A new class of nitric oxide-releasing derivatives of cetirizine; pharmacological profile in vascular and airway smooth muscle preparations.

BACKGROUND AND PURPOSE: The pharmacological properties of compounds NCX 1512 and NCX 1514, synthesized by linking the histamine H1-receptor antagonist cetirizine to NO-releasing spacer groups, are reported. The aim was to establish if the compounds retained the antihistamine action of the parent compound, to assess their efficacy as NO donors and to test if they had broader antiallergic activity than cetirizine in the lung. EXPERIMENTAL APPROACH: Antihistamine activity of NCX 1512 and NCX 1514 was investigated in vitro in the guinea pig ileum, in tracheal rings (GPTR) and lung parenchymal strips (GPLP) of the guinea-pig. The NO-releasing capacity was investigated in vascular preparations; the isolated rabbit and guinea-pig aorta and guinea-pig pulmonary artery. Kinetics of NO release were assessed in a rat whole blood assay. KEY RESULTS: Both NCX 1512 and NCX 1514 retained activity as H1-receptor antagonists in the guinea pig ileum and airway preparations. The NO-releasing NCX compounds relaxed the rabbit aorta, an action prevented by the guanylyl cyclase inhibitor ODQ (10 microM). NCX 1512 and NCX 1514 did not relax the antigen (ovalbumin) pre-contracted GPTR, whereas the NO donors NCX 2057 and DEA-NONOate relaxed guinea-pig pre-contracted vascular and tracheal preparations. Cetirizine (1-100 microM) and NCX 1512 (1-100 microM) reduced the cumulative (0.01-100 microg ml(-1)) ovalbumin-induced constriction in GPTR, but had no significant effect in GPLP. CONCLUSIONS AND IMPLICATIONS: NCX 1512 and NCX 1514 act as antihistamines and NO donors. However, there was no improved effect compared to cetirizine on antigen-induced constriction of the central and peripheral lung.

Depletion of Vbeta5.2/5.3 T cells with a humanized antibody in patients with multiple sclerosis.

A potentially pathogenic expansion of T cells expressing T cell receptor (TCR) Vbeta5.2/5.3 has been demonstrated in patients with multiple sclerosis (MS). A humanized antibody (ATM-027) directed against these T cells has been developed to further investigate the role of this subpopulation of T cells in MS. The pharmacokinetics/dynamics and safety of ATM-027 (0.3-300 mg intravenously over 30 min) were investigated in 14 patients with MS. The effect of treatment on cytokine expression and autoreactivity to peptides of myelin basic protein (MBP) was also studied. ATM-027 was well tolerated and raised no safety concerns. Clearance of the antibody was low and elimination half-life was approximately 3 weeks. The majority of the target Vbeta5.2/5.3 expressing T cells were depleted for at least 18 months. The small remaining fraction of target cells showed a marked decrease in their TCR expression, which was recovered within 8 months. The numbers of peripheral blood mononuclear cells (PBMCs) with spontaneous expression of IFN-gamma was decreased at 72 h and 8 weeks after treatment, whilst no clear effects on TNF-alpha, IL-4, IL-10, TGF-beta expression were observed. There was also a significant decrease in the number of PBMCs producing IFN-gamma in response to MBP peptide 80-102. We conclude that long-term depletion of T cells expressing defined Vbeta subgroups in MS patients is feasible using selective immunotherapy. The selective depletion of Vbeta5.2/5.3 expressing T cells in this study resulted in a decrease in potentially disease promoting anti-MBP reactivity and pro-inflammatory cytokine production.

Uncoupled regulation of leukotriene C4 synthase in platelets from aspirin-intolerant asthmatics and healthy volunteers after aspirin treatment.

BACKGROUND: We have reported that thromboxane A2 induces suppression of leukotriene (LT) C4 synthase activity in human platelets. AIM: In the present study, we describe a mechanism whereby aspirin treatment can lead to increased formation of LTC4, which is a potent bronchoconstrictor and inflammatory mediator. This mechanism is also demonstrated to be present in platelets from aspirin-intolerant asthmatics (AIA). METHODS: The effect of arachidonic acid or platelet agonists on LTC4 synthase activity was investigated in platelets obtained from healthy volunteers, aspirin-intolerant asthmatics or aspirin-tolerant asthmatics after in vivo treatment or in vitro pre-incubation with aspirin. RESULTS: Incubation of normal platelets with arachidonic acid or collagen provoked approximately 50% reduction of platelet LTC4 synthase activity, as determined by the conversion of LTA4 to LTC4. However, the inhibitory effect of arachidonic acid or collagen was not observed after oral administration of aspirin prior to collection of the platelets. Arachidonic acid-induced inhibition of LTC4 synthase activity was totally abolished in platelets collected from peripheral blood already 30 min after aspirin ingestion but was fully restored in platelets collected 3 to 7 days after the administration of aspirin. Treatment of platelet suspensions with aspirin in vitro dose-dependently counteracted the suppressive effect of arachidonic acid on LTC4 formation, with total reversal at approximately 40 microm. In contrast, the major aspirin metabolite, salicylic acid did not alter arachidonic acid-induced reduction of LTC4 synthase activity. Similarly, LTC4 synthase activity in platelets from AIA and aspirin-tolerant asthmatics (ATA) was reduced by approximately 50% after pre-treatment with arachidonic acid in vitro. Again the inhibitory effect was abolished when platelets were pre-incubated in the presence of aspirin. CONCLUSION: The results indicate that oral aspirin administration can lead to uncoupling of thromboxane A2-dependent negative feedback mechanisms, which may normally restrict the production of cysteinyl leukotrienes. This mechanism can be of potential interest in aspirin-induced asthma.

