RESUMEN
Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts, no new antibiotic class with activity against Gram-negative bacteria has been approved in over 50 years. LepB inhibitors (LepBi) based on the arylomycin class of natural products are a novel class of antibiotics and function by inhibiting the bacterial type I signal peptidase (SPase) in Gram-negative bacteria. One critical aspect of LepBi development involves optimization of the membrane-anchored lipophilic portion of the molecule. We therefore developed an approach that assesses the effect of this portion on the complicated equilibria of plasma protein binding, crossing the outer membrane of Gram-negative bacteria and anchoring in the bacterial inner membrane to facilitate SPase binding. Our findings provide important insights into the development of antibacterial agents where the target is associated with the inner membrane of Gram-negative bacteria.
RESUMEN
Oral administration is the most popular and convenient route for drug delivery, yet the success of oral drug delivery is dependent on the ADME properties of the drug. Among those ADME properties, permeability is considered one of the key attributes for successful oral drug absorption. Hence, the utilization of permeability enhancers to improve drug oral absorption is an important area of research in drug delivery. A multitude of data suggests that sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) is an effective permeability enhancer. Despite its success, the mechanism of how SNAC works to enhance the oral absorption of compounds is poorly understood. To better understand how SNAC worked, we investigated the hypothesis of SNAC promotes lymphatic absorption of target compounds. In this study, cyanocobalamin was used as the model compound and mesenteric lymph duct cannulated rats were used to investigate its absorption with or without SNAC. The present study demonstrated that SNAC enhanced the lymphatic absorption of cyanocobalamin when the two were co-dosed in rats. Furthermore, levels of SNAC in lymph fluid and the systemic circulation were higher when co-dosed with cyanocobalamin.
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Caprilatos , Sodio , Ratas , Animales , Preparaciones Farmacéuticas , Administración Oral , Vitamina B 12 , PermeabilidadRESUMEN
The "free drug hypothesis" assumes that, in the absence of transporters, the steady state free plasma concentrations equal to that at the site of action that elicit pharmacologic effects. While it is important to utilize the free drug hypothesis, exceptions exist that the free plasma exposures, either at Cmax, Ctrough, and Caverage, or at other time points, cannot represent the corresponding free tissue concentrations. This "drug concentration asymmetry" in both total and free form can influence drug disposition and pharmacological effects. In this review, we first discuss options to assess total and free drug concentrations in tissues. Then various drug design strategies to achieve concentration asymmetry are presented. Last, the utilities of tissue concentrations in understanding exposure-effect relationships and translational projections to humans are discussed for several therapeutic areas and modalities. A thorough understanding in plasma and tissue exposures correlation with pharmacologic effects can provide insightful guidance to aid drug discovery.
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Descubrimiento de Drogas , Plasma , Humanos , Proteínas de Transporte de MembranaRESUMEN
BACKGROUND: Janus Kinases (JAKs) mediate activity of many asthma-relevant cytokines. GDC-0214, an inhaled small molecule JAK1 inhibitor, has previously been shown to reduce fractional exhaled nitric oxide (FeNO) in patients with mild asthma, but required an excessive number of inhalations. AIM: To assess whether GDC-4379, a new inhaled JAK inhibitor, reduces FeNO and peripheral biomarkers of inflammation. METHODS: This study assessed the activity of GDC-4379 in a double-blind, randomized, placebo-controlled, Phase 1 study in patients with mild asthma. Participants included adults (18-65y) with a diagnosis of asthma for ≥6 months, forced expiratory volume in 1 s (FEV1)> 70% predicted, FeNO >40 ppb, using as-needed short-acting beta-agonist medication only. Four sequential, 14-day, ascending-dose cohorts (10 mg QD, 30 mg QD, 40 mg BID, and 80 mg QD) of 12 participants each were randomized 2:1 to GDC-4379 or placebo. The primary activity outcome was percent change from baseline (CFB) in FeNO to Day 14 compared to the pooled placebo group. Safety, tolerability, pharmacokinetics, and pharmacodynamic biomarkers, including blood eosinophils, serum CCL17, and serum CCL18, were also assessed. RESULTS: Of 48 enrolled participants, the mean age was 25 years and 54% were female. Median (range) FeNO at baseline was 79 (41-222) ppb. GDC-4379 treatment led to dose-dependent reductions in FeNO. Compared to placebo, mean (95% CI) percent CFB in FeNO to Day 14 was: -6 (-43, 32) at 10 mg QD, -26 (-53, 2) at 30 mg QD, -55 (-78, -32) at 40 mg BID and -52 (-72, -32) at 80 mg QD. Dose-dependent reductions in blood eosinophils and serum CCL17 were also observed. Higher plasma drug concentrations corresponded with greater FeNO reductions. No serious AEs occurred. The majority of AEs were mild to moderate. The most common AEs were headache and oropharyngeal pain. Minor changes in neutrophils were noted at 80 mg QD, but were not considered clinically meaningful. CONCLUSIONS: In patients with mild asthma, 14-day treatment with GDC-4379 reduced FeNO levels and peripheral biomarkers of inflammation. Treatment was well tolerated without any major safety concerns. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY: ACTRN12619000227190.
