The effect of lipoxygenase inhibitors and leukotriene antagonists on anaphylaxis

The experiments whose results are reported here were carried out with the aim of showing a possible role for lipoxygenase products in the modulation of the Schultz-Dale reaction. For this purpose, the actions of nordihydroguaieretic acid (NDGA) and of FPL 55712 were tested during anaphylaxis in guinea-pig ileum and tracheain vitro. Isolated preparations from guinea-pigs, which had been subcutaneously sensitized with ovalbumin and incomplete Freund adjuvant, were challenged with increasing concentrations of antigen; in preparations isolated from the same animal an antigen-concentration anaphylactic-reaction curve was performed in the presence of the drugs. NDGA 3.3×10^–6 M was capable of inhibiting anaphylaxis in the trachea to a maximum extent of 40% but it did not affect anaphylactic reaction in the intestinal smooth muscle. FPL 55712 2×10⁶ M did not exert any activity on anaphylaxis in either preparations. The difference between SRS-A and histamine as mediators of anaphylaxis in the tissue preparations used could explain the fact that NDGA acted on the trachea alone.     

Assessment of thein vivo biochemical efficacy of orally active leukotriene biosynthesis inhibitors

In man, the therapeutic effectiveness of specific inhibitors of leukotriene (LT) biosynthesis against allergen-induced bronchoconstriction appears to be related to thein vivo biochemical efficacy of these compounds, as measured by inhibition of whole blood LTB₄ generation (upon A23187 stimulus) and, particularly, urinary LTE₄ excretion. Accordingly, we have assessed the ability of two clinically documented LT biosynthesis inhibitors, zileuton and MK-886, and the structurally novel 5-lipoxygenase activatig protein antagonist, MK-0591, to inhibit the production of these inflammatory arachidonic acid metabolites in laboratory dogs. Zileuton (2 mg/kg) was extremely bioavailable in dogs (>10 M plasma concentrations), and inhibited the A23187-inducedex vivo production of LTB₄ by venous blood by >90%, in concordance with its potency in canine bloodin vitro (IC₅₀=1.1 M). Despite this degree of inhibition in whole blood, urinary LTE₄ excretion was reduced by only 52%, a profile of activity similar to that seen in clinical studies. MK-886 was less well absorbed, with plasma concentrations of 3 M being achieved only at 25 mg/kg. These levels resulted in <45% inhibition of LTB₄ production, but a significant (p<0.05) 47% inhibition of urinary LTE₄ excretion. MK-0591 was similarly bioavailable (compared with MK-886), but 10-fold more activein vivo as a 2 mg/kg dose resulted in 41–62% inhibition of urinary LTE₄ excretion (p<0.05 vs controls;n=4, 28). Significant inhibition ofex vivo LTB₄ synthesis was also observed at this dose (49%), in accord with peak plasma concentrations of 0.5 M and anin vitro potency of 0.2–0.4 M (IC₅₀) in whole blood from these animals. At higher dose (10 mg/kg), MK-0591 inhibited LTE₄ excretion by 69%, with 88% inhibition of the LT biosynthetic capacity of whole blood. These data demonstrate that the biochemical efficacy of structurally diverse leukotriene biosynthesis inhibitors can be assessedin vivo in normal laboratory dogs. Such measurements, combined with bioavailability data from other species, may be useful for predicting biochemical activity in man.     

Phlogistic activity of leukotriene D4 in the mouse

An investigation of the phlogistic activity of LTD₄ in the mouse was accomplished by examination of its ability to cause increased capillary permeability and edema formation following subcutaneous administration. It was observed that nanogram quantities of LTD₄ caused edema and increased capillary permeability in a dose-related manner. The increase in capillary permeability was not inhibited by pretreatment with indomethacin and thus was unrelated to the production of cyclooxygenase products. These data suggest that LTD₄ can mediate the edematous phase of the inflammatory response in the mouse and illustrate the sensitivity of this species to LTD₄.     

Comparison of the effect between leukotriene B4 and leukotriene B5 on the induction of interleukin 1-like activity and calcium mobilizing activity in human blood monocytes

Leukotriene B₄ (LTB₄), one of 5-lipoxygenase (5-LO) products of arachidonic acid, was reported to be more potent than leukotriene B₅ (LTB₅), one of 5-LO products of eicosapentaenoic acid, in the activation of neutrophil functions through the differential potency between these leukotrienes in calcium mobilization. So we compared the effect of LTB₄ and LTB₅ on the induction of interleukin 1 (IL-1)-like activity and calcium mobilization in human blood monocytes. LTB₄ significantly augmented IL-1-like activity in monocytes, however, LTB₅ had no significant effect. Also LTB₄ was apparently more potent than LTB₅ in inducing calcium mobilization in fura-2-loaded monocytes. IL-1 production in monocytes was reported to be partly dependent on cytosolic free calcium. These results, therefore, may indicate that the different activity between LTB₄ and LTB₅ in the enhancement of IL-1-like activity could be partly ascribed to the different potency in inducing calcium mobilization between LTB₄ and LTB₅ in human blood monocytes.     

