antigen concentration- and time-dependent, and LT synthesis was observable in non-perfused lungs and in lungs perfused free of blood. Maximum sensitization occurred at 1-2 days post i.p. administration of anti-OA IgG1 and was maintained up to 7 days. Measurement of anti-OA IgG1 using an enzyme-linked immunosorbent assay demonstrated that circulating antibody levels were 2-6 micrograms/ml at the doses which caused sensitization. The level of anti-OA IgG1 found in passively sensitized animals was at least 100-fold less than that found in actively-sensitized guinea-pigs despite the similar magnitude in LTD4/LTB4 synthesized and the amount of histamine released. Using purified antibody, the results demonstrate that in guinea-pigs, IgG1 can play a prominent role in regulating lung LT synthesis and histamine release, and that microgram per ml circulating levels of this antibody are sufficient to sensitize naive lungs.