RESUMEN
Mutant BRAFV600E is one of the most common oncogenic drivers in metastatic melanoma. While first generation BRAFV600E inhibitors are capable of controlling tumors systemically, they are unable to adequately treat tumors that have metastasized to the brain due to insufficient penetration across the blood-brain barrier (BBB). Through a combination of structure-based drug design (SBDD) and the optimization of physiochemical properties to enhance BBB penetration, we herein report the discovery of the brain-penetrant BRAFV600E inhibitor PF-07284890 (ARRY-461). In mice studies, ARRY-461 proved to be highly brain-penetrant and was able to drive regressions of A375 BRAFV600E tumors implanted both subcutaneously and intracranially. Based on compelling preclinical safety and efficacy studies, ARRY-461 was progressed into a Phase 1 A/B clinical trial (NCT04543188).
Asunto(s)
Antineoplásicos , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Humanos , Animales , Ratones , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Femenino , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/patología , Relación Estructura-Actividad , Ratas , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , MasculinoRESUMEN
HER2 overexpression and amplification have been identified as oncogenic drivers, and the development of therapies to treat tumors harboring these markers has received considerable attention. Activation of HER2 signaling and subsequent cell growth can also be induced by HER2 mutations, including the common YVMA insertion in exon 20 within the kinase domain. Enhertu is currently the only approved treatment for HER2 mutant tumors in NSCLC. TKIs tested in this space have suffered from off-target activity, primarily due to EGFRWT inhibition or attenuated activity against HER2 mutants. The goal of this work was to identify a TKI that would provide robust inhibition of oncogenic HER2WT and HER2 mutants while sparing EGFRWT activity. Herein, we describe the development of a potent, covalent inhibitor of HER2WT and the YVMA insertion mutant while providing oral bioavailability and avoiding the inhibition of EGFRWT.
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Inhibidores de Proteínas Quinasas , Receptor ErbB-2 , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Animales , Descubrimiento de Drogas , Mutación , Línea Celular Tumoral , Relación Estructura-Actividad , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Ratones , Ratas , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismoRESUMEN
RAF inhibitors have transformed treatment for patients with BRAFV600-mutant cancers, but clinical benefit is limited by adaptive induction of ERK signaling, genetic alterations that induce BRAFV600 dimerization, and poor brain penetration. Next-generation pan-RAF dimer inhibitors are limited by a narrow therapeutic index. PF-07799933 (ARRY-440) is a brain-penetrant, selective, pan-mutant BRAF inhibitor. PF-07799933 inhibited signaling in vitro, disrupted endogenous mutant-BRAF:wild-type-CRAF dimers, and spared wild-type ERK signaling. PF-07799933 ± binimetinib inhibited growth of mouse xenograft tumors driven by mutant BRAF that functions as dimers and by BRAFV600E with acquired resistance to current RAF inhibitors. We treated patients with treatment-refractory BRAF-mutant solid tumors in a first-in-human clinical trial (NCT05355701) that utilized a novel, flexible, pharmacokinetics-informed dose escalation design that allowed rapid achievement of PF-07799933 efficacious concentrations. PF-07799933 ± binimetinib was well-tolerated and resulted in multiple confirmed responses, systemically and in the brain, in patients with BRAF-mutant cancer who were refractory to approved RAF inhibitors. Significance: PF-07799933 treatment was associated with antitumor activity against BRAFV600- and non-V600-mutant cancers preclinically and in treatment-refractory patients, and PF-07799933 could be safely combined with a MEK inhibitor. The novel, rapid pharmacokinetics (PK)-informed dose escalation design provides a new paradigm for accelerating the testing of next-generation targeted therapies early in clinical development.