Receptor-mediated regulation of leukotriene C4 synthase activity in human platelets.

Human platelets possess a specific membrane-bound leukotriene (LT) C4 synthase, which catalyzes the conversion of LTA4 to LTC4. Stimulation of the receptors for thrombin, collagen or thromboxane A2 provoked inhibition of this enzyme, as judged by suppressed transformation of exogenous LTA4 to LTC4. Similarly, direct activation of protein kinase (PK) C with nanomolar concentrations of 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited the production of LTC4. Kinetic studies demonstrated that the inhibition induced by thrombin and PMA was non-competitive. Elevation of intracellular cAMP levels with carbacyclin did not affect basal LTC4 formation, but abolished the attenuation of platelet LTC4 synthase activity induced by the thromboxane receptor agonist U-46619. The unselective protein kinase inhibitor staurosporine prevented both receptor-mediated and PMA-induced suppression of LTC4 formation. In contrast, two selective PKC inhibitors, Ro 31-8220 and GF 109203X, reversed the inhibitory effect provoked by PMA, but failed to prevent thrombin-induced inhibition. Furthermore, the protein tyrosine phosphatase inhibitor, sodium orthovanadate, induced dose-dependent inhibition of LTC4 production in platelet sonicates. In conclusion, receptor-mediated activation of human platelets leads to decreased LTC4 synthase activity via phosphoregulation. Although the present results demonstrate that platelet LTC4 synthase can be regulated via PKC-dependent events, alternative mechanisms appears to be involved in the physiological regulation of this enzyme. The findings suggest the possible importance of protein tyrosine phosphorylations in this process.

Phorbol ester-induced suppression of leukotriene C4 synthase activity in human granulocytes.

The effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), on the metabolism of exogenous leukotriene (LT)A4 in human granulocytes was investigated. After incubation with LTA4 decreased levels of LTC4 but not LTB4 were observed in granulocyte suspensions pretreated with PMA. This finding could in part be ascribed to oxidative metabolism of LTC4, since PMA induced a rapid degradation of exogenously added LTC4. After blocking of LTC4 metabolism with the H2O2 scavenger catalase, a PMA-provoked suppression of the conversion of LTA4 to LTC4 was observed, indicating PKC-dependent regulation of LTC4 synthase activity. This effect, as well as PMA-induced degradation of LTC4 was prevented by specific protein kinase C inhibitors.

Ethanol affects leukotriene generation and leukotriene-induced functional responses in human polymorphonuclear granulocytes.

Since ethanol has been shown to inhibit the inflammatory response, we evaluated whether ethanol affected generation of leukotrienes in polymorphonuclear granulocytes (PMN) in vitro. Using the calcium ionophore A23187 as stimulus, the leukotriene B4 (LTB4) and leukotriene C4 (LTC4) generation were dose-dependently impaired by ethanol. No significant difference in the levels of the omega-oxidized metabolites was observed. However, the total LTB4 production (LTB4 plus omega-oxidized metabolites) was significantly decreased in the samples treated with ethanol. Furthermore, ethanol also modulated LTB4-induced functional responses. PMN aggregation, oxidative metabolism and elastase release were all inhibited in the presence of 1% ethanol (to 74 +/- 15%, 50 +/- 4% and 57 +/- 3% of controls, respectively). However, ethanol had no effect on intracellular calcium mobilization or on the change of the PMN membrane potential induced by either LTB4 or A23187. Thus, a possible mechanism for the reduced functional PMN responses in the presence of ethanol might be impaired generation of leukotrienes, but it is conceivable that ethanol impairs also other steps of the stimulus response coupling since the LTB4-induced functional responses were inhibited.

Stimulation of lipoxin synthesis from leukotriene A4 by endogenously formed 12-hydroperoxyeicosatetraenoic acid in activated human platelets.

