Effects of KP-496, a novel dual antagonist of leukotriene D4 and thromboxane A2 receptors on nasal blockage in guinea pig models of allergic rhinitis

OBJECTIVE AND DESIGN: KP-496 is a novel dual antagonist for leukotriene (LT) D(4) and thromboxane (TX) A(2) receptors. We investigated effects of KP-496 on antigeninduced nasal blockage in 2 guinea pig models of allergic rhinitis. SUBJECTS: Male Hartley guinea pigs were used. TREATMENT: Animals were actively sensitized with ovalbumin (OVA) or Japanese cedar pollen, and were then repeatedly challenged with OVA or pollen, respectively. KP-496 (0.003 %-0.05 %) was intranasally administered 0.5 or 1 h before and 2 h after an antigen challenge. METHODS: As an indicator of nasal blockage, specific airway resistance was measured using a double-flow plethysmograph system. Statistical analyses were performed with Dunnett's test (OVA model) or t-test (pollen model). RESULTS: Although early phase response was not affected by even a high dose (0.03 %) of KP-496, late phase nasal blockage (1.68 +/- 0.26) was inhibited by 0.01 % (0.87 +/- 0.19; p <0.05) and 0.03 % (0.44 +/- 0.12; p <0.01) of KP-496 in the OVA model. On the other hand, both early (5.60 +/- 0.77) and late phase responses (7.90 +/- 1.70) were inhibited by 0.05 % KP-496 to 2.68 +/- 0.84 (p <0.05) and 2.71 +/- 0.83 (p <0.05), respectively, in the pollen model, in which nasal hyperresponsiveness had been acquired by multiple challenges. CONCLUSIONS: KP-496 may be clinically effective for nasal blockage in allergic rhinitis.     

Itraconazole-mediated inhibition of calcium entry into platelet-activating factor-stimulated human neutrophils is due to interference with production of leukotriene B4

The primary objective of this study was to probe the involvement of leukotriene B(4) (LTB(4)) in itraconazole (0.1-5 microM)-mediated inhibition of Ca(2+) uptake by chemoattractant-activated human neutrophils. Following exposure of the cells to platelet-activating factor (PAF, 200 nM), LTB(4) was measured by immunoassay, while neutrophil cytosolic Ca(2+) concentrations were determined by a fura-2/AM-based spectrofluorimetric procedure. Activation of neutrophils was accompanied by an abrupt and sustained (for about 1 min) elevation in cytosolic Ca(2+) which was associated with increased generation of LTB(4), both of which were attenuated significantly by itraconazole at 0.5 microM and higher. The inhibitory effect of the anti-mycotic on Ca(2+) uptake by PAF-activated cells was mimicked by an LTB(4) antibody, as well as by LY255283 (1 microM) and MK886 (0.5 microM), an antagonist of LTB(4) receptors and an inhibitor of 5'-lipoxygenase-activating protein, respectively, while addition of itraconazole to purified 5'-lipoxygenase resulted in inhibition of enzyme activity. A mechanistic relationship between itraconazole-mediated inhibition of LTB(4) production and Ca(2+) influx was also supported by the observation that pulsed addition of purified LTB(4) to PAF-activated neutrophils caused substantial restoration of Ca(2+) uptake by cells treated with the anti-mycotic. Taken together, these observations suggest that the potentially beneficial anti-inflammatory interactions of itraconazole with activated neutrophils result from interference with production of LTB(4), with consequent attenuation of a secondary LTB(4)-mediated wave of Ca(2+) uptake by the cells.     

