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From previous work, the primary functional groups, i.e. side chains, of the vasoactive intestinal peptide which are responsible for interaction with the VIP receptor have been identified. One of these sites, the side chain of tyrosine22 is essential for high receptor affinity. The present work aims to examine this site in greater detail. Several Boc-substituted-phenylalanine derivatives were prepared and incorporated into VIP analogs as replacement for tyrosine22. These analogs, of the form Ac-[Lys12,Nle17,X22,Val26,Thr28]-VIP, were assayed as smooth muscle relaxants and found to be full agonists of native VIP. Most of the analogs, however, proved to be less potent than the parent analog by up to 300-fold. A few analogs, all possessing electron-donating substituents, retained nearly full potency. Two compounds, 3-F,4-OH-Phe, 42 and 3-OCH3,4-OH-Phe, 43, were found to be 1.5- and 3.4-fold more potent than the parent compound, which equates to being 8.9- and 20-fold more potent than native VIP. Compound 43 was also found to be active as a bronchodilator in vivo in guinea pigs, with slightly over 2-fold enhanced potency and a significantly longer duration of action (> 20 min) when compared to the parent compound (5 min). The physical characteristics of the various substituents and their effect on biological activity are discussed with a brief analysis by QSAR techniques.

A series of essential fatty acids and fatty acid derivatives were evaluated for their ability to inhibit [3H] leukotriene B4 (LTB4) binding to pig neutrophil membranes. The fatty acids varied in chain length, extent of unsaturation, position of unsaturation, and isomerization. Generally, fatty acids with two or more unsaturated sites and chain lengths of 18-22 were potent inhibitors of [3H]LTB4 binding; both n-3 and n-6 fatty acids were inhibitory. The most potent compounds tested were homogammalinolenic acid and ricinelaidic acid which gave Ki values of 1 microM and 2 microM in the binding assay. Ricinelaidic acid was also tested for its ability to inhibit LTB4-mediated chemotaxis (IC50 = 10 microM) and LTB4-induced calcium fluxes (IC50 = 7 microM) in isolated human neutrophils. Ricinelaidic acid did not show agonist activity in these assays. In an in vivo model of LTB4-induced bronchoconstriction, ricinelaidic acid and homogammalinolenic acid gave 46% and 53% inhibition, respectively, at a 1 mg/kg i.v. dose. These results indicate that essential fatty acids are LTB4 receptor antagonists, which may account in part for their reported anti-inflammatory activities.

Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N-terminus, one compound, Ac-[D-Cys6,D-Cys11,Lys12,Nle17,Val26,Th r28]-VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N-terminal region were found to be inactive. A second analog, Ac-[Lys12,Cys17,Val26,Cys28]-VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (> 40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor-active conformation of VIP.

A number of indoles containing the 2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl side chain have been prepared by standard methods. Alternate, novel syntheses of indole-2-carboxamides and indole-2-carbonitriles have been developed. The title compound, 7e, was found to be a potent inhibitor of bovine prostaglandin synthetase in vitro and to lower serum prostaglandin levels after oral or intraperitoneal administration to rats. Consistent with prostaglandin synthetase inhibition, 7e prevented arachidonic acid induced diarrhea in mice and also collagen, ADP, or epinephrine induced platelet aggregation in human platelet-rich plasma. In contrast to many prostaglandin synthetase and platelet-aggregation inhibitors, 7e had neither ulcerogenicity nor systemic antiinflammatory activity in rats.

The syntheses and biological activity of (all Z )-7,7-dimethyl-5-8,- 11,14-eicosatetraenoic acid, (all Z )-7,7,-dimethyl-5,8,11-eicosatrienoic acid, ( Z , Z -7,7-dimethyl-5,8-eicosadienoic acid, (all Z )-10,10-dimetyl- 5,8,11,14-eicosatetraenoic acid, (all Z -10,10-dimethyl-5,8,11-eicosatrienoic acid, and rac .-( Z , Z -15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid are described. These arachidonic acid analogs are all inhibitors of ionophore-induced SRS-A biosynthesis in rat peritoneal cells. Their mode of action may involve inhibition of phospholipase A2 rather than Δ5-lipoxygenase. These compounds failed to exhibit significant activity in an in vivo model designed to detect inhibitors of antigen-induced, leukotriene-mediated bronchoconstriction is sensitized guinea pigs.

