Fakhrul Ahsan

Inhalable Large Porous Microspheres of Low Molecular Weight Heparin: In Vitro and In Vivo Evaluation

This study tests the feasibility of large porous particles as long-acting carriers for pulmonary delivery of low molecular weight heparin (LMWH). Microspheres were prepared with a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA), by a double-emulsion-solvent-evaporation technique. The drug entrapment efficiencies of the microspheres were increased by modifying them with three different additivespolyethyleneimine (PEI), Span 60 and stearylamine. The resulting microspheres were evaluated for morphology, size, zeta potential, density, in vitro drug-release properties, cytotoxicity, and for pulmonary absorption in vivo. Scanning electron microscopic examination suggests that the porosity of the particles increased with the increase in aqueous volume fraction. The amount of aqueous volume fraction and the type of core-modifying agent added to the aqueous interior had varying degrees of effect on the size, density and aerodynamic diameter of the particles. When PEI was incorporated in the internal aqueous phase, the entrapment efficiency was increased from 16.22+/-1.32% to 54.82+/-2.79%. The amount of drug released in the initial burst phase and the release-rate constant for the core-modified microspheres were greater than those for the plain microspheres. After pulmonary administration, the half-life of the drug from the PEI- and stearylamine-modified microspheres was increased by 5- to 6-fold compared to the drug entrapped in plain microspheres. The viability of Calu-3 cells was not adversely affected when incubated with the microspheres. Overall, the data presented here suggest that the newly developed porous microspheres of LMWH have the potential to be used in a form deliverable by dry-powder inhaler as an alternative to multiple parenteral administrations of LMWH.

Aerosolized PLA and PLGA nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis B vaccine.

Porous poly(L-lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) nanoparticles were tested for pulmonary delivery of hepatitis B vaccine. In particular, the effects of particle size and hydrophobicity on mucosal and cell-mediated immune responses were investigated. Three formulations of PLA and PLGA nanoparticles containing a fixed amount of hepatitis B surface antigen (HBsAg) were prepared by a double-emulsion-solvent-evaporation method and characterized for surface morphology, charge, size, density and in vitro release. The immune responses were studied by measuring secretory IgA levels in mucosal fluids and quantitating cytokine levels in rat spleen homogenates. Particle uptake was studied in rat alveolar macrophages. Scanning electron microscopy revealed particles with smooth surfaces. Zeta potential measurements indicated that the particles carried negative surface charges. The antigen was continuously released for 42 days in phosphate buffer. Hydrophobic particles >500 nm elicited a more robust increase in secretary IgA, interleukin-2 and interferon-γ levels compared to hydrophilic particles <500 nm. Large hydrophobic particles were more efficiently internalized by rat alveolar macrophages compared to smaller hydrophilic particles. Calu-3 cell viability studies indicate that the viability of cells is not affected by nanoparticulate formulations. This study demonstrates that inhalable nanoparticles of HBsAg produce an enhancement of immune responses.

PEG-PLGA based large porous particles for pulmonary delivery of a highly soluble drug, low molecular weight heparin.

This study was designed to evaluate the feasibility of PEG-PLGA copolymers as carriers for pulmonary delivery of a highly soluble drug. We attempt to address the limitations of low entrapment efficiencies and poor release profiles that are associated with the use of conventional PLGAs. We have used low molecular weight heparin (LMWH) as a model for highly soluble and ionizable drugs and a 3 × 3 full factorial design to prepare nine formulations. We considered polymer type and percent NaCl in external phase as two independent variables at three different levels; the levels for polymer type were PLGA, PEG-PLGA and PLGA-PEG-PLGA and that for percent NaCl were 0%, 5% and 8%. Formulations were characterized for various physical properties, respirability, drug release, and evaluated for in vivo absorption, alveolar uptake, and safety. Statistical analyses suggest that both polymer type and salt concentration influenced the morphology and micromeritic properties of the particles. Compared with PLGA, PEG-PLGA copolymers produced inherently larger porous particles with high drug entrapment and a greater extent of drug release. Moreover, addition of NaCl in the external phase maximized drug entrapment but minimized burst release and produced smaller and denser particles. Fluorescent PEG-PLGA particles showed reduced uptake by alveolar macrophages, and exhibited a uniform distribution in the lungs compared with PLGA particles. Further, ~85% of the particles were cleared off the lungs within 6 days. Intratracheally administered PEG-PLGA based optimized formulation exhibited a biological half-life of 18.64 h, which was ~4.5 times longer than plain LMWH. No cytotoxic effect was observed when bronchial epithelial cells were incubated with PEG-PLGA based formulations. Similarly, no increase in the injury markers was observed in the bronchoalveolar lavage fluids collected from rats treated with PEG-PLGA particles of LMWH. Overall, this study suggests that PEG-PLGA block copolymers have the potential to be developed as efficient and biocompatible carriers for pulmonary delivery of highly water-soluble drugs.

Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses

This study investigates the efficacy of surface-modified microspheres of hepatitis B surface antigen (HBsAg) in eliciting systemic and mucosal immune responses. Positively charged poly(D,L-lactic-co-glycolic acid) microspheres were prepared by a double-emulsion solvent-evaporation method with cationic agents--stearylamine and polyethylenimine--in the external aqueous phase. Formulations were characterized for morphology, size, density, aerodynamic diameter, entrapment efficiency and in vitro drug-release profile. Immunization was performed after pulmonary administration of the formulations to female Sprague-Dawley rats and the immune response was monitored by measuring IgG levels in serum and secretory (sIgA) levels in salivary, vaginal and bronchoalveolar lavage fluids. The cell-mediated immune response was studied by measuring cytokine levels in spleen homogenates, and a cytotoxicity study was performed with Calu-3 cell line. The aerodynamic diameter of the particles was within the respirable range, with the exception of stearylamine-modified particles. Zeta potential values moved from negative (-6.76 mV) for unmodified formulations to positive (+0.515 mV) for polyethylenimine-modified particles. Compared to unmodified formulations, polyethylenimine-based formulations showed continuous release of antigen over a period of 28-42 days and increased levels of IgG in serum and sIgA in salivary, vaginal and bronchoalveolar lavage. Further, cytokine levels-interferon gamma and interleukin-2-were increased in spleen homogenates. The viability of Calu-3 cells was not adversely affected by the microparticles. In summation, this study establishes that positive surface charges on poly(D,L-lactic-co-glycolic acid) particles containing HBsAg enhances both the systemic and mucosal immune response upon immunization via the respiratory route.

In vitro, in vivo and ex vivo models for studying particle deposition and drug absorption of inhaled pharmaceuticals

Delivery of therapeutic agents via the pulmonary route has gained significant attention over the past few decades because this route of administration offers multiple advantages over traditional routes that include localized action, non-invasive nature and favorable lung-to-plasma ratio. However, assessment of post administration behavior of inhaled pharmaceuticals-such as deposition of particles over the respiratory airways, interaction with the respiratory fluid and movement across the air-blood barrier-is challenging because the lung is a very complex organs that is composed of airways with thousands of bifurcations with variable diameters. Thus, much effort has been put forward to develop models that mimic human lungs and allow evaluation of various pharmaceutical and physiological factors that influence the deposition and absorption profiles of inhaled formulations. In this review, we sought to discuss in vitro, in vivo and ex vivo models that have been extensively used to study the behaviors of airborne particles in the lungs and determine the absorption of drugs after pulmonary administration. We have provided a summary of lung cast models, cascade impactors, noninvasive imaging, intact animals, cell culture and isolated perfused lung models as tools to evaluate the distribution and absorption of inhaled particles. We have also outlined the limitations of currently used models and proposed future studies to enhance the reproducibility of these models.