Involvement of endogenous leukotriene B4 and platelet-activating factor in polymorphonuclear leucocyte recruitment to dermal inflammatory sites in rats

A critical role for leukotriene B(4) (LTB(4)) and/or platelet-activating factor (PAF) in regulating polymorphonuclear cell (PMN) trafficking to inflammatory sites has been reported in a number of experimental inflammatory models. In vitro, newly synthesized LTB(4) and PAF were shown to act in an autocrine/paracrine or intracrine fashion to enhance intracellular arachidonic acid availability and leukotriene biosynthesis. This suggested potentially cooperative effects of these lipid mediators in regulating PMN extravasation. The present study aimed to elucidate whether endogenous LTB(4) and PAF may both act to regulate plasma extravasation and PMN trafficking to inflammatory sites in experimental inflammation. With this aim, we have used selective and potent PAF and LTB(4) receptor antagonist pretreatments in dermal and pulmonary inflammation models in rats. Our results show additive inhibitory effects of dual LTB(4) and PAF receptor blockade in either PAF- or LTB(4)-elicited cutaneous PMN accumulation compared to single-drug administration. Furthermore, the combined administration of the drugs inhibited the PMN accumulation induced by the chemically unrelated soluble agonists tumour necrosis factor-alpha and C5a. Finally, in a model of pulmonary inflammation induced by the intravenous injection of Sephadex beads, lung neutrophilia was reduced by 63% following the administration of LTB(4) and PAF antagonists, in contrast with the lack of effect of single drug administration. Our results strongly support a role of both endogenous LTB(4) and PAF in regulating PMN trafficking to inflammatory sites in various experimental conditions.     

Recent advances in clinical development of leukotriene B4 pathway drugs

The LTB4 pathway is an attractive target for therapeutic drug development. Two broad classes of drugs have been pursued: antagonists of the primary LTB4 receptors (BLT1 and BLT2) and inhibitors of LTA4 Hydrolase (LTA4H), the rate limiting enzyme in the production of LTB4. An initial wave of effort culminated in the 1990s. Over the past 15 years, a second wave of more selective drug candidates, including at least 5 BLT antagonists and 6 LTA4H inhibitors, have reached Phase 2 clinical trials. Despite the extensive efforts to discover and develop LTB4 pathway targeting drugs, only one has reached the market to date. Recently discovered complexities in the pathway and challenges in matching pathway intervention with therapeutic effect could explain the limited clinical success of LTB4 pathway drugs, even though there is a large body of scientific evidence linking LTB4 to human diseases and demonstrating efficacy of these compounds in a wide array of preclinical models. Herein, we describe the clinical programs for the most prominent recent examples from each broad class and discuss the clinical outcomes and their implications for future development of LTB4 pathway drugs.     

Lipoxygenase inhibitory activity of U-66,858 and its deacetylated metabolite U-68,244 in human whole blood

The inhibitory effects of the semi-quinone U-66,858 and its metabolite U-68,244 on the ionophore-induced formation of leukotriene B₄ (LTB₄) were examined in human whole blood (WB). Preincubation of U-66,858 and U-68,244 for 1 min prior to challenge of blood with calcium ionophore A23187 resulted in IC₅₀ values of 1080±644 and 820±442 nmol/L, respectively (NS). After 60 min preincubation, values were 250±85 and 270±79 nmol/L (NS). The activity of the lipoxygenase inhibitor AA-861 in this system was similar to that of U-66,858, while vitamin K and the sulphate conjugate of U-66,858 showed significant inhibition of LTB₄ release only at micromolar concentrations. U-66,858 exhibited significant inhibition of thromboxane A₂ release (p<0.02) in a comparative study with the known cyclooxygenase (CO) inhibitor flurbiprofen. The metabolism of U-66,858 in contact with WB at 37°C was monitored for 70 min using [¹⁴C]-labelled drug and reverse-phase HPLC, the majority of recovered radioactivity no longer in the form of U-66,858 being accounted for by U-68,244 and polar conjugates of U-66,858. Thus, U-66,858 is a potent inhibitor of LTB₄ production in human whole blood and undergoes deacetylation to an initial metabolite with similar pharmacological potency. However, other metabolites of U-66,858 such as the sulphate conjugate, are relatively weak inhibitors of 5-lipoxygenase (5-LO) under similar conditions.     

