RESUMEN
The kinetic clarification of lung disposition for inhaled drugs in humans via pharmacokinetic (PK) modeling aids in their development and regulation for systemic and local delivery, but remains challenging due to its multiplex nature. This study exercised our lung delivery and disposition kinetic model to derive the kinetic descriptors for the lung disposition of four drugs [calcitonin, tobramycin, ciprofloxacin and fluticasone propionate (FP)] inhaled via different inhalers from the published PK profile data. With the drug dose delivered to the lung (DTL) estimated from the corresponding γ-scintigraphy or in vivo predictive cascade impactor data, the model-based curve-fitting and statistical moment analyses derived the rate constants of lung absorption (ka ) and non-absorptive disposition (knad ). The ka values differed substantially between the drugs (0.05-1.00 h-1 ), but conformed to the lung partition-based membrane diffusion except for FP, and were inhaler/delivery/deposition-independent. The knad values also varied widely (0.03-2.32 h-1 ), yet appeared to be explained by the presence or absence of non-absorptive disposition in the lung via mucociliary clearance, local tissue degradation, binding/sequestration and/or phagocytosis, and to be sensitive to differences in lung deposition. For FP, its ka value of 0.2 h-1 was unusually low, suggesting solubility/dissolution-limited slow lung absorption, but was comparable between two inhaler products. Thus, the difference in the PK profile was attributed to differences in the DTL and the knad value, the latter likely originating from different aerosol sizes and regional deposition in the lung. Overall, this empirical, rather simpler model-based analysis provided a quantitative kinetic understanding of lung absorption and non-absorptive disposition for four inhaled drugs from PK profiles in humans.
Asunto(s)
Calcitonina/farmacocinética , Ciprofloxacina/farmacocinética , Fluticasona/farmacocinética , Pulmón/metabolismo , Modelos Biológicos , Tobramicina/farmacocinética , Administración por Inhalación , Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacocinética , Disponibilidad Biológica , Calcitonina/administración & dosificación , Hormonas y Agentes Reguladores de Calcio/administración & dosificación , Hormonas y Agentes Reguladores de Calcio/farmacocinética , Ciprofloxacina/administración & dosificación , Fluticasona/administración & dosificación , Humanos , Distribución Tisular , Tobramicina/administración & dosificaciónRESUMEN
PURPOSE: To establish an in vivo-relevant Transwell dish-based dissolution test system for the "respirable" aerosols of inhaled corticosteroids (ICSs) using marketed inhaler products. METHODS: "Respirable" ≤ 5.8 or 6.5 µm aerosols of 7 ICSs from 11 inhaler products were collected onto the filter membranes under the modified assembly of the cascade impactor. Their dissolution in 10 ml of the simulated lung lining fluid (sLLF) was determined over time in the Transwell dish at 37°C and ~100% relative humidity in the presence of subsequent diffusive permeation across the Transwell's supporting membrane. RESULTS: While three ICSs with high-to-intermediate solubility enabled the first-order "sink" and complete dissolution in 6 h, 4 ICSs with poor solubility including fluticasone propionate (FP) resulted in the pseudo-zero-order "non-sink", slow and limited dissolution. The aerosol dissolution rate constants (kdiss) were derived, well-correlated with the solubility. For FP, but not for highly-soluble flunisolide (FN), dissolution was kinetically aerosol mass-dependent. However, for a given ICS, dissolution profiles were indistinguishable between the formulations and products upon comparable aerosol mass collection. CONCLUSIONS: The in vivo-relevant Transwell dish-based "respirable" aerosol dissolution test system was developed, kinetically discriminative in accordance with the ICS solubility, but indistinguishable for a given ICS between the marketed products.
