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Tamoxifen citrate (TMC), a non-steroidal antiestrogen drug used for the treatment of breast cancer, was loaded in a block copolymer of maltoheptaose-b-polystyrene (MH-b-PS) nanoparticles, a potential drug delivery system to optimize oral chemotherapy. The nanoparticles were obtained from self-assembly of MH-b-PS using the standard and reverse nanoprecipitation methods. The MH-b-PS@TMC nanoparticles were characterized by their physicochemical properties, morphology, drug loading and encapsulation efficiency, and release kinetic profile in simulated intestinal fluid (pH 7.4). Finally, their cytotoxicity towards the human breast carcinoma MCF-7 cell line was assessed. The standard nanoprecipitation method proved to be more efficient than reverse nanoprecipitation to produce nanoparticles with small size and narrow particle size distribution. Moreover, tamoxifen-loaded nanoparticles displayed spherical morphology, a positive zeta potential and high drug content (238.6 ± 6.8 µg mL-1) and encapsulation efficiency (80.9 ± 0.4 %). In vitro drug release kinetics showed a burst release at early time points, followed by a sustained release profile controlled by diffusion. MH-b-PS@TMC nanoparticles showed higher cytotoxicity towards MCF-7 cells than free tamoxifen citrate, confirming their effectiveness as a delivery system for administration of lipophilic anticancer drugs.
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Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Glucanos , Nanopartículas/química , Poliestirenos , Tamoxifeno/administração & dosagem , Neoplasias da Mama , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fenômenos Químicos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Composição de Medicamentos , Liberação Controlada de Fármacos , Feminino , Glucanos/química , Humanos , Cinética , Modelos Teóricos , Estrutura Molecular , Tamanho da Partícula , Poliestirenos/química , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Tamoxifeno/químicaRESUMO
Colistimethate sodium (CMS) for treatment of lung infections in cystic fibrosis patient was transformed into a dry powder for inhalation by spray drying. Design of Experiment was applied for understanding the role of the spray-drying process parameters on the critical quality attributes of the CMS spray-dried (SD) powders and agglomerates thereof. Eleven experimental SD microparticle powders were constructed under different process conditions according to a central composite design. The SD microparticles were then agglomerated in soft pellets. Eleven physico-chemical characteristics of SD CMS microparticle powders or agglomerates thereof were selected as critical quality attributes. The yield of SD process was higher than 75%. The emitted fraction of agglomerates from RS01 inhaler was 75-84%, and the fine particle fraction (particles <5 µm) was between 58% and 62%. The quality attributes of CMS SD powders and respective agglomerates that were significantly influenced by spray-drying process parameters were residual solvent and drug content of the SD microparticles as well as bulk density and respirable dose of the agglomerates. These attributes were also affected by the combination of the process variables. The air aspiration rate was found as the most positively influential on drug and solvent content and respirable dose. The residual solvent content significantly influenced the powder bulk properties and aerodynamic behavior of the agglomerates, i.e. quality attributes that govern drug metering in the device and the particles lungs deposition. Agglomerates of CMS SD microparticles, in combination with RS01 DPI, showed satisfactory results in terms of dose emitted and fine particle fraction.
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Colistina/análogos & derivados , Fibrose Cística/tratamento farmacológico , Infecções/tratamento farmacológico , Pulmão/efeitos dos fármacos , Pós/química , Pós/farmacologia , Administração por Inalação , Aerossóis/química , Aerossóis/farmacologia , Colistina/química , Composição de Medicamentos/métodos , Inaladores de Pó Seco , Humanos , Tamanho da Partícula , Solventes/químicaRESUMO
INTRODUCTION: Recent discoveries in the field of lung microbiota have enabled the investigation of new therapeutic interventions involving the use of inhaled probiotics. AREAS COVERED: This review provides an overview of what is known about the correlation between airway dysbiosis and the development of local and systemic diseases, and how this knowledge can be exploited for therapeutic interventions. In particular, the review focused on attempts to formulate probiotics that can be deposited directly on the airways. EXPERT OPINION: Despite considerable progress since the emergence of respiratory microbiota restoration as a new research field, numerous clinical implications and benefits remain to be determined. In the case of local diseases, once the pathophysiology is understood, manipulating the lung microbiota through probiotic administration is an approach that can be exploited. In contrast, the effect of pulmonary dysbiosis on systemic diseases remains to be clarified; however, this approach could represent a turning point in their treatment.
