Purpose: CAR, a cyclic peptide (CARSKNKDC), is known to translocate various small drug molecules into the cell cytoplasm. We have previously shown that CAR accumulates on pulmonary arterial endothelial cells upon binding with cell surface heparan sulfate that is overexpressed in the lung vasculature afflicted with pulmonary arterial hypertension (PAH), a progressive disease characterized by elevate pulmonary arterial pressure. Here, we tested the hypothesis that an isolated perfused rat lung (IPRL) model generates consistent and accurate information on lung specific delivery of targetable and inhaled formulations. We tested this assumption by formulating fasudil, a Rho-kinase inhibitor, in CAR-modified liposomes using lungs from both PAH-afflicted and healthy rats.
Methods: We first prepared liposomal fasudil by a thin film hydration-extrusion method, and encapsulated the drug by active loading and then conjugated CAR on the surface of liposomes. In vitro release profiles were evaluated in phosphate buffer saline using molecular weight cutoff (3.5K) cassettes. After assessing the formulations for various physicochemical properties, we studied drug accumulation in the lungs, in-vivo and ex-vivo. We used a Sugen/hypoxia to develop PAH rats. Collecting lungs from both healthy and PAH rats, we cannulated the pulmonary vein and artery, and the trachea, and finally placed the cannulated lungs in an artificial thoracic chamber. We then administered the drug or the formulations via the cannulated pulmonary artery or trachea and thus to mimic the intravenous and pulmonary routes, respectively. Using a validated LC-MS/MS method, we measured the drug in the perfusates for two hours after dosing. We then removed the lung from the thoracic chamber, homogenized and analyzed the drug content in the homogenates too. In vivo absorption profiles of the fasudil in unmodified liposome (plain liposome), CAR pretreatment followed by fasudil in unmodified liposomes (CAR then plain liposome), and fasudil in CAR modified liposomes (modified liposome) were evaluated using PAH induced male Sprague−Dawley rats. We studied the pharmacological efficacy of plain liposome, CAR the plain liposome, and modified liposome in two models: ex vivo efficacy through IPRL and pressure transducer P75, and in vivo efficacy using SUGEN 5416/hypoxia-induced rodent models of PAH.
Results: We first optimized the entrapment efficiency of fasudil in liposomes using passive and active loading methods and by varying lipid formation and hydration condition. We increased entrapment efficiency 1.5 folds (Fig. 1A). Fasudil was released in a controlled pattern from the unmodified and CAR modified liposomes with a cumulative release of 93.95±6.22 and 83.87±4.58% drug, respectively (Fig. 1B), which is not different significantly.
IPRL data showed that the accumulation of fasudil in CAR modified liposome after IT administration over PAH rats was significantly greater that other groups; it shows only 1.5 µg of drug in the perfusate (around 96% accumulation in the lung), however plain liposome and CAR then plain liposome shows less fasudil accumulation after IT administration. Fig. 2 showed fasudil accumulation in PAH lungs were meaningfully higher that healthy lungs. Finally, this figure clarified that IT administration increase drug accumulation in the lungs than IV administration. We quantified the amount of fasudil remained in the lung by homogenizing the tissue. The amount of fasudil remaining in the lungs treated with CAR modified liposomes were 2 and 4 times higher than lungs received plain liposome and CAR then plain liposomes (Fig. 3A and B). In vivo absorption profiles confirmed that inhalation CAR modified liposomes improved the pharmacokinetic of Fasudil. The plasme half-life of fasudil in plain liposomes after IT administration were 4.7 and 6.9 hr, which were increased by 3-4-fold upon IT administration in modified liposome Fig. 3F.
Ex vivo efficacy suggested that fasudil in modified liposome decrease mPAP (mean pulmonary arterial pressure) much higher (85% reduction) than other groups (Fig. 3C and D). In vivo model also showed 60% mPAP reduction for 10 hours without significant change of systemic blood pressure (Fig. 3E).
IPRL, lung homogenate, and in-vivo data showed that IT administration limit the drugs’ systemic exposure and enhance fasudil residence time the lung. Plain drugs absorbed very fast via the lung epithelium, whereas the drug in the formulations remained in the lungs for a much longer period. Although the concentration of the plain drug increased in the perfusate over time, the concentrations of the drug released from liposomes plateaued, suggesting an enhanced retention in the lungs.
Conclusion: This study establishes that IPRL model can be used for assessing drug distribution in the lung both after intravenous and intratracheal administrations. It proved that CAR-conjugated inhalable liposomal fasudil offers favorable pharmacokinetics and noninvasive, controlled release and pulmonary preferential treatment of PAH.
Ahmed Alobaida– Graduate Student/Research Assistant Ph.D. candidate, Texas Tech University Health Sciences Center, Amarillo
Taslim Al-Hilal– Post Doctorate Research Associate, Texas Tech University Health Sciences Center, Amarillo, Texas
Fakhrul Ahsan– Texas Tech University Health Sciences Center, Amarillo, Texas