Asunto(s)
Resistencia a Antineoplásicos , Mutación , Neoplasias , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Ratones , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Masculino , Persona de Mediana Edad , Bencimidazoles/farmacocinética , Bencimidazoles/uso terapéutico , Bencimidazoles/administración & dosificación , Bencimidazoles/farmacología , Anciano , Adulto , Línea Celular TumoralRESUMEN
Mesenchymal-epithelial transition factor (MET) is a receptor tyrosine kinase that serves a critical function in numerous developmental, morphogenic, and proliferative signaling pathways. If dysregulated, MET has been shown to be involved in the development and survival of several cancers, including non-small cell lung cancer (NSCLC), renal cancer, and other epithelial tumors. Currently, the clinical efficacy of FDA approved MET inhibitors is limited by on-target acquired resistance, dose-limiting toxicities, and less than optimal efficacy against brain metastasis. Therefore, there is still an unmet medical need for the development of MET inhibitors to address these issues. Herein we report the application of structure-based design for the discovery and development of a novel class of brain-penetrant MET inhibitors with enhanced activity against clinically relevant mutations and improved selectivity. Compound 13 with a MET D1228N cell line IC50 value of 23 nM showed good efficacy in an intracranial tumor model and increased the median overall survival of the animals to 100% when dosed orally at 100 mg/kg daily for 21 days.
Asunto(s)
Antineoplásicos , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-met , Pirazoles , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Humanos , Animales , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/farmacología , Pirazoles/química , Pirazoles/síntesis química , Línea Celular Tumoral , Relación Estructura-Actividad , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Pirazinas/farmacología , Pirazinas/síntesis química , Pirazinas/química , Pirazinas/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Ratones , Mutación , RatasRESUMEN
Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstrated by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd>3l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd=1.0l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Femenino , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Masculino , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-met/metabolismo , Distribución Aleatoria , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
Rationally targeted therapies have transformed cancer treatment, but many patients develop resistance through bypass signaling pathway activation. PF-07284892 (ARRY-558) is an allosteric SHP2 inhibitor designed to overcome bypass-signaling-mediated resistance when combined with inhibitors of various oncogenic drivers. Activity in this setting was confirmed in diverse tumor models. Patients with ALK fusion-positive lung cancer, BRAFV600E-mutant colorectal cancer, KRASG12D-mutant ovarian cancer, and ROS1 fusion-positive pancreatic cancer who previously developed targeted therapy resistance were treated with PF-07284892 on the first dose level of a first-in-human clinical trial. After progression on PF-07284892 monotherapy, a novel study design allowed the addition of oncogene-directed targeted therapy that had previously failed. Combination therapy led to rapid tumor and circulating tumor DNA (ctDNA) responses and extended the duration of overall clinical benefit. SIGNIFICANCE: PF-07284892-targeted therapy combinations overcame bypass-signaling-mediated resistance in a clinical setting in which neither component was active on its own. This provides proof of concept of the utility of SHP2 inhibitors in overcoming resistance to diverse targeted therapies and provides a paradigm for accelerated testing of novel drug combinations early in clinical development. See related commentary by Hernando-Calvo and Garralda, p. 1762. This article is highlighted in the In This Issue feature, p. 1749.