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OBJECTIVES: The aim of the project was to develop and characterise powders containing a probiotic (Lactiplantibacillus plantarum [Lpb. plantarum], Lacticaseibacillus rhamnosus, or Lactobacillus acidophilus) to be administered to the lung for the containment of pathogen growth in patients with lung infections. METHODS: The optimised spray drying process for the powder manufacturing was able to preserve viability of the bacteria, which decreased of only one log unit and was maintained up to 30 days. RESULTS: Probiotic powders showed a high respirability (42%-50% of particles had a size < 5 µm) suitable for lung deposition and were proven safe on A549 and Calu-3 cells up to a concentration of 107 colony-forming units/mL. The Lpb. plantarum adhesion to both cell lines tested was at least 10%. Surprisingly, Lpb. plantarum powder was bactericidal at a concentration of 106 colony-forming units/mL on P. aeruginosa, whereas the other two strains were bacteriostatic. CONCLUSION: This work represents a promising starting point to consider a probiotic inhalation powder a value in keeping the growth of pathogenic microflora in check during the antibiotic inhalation therapy suspension in cystic fibrosis treatment regimen. This approach could also be advantageous for interfering competitively with pathogenic bacteria and promoting the restoration of the healthy microbiota.
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Lactobacillales , Probióticos , Infecções por Pseudomonas , Humanos , Pseudomonas aeruginosa , Pós , Antibacterianos/farmacologiaRESUMO
Polyelectrolyte-drug complexes are interesting alternatives to improve unfavorable drug properties. Vancomycin (VAN) is an antimicrobial used in the treatment of methicillin-resistant Staphylococcus aureus pulmonary infections in patients with cystic fibrosis. It is generally administered intravenously with a high incidence of adverse side effects, which could be reduced by intrapulmonary administration. Currently, there are no commercially available inhalable formulations containing VAN. Thus, the present work focuses on the preparation and characterization of an ionic complex between hyaluronic acid (HA) and VAN with potential use in inhalable formulations. A particulate-solid HA-VAN25 complex was obtained by spray drying from an aqueous dispersion. FTIR spectroscopy and thermal analysis confirmed the ionic interaction between HA and VAN, while an amorphous diffraction pattern was observed by X-ray. The powder density, geometric size and morphology showed the suitable aerosolization and aerodynamic performance of the powder, indicating its capability of reaching the deep lung. An in vitro extended-release profile of VAN from the complex was obtained, exceeding 24 h. Microbiological assays against methicillin-resistant and -sensitive reference strains of Staphylococcus aureus showed that VAN preserves its antibacterial efficacy. In conclusion, HA-VAN25 exhibited interesting properties for the development of inhalable formulations with potential efficacy and safety advantages over conventional treatment.
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BACKGROUND: The lack of disease-modifying drugs is one of the major unmet needs in patients with heart failure (HF). Peptides are highly selective molecules with the potential to act directly on cardiomyocytes. However, a strategy for effective delivery of therapeutics to the heart is lacking. OBJECTIVES: In this study, the authors sought to assess tolerability and efficacy of an inhalable lung-to-heart nano-in-micro technology (LungToHeartNIM) for cardiac-specific targeting of a mimetic peptide (MP), a first-in-class for modulating impaired L-type calcium channel (LTCC) trafficking, in a clinically relevant porcine model of HF. METHODS: Heart failure with reduced ejection fraction (HFrEF) was induced in Göttingen minipigs by means of tachypacing over 6 weeks. In a setting of overt HFrEF (left ventricular ejection fraction [LVEF] 30% ± 8%), animals were randomized and treatment was started after 4 weeks of tachypacing. HFrEF animals inhaled either a dry powder composed of mannitol-based microparticles embedding biocompatible MP-loaded calcium phosphate nanoparticles (dpCaP-MP) or the LungToHeartNIM only (dpCaP without MP). Efficacy was evaluated with the use of echocardiography, invasive hemodynamics, and biomarker assessment. RESULTS: DpCaP-MP inhalation restored systolic function, as shown by an absolute LVEF increase over the treatment period of 17% ± 6%, while reversing cardiac remodeling and reducing pulmonary congestion. The effect was recapitulated ex vivo in cardiac myofibrils from treated HF animals. The treatment was well tolerated, and no adverse events occurred. CONCLUSIONS: The overall tolerability of LungToHeartNIM along with the beneficial effects of the LTCC modulator point toward a game-changing treatment for HFrEF patients, also demonstrating the effective delivery of a therapeutic peptide to the diseased heart.
