Inhaled sildenafil as an alternative to oral sildenafil in the treatment of pulmonary arterial hypertension (PAH)

Abstract

The practice of treating PAH patients with oral or intravenous sildenafil suffers from the limitations of short dosing intervals, peripheral vasodilation, unwanted side effects, and restricted use in pediatric patients. In this study, we sought to test the hypothesis that inhalable poly(lactic-co-glycolic acid) (PLGA) particles of sildenafil prolong the release of the drug, produce pulmonary specific vasodilation, reduce the systemic exposure of the drug, and may be used as an alternative to oral sildenafil in the treatment of PAH. Thus, we prepared porous PLGA particles of sildenafil using a water-in-oil-in-water double emulsion solvent evaporation method with polyethyleneimine (PEI) as a porosigen and characterized the formulations for surface morphology, respirability, in-vitro drug release, and evaluated for in vivo absorption, alveolar macrophage uptake, and safety. PEI increased the particle porosity, drug entrapment, and produced drug release for 36h. Fluorescent particles showed reduced uptake by alveolar macrophages. The polymeric particles were safe to rat pulmonary arterial smooth muscle cell and to the lungs, as evidenced by the cytotoxicity assay and analyses of the injury markers in the bronchoalveolar lavage fluid, respectively. Intratracheally administered sildenafil particles elicited more pulmonary specific and sustained vasodilation in SUGEN-5416/hypoxia-induced PAH rats than oral, intravenous, or intratracheal plain sildenafil did, when administered at the same dose. Overall, true to the hypothesis, this study shows that inhaled PLGA particles of sildenafil can be administered, as a substitute for oral form of sildenafil, at a reduced dose and longer dosing interval.

Keywords: Controlled release; Inhalation; PLGA; Phosphodiesterase 5; Pulmonary arterial hypertension; Sildenafil.

Figure 1

The morphology of the sildenafil particles prepared with different PLGA co-polymers and varying amounts of PEI in the internal aqueous phase. For compositions of the particles, please see Table 1.

Figure 2

Size (A), mass median aerodynamic diameters (B) and zeta potentials (C) of PLGA particles of sildenafil (n=3). For compositions of the particles, please see Table 1. *p<0.05, §p<0.005, Ƶp<0.001, ¥p<0.0001.

Figure 3

Drug entrapment efficiency of sildenafil particles prepared with different PLGA co-polymers and varying concentration PEI used in the internal aqueous phase (A). Cumulative release of sildenafil from particles prepared with PLGA 50:50 (B); and PLGA 75:25 (C) (n=3) *p<0.05, §p<0.005, Ƶp<0.001, ¥p<0.0001.

Figure 4

Uptake of the different formulations by the rat alveolar macrophages: qualitative analysis with fluorescent microscopy (A), quantitative analysis using flow cytometer (B) (n=3) *p<0.05.

Figure 5

The safety of the sildenafil particulate formulations: cytotoxicity when treated with rat pulmonary arterial smooth muscle cells (A); the enzymatic markers in the bronchoalveolar lavage fluid after intra-tracheal administration (B); release of inflammatory cytokines IL6(C) and TNF-alpha (D) 8 hours after intra-tracheal administration (n=3) *p<0.05, §p<0.005, Ƶp<0.001.

Figure 6

Changes in plasma concentration of sildenafil after pulmonary administration of 0.5 mg/kg sildenafil in the following forms: (i) plain intravenous, (ii) plain intra-tracheal, (iii) plain oral and (i) PLGA particles of sildenafil intra-tracheal (n=3–4). Analysis was performed using two-way repeated measure ANOVA with point-to-point multiple comparisons. Statistical significance was presented when plain intratracheal sildenafil are compared with intratracheal PLGA particles; §p<0.005, Ƶp<0.001, ¥p<0.0001.

Figure 7

Changes in the mean pulmonary arterial pressure (mPAP) and the mean systemic arterial pressure (mSAP) in SUGEN-5416/hypoxia induced PAH animals after administration of 0.5 mg/kg sildenafil in the following forms: no drug, saline (A), plain intravenous (B), plain intra-tracheal (C), plain oral (D), PLGA particles of sildenafil, intra-tracheal (E) (n=3–4). The plots comparing the changes in mPAP (F) and mSAP (G), when treated with various forms of sildenafil. Data represent mean ± SD. The differences between mPAP and mSAP of sildenafil-PLGA-particle groups and that among various treatments were analyzed by two-way repeated-measure ANOVA; *p<0.05, §p<0.005, Ƶp<0.001, ¥p<0.0001. ‡ shows statistically significant differences (at p<0.05) between sildenafil-PLGA-particle treated group and other treatment groups.