Asunto(s)
Corticoesteroides/administración & dosificación , Inhaladores de Polvo Seco , Inhaladores de Dosis Medida , Administración por Inhalación , Administración Oral , Aerosoles , Fluocinolona Acetonida/análogos & derivados , Fluocinolona Acetonida/química , Fluticasona/química , Cinética , Permeabilidad , Cemento de Policarboxilato/química , Solubilidad , Temperatura , Factores de TiempoRESUMEN
Emphysema causes progressive and life-threatening alveolar structural destruction/loss, yet remains irreversible and incurable to date. Impaired vascular endothelial growth factor (VEGF) signaling has been proposed as a new pathogenic mechanism, and if so, VEGF recovery may enable reversal of emphysema. Thus, we hypothesized that salvianolic acid B (Sal-B), a polyphenol in traditional Chinese herbal danshen, is an alveolar structural recovery agent for emphysema by virtue of VEGF stimulation/elevation via activation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), as stimulating lung cell proliferation and migration, and protecting against lung cell death. Using in vitro human lung microvascular endothelial (HMVEC-L) and alveolar epithelial (A549) cell systems, Sal-B was examined for 1) stimulation of cell proliferation by the MTT and BrdU assays; 2) promotion of cell migration by the scratch wound closure assay; 3) protection against emphysema-like induced cell death by the trypan blue exclusion and flow cytometry assays; and 4) mechanistic involvement of JAK2/STAT3/VEGF in these activities. Sal-B was also spray-dosed to the lungs of healthy rats for two weeks to verify the lung's STAT3 activation and VEGF elevation by western blot, as well as the absence of functional and morphological abnormalities. All the in vitro cell-based activities were concentration-dependent. At 25⯵M, Sal-B 1) stimulated cell proliferation by 1.4-2.6-fold; 2) promoted migratory cell wound closure by 1.5-1.7-fold; and 3) protected against cell death induced with H2O2 (oxidative stress) and SU5416 (VEGF receptor blockade) by 49-86%. JAK2 and STAT3 inhibitors and VEGF receptor antagonist each opposed these Sal-B's activities by over 65%, suggesting the mechanistic involvement of JAK2/STAT3 activation and VEGF stimulation/elevation. In rats, Sal-B at 0.2â¯mg/kg enabled 1.9 and 1.5-fold increased STAT3 phosphorylation and VEGF elevation in the lungs, respectively, while causing no functional and morphological abnormalities. Hence, Sal-B was projected to be a new class of anti-emphysema agent capable of reversing alveolar structural destruction/loss via JAK2/STAT3/VEGF-dependent stimulation of lung cell proliferation and migration, and inhibition of induced lung cell death.
Asunto(s)
Benzofuranos/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Enfisema Pulmonar/tratamiento farmacológico , Células A549 , Animales , Benzofuranos/administración & dosificación , Muerte Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Peróxido de Hidrógeno/administración & dosificación , Pulmón/citología , Pulmón/efectos de los fármacos , Masculino , Enfisema Pulmonar/fisiopatología , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Emphysema progressively destroys alveolar structures, leading to disability and death, yet remains irreversible and incurable to date. Impaired vascular endothelial growth factor (VEGF) signaling is an emerging pathogenic mechanism, thereby proposing a hypothesis that VEGF stimulation/elevation enables recovery from alveolar structural destruction and loss of emphysema. Our previous in vitro study identified that salvianolic acid B (Sal-B), a polyphenol of traditional Chinese herbal danshen, stimulated lung cell proliferation and migration, and protected against induced lung cell death, by virtue of signal transducer and activator of transcription 3 (STAT3) activation and VEGF stimulation/elevation. Thus, this study examined Sal-B for in vivo therapeutic reversal of established emphysema in two rat models. Emphysema was induced with porcine pancreatic elastase (PPE) and cigarette smoke extract (CSE), and established by day 21. Sal-B was then spray-dosed to the lung three times weekly for three weeks. Functional treadmill exercise endurance; morphological airspace enlargement and alveolar destruction; apoptosis, cell proliferation and tissue matrix proteins; phosphorylated STAT3 (pSTAT3) and VEGF expressions; neutrophil accumulation; and lipid peroxidation were determined. In both models, Sal-B at 0.2â¯mg/kg significantly reversed impaired exercise endurance by 80 and 64%; airspace enlargement [mean linear intercept (MLI)] by 56 and 67%; and alveolar destructive index (%DI) by 63 and 66%, respectively. Induced apoptosis activity [cleaved caspase-3] was normalized by 94 and 82%; and cell proliferation activity [proliferative cell nuclear antigen (PCNA)] was stimulated by 1.6 and 2.1-fold. In the PPE-induced model, Sal-B reduced induction of lung's matrix metalloproteinase (MMP)-9 and MMP-2 activities by 59 and 94%, respectively, and restored pSTAT3 and VEGF expressions to the healthy lung levels, while leaving neutrophil accumulation unchecked [myeloperoxidase (MPO) activity]. In the CSE-induced model, Sal-B elevated pSTAT3 and VEGF expressions both by 1.8-fold over the healthy lung levels, and normalized induced lipid peroxidation [malondialdehyde (MDA) activity] by 68%. These results provide an in vivo proof-of-concept for Sal-B as one of the first anti-emphysema agents enabling reversal of alveolar structural destruction and loss via local lung treatment by virtue of its STAT3 activation and VEGF stimulation.
