Novel respirable powder formulation: design, aerosolization and permeation studies through pulmonary epithelial cell line and mucus models.

2013 - 2014Direct administration of drugs to the lungs has been used for millennia as a major treatment for a number of diseases. Origin of the inhalation therapies can be found 2000 B.C in India, where people were used to smoke Atropa belladonna leaves to suppress cough. In 1986, researchers at Genentech Inc. (San Francisco, USA) discovered that the hormone of the human growth was naturally absorbed into systemic circulation of rats after its instillation into their lungs. Thus, the development of new inhalation medicines for both local and systemic administration raised a growing interest of academic and industrial researchers in the last 30 years. The first breakthrough for the treatment of a chronic systemic disease via inhalation was the inhaled insulin (Exubera®, Pfizer, New York, USA), available in USA from 2006 to 2007, then withdrawn from the market for economic reasons. The idea that serious diseases, such as diabetes, could be treated by pulmonary administration was going to be abandoned, until FDA (2014) decided to approve a new form of inhalable insulin, Afrezza® (Sanofi and MannKind, USA), obtained by a synergy of an innovative inhaler device DreamboatTM and the Technosphere® technology. Although pulmonary route is currently being exploited in ways never imagined before, local pulmonary drug delivery remains the preferred route for the administration of drugs to treat lung diseases, including tuberculosis, asthma, COPD and Cystic Fibrosis (CF). A drug administered by the pulmonary route directly targets the airways with minimized systemic side effects, rapid pharmacologic response and reduction in the required dose. Traditional inhalers, namely MDIs (Metered Dose Inhalers), incorporate a propellant into the formulation, which provides the energy for aerosolization upon actuation. The MDIs major drawback is the need that patient must well coordinate both inhalation and actuation. Solvent- and propellant-free DPIs (dry powder inhaler) are breath-actuated, hence removing the coordination requirement above. Moreover, it has to be noted that an inhaler must 1) allow powder dispersion upon inhalation at reasonable flow rates, 2) have flow-rate-independent performances. As the dry powder formulation and the device have to be intrinsically linked to obtain a unique inhalation product, DPI is considered one of the most complex pharmaceutical product. It is well known that a good deposition into the lung requires particles with an aerodynamic diameter in the range 1 to 5 µm. Different technologies are available to successfully produce inhalation medicines with desirable characteristic, including particle shape, size, adhesiveness, morphology and roughness. However, no standardized methods and correlated regulatory requirements are available to predict the fate of particles after the lung deposition. Hence, the old concept of pulmonary drug delivery, which states that “efficient aerosol generation and particle deposition in the lung are the main and only challenges for effective inhalation therapy”, is no longer valid (Ruge et al. 2013). The lack of standardized methods for the dissolution testing hinders a complete knowledge of the processes occurring after particles deposition in the respiratory tract. The Biopharmaceutics Classification System (BCS) established by Amidon and co-workers (Amidon et al. 1995) for the gastrointestinal absorption, predicting the in vivo pharmacokinetics of the drugs, is not transferable to pulmonary case. Lung administration requires an ad hoc study taking into account the lung specific biology (metabolism, clearance, mucus and surfactant) as well as the characteristics of formulation and solubility of drugs, as those parameters affect the pulmonary bioavailability. Moreover, in some pathologies, such as cystic fibrosis (CF), the presence of a thick viscid mucus may reduce the efficacy of the inhalation therapy. Thus, the study of drug–mucus interaction is a crucial step in CF to check the ability of the drug to penetrate and distribute through airways surface fluids. In the last decade, the Research Group in Pharmaceutical Technology of the University of Salerno has been involved in developing new dry powders for inhalation. Currently, the Group has active projects in this area addressing topics such as development of DPIs containing antibiotic, anti-inflammatory and antioxidant drugs. In this frame, the aim of the present PhD project was to design inhalable powder-based formulations that could improve the treatment of pulmonary diseases, mainly cystic fibrosis. Then, the first step of the project was to formulate in a respirable form the Ketoprofene lysine salt, a nonsteroidal anti-inflammatory drug (NSAID) using the well-known Mini Spray Drying and the innovative Nano Spray Drying technology, and to evaluate limits and strengths of these different techniques. Moreover, the research focused on in vitro assays to evaluate the aerodynamic behavior through the respiratory system of the produced powder, using the monodose DPI as device for the powder aerosolization. In the second part of the project, an in vitro method based on Franz-type diffusion equipment was proposed to predict the fate of drugs after deposition and to study drug dissolution/permeation processes. Moreover, to better mimic the pulmonary environment, permeation properties of the drug were evaluated through artificial and/or native CF mucus layer. To this purpose, a mucus model was prepared taking in account physico-chemical composition and rheological behavior of CF bronchial sputum. The final part of the project was performed at the Woolcock Institute of Medical Research in Sydney, under the supervision of Professors Daniela Traini and Paul M. Young. The research was aimed to study permeation processes of several antibiotics across Calu-3 cell line to obtain key information for the future formulation of inhaled products. Specific objectives of the project were: • i) design and development of Dry Powder Inhalers containing Ketoprofene lysine salt micronized powders by Mini and Nano spray drying production; ii) optimization of the aerodynamic characteristics of the powders, through the use of selected and safe excipients (amino acids) able to improve the powder flow properties and dispersion which, in turn, may increase lung deposition of the drugs (SECTION A). • i) optimization and development of a model of Cystic Fibrosis artificial mucus for the permeation experiments; ii) rheological characterization of Cystic Fibrosis mucus patients; iii) permeation studies of developed formulations through both artificial and native CF mucus (SECTION B). • investigation of the correlation between physico-chemical properties of different antibiotics, such as molecular weight, solubility, LogP and calculated permeability and their transport across Calu-3 cell line (SECTION C).[edited by author]XIII n.s

