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The dissolution of inhaled drug particles in the lungs is a challenge to model using biorelevant methods in terms of (i) collecting a respirable emitted aerosol fraction and dose, (ii) presenting this to a small volume of medium that is representative of lung lining fluid, and (iii) measuring the low concentrations of drug released. We report developments in methodology for each of these steps and utilize mechanistic in silico modeling to evaluate the in vitro dissolution profiles in the context of plasma concentration-time profiles. The PreciseInhale aerosol delivery system was used to deliver Flixotide aerosol particles to Dissolv It apparatus for measurement of dissolution. Different media were used in the Dissolv It chamber to investigate their effect on dissolution profiles, these were (i) 1.5% poly(ethylene oxide) with 0.4% l-alphaphosphatidyl choline, (ii) Survanta, and (iii) a synthetic simulated lung lining fluid (SLF) based on human lung fluid composition. For fluticasone proprionate (FP) quantification, solid phase extraction was used for sample preparation with LC-MS/MS analysis to provide an assay that was fit for purpose with a limit of quantification for FP of 312 pg/mL. FP concentration-time profiles in the flow-past perfusate were similar irrespective of the medium used in the Dissolv It chamber (â¼0.04-0.07%/min), but these were significantly lower than transfer of drug from air-to-perfusate in isolated perfused lungs (0.12%/min). This difference was attributed to the Dissolv It system representing slower dissolution in the central region of the lungs (which feature nonsink conditions) compared to the peripheral regions that are represented in the isolated lung preparation. Pharmacokinetic parameters ( Cmax, Tmax, and AUC0-∞) were estimated from the profiles for dissolution in the different lung fluid simulants and were predicted by the simulation within 2-fold of the values reported for inhaled FP (1000 µg dose) administered via Flixotide Evohaler 250 µg strength inhaler in man. In conclusion, we report methods for performing biorelevant dissolution studies for orally inhaled products and illustrate how they can provide inputs parameters for physiologically based pharmacokinetic (PBPK) modeling of inhaled medicines.
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Simulação por Computador , Liberação Controlada de Fármacos , Fluticasona/química , Modelos Biológicos , Nebulizadores e Vaporizadores , Administração por Inalação , Administração Oral , Aerossóis/química , Animais , Cromatografia Líquida , Feminino , Fluticasona/administração & dosagem , Pulmão/metabolismo , Modelos Animais , Perfusão , Ratos , Solubilidade , Espectrometria de Massas em TandemRESUMO
INTRODUCTION: Acute exposure to organic dust (OD) in pig barns induces intense airway inflammation with neutrophilia and hyperresponsiveness. This reaction is likely associated with increased cholinergic activity. Therefore, the involvement of cholinergic mechanisms in the reaction to acute exposure of OD was investigated in mice using the long-acting muscarinic antagonist tiotropium. METHODS: BALB/c mice received tiotropium (2-200â¯ng) intranasally on day 1 of the study. On days 2-4, mice received vehicle or OD (25⯵g) intranasally. Airway hyperresponsiveness to methacholine was assessed 24â¯h following the last OD exposure. Bronchoalveolar lavage (BAL) fluid, lung tissue and blood were collected for analyses. RESULTS: Organic dust elevated airway responsiveness to methacholine compared with controls (PBS) assessed as Newtonian resistance (1.5⯱â¯0.1 vs 0.9⯱â¯0.1â¯cmâ¯H2O x s/mL), tissue damping (12.4⯱â¯1.4 vs 8.9⯱â¯0.9â¯cmâ¯H2Oâs/mL) and tissue elastance (41.1⯱â¯5.3 vs 27.2⯱â¯2.5â¯cmâ¯H2Oâs/mL). Tiotropium (200â¯ng) decreased the Newtonian resistance and tissue damping after exposure to PBS or OD. Organic dust exposure increased inflammatory cells in BAL fluid by almost 400%, mainly due to neutrophil influx, which was unaffected by tiotropium. Organic dust increased levels of mainly Th1 mediators. Tiotropium treatment attenuated OD-induced release of IL-2, IL-4 and IL-6. CONCLUSIONS: Tiotropium decreased the OD-induced increase of specific cytokines without influencing the OD-induced increase of airway responsiveness and neutrophil infiltration into the lungs. We conclude that the cholinergic pathway contributes to the pro-inflammatory effects caused by inhalation of OD from pig barns.