Asunto(s)
Neoplasias Pulmonares , Proteínas Tirosina Quinasas , Humanos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Oncogenes , Atención Dirigida al PacienteRESUMEN
Pulegone is the major constituent of pennyroyal oil, a folkloric abortifacient that is associated with hepatotoxicity and, in severe cases, death. Cytochrome P450-mediated oxidation of pulegone generates menthofuran, which is further oxidized to form electrophilic reactive intermediates, menthofuran epoxide and the ring-opened γ-ketoenal, both of which can form adducts to hepatocellular proteins. Modification of hepatocellular proteins by the electrophilic reactive intermediates of menthofuran has been implicated in hepatotoxicity caused by pennyroyal oil. Herein, we describe the identification of several proteins that are the likely targets of menthofuran-derived reactive metabolites. These proteins were isolated from the livers of rats treated with a hepatotoxic dose of menthofuran by two-dimensional gel electrophoresis (2D-gel) separation and detected by Western blot analysis using an antiserum developed to detect protein adducts resulting from menthofuran bioactivation. The antibody-reacting proteins were excised from the 2D-gel and subjected to tryptic digestion for analysis of peptide fragments by LC-MS/MS. Although 10 spots were detected by Western blot analysis, only 4 were amenable to characterization by LC-MS/MS: serum albumin, mitochondrial aldehyde dehydrogenase (ALDH2), cytoplasmic malate dehydrogenase (MDH1), and mitochondrial ATP synthase subunit d. No direct adduct was detected, and, therefore, we complemented our analysis with enzyme activity determination. ALDH2 activity decreased by 88%, and ATP synthase complex V activity decreased by 34%, with no activity changes to MDH1. Although the relationship between these reactive metabolite adducted proteins and hepatotoxicity is not clear, these targeted enzymes are known to play critical roles in maintaining cellular homeostasis.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Malato Deshidrogenasa/metabolismo , Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Monoterpenos/farmacología , Albúmina Sérica/metabolismo , Aldehído Deshidrogenasa/antagonistas & inhibidores , Aldehído Deshidrogenasa Mitocondrial , Animales , Citoplasma/efectos de los fármacos , Citoplasma/enzimología , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Hígado/química , Malato Deshidrogenasa/antagonistas & inhibidores , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Chromatin immunoprecipitation (ChIP) studies were conducted in human hepatocytes treated with rifampicin in order to identify new pregnane-X receptor (PXR) target genes. Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified. Validation experiments identified several new drug disposition genes with PXR binding sites. Of these, only CYP4F12 demonstrated increased binding in the presence of rifampicin. The role of PXR in the basal and inductive response of CYP4F12 was confirmed in hepatocytes in which PXR was silenced. We also assessed the association of PXR-coactivators and -corepressors with known and newly identified PXREs. Both PXR and the steroid receptor coactivator (SRC-1) were found to bind to PXREs in the absence of rifampicin, although binding was stronger after rifampicin treatment. We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein. In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.
Asunto(s)
Hepatocitos/metabolismo , Regiones Promotoras Genéticas , Receptores de Esteroides/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Adulto , Sitios de Unión , Inmunoprecipitación de Cromatina , Femenino , Hepatocitos/efectos de los fármacos , Humanos , Persona de Mediana Edad , Receptor X de Pregnano , Interferencia de ARN , Receptores de Esteroides/antagonistas & inhibidores , Receptores de Esteroides/genética , Elementos de Respuesta , Rifampin/farmacología , Activación TranscripcionalRESUMEN
Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target's resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRASG12C that bind in the switch-II pocket of KRAS and make a covalent bond to cysteine 12. Using structure-based drug design in conjunction with a focused in vitro absorption, distribution, metabolism and excretion screening approach, analogues were synthesized to increase the potency and reduce metabolic liabilities of this series. The discovery of the clinical development candidate MRTX849 as a potent, selective covalent inhibitor of KRASG12C is described.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Humanos , Ratones , Modelos Moleculares , Mutación , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Future treatments for chronic hepatitis C virus (HCV) infection are likely to include agents that target viral components directly. Here, the preclinical characteristics of ITMN-191, a peptidomimetic inhibitor of the NS3/4A protease of HCV, are described. ITMN-191 inhibited a reference genotype 1 NS3/4A protein in a time-dependent fashion, a hallmark of an inhibitor with a two-step binding mechanism and a low dissociation rate. Under preequilibrium conditions, 290 pM ITMN-191 half-maximally inhibited the reference NS3/4A protease, but a 35,000-fold-higher concentration did not appreciably inhibit a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Subnanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 4, 5, and 6, while single-digit nanomolar potency was observed against NS3/4A from genotypes 2b and 3a. Dilution of a preformed enzyme inhibitor complex indicated ITMN-191 remained bound to and inhibited NS3/4A for more than 5 h after its initial association. In cell-based potency assays, half-maximal reduction of genotype 1b HCV replicon RNA was afforded by 1.8 nM; 45 nM eliminated the HCV replicon from cells. Peginterferon alfa-2a displayed a significant degree of antiviral synergy with ITMN-191 and reduced the concentration of ITMN-191 required for HCV replicon elimination. A 30-mg/kg of body weight oral dose administered to rats or monkeys yielded liver concentrations 12 h after dosing that exceeded the ITMN-191 concentration required to eliminate replicon RNA from cells. These preclinical characteristics compare favorably to those of other inhibitors of NS3/4A in clinical development and therefore support the clinical investigation of ITMN-191 for the treatment of chronic hepatitis C.