Human platelets are devoid of 5-lipoxygenase activity but convert exogenous leukotriene A4 (LTA4) either by a specific LTC4 synthase to leukotriene C4 or via a 12-lipoxygenase mediated reaction to lipoxins. Unstimulated platelets mainly produced LTC4, whereas only minor amounts of lipoxins were formed. Platelet activation with thrombin, collagen or ionophore A23187 increased the conversion of LTA4 to lipoxins and decreased the leukotriene production. Maximal effects were observed after incubation with ionophore A23187, which induced synthesis of comparable amounts of lipoxins and cysteinyl leukotrienes (LTC4, LTD4 and LTE4). Chelation of intra- and extracellular calcium with quin-2 and EDTA reversed the ionophore A23187-induced stimulation of lipoxin synthesis from LTA4 and inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from endogenous substrate. However, calcium did not affect the 12-lipoxygenase activity in the 100,000 x g supernatant of sonicated platelet suspensions. Furthermore, the stimulatory effect on lipoxin formation induced by platelet agonists could be mimicked in intact platelets by the addition of low concentrations of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results indicate that the elevated lipoxin synthesis during platelet activation is due to stimulated 12-lipoxygenase activity induced by endogenously formed 12-HPETE.

Formation and effects of leukotrienes and lipoxins in human bone marrow.

The present results demonstrate leukotriene and lipoxin synthesis in human bone marrow and link these findings to biological effects in the same tissue. However, the mechanisms behind the described effects on myeloid progenitor cell growth are presently unknown. It is conceivable that both leukotrienes and lipoxins may act through modulation of endogenous cytokine production. However, it should be noted, that these lipoxygenase products totally failed to induce colony growth in the absence of GM-CSF. Moreover, the role of lipoxins in the bone marrow needs to be further clarified, since LXA4 induced both synergistic (with GM-CSF) and antagonistic (with LTC4) effects on progenitor cell growth. A possible pathophysiological role for leukotrienes and lipoxins may be suggested in chronic myelogenous leukemia. Thus, the capacity of hematological cells from CML patients to synthesize LTC4 was significantly increased. In addition, we have recently reported that CML platelets possessed a markedly decreased ability to participate in transcellular synthesis of the potential inhibitors of myelopoiesis, LXA4 and 5(S),12(S)-diHETE (Stenke et al., 1991b). Moreover, the production of these compounds was totally abolished in platelets obtained from CML patients in blastic crisis. Further studies should aim at defining the mechanisms behind the regulatory actions of leukotrienes and lipoxins in normal and leukemic human myelopoiesis.

Conversion of 5,6-dihydroxyeicosatetraenoic acids. A novel pathway for lipoxin formation by human platelets.

Leukotriene A4 may be metabolized to 5(S),6(R)- and 5(S),6(S)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acids by enzymatic or non-enzymatic hydrolysis. Incubation of human platelet suspensions with these dihydroxy acids led to the formation of lipoxin A4 and 6(S)-lipoxin A4 via lipoxygenation at C-15. Furthermore, human platelets converted the two 5(R),6(S)- and 5(R),6(R)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acids to tetraene-containing trihydroxyeicosatetraenoic acids. In contrast, leukotrienes C4, D4 and E4 were not transformed to cysteinyl-lipoxins. Time-course studies of leukotriene A4 metabolism in human platelet suspensions indicated lipoxin formation via two pathways: (i) direct conversion of leukotriene A4, leading to formation of the lipoxin intermediate 15-hydroxy-leukotriene A4; and (ii) 15-lipoxygenation of the 5(S),6(R)- and 5(S),6(S)-dihydroxyeicosatetraenoic acids. The results demonstrate that lipoxygenation at C-15 of 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids may be an alternative novel pathway for platelet-dependent lipoxin formation.

Deficient lipoxin synthesis: a novel platelet dysfunction in myeloproliferative disorders with special reference to blastic crisis of chronic myelogenous leukemia.

The capacity to convert exogenous leukotriene A4 to lipoxins (LXs) was investigated in platelet suspensions from patients with myeloproliferative disorders (MPD) (n = 22) and healthy control subjects (n = 14). Platelets isolated from the controls produced mainly LXA4, but also 6(S)-LXA4 and the all-trans isomers of lipoxins A4 and B4, as determined by high-performance liquid chromatography and computerized UV spectroscopy. In comparison to control levels, the mean LX synthesis was significantly lower in platelets from the MPD patients (438.7 +/- 62.8 and 157.4 +/- 31.2 pmol LXA4 per 10(9) platelets, respectively; mean +/- SEM; P = .0001). Platelets from six of the patients showed a particularly low capacity to produce LXs, resulting in LX levels below the detection limit or less than 7% of mean control levels. Notably, all these patients were in blastic crisis of chronic myelogenous leukemia (CML). This severely deficient LX production was paralleled by a dramatically attenuated conversion of arachidonic acid to 12-HETE (12-hydroxyheptadecatrienoic acid), a product formed via the prostaglandin endoperoxide synthase pathway, was normal. In addition, longitudinal studies of CML patients showed that blastic metamorphosis was associated with a markedly reduced capability to synthesize LXs, while this capacity improved after retransformation into a second chronic phase. The results reveal deficient LX synthesis as a novel platelet dysfunction in MPD, particularly in blastic crisis of CML in which an essentially abolished 12-lipoxygenase activity may be a general phenomenon.