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Insuficiência Cardíaca , Animais , Doença Crônica , Pulmão , Peptídeos , Volume Sistólico , Suínos , Porco Miniatura , Função Ventricular EsquerdaRESUMO
INTRODUCTION: The upper respiratory tract is a major route of infection for COVID-19 and other respiratory diseases. Thus, it appears logical to exploit the nose as administration site to prevent, fight, or minimize infectious spread and treat the disease. Numerous nasal products addressing these aspects have been considered and developed for COVID-19. AREAS COVERED: This review gives a comprehensive overview of the different approaches involving nasal delivery, i.e., nasal vaccination, barrier products, and antiviral pharmacological treatments that have led to products on the market or under clinical evaluation, highlighting the peculiarities of the nose as application and absorption site and pointing at key aspects of nasal drug delivery. EXPERT OPINION: From the analysis of nasal delivery strategies to prevent or fight COVID-19, it emerges that, especially for nasal immunization, formulations appear the same as originally designed for parenteral administration, leading to suboptimal results. On the other hand, mechanical barrier and antiviral products, designed to halt or treat the infection at early stage, have been proven effective but were rarely brought to the clinics. If supported by robust and targeted product development strategies, intranasal immunization and drug delivery can represent valid and sometimes superior alternatives to more conventional parenteral and oral medications.
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COVID-19 , Mucosa Nasal , Humanos , COVID-19/prevenção & controle , Administração Intranasal , Sistemas de Liberação de Medicamentos , Antivirais/uso terapêuticoRESUMO
The correct use of dry powder inhalers by the patients is essential to ensure effective treatment and management of the disease. The purpose of the work was to assess the consequence of inhaler misuse in terms of emitted dose and aerodynamic parameters. One reservoir multidose device (Foster-NEXThaler®) and one pre-dosed device (Relvar-Ellipta®), both sharing the "open, inhale and close" procedure, were the subject of the study. NEXThaler activated at different degrees of inclination showed a consistent dose delivery for both the drugs included in the formulation (beclometasone dipropionate/formoterol fumarate). Contrary, Ellipta showed a decrease of the emitted dose for both fluticasone furoate (FluF) and vilanterol trifenatate (VT) when the device was operated facing downward (-14% at 45° and -22% at 90°). Similarly, the delivered dose of NEXThaler was unaffected by an accidental fall, while Ellipta released FluF and VT doses 50% lower than control values. The presence of the dose protector in NEXThaler offers the advantage of retaining the powder if the inhaler is subjected to incorrect manipulations. Both products proved to be reliable in double activation. Finally, simulation exhalation conditions impaired, although not significantly, the aerodynamic profile of the two products.
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Beclometasona , Doença Pulmonar Obstrutiva Crônica , Humanos , Administração por Inalação , Fumarato de Formoterol , Inaladores de Pó Seco , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , BroncodilatadoresRESUMO
It has long been accepted that suspension pressurized metered-dose inhalers (pMDIs) must be shaken if a correct dose is to be delivered, if not, it will usually be higher than the label claim. The purpose of this work was to investigate the influence of the device being unshaken, shaken and after a period of delay in pMDI actuation on the Fine Particle Mass (<5 µm), Extra Fine Particle Mass (<2 µm) and MMAD. Solution and suspension commercial pMDIs containing one, two or three components were used in the study. Most of the suspension pMDIs produced variable amounts of respirable size drug following the shake-fire delays tested in terms of the label claim dose. The effect was even more critical if the inhaler was not shaken and the FPM was found to be between -82â¯% for Symbicort and 363â¯% for Ventolin compared with the control values. In the case of MMAD measurements, Seretide and Serzyl inhalers showed the largest change from around 3⯵m to 4.2-5.1⯵m when not shaken. Conversely, the FPM and MMAD for the solution aerosols remained unchanged whether or not they were shaken or when a progressive increase in the delay in actuation after shaking was employed.