Nanospray Drying as a Novel Technique for the Manufacturing of Inhalable NSAID Powders

The aim of this research was to evaluate the potential of the nanospray drier as a novel apparatus for the manufacturing of a dry powder for inhalation containing ketoprofen lysinate, a nonsteroidal anti-inflammatory drug able to control the inflammation in cystic fibrosis patients. We produced several ketoprofen lysinate and leucine powder batches by means of nanospray dryer, studying the influence of process parameters on yield, particle properties (size distribution and morphology), and, mainly, aerodynamic properties of powders. Micronized particles were prepared from different hydroalcoholic solutions (alcohol content from 0 to 30% v/v) using ketoprofen in its lysine salt form and leucine as dispersibility enhancer in different ratios (from 5 to 15% w/w) with a total solid concentration ranging from 1 to 7% w/v. Results indicated that the spray head equipped with a 7 µm nozzle produced powders too big to be inhaled. The reduction of nozzle size from 7 to 4 µm led to smaller particles suitable for inhalation but, at the same time, caused a dramatic increase in process time. The selection of process variables, together with the nozzle pretreatment with a surfactant solution, allowed us to obtain a free flowing powder with satisfying aerosol performance, confirming the usefulness of the nanospray drier in the production of powder for inhalation

Antibiotic transport across bronchial epithelial cells: Effects of molecular weight, LogP and apparent permeability

Purpose The first step in developing a new inhalable formulation for the treatment of respiratory diseases is to understand the mechanisms involved in the absorption of drugs after lung deposition. This information could be important for the treatment of bacterial infection in the lung, where low permeability would probably be beneficial, or a systemic infection, where high permeability would be desirable. The goal of this study was to evaluate the transport of several antibiotics (ciprofloxacin, azithromycin, moxifloxacin, rifampicin, doxycycline and tobramycin) across human bronchial airway epithelium and to study the influence of molecular weight and LogP on the apparent permeability. Methods The experiments were conducted using Calu-3 cells seeded in the apical compartment of 24-well Transwell® inserts. The antibiotics transport was measured in both apical to basolateral (A-B) and basolateral to apical (B-A) directions and the apparent permeability of each antibiotic was calculated. Results The A-B transport of ciprofloxacin and rifampicin was independent of the initial concentration in the donor compartment, suggesting the involvement of active transporters in their absorption. Moxifloxacin, doxycycline, azithromycin and tobramycin presented a low absorptive permeation in the A-B direction, indicating that these substances could be substrate for efflux pumps. Generally, all antibiotics studied showed low permeabilities in the B-A direction. Conclusions These findings suggest that the inhalation route would be favorable for delivering these specific antibiotics for the treatment of respiratory infection, compared with present oral or intravenous administration

Nanospray Drying as a Novel Technique for the Manufacturing of Inhalable NSAID Powders