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Asma , Inhibidores de las Cinasas Janus , Adulto , Asma/tratamiento farmacológico , Australia , Biomarcadores , Pruebas Respiratorias , Femenino , Humanos , Inflamación/tratamiento farmacológico , Inhibidores de las Cinasas Janus/efectos adversos , Masculino , Óxido NítricoRESUMEN
Several inflammatory cytokines that promote inflammation and pathogenesis in asthma signal through the Janus kinase 1 (JAK1) pathway. This phase I, randomized, placebo-controlled trial assessed the pharmacokinetics and safety of single and multiple ascending doses up to 15 mg twice daily for 14 days of a JAK1 inhibitor, GDC-0214, in healthy volunteers (HVs; n = 66). Doses were administered with a dry powder, capsule-based inhaler. An accompanying open-label gamma scintigraphy study in HVs examined the lung deposition of a single dose of inhaled Technetium-99m (99m Tc)-radiolabeled GDC-0214. GDC-0214 plasma concentrations were linear and approximately dose-proportional after both single and multiple doses. Peak plasma concentrations occurred at 15-30 min after dosing. The mean apparent elimination half-life ranged from 32 to 56 h across all single and multiple dose cohorts. After single and multiple doses, all adverse events were mild or moderate, and none led to treatment withdrawal. There was no clear evidence of systemic toxicity due to JAK1 inhibition, and systemic exposure was low, with plasma concentrations at least 15-fold less than the plasma protein binding-corrected IC50 of JAK1 at the highest dose. Scintigraphy showed that approximately 50% of the emitted dose of radiolabeled GDC-0214 was deposited in the lungs and was distributed well to the peripheral airways. 99m Tc-radiolabeled GDC-0214 (1 mg) exhibited a mean plasma Cmax similar to that observed in phase I at the same dose level. Overall, inhaled GDC-0214 exhibited pharmacokinetic properties favorable for inhaled administration.
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Pulmón , Área Bajo la Curva , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Voluntarios Sanos , Humanos , Pulmón/diagnóstico por imagen , CintigrafíaRESUMEN
Age, hypercholesterolemia, and vitamin D deficiency are risk factors that increase the brain accumulation of pathogenic ß-amyloid peptides (40 and 42), precursors leading to Alzheimer's disease (AD) in humans. The relative changes accompanying aging, high cholesterol, and/or treatment of calcitriol, active vitamin D receptor (VDR) ligand, under normal physiology are unknown. We examined these relative changes in C57BL/6 mice of ages 2, 4-8, and more than 10 months old, which were fed a normal or high fat / high cholesterol diet and treated with calcitriol, active ligand of the vitamin D receptor (0 or 2.5 µg/kg ×4, intraperitoneally, every other day to elicit cholesterol lowering in liver). Aß40 but not Aß42 accumulation in brain and lower P-glycoprotein (P-gp) and neprilysin protein expressions for Aß efflux and degradation, respectively, were found to be associated with aging. But there was no trend for BACE1 (ß-secretase 1, a cholesterol-sensitive enzyme) toward Aß synthesis with age. In response to calcitriol treatment, P-gp was elevated, mitigating partially the age-related changes. Although age-dependent decreasing trends in mRNA expression levels existed for Cyp46a1, the brain cholesterol processing enzyme, whose inhibition increases BACE1 and ApoE to facilitate microglia Aß degradation, mRNA changes for other cholesterol transporters: Acat1 and Abca1, and brain cholesterol levels remained unchanged. There was no observable change in the mRNA expression of amyloid precursor protein (APP) and the influx (RAGE) and efflux (LRP1) transporters with respect to age, diet, or calcitriol treatment. Overall, aging poses as a risk factor contributing to Aß accumulation in brain, and VDR-mediated P-gp activation partially alleviates the outcome.