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Broncodilatadores , Inaladores Dosimetrados , Administração por Inalação , Aerossóis , Albuterol , Suspensões , Tamanho da Partícula , Desenho de EquipamentoRESUMO
This work illustrates the development of a dry inhalation powder of cyclosporine-A for the prevention of rejection after lung transplantation and for the treatment of COVID-19. The influence of excipients on the spray-dried powder's critical quality attributes was explored. The best-performing powder in terms of dissolution time and respirability was obtained starting from a concentration of ethanol of 45% (v/v) in the feedstock solution and 20% (w/w) of mannitol. This powder showed a faster dissolution profile (Weibull dissolution time of 59.5 min) than the poorly soluble raw material (169.0 min). The powder exhibited a fine particle fraction of 66.5% and an MMAD of 2.97 µm. The inhalable powder, when tested on A549 and THP-1, did not show cytotoxic effects up to a concentration of 10 µg/mL. Furthermore, the CsA inhalation powder showed efficiency in reducing IL-6 when tested on A549/THP-1 co-culture. A reduction in the replication of SARS-CoV-2 on Vero E6 cells was observed when the CsA powder was tested adopting the post-infection or simultaneous treatment. This formulation could represent a therapeutic strategy for the prevention of lung rejection, but is also a viable approach for the inhibition of SARS-CoV-2 replication and the COVID-19 pulmonary inflammatory process.
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Recently, there has been increasing interest in developing biocompatible inhalable nanoparticle formulations, as they have enormous potential for treating and diagnosing lung disease. In this respect, here, we have studied superparamagnetic iron-doped calcium phosphate (in the form of hydroxyapatite) nanoparticles (FeCaP NPs) which were previously proved to be excellent materials for magnetic resonance imaging, drug delivery and hyperthermia-related applications. We have established that FeCaP NPs are not cytotoxic towards human lung alveolar epithelial type 1 (AT1) cells even at high doses, thus proving their safety for inhalation administration. Then, D-mannitol spray-dried microparticles embedding FeCaP NPs have been formulated, obtaining respirable dry powders. These microparticles were designed to achieve the best aerodynamic particle size distribution which is a critical condition for successful inhalation and deposition. The nanoparticle-in-microparticle approach resulted in the protection of FeCaP NPs, allowing their release upon microparticle dissolution, with dimensions and surface charge close to the original values. This work demonstrates the use of spray drying to provide an inhalable dry powder platform for the lung delivery of safe FeCaP NPs for magnetically driven applications.
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Vaccines are one of the greatest medical achievements of modern medicine. The nasal mucosa represents an effective route of vaccination for both mucosal immunity and peripheral, being at the same time an inductive and effector site of immunity. In this paper, the innovative and patented compositions and manufacturing procedures of nanomaterials have been studied using the peerreviewed research literature. Nanomaterials have several properties that make them unique as adjuvant for vaccines. Nanoadjuvants through the influence of antigen availability over time affect the immune response. Namely, the amount of antigen reaching the immune system or its release over prolonged periods of time can be effectively increased by nanoadjuvants. Mucosal vaccines are an interesting alternative for immunization of diseases in which pathogens access the body through these epithelia. Nanometric adjuvants are not only a viable approach to improve the efficacy of nasal vaccines but in most of the cases they represent the core of the intellectual property related to the innovative vaccine.
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Vacinas , Adjuvantes Imunológicos , Mucosa Nasal , Imunização , Imunidade nas Mucosas , AntígenosRESUMO
Pulmonary drug delivery is currently the focus of research and development because of its potential to produce maximum therapeutic benefit to patients by directing the drug straight to the lung disease site. Among all the available delivery options, one popular, proven and convenient inhaler device is the capsule-based dry powder inhaler (cDPI) for the treatment of an increasingly diverse range of diseases. cDPIs use a hard capsule that contains a powder formulation which consists of a mixture of a micronized drug and a carrier usually the lactose, known for its good lung tolerance. The capsule is either inserted into the device during manufacturer or by the patient prior to use. After perforating, opening or cut the capsule in the device, patients take a deep and rapid breath to inhale the powder, using air as the vector of drug displacement. The system is simple, relatively cheap and characterized by a lower carbon footprint than that of pressurized metered dose inhalers. This article reviews cDPI technology, focusing particularly on the importance of capsule characteristics and their function as a drug reservoir in cDPIs.