Asunto(s)
Antivirales , Proteínas Portadoras/antagonistas & inhibidores , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Inhibidores de Proteasas , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Animales , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Hepacivirus/genética , Hepacivirus/fisiología , Humanos , Interferón alfa-2 , Interferón-alfa/farmacología , Péptidos y Proteínas de Señalización Intracelular , Hígado/metabolismo , Macaca fascicularis , Polietilenglicoles/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Ratas , Proteínas Recombinantes , Replicación Viral/efectos de los fármacosRESUMEN
PRO304186, a humanized monoclonal antibody targeting soluble interleukin-17 A and F, was developed for autoimmune and inflammatory disease indications. When administered to cynomolgus monkeys PRO304186 induced unexpected adverse effects characterized by clinical signs of hematemesis, hematochezia, and moribundity. Pathology findings included hemorrhage throughout the gastrointestinal tract without any evidence of vascular wall damage or inflammatory cellular infiltration. Mechanistic investigation of these effects revealed mild elevations of serum MCP-1 and IL-12/23 but without a classical proinflammatory profile in PRO304186-treated animals. In vitro studies demonstrated off-target effects on vascular endothelial cells including activation of nitric oxide synthase leading to production of nitric oxide (NO) accompanied by increased mitochondrial membrane depolarization, glutathione depletion, and increased paracellular permeability. Additionally, endothelial cell-PRO304186-conditioned medium reduced myosin light chain phosphorylation in vascular smooth muscle cells. Furthermore, an ex vivo study utilizing segments from cynomolgus aorta and femoral artery confirmed PRO304186-induced endothelium-dependent smooth muscle relaxation and vasodilation mediated via NO. Finally, a single dose of PRO304186 in cynomolgus monkeys induced a rapid and pronounced increase in NO in the portal circulation that preceded a milder elevation of NO in the systemic circulation and corresponded temporally with systemic hypotension; findings consistent with NO-mediated vasodilation leading to hypotension. These changes were associated with non-inflammatory, localized hemorrhage in the gastrointestinal tract consistent with hemodynamic vascular injury associated with intense local vasodilation. Together, these data demonstrate that PRO304186-associated toxicity in monkeys was due to an off-target effect on endothelium that involved regional NO release resulting in severe systemic vasodilation, hypotension, and hemorrhage.
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Anticuerpos Monoclonales Humanizados/toxicidad , Arterias/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Hemorragia Gastrointestinal/inducido químicamente , Hipotensión/inducido químicamente , Óxido Nítrico/metabolismo , Vasodilatación/efectos de los fármacos , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Arterias/metabolismo , Arterias/fisiopatología , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Femenino , Hemorragia Gastrointestinal/metabolismo , Hemorragia Gastrointestinal/fisiopatología , Hematemesis/inducido químicamente , Hematemesis/metabolismo , Hematemesis/fisiopatología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Hipotensión/metabolismo , Hipotensión/fisiopatología , Interleucina-17/antagonistas & inhibidores , Interleucina-17/inmunología , Interleucina-17/metabolismo , Macaca fascicularis , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Factores de TiempoRESUMEN
MEK, a kinase downstream of Ras and Raf oncogenes, constitutes a high priority target in oncology research. MEK small molecule inhibitors cause soft tissue mineralization in rats secondary to serum inorganic phosphorus (iP) elevation, but the molecular mechanism for this toxicity remains undetermined. We performed investigative studies with structurally distinct MEK inhibitors GEN-A and PD325901 (PD-901) in Sprague-Dawley rats. Our data support a mechanism that involves FGF-23 signal blockade in the rat kidney, causing transcriptional upregulation of 25-hydroxyvitamin D(3) 1-alpha-hydroxylase (Cyp27b1), the rate-limiting enzyme in vitamin D activation, and downregulation of 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1), the enzyme that initiates the degradation of the active form of vitamin D. These transcriptional changes increase serum vitamin D levels, which in turn drive the increase in serum iP, leading to soft tissue mineralization in the rat.