Clinical cases of acquired coagulation inhibitors

The acquired coagulation factor inhibitors are classified into alloantibodies, which appear in association with supplementary treatment for congenital coagulation factor deficiency, and autoantibodies, which are spontaneously produced. We report here 2 cases of acquired factor VIII inhibitor and 1 case of factor V inhibitor. Case 1: A 52-year-old woman noted swelling of the right parotid region in March 1988. Though contrast examination was scheduled, she was admitted for detailed examination due to a markedly prolonged coagulation time. An APTT correction test suggested that decreased factor VIII activity was due to the presence of an inhibitor. Since antinuclear antibody and SS-A antibody were positive and infiltration by lymphocytes in the salivary gland acini in a lip biopsy specimen was detected, Sj?gren's syndrome was diagnosed. Case 2: A 33-year-old woman had normal delivery of her second child in February 1998. In June 1998, she suffered slight contusion in the left lower limb. The affected site became swollen and painful, making walking difficult. Since both upper limbs became markedly swollen after 1 week, she visited our hospital. Prolonged APTT and a marked decrease in factor VIII activity were observed. Factor VIII inhibitor titer was high at 19 Bethesda units. Case 3: A 64-year-old man had had asymptomatic macroscopic hematuria since the beginning of August 1998 but was placed under observation since no abnormal findings were observed on various imaging tests. However, he was admitted to Osaka City General Medical Center because of vesicular tamponade. Factor V activity was markedly decreased to 1.0%. PT correction test suggested that decreased factor V activity was due to the presence of an inhibitor. The underlying disease could not be determined in this case. In patients with acquired coagulation inhibitors, bleeding symptoms are reported to be mild in many cases, and severe bleeding is rare. However, cases of death without severe bleeding or underlying disease have also been reported, indicating that the prompt diagnosis and treatment of this condition are necessary.     

Clinical studies with matrix metalloproteinase inhibitors

Matrix metalloproteinases (MMPs) have been implicated in the invasive growth and spread of tumours. Potent and selective inhibitors of MMPs have been synthesized. These inhibitors can prevent tumour invasion in vitro and have suppressed tumour growth and metastasis in animal cancer models. Several MMP inhibitors have now reached clinical trials. Phase I studies indicate that the inhibitors can be given by mouth and that over the short term they are well tolerated. Longer, randomised studies in cancer patients are now underway.     

Clinical results with direct thrombin inhibitors

Direct thrombin inhibitors inactivate thrombin without the need for antithrombin and some inactivate not only thrombin but also fibrin-bound thrombin. Hirudin has been shown to be more effective than low-dose unfractionated heparin and low molecular weight heparin for the prevention of deep vein thrombosis in high-risk orthopaedic patients. Major studies are assessing the value of direct thrombin inhibitors in patients with acute coronary syndromes. Currently, argatroban is the drug of choice in patients with heparin-induced thrombocytopenia.     

Clinical experience with cyclooxygenase-2 inhibitors

Increasing amounts of experimental and clinical data support the role of selective cyclooxygenase (COX)-2 inhibition in anti-inflammatory processes and the role of COX-1 inhibition in increasing the frequency of side effects. This article reviews the regulation of COX-2 in inflammatory processes based on in vitro and in vivo work. In addition, it summarizes the various in vitro assays used to classify the new generation of selective and highly selective inhibitors of COX-2, since prior categorization of NSAIDs does not satisfactorily encompass the COX-2 concept. Finally, the latest published clinical data of new selective and highly selective inhibitors of COX-2 (meloxicam, nimesulide, etodolac, celecoxib and MK966) are discussed.     