Asunto(s)
Benzofuranos/farmacología , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Enfisema Pulmonar/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Masculino , Elastasa Pancreática/administración & dosificación , Alveolos Pulmonares/patología , Enfisema Pulmonar/fisiopatología , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/metabolismo , Humo/efectos adversos , Porcinos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Induced lung cell death and impaired hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) signaling are proposed as a pathobiologic mechanism for alveolar structural destruction and loss in emphysema. We hypothesized that our sulfated dehydropolymer of caffeic acid, CDSO3, exerts anti-cell death activities and therapeutic interventions in emphysema by virtue of Fe2+ chelation-based HIF-1α/VEGF stabilization and elevation. The Fe2+ chelating activity was determined in the chromogenic ferrozine-Fe2+ chelation inhibitory assay. The in vitro anti-cell death activities and their Fe2+ and HIF-1α dependence were assessed against a range of emphysematous insults in the lung endothelial (HMVEC-L) and epithelial (A549) cells. CDSO3 was spray-dosed to the lung for three weeks (day 1-21) in an in vivo rat model of apoptotic emphysema induced with a VEGF receptor antagonist SU5416. Post-treatment treadmill exercise endurance, airspace enlargement, and several lung biomarkers/proteins were measured. CDSO3 was a potent Fe2+ chelating molecule. At 10 µM, CDSO3 inhibited HMVEC-L and A549 cell death induced by histone deacetylase inhibition with trichostatin A, VEGF receptor blockade with SU5416, and cigarette smoke extract by 65-99%, which were all significantly opposed by addition of excess Fe2+ or HIF-1α inhibitors. As a potent elastase inhibitor and antioxidant, CDSO3 also inhibited elastase- and H2O2-induced cell death by 92 and 95%, respectively. In the rat model of SU5416-induced apoptotic emphysema, CDSO3 treatment at 60 µg/kg 1) produced 61-77% interventions against exercise endurance impairment, airspace enlargement [mean linear intercept] and oxidative lung damage [malondialdehyde activity]; 2) normalized the apoptotic marker [cleaved caspase-3]; 3) stimulated the VEGF signaling [VEGF receptor 2 phosphorylation] by 1.4-fold; and 4) elevated the HIF-1α and VEGF expression by 1.8- and 1.5-fold, respectively. All of these were consistent with CDSO3's Fe2+ chelation-based HIF-1α/VEGF stabilization and elevation against their pathobiologic deficiency, inhibiting lung cell death and development of apoptotic emphysema.
Asunto(s)
Ácidos Cafeicos/farmacología , Muerte Celular/efectos de los fármacos , Pulmón/efectos de los fármacos , Enfisema Pulmonar/tratamiento farmacológico , Células A549 , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Ácidos Cafeicos/química , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Indoles/farmacología , Pulmón/citología , Pulmón/metabolismo , Masculino , Enfisema Pulmonar/patología , Pirroles/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Prostacyclin and its analogues improve cardiac output and functional capacity in patients with pulmonary arterial hypertension (PAH); however, the underlying mechanism is not fully understood. We hypothesised that prostanoids have load-independent beneficial effects on the right ventricle (RV). Angio-obliterative PAH and RV failure were induced in rats with a single injection of SU5416 followed by 4 weeks of exposure to hypoxia. Upon confirmation of RV dysfunction and PAH, rats were randomised to 0.1 µg·kg(-1) nebulised iloprost or drug-free vehicle, three times daily for 2 weeks. RV function and treadmill running time were evaluated pre- and post-iloprost/vehicle treatment. Pulmonary artery banded rats were treated 8 weeks after surgery to allow for significant RV hypertrophy. Inhaled iloprost significantly improved tricuspid annulus plane systolic excursion and increased exercise capacity, while mean pulmonary artery pressure and the percentage of occluded pulmonary vessels remained unchanged. Rats treated with iloprost had a striking reduction in RV collagen deposition, procollagen mRNA levels and connective tissue growth factor expression in both SU5416/hypoxia and pulmonary artery banded rats. In vitro, cardiac fibroblasts treated with iloprost showed a reduction in transforming growth factor (TGF)-ß1-induced connective tissue growth factor expression, in a protein kinase A-dependent manner. Iloprost decreased TGF-ß1-induced procollagen mRNA expression as well as cardiac fibroblast activation and migration. Iloprost significantly induced metalloproteinase-9 gene expression and activity and increased the expression of autophagy genes associated with collagen degradation. Inhaled iloprost improves RV function and reverses established RV fibrosis partially by preventing collagen synthesis and by increasing collagen turnover.