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Envejecimiento/fisiología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo , Calcitriol/farmacología , Receptores de Calcitriol/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Apolipoproteínas E/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/enzimología , Encéfalo/metabolismo , Encéfalo/patología , Colesterol 24-Hidroxilasa/metabolismo , Hipercolesterolemia/metabolismo , Ratones , Ratones Endogámicos C57BL , Vitaminas/farmacologíaRESUMEN
Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.
RESUMEN
Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.
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Neoplasias/tratamiento farmacológico , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinonas/uso terapéutico , Quinasas raf/antagonistas & inhibidores , Animales , Azetidinas/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Combinación de Medicamentos , Sinergismo Farmacológico , Femenino , Humanos , Células de Riñón Canino Madin Darby , Ratones Desnudos , Estructura Molecular , Mutación , Compuestos de Fenilurea/química , Compuestos de Fenilurea/metabolismo , Piperidinas/uso terapéutico , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Quinazolinonas/química , Quinazolinonas/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
BACKGROUND: The Janus kinase (JAK) pathway mediates the activity of many asthma-relevant cytokines, including IL-4 and IL-13. GDC-0214 is a potent, inhaled, small-molecule JAK inhibitor being developed for the treatment of asthma. OBJECTIVE: We sought to determine whether GDC-0214 reduces fractional exhaled nitric oxide (Feno), a JAK1-dependent biomarker of airway inflammation, in patients with mild asthma. METHODS: We conducted a double-blind, randomized, placebo-controlled, phase 1 proof-of-activity study in adults with mild asthma and Feno higher than 40 parts per billion (ppb). Subjects were randomized 2:1 (GDC-0214:placebo) into 4 sequential ascending-dose cohorts (1 mg once daily [QD], 4 mg QD, 15 mg QD, or 15 mg twice daily). All subjects received 4 days of blinded placebo, then 10 days of either active drug or placebo. The primary outcome was placebo-corrected percent reduction in Feno from baseline to day 14. Baseline was defined as the average Feno during the blinded placebo period. Pharmacokinetics, safety, and tolerability were also assessed. RESULTS: Thirty-six subjects (mean age, 28 years; 54% females) were enrolled. Mean Feno at baseline across all subjects was 93 ± 43 ppb. At day 14, placebo-corrected difference in Feno was -23% (95% CI, -37.3 to -9) for 15 mg QD and -42% (95% CI, -57 to -27.4) for 15 mg twice daily. Higher plasma exposure was associated with greater Feno reduction. No dose-limiting adverse events, serious adverse events, or treatment discontinuations occurred. There were no major imbalances in adverse events or laboratory findings, or evidence of systemic JAK inhibition. CONCLUSIONS: GDC-0214, an inhaled JAK inhibitor, caused dose-dependent reductions in Feno in mild asthma and was well tolerated without evidence of systemic toxicity.