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Antagonistas Colinérgicos/farmacologia , Inflamação/tratamento farmacológico , Hipersensibilidade Respiratória/tratamento farmacológico , Brometo de Tiotrópio/farmacologia , Animais , Líquido da Lavagem Broncoalveolar , Antagonistas Colinérgicos/administração & dosagem , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Poeira , Feminino , Inflamação/etiologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/efeitos dos fármacos , Hipersensibilidade Respiratória/etiologia , Suínos , Brometo de Tiotrópio/administração & dosagemRESUMO
Indoor air pollution has caused increasing concern in recent years. As we spend most of our lives indoors, it is crucial to understand the health effects caused by indoor air pollution. Household dust serve as good proxy for accessing indoor air pollution, especially smaller dust particles that can pass into the lungs are of interest. In this study we present an efficient method for the isolation of dust particles in the respirable size range. The respirable fraction was recovered from vacuum cleaner bags, separated by stepwise sieving, followed by characterization for size, morphology, surface area, organic content and elemental composition. The respirable fraction was obtained in a yield of 0.6% with a specific surface area of 2.5m2/g and a Mass Median Aerodynamic Diameter of 3.73 ± 0.15µm. Aluminum and zink were the dominating metals measured in the dust, whereas the major mineral components were found to be silicon dioxide and calcium carbonate. The fraction of organic matter in the dust was measured to be 69 ± 1%. The organic matrix contained bacterial and fungi and a presence of skin fragments. We present here an efficient and fast method for the isolation of dust particles in the respirable size range. That is of considerable value due to the need for large quantities of respirable particle fractions to conduct toxicological studies and risk assessment work.
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Poluição do Ar em Ambientes Fechados , Poeira , Poluição do Ar em Ambientes Fechados/análise , Monitoramento Ambiental , Habitação , Tamanho da PartículaRESUMO
BACKGROUND: Diesel exhaust particles (DEP) are a major component of outdoor air pollution. DEP mediated pulmonary effects are plausibly linked to inflammatory and oxidative stress response in which macrophages (MQ), epithelial cells and their cell-cell interaction plays a crucial role. Therefore, in this study we aimed at studying the cellular crosstalk between airway epithelial cells with MQ and MQ polarization following exposure to aerosolized DEP by assessing inflammation, oxidative stress, and MQ polarization response markers. METHOD: Lung mucosa models including primary bronchial epithelial cells (PBEC) cultured at air-liquid interface (ALI) were co-cultured without (PBEC-ALI) and with MQ (PBEC-ALI/MQ). Cells were exposed to 12.7 µg/cm2 aerosolized DEP using XposeALI®. Control (sham) models were exposed to clean air. Cell viability was assessed. CXCL8 and IL-6 were measured in the basal medium by ELISA. The mRNA expression of inflammatory markers (CXCL8, IL6, TNFα), oxidative stress (NFKB, HMOX1, GPx) and MQ polarization markers (IL10, IL4, IL13, MRC1, MRC2 RETNLA, IL12 andIL23) were measured by qRT-PCR. The surface/mRNA expression of TLR2/TLR4 was detected by FACS and qRT-PCR. RESULTS: In PBEC-ALI exposure to DEP significantly increased the secretion of CXCL8, mRNA expression of inflammatory markers (CXCL8, TNFα) and oxidative stress markers (NFKB, HMOX1, GPx). However, mRNA expressions of these markers (CXCL8, IL6, NFKB, and HMOX1) were reduced in PBEC-ALI/MQ models after DEP exposure. TLR2 and TLR4 mRNA expression increased after DEP exposure in PBEC-ALI. The surface expression of TLR2 and TLR4 on PBEC was significantly reduced in sham-exposed PBEC-ALI/MQ compared to PBEC-ALI. After DEP exposure surface expression of TLR2 was increased on PBEC of PBEC-ALI/MQ, while TLR4 was decreased in both models. DEP exposure resulted in similar expression pattern of TLR2/TLR4 on MQ as in PBEC. In PBEC-ALI/MQ, DEP exposure increased the mRNA expression of anti-inflammatory M2 macrophage markers (IL10, IL4, IL13, MRC1, MRC2). CONCLUSION: The cellular interaction of PBEC with MQ in response to DEP plays a pivotal role for MQ phenotypic alteration towards M2-subtypes, thereby promoting an efficient resolution of the inflammation. Furthermore, this study highlighted the fact that cell-cell interaction using multicellular ALI-models combined with an in vivo-like inhalation exposure system is critical in better mimicking the airway physiology compared with traditional cell culture systems.