Asunto(s)
Hipertrofia Ventricular Derecha/tratamiento farmacológico , Iloprost/uso terapéutico , Animales , Colágeno/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ecocardiografía , Fibroblastos/citología , Fibrosis , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Hemodinámica , Hipertensión Pulmonar/inducido químicamente , Hipoxia/fisiopatología , Indoles , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Microscopía de Contraste de Fase , Condicionamiento Físico Animal , Procolágeno/metabolismo , Pirroles , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta1/metabolismo , Vasodilatadores/uso terapéutico , Función Ventricular DerechaRESUMEN
BACKGROUND: Although emphysema destroys alveolar structures progressively and causes death eventually, no drug has been discovered to prevent, intervene, and/or resolve this life-threatening disease. We recently reported that sulfated caffeic acid dehydropolymer CDSO3 is a novel potent triple-action inhibitor of elastolysis, oxidation, and inflammation in vitro, and therefore, a potential anti-emphysema agent. However, the in vivo therapeutic potency, duration and mode of actions, and effective route remain to be demonstrated. METHODS: Emphysema was induced in rats with human sputum elastase (HSE) combined with cigarette smoke extract (CSE). CDSO3 at 5, 30, or 100 µg/kg was dosed to the lung or injected subcutaneously at 2, 6, or 24 h before or 1 or 24 h or 1 week after the HSE/CSE instillation. At 1 h or 48 h or on day 21-22 or day 28, lungs were examined for airway-to-blood injurious barrier damage; their elastolytic, oxidative, and inflammatory activities; lung luminal leukocytes infiltration; functional treadmill exercise endurance; and/or morphological airspace enlargement. RESULTS: CDSO3, when dosed to the lung at 30 or 100 µg/kg, but not via systemic subcutaneous injection, significantly (43-93 %) attenuated HSE/CSE-induced (1) barrier damage measured by luminal hemorrhage and protein leak; (2) elastolytic, oxidative, and inflammatory activities measured with elastase, reduced glutathione, and TNFα levels, respectively; (3) luminal neutrophil infiltration and tissue myeloperoxidase activity; (4) functional impairment of exercise endurance; and (5) airspace enlargement, in both preventive and interventional dosing protocols. Notably, the effects were shown to last for 24 h at the greater 100-µg/kg dose, and the 1-week-delayed administration was also capable of attenuating the development of emphysema. CONCLUSIONS: CDSO3 is a novel, potent, long-acting, nonpeptidic macromolecule that inhibits HSE/CSE-induced elastolysis, oxidation, and inflammation in the lung and thereby attenuates the development of emphysema in rats, in both preventive and interventional manners, when administered locally to the lung.
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Ácidos Cafeicos/farmacología , Pulmón/efectos de los fármacos , Elastasa Pancreática , Enfisema Pulmonar/prevención & control , Inhibidores de Serina Proteinasa/farmacología , Humo , Fumar , Ésteres del Ácido Sulfúrico/farmacología , Animales , Antiinflamatorios/administración & dosificación , Antioxidantes/administración & dosificación , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Ácidos Cafeicos/administración & dosificación , Modelos Animales de Enfermedad , Esquema de Medicación , Glutatión/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Inyecciones Subcutáneas , Pulmón/enzimología , Pulmón/patología , Pulmón/fisiopatología , Masculino , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/metabolismo , Enfisema Pulmonar/enzimología , Enfisema Pulmonar/etiología , Enfisema Pulmonar/patología , Enfisema Pulmonar/fisiopatología , Ratas Sprague-Dawley , Inhibidores de Serina Proteinasa/administración & dosificación , Esputo/enzimología , Ésteres del Ácido Sulfúrico/administración & dosificación , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
No molecule has been found to be effective against emphysema to date primarily because of its complex pathogenesis that involves elastolysis, oxidation and inflammation. We here describe novel unsulfated or sulfated low molecular weight lignins (LMWLs) chemo-enzymatically prepared from 4-hydroxycinnamic acids monomers, as the first potent triple-action inhibitors of neutrophil elastase, oxidation and inflammation. The inhibitory potencies of three different cinnamic acid-based LMWLs were determined in vitro using chromogenic substrate hydrolysis assays, radical scavenging and lung cellular oxidative biomarker reduced glutathione (rGSH) assays, and lung cellular inflammatory biomarker NFκB and IL-8 assays, respectively. Each LWML uniquely displayed triple-action inhibition, among which CDSO3, a sulfated caffeic acid-based LMWL, was most potent. The half-maximal anti-human neutrophil elastase (HNE) potency of CDSO3 was 0.43 µM. This high potency arose from lignin-like oligomerization, which was further potentiated by 6.6-fold due to sulfation. Mechanistically, this elastase inhibition was of mixed-type, time-dependent and more selective to positively charged elastases. The half-maximal anti-oxidative potency of CDSO3 was 3.52 µM, 4.8-fold potentiated from that of the monomer, caffeic acid (CA). In contrast, the half-maximal inhibitory potency to TNFα-induced inflammation was 5-10 µM, despite no activity with the monomer. More intriguingly, this anti-inflammatory activity was essentially identical with different stimuli, okadaic acid and hydrogen peroxide (H(2)O(2)), which implied that CDSO3 acts directly on inflammatory cascades within the cells. Overall, oligomerization and sulfation produced or significantly potentiated the activity, in comparison to the monomer. Thus, sulfated and unsulfated LMWLs are novel non-peptidic 2.8-4.1 kDa macromolecules that exhibit for the first time potent triple inhibitory activity against elastase, oxidation and inflammation, the three major pathogenic mechanisms known to cause emphysema.