The aim of this research was to evaluate the potential of the nanospray drier as a novel apparatus for the manufacturing of a dry powder for inhalation containing ketoprofen lysinate, a nonsteroidal anti-inflammatory drug able to control the inflammation in cystic fibrosis patients. We produced several ketoprofen lysinate and leucine powder batches by means of nanospray dryer, studying the influence of process parameters on yield, particle properties (size distribution and morphology), and, mainly, aerodynamic properties of powders. Micronized particles were prepared from different hydroalcoholic solutions (alcohol content from 0 to 30% v/v) using ketoprofen in its lysine salt form and leucine as dispersibility enhancer in different ratios (from 5 to 15% w/w) with a total solid concentration ranging from 1 to 7% w/v. Results indicated that the spray head equipped with a 7m nozzle produced powders too big to be inhaled. The reduction of nozzle size from 7 to 4m led to smaller particles suitable for inhalation but, at the same time, caused a dramatic increase in process time. The selection of process variables, together with the nozzle pretreatment with a surfactant solution, allowed us to obtain a free flowing powder with satisfying aerosol performance, confirming the usefulness of the nanospray drier in the production of powder for inhalation

Non-steroidal anti-inflammatory drug for pulmonary administration: Design and investigation of ketoprofen lysinate fine dry powders

Pulmonary inflammation is an important therapeutic target in cystic fibrosis (CF) patients, aiming to limit and delay the lung damage. The purpose of the present research was to produce respirable engineered particles of ketoprofen lysinate, a non-steroidal anti-inflammatory drug able to fight lung inflammatory status by direct administration to the site of action. Micronized drug powders containing leucine as dispersibility enhancer were prepared by co-spray drying the active compound and the excipient from water or hydro-alcoholic feeds. Microparticles were fully characterized in terms of process yield, particle size distribution, morphology and drug content. The ability of the drug to reach the deepest airways after aerosolization of spray-dried formulations was evaluated by Andersen cascade impactor, using the monodose DPI as device. In order to investigate the behaviour of the drug once in contact with lung fluid, an artificial CF mucus was prepared. Drug permeation properties were evaluated interposing the mucus layer between the drug and a synthetic membrane mounted in Franz-type diffusion cells. Finally, the effect of the engineered particles on vitality of human airway epithelial cells of patients homozygous for ΔF 508 CF (CuFi1) was studied and compared to that of raw active compound. Results indicated that powders engineering changed the diameter and shape of the particles, making them suitable for inhalation. The mucus layer in the donor compartment of vertical diffusion cells slowed down drug dissolution and permeation, leucine having no influence. Cell proliferation studies evidenced that the spray drying process together with the addition of leucine reduced the cytotoxic effect of ketoprofen lysine salt as raw material, making the ketoprofen lysinate DPI a very promising product for the inflammation control in CF patients

Prilling for the development of multi-particulate colon drug delivery systems: Pectin vs. pectin–alginate beads

This paper proposes a multi-particulate drug delivery system produced by prilling technique in com-bination with an enteric coating. Optimization of process parameters, such as feed viscosity at nozzle, selection of cross-linker, pH of the gelling solution and cross-linking time, allows to obtain beads with strong gelled matrix. Results showed that dextran/piroxicam beads demonstrated high encapsulation efficiency, very narrow dimensional distribution and high sphericity. Coated beads retained shape and narrow size distribution of the uncoated particles. Moreover, the strength of the produced Zn2+–pectinate beads allows to reduce Eudragit® coating thickness. Piroxicam loaded multi-particulate systems show an interesting prolonged drug release in intestinal fluids. Hence, such platforms could be proposed for the treatment of inflammatory bowel diseases

Gentamicin and leucine inhalable powder: What about antipseudomonal activity and permeation through cystic fibrosis mucus?

The aim of this study was to evaluate the permeation properties of gentamicin (G) in a novel dry powder form for inhalation through an artificial mucus model. Moreover, since respiratory infections sustained by Pseudomonas are a major cause of sickness and death in CF patients, the susceptibility of P. aeruginosa to engineered G powders was investigated. Micronized G and G/leucine (85:15) formulations were produced by co-spray-drying, using process parameters and conditions previously set. Powders were characterized in terms of yield, drug content and aerodynamic profiles, analyzed by Andersen Cascade Impactor. Different mucus models were prepared, showing composition and viscosity similar to those of the native CF mucus. To investigate the impact on drug permeation, Franz-type vertical diffusion cells were used; the powders were applied directly on a synthetic membrane with or without the interposition of the artificial mucus layer. In buffer, gentamicin showed a diffusion controlled release; the presence of leucine reduced powder wettability and, consequently, the permeation rate. Otherwise, mucus delayed drug permeation from both G and G/leucine formulations, with a faint influence of the aminoacid. Antimicrobial tests revealed that G/leu engineered particles are able to preserve the antipseudomonal activity, even in presence of the mucus