RESUMO
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.
Assuntos
Antineoplásicos , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-met , Pirazóis , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Humanos , Animais , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/farmacologia , Pirazóis/química , Pirazóis/síntese química , Linhagem Celular Tumoral , Relação Estrutura-Atividade , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Descoberta de Drogas , Pirazinas/farmacologia , Pirazinas/síntese química , Pirazinas/química , Pirazinas/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Camundongos , Mutação , RatosRESUMO
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).
Assuntos
Antineoplásicos , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Humanos , Animais , Camundongos , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Relação Estrutura-Atividade , Ratos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , MasculinoRESUMO
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.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Mutação , Neoplasias , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Camundongos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Neoplasias/tratamento farmacológico , Neoplasias/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Masculino , Pessoa de Meia-Idade , Benzimidazóis/farmacocinética , Benzimidazóis/uso terapêutico , Benzimidazóis/administração & dosagem , Benzimidazóis/farmacologia , Idoso , Adulto , Linhagem Celular TumoralRESUMO
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.
Assuntos
Inibidores de Proteínas Quinases , Receptor ErbB-2 , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Animais , Descoberta de Drogas , Mutação , Linhagem Celular Tumoral , Relação Estrutura-Atividade , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Camundongos , Ratos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismoRESUMO
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.
Assuntos
Neoplasias Pulmonares , Proteínas Tirosina Quinases , Humanos , Proteínas Tirosina Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Oncogenes , Assistência Centrada no PacienteRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Camundongos , Modelos Moleculares , Mutação , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Anticorpos Monoclonais Humanizados/toxicidade , Artérias/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Hemorragia Gastrointestinal/induzido quimicamente , Hipotensão/induzido quimicamente , Óxido Nítrico/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Artérias/metabolismo , Artérias/fisiopatologia , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Hemorragia Gastrointestinal/metabolismo , Hemorragia Gastrointestinal/fisiopatologia , Hematemese/induzido quimicamente , Hematemese/metabolismo , Hematemese/fisiopatologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipotensão/metabolismo , Hipotensão/fisiopatologia , Interleucina-17/antagonistas & inibidores , Interleucina-17/imunologia , Interleucina-17/metabolismo , Macaca fascicularis , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Fatores de TempoRESUMO
PURPOSE: Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1-induced thrombocytopenia. EXPERIMENTAL DESIGN: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H(3)]-DM1 and (125)I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR. RESULTS: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure. CONCLUSIONS: These data support the hypothesis that T-DM1-induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs.
Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Antineoplásicos/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Maitansina/análogos & derivados , Trombocitopenia/patologia , Ado-Trastuzumab Emtansina , Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias da Mama/complicações , Neoplasias da Mama/patologia , Diferenciação Celular/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/efeitos adversos , Maitansina/administração & dosagem , Maitansina/efeitos adversos , Megacariócitos/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Plasma Rico em Plaquetas/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Trombocitopenia/induzido quimicamente , Trombocitopenia/etiologia , TrastuzumabRESUMO
Improved endurance exercise performance in adult humans after sprint interval training (SIT) has been attributed to mitochondrial biogenesis. However, muscle protein synthesis (MPS) and mitochondrial biogenesis during SIT have not been measured, nor have sex-specific differences. We hypothesized that males and females would have similar rates of MPS, mitochondrial biogenesis, and synthesis of individual proteins during SIT. Deuterium oxide (D2O) was orally administered to 21 adults [11 male, 10 female; mean age, 23±1 yr; body mass index (BMI), 22.8±0.6 kg/m(2); mean± SE] for 4 wk, to measure protein synthesis rates while completing 9 sessions of 4-8 bouts of 30 s duration on a cycle ergometer separated by 4 min of active recovery. Samples of the vastus lateralis were taken before and 48 h after SIT. SIT increased maximum oxygen uptake (VO(2max), males 43.4±2.1-44.0±2.3; females 39.5±0.9-42.5±1.3 ml/kg/min; P=0.002). MPS was greater in the males than in the females in the mixed (~150%; P < 0.001), cytosolic (~135%; P=0.038), and mitochondrial (~135%; P=0.056) fractions. The corresponding ontological clusters of individual proteins were significantly greater in the males than in the females (all P<0.00001). For the first time, we document greater MPS and mitochondrial biogenesis during SIT in males than in females and describe the synthetic response of individual proteins in humans during exercise training.
Assuntos
Exercício Físico/fisiologia , Mitocôndrias Musculares/metabolismo , Proteínas Musculares/biossíntese , Caracteres Sexuais , Óxido de Deutério , Feminino , Humanos , Masculino , Proteínas Mitocondriais/biossíntese , Consumo de Oxigênio/fisiologia , Educação Física e Treinamento , Resistência Física/fisiologia , Músculo Quadríceps/metabolismo , Adulto JovemRESUMO
Having an understanding of drug tissue accumulation can be informative in the assessment of target organ toxicities; however, obtaining tissue drug levels from toxicology studies by bioanalytical methods is labor-intensive and infrequently performed. Additionally, there are no described methods for predicting tissue drug distribution for the experimental conditions in toxicology studies, which typically include non-steady-state conditions and very high exposures that may saturate several processes. The aim was the development of an algorithm to provide semiquantitative and quantitative estimates of tissue-to-plasma concentration ratios (Kp ) for several tissues from readily available parameters of pharmacokinetics (PK) such as volume of distribution (Vd ) and clearance of each drug, without performing tissue measurement in vivo. The computational approach is specific for the oral route of administration and non-steady-state conditions and was applied for a dataset of 29 Genentech small molecules such as neutral compounds as well as weak and strong organic bases. The maximum success rate in predicting Kp values within 2.5-fold error of observed Kp values was 82% at low doses (<100 mg/kg) in preclinical species. Prediction accuracy was relatively lower with saturation at high doses (≥100 mg/kg); however, an approach to perform low-to-high dose extrapolations of Kp values was presented and applied successfully in most cases. An approach for the interspecies scaling was also applied successfully. Finally, the proposed algorithm was used in a case study and successfully predicted differential tissue distribution of two small-molecule MET kinase inhibitors, which had different toxicity profiles in mice. This newly developed algorithm can be used to predict the partition coefficients Kp for small molecules in toxicology studies, which can be leveraged to optimize the PK drivers of tissue distribution in an attempt to decrease drug tissue level, and improve safety margins.
