Tunable cavity optomechanics with ultracold atoms.

We present an atom-chip-based realization of quantum cavity optomechanics with cold atoms localized within a Fabry-Perot cavity. Effective subwavelength positioning of the atomic ensemble allows for tuning the linear and quadratic optomechanical coupling parameters, varying the sensitivity to the displacement and strain of a compressible gaseous medium. We observe effects of such tuning on cavity optical nonlinearity and optomechanical frequency shifts, providing their first characterization in the quadratic-coupling regime.

Optimization of the potency and duration of action of N-hydroxyurea 5-lipoxygenase inhibitors.

As in vitro glucuronidation assay and several biochemical assays were utilized to discover potent new N-hydroxyurea-containing 5-lipoxygenase inhibitors with long durations of action. The best of these, A-78773, is a racemic mixture of two enantiomers. These enantiomers were purified and the R(+)-enantiomer A-79175 was found to be superior to the S(-)-enantiomer with respect to in vitro metabolism and duration of action in the monkey. A-79175 was a potent selective inhibitor of 5-hydroxyeicosatetraenoic acid formation in rat basophilic leukemic homogenates (IC50 = 54 nM) and of calcium ionophore-induced leukotriene B4 (LTB4) formation in purified human polymorphonuclear leukocytes (IC50 = 25 nM) and human whole blood (IC50 = 80 nM). The compound inhibited LT formation in the rat with oral ED50s of 1 to 2 mg/kg. It also was a potent inhibitor of edema and inflammatory cell influx in rats and mice. A-79175 was resistant to glucuronidation and had an elimination half-life of nearly 9 hr in cynomolgus monkeys. A-79175 inhibited ex vivo LTB4 formation by monkeys for extended periods. A single 0.5-mg/kg oral dose gave > 50% inhibition of calcium ionophore-induced LTB4 formation ex vivo for 12 hr. A good correlation was found between the elimination half-life for A-78773 and its enantiomers in cynomolgus monkeys and humans. These data indicate that A-79175 is a promising long-acting agent that should be useful to delineate the importance of LTs in animal and human studies.

Clinical activity of leukotriene inhibitors.

Data from the emerging clinical trials with compounds such as zileuton, ICI 204,219, Bay X1005, MK571, MK679, and MK591 are demonstrating the importance of the leukotrienes as mediators of asthma and possibly other diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. One of the major questions facing the asthma community is how much improvement in the FEV1 is needed to improve the quality of life of the asthmatic patient. Comparing the various approaches to asthma treatment, there is typically 15-20% improvement in the lung function with inhaled steroids. Leukotriene interventions apparently will improve lung function to similar levels as with inhaled steroids, and thus may offer an alternative to steroids. Like the steroids, zileuton appears to also reduce the inflammatory cell influx into the antigen-challenged site, which may have the long-term effect of reversing some of the tissue alterations that occur as a result of the inflammation seen with asthma. Importantly, the reported experience to date has shown that the leukotriene modulators do not have the same side-effects as the current therapies, and thus offer the hope that both safe and effective treatment may be derived from this approach. The clinical data reported do not yet define a preferred approach to the modulation of leukotriene pathology. As more studies are published in other diseases the broad spectrum use of these inhibitors will become known.

Stereoselective metabolism of the 5-lipoxygenase inhibitor A-78773.

