RESUMO
Pharmacokinetics (PK) of antisense oligonucleotides (ASOs) is characterized by rapid distribution from plasma to tissue and slow terminal plasma elimination driven by re-distribution from tissue. Quantitative understanding of tissue PK and RNA knockdown for various ASO chemistries, conjugations, and administration routes is critical for successful drug discovery. Here, we report concentration-time and RNA knockdown profiles for a gapmer ASO with locked nucleic acid ribose chemistry in mouse liver, kidney, heart, and lung after subcutaneous and intratracheal administration. Additionally, the same ASO with liver targeting conjugation (galactosamine-N-acetyl) is evaluated for subcutaneous administration. Data indicate that exposure and knockdown differ between tissues and strongly depend on administration route and conjugation. In a second study, we show that tissue PK is similar between the three different ribose chemistries locked nucleic acid, constrained ethyl and 2'-O-methoxyethyl, both after subcutaneous and intratracheal administration. Further, we show that the half-life in mouse liver may vary with ASO sequence. Finally, we report less than dose-proportional increase in liver concentration in the dose range of 3-30 µmol/kg. Overall, our studies contribute pivotal data to support design and interpretation of ASO in vivo studies, thereby increasing the probability of delivering novel ASO therapies to patients.
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The contribution of neutrophils and CXC chemokines to the pathogenesis of bronchopulmonary dysplasia is not well defined. The transgenic expression of IL-1ß in the pulmonary epithelium causes lung inflammation and disrupts alveolar development in infant mice. To study the hypothesis that CXC chemokine receptor-2 (CXCR2) is a mediator of inflammatory lung injury, we compared lung development in IL-1ß-expressing mice with wild-type (IL-1ß/CXCR2(+/+)) or null (IL-1ß/CXCR2(-/-)) CXCR2 loci. CXCR2 deficiency abolished the transmigration of neutrophils into the alveolar lumen in IL-1ß-expressing mice, but did not alter the number of neutrophils in the parenchyma. The deletion of CXCR2 increased the alveolar chord length and reduced the survival of mice when IL-1ß was expressed from the pseudoglandular to the alveolar stages. The capillary configuration was highly abnormal in both IL-1ß/CXCR2(+/+) and IL-1ß/CXCR2(-/-) lungs, but in very different ways. The cellular area of the parenchyma and the total capillary area of IL-1ß/CXCR2(+/+) and IL-1ß/CXCR2(-/-) mice were smaller than those of control/CXCR2(+/+) and control/CXCR2(-/-) mice, but the ratio of capillary area to cellular area was similar in all four genotypes. When IL-1ß was expressed during the saccular stage, IL-1ß/CXCR2(-/-) mice had smaller alveolar chord lengths and better survival than did IL-1ß/CXCR2(+/+) mice. Independent of the timing of IL-1ß expression, IL-1ß increased alveolar septal thickness in mice with wild-type CXCR2 loci, but not in CXCR2 null mice. Depending on the developmental stage at the time of the inflammatory insult, inhibition of the CXCR2 pathway may exert opposite effects on alveolar septation in the neonatal lung.
Assuntos
Displasia Broncopulmonar/metabolismo , Pulmão/metabolismo , Receptores de Interleucina-8B/fisiologia , Animais , Apoptose , Displasia Broncopulmonar/patologia , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Proliferação de Células , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Humanos , Recém-Nascido , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Pulmão/irrigação sanguínea , Pulmão/crescimento & desenvolvimento , Pulmão/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Microvasos/patologia , Morfogênese , Neovascularização Fisiológica , Infiltração de Neutrófilos , Alvéolos Pulmonares/irrigação sanguínea , Alvéolos Pulmonares/crescimento & desenvolvimento , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Bronchopulmonary dysplasia (BPD) is a common inflammatory lung disease in premature infants. To study the hypothesis that the sensitivity of the lung to inflammatory injury depends on the developmental stage, we studied postnatal lung development in transgenic mice expressing human IL-1ß (hIL-1ß) in the lungs during the late canalicular-early saccular, saccular, or late saccular-alveolar stage. Overexpression of hIL-1ß in the saccular stage caused arrest in alveolar development, airway remodeling, and goblet cell hyperplasia in the lungs as well as poor growth and survival of infant mice. Overexpression of hIL-1ß during the late canalicular-early saccular stage did not adversely affect lung development, growth, or survival of the pups. Mice expressing hIL-1ß from the late saccular to alveolar stage had smaller alveolar chord length, thinner septal walls, less airway remodeling and mucus metaplasia, and better survival than mice expressing hIL-1ß during the saccular stage. Human IL-1ß overexpression in the saccular stage was sufficient to cause a BPD-like illness in infant mice, whereas the lung was more resistant to hIL-1ß-induced injury at earlier and later developmental stages.
