ABSTRACT
BACKGROUND: Disruption of fibrinolytic homeostasis participates in the pathogenesis of severe lung diseases like acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF) and plastic bronchitis. We have developed a pulmonary formulation of tissue plasminogen activator (pf-tPA) that withstands nebulization and reaches the lower airways. OBJECTIVE: Since treatment of ARDS, IPF and plastic bronchitis will require repeated administration of pf-tPA, the purpose of this study was to determine the safety of prolonged, repeated administration of pf-mouse tPA (pf-mtPA) to the lungs of healthy mice. METHODS: Male and female B6C3F1 mice received one of two intratracheal (IT) doses of either nebulized pf-mtPA or sterile saline twice daily for 28 days. Weekly blood samples were collected to estimate hematocrit. Following the dosing period, animals were sacrificed for gross necropsy, the acquisition of bronchoalveolar lavage fluid (BALF), and histological assessment of the lungs and other major organs. RESULTS: The low dose of pf-mtPA was well tolerated by both female and male mice. However, female and male mice that received the high dose experienced a 16% and 8% incidence, respectively, of fatal pulmonary hemorrhage. Although male mice had a lower incidence of bleeding, these events occurred at lower mean (+/-S.E.) doses (1.06+/-0.02mg/kg/d) of pf-mtPA compared with females (1.48+/-0.03mg/kg/d, p<0.001). In addition, male mice had higher BALF mtPA concentrations. Bleeding occurred six and 12 days in male and female mice, respectively, after the initiation of dosing suggesting that mtPA accumulated in the lungs. CONCLUSION: This study established a safe dose range and demonstrated the feasibility of prolonged, repeated dosing of pf-tPA. High doses (> or =1mg/kg/d) were associated with pulmonary hemorrhage that may be due, in part, to accumulation of drug in the lungs.
Subject(s)
Fibrinolytic Agents/toxicity , Hemorrhage/chemically induced , Lung/drug effects , Tissue Plasminogen Activator/toxicity , Administration, Inhalation , Animals , Bronchoalveolar Lavage Fluid/chemistry , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Delivery Systems , Female , Fibrinolytic Agents/administration & dosage , Fibrinolytic Agents/pharmacokinetics , Lung/metabolism , Lung Diseases/drug therapy , Male , Mice , Sex Factors , Tissue Distribution , Tissue Plasminogen Activator/administration & dosage , Tissue Plasminogen Activator/pharmacokineticsABSTRACT
Cell adhesion molecules are critical in monocyte (MN) recruitment in immune-mediated and hematologic diseases. We investigated the novel role of recombinant human migration inhibitory factor (rhMIF) in up-regulating vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and their signaling pathways in human MNs. rhMIF-induced expression of VCAM-1 and ICAM-1 was significantly higher compared with nonstimulated MNs. rhMIF induced MN VCAM-1 and ICAM-1 expression in a concentration-dependent manner (P < .05). Antisense oligodeoxynucleotides (ODNs) and inhibitors of Src, PI3K, p38, and NFkappaB significantly reduced rhMIF-induced MN VCAM-1 and ICAM-1 expression (P < .05). However, Erk1/2 and Jak2 were not involved. Silencing RNA directed against MIF, and inhibitors of Src, PI3K, NFkappaB, anti-VCAM-1, and anti-ICAM-1 significantly inhibited rhMIF-induced adhesion of HL-60 cells to human dermal microvascular endothelial cells (HMVECs) or an endothelial cell line, HMEC-1, in cell adhesion assays, suggesting the functional significance of MIF-induced adhesion molecules (P < .05). rhMIF also activated MN phospho-Src, -Akt, and -NFkappaB in a time-dependent manner. rhMIF induced VCAM-1 and ICAM-1 up-regulation in 12 hours via Src, PI3K, and NFkappaB as shown by Western blotting and immunofluorescence. MIF and MIF-dependent signaling pathways may be a potential target for treating diseases characterized by up-regulation of cell adhesion molecules.