Anti-CD45 antibody enhances lipoxygenase pathway of human naïve mononuclear cells and cyclooxygenase pathway of neutrophils

Objective and Design: Anti-CD45 antibody exhibits multiple biological effects on human mononuclear cells (MNC) and polymorphonuclear neutrophils (PMN). We intended to determine whether anti-CD45 antibody could affect arachidonic acid metabolism and thereby, the interactions between human na?ve MNC and PMN. Materials and Methods: Human na?ve MNC and PMN were incubated with monoclonal anti-human CD45 IgG F(ab’)₂ antibody or non-specific IgG F(ab’)₂ for 30 min. The mRNA expression of cyclooxygenase type 1 (COX-1), type 2 (COX-2), 5-lipoxygenase (5-LOX) and leukotriene A₄ hydrolase (LTA₄ hydrolase) in both cells was detected by RT-PCR and quantified by densitometric determination. The presence of COX-1 and COX-2 molecules in the cells was detected by Western blot. The concentration of PGE₂ and LTB₄ in cultured supernatants was measured by EIA kits. Results: Anti-CD45 IgG F(ab’)₂ up-regulated LTA₄ hydrolase mRNA expression and LTB₄ production, but down-regulated COX-1 and COX-2 mRNA expression and PGE2 production, of na?ve MNC compared to non-specific IgG F(ab’)₂. In contrast, a reverse modulation by the specific antibody on PMN was observed including up-regulation of cyclooxygenase pathway and down-regulation of lipoxygenase pathway. Conclusions: A novel activity of anti-CD45 with reverse modulation on cyclooxygenase/lipoxygenase pathways was found such that the expression of COX-1 and COX-2 in PMN, and 5-LOX and LTA₄ hydrolase in MNC were enhanced. Received 4 May 2005; returned for revision 29 June 2005; returned for final revision 12 October 2005; accepted by M. Katori 16 November 2005     

缺血预处理通过抑制白三烯C4生成减轻大鼠肝缺血再灌注损伤

 目的：研究缺血预处理（IP）减轻大鼠肝脏缺血再灌注（I/R）损伤是否涉及前炎症因子白三烯C4(LTC4)。方法：健康成年雄性SD大鼠随机分为3组(每组6只)。I/R组： 采用大鼠部分（70%左右）肝脏缺血60 min再灌注5 h模型,缺血前15 min开始至复灌5 h经颈外静脉输注生理盐水（3 mL·kg-1·min-1）;假手术组：只麻醉开腹,不阻断肝脏血流;IP组：在I/R前先阻断肝左、中叶血流10 min,然后开放血流10 min,余步骤同I/R模型组。应用反相高效液相色谱法（RP-HPLC）检查肝组织LTC4含量,同时生化检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）活性和肝组织谷胱甘肽（GSH）含量,以及HE染色法检查肝组织学损伤。结果：肝脏IP处理完全逆转了再灌注5 h所致肝组织LTC4含量增加（P<0.05）;同时增加肝组织GSH含量,明显降低血清ALT和AST活性（P<0.05）,并减轻肝脏组织结构损伤。结论：IP处理减少肝脏I/R期间LTC4堆积,同时伴随血清肝酶释放减少和肝组织结构损伤降低,以及保护肝组织氧化还原状态,表明IP的有利影响可能涉及其在肝脏I/R损伤期间抑制LTC4生成。     

Simultaneous presence of platelet activating factor,leukotriene B4, prostaglandin F1α and F2α in the supernatant of human neutrophils treated with phospholipase A2 of human monocytes

Summary The simultaneos presence of platelet-activating factor, leukotriene B₄, prostaglandin F₁ and F₂ are detectable in the supernatant of human neutrophil granulocytes treated with phospholipase A₂ of human monocytes. This enzyme is suspected to play an important role in the pathomechanism of inflammation.     