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Antiasmáticos/uso terapéutico , Asma/tratamiento farmacológico , Inhibidores de las Cinasas Janus/uso terapéutico , Óxido Nítrico/metabolismo , Adolescente , Adulto , Antiasmáticos/sangre , Antiasmáticos/farmacocinética , Antiasmáticos/farmacología , Asma/metabolismo , Método Doble Ciego , Espiración , Femenino , Humanos , Inhibidores de las Cinasas Janus/sangre , Inhibidores de las Cinasas Janus/farmacocinética , Inhibidores de las Cinasas Janus/farmacología , Masculino , Adulto JovenRESUMEN
Cyclodextrins are widely used pharmaceutical excipients, particularly for insoluble compounds dosed orally, such as the oral solution of itraconazole, which is frequently used in clinical drug-drug interaction studies to inhibit cytochrome P450 3A. Since cyclodextrins act by forming inclusion complexes with their coformulated drug, they could have an unintended consequence of affecting absorption if they form a strong complex with the potential victim drug in an itraconazole drug-drug interaction study. This observation was made in a drug-drug interaction study with the Bruton's tyrosine kinase (BTK) inhibitor fenebrutinib and itraconazole, in which, relative to the control group, the expected increase in fenebrutinib maximum plasma concentration (Cmax ) was not observed in the itraconazole group, and a delay in time to reach maximum plasma concentration (Tmax ) was observed in the itraconazole group. The in vitro binding constant between fenebrutinib and hydroxypropyl-ß-cyclodextrin was determined to be 2 × 105 M-1 , and the apparent permeability of fenebrutinib across a Madin-Darby canine kidney cell monolayer decreased in a cyclodextrin concentration-dependent manner. This observation was confirmed in vivo, in a pentagastrin-pretreated dog model, in which fenebrutinib was administered with or without cyclodextrin; a reduction in Cmax , a prolonged Tmax , and increased fenebrutinib recovery in feces replicated the previous observation in healthy volunteers and supported the hypothesis that complexation with cyclodextrin decreased rate and extent of fenebrutinib absorption. Physiologically-based pharmacokinetic modeling was used to translate the in vitro effect of cyclodextrin on fenebrutinib apparent permeability to the in vivo effect on absorption, which was then confirmed using the in vivo dog pharmacokinetic data.
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2-Hidroxipropil-beta-Ciclodextrina/efectos adversos , Excipientes/administración & dosificación , Absorción Intestinal/efectos de los fármacos , Itraconazol/efectos adversos , Piperazinas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinética , Piridonas/farmacocinética , 2-Hidroxipropil-beta-Ciclodextrina/administración & dosificación , Administración Oral , Adolescente , Adulto , Animales , Perros , Esquema de Medicación , Interacciones Farmacológicas , Excipientes/toxicidad , Heces/química , Femenino , Humanos , Itraconazol/administración & dosificación , Células de Riñón Canino Madin Darby , Masculino , Persona de Mediana Edad , Modelos Animales , Permeabilidad , Piperazinas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridonas/administración & dosificación , Adulto JovenRESUMEN
Fenebrutinib is a CYP3A substrate and time-dependent inhibitor, as well as a BCRP and OATP1B transporter inhibitor in vitro. Physiologically-based pharmacokinetic (PBPK) modeling strategies with the ultimate goal of understanding complex drug-drug interactions (DDIs) and proposing doses for untested scenarios were developed. The consistency in the results of two independent approaches, PBPK simulation and endogenous biomarker measurement, supported that the observed transporter DDI is primarily due to fenebrutinib inhibition of intestinal BCRP, rather than hepatic OATP1B. A mechanistic-absorption model accounting for the effects of excipient complexation with fenebrutinib was used to rationalize the unexpected observation of itraconazole-fenebrutinib DDI (maximum plasma concentration (Cmax ) decreased, and area under the curve (AUC) increased). The totality of the evidence from sensitivity analysis and clinical and nonclinical data suggested that fenebrutinib is likely a sensitive CYP3A substrate. This advanced PBPK application allowed the use of model-informed approach to facilitate the development of concomitant medication recommendations for fenebrutinib without requiring additional clinical DDI studies.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Inhibidores del Citocromo P-450 CYP3A/farmacocinética , Citocromo P-450 CYP3A/metabolismo , Intestinos/efectos de los fármacos , Transportador 1 de Anión Orgánico Específico del Hígado/antagonistas & inhibidores , Hígado/efectos de los fármacos , Modelos Biológicos , Proteínas de Neoplasias/antagonistas & inhibidores , Piperazinas/farmacocinética , Piridonas/farmacocinética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Biotransformación , Ensayos Clínicos Fase I como Asunto , Simulación por Computador , Inhibidores del Citocromo P-450 CYP3A/efectos adversos , Inhibidores del Citocromo P-450 CYP3A/química , Perros , Composición de Medicamentos , Desarrollo de Medicamentos , Interacciones Farmacológicas , Excipientes/química , Humanos , Hígado/metabolismo , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de Neoplasias/metabolismo , Piperazinas/efectos adversos , Piperazinas/química , Piridonas/efectos adversos , Piridonas/química , Estudios RetrospectivosRESUMEN
Oral administration is the preferred route for drug delivery and its success is highly dependent on a compound's ADME properties, of which, permeability plays a major role. Therefore, permeability enhancers are an attractive area of research in the pharmaceutical industry. Recent data suggest that sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) is an effective permeability enhancer, yet the pharmacokinetic (PK) and systemic effects of SNAC are poorly understood, specifically its oral bioavailability and systemic effects on distribution, which could influence the safety of certain drugs. To answer these questions, both in vitro and in vivo studies were conducted. Of 3 permeability enhancers (SNAC, 4-CNAB, and 5-CNAC), SNAC was found to have the greatest effect on the absorption of cyanocobalamin in rats. It was also found that SNAC is orally bioavailable (almost 40%) when dosed to rats. Based on these findings, tool compounds were co-dosed in rats to further evaluate the systemic effects of SNAC. Oral co-dosing of SNAC with an intravenous infusion of 2 poorly brain penetrant compounds, quinidine, and gabapentin, did not increase brain ISF: plasma ratio or total brain:plasma ratio for either of these compounds, implying that SNAC is effective only in the intestine at pharmacologically relevant concentrations.
