CAR, a Homing Peptide, Prolongs Pulmonary Preferential Vasodilation by Increasing Pulmonary Retention and Reducing Systemic Absorption of Liposomal Fasudil

Abstract

Here, we sought to elucidate the role of CAR (a cyclic peptide) in the accumulation and distribution of fasudil, a drug for pulmonary arterial hypertension (PAH), in rat lungs and in producing pulmonary specific vasodilation in PAH rats. As such, we prepared liposomes of fasudil and CAR-conjugated liposomal fasudil and assessed the liposomes for CAR conjugation, physical properties, entrapment efficiencies, in vitro release profiles, and stabilities upon incubation in cell culture media, storage, and aerosolization. We also studied the cellular uptake of fasudil in different formulations, quantified heparan sulfate (HS) in pulmonary arterial smooth muscle cells (PASMCs), and investigated the distribution of the liposomes in the lungs of PAH rats. We assessed the drug accumulation in a close and recirculating isolated perfused rat lung model and studied the pharmacokinetics and pharmacological efficacy of the drug and formulations in Sugen/hypoxia-induced PAH rats. The entrapment efficiency of the liposomal fasudil was 95.5 ± 4.5%, and the cumulative release was 93.95 ± 6.22%. The uptake of CAR liposomes by pulmonary arterial cells and their distribution and accumulation in the lungs were much greater than those of no-CAR-liposomes. CAR-induced increase in the cellular uptake was associated with an increase in HS expression by rat PAH-PASMCs. CAR, when conjugated with liposomal fasudil and given via an intratracheal instillation, extended the elimination half-life of the drug by four-fold compared with fasudil-in-no-CAR-liposomes given via the same route. CAR-conjugated liposomal fasudil, as opposed to fasudil-in-no-CAR-liposomes and CAR pretreatment followed by fasudil-in-no-CAR-liposomes, reduced the mean pulmonary arterial pressure by 40-50% for 6 h, without affecting the mean systemic arterial pressure. On the whole, this study suggests that CAR aids in concentrating the drug in the lungs, increasing the cellular uptake, extending the half-life of fasudil, and eliciting a pulmonary-specific vasodilation when the peptide remains conjugated on the liposomal surface, but not when CAR is given as a pretreatment or alone as an admixture with the drug.

Keywords: fasudil; isolated perfused rat lung; liposomes; peptide as a targeting moiety; pulmonary hypertension.

Figure 1.

Structure of targeted CAR-conjugated liposome containing fasudil.

Figure 2.

Chemical reactions involved in the conjugation of CAR peptide with the amino groups of the lipids of liposomes.

Figure 3.

IPRL system showing the routes of administration, reservoir for sampling and thoracic chamber. Arrow 1 and 2 show IV- and inhalation-mimicking routes, respectively.

Figure 4.

Changes in (A) size and (B) entrapment efficiency upon storage of various liposomal formulations for 28 days at 4 °C. In vitro release profiles of (C) fasudil-in-no-CAR and fasudil-in-CAR-liposomes in PBS at 37 °C (data represent mean ± SD, n = 3).

Figure 5.

Fasudil concentration in the perfusate after the treatments with the plain drug and various liposomal formulations of the drug given via the IV-mimicking route (cannulated pulmonary artery) to (A,C) sham and (B,D) PAH lungs. The dose of fasudil was 3 mg/kg (rat body weight) or liposomes containing equivalent amounts of fasudil and the dose of CAR was 0.5 mg/kg (data represent mean ± SD, n = 3).

Figure 6.

Fasudil concentration in the perfusate after the treatments with the plain drug and various liposomal formulations of the drug given via the inhalation-mimicking route (IT instillation) to (A,C) sham and (B,D) PAH lungs. The dose of fasudil was 3 mg/kg (rat body weight) or liposomes containing equivalent amount of fasudil and the dose of CAR was 0.5 mg/kg (data represent mean ± SD, n = 3).

Figure 7.

Fasudil in the lung homogenates after treating the lungs with plain fasudil, CAR pretreatment, and liposomal formulations of fasudil given via (A) IV- and (B) IT-mimicking routes to sham and PAH lungs. The dose of fasudil was 3 mg/kg (rat body weight) or liposomes containing equivalent amounts of fasudil and the dose of CAR was 0.5 mg/kg (data represent mean ± SD, n = 3, *p < 0.05).

Figure 8.

Fasudil concentration in the cell lysate. Plain fasudil or various liposomal formulations of fasudil were incubated with EC, SMCs, and FBCs collected from the pulmonary arteries of Sugen-hypoxia-induced PAH and control rats (data represent mean ± SD, n = 3; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.001).

Figure 9.

Concentration of HS in the cell lysate of SMCs collected from the pulmonary arteries of Sugen-hypoxia-induced PAH and control rats, (data represent mean ± SD, n = 3; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.001).

Figure 10.

Representative fluorescence microscopy images showing the uptake of liposomes by PAH-SMCs after (A) immediate and (B) 24 h of incubation of (i) fluorescent liposomes without CAR peptide and (II) fluorescent CAR-conjugated liposomes in cell culture media. Green, beta actin; blue, DAPI; red, rhodamine B-labeled liposomes. The rightmost panel shows the overlay.

Figure 11.

Images of the lungs treated with fluorescent liposomes: (A) whole lung: (i) no treatment, (II) liposomes without CAR, and (III) CAR-conjugated liposomes. (B) Clearance of various formulations of the liposomes from PAH-induced rat lungs after IT administration. (C) Images of lung tissue sections collected 24 h after administration of fluorescent particles: (I) liposomes without CAR, and (II) CAR-conjugated liposomes (green, anti-prosurfactant protein C; blue, DAPI; red, rhodamine B-labeled liposomes). (Data represent mean ± SD, n = 3; *p < 0.05).

Figure 12.

Absorption profiles of fasudil and liposomal formulations of fasudil given intravenously to (A) sham and (B) PAH rats and those given intratracheally to (C) sham and (D) PAH rats. The dose of fasudil was 3 mg/kg (rat body weight) or liposomes containing equivalent amount of fasudil and the dose of CAR was 0.5 mg/kg (data represent mean ± SD, n = 3).

Figure 13.

Effect of liposomal fasudil on the ex vivo PAP of lungs collected from Sugen/hypoxia PAH rats, determined using IPRL model. The percent reduction of initial ex vivo PAP after administration of various forms of liposomal fasudil via (A) IV- and (B) IT-mimicking routes. The dose of fasudil was 3 mg/kg (rat body weight) or liposomes containing equivalent amount of fasudil and the dose of CAR was 0.5 mg/kg (data represent mean ± SD, n = 3).

Figure 14.

Effect of liposomal fasudil on the pulmonary hemodynamics in Sugen/hypoxia PAH rats after administration of (A) fasudil in no-CAR-liposomes, (B) CAR pretreatment followed by fasudil in no-CAR-liposomes and (C) fasudil in CAR-liposomes. (D) Targeting indices of fasudil after IT administration in various forms. The dose of fasudil was 3 mg/kg (rat body weight) or liposomes containing equivalent amount of fasudil and the dose of CAR was 0.5 mg/kg (data represent mean ± SD, n = 3; *p < 0.05, ^§p < 0.005, Ƶp < 0.001).