RESUMEN
The metabolic fate of the human hepatotoxin fenclozic acid ([2-(4-chlorophenyl)-1,3-thiazol-4-yl]acetic acid) (Myalex) was studied in normal and bile-cannulated chimeric mice with a humanized liver, following oral administration of 10 mg/kg. This in vivo animal model was investigated to assess its utility to study "human" metabolism of fenclozic acid, and in particular to explore the formation of electrophilic reactive metabolites (RMs), potentially unique to humans. Metabolism was extensive, particularly involving the carboxylic acid-containing side chain. Metabolism resulted in the formation of a large number of metabolites and involved biotransformation via both oxidative and conjugative routes. The oxidative metabolites detected included a variety of hydroxylations as well as cysteinyl-, N-acetylcysteinyl-, and cysteinylglycine metabolites. The latter resulted from the formation of glutathione adducts/conjugates providing evidence for the production of RMs. The production of other classes of RMs included acyl-glucuronides, and the biosynthesis of acyl carnitine, taurine, glutamine, and glycine conjugates via potentially reactive acyl-CoA intermediates was also demonstrated. A number of unique "human" metabolites, e.g., those providing evidence for side-chain extension, were detected in the plasma and excreta of the chimeric liver-humanized mice that were not previously characterised in, e.g., the excreta of rat and C57BL/6 mice. The different pattern of metabolism seen in these chimeric mice with a humanized liver compared to the conventional rodents may offer clues to the factors that contributed to the drug-induced liver injury seen in humans.
Asunto(s)
Hígado/metabolismo , Tiazoles/farmacocinética , Administración Oral , Animales , Bilis/efectos de los fármacos , Bilis/metabolismo , Quimera , Heces , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Ratones SCID , Tiazoles/administración & dosificación , Tiazoles/sangre , Distribución TisularRESUMEN
As part of the drug discovery and development process, it is important to understand the human metabolism of a candidate drug prior to clinical studies. Preclinical in vitro and in vivo experiments across species are conducted to build knowledge concerning human circulating metabolites in preparation for clinical studies; therefore, the quality of these experiments is critical. Within AstraZeneca, all metabolite identification (Met-ID) information is stored in a global database using ACDLabs software. In this study, the Met-ID information derived from in vitro and in vivo studies for 27 AstraZeneca drug candidates that underwent human absorption, distribution, metabolism, and excretion studies was extracted from the database. The retrospective analysis showed that 81% of human circulating metabolites were previously observed in preclinical in vitro and/or in vivo experiments. A detailed analysis was carried out to understand which human circulating metabolites were not captured in the preclinical experiments. Metabolites observed in human hepatocytes and rat plasma but not seen in circulation in humans (extraneous metabolites) were also investigated. The majority of human specific circulating metabolites derive from multistep biotransformation reactions that may not be observed in in vitro studies within the limited time frame in which cryopreserved hepatocytes are active. Factors leading to the formation of extraneous metabolites in preclinical studies seemed to be related to species differences with respect to transporter activity, secondary metabolism, and enzyme kinetics. This retrospective analysis assesses the predictive value of Met-ID experiments and improves our ability to discriminate between metabolites expected to circulate in humans and irrelevant metabolites seen in preclinical studies.
Asunto(s)
Preparaciones Farmacéuticas/metabolismo , Animales , Perros , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Hepatocitos/metabolismo , Humanos , Macaca fascicularis , Masculino , Ratones , Conejos , Ratas , Estudios RetrospectivosRESUMEN
Although idiosyncratic adverse drug reactions are rare, they are still a major concern to patient safety. Reactive metabolites are widely accepted as playing a pivotal role in the pathogenesis of idiosyncratic adverse drug reactions. While there are today well established strategies for the risk assessment of stable metabolites within the pharmaceutical industry, there is still no consensus on reactive metabolite risk assessment strategies. This is due to the complexity of the mechanisms of these toxicities as well as the difficulty in identifying and quantifying short-lived reactive intermediates such as reactive metabolites. In this review, reactive metabolite risk and hazard assessment approaches are discussed, and their pros and cons highlighted. We also discuss the nature of idiosyncratic adverse drug reactions, using acetaminophen and nefazodone to exemplify the complexity of the underlying mechanisms of reactive metabolite mediated hepatotoxicity. One of the key gaps moving forward is our understanding of and ability to predict the contribution of immune activation in idiosyncratic adverse drug reactions. Sections are included on the clinical phenotypes of immune mediated idiosyncratic adverse drug reactions and on the present understanding of immune activation by reactive metabolites. The advances being made in microphysiological systems have a great potential to transform our ability to risk assess reactive metabolites, and an overview of the key components of these systems is presented. Finally, the potential impact of systems pharmacology approaches in reactive metabolite risk assessments is highlighted.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Acetaminofén/metabolismo , Acetaminofén/toxicidad , Analgésicos no Narcóticos/metabolismo , Analgésicos no Narcóticos/toxicidad , Animales , Antidepresivos de Segunda Generación/metabolismo , Antidepresivos de Segunda Generación/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hipersensibilidad a las Drogas/diagnóstico , Hipersensibilidad a las Drogas/metabolismo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Piperazinas , Medición de Riesgo/métodos , Triazoles/metabolismo , Triazoles/toxicidadRESUMEN
