Pharmacologic actions of the second generation leukotriene B4 receptor antagonist LY29311: in vivo pulmonary studies.

We examined the in vivo actions of LY293111 sodium (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]pro poxy]phenoxy] benzoic acid sodium salt). Guinea pigs were used to evaluate the effect of this agent on (1) acute airway obstruction produced by intravenous leukotriene B4, (2) pulmonary granulocyte infiltration and delayed onset airway obstruction resulting from a 4-h leukotriene B4 inhalation and (3) lung inflammation after aerosol challenge with the divalent cationic ionophore A23187 (6S-[6alpha(2S*,3S*),8beta(R*),9beta,11alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)e thyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazole carboxylic acid). Airway obstruction was quantitated using pulmonary gas trapping measurements and lung inflammation was evaluated by bronchoalveolar lavage (BAL) and histology. LY293111 sodium produced a dose-related inhibition of acute leukotriene B4-induced airway obstruction when administered i.v. (ED50=14 microg/kg) or p.o. (ED50=0.4 mg/kg). In contrast, LY293111 sodium did not inhibit the pulmonary gas trapping caused by aerosols of histamine, leukotriene D4, or the thromboxane mimetic U46619 (15 [(S)-hydroxy11a,9a-(epoxymethano)prosta-5Z,13E-dienoic acid]). Oral LY293111 sodium inhibited leukotriene B4-induced bronchoalveolar lavage granulocyte infiltration and delayed onset airway obstruction at doses as low as 0.3 mg/kg. In A23187-challenged animals, pulmonary inflammation was markedly inhibited at 1 h, but not 2 h and 4 h post-exposure. We conclude that LY293 11 sodium is a selective leukotriene B4 receptor antagonist with potent pulmonary anti-inflammatory activity.

Synthesis and biological evaluation of analogues of bacterial lipid I.

Bacterial Lipid I analogues containing different anomeric groups at the muramic acid moiety were synthesized and screened in MurG enzyme assays run in the presence and absence of cell wall membranes. The results obtained in this study help elucidate the role of the lipid diphosphate in the recognition of Lipid I by MurG.

Pharmacologic actions of the second-generation leukotriene B4 receptor antagonist LY293111: in vitro studies.

The in vitro actions were investigated of LY293111, a potent and selective leukotriene B4 (LTB4) receptor antagonist, on human neutrophils, human blood fractions, guinea pig lung membranes, and guinea pig parenchymal and tracheal strips. The IC50 for inhibiting [3H]LTB4 binding to human neutrophils was 17.6 +/- 4.8 nM. LY293111 inhibited LTB4-induced human neutrophil aggregation (IC50 = 32 +/- 5 nM), luminol-dependent chemiluminescence (IC50 = 20 +/- 2 nM), chemotaxis (IC50 = 6.3 +/- 1.7 nM), and superoxide production by adherent cells (IC50 = 0.5 nM). Corresponding responses induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine were inhibited by 100-fold higher concentrations of LY293111. LTB4 binding to guinea pig tissues and subsequent activation were also inhibited. The Ki for inhibition of [3H]LTB4 binding to lung membranes was 7.1 +/- 0.8 nM; IC50 for preventing binding of [3H]LTB4 to spleen membranes was 65 nM. The compound inhibited LTB4-induced contraction of guinea pig lung parenchyma. At 10 nM, LY293111 caused a parallel rightward shift of the LTB4 concentration-response curve. At higher concentrations, plots were shifted in a nonparallel manner, and maximum responses were depressed. LY293111 did not prevent antigen-stimulated contraction of sensitized trachea strips. At micromolar concentrations, LY293111 inhibited production of LTB4 and thromboxane B2 by plasma-depleted human blood stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine and thrombin. In addition, at these higher concentrations, formation of LTB4 by A23187-activated whole blood and conversion of arachidonic acid to LTB4 by a human neutrophil cytosolic fraction were inhibited. In summary, LY293111 is a second-generation LTB4 receptor antagonist with much improved potency in a variety of functional assay systems.

Recent developments in solid- and solution-phase methods for generating carbohydrate libraries.

Carbohydrates and glycoconjugates have attracted a great deal of attention in recent years because of the key roles they play in biological processes. The development of carbohydrate-based libraries is a crucial step towards investigating the details of carbohydrate-mediated interactions, exploring the molecular diversity intrinsic to carbohydrates and identifying novel therapeutically interesting carbohydrate-containing drugs. This review describes recent advancements in solid- and solution-phase methods that relate to the generation of carbohydrate and glycoconjugate libraries. Special attention will be dedicated to the increasing use of carbohydrates as molecular scaffolds for library synthesis.

Intestinal transport of gentamicin with a novel, glycosteroid drug transport agent.