Assuntos
Preparações Farmacêuticas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacocinética , Algoritmos , Animais , Cães , Camundongos , Modelos Biológicos , Ratos , Distribuição TecidualRESUMO
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.
Assuntos
Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/toxicidade , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Feminino , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-met/metabolismo , Distribuição Aleatória , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologiaRESUMO
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.
Assuntos
Aldeído Desidrogenase/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Malato Desidrogenase/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Monoterpenos/farmacologia , Albumina Sérica/metabolismo , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído-Desidrogenase Mitocondrial , Animais , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Fígado/química , Malato Desidrogenase/antagonistas & inibidores , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Fibroblast growth factor 19 (FGF19) represses cholesterol 7α-hydroxylase (Cyp7α1) and inhibits bile acid synthesis in vitro and in vivo. Previous studies have shown that anti-FGF19 antibody treatment reduces growth of colon tumor xenografts and prevents hepatocellular carcinomas in FGF19 transgenic mice and thus may be a useful cancer target. In a repeat dose safety study in cynomolgus monkeys, anti-FGF19 treatment (3-100 mg/kg) demonstrated dose-related liver toxicity accompanied by severe diarrhea and low food consumption. The mechanism of anti-FGF19 toxicity was investigated using in vitro and in vivo approaches. Our results show that anti-FGF19 antibody had no direct cytotoxic effect on monkey hepatocytes. Anti-FGF19 increased Cyp7α1, as expected, but also increased bile acid efflux transporter gene (bile salt export pump, multidrug resistant protein 2 [MRP2], and MRP3) expression and reduced sodium taurocholate cotransporting polypeptide and organic anion transporter 2 expression in liver tissues from treated monkeys and in primary hepatocytes. In addition, anti-FGF19 treatment increased solute transporter gene (ileal bile acid-binding protein, organic solute transporter α [OST-α], and OST-ß) expression in ileal tissues from treated monkeys but not in Caco-2 cells. However, deoxycholic acid (a secondary bile acid) increased expression of FGF19 and these solute transporter genes in Caco-2 cells. Gas chromatography-mass spectrometry analysis of monkey feces showed an increase in total bile acids and cholic acid derivatives. These findings suggest that high doses of anti-FGF19 increase Cyp7α1 expression and bile acid synthesis and alter the expression of bile transporters in the liver resulting in enhanced bile acid efflux and reduced uptake. Increased bile acids alter expression of solute transporters in the ileum causing diarrhea and the enhanced enterohepatic recirculation of bile acids leading to liver toxicity.
Assuntos
Anticorpos/imunologia , Ácidos e Sais Biliares/biossíntese , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Íleo/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Fatores de Crescimento de Fibroblastos/imunologia , Perfilação da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/metabolismo , Absorção Intestinal , Macaca fascicularisRESUMO
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.
Assuntos
Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Rim/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fósforo/sangue , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Animais , Cálcio/sangue , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Homeostase/efeitos dos fármacos , Rim/enzimologia , Rim/metabolismo , Masculino , Estrutura Molecular , Hormônio Paratireóideo/sangue , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Espectrometria de Massas em Tandem , Vitamina D/sangueRESUMO
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.
Assuntos
Cromatografia Líquida , Inibidores das Enzimas do Citocromo P-450 , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Microssomos Hepáticos/efeitos dos fármacos , Espectrometria de Massas em Tandem , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , Técnicas In Vitro , Isoenzimas , Cinética , Microssomos Hepáticos/enzimologia , Sondas Moleculares/metabolismo , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
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.
Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP3A/biossíntese , Descoberta de Drogas/métodos , Hepatócitos/enzimologia , Oxirredutases N-Desmetilantes/biossíntese , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Receptor Constitutivo de Androstano , Citocromo P-450 CYP2B6 , Indução Enzimática , Hepatócitos/efeitos dos fármacos , Humanos , Ligantes , Receptor de Pregnano X , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Espectrometria de Massas em Tandem , Xenobióticos/farmacologiaRESUMO
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.
Assuntos
Hepatócitos/metabolismo , Regiões Promotoras Genéticas , Receptores de Esteroides/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Adulto , Sítios de Ligação , Imunoprecipitação da Cromatina , Feminino , Hepatócitos/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Receptor de Pregnano X , Interferência de RNA , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/genética , Elementos de Resposta , Rifampina/farmacologia , Ativação TranscricionalRESUMO
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.
Assuntos
Antivirais , Proteínas de Transporte/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Inibidores de Proteases , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/metabolismo , Macaca fascicularis , Polietilenoglicóis/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Ratos , Proteínas Recombinantes , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Técnicas de Química Combinatória/métodos , Perfilação da Expressão Gênica/métodos , Regiões Promotoras Genéticas , Receptores de Esteroides/metabolismo , Benzopiranos/farmacologia , Células CACO-2 , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Regulação da Expressão Gênica , Humanos , Inativação Metabólica/genética , Intestinos/efeitos dos fármacos , Modelos Biológicos , Receptor de Pregnano X , Rifampina/farmacologia , Especificidade por Substrato/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
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.