The plasma half-life of hirudin is 60 min after IV injection, and 120 min after subcutaneous injection. Hirudin is cleared via the kidneys and should not be used in patients with renal insufficiency. The anticoagulant effect of hirudin can be monitored using the activated partial thromboplastin time (APTT).

Recombinant hirudin (lepirudin) is licensed for the treatment of arterial or venous thrombosis complicating heparin-induced thrombocytopenia, and as an alternative for heparin for cardiopulmonary bypass surgery in these patients. Hirudin has been evaluated in acute coronary syndromes and, to a lesser extent, for the prevention and treatment of venous thrombosis. The results for each of these indications will be discussed separately.

Unstable angina. The largest phase II study to compare hirudin with heparin, the Organization to Assess Strategies for Ischemic Syndromes (OASIS)-1 pilot trial, randomized 909 patients with unstable angina or non-ST-elevation MI to receive a 72-h infusion of hirudin, in either a low dose or medium dose, or to heparin. Doses of hirudin and heparin were adjusted to maintain the APTT between 60 and 100 s. Compared with heparin, hirudin produced a promising reduction in the primary outcome, a composite of cardiovascular death, MI, or refractory angina at 7 days (odds ratio [OR] 0.57; 95% confidence interval [CI], 0.32 to 1.02) and a significant reduction in the secondary outcome, a composite of death, MI, or refractory or severe angina requiring revascularization at 7 days (OR, 0.49; 95% CI, 0.27 to 0.86). Major bleeding occurred in about 1% of patients in both treatment groups and was not significantly higher in those who received hirudin (OR, 0.86; 95% CI, 0.23 to 3.19). Minor bleeding, however, was more frequent in patients given a medium or low dose of hirudin than it was in those treated with heparin (21.3%, 16.2%, and 10.5%, respectively), and the differences were statistically significant (medium-dose or low-dose hirudin, p = 0.033; heparin, p = 0.001).

The results of the OASIS-1 trial prompted the OASIS-2 trial, a phase III trial that randomized 10,141 patients with unstable angina or non-ST-elevation MI to receive a 72-h infusion of medium-dose hirudin or heparin. During treatment, hirudin produced a significant reduction in the composite end point of death or MI compared with heparin (2.0% and 2.6%, respectively; OR, 0.76, 95% CI, 0.59-0.99). Although the primary outcomes, a composite of death or MI at 7 days (3.6% and 4.2%, respectively; OR, 0.84; 95% CI, 0.69 to 1.02) and 35 days (6.8% and 7.7%, respectively; OR, 0.87; 95% CI, 0.75 to 1.01) were not significantly different between the two groups, the absolute risk reduction in death or MI produced by hirudin during the 72-h treatment period was maintained at 7 and 35 days. Major bleeding occurred more frequently with hirudin therapy than with heparin therapy (1.2% and 0.7%, respectively; OR, 1.73; 95% CI, 1.13 to 2.63), but the rates of life-threatening bleeding were similar (0.4% in both groups).

In an open-label, dose-escalation study, soluble thrombomodulin attenuated coagulation abnormalities in patients with disseminated intravascular coagulation. Soluble thrombomodulin has recently been evaluated in a phase II dose-ranging study in patients undergoing elective hip arthroplasty. Patients were given thrombomodulin subcutaneously (dose, 0.3 or 0.45 mg/kg) 2 to 4 h after surgery. Those patients receiving the lower dose of thrombomodulin received a second injection 5 days later. The primary end point, a composite of venographically detected DVT and symptomatic venous thromboembolism, occurred in 4.3% of the 94 patients who received the lower dose of soluble thrombomodulin and in none of the 99 patients receiving the higher dose. Major bleeding occurred in 1.6% and 5.7%, respectively, of patients receiving the low dose or high dose of soluble thrombomodulin. Phase III clinical trials are necessary to compare soluble thrombomodulin with other forms of thromboprophylaxis, such as low-molecular-weight heparin or fondaparinux.

3.2 Inhibitors of fibrin formation

Thrombin, the enzyme that converts fibrinogen to fibrin, can be inhibited indirectly or directly. Indirect thrombin inhibitors act by catalyzing antithrombin and/or heparin cofactor II. In contrast, direct inhibitors bind directly to thrombin and block its interaction with substrates. All of the new agents that block fibrin formation are direct thrombin inhibitors.