Assuntos
Displasia Broncopulmonar/patologia , Modelos Animais de Doenças , Lesão Pulmonar/patologia , Animais , Apoptose/fisiologia , Proliferação de Células , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Pulmão/anatomia & histologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Taxa de SobrevidaRESUMO
INTRODUCTION: The lungs possess many xenobiotic metabolizing enzymes which influence the pharmacokinetics and safety of inhaled medicines. Anticipating metabolism in the lungs provides an opportunity to optimize new inhaled medicines and overcome challenges in their development. AREAS COVERED: This article summarizes current knowledge on xenobiotic metabolizing enzymes in the lungs. The impact of metabolism on inhaled medicines is considered with examples of how this impacts small molecules, biologics and macromolecular formulation excipients. Methods for measuring and predicting xenobiotic lung metabolism are critically reviewed and the potential for metabolism to influence inhalation toxicology is acknowledged. EXPERT OPINION: Drugs can be optimized by molecular modification to (i) reduce systemic exposure using a 'soft drug' approach, (ii) improve bioavailability by resisting metabolism, or (iii) use a prodrug approach to overcome pharmacokinetic limitations. Drugs that are very labile in the lungs may require a protective formulation. Some drug carriers being investigated for PK-modification rely on lung enzymes to trigger drug release or biodegrade. Lung enzyme activity varies with age, race, smoking status, diet, drug exposure and preexisting lung disease. New experimental technologies to study lung metabolism include tissue engineered models, improved analytical capability and in silico models.
Assuntos
Sistemas de Liberação de Medicamentos , Pulmão/metabolismo , Xenobióticos/metabolismo , Administração por Inalação , Animais , Disponibilidade Biológica , Simulação por Computador , Portadores de Fármacos/química , Humanos , Pulmão/enzimologia , Pneumopatias/fisiopatologia , Pró-Fármacos , Engenharia Tecidual , Xenobióticos/administração & dosagem , Xenobióticos/efeitos adversosRESUMO
Little is known about the influence of maternal inflammation on neonatal outcome. Production of IL-1beta in the lungs of newborn infants is associated with bronchopulmonary dysplasia. Using bitransgenic (bi-TG) mice in which human (h) IL-1beta is expressed with a doxycycline-inducible system controlled by the Clara cell secretory protein promoter, we have shown that hIL-1beta expression causes a bronchopulmonary dysplasia-like illness in infant mice. To study the hypothesis that maternal hIL-1beta production modifies the response of the newborn to hIL-1beta, doxycycline was administered to bi-TG and control dams from Embryonic Day 0, inducing production of hIL-1beta by the bi-TG dams before hIL-1beta production started in their bi-TG fetuses, or from Embryonic Day 15, inducing simultaneous production of hIL-1beta by both the bi-TG dams and their bi-TG fetuses. In addition to the lungs, hIL-1beta was expressed at low levels in the uteri of bi-TG dams. Maternal inflammation preceding fetal inflammation increased the survival and growth of hIL-1beta-expressing pups, enhanced alveolarization, and protected the airways against remodeling and goblet cell hyperplasia. Maternal hIL-1beta production preceding fetal hIL-1beta production caused silencing of several inflammatory genes, including CXC and CC chemokines, murine IL-1beta, serum amyloid A3, and Toll-like receptors 2 and 4, and suppressed the expression of chitinase-like lectins Ym1 and Ym2 in the lungs of infant mice. Maternal inflammation protects the newborn against subsequent hIL-1beta-induced lung inflammation and injury. In contrast, induction of hIL-1beta production simultaneously in bi-TG dams and their fetuses offered no protection against inflammatory lung disease in the neonate.