Based on the knowledge that glucuronidation was a major route of metabolism of the N-hydroxyurea class of 5-lipoxygenase inhibitors, a simple in vitro glucuronidation assay was established using liver microsomes from various species, including man. Compounds that were potent inhibitors of 5-LO and showed a reduced metabolic liability in vitro were then characterized more extensively in experimental animals. This prudent usage of in vitro glucuronidation proved to be highly efficient and was indispensable in the identification of A-78773, a potent new long-acting 5-LO inhibitor. Further studies with liver microsomes revealed that glucuronidation of A-78773 was stereoselective and that the R(+) enantiomer was considerably more resistant to conjugation than the S(-) stereoisomer. Pharmacokinetic studies in experimental animals and humans confirmed the greater metabolic stability of the R(+) enantiomer. A single 400-mg oral dose of A-78773 inhibited ex vivo leukotriene biosynthesis for more than 24 hours. Since 78% of the drug plasma AUC following A-78773 administration was accounted for by the R(+) enantiomer, it is reasonable to assume that the majority of the leukotriene inhibition caused by the racemate is attributable to the R(+) enantiomer, A-79175, particularly at the later times. The equivalent 5-lipoxygenase inhibitory potency coupled with the superior pharmacokinetic profile of the R(+) enantiomer, A79175, compared to the S(-) enantiomer, A-79176, indicate that the development of this compound may be preferable to the racemate A-78773.

The 5-lipoxygenase inhibitory activity of zileuton in in vitro and in vivo models of antigen-induced airway anaphylaxis.

Leukotrienes are biologically active lipid mediators capable of producing airway inflammation, hyperresponsiveness and bronchoconstriction. The first enzyme in the metabolic pathway of arachidonic acid leading to the leukotrienes is 5-lipoxygenase (5-LO). A selective and potent 5-LO inhibitor, zileuton (N-1(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea, A-64077) was evaluated in models of airway anaphylaxis, where leukotrienes are a major component. In vitro, zileuton inhibited antigen-induced contractions of guinea-pig tracheal strips (GPTS) from actively sensitized animals with an IC50 of 6 microM. Similar results were obtained in human bronchial strips passively sensitized to IgE. Zileuton had little or no effect on contractions elicited by acetylcholine, prostaglandin D2 (PGD2), or the thromboxane agonist, U-44069. In anesthetized sensitized guinea-pigs pretreated with meclofenamic acid and mepyramine, a single aerosol exposure of antigen produced a substantial decrease in dynamic lung compliance (Cdyn). These profound changes in lung function were dose-dependently inhibited by orally administered zileuton (ED50 = 12 mg/kg). These results demonstrate that zileuton is a potent, selective inhibitor of in vitro contraction of GPTS and antigen-induced bronchoconstriction in vivo. These data also confirm the participation of 5-LO products in these models of airway anaphylaxis and suggest the usefulness of the guinea-pig for identifying and characterizing the pulmonary effects of 5-LO inhibitors.

Quantitative structure-activity relationships of 5-lipoxygenase inhibitors. Inhibitory potency of pyridazinone analogues.

Quantitative structure-activity relationships (QSAR) of 72 1-phenyltetrahydropyridazin-3(2H)-one (I) analogues are examined for the inhibitory potency (IC50) of 5-lipoxygenase in a broken cell. The potency is increased by lipophilic substituents at the 3'- and 4'-positions. Substituents with positive F value at the 4'-position also increase the potency, while substituents at the 3'-position with a positive R value decrease it. The potency also decreases as the size of the 2'- and/or 4'-substituents increases. Thioketone analogues are about 5 times more potent than the corresponding carbonyl analogues.

Preclinical and clinical activity of zileuton and A-78773.

The importance of leukotrienes as mediators of inflammation and bronchoconstriction was examined with two recently described 5-lipoxygenase inhibitors, zileuton and A-78773. Preclinical evaluation of these two molecules indicates that they are potent, selective, direct, reversible inhibitors of 5-lipoxygenase with activity in a variety of purified cells and in more complex biological systems such as whole blood, lung fragments, and tracheal tissues. In various animals models of inflammation and allergy, the molecules inhibited edema, inflammatory cell influx, and bronchospasm. These observations are consistent with the recent clinical success of zileuton in treating asthma and inflammatory bowel disease. In all preclinical systems tested thus far, A-78773 is more potent and longer acting than zileuton, indicating that the molecule could be even more effective in the clinic than zileuton and that both molecules are useful tools in defining the role of leukotrienes in preclinical and clinical settings.

The properties of A-69412: a small hydrophilic 5-lipoxygenase inhibitor.