Adenosine inhibits guinea-pig lung adenylate cyclase (ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1) through a ‘P’ type regulatory site. The inhibition is of a non-competitive type. Divalent cations which activate the enzyme (Mg2+ and Mn2+) and also those which inhibit (Ca2+) increase the inhibitory potency of ‘P’ site analogs at this site. Guanine nucleotides also increase the inhibitory potency at this regulatory site but this does not appear to be directly related to the ability of the guanine nucleotides to activate the enzyme. Other regulators of lung adenylate cyclase, epinephrine and isoproterenol, do not affect the adenosine inhibitory process when examined at physiological concentrations. These studies demonstrate that two types of ligand which regulate the catalytic activity of the lung adenylate cyclase (metal ions and guanine nucleotides) also have a role in regulating the inhibition of the enzyme by adenosine.

Adenosine potentiates the in vitro antigen-induced release of histamine from chopped lung tissue prepared from a guinea pig actively sensitized to egg albumin. By studying the activity of a variety of adenosine analogs, the potentiation process in lung has been characterized as occurring through an ‘R’ adenosine receptor site. Similar studies on ionophore A23187-induced release of histamine from purified rat peritoneal mast cells have demonstrated that an ‘R’ adenosine receptor site is also associated with adenosine potentiation in this system. Since adenosine inhibits adenylate cyclase activity in a homogenate of guinea pig lung, and since decreases in cyclic AMP levels are thought to be associated with mediator release, the regulatory site involved in the enzyme inhibition process was also characterized to determine if, in lung, the ability of adenosine to inhibit adenylate cyclase is associated with potentiation of mediator release. It was found, however, that inhibition of adenylate cyclase occurs through a ‘P’ site, suggesting that the two processes are not related phenomena. Theophylline blocks the effect of adenosine on the antigen-induced release of histamine from guinea pig lung. This is in accord with previous data demonstrating that theophylline is an ‘R’ site antagonist. This observation also supports the idea that part of the pharmacological action of theophylline in human bronchial asthma may be through inhibition of the ability of adenosine to potentiate mediator release.

1 Pharmacological modulation of antigen-induced anaphylaxis in actively sensitized guinea-pigs with intravenously administered indomethacin (10 mg/kg), pyrilamine (2.0 mg/kg) and propranolol (0.1 mg/kg) resulted in a delayed onset, slowly developing bronchoconstriction indicative of a slow-reacting substance of anaphylaxis (SRS-A) response. 2 Measurements of pulmonary mechanics on the drug-pretreated animals challenged with ovalbumin demonstrated a more prominent effect on dynamic compliance than resistance. This is consistent with the more potent effects of SRS-A on peripheral rather than central airways. 3 The slowly developing bronchoconstriction obtained after treatment with indomethacin, pyrilamine and propranolol was inhibited by the standard SRS-A antagonist, FPL 55712 and the SRS-A synthesis inhibitors, phenidone, BW 755C and nordihydroguaiaretic acid. 4 Plasma SRS-A levels were determined in guinea-pigs following antigen challenge. The appearance of SRS-A in the plasma preceded the onset of bronchoconstriction and SRS-A levels remained elevated throughout its development. Coincident with the inhibition of bronchoconstriction by the SRS-A synthesis inhibitor, phenidone, was a dose-dependent reduction in plasma SRS-A. The intravenous ED50 in each case was 4 mg/kg. 5 This model of antigen-induced SRS-A-mediated bronchoconstriction should prove useful for the in vivo evaluation and development of therapeutics which regulate the synthesis of SRS-A.