The effect of leukotriene antagonists,lipoxygenase inhibitors and selected standards on leukotriene-mediated allergic bronchospasm in guinea pigs

Leukotrienes (LT) C₄, D₄, and E₄ are major contributors to the pathobiology of human bronchial asthma. Therefore, it is likely that compounds that antagonize the action or inhibit the formation of LTs will be useful therapeutic agents. We have studied the effects of LT antagonists, 5-lipoxygenase inhibitors and selected standards in a model of LT-mediated allergic bronchospasm in guinea pigs. Sensitized animals were pretreated with mepyramine, indomethacin and propranolol to eliminate the influence of histamine, prostaglandins, thromboxanes and circulating catecholamines. In these animals, inhalation of antigen resulted in a bronchospasm consistent with a LT-mediated response that was slow in onset, of long duration and was inhibited by the selective LTD₄, antagonists FPL-55712, LY-171,883 and ICI-198,615 ICI-198, 615 was approximately 50-times more potent than FPL-55712 by the intravenous and intratracheal routes. However, of thirteen compounds known to inhibit 5-lipoxygenase and LT biosynthesisin vitro only phenidone, piriprost and AA-861 were active in thisin vivo model. The allergic bronchospams was inhibited by bronchodilators (e.g. PGE₂, aminophylline and forskolin) and by some mast cell stabilizers, but was otherwise insensitive to other pharmacological classes of compounds including calcium channel blockers and antagonists of serotonin, acetylcholine and platelet-activating factor. This model seems useful and reasonably selective for the evaluation of new antianaphylactic compounds that are LT antagonists. The inactivity of many 5-lipoxygenase inhibitors in this model suggests they do not inhibit LT formationin vivo.     

Proteinase inhibitors from Streptomyces with antiviral activity

An extensive screening study for the production of proteolytic inhibitors has been carried out on 75 Streptomyces strains. It was found that 18 of the strains and/or their variants (24%) produced proteinaceous substances, which belonged to the group of typical serine protease inhibitors. 23 samples were tested for inhibitory activity on the replication of influenza virus A/Germany/34, strain Rostock (H7N1) (A/Rostock) in chicken embryonic fibroblast (CEF) cells. Eleven of the tested samples (52.2%) significantly inhibited viral growth. Further the specific inhibitory effect on the replication of influenza virus A/Aichi/2/68 (H3N2) (A/Aichi) in Madin-Darby canine kidney (MDCK) cells and on the growth of herpes simplex virus type 1, strain DA (HSV-1) in Madin-Darby bovine kidney (MDBK) cells was tested. Nine samples significantly inhibited A/Aichi and four - HSV-1. The most effective inhibitors, produced by Streptomyces sp. 225b (SS 225b) and Streptomyces chromofuscus 34-1 (SS 34-1) protected mice from mortality in the experimental influenza A/Aichi virus infection.     

Effects of auranofin on leukotriene production and leukotriene stimulated neutrophil function

Arachidonic acid is metabolized in neutrophils by lipoxygenase to leukotrienes, which are suggested to play a central role in inflammation. The antirheumatic drug auranofin (4 g/ml) was found not to inhibit neutrophil production of the lipoxygenase products 5-HETE, 15-HETE and LTB₄,in vitro when stimulated with the calcium ionophore A23187. Auranofin, however, modulated neutrophil aggregation, enzyme release and chemotaxis induced by LTB₄. The results suggest that auranofin may exert some of its antirheumatic effects through affecting neutrophil responses to leukotrienes.Supported by grants from: Swedish National association against Rheumatism, King Gustav V 80 years Fund, Tore Nilssons Fund, P and A Hedlunds Fund, Swedish Society for Medical Research, Förenade Liv Mutual Group Life Insurance Company, Stockholm, Sweden.     

In vitro antifilarial activity of glutathione S-transferase inhibitors

Female adult bovine filarial worms Setaria digitata were extracted with phosphate-buffered saline (pH 7.4) and glutathione S-transferase (GST) activity and protein content were determined. The protein content, GST enzyme activity, and specific activity were 10.61 ± 3.41 mg ml⁻¹, 0.09 ± 0.019 μmol min⁻¹ ml⁻¹, and 0.009 ± 0.002 μmol min⁻¹ mg⁻¹ protein, respectively. The GST inhibition studies were performed with and without the inhibitors resulted from earlier molecular docking studies viz., ethacrynic acid, plumbagin, and curcumin for which the IC₅₀ values were 19.42, 51.41, and 114.86 μM, respectively. The in vitro macrofilaricidal activity of these molecules was studied by worm motility and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay at 24- and 48-h incubation. Plumbagin and ethacrynic acid showed 100% inhibition in worm motility at lower concentrations of 3.19 and 6.6 μM, respectively, at 48-h incubation while curcumin was effective at 54.29 μM. In MTT reduction assay, the ED₅₀ values (50% inhibition in formazan formation) for plumbagin, ethacrynic acid, and curcumin at 48-h incubation were 1.20, 2.48, and 19.86 μM, respectively. MTT reduction assay showed that plumbagin was the most effective in killing the adult S. digitata worms followed by ethacrynic acid and curcumin. In conclusion, all the three molecules selected by molecular modeling and docking studies inhibited the GST enzyme isolated from S. digitata and exhibited macrofilaricidal activity in vitro.