Purpose-This study was conducted to assess the feasibility of a pulmonary formulation of tissue plasminogen activator (tPA) for nebulization into the airway by measuring protein stability, biologic activity, particle size, and estimating human lung distribution.Methods-Formulations were derived by varying the surfactant and protein concentrations. Protein stability and recovery of each nebulized tPA formulation were assessed by ultraviolet spectroscopy. Formulations that met protein stability feasibility criteria were assessed for biologic and fibrinolytic activities. Biologic activity was determined by their ability to inhibit superoxide anion production by human neutrophils. Fibrinolytic activity was assessed by the cleavage of plasminogen to plasmin. Aerodynamic properties were assessed using a cascade impactor, and an estimation of human airway deposition was made via a human lung replica.Results-Twenty-seven tPA formulations were initially assessed, 15 of which met protein stability criteria. Subsequently, three of these formulations maintained biologic and fibrinolytic activities. These formulations exhibited particle sizes of 2.4-3.1 μm, and had respirable doses ≥65%. A formulation of 1 mg mL −1 tPA and 0.1% Tween 80 exhibited a 45% deposition in the lower airways of a human lung replica.Conclusions-A suitable pulmonary tPA formulation was identified that, following nebulization, maintained protein stability as well as biologic and fibrinolytic activities, and resulted in an optimal respirable dose and human airway deposition. This formulation may be applicable in the treatment of lung diseases, such as acute respiratory distress syndrome by permitting targeted pulmonary delivery of a therapeutic protein to the lungs.
Summary
1.Tissue plasminogen activator (tPA) has both fibrinolytic and anti-inflammatory activity.These properties may be useful in treating inflammatory lung diseases, such as acute respiratory distress syndrome (ARDS).
2.We have previously demonstrated the feasibility of targeted pulmonary delivery of tPA. As part of our research to develop a clinically viable pulmonary formulation of tPA, we assessed the tolerability and incidence of haemorrhage associated with the administration of a pulmonary formulation of mouse tPA (pf-mtPA).
3.Intratracheal doses of nebulized pf-mtPA or sterile saline were administered with increasing frequency to male and female B6C3F1 mice. After dosing, the mice entered a recovery period, after which they were killed and their lungs were lavaged and harvested. Postmortem gross necropsy was performed and all major organs were assessed histologically for haemorrhage. The bronchoalveolar lavage fluid was assessed for markers of lung injury.
4.Mouse tPA that was formulated to mimic a previously characterized human pf-tPA was well tolerated when given intratracheally with increasing dosing frequency. The administration of pf-mtPA did not result in any detectable haemorrhagic-related events or signs of lung injury.
5.The results of the present longitudinal study demonstrate that a maximally feasible dose of pf-mtPA (3 mg/kg) can be given frequently over a short period of time (12 h) without haemorrhagic complications. Although these data were generated in a healthy mouse model, they provide support for the continued evaluation of pf-tPA for the treatment of pulmonary diseases, such as ARDS.
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