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Enfisema/tratamiento farmacológico , Lignina/farmacología , Proteínas Inhibidoras de Proteinasas Secretoras/farmacología , Heparina de Bajo-Peso-Molecular/análogos & derivados , Humanos , Interleucina-8/metabolismo , Peso Molecular , FN-kappa B/metabolismoRESUMEN
To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ciclopropanos/química , Citosina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Cristalografía por Rayos X , Citosina/síntesis química , Citosina/química , Citosina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Conformación Molecular , Relación Estructura-ActividadRESUMEN
Selective CB2 agonists have the potential for treating pain without central CB1-mediated adverse effects. Screening efforts identified 1,2-dihydro-3-isoquinolone 1; however, this compound has the drawbacks of being difficult to synthesize with two asymmetric carbons on an isoquinolone scaffold and of having a highly lipophilic physicochemical property. To address these two major problems, we designed the 2-pyridone-based lead 15a, which showed moderate affinity for CB2. Optimization of 15a led to identification of 39f with high affinity for CB2 and selectivity over CB1. Prediction of the binding mode of 39f in complex with an active-state CB2 homology model provided structural insights into its high affinity for CB2.
Asunto(s)
Diseño de Fármacos , Piridonas/química , Piridonas/farmacología , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/metabolismo , Dominio Catalítico , Humanos , Simulación del Acoplamiento Molecular , Piridonas/síntesis química , Receptor Cannabinoide CB2/química , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The CB2 receptor has emerged as a potential target for the treatment of pruritus as well as pain without CB1-mediated side effects. We previously identified 2-pyridone derivatives 1 and 2 as potent CB2 agonists; however, this series of compounds was found to have unacceptable pharmacokinetic profiles with no significant effect in vivo. To improve these profiles, we performed further structural optimization of 1 and 2, which led to the discovery of bicyclic 2-pyridone 18e with improved CB2 affinity and selectivity over CB1. In a mouse pruritus model, 18e inhibited compound 48/80 induced scratching behavior at a dose of 100 mg/kg. In addition, the docking model of 18e with an active-state CB2 homology model indicated the structural basis of its high affinity and selectivity over CB1.
Asunto(s)
Antipruriginosos/síntesis química , Compuestos Bicíclicos con Puentes/síntesis química , Prurito/tratamiento farmacológico , Piridonas/síntesis química , Receptor Cannabinoide CB2/agonistas , Administración Oral , Animales , Antipruriginosos/farmacocinética , Antipruriginosos/farmacología , Conducta Animal/efectos de los fármacos , Compuestos Bicíclicos con Puentes/farmacocinética , Compuestos Bicíclicos con Puentes/farmacología , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Ratones , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Prurito/metabolismo , Prurito/fisiopatología , Piridonas/farmacocinética , Piridonas/farmacología , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB2/química , Receptor Cannabinoide CB2/metabolismo , Relación Estructura-ActividadRESUMEN
Full details of the total synthesis of pacidamycin D (4) and its 3'-hydroxy analogue 32 are described. The chemically labile Z-oxyacyl enamide moiety is the most challenging chemical structure found in uridylpeptide natural products. Key elements of our approach to the synthesis of 4 include the efficient and stereocontrolled construction of the Z-oxyvinyl halides 6 and 7 and their copper-catalyzed cross-coupling with the tetrapeptide carboxamide 5, a thermally unstable compound containing a number of potentially reactive functional groups. This synthetic route also allowed us to easily prepare 3'-hydroxy analogue 32. The assemblage by cross-coupling of the Z-oxyvinyl halide 6 and the carboxamide 5 at a late stage of the synthesis provided ready access to a range of uridylpeptide antibiotics and their analogues, despite their inherent labile nature with potential epimerization, simply by altering the tetrapeptide moiety.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Técnicas de Química Sintética/métodos , Péptidos/síntesis química , Péptidos/farmacología , Nucleósidos de Pirimidina/síntesis química , Nucleósidos de Pirimidina/farmacología , Antibacterianos/química , Catálisis , Cobre/química , Pruebas de Sensibilidad Microbiana , Péptidos/química , Pseudomonas aeruginosa/efectos de los fármacos , Nucleósidos de Pirimidina/químicaRESUMEN
The second-generation synthesis of 3'-hydroxypacidamycin D (2) has been accomplished via an Ugi-four component reaction at a late stage of the synthesis. This approach provided ready access to a range of analogues including diastereomers of the diaminobutylic acid residue and hybrid-type analogues of mureidomycins. Biological evaluations of these analogues indicated that the stereochemistry at the diaminobutylic acid residue has a crucial impact on both the MraY biochemical inhibition and whole-cell antibacterial activity.