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Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Riñón/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Fósforo/sangre , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Animales , Calcio/sangre , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/sangre , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Homeostasis/efectos de los fármacos , Riñón/enzimología , Riñón/metabolismo , Masculino , Estructura Molecular , Hormona Paratiroidea/sangre , Inhibidores de Proteínas Quinasas/química , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Espectrometría de Masas en Tándem , Vitamina D/sangreRESUMEN
A validated method for the simultaneous characterization of xenobiotic compound-mediated inhibition of seven major cytochrome P450 (CYP) enzymes in pooled human liver microsomes through the use of specific CYP probe substrates (cocktail assay) with low protein content, and a rapid, three minute LC-MS/MS analytical method is described. The specific CYP substrates used in this cocktail assay included phenacetin (CYP1A2), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4/5). The LC-MS method incorporated the aforementioned seven CYP substrates along with their respective major metabolites, and one internal standard, labetalol. In a cross-validation analysis, the concentrations of each CYP probe substrate in the assay had minimal effect (i.e., inhibition or activation) on the other CYP activities. Furthermore, the assay conditions for the multiple probe substrate, ie., cocktail assay, were validated against the single probe substrate assay using 18 compounds with known CYP inhibition liabilities and 10 proprietary compounds. The inhibitory constant (Ki) determined with this cocktail assay was highly correlated (R(2) ≥ 0.77 for each individual probe substrate) with that of the single probe substrate assay for all 27 CYP inhibitors. This seven CYP inhibition cocktail assay has increased the efficiency to assess compounds for inhibition of the major CYP isoforms in a high throughput, drug discovery setting.
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Cromatografía Liquida , Inhibidores Enzimáticos del Citocromo P-450 , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Microsomas Hepáticos/efectos de los fármacos , Espectrometría de Masas en Tándem , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Humanos , Técnicas In Vitro , Isoenzimas , Cinética , Microsomas Hepáticos/enzimología , Sondas Moleculares/metabolismo , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
INTRODUCTION: The human nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) are known to regulate gene expression of the cytochrome P450 (CYP) enzymes, 3A4, 2B6, and 1A2, respectively. In conventional CYP induction studies, the activity of each CYP enzyme is assessed in a separate incubation with the appropriate marker substrate. The objective of this study was to assess, simultaneously, the induction of CYP3A4, CYP2B6, and CYP1A2 activity in cultured human hepatocytes treated with various prototypical ligands of PXR, CAR, and AhR by utilizing an optimized substrate cocktail, as well as a rapid, sensitive liquid chromatography-mass spectrometry method. METHODS: To evaluate the xenobiotic-mediated induction of hepatocellular gene expression, the prototypical inducers rifampicin (10 µM) and phenobarbital (1 mM) were used for CYP3A4, CITCO (1 µM) and artemisinin (50 µM) were used for CYP2B6, and 3-methylcholanthrene (1 µM) and omeprazole (50 µM) were utilized for induction of CYP1A2. Primary human hepatocytes were treated with each compound for 48h, followed by a 30-min incubation of the hepatocyte culture along with the addition of three marker substrates for specific CYP activity: midazolam (CYP3A4; 5 µM), bupropion (CYP2B6; 50 µM), and phenacetin (CYP1A2; 100µM). The assessment of CYP activity was performed with a rapid, sensitive liquid chromatography-tandem mass spectrometry method which simultaneously assessed activity of CYP3A4, CYP2B6, and CYP1A2 in a single 3-min method by examining the formation of the probe substrate metabolites, 1'-hydroxymidazolam, hydroxybupropion, and acetaminophen, respectively. RESULTS: The average fold-induction of CYP3A4, CYP2B6, and CYP1A2 activity was comparable between the cocktail and the conventional assay. DISCUSSION: The combination of three marker substrates in a single 30-min incubation, in addition to a rapid, sensitive LC-MS/MS method, resulted in an efficient and robust method for assessing cytochrome P450 induction as compared to the conventional methodology.
