Targeted activation of human ether-à-go-go-related gene channels rescues electrical instability induced by the R56Q+/- long QT syndrome variant.

AIMS: Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. METHODS AND RESULTS: The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. CONCLUSION: Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs.

Advanced Microparticulate/Nanoparticulate Respirable Dry Powders of a Selective RhoA/Rho Kinase (Rock) Inhibitor for Targeted Pulmonary Inhalation Aerosol Delivery.

Pulmonary hypertension (PH) is a progressive disease that eventually leads to heart failure and potentially death for some patients. There are many unique advantages to treating pulmonary diseases directly and non-invasively by inhalation aerosols and dry powder inhalers (DPIs) possess additional unique advantages. There continues to be significant unmet medical needs in the effective treatment of PH that target the underlying mechanisms. To date, there is no FDA-approved DPI indicated for the treatment of PH. Fasudil is a novel RhoA/Rho kinase (ROCK) inhibitor that has shown great potential in effectively treating pulmonary hypertension. This systematic study is the first to report on the design and development of DPI formulations comprised of respirable nanoparticles/microparticles using particle engineering design by advanced spray drying. In addition, comprehensive physicochemical characterization, in vitro aerosol aerosol dispersion performance with different types of human DPI devices, in vitro cell-drug dose response cell viability of different human respiratory cells from distinct lung regions, and in vitro transepithelial electrical resistance (TEER) as air-interface culture (AIC) demonstrated that these innovative DPI fasudil formulations are safe on human lung cells and have high aerosol dispersion performance properties.

Design and Comprehensive Characterization of Tetramethylpyrazine (TMP) for Targeted Lung Delivery as Inhalation Aerosols in Pulmonary Hypertension (PH): In Vitro Human Lung Cell Culture and In Vivo Efficacy.

This is the first study reporting on the design and development innovative inhaled formulations of the novel natural product antioxidant therapeutic, tetramethylpyrazine (TMP), also known as ligustrazine. TMP is obtained from Chinese herbs belonging to the class of Ligusticum. It is known to have antioxidant properties. It can act as a Nrf2/ARE activator and a Rho/ROCK inhibitor. The present study reports for the first time on the comprehensive characterization of raw TMP (non-spray dried) and spray dried TMP in a systematic manner using thermal analysis, electron microscopy, optical microscopy, and Raman spectroscopy. The in vitro aerosol dispersion performance of spray dried TMP was tested using three different FDA-approved unit-dose capsule-based human dry powder inhaler devices. In vitro human cellular studies were conducted on pulmonary cells from different regions of the human lung to examine the biocompatibility and non-cytotoxicity of TMP. Furthermore, the efficacy of inhaled TMP as both liquid and dry powder inhalation aerosols was tested in vivo using the monocrotaline (MCT)-induced PH rat model.

Advanced therapeutic inhalation aerosols of a Nrf2 activator and RhoA/Rho kinase (ROCK) inhibitor for targeted pulmonary drug delivery in pulmonary hypertension: design, characterization, aerosolization, in vitro 2D/3D human lung cell cultures, and in vivo efficacy.

Inhalable nanostructured microparticles of simvastatin, a Nrf2 activator and RhoA/Rho kinase (ROCK) inhibitor, were rationally designed for targeted pulmonary delivery as dry powder inhalers (DPIs) for the treatment of pulmonary hypertension (PH). Advanced particle engineering design technology was employed to develop inhalable dry powders using different dilute feed concentrations and spray drying pump rates. Several analytical techniques were used comprehensively to characterize the physicochemical properties of the resulting powders. Scanning electron microscopy (SEM) was used to visualize particle morphology (shape), surface structure, size, and size distribution. Karl Fischer titration (KFT) was employed to quantify the residual water content in the powders. X-ray powder diffraction (XRPD) was used to determine crystallinity. Hot-stage microscopy (HSM) under cross-polarizing lens was used to observe the presence or absence of birefringence characteristic of crystallinity. Differential scanning calorimetry (DSC) was employed to quantify thermotropic phase behavior. Attenuated total reflectance (ATR)-Fourier-transform infrared (FTIR) spectroscopy and Raman spectroscopy were used to determine the molecular fingerprint of simvastatin powders before and after particle engineering design. In vitro aerosol dispersion performance was performed with three different Food and Drug Administration (FDA)-approved human DPI devices. Cell viability and transepithelial electrical resistance (TEER) were demonstrated using different in vitro human pulmonary cell two and three-dimensional models at the air-liquid interface, and in vivo safety in healthy rats by inhalation. Efficacy was demonstrated in the in vivo lamb model of PH. Four different inhalable powders of simvastatin were successfully produced. They possessed nanostructured surfaces and were in the inhalable size range. Simvastatin retained its crystallinity following particle engineering design. The more dilute feed concentration spray dried at the lower pump rate produced the smallest particles. All powders successfully aerosolized with all three DPI human devices. Inhaled simvastatin as an aerosol restored the endothelial function in the shunt lamb model of PH, as demonstrated by the reduction of pulmonary vascular resistance (PVR) in response to the endothelium-dependent vasodilator acetylcholine.The reviews of this paper are available via the supplemental material section.