Cyclodextrins in Nasal Delivery of Low-Molecular-Weight Heparins: In Vivo and in Vitro Studies

AbstractPurpose. To test the hypothesis that cyclodextrins reversibly enhance nasal absorption of low-molecular-weight heparins (LMWHs) and to investigate the mechanisms by which cyclodextrins enhance LMWH absorption via the nose. Methods. Absorption of LMWHs was studied by measuring plasma anti-factor Xa activity after nasal administration of various LMWH formulations to anesthetized rats. In vivo reversibility studies were performed to investigate if the effects of cyclodextrins are reversible and diminish with time. The absorption-enhancing mechanisms of cyclodextrins were investigated in cell culture model. The transport of enoxaparin and mannitol, changes in transepithelial electrical resistance (TEER), and distribution of tight junction protein ZO-1 were investigated. Results. Formulations containing 5% dimethyl-β-cyclodextrin (DMβCD) produced the highest increase in the bioavailability of LMWH preparations tested. In vivo reversibility studies with 5% DMβCD showed that the effect of the absorption enhancer at the site of administration diminished with time. Transport studies using 16HBE14o− cells demonstrated that the increase in the permeability of enoxaparin and mannitol, reduction in TEER, and the changes in the tight junction protein ZO-1 distribution produced by 5% DMβCD were much greater than those produced by β-cyclodextrin (βCD) or hydroxyl-propyl-β-cyclodextrin (HPβCD). Conclusions. Of the cyclodextrins tested, DMβCD was the most efficacious in enhancing absorption of LMWHs both in vivo and in vitro. The study also suggests that cyclodextrins enhance nasal drug absorption by opening of cell-cell tight junctions.

Liposomal fasudil, a rho-kinase inhibitor, for prolonged pulmonary preferential vasodilation in pulmonary arterial hypertension

Current pharmacological interventions for pulmonary arterial hypertension (PAH) require continuous infusions, multiple inhalations, or oral administration of drugs that act on various pathways involved in the pathogenesis of PAH. However, invasive methods of administration, short duration of action, and lack of pulmonary selectivity result in noncompliance and poor patient outcomes. In this study, we tested the hypothesis that encapsulation of an investigational anti-PAH molecule fasudil (HA-1077), a Rho-kinase inhibitor, into liposomal vesicles results in prolonged vasodilation in distal pulmonary arterioles. Liposomes were prepared by hydration and extrusion method and fasudil was loaded by ammonium sulfate-induced transmembrane electrochemical gradient. Liposomes were then characterized for various physicochemical properties. Optimized formulations were tested for pulmonary absorption and their pharmacological efficacy in a monocrotaline (MCT) induced rat model of PAH. The entrapment efficiency of optimized liposomal fasudil formulations was between 68.1±0.8% and 73.6±2.3%, and the cumulative release at 37°C was 98-99% over a period of 5 days. Compared to intravenous (IV) fasudil, a ~10 fold increase in the terminal plasma half-life was observed when liposomal fasudil was administered as aerosols. The t1/2 of IV fasudil was 0.39±0.12 h. and when given as liposomes via pulmonary route, the t1/2 extended to 4.71±0.72 h. One h after intratracheal instillation of liposomal fasudil, mean pulmonary arterial pressure (MPAP) was reduced by 37.6±5.7% and continued to decrease for about 3 h, suggesting that liposomal formulations produced pulmonary preferential vasodilation in MCT induced PAH rats. Overall, this study established the proof-of-principle that aerosolized liposomal fasudil is a feasible option for a non-invasive, controlled release and pulmonary preferential treatment of PAH.

Pulmonary delivery of low molecular weight heparins.

AbstractPurpose. To investigate if pulmonary delivery of low molecular weight heparin (LMWH) formulated with tetradecyl-β-maltoside (TDM) or dimethyl-β-cyclodextrin (DMβCD) could be a feasible alternative to subcutaneous injections for the treatment of pulmonary embolism. Methods. The pulmonary absorption of two LMWHs and unfractionated heparin formulated with TDM or DMβCD was studied in cell culture and rodent model. The in vitro study was performed by measuring the transport of radiolabeled enoxaparin and mannitol across human bronchial epithelial cells (Calu-3) in the presence or absence of varying concentrations of TDM or DMβCD. The changes in transepithelial electrical resistance (TEER) and enoxaparin metabolic stability were also investigated using Calu-3 cells. In vivo absorption studies were performed by measuring plasma anti-factor Xa activity after pulmonary administration of enoxaparin, dalteparin, or unfractionated heparin to anesthetized rats. Results. In vitro experiments conducted in Calu-3 cells suggest that the addition of TDM or DMβCD to the apical chamber results in a significant increase in 3H-enoxaparin and 14C-mannitol permeability and a decrease in TEER across the Calu-3 cell monolayer. Enoxaparin incubated in Calu-3 cell extracts was stable for 8 h. In vivo studies indicate that both TDM and DMβCD enhance pulmonary absorption of LMWH. However, TDM was found to be more potent than DMβCD in both in vitro transport and in vivo absorption studies. Conclusions. TDM and DMβCD enhance pulmonary absorption of LMWH both in vitro and in vivo, with TDM being more efficacious than DMβCD. Both agents increase drug transport by acting mainly on the membrane rather than interacting with the drug.