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Hidrocarburo de Aril Hidroxilasas/biosíntesis , Citocromo P-450 CYP1A2/biosíntesis , Citocromo P-450 CYP3A/biosíntesis , Descubrimiento de Drogas/métodos , Hepatocitos/enzimología , Oxidorreductasas N-Desmetilantes/biosíntesis , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B6 , Inducción Enzimática , Hepatocitos/efectos de los fármacos , Humanos , Ligandos , Receptor X de Pregnano , Receptores de Hidrocarburo de Aril/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad por Sustrato , Espectrometría de Masas en Tándem , Xenobióticos/farmacologíaRESUMEN
Detecting and understanding the potential for off-target pharmacological effects is critical in the optimization of lead compounds in drug discovery programs. Compound-mediated activation of the pregnane X receptor (PXR; NR1I2), a key regulator for drug metabolism genes, is often monitored to avoid potential drug-drug interactions. Two structural analogs, MRL-1 and MRL-2, were determined to be equivalent PXR activators in trans-activation assays. To differentiate these two PXR activators, their transcriptional effects were examined in PXR-sufficient (LS180) and PXR-deficient (Caco-2) adenocarcinoma cell lines. Both compounds regulated drug-management genes (e.g. CYP3A4, CYP2B6, UGT1A1 and ABCB1) in LS180 cells, but not in PXR-deficient Caco-2 cells. The potency of MRL-1 and MRL-2 on PXR activation was again equivalent as revealed by a set of 113 genes that were regulated by four prototypical PXR agonists (rifampicin, ritonavir, troglitazone and dexamethasone) in the LS180 cells. The specificity of the PXR signature genes was supported by the enrichment of putative PXR binding sites uncovered by sequence-based promoter analyses. Interestingly, an additional off-target activity of MRL-2 was suggested where sterol response element binding protein binding sites were found enriched in a subset of PXR signature genes. These genes, involved in cholesterol and fatty acid synthesis, were significantly regulated by ritonavir, chlorpromazine and MRL-2, which were linked to the manifestation of phospholipidosis. The present study demonstrates the utility of our approach in the differentiation and selection of lead compounds for drug development.
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Técnicas Químicas Combinatorias/métodos , Perfilación de la Expresión Génica/métodos , Regiones Promotoras Genéticas , Receptores de Esteroides/metabolismo , Benzopiranos/farmacología , Células CACO-2 , Células Cultivadas , Ensayos de Selección de Medicamentos Antitumorales/métodos , Regulación de la Expresión Génica , Humanos , Inactivación Metabólica/genética , Intestinos/efectos de los fármacos , Modelos Biológicos , Receptor X de Pregnano , Rifampin/farmacología , Especificidad por Sustrato/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
The multidrug resistance protein Mrp2 is an ATP-binding cassette (ABC) transporter mainly expressed in liver, kidney, and intestine. One of the physiological roles of Mrp2 is to transport bilirubin glucuronides from the liver into the bile. Current in vivo models to study Mrp2 are the transporter-deficient and Eisai hyperbilirubinemic rat strains. Previous reports showed hyperbilirubinemia and induction of Mrp3 in the hepatocyte sinusoidal membrane in the mutant rats. In addition, differences in liver cytochrome P450 and UGT1a levels between wild-type and mutant rats were detected. To study whether these compensatory mechanisms were specific to rats, we characterized Mrp2(-/-) mice. Functional absence of Mrp2 in the knockout mice was demonstrated by showing increased levels of bilirubin and bilirubin glucuronides in serum and urine, a reduction in biliary excretion of bilirubin glucuronides and total glutathione, and a reduction in the biliary excretion of the Mrp2 substrate dibromosulfophthalein. To identify possible compensatory mechanisms in Mrp2(-/-) mice, the expression levels of 98 phase I, phase II, and transporter genes were compared in liver, kidney, and intestine of male and female Mrp2(-/-) and control mice. Unlike in Mrp2 mutant rats, no induction of Mrp3 in Mrp2(-/-) mice was detected. However, Mrp4 mRNA and protein in liver and kidney were increased approximately 6- and 2-fold, respectively. Phenotypic analysis of major cytochrome P450-mediated activities in liver microsomes did not show differences between wild-type and Mrp2(-/-) mice. In conclusion, Mrp2(-/-) mice are a new valuable tool to study the role of Mrp2 in drug disposition.