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Brônquios/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Emissões de Veículos/toxicidade , Brônquios/citologia , Brônquios/imunologia , Brônquios/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Humanos , Inflamação , Macrófagos Alveolares/metabolismo , Estresse Oxidativo/imunologia , Cultura Primária de Células , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismoRESUMO
BACKGROUND: In development of inhaled drugs- and formulations the measured concentration in the systemic circulation is often used as a surrogate for local dosimetry in the lungs. To further elucidate regional differences in the fate of drugs in the lungs, different aerodynamic sizes of aerosols have been used to target major airway regions. An alternative approach to achieve regional targeting of aerosols, is to use a defined aerosol bolus together with a bolus breath hold strategy. A small volume of test aerosol is intercalated and stopped at different penetration depths, to achieve increased drug deposition at chosen lung locations. Drug permeation from the lung regions is then investigated by repeatedly sampling venous blood from the systemic circulation. The PreciseInhale® (PI) exposure platform was developed to allow generation of aerosols from different sources, including clinical inhalers, into a holding chamber, for subsequent use with alternative exposure modules in vitro and in vivo. In the current first-in-human study was investigated the feasibility of a new clinical exposure module added to the PI system. By extracting aerosol puffs from a medical inhaler for subsequent delivery to volunteers, it was possible to administer whole lung exposures, as well as regional targeting exposures. METHODS: Aerosols containing 250 µg/25 µg fluticasone propionate (FP)/salmeterol xinafoate (SMX) were automatically actuated and extracted from the pressurized Metered Dose Inhaler (pMDI) Evohaler Seretide forte into the PI system's holding chamber, then administered to the healthy volunteers using controlled flowrate and volume exposure cycles. Two main comparisons were made by measuring the systemic PK response: I. One label dose directly from the inhaler to the subject was compared to the same dose extracted from the pMDI into the PI system and then administered to the subject. II A small aerosol bolus at a penetration level in the central airways was compared to a small aerosol bolus at a penetration level in the peripheral lung. RESULTS AND CONCLUSIONS: When one inhaler dose was administered via the PI system, the absorbed dose, expressed as AUC24, was approximately twice as high and the CV was less than half, compared to direct inhalation from the same pMDI. Bolus breath hold targeting of drugs from the same aerosol mixture to the peripheral lung and the central airways showed a difference in their appearance in the systemic circulation. Normalized to the same deposited dose, SMX had a 57 % higher Cmax in the peripheral lung compared to the central airways. However, from 6 to 24 h after dosing the systemic concentrations of SMX from both regions were quite similar. FP had parallel concentrations curves with a 23 % higher AUC24 in the peripheral lung with no noticeable elevation around Cmax. The permeability of these two substances from similar sized aerosols was indeed higher in the thinner air/blood barriers of the peripheral lung compared to the central airways, but differences as measured on the venous side of the circulation were not dramatic. In conclusion, the PI system provided better control of actuation, aspiration, and dispensation of aerosols from the clinical inhaler and thereby delivered higher quality read outs of pharmacokinetic parameters such as tmax, Cmax, and AUC. Improved performance, using PI system, can likely also be employed for studying regional selectivity of other responses in the lungs, for use in drug development.
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Traditionally, developing inhaled drug formulations relied on trial and error, yet recent technological advancements have deepened the understanding of 'inhalation biopharmaceutics' i.e. the processes that occur to influence the rate and extent of drug exposure in the lungs. This knowledge has led to the development of new in vitro models that predict the in vivo behavior of drugs, facilitating the enhancement of existing formulation and the development of novel ones. Our prior research examined how simulated lung fluid (SLF) affects the solubility of inhaled drugs. Building on this, we aimed to explore drug dissolution and permeability in lung mucosa models containing mucus. Thus, the permeation of four active pharmaceutical ingredients (APIs), salbutamol sulphate (SS), tiotropium bromide (TioBr), formoterol fumarate (FF) and budesonide (BUD), was assayed in porcine mucus covered Calu-3 cell layers, cultivated at an air liquid interface (ALI) or submerged in a liquid covered (LC) culture system. Further analysis on BUD and FF involved their transport in a mucus-covered PAMPA system. Finally, their dissolution post-aerosolization from Symbicort® was compared using 'simple' Transwell and complex DissolvIt® apparatuses, alone or in presence of porcine mucus or polymer-lipid mucus simulant. The presence of porcine mucus impacted both permeability and dissolution of inhaled drugs. For instance, permeability of SS was reduced by a factor of ten in the Calu-3 ALI model while the permeability of BUD was reduced by factor of two in LC and ALI setups. The comparison of dissolution methodologies indicated that drug dissolution performance was highly dependent on the setup, observing decreased release efficiency and higher variability in Transwell system compared to DissolvIt®. Overall, results demonstrate that relatively simple methodologies can be used to discriminate between formulations in early phase drug product development. However, for more advanced stages complex methods are required. Crucially, it was clear that the impact of mucus and selection of its composition in in vitro testing of dissolution and permeability should not be neglected when developing drugs and formulations intended for inhalation.
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Albuterol , Budesonida , Liberação Controlada de Fármacos , Fumarato de Formoterol , Muco , Permeabilidade , Brometo de Tiotrópio , Muco/metabolismo , Administração por Inalação , Suínos , Animais , Budesonida/farmacocinética , Budesonida/administração & dosagem , Budesonida/química , Fumarato de Formoterol/administração & dosagem , Fumarato de Formoterol/farmacocinética , Humanos , Albuterol/administração & dosagem , Albuterol/farmacocinética , Albuterol/química , Brometo de Tiotrópio/administração & dosagem , Brometo de Tiotrópio/farmacocinética , Brometo de Tiotrópio/química , Solubilidade , Linhagem Celular , Broncodilatadores/administração & dosagem , Broncodilatadores/farmacocinética , Broncodilatadores/química , Pulmão/metabolismo , Composição de Medicamentos/métodosRESUMO
Indoor environments may impact human health due to chemical pollutants in the indoor air and house dust. This study aimed at comparing the bioavailability and distribution of PFOA following both an inhalation and an oral exposure to PFOA coated house dust in rats. In addition, extractable organofluorine (EOF) was measured in different tissue samples to assess any potential influence of other organofluorine compounds in the experimental house dust. Blood samples were collected at sequential time points after exposure and at the time of termination; the lungs, liver, and kidney were collected for quantification of PFOA and EOF. The concentration of PFOA in plasma increased rapidly in both exposure groups attaining a Cmax at 3 h post exposure. The Cmax following inhalation was four times higher compared to oral exposures. At 48 h post exposure, the levels of PFOA in the plasma, liver, and kidney were twice as high from inhalation exposures. This shows that PFOA is readily bioavailable and has a rapid systemic distribution following an inhalation or oral exposure to house dust coated with PFOA. The proportion of PFOA to EOF corresponded to 65-71% and 74-87% in plasma and tissues, respectively. The mass balance between EOF and target PFOA indicates that there might be other unknown PFAS precursor and/or fluorinated compounds that co-existed in the house dust sample that can have accumulated in rats.
