Inhalable Nanoparticles/Microparticles of an AMPK and Nrf2 Activator for Targeted Pulmonary Drug Delivery as Dry Powder Inhalers

Abstract

Metformin is an activator of the AMPK and Nrf2 pathways which are important in the pathology of several complex pulmonary diseases with unmet medical needs. Organic solution advanced spray drying in the absence of water in closed-mode was used to design and develop respirable dry powders. Following comprehensive characterization, the influence of physicochemical properties was correlated with performance as aerosols using inertial impaction and three different human dry powder inhaler (DPI) devices varying in device properties. In vitro cell assays were conducted to test safety in 2D human pulmonary cell lines and in 3D small airway epithelia comprising primary cells at the air-liquid interface (ALI). In addition, in vitro transepithelial electrical resistance (TEER) was carried out. Metformin remained crystalline following advanced spray drying under these conditions. All SD powders consisted of nanoparticles/microparticles in the solid state. In vitro aerosol dispersion performance showed high aerosolization for all SD metformin powders with all DPI devices tested. High emitted dose for all powders with all three DPI devices was measured. Differences in other aerosol performance parameters and the interplay between the properties of different formulations produced at specific pump rates and the three different DPI devices were correlated with spray drying pump rate and device properties. Safety over a wide metformin dose range was also demonstrated in vitro. Aerosol delivery of metformin nanoparticles/microparticles has the potential to be a new "first-in-class" therapeutic for the treatment of a number of pulmonary diseases including pulmonary vascular diseases such as pulmonary hypertension.

Keywords: 2D/3D human lung cell cultures; advanced spray drying; in vitro; nanotechnology; respiratory drug delivery.

Fig. 1

SEM micrographs of a raw metformin HCl, b SD metformin (25% PR), c SD metformin (50% PR), d SD metformin (75% PR), and e SD metformin (100% PR)

Fig. 2

XRPD diffraction patterns of a raw metformin HCl, b SD metformin (25% PR), c SD metformin (50% PR), d SD metformin (75% PR), e SD metformin (100% PR), and f all

Fig. 3

DSC thermograms of a raw metformin HCl, b SD metformin (25% PR), c SD metformin (50% PR), d SD metformin (75% PR), e SD metformin (100% PR), and f all

Fig. 4

Representative HSM images of a raw metformin HCl and b SD metformin (25% PR). Scale bar = 10 μm

Fig. 5

In vitro aerosol dispersion performance for various SD Met powders with three different human DPI devices: a Aerolizer®, b NeoHaler™, and c HandiHaler®. (n = 3, mean ± SD)

Fig. 6

In vitro cell viability on human pulmonary cell lines a H358 and b A549, and cells after 72 h of exposure to different concentrations of raw metformin HCl and SD metformin. (n = 6, mean ± SD)

Fig. 7

In vitro transepithelial electrical resistance (TEER) analysis of Calu-3 human lung bronchial epithelial cell line at the air-liquid interface (ALI) exposed to 1,000 micromolar concentration of raw metformin HCl and SD metformin using a Penn-Century MicroSprayer® Aerosolizer Model IA-1B (n = 3, mean ± SD)

Fig. 8

In vitro cell viability for SmallAir™ 3D human pulmonary primary cells at the air-liquid interface (ALI) after 72 h of exposure to SD metformin (25% PR). (n = 3, mean ± SD)

Fig. 9

In vitro transepithelial electrical resistance (TEER) analysis of SmallAir™ human pulmonary primary cells at the air-liquid interface (ALI) exposed to 1,000 micromolar concentration of SD metformin at the ALI using a micropipette. (n = 3, mean ± SD)

Fig. 10

3D surface response plots by Design-Expert® 8.0.7.1 software (Stat-Ease Corporation, Minneapolis, MN, USA) displaying the influence and interplay of pump rate and different DPI devices on in vitro aerosol dispersion parameters for SD metformin dry powder formulations for a ED, b RF, c FPF, and d MMAD