Formation and proliferative effects of lipoxins in human bone marrow.

Lipoxins A4 and B4 together with the all-trans lipoxin (LX) isomers were produced by normal human bone marrow cell suspensions after incubation with ionophore A23187. Both LXA4 and LXB4 enhanced the growth of myeloid progenitor cells in semisolid agar in the presence of suboptimal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lipoxin A4 at 10(-10) M stimulated the colony formation in 13 out of 15 tested human bone marrows with a mean (+/- SEM) increase of 47 +/- 11% (p = 0.001). A similar stimulatory effect was observed after addition of LXB4 (10(-10) M). The monohydroxyeicosatetraenoic acids 5-, 12- and 15-HETE did not affect colony growth. In addition, LXA4 (10(-8) M) efficiently counteracted the increased colony formation induced by leukotriene C4 (10(-10) M), suggesting an antagonistic relationship between these lipoxygenase products. The results support a role for lipoxins in the regulation of human myelopoiesis.

On the mechanism of transcellular lipoxin formation in human platelets and granulocytes.

Endogenous arachidonic acid was converted to lipoxins A4, B4 and (6S)-lipoxin A4, in ionophore-A23187-stimulated mixtures of human platelets and granulocytes, while no lipoxins were formed when these cells were incubated separately. However, pure platelet suspensions transformed exogenous leukotriene A4 to lipoxins, including lipoxin A4 and (6S)-lipoxin A4, but not lipoxin B4. This compound was produced exclusively in the presence of granulocytes. A common unstable tetraene intermediate in lipoxin formation, 15-hydroxy-leukotriene A4 [5(6)-epoxy-15-hydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid], was indicated by trapping experiments with methanol. Thus, identical profiles of less polar tetraene-containing derivatives were formed from leukotriene A4 in platelet suspensions, from exogenous 15-hydroxyeicosatetraenoic acid in granulocyte suspensions and from endogenous substrate in mixed platelet/granulocyte suspensions. Evidence for the involvement of 12-lipoxygenase in platelet-dependent lipoxin formation was obtained. Thus, lipoxin synthesis from leukotriene A4 and 12-hydroxyeicosatetraenoic acid production from arachidonic acid by human platelets was equally inhibited by 15-hydroxyeicosatetraenoic acid with 50% inhibition obtained at 7.0 microM and 8.2 microM, respectively. In experiments with subcellular preparations from platelets, lipoxin synthesis was observed in both the particulate and soluble fraction and was paralleled by the 12-lipoxygenase activity. Furthermore, lipoxin formation from leukotriene A4 in platelet sonicates was dose-dependently inhibited by exogenous arachidonic acid. Finally, 12-lipoxygenase-deficient platelets from a patient with chronic myelogenous leukemia were totally unable to produce lipoxins from exogenous or granulocyte-derived leukotriene A4. It is concluded that the transcellular lipoxin synthesis is dependent on the platelet 12-lipoxygenase and proceeds via the unstable intermediate, 15-hydroxy-leukotriene A4. This tetraene epoxide is transformed to lipoxin B4 by a granulocyte epoxide hydrolase activity or to lipoxin A4 and lipoxins A4/B4 isomers by enzymatic or nonenzymatic hydrolysis.

Lipoxin formation in human nasal polyps and bronchial tissue.

Chopped human nasal polyps and bronchial tissue produced lipoxin A4 and isomers of lipoxins A4 and B4, but not lipoxin B4, after incubation with exogenous leukotriene A4. In addition, these tissues transformed arachidonic acid to 15-hydroxyeicosatetraenoic acid. The capacity per gram of tissue to produce lipoxins and 15-hydroxyeicosatetraenoic acid was 3-5-times higher in the nasal polyps. Neither tissue produced detectable levels of lipoxins or leukotrienes after incubation with ionophore A23187 and arachidonic acid. Co-incubation of nasal polyps and polymorphonuclear granulocytes with ionophore A23187 led to the formation of lipoxins, including lipoxins A4 and B4. The results indicate the involvement of an epithelial 15-lipoxygenase in lipoxin formation in human airways.