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Poluentes Ambientais , Fluorocarbonos , Humanos , Ratos , Animais , Poeira/análise , Fluorocarbonos/análise , Disponibilidade Biológica , Poluentes Ambientais/análiseRESUMO
There is mounting evidence that shows the association between chronic exposure to air pollutants (particulate matter and gaseous) and onset of various respiratory impairments. However, the corresponding toxicological mechanisms of mixed exposure are poorly understood. Therefore, in this study, we aimed to establish a repeated exposure setting for evaluating the pulmonary toxicological effects of diesel exhaust particles (DEP), nitrogen dioxide (NO2), and sulfur dioxide (SO2) as representative criterial air pollutants. Single, combined (DEP with NO2 and SO2), and repeated exposures were performed using physiologically relevant human bronchial mucosa models developed at the air−liquid interface (bro-ALI). The bro-ALI models were generated using human primary bronchial epithelial cells (3−4 donors; 2 replicates per donor). The exposure regime included the following: 1. DEP (12.5 µg/cm2; 3 min/day, 3 days); 2. low gaseous (NO2: 0.1 ppm + SO2: 0.2 ppm); (30 min/day, 3 days); 3. high gaseous (NO2: 0.2 ppm + SO2: 0.4 ppm) (30 min/day, 3 days); and 4. single combined (DEP + low gaseous for 1 day). The markers for pro-inflammatory (IL8, IL6, NFKB, TNF), oxidative stress (HMOX1, GSTA1, SOD3,) and tissue injury/repair (MMP9, TIMP1) responses were assessed at transcriptional and/ or secreted protein levels following exposure. The corresponding sham-exposed samples under identical conditions served as the control. A non-parametric statistical analysis was performed and p < 0.05 was considered as significant. Repeated exposure to DEP and single combined (DEP + low gaseous) exposure showed significant alteration in the pro-inflammatory, oxidative stress and tissue injury responses compared to repeated exposures to gaseous air pollutants. The study demonstrates that it is feasible to predict the long-term effects of air pollutants using the above explained exposure system.
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Thermostable dry powder inhaler (DPI) formulations with high aerosol performance are attractive inhalable solid dosage forms for local treatment of inflammatory lung diseases. We recently demonstrated that lipidoid-polymer hybrid nanoparticles (LPNs) loaded with small interfering RNA (siRNA) directed against tumor necrosis factor alpha (TNF-α) mediate efficient intracellular siRNA delivery and reduce inflammation in vivo. Here, we show that mixtures of the stabilizing excipients trehalose (Tre) and dextran (Dex), in combination with the shell-forming dispersion enhancer leucine (Leu), stabilize TNF-α siRNA-loaded LPNs during spray drying into nanocomposite microparticles, and result in DPI formulations with high aerosol performance. At low Leu content (0 to 10%, w/w), the DPI formulations were amorphous, and exhibited poor aerosol performance. When the Leu content was increased from 20 to 60% (w/w), the surface content of Leu increased from 39.2 to 68.1 mol%, and the flowability was significantly improved. Microscopy analysis suggest that the improved powder dispersibility is the result of a wrinkled surface morphology, which reduces the surface area available for interparticle interactions. Increasing the Leu content further (to above 10%, w/w) did not influence the aerosol performance, and the aerosol yield was maximal at 30-40% Leu (w/w). Formulations containing 40% Leu and a Tre:Dex ratio of 10:90 (w/w) displayed a high fine particle fraction and aerosol properties suitable for inhalation. The chemical integrity of TNF-α siRNA was preserved in the solid state, and biodistribution studies in mice showed that pulmonary administration of DPI formulations with high aerosol performance resulted in homogenous deep lung deposition. Our results demonstrate that at optimal ratios, ternary excipient mixtures of Leu, Tre and Dex protect TNF-α siRNA-loaded LPNs during spray drying. Hence, this study shows that microparticles with an amorphous Tre/Dex matrix and a crystalline Leu shell efficiently stabilize the nanocomposite LPNs in the solid state, and ensure aerosol properties suitable for inhalation.