Pulmonary absorption of insulin mediated by tetradecyl-β-maltoside and dimethyl-β-cyclodextrin

AbstractPurpose. To determine if tetradecyl-β-maltoside (TDM) and dimethyl-β-cyclodextrin (DMβCD) enhance pulmonary absorption of insulin and to investigate if they do so by a reversible action on respiratory epithelium. Methods. Insulin formulated with saline, TDM, or DMβCD was administered intratracheally, after laryngoscopic visualization, as a spray to anesthetized rats. Reversibility studies were conducted in intact rats by administering insulin at different time points after administration of TDM or DMβCD. The pharmacodynamics and pharmacokinetics of insulin formulations were assessed by measuring plasma glucose and plasma insulin concentrations. Results. When insulin formulated with increasing concentrations (0.06-0.25%) of TDM or DMβCD were administered to anesthetized rats, there was a concentration-dependent decrease in plasma glucose and increase in plasma insulin concentrations. The relative bioavailability of insulin formulations containing TDM was higher (0.34-0.84%) than that of formulations containing DMβCD (0.19-0.48%). When insulin was administered 120 min after an agent was administered, in the reversibility study, no significant change in plasma glucose and insulin levels occurred compared to control. Conclusions. Both TDM and DMBCD enhance pulmonary absorption of insulin, with TDM being more efficacious than DMβCD in enhancing insulin absorption via pulmonary administration. The effects of TDM and DMβCD on respiratory epithelium are reversible, and the epithelium reestablishes its normal physiologic barrier 120 min after exposure to these agents.

Influence of PEI as a core modifying agent on PLGA microspheres of PGE1, a pulmonary selective vasodilator

This study tests the hypothesis that large porous poly (lactic-co-glycolic acid) (PLGA) microparticles modified with polyethyleneimine (PEI) are viable carriers for pulmonary delivery of prostaglandin E(1) (PGE(1)) used in the treatment of pulmonary arterial hypertension (PAH), a pulmonary vascular disorder. The particles were prepared by a double-emulsion solvent evaporation method with PEI-25 kDa in the internal aqueous phase to produce an osmotic pressure gradient. Polyvinyl alcohol (PVA) was used for external coating of the particles. The particles were examined for morphology, size, aerodynamic diameter, surface area, pore volume and in-vitro release profiles. Particles with optimal properties for inhalation were tested for in-vivo pulmonary absorption, metabolic stability in rat lung homogenates, and acute toxicity in rat bronchoalveolar lavage fluid and respiratory epithelial cells, Calu-3. The micromeritic data indicated that the PEI-modified particles of PGE(1) are optimal for inhalation. Incorporation of PEI in the formulations resulted in an increased entrapment efficiency - 83.26 ± 3.04% for particles with 1% PVA and 95.48 ± 0.46% for particles with 2% PVA. The amount of cumulative drug released into the simulated interstitial lung fluid was between 50.8 ± 0.76% and 55.36 ± 0.06%. A remarkable extension of the circulation half-life up to 6.0-6.5h was observed when the formulations were administered via the lungs. The metabolic stability and toxicity studies showed that the optimized formulations were stable at physiological conditions and relatively safe to the lungs and respiratory epithelium. Overall, this study demonstrates that large porous inhalable polymeric microparticles can be a feasible option for non-invasive and controlled release of PGE(1) for treatment of PAH.