A compound which inhibits leukotriene biosynthesis could be clinically useful in treating several allergic and inflammatory diseases. One site for such inhibition is at the enzyme 5-lipoxygenase. Most inhibitors of this enzyme thus far described are poorly bioavailable. A-69412 is a small, relatively hydrophilic compound of the N-hydroxyurea class, which exhibits minimal plasma protein binding (6-12%). The compound was found to be a potent long-acting inhibitor of leukotriene formation in vivo in the rat (oral ED50 = 5 mg/kg) and ex vivo in several species. In addition, the compound exhibits excellent bioavailability in dogs and monkeys with a relatively long elimination half-life in both the species (6 and 3 h, respectively). The biochemical activity and pharmacological profile of A-69412 indicates its potential utility in asthma and ulcerative colitis, and possibly other inflammatory and allergic conditions.

A-78773: a selective, potent 5-lipoxygenase inhibitor.

The potency and selectivity of A-78773, a newly discovered 5-lipoxygenase inhibitor, were examined. The compound was significantly more potent than zileuton in inhibiting leukotriene formation in cell free lysates and in isolated human neutrophils. A-78773 inhibited a RBL cell lysate 5-lipoxygenase at concentrations 2 orders of magnitude lower than those required to inhibit rabbit reticulocyte 15-lipoxygenase or human platelet 12-lipoxygenase. The compound was also a potent, long lasting, orally active inhibitor of leukotriene formation ex vivo in dogs and in vivo in the rat. In experiments where leukotriene formation was completely inhibited, no increase in eicosanoids from other pathways was observed. A-78773 should prove to be a valuable clinical tool in treating leukotriene mediated diseases.

The discovery and development of zileuton: an orally active 5-lipoxygenase inhibitor.

The enzyme 5-lipoxygenase is a key target in the effort to discover drugs which inhibit the pathophysiology associated with the formation of leukotrienes. The research efforts of these laboratories have focused on the discovery of direct enzyme inhibitors of 5-lipoxygenase. In particular, compounds with hydroxamate or N-hydroxyurea functionalities have proven to be potent inhibitors of leukotriene biosynthesis in vitro and more importantly in vivo. One of these compounds, zileuton (N-(1-benzo-[b]-thien-2-ylethyl)-N-hydroxyurea) has been shown recently to be an effective leukotriene inhibitor in man. The critical approaches and breakthroughs in the discovery and development of zileuton are described. In addition, some recent results with zileuton in animals and man are detailed.

Inhibition of leukotriene biosynthesis in the rat peritoneal cavity.

In the search for a model of leukotriene (LT) production to provide a method to determine in vivo 5-lipoxygenase (5-LO) inhibitory activity by various compounds, a passive anaphylactic reaction in the rat peritoneal cavity was examined, refined and characterized. The reaction, produced by passive sensitization with an i.p. injection of rabbit anti-bovine serum albumin (anti-BSA) followed by an i.p. injection of BSA, resulted in the biosynthesis of large amounts of sulfidopeptide LTs measurable by immunoassay or by reversed phase high performance liquid chromatography. The oral activity of several 5-LO inhibitors has been examined using this model. An example of these is zileuton (Abbott-64077), a potent 5-lipoxygenase inhibitor now under clinical evaluation. Zileuton inhibited sulfidopeptide LT biosynthesis in the rat peritoneal cavity in a dose-dependent manner (ED50 = 3 mg/kg). WY-49,232, MK-866, BW A4C and phenidone also produced good activity with ED50 values of 6, 8, 11 and 17 mg/kg, respectively. This modified rat peritoneal anaphylaxis model appears to be a valuable tool for establishing in vivo activity of 5-LO inhibitors.

4-hydroxythiazole inhibitors of 5-lipoxygenase.