Direct thrombin inhibitors have properties that give them potential mechanistic advantages over indirect thrombin inhibitors, such as heparin. First, because direct thrombin inhibitors do not bind to plasma proteins, they produce a more predictable anticoagulant response. Second, unlike heparin, direct thrombin inhibitors do not bind to PF4. Consequently, the anticoagulant activity of direct thrombin inhibitors is unaffected by the large quantities of PF4 released in the vicinity of platelet-rich thrombi. Finally, direct thrombin inhibitors inactivate fibrin-bound thrombin, as well as fluid-phase thrombin.

Three parenteral direct thrombin inhibitors (ie, hirudin, argatroban, and bivalirudin) have been licensed in North America for limited indications. Hirudin and argatroban are approved for the treatment of patients with heparin-induced thrombocytopenia, whereas bivalirudin is licensed as an alternative to heparin in patients undergoing percutaneous coronary interventions. Ximelagatran, a prodrug of melagatran, is the first orally available direct thrombin inhibitor.

3.2.2 Hirudin

A 65-amino acid polypeptide originally isolated from the salivary glands of the medicinal leech Hirudo medici-nalis, hirudin is now available in recombinant form. Hirudin inhibits thrombin in a bivalent fashion. Its globular amino-terminal domain interacts with the active site of thrombin, whereas its anionic carboxy-terminal tail binds to exosite 1 on thrombin, the substrate recognition site. The hirudin/thrombin complex is essentially irreversible, which is a potential drawback because there is no specific antidote.

Abbreviations: ALI = air-liquid interface; AP = apical; BL = basolateral; CCK-8 = cell counting kit-8; CF = cystic fibrosis; DEX = dexamethasone; DMSO = dimethylsulfoxide; DPB = diffuse panbronchiolitis; ERK = extracellular-regulated protein kinase; ETT = endotrachial tube; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; HRP = horseradish peroxidase; JNK = c-Jun NH₂– terminal protein kinase; LPS = Lipopolysaccharide; MAPK = mitogen-activated protein kinase; MCT = mucociliary transport; MEK = mitogen-activated protein kinase/extracellular-regulated protein kinase kinase; NF-kB = nuclear factor-кВ; NHBE = normal human bronchial epithelial; PD98059 = 2′-amino-3′-methoxyflavone; SAPK = stress-activated protein kinase; TLR4 = toll-like receptor 4.

In response to bacterial invasion, mucociliary clearance is stimulated, and inflammatory mediators and cytokines are secreted as a defense, but these also can damage the airway. Among epithelial-derived pleiotropic cytokines, IL-8, a member of the cysteine-X-cysteine chemokine family, acts as one of the most potent neutrophil chemoattractants. Neutrophil-dominated inflammation is a characteristic of COPD, diffuse panbronchiolitis (DPB), and cystic fibrosis (CF). IL-8 is produced by airway epithelial cells. Increased IL-8 in sputum and BAL fluid is associated with the severity of DPB and CF,-  and there is increased IL-8 gene expression in the bronchial epithelium of subjects with severe asthma and COPD.

Proinflammatory cytokines, bacterial flagellin, and lipopolysaccharide (LPS) can increase IL-8 production by normal human bronchial epithelial (NHBE) cells. Among the many agents present in organic dusts, LPS is a major inducer of the inflammatory reaction.¹⁰ LPS binds to toll-like receptor 4 (TLR4), which activates intracellular signaling pathways, including the nuclear factor-kB (NK-kB) pathway, phosphatidylinositol 3 kinase, and mitogen-activated protein kinase (MAPK) pathways. Three MAPK pathways contribute to IL-8 gene expression: the extracellular-regulated protein kinase (ERK), c-Jun NH₂-terminal protein kinase (JNK), and p38 MAPK cascades. The relative degree of activation of each pathway and the functional consequences differ among cell types and experimental systems.

Macrolide antibiotics decrease neutrophils and IL-8 concentration in BAL of subjects with DPB- and sputum IL-8 concentration in CF. Macrolides can inhibit IL-8 release from airway epithelial cells in culture through inactivation of ERK or NK-kB.