Assuntos
Displasia Broncopulmonar/prevenção & controle , Interleucina-1beta/biossíntese , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/fisiopatologia , Lesão Pulmonar Aguda/prevenção & controle , Animais , Animais Recém-Nascidos , Sequência de Bases , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/patologia , Displasia Broncopulmonar/fisiopatologia , Primers do DNA/genética , Modelos Animais de Doenças , Doxiciclina/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Idade Gestacional , Humanos , Recém-Nascido , Inflamação/complicações , Inflamação/genética , Inflamação/patologia , Inflamação/fisiopatologia , Interleucina-1beta/genética , Masculino , Troca Materno-Fetal , Camundongos , Camundongos Transgênicos , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/patologia , Complicações na Gravidez/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Significant pulmonary metabolism of inhaled drugs could have drug safety implications or influence pharmacological effectiveness. To study this in vitro, lung microsomes or S9 are often employed. Here, we have determined if rat and human lung microsomes are fit for purpose or whether it is better to use specific cells where drug-metabolizing enzymes are concentrated, such as alveolar type II (ATII) cells. Activities for major hepatic and pulmonary human drug-metabolizing enzymes are assessed and the data contextualized towards an in vivo setting using an ex vivo isolated perfused rat lung model. Very low rates of metabolism are observed in incubations with human ATII cells when compared to isolated hepatocytes and fewer of the substrates are found to be metabolized when compared to human lung microsomal incubations. Reactions selective for flavin-containing monooxygenases (FMOs), CYP1B1, CYP2C9, CYP2J2, and CYP3A4 all show significant rates in human lung microsomal incubations, but all activities are higher when rat lung microsomes are used. The work also demonstrates that a lung microsomal intrinsic clearance value towards the lower limit of detection for this parameter (3 µL/min/mg protein) results in a very low level of pulmonary metabolic clearance during the absorption period, for a drug dosed into the lung in vivo.
RESUMO
Both initiation and suppression of inflammation are hallmarks of the immune response. If not balanced, the inflammation may cause extensive tissue damage, which is associated with common diseases, e.g., asthma and atherosclerosis. Anti-inflammatory drugs come with side effects that may be aggravated by high and fluctuating drug concentrations. To remedy this, an anti-inflammatory drug should have an appropriate pharmacokinetic half-life or better still, a sustained anti-inflammatory drug response. However, we still lack a quantitative mechanistic understanding of such sustained effects. Here, we study the anti-inflammatory response to a common glucocorticoid drug, dexamethasone. We find a sustained response 22 hours after drug removal. With hypothesis testing using mathematical modeling, we unravel the underlying mechanism-a slow release of dexamethasone from the receptor-drug complex. The developed model is in agreement with time-resolved training and testing data and is used to simulate hypothetical treatment schemes. This work opens up for a more knowledge-driven drug development to find sustained anti-inflammatory responses and fewer side effects.
Assuntos
Anti-Inflamatórios/farmacocinética , Dexametasona/farmacocinética , Dexametasona/uso terapêutico , Inflamação/tratamento farmacológico , Macrófagos Alveolares/efeitos dos fármacos , Modelos Biológicos , Animais , RatosRESUMO
Background: For the treatment of respiratory disease, inhaled drug delivery aims to provide direct access to pharmacological target sites while minimizing systemic exposure. Despite this long-held tenet of inhaled therapeutic advantage, there are limited data of regional drug localization in the lungs after inhalation. The aim of this study was to investigate the distribution and retention of different chemotypes typifying available inhaled drugs [slowly dissolving neutral fluticasone propionate (FP) and soluble bases salmeterol and salbutamol] using mass spectrometry imaging (MSI). Methods: Salmeterol, salbutamol, and FP were simultaneously delivered by inhaled nebulization to rats. In the same animals, salmeterol-d3, salbutamol-d3, and FP-d3 were delivered by intravenous (IV) injection. Samples of lung tissue were obtained at 2- and 30-minute postdosing, and high-resolution MSI was used to study drug distribution and retention. Results: IV delivery resulted in homogeneous lung distribution for all molecules. In comparison, while inhalation also gave rise to drug presence in the entire lung, there were regional chemotype-dependent areas of higher abundance. At the 30-minute time point, inhaled salmeterol and salbutamol were preferentially retained in bronchiolar tissue, whereas FP was retained in all regions of the lungs. Conclusion: This study clearly demonstrates that inhaled small molecule chemotypes are differentially distributed in lung tissue after inhalation, and that high-resolution MSI can be applied to study these retention patterns.