Anti-allergic effects of sinomenine by inhibition of prostaglandin D2 and leukotriene C4 in mouse bone marrow-derived mast cells

Sinomenine is an alkaloid compound and a prominent anti-allergic agent found in the root of the climbing plant Sinomenium acutum. However, its effects on the bone marrow-derived mast cell (BMMC) mediated allergy and inflammation mechanism remain unknown. In this study, the biological effects of sinomenine were evaluated while focusing on its effects on the allergic mediator in PMA plus A23187-stimulated BMMCs. An investigation was also conducted to determine its effects on the production of several allergic mediators including interleukin-6 (IL-6), prostaglandin D₂ (PGD₂), leukotriene C₄ (LTC₄), β-Hexosaminidase (β-Hex), and cyclooxygenase-2 (COX-2) protein. The results revealed that sinomenine inhibited the PMA plus A23187-induced production of IL-6, PGD₂, LTC₄, β-Hex, and COX-2 protein. Taken together, these findings indicate that sinomenine has the potential for use in the treatment of allergy.     

Tacrolimus reduces urinary excretion of leukotriene E(4) and inhibits aspirin-induced asthma to threshold dose of aspirin

BACKGROUND: The exact mechanism of aspirin-induced asthma is not clear. It has been postulated that precipitation of asthma attacks by aspirin is linked to inhibition of COX activity and massive release of cysteinyl leukotriene into the airway. Tacrolimus, a macrolide-derived immunosuppressant, is used for immunosuppression in organ transplantation and also for allergic diseases such as atopic dermatitis. OBJECTIVE: We evaluated the effects of tacrolimus in aspirin-induced asthma by using a double-blind, crossover study design. METHODS: Twelve patients with aspirin-induced asthma (male:female, 3:9; mean age +/- SD, 36.7 +/- 7.2 years) received either tacrolimus (0.1 mg/kg) or placebo 2 hours before the threshold dose of oral aspirin. RESULTS: In the placebo arm, oral aspirin significantly decreased FEV 1 concomitant with significant increases in sputum eosinophilic cationic protein and urinary leukotriene E(4) levels. Tacrolimus significantly inhibited bronchoconstriction and abrogated aspirin-induced increase in both sputum eosinophilic cationic protein and urinary leukotriene E(4) levels. CONCLUSION: The current study suggested that tacrolimus inhibited bronchoconstriction to a threshold dose of aspirin by inhibition of cysteinyl leukotriene excretion.     

Isolectin B4 binding in populations of rat trigeminal ganglion cells

We have recently classified dissociated trigeminal ganglion cells into nine types using electrophysiological current signatures. In the present study, we investigated the relationship between isolectin B₄ (IB₄) binding and the cell types in rat trigeminal ganglion cells. We found that IB₄ was bound to all type 2 cells and more than 70% of cell types 1 and 13; however, it was bound to less than 20% of cell types 7 and 8 and did not bind at all to cell types 3–5 and 9. Thus, each trigeminal ganglion cell type showed high homogeneity in IB₄ binding. These results correspond to reported IB₄ binding profiles in the matched dorsal root ganglion cell types, except for types 5 and 7.     

Vitamin B12-Gehalt im normalen Leberstanzcylindergewebe

Zusammenfassung Die Bestimmung des Vitamin B₁₂-Gehaltes von 220 Leberstanzcylindern lebergesunder Individuen ergab einen annähernden Normwert von 10 ng/mg Biuretprotein der Leber. Die individuelle Schwankungsbreite (0,7–79 ng/mg) ist ähnlich wie beim Serumspiegel (200 bis 800 pg/ml) sehr groß, während der individuelle Vitamin B₁₂-Gehalt der normalen Leber über größere Zeiträume kaum wesentlichen Schwankungen unterliegt. Im Gegensatz zu dieser Beobachtung ist die individuelle Schwankungsbreite einer über längere Zeit gleichmäßig ernährten Rattenpopulation nur unbedeutend. Möglicherweise werden die Verhältnisse beim Menschen durch unterschiedliche Eßgewohnheiten oder vorausgehende Gesundheitsstörungen geprägt.     