PURPOSE: The objective was to investigate the ability of a glycosteroid (TC002) to increase the oral bioavailability of gentamicin. METHODS: Admixtures of gentamicin and TC002 were administered to the rat ileum by injection and to dogs by ileal or jejunal externalized ports, or PO. Bioavailability of gentamicin was determined by HPLC. 3H-TC002 was injected via externalized cannulas into rat ileum or jejunum, or PO and its distribution and elimination was determined. The metabolism of TC002 in rats was evaluated by solid phase extraction and HPLC analysis of plasma, urine and feces following oral or intestinal administration. RESULTS: The bioavailability of gentamicin was substantially increased in the presence of TC002 in both rats and dogs. The level of absorption was dependent on the concentration of TC002 and site of administration. Greatest absorption occurred following ileal orjejunal administration. TC002 was significantly more efficacious than sodium taurocholate, but similar in cytotoxicity. TC002 remained primarily in the GI tract following oral or intestinal administration and cleared rapidly from the body. It was only partly metabolized in the GI tract, but was rapidly and completely converted to its metabolite in plasma and urine. CONCLUSIONS: TC002 shows promise as a new drug transport agent for promoting intestinal absorption of polar molecules such as gentamicin.

Design of compounds that increase the absorption of polar molecules.

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.

Carbohydrate-based combinatorial libraries.

Carbohydrate-based combinatorial libraries are tremendously valuable for studying the role of sugars in biology and for expanding accessible molecular diversity needed in broad-based drug screening programs. This review discusses the issues that are relevant to the successful implementation of comprehensive carbohydrate-based combinatorial library programs. In addition, details of oligosaccharide and glycoconjugate libraries constructed using both solid phase and solution phase strategies are presented.

Cationic facial amphiphiles: a promising class of transfection agents.

A promising class of compounds for DNA transfection have been designed by conjugating various polyamines to bile-acid-based amphiphiles. Formulations containing these compounds were tested for their ability to facilitate the uptake of a beta-galactosidase reporter plasmid into COS-7 cells. Dioleoyl phosphatidyl ethanolamine (DOPE) formulations of some of the compounds were several times better than Lipofectin at promoting DNA uptake. The most active compounds contained the most hydrophilic bile acid components. The activity is clearly not related to affinity for DNA: the hydrophobic bile acid conjugates were found to form stable complexes with DNA at lower charge ratios than the hydrophilic conjugates. We suggest that the high activity of the best compounds is related to their facial amphiphilicity, which may confer an ability to destabilize membranes. The success of these unusual cationic transfection agents may inspire the design of even more effective gene delivery agents.

Aerosolized LTB4 produces delayed onset increases in pulmonary gas trapping.

Airway obstruction, as measured by increases in postmortem pulmonary gas trapping, and lung inflammatory changes were examined in guinea pigs exposed for up to 4 h to aerosols of leukotriene B4 (LTB4) or its non-chemotactic isomer, 6-trans-12-epi-LTB4. Airway obstruction and cytological responses in isomer-exposed animals were similar to those of unexposed control animals. LTB4-exposed animals had minimal inflammatory changes at 0.5 h but became dyspneic by 2 h and had increased airway obstruction, bronchoalveolar lavage neutrophils and eosinophils, and pulmonary tissue granulocyte scores. The LTB4-induced effects at 4 h were similar to those 2 h, except for further increase in BAL neutrophils and eosinophils. LTB4-induced airway obstructive and inflammatory changes were prevented by pretreatment with the LTB4 receptor antagonist SC-41930, but were unaffected by indomethacin. Thus, prolonged LTB4 inhalation can produce delayed onset airway obstruction that is stereospecific, cyclooxygenase-independent, and temporally associated with the influx of granulocytes into lung airways.

Pulmonary responses of the guinea pig to inhaled A23187: a brief review.

The pulmonary effects of inhaled A23187 are reviewed. Guinea pigs challenged with this divalent cationic ionophore rapidly develop airway obstruction, which is maintained for at least 4 h. Pulmonary inflammation and increased airway responsiveness are also observed. Pharmacologic manipulations suggest that these actions are due to the release of multiple mediators. We have found A23187 challenge to be valuable as an approach for testing potential asthma drugs.

Flow cytometric evaluation of the effects of leukotriene B4 receptor antagonists (LY255283 and SC-41930) on calcium mobilization and integrin expression of activated human neutrophils.

Leukotriene B4 (LTB4) is a naturally occurring eicosanoid mediator which chemoattracts and stimulates human neutrophils to an activated state. In an attempt to identify novel antiinflammatory drugs, synthetic LTB4 receptor antagonists have been developed in several laboratories. In this study, the effects of two such LTB4 receptor antagonists were examined for their influences on two elements of human neutrophil activation using flow cytometric techniques. Quantitative flow cytometric assays of human neutrophil intracellular calcium mobilization and up-regulation of integrin (CD11b/CD18) cell surface expression were developed and used to determine the potency and selectivity of compounds LY255283 and SC-41930 on these activities. Our results indicate that both compounds preferentially block these functions of LTB4-induced human neutrophil activation in a concentration dependent manner and fall in the 1-2 microM range of antagonist activity. Compound SC-41930 was approximately twice as potent as LY255283 in blocking the targeted agonist effects. Both compounds were approximately 100-fold less potent in blocking the same functions of interleukin-8-induced human neutrophil activation.