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Bilirrubina/análogos & derivados , Expresión Génica , Proteínas de Transporte de Membrana/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Animales , Bilis/metabolismo , Bilirrubina/sangre , Bilirrubina/orina , Sistema Enzimático del Citocromo P-450/metabolismo , Femenino , Glutatión/metabolismo , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Sulfobromoftaleína/farmacocinéticaRESUMEN
Many phase I and II microsomal enzyme inducers share common mechanisms of transcriptional activation and thus share a similar battery of genes that are coordinately regulated. Many phase II metabolites are thought to be transported out of cells by multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3). The purpose of this study was to determine the organ distribution of these three transporters in rat, and whether they are coordinately regulated with phase I and II drug-metabolizing enzymes. Therefore, Mrp1, 2, and 3 mRNAs were quantified using branched DNA signal amplification in multiple tissues and in tissues from rats that were treated with 18 chemicals thought to induce drug-metabolizing enzymes by six different transcription activation mechanisms [aryl-hydrocarbon receptor ligands, constitutive androstane receptor (CAR) activators, pregnane-X-receptor ligands, peroxisome proliferator activator receptor ligands, electrophile response element (EpRE) activators, and CYP2E1 inducers]. It was found that Mrp1 was expressed at a high level in kidney, lung, intestine, and brain, with low expression in liver. Mrp2 was highly expressed in liver and duodenum, and Mrp3 was highly expressed throughout the intestine but very low in liver. Microsomal enzyme inducers did not markedly increase the expression of Mrp1 or Mrp2. However, Mrp3 expression was significantly increased by each of the CAR activators and an EpRE activator in liver. Mrp3 was not similarly induced in kidney and large intestine, demonstrating that the coordinate inducibility of Mrp3 is specific to the liver. We conclude that rat hepatic Mrp3 is induced by CAR activators, thus enhancing the vectoral excretion of some phase II metabolites from the liver to the blood.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Andrógenos , Androstanos/metabolismo , Hígado/metabolismo , Proteínas de Transporte de Membrana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , ARN Mensajero/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Inducción Enzimática/efectos de los fármacos , Femenino , Genes MDR/genética , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Sondas de Oligonucleótidos/farmacología , Dibenzodioxinas Policloradas/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Distribución Tisular , Xenobióticos/farmacologíaRESUMEN
To date, organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is known as a liver-specific and sodium-independent transporter that mediates transport of a variety of compounds. The purpose of this study was to determine whether Oatp4 mRNA expression is specific to the liver compared with Oatp1, 2, 3, or 5. In addition, the effect of gender and age was determined by assessing the expression of Oatp4 mRNA during the postnatal development of rats. Furthermore, to determine whether Oatp4 gene expression is coordinately modulated by drug-metabolizing enzyme inducers, male rats were administered chemicals known to induce the expression of drug-metabolizing enzymes through six mechanisms: the aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, peroxisome proliferator-activated receptor, electrophile response element, or CYP2E1 inducers. The levels of Oatp1, 2, 3, 4, and 5 mRNA were measured using the branched DNA signal amplification technique. The tissue distribution of Oatp4 was almost exclusively expressed in liver in contrast to Oatp1, 2, 3, and 5. The hepatic expression of Oatp4 was low in newborn rats and increased gradually to the adult level with no significant difference between genders. The expression of Oatp4 was not consistently induced by any of the six groups of enzyme inducers. These findings continue to suggest that Oatp4 is expressed specifically in the liver. The preference of Oatp4 for endogenous compounds coupled with its refractory response to known drug-metabolizing enzyme inducers suggests that Oatp4 may be largely responsible for the homeostasis of endogenous rather than exogenous chemicals, including pharmaceuticals.