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Caprilatos , Preparaciones Farmacéuticas , Administración Oral , Animales , Permeabilidad , Ratas , SodioRESUMEN
Mechanistic understanding of complex clinical drug-drug interactions (DDIs) with potential involvement of multiple elimination pathways has been challenging, especially given the general lack of specific probe substrates for transporters. Here, we conducted a clinical DDI study to evaluate the interaction potential of fenebrutinib using midazolam (MDZ; CYP3A), simvastatin (CYP3A and OATP1B), and rosuvastatin (BCRP and OATP1B) as probe substrates. Fenebrutinib (200 mg) increased the area under the curve (AUC) of these probe substrates twofold to threefold. To evaluate the mechanism of the observed DDIs, we measured the concentration of coproporphyrin I (CP-I) and coproporphyrin III (CP-III), endogenous biomarkers of OATP1B. There was no change in CP-I or CP-III levels with fenebrutinib, suggesting that the observed DDIs were caused by inhibition of CYP3A and BCRP rather than OATP1B, likely due to increased bioavailability. This is the first published account using an endogenous transporter biomarker to understand the mechanism of complex DDIs involving multiple elimination pathways.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Citocromo P-450 CYP3A/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Adulto , Área Bajo la Curva , Biomarcadores/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas , Femenino , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado/efectos de los fármacos , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Masculino , Midazolam/farmacocinética , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Piperazinas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridonas/administración & dosificación , Rosuvastatina Cálcica/farmacocinética , Simvastatina/farmacocinética , Adulto JovenRESUMEN
The pan-proteasome inhibitor bortezomib demonstrated clinical efficacy in off-label trials of Systemic Lupus Erythematosus. One potential mechanism of this clinical benefit is from the depletion of pathogenic immune cells (plasmablasts and plasmacytoid dendritic cells). However, bortezomib is cytotoxic against nonimmune cells, which limits its use for autoimmune diseases. An attractive alternative is to selectively inhibit the immune cell-specific immunoproteasome to deplete pathogenic immune cells and spare nonhematopoietic cells. Here, we disclose the development of highly subunit-selective immunoproteasome inhibitors using insights obtained from the first bona fide human immunoproteasome cocrystal structures. Evaluation of these inhibitors revealed that immunoproteasome-specific inhibition does not lead to immune cell death as anticipated and that targeting viability requires inhibition of both immuno- and constitutive proteasomes. CRISPR/Cas9-mediated knockout experiments confirmed upregulation of the constitutive proteasome upon disruption of the immunoproteasome, protecting cells from death. Thus, immunoproteasome inhibition alone is not a suitable approach to deplete immune cells.
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Diseño de Fármacos , Inmunidad Celular/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/síntesis química , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Humanos , Inmunidad Celular/fisiología , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/farmacología , Estructura Terciaria de ProteínaRESUMEN
Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.