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Inaladores de Pó Seco , Nanopartículas , Administração por Inalação , Aerossóis , Animais , Excipientes/química , Leucina/química , Camundongos , Nanopartículas/química , Tamanho da Partícula , Pós , RNA Interferente Pequeno , Distribuição Tecidual , Trealose , Fator de Necrose Tumoral alfaRESUMO
The composition, morphology and dissolution profile of particles and micro-sized agglomerates delivered upon inhalation may have a significant impact on the product clinical effect. However, although several efforts are ongoing, a methodology that considers deposition structures and dissolution performance evaluation in a biorelevant set-up is not yet standardized. The goal of this work is to apply a collection and dissolution methodology able to discriminate dry powder inhaler (DPI) formulations in terms of deposition structures and dissolution profile in vitro. Hence, Fluticasone Propionate (FP) engineered particles and formulated products (used as a case study) were collected employing a breath simulator and characterized regarding (i) aerodynamic particle size distribution; (ii) deposited microstructures; and (iii) dissolution/absorption profiles using the DissolvIt® bio-relevant dissolution equipment. The results indicated that the particle engineering technology had an impact on the generated and deposited microstructures, here associated to the differences on surface properties of jet milled and wet polished particles quantified by the specific surface area. Differences on surface properties modulate particle interactions, resulting in agglomerates of drug substance and excipient upon actuation with significant different morphologies, observed by microscope, as well as quantified by Marple cascade impactor. These observations allow for a further understanding of the DPI aerosolization and deposition mechanisms. The dissolution and absorption assessment indicates that the presence of lactose may accelerate the drug substance dissolution kinetics, and the FP dissolution can be significantly enhanced when formulated as a spray-dried dispersion particle. Ultimately, the results suggest dissolution testing can be an essential tool to both optimize an innovator DPI and de-risk generics development.
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Inaladores de Pó Seco , Administração por Inalação , Aerossóis , Tamanho da Partícula , Pós , SolubilidadeRESUMO
Background: The lower respiratory tract of the landrace pig has close anatomical and physiological similarities with that of the human, and hence, for inhalation studies this species is well suited for biopharmaceutical research. Methods: The objective of this study was to evaluate pharmacokinetics in pigs following one dose of Diskus™ Seretide™ forte device, labeled 500/50 fluticasone propionate (FP) and salmeterol xinafoate (SX), respectively. The PreciseInhale™ (PI) instrument was used to actuate the inhaler for in vitro testing and aerosol dosing to pigs. In vitro, the aerosol was characterized with a cascade impactor with respect to mass median aerodynamic diameter, geometric standard deviation, and fine particle dose. In vivo, dry powder inhalation exposure was delivered as a short bolus dose, to anesthetized and mechanically ventilated landrace pigs. In addition to plasma PK, PK assessment of airway epithelial lining fluid (ELF) was used in this study. ELF of the depth of three to fourth airway generation of the right lung was accessed using standard bronchoscopy and a synthetic absorptive matrix. Results and Conclusions: Dry powder inhalation exposures with good consistency and well characterized aerosols to the pig lung were achieved by the use of the PreciseInhale™ instrument. Drug concentrations of ELF for both FP and SX were demonstrated to be four to five orders of magnitude higher than its corresponding systemic plasma drug concentrations. Clinical PK following inhalation of the same dose was used as benchmark, and the clinical study did demonstrate similar plasma PK profiles and drug exposures of both FP and SX as the current pig study. Two factors explain the close similarity of PK (1) similiar physiology between species and (2) the consistency of dosing to animals. To conclude, our study demonstrated the utility and translational potential of conducting PK studies in pigs in the development of inhaled pharmaceuticals.
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Inaladores de Pó Seco , Respiração Artificial , Administração por Inalação , Animais , Fluticasona , Combinação Fluticasona-Salmeterol , Pulmão , Xinafoato de Salmeterol , SuínosRESUMO
INTRODUCTION: Clinical studies have shown that inhaled corticosteroids can induce rapid vasoconstriction in the airways, leading to decreased mucosal blood flow. The aim of this study was to investigate whether vasoconstriction of the pulmonary circulation after short inhalation of a corticosteroid can be detected in the isolated and perfused rat lung (IPL) - a model which could serve as a substitute or a complement to clinical models. METHODS: IPLs were briefly exposed to dry powder aerosol of budesonide. The pulmonary perfusate flow rate was assessed during 100min post-exposure. A reduction in perfusion flow rate was interpreted as vasoconstriction. MAIN RESULTS: Vasoconstriction was more pronounced after brief inhalation of 10 and 50microg budesonide than 2microg. The onset of vasoconstriction became statistically significant within 10-40min after inhalation. Co-administration of a selective alpha(1)-adrenoceptor antagonist (prazosin 50nM added to the perfusate) reduced vasoconstriction by approximately 50% during 100min of perfusion (p=0.003). CONCLUSIONS: Inhaled budesonide rapidly induces pulmonary vasoconstriction suggesting a nongenomic mechanism probably related to disposition of noradrenaline at the neuro-muscular junction. This ex vivo model could serve as a substitute or a complement to clinical models for investigating rapid effects of glucocorticoid receptor agonists on the pulmonary/bronchial circulation.
