RESUMEN
Monkeys have been proposed as an animal model to predict the magnitude of human clinical drug-drug interactions caused by CYP3A4 enzyme induction. To evaluate whether the cynomolgus monkey can be an effective in vivo model, human CYP3A4 inducers were evaluated both in vitro and in vivo. First, a full-length pregnane X receptor (PXR) was cloned from the cynomolgus monkey, and the sequence was compared with those of rhesus monkey and human PXR. Cynomolgus and rhesus monkey PXR differed by only one amino acid (A68V), and both were highly homologous to human PXR (approximately 96%). When the transactivation profiles of 30 compounds, including known inducers of CYP3A4, were compared between cynomolgus and human PXR, a high degree of correlation with EC(50) values was observed. These results suggest that cynomolgus and human PXR respond in a similar fashion to these ligands. Second, two known human CYP3A4 inducers, rifampicin and hyperforin, were tested in monkey and human primary hepatocytes for induction of CYP3A enzymes. Both monkey and human hepatocytes responded similarly to the inducers and resulted in increased RNA and enzyme activity changes of CYP3A8 and CYP3A4, respectively. Lastly, in vivo induction of CYP3A8 by rifampicin and hyperforin was shown by significant reductions of midazolam exposure that were comparable with those in humans. These results show that the cynomolgus monkey can be a predictive in vivo animal model of PXR-mediated induction of human CYP3A4 and can provide a useful assessment of the resulting pharmacokinetic changes of affected drugs.
Asunto(s)
Citocromo P-450 CYP3A/biosíntesis , Hepatocitos/metabolismo , Macaca fascicularis , Receptores de Esteroides/metabolismo , Xenobióticos/farmacocinética , Adulto , Secuencia de Aminoácidos , Animales , Compuestos Bicíclicos con Puentes/sangre , Compuestos Bicíclicos con Puentes/farmacocinética , Compuestos Bicíclicos con Puentes/farmacología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Clonación Molecular , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas/genética , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/genética , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Hypericum/química , Macaca mulatta , Masculino , Midazolam/sangre , Midazolam/metabolismo , Midazolam/farmacocinética , Persona de Mediana Edad , Modelos Animales , Datos de Secuencia Molecular , Floroglucinol/análogos & derivados , Floroglucinol/sangre , Floroglucinol/farmacocinética , Floroglucinol/farmacología , Extractos Vegetales/sangre , Extractos Vegetales/farmacocinética , Receptor X de Pregnano , Receptores de Esteroides/genética , Rifampin/sangre , Rifampin/farmacocinética , Rifampin/farmacología , Homología de Secuencia de Aminoácido , Terpenos/sangre , Terpenos/farmacocinética , Terpenos/farmacología , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , TransfecciónRESUMEN
The identification of a new series of selective nonsteroidal progesterone receptor (PR) agonists is reported. Using a high-throughput screening assay based on the measurement of transactivation of a mouse mammary tumor virus promoter-driven luciferase reporter (MMTV-Luc) in human breast cancer T47D cells, a benzimidazole-2-thione analog was identified. Compound 1 showed an apparent EC50 of 53 nM and efficacy of 93% with respect to progesterone. It binds to PR with high affinity (Ki nM), but had no or very low affinity for other steroid hormone receptors. Structure-activity relationship studies of a series of benzimidazole-2-thione analogs revealed critical positions for high PR binding affinity and transactivation potency as well as receptor selectivity, as exemplified by 25. Compound 25 binds to human PR with high affinity (Ki nM) and had at least > 1000-fold selectivity for PR versus other steroid receptors. Molecular modeling studies suggested that these agonists overlap favorably with progesterone in the ligand-binding domain of PR. In T47D cells, compound 25 acted as a full agonist in the MMTV-Luc reporter assay, as well as in the induction of endogenous alkaline phosphatase activity with apparent EC50 values of 4 and 9 nM, respectively. In the immature rat model, compound 25 provided a significant suppression of estrogen-induced endometrium hypertrophy as measured by luminal epithelial height. In contrast, compound 25 was inactive in the luteinizing hormone release assay in young ovariectomized rats. These benzimidazole-2-thione analogs constitute a new series of nonsteroidal PR agonists with an excellent steroid receptor selectivity profile. The differential activities observed in the in vivo progestogenic assays in rat models suggest that these analogs can act as selective PR modulators.
Asunto(s)
Bencimidazoles/farmacología , Imidazoles/farmacología , Receptores de Progesterona/agonistas , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/farmacología , Animales , Bencimidazoles/síntesis química , Bencimidazoles/química , Bencimidazoles/metabolismo , Unión Competitiva/efectos de los fármacos , Línea Celular Tumoral , Femenino , Genes Reporteros , Humanos , Imidazoles/síntesis química , Imidazoles/química , Imidazoles/metabolismo , Hormona Luteinizante/metabolismo , Acetato de Medroxiprogesterona/metabolismo , Acetato de Medroxiprogesterona/farmacología , Modelos Moleculares , Conformación Molecular , Progesterona/metabolismo , Progesterona/farmacología , Unión Proteica , Ratas , Ratas Sprague-Dawley , Receptores de Progesterona/metabolismo , Compuestos de Sulfhidrilo/síntesis química , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Activación Transcripcional/efectos de los fármacos , Útero/efectos de los fármacos , Útero/metabolismoRESUMEN
The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.
Asunto(s)
Antivirales/análisis , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/crecimiento & desarrollo , Ensayos Analíticos de Alto Rendimiento/métodos , Farmacorresistencia Viral/efectos de los fármacos , Genoma Viral/genética , Hepacivirus/genética , Humanos , Reproducibilidad de los Resultados , Replicación Viral/efectos de los fármacosRESUMEN
From a trial comparing interventions to improve adherence to antiretroviral therapy-directly administered antiretroviral therapy (DAART) or an intensive adherence case management (IACM)-to standard of care (SOC), for HIV-infected participants at public HIV clinics in Los Angeles County, California, we examined the cost of adherence programs and associated health care utilization. We assessed differences between DAART, IACM, and SOC in the rate of hospitalizations, hospital days, and outpatient and emergency department visits during an average of 1.7 years from study enrollment, beginning November 2001. We assigned costs to health care utilization and program delivery. We calculated incremental costs of DAART or IACM v SOC, and compared those costs with savings in health care utilization among participants in the adherence programs. IACM participants experienced fewer hospital days compared with SOC (2.3 versus 6.7 days/1000 person-days, incidence rate ratio [IRR]: 0.34, 97.5% confidence interval [CI]: 0.13-0.87). DAART participants had more outpatient visits than SOC (44.2 versus 31.5/1000 person-days, IRR: 1.4; 97.5% CI: 1.01-1.95). Average per-participant health care utilization costs were $13,127, $8,988, and $14,416 for DAART, IACM, and SOC, respectively. Incremental 6-month program costs were $2,120 and $1,653 for DAART and IACM participants, respectively. Subtracting savings in health care utilization from program costs resulted in an average net program cost of $831 per DAART participant; and savings of $3,775 per IACM participant. IACM was associated with a significant decrease in hospital days compared to SOC and was cost saving when program costs were compared to savings in health care utilization.