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Pulmonary delivery of chitosan nanoparticles is met with nanoparticle agglomeration and exhalation. Admixing lactose-based microparticles (surface area-weighted diameter~5 µm) with nanoparticles mutually reduces particle agglomeration through surface adsorption phenomenon. Lactose-polyethylene glycol (PEG) microparticles with different sizes, morphologies and crystallinities were prepared by a spray drying method using varying PEG molecular weights and ethanol contents. The chitosan nanoparticles were similarly prepared. In vitro inhalation performance and peripheral lung deposition of chitosan nanoparticles were enhanced through co-blending with larger lactose-PEG microparticles with reduced specific surface area. These microparticles had reduced inter-microparticle interaction, thereby promoting microparticle-nanoparticle interaction and facilitating nanoparticles flow into peripheral lung.
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To overcome some of the shortfalls of the types of dissolution testing currently used for pulmonary products, a new custom-built dissolution apparatus has been developed. For inhalation products, the main in vitro characterisation required by pharmacopoeias is the deposition of the active pharmaceutical ingredient in an impactor to estimate the dose delivered to the target site, i.e., the lung. Hence, the collection of the respirable dose (<5 µm) also appears to be an essential requirement for the study of the dissolution rate of particles, because it results as being a relevant parameter for the pharmacological action of the powder. In this sense, dissolution studies could become a complementary test to the routine testing of inhaled formulation delivered dose and aerodynamic performance, providing a set of data significant for product quality, efficacy and/or equivalence. In order to achieve the above-mentioned objectives, an innovative dissolution apparatus (RespiCell™) suitable for the dissolution of the respirable fraction of API deposited on the filter of a fast screening impactor (FSI) (but also of the entire formulation if desirable) was designed at the University of Parma and tested. The purpose of the present work was to use the RespiCell dissolution apparatus to compare and discriminate the dissolution behaviour after aerosolisation of various APIs characterised by different physico-chemical properties (hydrophilic/lipophilic) and formulation strategies (excipients, mixing technology).
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Pleural mesothelioma is a lung diffuse tumor, whose complete resection is unlikely. Consequently, metastases reappear where the primary tumor was removed. This paper illustrates the orphan medicine designation procedure of an intracavitary cisplatin film and related pharmaceutical development aspects requested by the European Medicines Agency (EMA) in its Scientific Advice. Since cisplatin pharmacokinetics from the implanted film in sheep resulted substantially modified compared to intravenous administration, the formation of a cisplatin/hyaluronan complex had been hypothesized. Here, the interaction between sodium hyaluronate (NaHA) and cisplatin (CisPt) was demonstrated. Size exclusion chromatography qualitatively evidenced the complex in the film-forming mixture, only showing the NaHA peak. Atomic absorption spectroscopy of the corresponding fraction revealed platinum, confirming the interaction. Reverse phase HPLC quantified about 5% free cisplatin in the film-forming mixture, indirectly meaning that 95% was complexed. Finally, a study of CisPt release from the film assessed how CisPt/NaHA complex affected drug availability. In water, a medium without chloride ions, there was no release and the film remained intact for 48 h and longer, whereas the placebo film dissolved in 15 min. In 0.9% NaCl medium, the film became more soluble, dissolving within 3-4 h. However, cisplatin release was still controlled by the existing complex in solution until chloride ions displaced it. While the film modified its dissolution with aging, CisPt release remained unaffected (90% released in 48 h).
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Glycyrrhizic acid and its hydrolyzed metabolite 18ß-glycyrrhetinic acid, obtained from the plant Glycyrrhiza glabra, have numerous pharmacological activities, such as anti-inflammatory, anti-ulcerative, antiallergic, immunomodulatory, antiviral, antitumor, hepatoprotective, and antioxidant effects, and others. In addition to the pharmacological activities, in the 1980s, an interaction and uptake of these molecules by the liver was verified, which was later confirmed by other studies through the discovery of specific receptors in the hepatocytes. The presence of these specific receptors in the liver led to vectorization and delivery of drugs, by the introduction of glycyrrhizic acid or glycyrrhetinic acid on the surface of nanosystems, for the treatment of liver diseases. This review describes experimental evidence of vectorization by conjugating glycyrrhizic acid or glycyrrhetinic acid to nanosystems and delivery of antitumor drugs for the treatment of liver cancer and also describes the techniques used to perform this conjugation. We have shown that due to the existence of specific receptors for these molecules, in addition to the targeting of nanosystems to hepatocytes, nanosystems having glycyrrhizic acid or glycyrrhetinic acid on their surface had the same therapeutic effect in a significantly lower dose compared to the free drug and unconjugated nanosystems, with consequent reduction of side effects and toxicity.