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Broncodilatadores/farmacologia , Budesonida/farmacologia , Circulação Pulmonar/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Administração por Inalação , Animais , Budesonida/administração & dosagem , Feminino , Lactose/farmacologia , Norepinefrina/metabolismo , Perfusão , Prazosina/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Background: Many substances used in inhalation research are water soluble and can be administered as nebulized solutions. Typical examples are therapeutic, small-molecular agents, or macromolecules. Another category is a number of water-soluble agents used for airway diagnostics or disease modeling. Mesh nebulizers have facilitated well-controlled liquid aerosol exposures. Meanwhile, a benchtop inhalation platform, PreciseInhale, was developed for providing small-scale, well-controlled aerosol exposures in preclinical configurations. The purpose of the current research was to adapt the Aerogen mesh nebulizer to work within the PreciseInhale system for both cell culture and rodent exposures. Methods: The wet aerosols produced with the Aerogen Pro nebulizer were dried out in an aerosol holding chamber by supplying dry carrier air, which was provided by passing the incoming ambient air through a column with silica gel. The nebulizer was installed in an aerosol holding chamber between an upstream flow-rate pneumotach and a downstream aerosol monitor. By pulsing, the nebulizer output was reduced to 1%-10% of continuous operation to better match the exposure ventilation requirements. Additional drying was obtained by mantling the holding chamber with dried paper. Results and Conclusions: The nebulizer output was reduced to 3-30 µL/min and dried out before reaching the in vitro or in vivo exposure modules. Using solute concentrations in the range of 0.5%-2% (w/w), dried aerosols were produced with a mass median aerodynamic diameter of 1.5-2.0 µm, compared to the 4-5 µm droplets emitted by the nebulizer. Controlling the Aerogen nebulizer under a reduced output scheme within the PreciseInhale platform gave two major advantages: (i) by reducing aerosol output to better match exposure flow rates of single rodents, increased airway deposition yields were obtained in a range of 1%-10% relative to the nebulized amount of test substance and (ii) shrinking aerosol particle sizes through drying improved the peripheral lung deposition of test aerosols.
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Aerossóis , Sistemas de Liberação de Medicamentos , Nebulizadores e Vaporizadores , Preparações Farmacêuticas/administração & dosagem , Administração por Inalação , Animais , Células Cultivadas , Desenho de Equipamento , Espaçadores de Inalação , Tamanho da Partícula , Preparações Farmacêuticas/química , Ratos , Roedores , SolubilidadeRESUMO
Relevant in vitro assays that can simulate exposure to nanoparticles (NPs) via inhalation are urgently needed. Presently, the most common method employed is to expose lung cells under submerged conditions, but the cellular responses to NPs under such conditions might differ from those observed at the more physiological air-liquid interface (ALI). The aim of this study was to investigate the cytotoxic and inflammatory potential of CeO2 NPs (NM-212) in a co-culture of A549 lung epithelial cells and differentiated THP-1 cells in both ALI and submerged conditions. Cellular dose was examined quantitatively using inductively coupled plasma mass spectrometry (ICP-MS). The role of serum and LPS-priming for IL-1ß release was further tested in THP-1 cells in submerged exposure. An aerosol of CeO2 NPs was generated by using the PreciseInhale® system, and NPs were deposited on the co-culture using XposeALI®. No or minor cytotoxicity and no increased release of inflammatory cytokines (IL-1ß, IL-6, TNFα, MCP-1) were observed after exposure of the co-culture in ALI (max 5 µg/cm2) or submerged (max 22 µg/cm2) conditions. In contrast, CeO2 NPs cause clear IL-1ß release in monocultures of macrophage-like THP-1, independent of the presence of serum and LPS-priming. This study demonstrates a useful approach for comparing effects at various in-vitro conditions.
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Carbon nanoparticles (CNP) are generated by incomplete combustion of diesel engines. Several epidemiological studies associated higher susceptibility to particulate matter related adverse respiratory outcomes with preexisting conditions like chronic bronchitis (CB). Therefore, we compared the effect of CNP exposure on primary bronchial epithelial cells (PBEC) developed in air-liquid interface (ALI) models of normal versus CB-like-mucosa.PBEC cultured at ALI represented normal mucosa (PBEC-ALI). To develop CB-like-mucosa (PBEC-ALI/CB), 1 ng/ml interleukin-13 was added to the basal media of PBEC-ALI culturing. PBEC-ALI and PBEC-ALI/CB were exposed to sham or to aerosolized CNP using XposeALI® system. Protein levels of CXCL-8 and MMP-9 were measured in the basal media using ELISA. Transcript expression of pro-inflammatory (CXCL8, IL6, TNF, NFKB), oxidative stress (HMOX1, SOD3, GSTA1, GPx), tissue injury/repair (MMP9/TIMP1) and bronchial cell type markers (MUC5AC, CC10) were assessed using qRT-PCR.Increased secretion of CXCL-8 and MMP-9 markers was detected 24 h post-exposure in both PBEC-ALI and PBEC-ALI/CB with more pronounced effect in the later. Pro-inflammatory and tissue injury markers were increased at both 6 h and 24 h post-exposure in PBEC-ALI/CB. Oxidative stress markers exhibited similar responses at 6 h and 24 h post-exposure in PBEC-ALI/CB. The club cell specific marker CC10 was increased by 300 fold in PBEC-ALI/CB and 20 fold in PBEC-ALI following CNP exposure.Our data indicates an earlier and stronger reaction of pro-inflammatory, oxidative stress and tissue injury markers in PBEC-ALI/CB models compared to PBEC-ALI models following CNP exposure. The findings may provide insight into the plausible mechanisms of higher susceptibility among predisposed individuals to nanoparticle exposure.