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Antibacterianos/clasificación , Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Biocatálisis/efectos de los fármacos , Productos Biológicos/clasificación , Productos Biológicos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Escherichia coli/enzimología , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/patogenicidad , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/patogenicidad , Lisina/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/química , Porinas , Unión Proteica , Dominios Proteicos , Serina Endopeptidasas , Especificidad por SustratoRESUMEN
Intracerebral microdialysis is a powerful technique for quantifying unbound brain extracellular fluid concentrations of drugs and drug candidates over time in live, conscious, freely moving animals. The results provide crucial information on the brain penetrance of the administered agent. This unit details the surgical procedures, study planning and execution, and data analysis required for a brain microdialysis study following intravenous infusion of a test substance to steady state. The accumulated data makes possible the determination of the area under the concentration-time curve ratio and steady-state concentration ratio for unbound compound in rat brain extracellular fluid as compared to that in plasma (Kpu,u ). In vitro determination of the compound recovery from the probe, a critical parameter in a microdialysis study, is considered as well. © 2018 by John Wiley & Sons, Inc.
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Encéfalo/metabolismo , Líquido Extracelular/metabolismo , Microdiálisis , Animales , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/metabolismo , RatasRESUMEN
Microdialysis is a powerful technique allowing for real-time measurement of unbound drug concentrations in brain interstitial fluid in conscious animals. Use of microdialysis in drug discovery is limited by high resource requirement and low throughput, but this may be improved by cassette dosing. Administering multiple compounds intravenously of diverse physiochemical properties, it is often very challenging and time consuming to identify a vehicle that can dissolve all of the compounds. To overcome this limitation, the present study explores the possibility of administering a cassette dose of nine diverse compounds (carbamazepine, citalopram, desmethylclozapine, diphenhydramine, gabapentin, metoclopramide, naltrexone, quinidine, and risperidone) in suspension, rather than in solution, by intraperitoneal and subcutaneous routes, and determining if this is a viable option for assessing blood-brain barrier penetration in microdialysis studies. Repeated hourly subcutaneous dosing during the 6-hour microdialysis study allowed for the best attainment of distributional equilibrium between brain and plasma, resulting in less than a 2-fold difference in the unbound brain to unbound plasma concentration ratio for the cassette dosing method versus discrete dosing. Both subcutaneous and intraperitoneal repeated dosing can provide a more practical substitute for intravenous dosing in determining brain penetration of a cassette of diverse compounds in brain microdialysis studies. The results from the present study demonstrate that dosing compounds in suspension represents a practical approach to eliminating the technical challenge and labor-intensive step of preparation of solutions of a mixture of compounds and will enable the use of the cassette brain microdialysis method in a central nervous system drug discovery setting.
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Preparaciones Farmacéuticas/administración & dosificación , Animales , Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Líquido Extracelular/metabolismo , Inyecciones Intraperitoneales , Masculino , Microdiálisis/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
PURPOSE: Since the vitamin D receptor (VDR) was found to up-regulate cerebral P-glycoprotein expression in vitro and in mice, we extend our findings to rats by assessing the effect of rat Vdr activation on brain efflux of quinidine, a P-gp substrate that is eliminated primarily by cytochrome P450 3a. METHODS: We treated rats with vehicle or the active VDR ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] (4.8 or 6.4 nmol/kg i.p. every 2nd day × 4) and examined P-gp expression and cerebral quinidine disposition via microdialysis in control and treatment studies conducted longitudinally in the same rat. RESULTS: The 6.4 nmol/kg 1,25(OH)2D3 dose increased cerebral P-gp expression 1.75-fold whereas hepatic Cyp3a remained unchanged. Although there was no change in systemic clearance elicited by 1,25(OH)2D3, brain extracellular fluid quinidine concentrations were lower in treated rats. We noted that insertion of indwelling catheters increased plasma protein binding of quinidine and serial sampling decreased the blood:plasma concentration ratio, factors that alter distribution ratios in microdialysis studies. After appropriate correction, KECF/P,uu and KECF/B,uu, or ratios of quinidine unbound concentrations in brain extracellular fluid to plasma or blood at steady-state, were more than halved. CONCLUSION: We demonstrate that VDR activation increases cerebral P-gp expression and delimits brain penetration of P-gp substrates.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Calcitriol/farmacología , Permeabilidad Capilar/efectos de los fármacos , Microdiálisis , Quinidina/metabolismo , Receptores de Calcitriol/agonistas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Estado de Conciencia , Citocromo P-450 CYP3A/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Microsomas Hepáticos/enzimología , Unión Proteica , Quinidina/sangre , Ratas Sprague-Dawley , Receptores de Calcitriol/metabolismo , Regulación hacia ArribaRESUMEN
We demonstrate a role of the vitamin D receptor (VDR) in reducing cerebral soluble and insoluble amyloid-ß (Aß) peptides. Short-term treatment of two human amyloid precursor protein-expressing models, Tg2576 and TgCRND8 mice, with 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], the endogenous active ligand of VDR, resulted in higher brain P-glycoprotein (P-gp) and lower soluble Aß levels, effects negated with coadministration of elacridar, a P-gp inhibitor. Long-term treatment of TgCRND8 mice with 1,25(OH)2D3 during the period of plaque formation reduced soluble and insoluble plaque-associated Aß, particularly in the hippocampus in which the VDR is abundant and P-gp induction is greatest after 1,25(OH)2D3 treatment, and this led to improved conditioned fear memory. In mice fed a vitamin D-deficient diet, lower cerebral P-gp expression was observed, but levels were restored on replenishment with VDR ligands. The composite data suggest that the VDR is an important therapeutic target in the prevention and treatment of Alzheimer's disease.