Effects of two leukotriene B4 (LTB4) receptor antagonists (LY255283 and SC-41930) on LTB4-induced human neutrophil adhesion and superoxide production.

Leukotriene B4 (LTB4) induces a number of functional changes in human neutrophils, including both superoxide release and CD11b/CD18 (Mo1)-mediated adherence to various substrates, such as keyhole limpet hemocyanin (KLH). These effects are both time- and concentration-dependent. Neutrophil adhesion was at least 10-fold more sensitive to the stimulatory action of LTB4 than superoxide production. Two LTB4 receptor antagonists, LY255283 (1-(5-ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)heptyloxy )- phenyl)ethanone) and the sodium salt of SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H- 1-benzopyran-2-carboxylic acid) were evaluated for effects on human neutrophil superoxide production and adhesion. Despite being more sensitive to LTB4-induced stimulation, neutrophil adhesion was at least 100-fold less sensitive to inhibition by LY255283 and SC-41930 than superoxide production. Both LTB4 receptor antagonists behaved similarly in these models. These compounds did not inhibit neutrophil responses induced by granulocyte/macrophage colony-stimulating factor (GM-CSF).

Enol lactone inhibitors of serine proteases. The effect of regiochemistry on the inactivation behavior of phenyl-substituted (halomethylene)tetra- and -dihydrofuranones and (halomethylene)tetrahydropyranones toward alpha-chymotrypsin: stable acyl enzyme intermediate.

We have found that alpha-aryl-substituted halo enol lactones (I and II) are effective mechanism-based inactivators for chymotrypsin. In this study, we have investigated, for comparative purposes, halo enol lactones with aryl functions situated beta and gamma to the lactone carbonyl group. We synthesized 4-phenyl-5(E)-(iodomethylidene)tetrahydro-2-furanone (1), 4-phenyl-5(E)-(iodomethylidene)dihydro-2-furanone (2), 4-phenyl-6(E)-(iodomethylidene)tetrahydro-2-pyranone (3), and 5-phenyl-6(E)-(iodomethylidene)tetrahydro-2-pyranone (4), using a halolactonization reaction to convert the appropriate phenyl-substituted acetylenic acid precursor into the corresponding 5(E)-(halomethylidene)furanone and 6(E)-(halomethylidene)pyranone system. The 4-phenylfuranone (1 and 2) and the 5-phenylpyranone (4) proved to be only reversible, competitive inhibitors. By contrast, the 4-phenyltetrahydropyranone (3) inactivated alpha-chymotrypsin in a time-dependent manner. This inactivation was very rapid but reversible, with regeneration of enzyme activity being spontaneous and hydrazine-accelerated, suggestive of the intermediacy of a stable acyl enzyme. Kinetic comparison of the iodomethylene lactone 3 with the corresponding protio lactone 25 indicates that the iodine accelerates the rate of chymotrypsin acylation but produces an acyl enzyme that is more hydrolytically labile than that formed from lactone 25. From the results of this study, we conclude that a phenyl group situated at C-3 (alpha to the lactone carbonyl group) in both the 5(E)-(iodomethylidene)tetrahydro-2-furanone (I) and 6(E)-(iodomethylidene)tetrahydro-2-pyranone (II) series is essential for their activity as mechanism-based irreversible inactivators of chymotrypsin. The corresponding beta-aryl-substituted lactones, by contrast, are potent acylating agents that lead to acyl enzymes of high stability.

Haloenol lactones. Potent enzyme-activated irreversible inhibitors for alpha-chymotrypsin.

Haloenol lactones can act as enzyme-activated irreversible inhibitors for alpha-chymotrypsin: acyl transfer to the active site serine releases a halomethyl ketone that remains tethered in the active site during the lifetime of the acyl enzyme, poised to alkylate an accessible nucleophilic residue. To investigate the structural determinants for chymotrypsin suicide inactivation with haloenol lactones, we prepared a series of nine lactones, differing in ring size (6-membered valerolactones and 5-membered butyrolactones) and in the nature of the aromatic substituent (phenyl and alpha-naphthyl), and the halogen (bromine and iodine). The inactivating behavior of these lactones is characterized by a binding constant (Ki) and three rate constants, for inactivation (k2), catalytic hydrolysis (kc), and spontaneous hydrolysis (kh). The six-membered valerolactones were much more potent inactivators than the butyrolactones, having both higher affinity and more rapid inactivation; the alpha-naphthyl-substituted lactones were also more effective, but the nature of the halogen had relatively little effect. The spontaneous rate of hydrolysis of all of these lactones is low. The turnovers per inactivation of these lactones vary from 91-1.7, with some of the alpha-naphthyl-substituted lactones approaching ideal behavior (stoichiometric inactivation). These studies indicate that several haloenol lactones are effective enzyme-activated irreversible inhibitors of chymotrypsin, and that their potency and efficiency depends markedly upon certain structural features of the lactone system.