Assuntos
Albuterol/farmacocinética , Fluticasona/farmacocinética , Pulmão/metabolismo , Xinafoato de Salmeterol/farmacocinética , Administração por Inalação , Albuterol/administração & dosagem , Animais , Broncodilatadores/administração & dosagem , Broncodilatadores/farmacocinética , Sistemas de Liberação de Medicamentos , Fluticasona/administração & dosagem , Pulmão/diagnóstico por imagem , Masculino , Espectrometria de Massas , Ratos , Ratos Wistar , Xinafoato de Salmeterol/administração & dosagem , Distribuição TecidualRESUMO
Treatment of respiratory disease with a drug delivered via inhalation is generally held as being beneficial as it provides direct access to the lung target site with a minimum systemic exposure. There is however only limited information of the regional localization of drug retention following inhalation. The aim of this study was to investigate the regional and histological localization of salmeterol retention in the lungs after inhalation and to compare it to systemic administration. Lung distribution of salmeterol delivered to rats via nebulization or intravenous (IV) injection was analyzed with high-resolution mass spectrometry imaging (MSI). Salmeterol was widely distributed in the entire section at 5 min after inhalation, by 15 min it was preferentially retained in bronchial tissue. Via a novel dual-isotope study, where salmeterol was delivered via inhalation and d3-salmeterol via IV to the same rat, could the effective gain in drug concentration associated with inhaled delivery relative to IV, expressed as a site-specific lung targeting factor, was 5-, 31-, and 45-fold for the alveolar region, bronchial sub-epithelium and epithelium, respectively. We anticipate that this MSI-based framework for quantifying regional and histological lung targeting by inhalation will accelerate discovery and development of local and more precise treatments of respiratory disease.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Brônquios/metabolismo , Broncodilatadores/administração & dosagem , Pulmão/metabolismo , Alvéolos Pulmonares/metabolismo , Mucosa Respiratória/metabolismo , Xinafoato de Salmeterol/administração & dosagem , Administração por Inalação , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacocinética , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Brônquios/citologia , Brônquios/diagnóstico por imagem , Brônquios/efeitos dos fármacos , Broncodilatadores/metabolismo , Broncodilatadores/farmacocinética , Broncodilatadores/farmacologia , Análise por Conglomerados , Deutério , Injeções Intravenosas , Pulmão/citologia , Pulmão/diagnóstico por imagem , Pulmão/efeitos dos fármacos , Masculino , Espectrometria de Massas , Imagem Molecular , Veículos Farmacêuticos/química , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Polissorbatos/química , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/diagnóstico por imagem , Alvéolos Pulmonares/efeitos dos fármacos , Ratos Wistar , Mucosa Respiratória/citologia , Mucosa Respiratória/diagnóstico por imagem , Mucosa Respiratória/efeitos dos fármacos , Absorção pelo Trato Respiratório , Xinafoato de Salmeterol/metabolismo , Xinafoato de Salmeterol/farmacocinética , Xinafoato de Salmeterol/farmacologia , Distribuição TecidualRESUMO
Modulating and optimizing the local pharmacokinetics of inhaled drugs by chemical design or formulation is challenged by the lack of predictive in vitro systems and in vivo techniques providing a detailed description of drug location in the lung. The present study investigated whether a new experimental setup of freshly prepared agarose-filled lung slices can be used to estimate lung retention in vitro, by comparing with in vivo lung retention after intratracheal instillation. Slices preloaded with inhaled ß-adrenergic compounds (salbutamol, formoterol, salmeterol, indacaterol or AZD3199) were incubated in a large volume of buffer (w/wo monensin to assess the role of lysosomal trapping), and the amount remaining in slices at different time points was determined with liquid chromatography-tandem mass spectrometry. The in vitro lung retention closely matched the in vivo lung retention (half-lives within 3-fold for 4/5 compounds), and monensin shortened the half-lives for all compounds. The results suggest that freshly prepared rat lungs slices can be used to predict lung retention and that slow kinetics of lysosomal trapping is a key mechanism by which retention in the lung and the effect duration of inhaled ß-adrenergic bronchodilators are prolonged.