Dapsone, a synthetic sulfone, has been used to treat leprosy, Pneumocystis jiroveci pneumonia, and malaria. Dapsone also is recognized as an antiinflammatory drug and has been used systemically and topically to treat skin diseases like dermatitis herpetiformis, which is characterized by neutrophil-dominated inflammation. We speculated that dapsone would inhibit IL-8 secretion by stimulated airway cells; therefore, we studied the effect of dap-sone on IL-8 secretion from NHBE cells stimulated with LPS and further investigated the signaling pathways involved. We then evaluated the effectiveness of dapsone in decreasing airway neutrophil recruitment and preserving mucociliary clearance when administered orally or as an aerosol to ferrets with airways that had been exposed to (ie, inflamed by) LPS.

Materials and Methods Cheap tadalafil Canada

Reagents

Dapsone (4,4′-diaminodiphenyl sulfone), LPS (Escherichia coli serotype 0111:B4), and all other reagents were purchased from Sigma-Aldrich Corp (St Louis, Missouri), unless otherwise indicated. PD98059 (2 – -amino-3′-methoxyflavone), an MAPK/ERK kinase (MEK) inhibitor (an upstream kinase of ERK1/2) was obtained from Calbiochem (La Jolla, California). Phospho- and nonphospho-specific ERK1/2, anti-p38 MAPK, anti-stress-activated protein kinase (SAPK)/JNK, and phospho-specific NF-kB p65 (Ser536) as well as anti-rabbit-IgG horseradish peroxidase (HRP) antibodies were purchased from Cell Signaling Technology, Inc (Beverly, Massachusetts). Dimethylsulfoxide (DMSO) was used as a solvent of dapsone, and the final concentration did not exceed 0.01% (v/v). Preliminary in vitro experiments showed that 0.01% DMSO medium had no significant effect on cell viability and IL-8 secretion for up to 72 h (data not shown).

NHBE cells (Lonza Walkersville, Inc; Walkersville, Maryland) were plated at 3,500 cells/cm- in culture dishes in bronchial epithelial cell growth medium supplemented with the single-quot kit (Lonza Walkersville, Inc) without antibiotics and cultured at 37°C in a 5% CO- incubator. We used endotoxin-free media (< 0.005 endotoxin units/mL) and second-passage cells for all experiments. Cells were grown to confluence for 6 days. Cultures without antibiotics were then transferred to six-well or 35-mm dishes coated with type 1 rat-tail collagen and seeded at 3,500 cells/cm². The medium was changed every 24 h. To avoid influence of growth factors on cell signaling and IL-8 secretion, cells were cultured in supplement-free bronchial epithelial cell basal medium for 24 h before stimulation. We evaluated cell response at the time of cell confluence rather than normalized to the relative number of cells because cell maturation could affect cell signaling and cytokine secretion, and at confluence, all cells are at similar growth stages.

For NHBE cell differentiation, cells were plated at 2.0 X 10⁵ cells/cm² onto polycarbonate inserts of 6.5-mm diameter, 0.4-pm pore size, and 10-pm thickness (Transwell Clear; Corning Costar; Cambridge, Massachusetts); coated with type 1 rat-tail collagen; and cultured with serum-free Dulbecco modified eagle medium:nutrient mixture F-12 medium containing 1.0% ITS-A (Invitrogen Corp; Carlsbad, California), 0.5 ng/mL recombinant human epidermal growth factor (Invitrogen Corp), 10 ng/mL triiodothyronine (MP Biomedicals LLC; Solon, Ohio), 0.5 mg/mL hydrocortisone (MP Biomedicals LLC), 1.0 X 10^2? M all-trans retinoic acid (Sigma-Aldrich Corp), 2.0 mg/mL bovine serum albumin (Sigma-Aldrich Corp), and 30 mg/mL bovine pituitary extract (Invitrogen Corp). After achieving confluence, the apical medium was removed, and cells were cultured with an air-liquid interface (ALI) method Canadian Generic viagra. The culture medium was changed every 48 h, and cells were maintained at 37°C in 5% CO- for 10 to 14 days.