Assuntos
Brônquios/efeitos dos fármacos , Bronquite Crônica/induzido quimicamente , Células Epiteliais/efeitos dos fármacos , Brônquios/citologia , Brônquios/metabolismo , Bronquite Crônica/patologia , Carbono/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Mucosa/efeitos dos fármacos , Nanopartículas , Estresse Oxidativo/efeitos dos fármacos , Material Particulado , Mucosa Respiratória/efeitos dos fármacosRESUMO
The surface area of the air/liquid interface in the lungs is substantial, so deposited doses of aerosol medicines per interface surface area when administered via the inhalation route is always quite low. However, in most in vitro systems used for dissolution testing of dry powder inhalables, the dose per surface area is generally much higher. The aim of this study was to investigate in one in vitro lung dissolution system, the DissolvIt, the manner in which the deposited dose per test surface area of drug particles influences the simulated dissolution- and absorption rate. Here we used the dissolution test method DissolvIt to investigate the influence on dissolution behavior by varying the deposited surface density of tested drugs. Dry powders of three different active pharmaceutical ingredients with different solubilities were used; salmeterol, budesonide and fluticasone propionate. It was found that by varying the dose density from 0.23 to 29⯵g/cm2 the dissolution- and absorption rate of test particles was affected for all three substances, with decreasing relative dissolution rates above certain dose limits. The effect was much more prominent with the least soluble fluticasone propionate. In contrast, in a real lung it has been shown that a tenfold increase of the even less soluble fluticasone furoate did not affect the pulmonary dissolution- and absorption as measured in the ex vivo isolated perfused rat lung. This indicates that the deposited particle dose on the test surface used must be carefully considered in all in vitro dissolution testing apparatuses used for inhalation drugs, especially when aiming for in vitro-in vivo correlations. Conclusive data show that in the DissolvIt system consistent normalized dissolution- and absorption data can be obtained if the deposition density of test substance are kept below 1⯵g/cm2 and the variability between the initial drug doses is smaller than 10-15% expressed as standard deviation.
Assuntos
Broncodilatadores/farmacocinética , Liberação Controlada de Fármacos , Pulmão/metabolismo , Modelos Biológicos , Mucosa Respiratória/metabolismo , Administração por Inalação , Aerossóis , Broncodilatadores/administração & dosagem , Budesonida/administração & dosagem , Budesonida/farmacocinética , Inaladores de Pó Seco , Fluticasona/administração & dosagem , Fluticasona/farmacocinética , Tamanho da Partícula , Pós , Xinafoato de Salmeterol/administração & dosagem , Xinafoato de Salmeterol/farmacocinética , SolubilidadeRESUMO
The inhalation route provides closer contact between the ambient environment and living cells than the other major routes-of-entry to the body. In addition, the ambient air transporting exogenous agents to the close proximity of living cells can maintain the reactivity of such agents until they deposit in the extracellular lining layer of the lungs at micrometer distances from the airway epithelium. While toxicity may often occur following the systemic distribution of exogenous substances via the respiratory tract, it is the situation when toxicity occurs at the airway portal-of-entry that pose a particular challenge in the mapping of the dosimetry. In such case, the volume of distribution of solutes before they may enter the airway cells is very small. Therefore exposures of the airway epithelium are more variable and can be much higher than exposures of viable cells of the skin or the gastro-intestinal tract at similar concentrations in the ambient environment. Especially with aerosol exposures, local concentrations around deposited particles can be exceedingly high. To simulate these exposures in a cell culture is a difficult task. However, in order to bridge the in vivo-in vitro gap two methods can be used: (1) Specially designed in vitro systems to better mimic the physiology/morphology of cells residing in the respiratory tract. (2) Mathematical models to analyse the toxicokinetics of the in vitro systems and extrapolate to the corresponding in vivo situation. Both strategies will be exemplified and discussed.