Gallic acid-l-leucine (GAL) conjugate enhances macrophage phagocytosis via inducing leukotriene B4 12-hydroxydehydrogenase (LTB4DH) expression

Timely clearance of apoptotic cells is an important step in the resolution of ongoing inflammation and the restoration of tissue integrity and function after acute myocardial infarction. Natural products gallic acid and l-leucine are well-documented for anti-inflammatory and anabolic effects. We synthesized gallic acid-l-leucine (GAL) conjugate via direct coupling gallic acid and l-leucine. The aim of the present study was to investigate the effect of GAL conjugate on the phagocytotic activity of macrophages. By using murine macrophage cell line RAW264.7 as an in vitro model, we evaluated the effect of GAL conjugate on the phagocytic uptake of fluorescently labeled latex beads and apoptotic cardiomyocyte H9c2 cells. We found that GAL conjugate enhanced the phagocytic activity of macrophage RAW264.7 cells in a concentration-dependent manner. Further mechanistic studies revealed that the effect of GAL conjugate on macrophage phagocytosis was positively correlated with the up-regulation of leukotriene B4 12-hydroxydehydrogenase (LTB4DH) expression at both mRNA and protein levels. By ESI–MS based lipidomics profiling, GAL conjugate increased the enzymatic activities of LTB4DH, leading to the formation of lipid metabolites including 12-oxo-LTB4, 13,14-dh-oxo-PGE2 and 13,14-dh-oxo-PGF2α. Interestingly, GAL conjugate failed to increase macrophage phagocytosis upon silencing of LTB4DH by specific siRNA. Moreover, it appeared that GAL conjugate induced LTB4DH expression via activating the Nrf2/HO-1 pathway. After Nrf-2 was silenced by specific siRNA, GAL conjugate no longer induced LTB4DH expression in the Nrf2-siRNA transfected cells. Taken together, our results suggest that GAL enhances macrophage phagocytosis via sequentially activating Nrf2 and up-regulating LTB4DH expression. Thus, GAL conjugate may serve as a lead compound for the development of new anti-inflammatory drugs.     

Serum from NSAID-treated patients attenuates the capacity of rat leukocytes to synthesize leukotriene B4

The synthesis of leukotriene B₄ by A23187-stimulated rat peritoneal leukocytes was studied in the presence of 0.1% normal human serum, serum from patients treated with NSAIDs for either an inflammatory (rheumatoid arthritis, RA) or a non-inflammatory condition (lumbar disc protrusion, LDP), and serum from RA patients drawn one week after withdrawal from NSAID treatment. The capacity for LTB₄ synthesis was significantly lower in the presence of serum from NSAID treated patients: thirty per cent less than observed in presence of normal serum in the RA group, and fifty per cent in the LDP group. When NSAIDs were withdrawn from RA patients, the LTB₄ production in presence of serum increased, but was not completely normalized after one week. These results indicate that NSAID treatment may down-regulate the capacity for leukotriene synthesis by an indirect effect.accepted by M. J. Parnham     

Synthesis and characterization of three Aflatoxin B1‐protein conjugates

Pure aflatoxin B, was obtained after extraction and chromatographic purification from cultures of A. flavus. Aflatoxin B, derivatives suitable for conjugate formation were synthesized. Intermediates and conjugates were characterized spectrophotometrically.     

Survey of aflatoxin B1 and ochratoxin A in commercial green coffee beans by high‐performance liquid chromatography linked with immunoaffinity chromatography

The natural occurrence of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) has been surveyed in 47 samples of commercial green coffee beans. Samples were collected in Nagoya City, Japan, during 1988–93. Using a combination of immunoaffinity chromatography and high‐performance liquid chromatography analysis, AFB₁ and OTA were quantified with detection limits of 2 ng kg^‐1 and 0.1 μg kg^‐1 respectively. Positive rates and levels of AFB₁and OTA were 32% and 2.0–32.9 ng kg^‐1, and 30% and 0.1–17.4 μg kg^‐1 respectively. The samples contaminated with AFB₁ or OTA were mainly imported from African and Asian countries, and several samples were found to be positive for these two mycotoxins for the first time in Japan, although their levels were low.