Design and development of innovative microparticulate/nanoparticulate inhalable dry powders of a novel synthetic trifluorinated chalcone derivative and Nrf2 agonist.

Chalcone derivatives are shown to possess excellent anti-inflammatory and anti-oxidant properties which are of great interest in treating respiratory diseases such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis (PF). This study successfully designed and developed dry powder inhaler (DPI) formulations of TMC (2-trifluoromethyl-2'-methoxychalone), a new synthetic trifluorinated chalcone and Nrf2 agonist, for targeted pulmonary inhalation aerosol drug delivery. An advanced co-spray drying particle engineering technique was used to design and produce microparticulate/nanoparticulate formulations of TMC with a suitable excipient (mannitol) as inhalable particles with tailored particle properties for inhalation. Raw TMC and co-spray dried TMC formulations were comprehensively characterized for the first time using scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) spectroscopy, thermal analysis, X-ray powder diffraction (XRPD), and molecular fingerprinting as dry powders by ATR-FTIR spectroscopy and Raman spectroscopy. Further, biocompatibility and suitability of formulations were tested with in vitro cellular transepithelial electrical resistance (TEER) in air-interface culture (AIC) using a human pulmonary airway cell line. The ability of these TMC formulations to perform as aerosolized dry powders was systematically evaluated by design of experiments (DOEs) using three different FDA-approved human inhaler devices followed by interaction parameter analyses. Multiple spray drying pump rates (25%, 75%, and 100%) successfully produced co-spray dried TMC:mannitol powders. Raw TMC exhibited a first-order phase transition temperature at 58.15 ± 0.38 °C. Furthermore, the results demonstrate that these innovative TMC dry powder particles are suitable for targeted delivery to the airways by inhalation.

Advanced spray dried proliposomes of amphotericin B lung surfactant-mimic phospholipid microparticles/nanoparticles as dry powder inhalers for targeted pulmonary drug delivery.

The purpose of this study was to design, develop and characterize inhalable proliposomal microparticles/nanoparticles of Amphotericin B (AmB) with synthetic phospholipids, dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) which are lung surfactant-mimic phospholipids. Organic solutions of AmB and phospholipids, were co-spray dried using an advanced closed-mode system and a high performance cyclone. Scanning electron microscopy (SEM) was employed to visualize the surface structure, morphology, and particles size. The residual water content of the proliposomes was quantified by Karl Fisher coulometric titration (KFT). Degree of crystallinity/non-crystallinity was measured by X-ray powder diffraction (XRPD). Phase behavior was measured by differential scanning calorimetry. The chemical composition by molecular fingerprinting was established using attenuated total reflectance (ATR)-Fourier-transform infrared (FTIR) spectroscopy. The amount of AmB loaded into the proliposomes was quantified using UV-VIS spectroscopy. The in vitro aerosol dispersion performance was conducted using the Next Generation Impactor (NGI) and the human dry powder inhaler (DPI) (Handihaler®) that is FDA-approved. Different human lung cell lines were employed to demonstrate in vitro safety as a function of dose and formulation. Smooth, spherical microparticles/nanoparticles were formed at medium and high spray drying pump rates and had low residual water content. A characteristic peak in the XRPD diffraction pattern as well as an endotherm in DSC confirmed the presence of the lipid bilayer structure characteristic in the DPPC/DPPG proliposomal systems. Superior in vitro aerosol performance was achieved with engineered microparticles/nanoparticles demonstrating suitability for targeted pulmonary drug delivery as inhalable dry powders. The in vitro cellular studies demonstrated that the formulated proliposomes are safe. These AmB proliposomes can be a better option for targeted treatment of severe pulmonary fungal infections.