4-Hydroxythiazoles have been identified as potent inhibitors of 5-lipoxygenase in vitro exhibiting IC50's of less than 1 microM. An investigation of structure-activity relationships showed that the most potent inhibitors of this series are the 5-phenyl derivatives. The corresponding thiazolidin-4-one analogues were found to be relatively inactive. The 4-hydroxythiazoles were active inhibitors against 5-lipoxygenase in both intact rat polymorphonuclear leukocytes and human whole blood. The compounds were also selective inhibitors of 5-lipoxygenase, displaying only weak activity against other related enzymes, cyclooxygenase and 12- and 15-lipoxygenase.

5-lipoxygenase inhibitory activity of zileuton.

Zileuton [N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyure] inhibited 5-hydroxyeicosatetraenoic acid synthesis by rat basophilic leukemia cell 20,000 x g supernatant and rat polymorphonuclear leukocytes (PMNL) (IC50 = 0.5 and 0.3 microM) respectively. It also inhibited leukotriene (LT)B4 biosynthesis by rat PMNL (IC50 = 0.4 microM), human PMNL (IC50 = 0.4 microM) and human whole blood (IC50 = 0.9 microM). Inhibition of human PMNL LTB4 biosynthesis was removed readily by a simple wash procedure. At concentrations up to 100 microM, the compound produced little or no inhibition of several related enzymes, such as platelet 12-lipoxygenase, soybean and rabbit reticulocyte 15-lipoxygenase and sheep seminal vesicle cyclooxygenase. At p.o. doses from 0.5 to 5 mg/kg in the dog, zileuton produced a rapid and sustained inhibition of ex vivo blood LTB4 biosynthesis which correlated with the pharmacokinetic behavior of the compound. In a similar ex vivo study in the rat, the compound displayed an p.o. ED50 of 2 mg/kg. Zileuton was highly effective in preventing 6-sulfidopeptide LT formation in the rat peritoneal cavity triggered by an antigen-antibody reaction with an ED50 of 3 mg/kg. In experimental models of inflammation, zileuton significantly reduced arachidonic-acid induced mouse ear edema (ED50 = 31 mg/kg) and also attenuated inflammatory cell accumulation in the rat pleural Arthus reaction. The effectiveness of this compound for preventing LT formation in vitro, ex vivo and in vivo suggests its utility for preventing the pathophysiological effects of the LTs and other 5-lipoxygenase products in animals and in humans.

In vivo characterization of hydroxamic acid inhibitors of 5-lipoxygenase.

The hydroxamic acid functionally can be incorporated into simple molecules to produce potent inhibitors of 5-lipoxygenase. The ability of many of these hydroxamates to inhibit leukotriene synthesis in vivo has been measured directly with a rat peritoneal anaphylaxis model. Despite their potent enzyme inhibitory activity in vitro, many orally dosed hydroxamic acids only weakly inhibited leukotriene synthesis in vivo. This discrepancy is attributable at least in part to the rapid metabolism of hydroxamates to the corresponding carboxylic acids, which are inactive against the enzyme. A study of the structural features that affect this metabolism revealed that 2-arylpropionohydroxamic acids are relatively resistant to metabolic hydrolysis. Several members of this class of hydroxamates are described that are orally active inhibitors of leukotriene synthesis.

Synthesis and 5-lipoxygenase inhibitory activity of 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid analogues.

A series of eicosatetraenes (2-24) were designed, synthesized, and evaluated in vitro for inhibitory activity against 5-lipoxygenase (20000g supernatant from homogenized rat basophilic leukemia cells). All compounds were found to be active with the potencies (IC50's) ranging from 0.19 to 97 microM. Compounds containing the hydroxamic acid functionality (10-12) exhibited the best activity (IC50 = 0.19-2.8 microM). The most potent inhibitor was 5-[(hydroxyamino)carbonyl]methyl]-6,8,11,14-eicosatetraenoic acid (11), which was 10 times more active than the C-1 hydroxamates of arachidonic acid or 5-HETE. Cyclization of the linear eicosanoids 2 and 14 in the C-1 to C-5 region produced compounds (21 and 24, respectively) with several-fold greater potency.