RESUMEN
The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72-96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Proteínas Proto-Oncogénicas c-met/inmunología , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/inmunología , Procesos de Crecimiento Celular/efectos de los fármacos , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/inmunología , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Epítopos Inmunodominantes/inmunología , Ratones , Ratones Desnudos , Morfogénesis/efectos de los fármacos , Células 3T3 NIH , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/genética , Transgenes/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Kinases are members of a major protein family targeted for drug discovery and development. Given the ubiquitous nature of many kinases as well as the broad range of pathways controlled by these enzymes, early safety assessments of small molecule inhibitors of kinases are crucial in identifying new molecules with sufficient therapeutic window for clinical development. Failure or attrition of drug candidates in late-stage pipelines due to hepatotoxicity is a significant challenge in the drug development field. Herein we provide detailed methods for the hepatocyte imaging assay technology (HIAT) and the bile flux imaging assay technology (BIAT) to evaluate drug-induced liver injury (DILI) potentials for drug candidates. Optimized culturing methods for primary human hepatocytes, both freshly isolated and prequalified cryopreserved cells, are also presented. The applications of these high-content cellular imaging technologies in the evaluation of p38 and Her2 kinase inhibitors are highlighted to illustrate the usefulness of the research methodology in a compound screening as well as mechanistic investigative setting.
Asunto(s)
Bilis/metabolismo , Inhibidores Enzimáticos/toxicidad , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Imagen Molecular/métodos , Fosfotransferasas/antagonistas & inhibidores , Pruebas de Toxicidad/métodos , Bioensayo/métodos , Células Cultivadas , Colágeno , Criopreservación , Combinación de Medicamentos , Humanos , Procesamiento de Imagen Asistido por Computador , Laminina , Microscopía Fluorescente , Proteoglicanos , Coloración y EtiquetadoRESUMEN
CP-724,714, a potent and selective orally active HER2 tyrosine kinase inhibitor, was discontinued from clinical development due to unexpected hepatotoxicity in cancer patients. Based on the clinical manifestation of the toxicity, CP-724,714 likely exerted its hepatotoxicity via both hepatocellular injury and hepatobiliary cholestatic mechanisms. The direct cytotoxic effect, hepatobiliary disposition of CP-724,714, and its inhibition of active canalicular transport of bile constituents were evaluated in established human hepatocyte models and in vitro transporter systems. CP-724,714 exhibited direct cytotoxicity using human hepatocyte imaging assay technology with mitochondria identified as a candidate organelle for its off-target toxicity. Additionally, CP-724,714 was rapidly taken up into human hepatocytes, partially via an active transport process, with an uptake clearance approximately fourfold higher than efflux clearance. The major human hepatic uptake transporter, OATP1B1, and efflux transporters, multidrug resistance protein 1 (MDR1) and breast cancer resistance protein, were involved in hepatobiliary clearance of CP-724,714. Furthermore, CP-724,714 displayed a concentration-dependent inhibition of cholyl-lysyl fluorescein and taurocholate (TC) efflux into canaliculi in cryopreserved and fresh cultured human hepatocytes, respectively. Likewise, CP-724,714 inhibited TC transport in membrane vesicles expressing human bile salt export pump with an IC(50) of 16 microM. Finally, CP-724,714 inhibited the major efflux transporter in bile canaliculi, MDR1, with an IC(50) of approximately 28 microM. These results suggest that inhibition of hepatic efflux transporters contributed to hepatic accumulation of drug and bile constituents leading to hepatocellular injury and hepatobiliary cholestasis. This study provides likely explanations for clinically observed adverse liver effects of CP-724,714.
Asunto(s)
Proteínas Portadoras/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/toxicidad , Hígado/metabolismo , Quinazolinas/farmacocinética , Quinazolinas/toxicidad , Receptor ErbB-2/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Bilis/metabolismo , Ácidos y Sales Biliares/metabolismo , Calcio/metabolismo , Células Cultivadas , Fluoresceínas/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado/antagonistas & inhibidores , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismoRESUMEN
Drug-induced liver injury (DILI) is the most common adverse event causing drug nonapprovals and drug withdrawals. Using drugs as test agents and measuring a panel of cellular phenotypes that are directly linked to key mechanisms of hepatotoxicity, we have developed an in vitro testing strategy that is predictive of many clinical outcomes of DILI. Mitochondrial damage, oxidative stress, and intracellular glutathione, all measured by high content cellular imaging in primary human hepatocyte cultures, are the three most important features contributing to the hepatotoxicity prediction. When applied to over 300 drugs and chemicals including many that caused rare and idiosyncratic liver toxicity in humans, our testing strategy has a true-positive rate of 50-60% and an exceptionally low false-positive rate of 0-5%. These in vitro predictions can augment the performance of the combined traditional preclinical animal tests by identifying idiosyncratic human hepatotoxicants such as nimesulide, telithromycin, nefazodone, troglitazone, tetracycline, sulindac, zileuton, labetalol, diclofenac, chlorzoxazone, dantrolene, and many others. Our findings provide insight to key DILI mechanisms, and suggest a new approach in hepatotoxicity testing of pharmaceuticals.
Asunto(s)
Hepatocitos/efectos de los fármacos , Células Cultivadas , Bases de Datos como Asunto , Reacciones Falso Positivas , Hepatocitos/patología , Humanos , Mitocondrias Hepáticas/fisiología , Preparaciones Farmacéuticas/clasificación , Farmacocinética , Curva ROCRESUMEN
Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, nefazodone was the most toxic compound of the three, trazodone had relatively modest effects, whereas buspirone showed the least toxicity. Nefazodone profoundly inhibited mitochondrial respiration in isolated rat liver mitochondria and in intact HepG2 cells where this was accompanied by simultaneous acceleration of glycolysis. Using immunocaptured oxidative phosphorylation (OXPHOS) complexes, we identified Complex 1, and to a lesser amount Complex IV, as the targets of nefazodone toxicity. No inhibition was found for trazodone, and buspirone showed 3.4-fold less inhibition of OXPHOS Complex 1 than nefazodone. In human hepatocytes that express cytochrome P450, isoform 3A4, after 24 h exposure, nefazodone and trazodone collapsed mitochondrial membrane potential, and imposed oxidative stress, as detected via glutathione depletion, leading to cell death. Our results suggest that the mitochondrial impairment imposed by nefazodone is profound and likely contributes to its hepatotoxicity, especially in patients cotreated with other drugs with mitochondrial liabilities.