Advanced design and development of nanoparticle/microparticle dual-drug combination lactose carrier-free dry powder inhalation aerosols.

Rationale: lactose is the only FDA-approved carrier for dry powder inhaler (DPI) formulations in the US. Lactose carrier-based DPI products are contraindicated in patients with a known lactose allergy. Hence, inhaler formulations without lactose will benefit lactose allergic asthmatics. Objectives: to rationally design and develop lactose carrier-free dry powder inhaler formulations of fluticasone propionate and salmeterol xinafoate that will benefit people with known lactose allergy. The study also aims at improving the aerosol deposition of the dry powder formulation through advanced particle engineering design technologies to create inhalable powders consisting of nanoparticles/microparticles. Methods: advanced DPI nanoparticle/microparticle formulations were designed, developed and optimized using organic solution advanced closed-mode spray drying. The co-spray dried (co-SD) powders were comprehensively characterized in solid-state and in vitro comparative analysis of the aerodynamic performance of these molecularly mixed formulations was conducted with the marketed formulation of Advair® Diskus® interactive physical mixture. Measurements and main results: comprehensive solid-state physicochemical characterization of the powders showed that the engineered co-SD particles were small and spherical within the size range of 450 nm to 7.25 µm. Improved fine particle fraction and lower mass median aerodynamic diameter were achieved by these DPI nanoparticles/microparticles. Conclusions: this study has successfully produced a lactose-free dry powder formulation containing fluticasone propionate and salmeterol xinafoate with mannitol as excipient engineered as inhalable DPI nanoparticles/microparticles by advanced spray drying. Further, co-spray drying with mannitol and using Handihaler® device can generate higher fine particle mass of fluticasone/salmeterol. Mannitol, a mucolytic agent and aerosol performance enhancer, is a suitable excipient that can enhance aerosol dispersion of DPIs.

Microparticulate/Nanoparticulate Powders of a Novel Nrf2 Activator and an Aerosol Performance Enhancer for Pulmonary Delivery Targeting the Lung Nrf2/Keap-1 Pathway.

This systematic and comprehensive study reports for the first time on the successful rational design of advanced inhalable therapeutic dry powders containing dimethyl fumarate, a first-in-class Nrf2 activator drug to treat pulmonary inflammation, using particle engineering design technology for targeted delivery to the lungs as advanced spray dried (SD) one-component DPIs. In addition, two-component co-spray dried (co-SD) DMF:D-Man DPIs with high drug loading were successfully designed for targeted lung delivery as advanced DPIs using organic solution advanced spray drying in closed mode. Regional targeted deposition using design of experiments (DoE) for in vitro predictive lung modeling based on aerodynamic properties was tailored based on composition and spray drying parameters. These findings indicate the significant potential of using D-Man in spray drying to improve particle formation and aerosol performance of small molecule with a relatively low melting point. These respirable microparticles/nanoparticles in the solid-state exhibited excellent aerosol dispersion performance with an FDA-approved human DPI device. Using in vitro predictive lung deposition modeling, the aerosol deposition patterns of these particles show the capability to reach lower airways to treat inflammation in this region in pulmonary diseases such as acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), pulmonary hypertension (PH), and pulmonary endothelial disease.

In Vitro Pulmonary Cell Culture in Pharmaceutical Inhalation Aerosol Delivery: 2-D, 3-D, and In Situ Bioimpactor Models.

BACKGROUND: The use of non-invasive inhaled aerosols for pulmonary drug delivery continues to grow. This is due to the many unique advantages this delivery route offers for the treatment of both local and systemic diseases. The physicochemical properties of the formulated drugs as well as the physiology of the lungs play a key role in both the deposition and absorption of the particles. The airway and the alveolar epithelium are targets for the treatment of respiratory diseases. However, particles have to overcome biological barriers before they reach their target and produce an effect. METHODS: In vitro aerosol dispersion performance (i.e. aerodynamic size and aerodynamic size distribution) of inhalable particles is quantified by inertial impaction, as required by regulatory agencies for an investigational pharmaceutical inhalation aerosol formulation to be approved for use in patients as a marketed pharmaceutical product. Using inertial impaction in conjunction with cell cultures of various pulmonary cells in situ as bioimpactors has unique aspects in correlating aerodynamic properties with pulmonary cellular behavior including viability and uptake. These can be as co-culture or in single culture, as 3-D multicellular spheroids or 2-D cellular monolayer using different conditions to grow them, such as air-liquid interface culture (ALI) or in liquid covered culture (LCC). RESULTS: evaluation of the currently available in vitro models and the challenges in developing reliable cellular tools to predict the deposition of inhalable particles in the lungs as a function of aerodynamic particle properties is presented in the manuscript. CONCLUSION: The mechanistic aerodynamic and biophysical properties of inhaled aerosol particles on the entire respiratory tract at the cellular level based on aerodynamic size and aerodynamic size distribution will be better understood with the development of in vitro methods which are described in this work.