Asunto(s)
Antibacterianos/síntesis química , Oligopéptidos/síntesis química , Nucleósidos de Pirimidina/síntesis química , Aldehídos/química , Antibacterianos/farmacología , Hidroxilación , Estructura Molecular , Oligopéptidos/farmacología , Pseudomonas/efectos de los fármacos , Nucleósidos de Pirimidina/farmacología , Relación Estructura-ActividadRESUMEN
PURPOSE: To develop a unique in vitro aqueous fluid-capacity-limited dissolution system for the kinetic assessment of respirable aerosol drug particles from inhaler products. METHODS: Aerosol particles of 5 inhaled corticosteroids (ICSs) from 7 inhaler products were collected in the aerodynamic 2.1-3.3 or 4.7-5.8 mum on the filter membranes using the Andersen cascade impactor. Each filter membrane was then placed onto the donor compartment of the Transwell(R) system, where addition of 0.04ml aqueous fluid initiated aerosol ICS dissolution and permeation across its supporting membrane at 37 degrees C and approximately 100% humidity. RESULTS: The % profiles of dissolution and permeation were apparent first-order or pseudo-zero-order, reaching varying 1.9-95.0% by 5 h. Their kinetics overall conformed to the ICS aqueous solubility. With increasing aerosol mass, however, the profiles decelerated, attributed to undissolved ICSs left by the limited dissolution fluid capacity. The profiles could be also product-specific, as beclomethasone dipropionate aerosols from QVAR dissolved faster than those from VANCERIL, whereas fluticasone propionate aerosols from two different inhaler products exhibited comparable profiles. The 2.1-3.3 mum aerosols dissolved faster than the 4.7-5.8 mum aerosols. CONCLUSIONS: Aerosol ICS dissolution into the limited aqueous fluid volume differed kinetically due to ICS solubility and aerosol mass, size, formulation and/or generation.
Asunto(s)
Aerosoles/química , Preparaciones Farmacéuticas/administración & dosificación , Administración por Inhalación , Corticoesteroides/administración & dosificación , Corticoesteroides/química , Química Farmacéutica , Filtración , Cinética , Membranas Artificiales , Nebulizadores y Vaporizadores , Tamaño de la Partícula , Permeabilidad , Preparaciones Farmacéuticas/química , Solubilidad , Soluciones , AguaRESUMEN
The protozoan Trypanosoma cruzi, the causative agent of Chagas' disease, can infect the heart, causing cardiac arrest frequently followed by death. To treat this disease, a potential molecular drug target is T. cruzi trans-sialidase (TcTS). However, inhibitors found to date are not strong enough to serve as a lead scaffold; most inhibitors reported thus far are derivatives of the substrate sialic acid or a transition state analogue known as 2,3-dehydro-3-deoxy-N-acetylneuraminic acid (DANA) with an IC(50) value of more than hundreds of micromolar. Since natural products are highly stereodiversified and often provide highly specific biological activity, we screened a natural product library for inhibitors of TcTS and identified promising flavonoid and anthraquinone derivatives. A structure-activity relationship (SAR) analysis of the flavonoids revealed that apigenin had the minimal and sufficient structure for inhibition. Intriguingly, the compound has been reported to possess trypanocidal activity. An SAR analysis of anthraquinones showed that 6-chloro-9,10-dihydro-4,5,7-trihydroxy-9,10-dioxo-2-anthracenecarboxylic acid had the strongest inhibitory activity ever found against TcTS. Moreover, its inhibitory activity appeared to be specific to TcTS. These compounds may serve as potent lead chemotherapeutic scaffolds against Chagas' disease.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glicoproteínas/antagonistas & inhibidores , Neuraminidasa/antagonistas & inhibidores , Trypanosoma cruzi/enzimología , Secuencia de Bases , Cartilla de ADN , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Neuraminidasa/genética , Neuraminidasa/aislamiento & purificación , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
The assessment and prediction of lung absorption and disposition are an increasingly essential preclinical task for successful discovery and product development of inhaled drugs for both local and systemic delivery. Hence, in vitro, ex vivo and in vivo preclinical methods of lung absorption continue to evolve with several technical, methodological and analytical refinements. As in vitro lung epithelial cell monolayer models, the air-liquid interface (ALI)-cultured Calu-3 cells have most frequently been used, but the NCI-H441 and hAELVi cells have now been proposed as the first immortalized human alveolar epithelial cells capable of forming highly-restricted monolayers. The primary ALI-cultured three-dimensional (3D) human lung cell barriers have also become available; efforts to incorporate aerosol drug deposition into the in vitro lung cell models continue; and stem cell-derived lung epithelial cells and "lung-on-a-chip" technology are emerging. The ex vivo isolated perfused rat lung (IPRL) methods have increasing been used, as they enable the kinetic determination of tissue/organ-level diffusive and membrane protein-mediated absorption and competing non-absorptive loss; the assessment of "pre-epithelial" aerosol biopharmaceutical events in the lung, such as dissolution and release; and the ex vivo-to-in vivo extrapolation and prediction. Even so, in vivo small rodent-based methods have been of mainstay use, while large animal-based methods find an additional opportunity to study region-dependent lung absorption and disposition. It is also exciting that human pharmacokinetic (PK) profiles and systemic exposures for inhaled drugs/molecules may be able to be predicted from these in vivo rodent PK data following lung delivery using kinetic modeling approach with allometric scaling. Overall, the value of these preclinical assessments appears to have shifted more to their translational capability of predicting local lung and systemic exposure in humans, in addition to rationalizing optimal inhaled dosage form and delivery system for drugs/molecules in question. It is critically important therefore to make appropriate selection and timely exploitation of the best models at each stage of drug discovery and development program for efficient progress toward product approval and clinical use.