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Hígado/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/biosíntesis , Factores de Edad , Animales , Femenino , Masculino , Transportadores de Anión Orgánico/biosíntesis , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/genética , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Factores Sexuales , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos , Distribución TisularRESUMEN
Multiple drug resistance (mdr) genes encode P-glycoprotein, which is responsible for resistance to some cancer chemotherapeutic drugs and efflux of xenobiotics of cells. Thus, mdr can protect organs from xenobiotics. In rats, there are two mdr1 genes capable of xenobiotic transport, mdr1a and mdr1b. The purpose of this study was to determine the tissue distribution of rat mdr1a and mdr1b mRNA and whether microsomal enzyme inducers that increase phase I and II drug-metabolizing enzymes coordinately regulate mdr1a and/or mdr1b. The mRNA levels of mdr1a and mdr1b were determined using branched-DNA signal amplification technology. The highest level of expression of mdr1a mRNA was observed in the gastrointestinal tract, with levels increasing, respectively, from duodenum, jejunum, and ileum to large intestine. Expression levels of mdr1a mRNA in the cerebral cortex, cerebellum, kidney, lung, and liver were less than one-tenth of that in the ileum. The tissue distribution of mdr1b mRNA was similar to mdr1a with highest expression in the gastrointestinal tract but only about 3-fold higher than in most other tissues. The induction of mdr1a and mdr1b mRNA transcripts in liver, kidney, and ileum by treatment of rats with 18 chemicals representing aryl hydrocarbon receptor ligands, constitutive androstane receptor ligands, pregnane X receptor ligands, peroxisome proliferator-activated receptor ligands, electrophile-response-element activators, and CYP4502E1 inducers was assessed. Hepatic, renal, and intestinal expression of mdr1a and mdr1b mRNA were not significantly altered by treatment of rats with any of these classes of ligands. In conclusion, the primary expression of rat mdr1 genes is in the gastrointestinal tract where they are thought to function to decrease the absorption of some xenobiotics. Rat mdr1 gene expression is not readily increased by microsomal enzyme inducers in rats through coordinate mechanisms with phase I and II drug-metabolizing enzymes.
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Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Resistencia a Múltiples Medicamentos/fisiología , Compuestos Orgánicos/farmacocinética , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Masculino , Compuestos Orgánicos/farmacología , Ratas , Ratas Sprague-Dawley , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
The purpose of the present study was to evaluate the effect of 1,7-phenanthroline (PH), which has been proposed to be a selective phase II enzyme inducer, on the gene expression of xenobiotic transporters, as well as hepatic and renal drug-metabolizing enzymes. After oral administration of PH for 3 days to male Sprague-Dawley rats, mRNA levels in liver (75 and 150 mg/kg doses) and kidney (75 mg/kg dose only) were determined using real-time quantitative polymerase chain reaction. At 150 mg/kg/day, PH treatment resulted in significant increases in hepatic mRNA levels of Mrp3 (36-fold), UGT1A6 (20-fold), UGT2B1 (4-fold), and quinone reductase (QR, 5-fold), compared with the vehicle-treated group. Similar increases in Mrp3 (99-fold), UGT1A6 (17-fold), UGT2B1 (3-fold), and QR (11-fold) mRNA levels were observed in the liver after PH treatment of rats at 75 mg/kg/day. In contrast, the expression levels of CYP2C11 and Oatp2 were decreased by approximately 80 and 50%, respectively. In addition, PH (75 mg/kg/day) elicited statistically significant changes in renal gene expression of CYP3A1, UGT1A6, QR, and Mrp3, but the magnitude of renal Mrp3 induction was less than 2-fold over control. Although PH is known to modulate hepatic glucuronidation in vivo, these data indicated that PH induced mRNA levels of the efflux transporter, Mrp3, which may also affect the disposition of xenobiotics.