Assuntos
Biomarcadores Farmacológicos/análise , Técnicas de Cultura de Células , Exposição por Inalação , Testes de Toxicidade/métodos , Xenobióticos/toxicidade , Alternativas aos Testes com Animais , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismoRESUMO
BACKGROUND: Preclinical evaluation of new chemical entities (NCEs) designed to be administered by inhalation route requires lung administration to rodents, especially in the discovery phase. Different administration methods have been used until now, but more efforts are required to obtain controlled and reproducible lung deposition when only small amounts of neat powder material are available. METHODS: The PreciseInhale platform used in the present study enables well-controlled powder aerosol exposures with only small amounts of micronized neat material, providing data on inhalation pharmacokinetic (PK) of NCEs at a very early stage. The DustGun aerosol technology uses compressed air to generate a respirable aerosol from milligram-amounts of powder that is delivered to one animal at a time. The new methodology was used to investigate the inhalation PK and lung retention in the rat of the novel Chiesi PDE4 inhibitor CHF6001 in three exposure models of the PreciseInhale platform: nose-only, intratracheally intubated rat, and the isolated, ventilated, and perfused rat lung. Results were compared with data from two other pulmonary delivery systems commonly used in preclinical studies: liquid instillation and powder insufflation. RESULTS: Administration of micronized CHF6001 using the PreciseInhale system yielded lung exposures in the same range as the other tested devices, but the reproducibility in lung deposition was improved. The initial amount of CHF6001 in lungs at the first sampling time point was close to the predetermined target dose. Tracheal deposition with PreciseInhale (0.36 ± 0.22 µg) was significantly less than with other tested delivery systems: PennCentury (23.7 ± 3.2 µg) and Airjet (25.6 ± 7.2 µg). CONCLUSIONS: The PreciseInhale platform enabled the administration of CHF6001 powder with good accuracy and reproducibility, with low tracheal deposition. The new platform can be used at an early discovery stage to obtain inhalatory PK data for respirable aerosols of neat NCE powder without excipients and with minimal use of dry powder formulation work.
Assuntos
Sistemas de Liberação de Medicamentos , Pulmão/metabolismo , Inibidores da Fosfodiesterase 4/farmacocinética , Sulfonamidas/farmacocinética , para-Aminobenzoatos/farmacocinética , Administração por Inalação , Aerossóis , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Masculino , Modelos Biológicos , Inibidores da Fosfodiesterase 4/administração & dosagem , Pós , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sulfonamidas/administração & dosagem , Tecnologia Farmacêutica/métodos , Distribuição Tecidual , Traqueia/metabolismo , para-Aminobenzoatos/administração & dosagemRESUMO
Phthalate esters, suspected endocrine disrupting chemicals, are used in a wide range of applications. Because phthalate esters are not covalently bound, they can easily leach into the indoor environment and associate to dust particles. Thus, exposure may occur through inhalation, ingestion, or contact with the skin. However, it is unclear to what degree indoor dust contributes to the daily intake of phthalate esters. This study investigates household dust as an exposure pathway for seven phthalate esters, the monoester MEHP, and the plasticizer DINCH. Household dust collected from children's sleeping rooms and from living rooms were analysed using gas and liquid chromatography tandem mass spectrometry. To compare two exposure pathways, different dust particle sizes were generated: a respirable fraction (<5⯵m) and an ingested particle fraction in the anticipated size range of skin adherence (<75⯵m). Modelling of dust inhalation and ingestion showed that the daily intake of dust-bound phthalate esters was likely to be 2 times (inhalation) to 12 times (ingestion) higher for 21-month-old children than for adults. These children's daily uptake of phthalate esters was 40-140 times higher through ingestion than inhalation. Furthermore, dust may be an exposure pathway for phthalate esters as well as for MEHP. Therefore, phthalate monoesters could be environmental contaminants of their own and need to be considered in health risk assessments.
Assuntos
Poluição do Ar em Ambientes Fechados/análise , Dietilexilftalato/análogos & derivados , Poeira/análise , Ingestão de Alimentos , Inalação , Ácidos Ftálicos/análise , Plastificantes/análise , Adulto , Pré-Escolar , Dietilexilftalato/análise , Dietilexilftalato/metabolismo , Exposição Ambiental/análise , Poluição Ambiental/análise , Humanos , Lactente , Ácidos Ftálicos/metabolismo , Medição de RiscoRESUMO
The main purpose of this work was to develop an in vitro method for simulating the dissolution and absorption of inhaled dry powder drugs that also mimics systemic pharmacokinetic data. A second purpose was to evaluate this method. DissolvIt® was developed as a simulation of the air-blood barrier of the upper airways, constituting: "airborne" particles deposited on a glass cover slip, a mucus simulant, a polycarbonate (basal) membrane, and a pumped albumin buffer simulating the pulmonary blood flow. The PreciseInhale® exposure system was used to aerosolize and deposit test formulations onto cover slips. The particle dissolution was observed by optical microscopy as particle disappearance, and it was started directly when the particles came into contact with the mucus simulant. Solute from the dissolving particles diffused through the barrier and was absorbed into the perfusate. The drug concentration in the perfusate over time and the remaining drug in the barrier at the end of the experiment were quantitated by using liquid chromatography-tandem mass spectrometry. Budesonide and fluticasone propionate generated different pharmacokinetic dissolution/absorption profiles in DissolvIt. This study indicates that DissolvIt simulates dissolution and absorption of drugs in the lung, and that DissolvIt also mimics pharmacokinetic profiles and parameters.