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Enfermedad de Alzheimer/complicaciones , Péptidos beta-Amiloides/metabolismo , Corteza Cerebral/metabolismo , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/etiología , Vitamina D/análogos & derivados , Vitaminas/uso terapéutico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Corteza Cerebral/efectos de los fármacos , Condicionamiento Psicológico/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Vitamina D/sangre , Vitamina D/uso terapéuticoRESUMEN
BACKGROUND & AIMS: Little is known about the effects of the vitamin D receptor (VDR) on hepatic activity of human cholesterol 7α-hydroxylase (CYP7A1) and cholesterol metabolism. We studied these processes in mice in vivo and mouse and human hepatocytes. METHODS: Farnesoid X receptor (Fxr)(-/-), small heterodimer partner (Shp)(-/-), and C57BL/6 (wild-type control) mice were fed normal or Western diets for 3 weeks and were then given intraperitoneal injections of vehicle (corn oil) or 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3; 4 doses, 2.5 µg/kg, every other day). Plasma and tissue samples were collected and levels of Vdr, Shp, Cyp7a1, Cyp24a1, and rodent fibroblast growth factor (Fgf) 15 expression, as well as levels of cholesterol, were measured. We studied the regulation of Shp by Vdr using reporter and mobility shift assays in transfected human embryonic kidney 293 cells, quantitative polymerase chain reaction with mouse tissues and mouse and human hepatocytes, and chromatin immunoprecipitation assays with mouse liver. RESULTS: We first confirmed the presence of Vdr mRNA and protein expression in livers of mice. In mice fed normal diets and given injections of 1,25(OH)2D3, liver and plasma concentrations of 1,25(OH)2D3 increased and decreased in unison. Changes in hepatic Cyp7a1 messenger RNA (mRNA) correlated with those of Cyp24a1 (a Vdr target gene) and inversely with Shp mRNA, but not ileal Fgf15 mRNA. Similarly, incubation with 1,25(OH)2D3 increased levels of Cyp24a1/CYP24A1 and Cyp7a1/CYP7A1 mRNA in mouse and human hepatocytes, and reduced levels of Shp mRNA in mouse hepatocytes. In Fxr(-/-) and wild-type mice with hypercholesterolemia, injection of 1,25(OH)2D3 consistently reduced levels of plasma and liver cholesterol and Shp mRNA, and increased hepatic Cyp7a1 mRNA and protein; these changes were not observed in Shp(-/-) mice given 1,25(OH)2D3 and fed Western diets. Truncation of the human small heterodimer partner (SHP) promoter and deletion analyses revealed VDR-dependent inhibition of SHP, and mobility shift assays showed direct binding of VDR to enhancer regions of SHP. In addition, chromatin immunoprecipitation analysis of livers from mice showed that injection of 1,25(OH)2D3 increased recruitment of Vdr and rodent retinoid X receptor to the Shp promoter. CONCLUSIONS: Activation of the VDR represses hepatic SHP to increase levels of mouse and human CYP7A1 and reduce cholesterol.