Asunto(s)
Aerosoles/administración & dosificación , Aerosoles/farmacocinética , Evaluación Preclínica de Medicamentos/métodos , Pulmón/metabolismo , Administración por Inhalación , Animales , Transporte Biológico , Línea Celular , Células Epiteliales/metabolismo , Humanos , Dispositivos Laboratorio en un Chip , RatasRESUMEN
BACKGROUND: Human factor XIa (FXIa) is an actively pursued target for development of safer anticoagulants. Our long-standing hypothesis has been that allosterism originating from heparin-binding site(s) on coagulation enzymes is a promising approach to yield safer agents. OBJECTIVES: To develop a synthetic heparin mimetic as an inhibitor of FXIa so as to reduce clot formation in vivo but not carry high bleeding risk. METHODS: We employed a gamut of methods involving synthetic chemistry, biophysical biochemistry, enzyme assays, blood and plasma coagulation assays, and in vivo thrombosis models in this work. RESULTS: Sulfated chiro-inositol (SCI), a non-saccharide mimetic of heparin, was synthesized in three steps in overall yields of >50%. SCI inhibited FXIa with potency of 280 nmol/L and preferentially engaged FXIa's heparin-binding site to conformationally alter its active site. SCI inhibition of FXIa could be rapidly reversed by common antidotes, such as protamine. SCI preferentially prolonged plasma clotting initiated with recalcification, rather than thromboplastin, alluding to its intrinsic pathway-based mechanism. Human blood thromboelastography indicated good ex vivo anticoagulation properties of SCI. Rat tail bleeding and maximum-dose-tolerated studies indicated that no major bleeding or toxicity concerns for SCI suggesting a potentially safer anticoagulation outcome. FeCl3 -induced arterial and thromboplastin-induced venous thrombosis model studies in the rat showed reduced thrombus formation by SCI at 250 µg/animal, which matched enoxaparin at 2500 µg/animal. CONCLUSIONS: Overall, SCI is a highly promising, allosteric inhibitor of FXIa that induces potent anticoagulation in vivo. Further studies are necessary to assess SCI in animal models mimicking human clinical indications.
Asunto(s)
Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Factor XIa/antagonistas & inhibidores , Heparina/farmacología , Inositol/farmacología , Imitación Molecular , Sulfatos/farmacología , Trombosis/prevención & control , Regulación Alostérica , Animales , Anticoagulantes/síntesis química , Anticoagulantes/toxicidad , Cloruros , Modelos Animales de Enfermedad , Factor XIa/metabolismo , Femenino , Compuestos Férricos , Hemorragia/inducido químicamente , Heparina/química , Heparina/toxicidad , Humanos , Inositol/análogos & derivados , Inositol/síntesis química , Inositol/toxicidad , Ratas Wistar , Medición de Riesgo , Sulfatos/síntesis química , Sulfatos/toxicidad , Trombosis/sangre , Trombosis/inducido químicamenteRESUMEN
As a promising long-acting inhaled formulation, liposomal ciprofloxacin (Lipo-CPFX) was characterized in the in vitro human lung epithelial Calu-3 cell monolayer system, compared to ciprofloxacin in solution (CPFX). Its modulated absorptive transport and uptake, and sustained inhibitory activity against induced pro-inflammatory interleukin-8 (IL-8) release were examined. The absorptive transport and uptake kinetics for Lipo-CPFX and CPFX were determined at 0.1-50â¯mg/ml in the Transwell system. The Lipo-CPFX transport was then challenged for mechanistic exploration via cell energy depletion, a reduced temperature, endocytosis and/or lipid fusion inhibition, and addition of excess non-loaded liposomes. The inhibitory activities of Lipo-CPFX and CPFX against lipopolysaccharide (LPS)-induced IL-8 release were assessed in a co-incubation or pre-incubation mode. In the tight Calu-3 cell monolayers, Lipo-CPFX yielded 15-times slower ciprofloxacin flux of absorptive transport and 5-times lower cellular drug uptake than CPFX. Its transport appeared to be transcellular; kinetically linear, proportional to encapsulated ciprofloxacin concentration; and consistent with the cell energy-independent lipid bilayer fusion mechanism. Lipo-CPFX was equipotent to CPFX in the anti-IL-8 releasing activity upon 24â¯h co-incubation with LPS. Additionally, Lipo-CPFX, but not CPFX, retained the anti-IL-8 releasing activity even 24â¯h after pre-incubation. In conclusion, Lipo-CPFX enabled slower absorptive lung epithelial cell transport and uptake of ciprofloxacin, apparently via the lipid bilayer fusion mechanism, and the sustained inhibitory activity against LPS-induced IL-8 release, compared to CPFX.