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Broncodilatadores/metabolismo , Pulmão/metabolismo , Lisossomos/metabolismo , Administração por Inalação , Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Animais , Broncodilatadores/administração & dosagem , Previsões , Pulmão/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos WistarRESUMO
The challenge of defining the concentration of unbound drug at the lung target site after inhalation limits the possibility to optimize target exposure by compound design. In this study, a novel rat lung slice methodology has been developed and applied to study drug uptake in lung tissue, and the mechanisms by which this occurs. Freshly prepared lung slices (500 µm) from drug-naive rats were incubated with drugs followed by determination of the unbound drug volume of distribution in lung (Vu,lung), as the total concentration of drug in slices divided by the buffer (unbound) concentration. Vu,lung determined for a set of inhaled drug compounds ranged from 2.21 mL/g for salbutamol to 2970 mL/g for dibasic compound A. Co-incubation with monensin, a modulator of lysosomal pH, resulted in inhibition of tissue uptake of basic propranolol to 13%, indicating extensive lysosomal trapping. Partitioning into cells was particularly high for the cation MPP+ and the dibasic compound A, likely because of the carrier-mediated transport and lysosomal trapping. The results show that different factors are important for tissue uptake and the presented method can be used for profiling of inhaled compounds, leading to a greater understanding of distribution and exposure of drug in the lung.
Assuntos
Broncodilatadores/farmacologia , Pulmão/efeitos dos fármacos , Sobrevivência de Tecidos/efeitos dos fármacos , Administração por Inalação , Albuterol/farmacologia , Animais , Relação Dose-Resposta a Droga , Pulmão/fisiologia , Masculino , Técnicas de Cultura de Órgãos , Propranolol/farmacologia , Ratos , Ratos Wistar , Sobrevivência de Tecidos/fisiologiaRESUMO
The role of inflammation in the pathogenesis of bronchopulmonary dysplasia (BPD) is not well understood. By using a transgenic mouse expressing the inflammatory cytokine interleukin (IL)-1beta in the lung, we have shown that perinatal expression of IL-1beta causes a BPD-like illness in infant mice. We have used this model to identify mechanisms by which inflammation causes neonatal lung injury. Increased matrix metalloproteinase (MMP)-9 activity is associated with BPD. MMP-9 deficiency worsens alveolar hypoplasia in IL-1beta-expressing newborn mice, suggesting that MMP-9 has a protective role in neonatal inflammatory lung injury. The beta6 integrin subunit, an activator of transforming growth factor-beta, is involved in adult lung disease. Absence of the beta6 integrin subunit improves alveolar development in IL-1beta-expressing mice, suggesting that the beta6 integrin subunit is a pathogenetic factor in inflammatory lung disease in the newborn. The authors of clinical studies who have examined maternal inflammation as a risk factor for BPD have found variable results. We have shown that maternal IL-1beta production preceding fetal IL-1beta production prevents lung inflammation, alveolar hypoplasia, and airway remodeling in newborn IL-1beta-expressing mice. Thus, maternal inflammation may protect the newborn lung against subsequent inflammatory injury. In contrast, when maternal and fetal production of IL-1beta are induced simultaneously, the development of IL-1beta-induced lung disease in the newborn is not prevented.