Inhalable nanoparticulate powders for respiratory delivery.

Nanoparticles are extensively studied for drug delivery and are proving to be effective in drug delivery and the diagnostic field. Drug delivery to lungs has its advantages over other routes of administration. Inhalable powders consisting of nanoparticles are gaining much interest in respiratory research and clinical therapy. Particle engineering technique is a key factor to develop inhalable formulations that can successfully deliver drug with improved therapeutic effect and enhanced targeting. Inhalable nanoparticles in the solid-state dry powders for targeted pulmonary delivery offer unique advantages and are an exciting new area of research. Nasal delivery of inhalable nanoparticulate powders is gaining research attention recently, particularly in vaccine applications, systemic drug delivery in the treatment of pain, and non-invasive brain targeting. Fundamental aspects and recent advancements along with future prospects of inhalable powders consisting of nanoparticles in the solid-state for respiratory delivery are presented. FROM THE CLINICAL EDITOR: The advance in nanotechnology has enabled the design of new drug delivery systems through inhalation, which has many advantages over traditional delivery systems. This comprehensive review describes and discusses the current status, drug design and modification for targeted delivery and challenges of the use of nanoparticles in the respiratory tract.

Dry powder inhalers in COPD, lung inflammation and pulmonary infections.

INTRODUCTION: The number of pulmonary diseases that are effectively treated by aerosolized medicine continues to grow. AREAS COVERED: These diseases include chronic obstructive pulmonary disease (COPD), lung inflammatory diseases (e.g., asthma) and pulmonary infections. Dry powder inhalers (DPIs) exhibit many unique advantages that have contributed to the incredible growth in the number of DPI pharmaceutical products. To improve the performance, there are a relatively large number of DPI devices available for different inhalable powder formulations. The relationship between formulation and inhaler device features on performance of the drug-device combination product is critical. Aerosol medicine products are drug-device combination products. Device design and compatibility with the formulation are key drug-device combination product aspects in delivering drugs to the lungs as inhaled powders. In addition to discussing pulmonary diseases, this review discusses DPI devices, respirable powder formulation and their interactions in the context of currently marketed DPI products used in the treatment of COPD, asthma and pulmonary infections. EXPERT OPINION: There is a growing line of product options available for patients in choosing inhalers for treatment of respiratory diseases. Looking ahead, combining nanotechnology with optimized DPI formulation and enhancing device design presents a promising future for DPI development.

Inhalable PEGylated Phospholipid Nanocarriers and PEGylated Therapeutics for Respiratory Delivery as Aerosolized Colloidal Dispersions and Dry Powder Inhalers.

Nanomedicine is making groundbreaking achievements in drug delivery. The versatility of nanoparticles has given rise to its use in respiratory delivery that includes inhalation aerosol delivery by the nasal route and the pulmonary route. Due to the unique features of the respiratory route, research in exploring the respiratory route for delivery of poorly absorbed and systemically unstable drugs has been increasing. The respiratory route has been successfully used for the delivery of macromolecules like proteins, peptides, and vaccines, and continues to be examined for use with small molecules, DNA, siRNA, and gene therapy. Phospholipid nanocarriers are an attractive drug delivery system for inhalation aerosol delivery in particular. Protecting these phospholipid nanocarriers from pulmonary immune system attack by surface modification by polyethylene glycol (PEG)ylation, enhancing mucopenetration by PEGylation, and sustaining drug release for controlled drug delivery are some of the advantages of PEGylated liposomal and proliposomal inhalation aerosol delivery. This review discusses the advantages of using PEGylated phospholipid nanocarriers and PEGylated therapeutics for respiratory delivery through the nasal and pulmonary routes as inhalation aerosols.