Asunto(s)
Ciprofloxacina/farmacología , Células Epiteliales/efectos de los fármacos , Interleucina-8/antagonistas & inhibidores , Liposomas/química , Liposomas/farmacología , Pulmón/efectos de los fármacos , Línea Celular , Ciprofloxacina/química , Células Epiteliales/metabolismo , Humanos , Membrana Dobles de Lípidos/metabolismo , Pulmón/metabolismoRESUMEN
Despite the interest in systemic delivery of therapeutic molecules including macromolecular proteins and peptides via the lung, the accurate assessment of their pulmonary biopharmaceutics is a challenging experimental task. This article reviews in vivo, in vitro and ex vivo models currently available for studying lung absorption and disposition for inhaled therapeutic molecules. The general methodologies are discussed with recent advances, current challenges and perspectives, especially in the context of their use in systemic pulmonary delivery research. In vivo approaches in small rodents continue to be the mainstay of assessment by virtue of the acquisition of direct pharmacokinetic data, more meaningful when attention is given to reproducible dosing and control of lung-regional distribution through use of more sophisticated lung-dosing methods, such as forced instillation, microspray, nebulization and aerosol puff. A variety of in vitro lung epithelial cell lines models and primary cultured alveolar epithelial (AE) cells when grown to monolayer status offer new opportunity to clarify the more detailed kinetics and mechanisms of transepithelial drug transport. While continuous cell lines, Calu-3 and 16HBE14o-, show potential, primary cultured AE cell models from rat and human origins may be of greater use, by virtue of their universally tight intercellular junctions that discriminate the transport kinetics of different therapeutic entities. Nevertheless, the relevance of using these reconstructed barriers to represent complex disposition of intact lung may still be debatable. Meanwhile, the intermediate ex vivo model of the isolated perfused lung (IPL) appears to resolve deficiencies of these in vivo and in vitro models. While controlling lung-regional distributions, the preparation alongside a novel kinetic modeling analysis enables separate determinations of kinetic descriptors for lung absorption and non-absorptive clearances, i.e., mucociliary clearance, phagocytosis and/or metabolism. This ex vivo model has been shown to be kinetically predictive of in vivo, with respect to macromolecular disposition, despite limitations concerning short viable periods of 2-3 h and likely absence of tracheobronchial circulation. Given the advantages and disadvantages of each model, scientists must make appropriate selection and timely exploitation of the best model at each stage of the research and development program, affording efficient progress toward clinical trials for future inhaled therapeutic entities for systemic delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Pulmón/metabolismo , Preparaciones Farmacéuticas/administración & dosificación , Absorción , Administración por Inhalación , Animales , Transporte Biológico , Células Cultivadas , Humanos , Modelos Animales , Distribución TisularRESUMEN
The complexity and rapid clearance mechanisms of lung tissue make it difficult to develop effective treatments for many chronic pathologies. We are investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. The main objectives of this study include effective decellularization of porcine lung tissue, development of a hydrogel from the porcine ECM, and characterization of the material's composition, mechanical properties, and ability to support cellular growth. Our evaluation of the decellularized tissue indicated successful removal of cellular material and immunogenic remnants in the ECM. The self-assembly of the lung ECM hydrogel was rapid, reaching maximum modulus values within 3 min at 37°C. Rheological characterization showed the lung ECM hydrogel to have a concentration dependent storage modulus between 15 and 60 Pa. The purpose of this study was to evaluate our novel ECM derived hydrogel and measure its ability to support 3D culture of MSCs in vitro and in vivo delivery of MSCs. Our in vitro experiments using human mesenchymal stem cells demonstrated our novel ECM hydrogel's ability to enhance cellular attachment and viability. Our in vivo experiments demonstrated that rat MSC delivery in pre-gel solution significantly increased cell retention in the lung over 24 h in an emphysema rat model. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1922-1935, 2016.