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Neuroinflammation in Alzheimer's disease (AD) revamped the role of a preventive therapeutic action of non steroidal anti-inflammatory drugs; flurbiprofen could delay AD onset, provided its access to brain is enhanced and systemic exposure limited. Nasal administration could enable direct drug access to central nervous system (CNS) via nose-to-brain transport. Here, we investigated the insufflation, deposition, dissolution, transmucosal permeation, and in vivo transport to rat brain of flurbiprofen from nasal powders combined in an active device. Flurbiprofen sodium spray-dried microparticles as such, or soft pellets obtained by agglomeration of drug microparticles with excipients, were intranasally administered to rats by the pre-metered insufflator device. Blood and brain were collected to measure flurbiprofen levels. Excipient presence in soft pellets lowered the metered drug dose to insufflate. Nevertheless, efficiency of powder delivery by the device, measured as emitted fraction, was superior with soft pellets than microparticles, due to their coarse size. Both nasal powders resulted into rapid flurbiprofen absorption. Absolute bioavailability was 33% and 58% for microparticles and pellets, respectively. Compared to intravenous flurbiprofen, the microparticles were more efficient than soft pellets at enhancing direct drug transport to CNS. Direct Transport Percentage index evidenced that more than 60% of the intranasal dose reached the brain via direct nose-to-brain transport for both powders. Moreover, remarkable drug concentrations were measured in the olfactory bulb after microparticle delivery. Bulb connection with the entorhinal cortex, from where AD initiates, makes flurbiprofen sodium administration as nasal powder worth of further investigation in an animal model of neuroinflammation.
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Flurbiprofeno , Insuflação , Administração Intranasal , Animais , Encéfalo , Sistemas de Liberação de Medicamentos , Nariz , RatosRESUMO
Inhalation of Calcium Phosphate nanoparticles (CaPs) has recently unmasked the potential of this nanomedicine for a respiratory lung-to-heart drug delivery targeting the myocardial cells. In this work, we investigated the development of a novel highly respirable dry powder embedding crystalline CaPs. Mannitol was selected as water soluble matrix excipient for constructing respirable dry microparticles by spray drying technique. A Quality by Design approach was applied for understanding the effect of the feed composition and spraying feed rate on typical quality attributes of inhalation powders. The in vitro aerodynamic behaviour of powders was evaluated using a medium resistance device. The inner structure and morphology of generated microparticles were also studied. The 1:4 ratio of CaPs/mannitol led to the generation of hollow microparticles, with the best aerodynamic performance. After microparticle dissolution, the released nanoparticles kept their original size.
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BACKGROUND: Pressurized metered-dose inhalers (pMDIs) include hydrofluoroalkane (HFA) propellant to generate a drug aerosol upon actuation and drugs can be formulated as solution or suspension. Suspended particles can cream or sediment depending on density differences between drug and propellant and shaking the pMDI is an essential step to ensure a uniform drug dose release. RESEARCH DESIGN AND METHODS: The effect of the delay (0, 10, 30, 60 seconds) in pMDI actuation after shaking and the effect of no-shaking during the canister life on the emitted dose (ED) for commercial solution and suspension pMDIs was investigated. RESULTS: The ED for solutions was unaffected by no-shaking or by the progressive increasing delay in actuation after shaking (between 77% and 97%). For all the suspension products, shaking was demonstrated to be critical to assure the close to nominal drug delivery. In detail, the actuation delay after shaking led to an increase up to 380% or a drop to 32% of ED in relation to the label claim with high variability. CONCLUSION: The drug delivered can vary widely for no-shaking and over different shake-fire delays with suspension pMDIs while solution formulations appear to remain stable.