RATIONALE: Structural information on metabolites obtained in relevant biological systems can have considerable impact on the design of new drug candidates. However, with demanding turnaround times, the amount of available structural information may become rate limiting. METHODS: The workflow for metabolite identification used in our laboratory was compared to a workflow using a software tool built for computer-assisted metabolite identification. The present study covered the in vitro metabolism of a diverse set of 65 in-house compounds. The compounds were profiled across three liver-based systems, 17 compounds were tested in human liver microsomes (HLM), 12 in rat hepatocytes (RHEP), and 36 in human hepatocytes (HHEP). RESULTS: For 92% of the metabolites reported, the exact match or Markush representations were in agreement between the two workflows. The major specific biotransformations in hepatocytes which formed the metabolites were aromatic or aliphatic hydroxylations (33%), N-dealkylations (15%) and glucuronidations (12%). CONCLUSIONS: The software was shown to perform well for structural elucidation of metabolites from both phase I and phase II metabolism where the focus was on quickly understanding the rate-limiting metabolic step(s).
Asunto(s)
Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Programas Informáticos , Animales , Descubrimiento de Drogas , Hepatocitos/metabolismo , Humanos , Microsomas Hepáticos/metabolismo , Estructura Molecular , RatasRESUMEN
Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Antagonistas de los Receptores de Endotelina/toxicidad , Isoxazoles/toxicidad , Fenilpropionatos/toxicidad , Piridazinas/toxicidad , Sulfonamidas/toxicidad , Tiofenos/toxicidad , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Bosentán , Línea Celular , Supervivencia Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Antagonistas de los Receptores de Endotelina/farmacocinética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Isoxazoles/farmacocinética , Mitocondrias/metabolismo , Estructura Molecular , Consumo de Oxígeno/fisiología , Fenilpropionatos/farmacocinética , Piridazinas/farmacocinética , Sulfonamidas/farmacocinética , Tiofenos/farmacocinética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Idiosyncratic adverse drug reactions (IADRs) in humans can result in a broad range of clinically significant toxicities leading to attrition during drug development as well as postlicensing withdrawal or labeling. IADRs arise from both drug and patient related mechanisms and risk factors. Drug related risk factors, resulting from parent compound or metabolites, may involve multiple contributory mechanisms including organelle toxicity, effects related to compound disposition, and/or immune activation. In the current study, we evaluate an in vitro approach, which explored both cellular effects and covalent binding (CVB) to assess IADR risks for drug candidates using 36 drugs which caused different patterns and severities of IADRs in humans. The cellular effects were tested in an in vitro Panel of five assays which quantified (1) toxicity to THLE cells (SV40 T-antigen-immortalized human liver epithelial cells), which do not express P450s, (2) toxicity to a THLE cell line which selectively expresses P450 3A4, (3) cytotoxicity in HepG2 cells in glucose and galactose media, which is indicative of mitochondrial injury, (4) inhibition of the human bile salt export pump, BSEP, and (5) inhibition of the rat multidrug resistance associated protein 2, Mrp2. In addition, the CVB Burden was estimated by determining the CVB of radiolabeled compound to human hepatocytes and factoring in both the maximum prescribed daily dose and the fraction of metabolism leading to CVB. Combining the aggregated results from the in vitro Panel assays with the CVB Burden data discriminated, with high specificity (78%) and sensitivity (100%), between 27 drugs, which had severe or marked IADR concern, and 9 drugs, which had low IADR concern, we propose that this integrated approach has the potential to enable selection of drug candidates with reduced propensity to cause IADRs in humans.
Asunto(s)
Profármacos/efectos adversos , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocromo P-450 CYP3A/metabolismo , Galactosa/farmacología , Glucosa/farmacología , Células Hep G2 , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Profármacos/metabolismo , Profármacos/toxicidad , Ratas , Ratas Sprague-Dawley , Factores de RiesgoRESUMEN
As part of the drug discovery and development process, it is important to understand the fate of the drug candidate in humans and the relevance of the animal species used for preclinical toxicity and pharmacodynamic studies. Therefore, various in vitro and in vivo studies are conducted during the different stages of the drug development process to elucidate the absorption, distribution, metabolism, and excretion properties of the drug candidate. Although state-of-the-art LC/MS techniques are commonly employed for these studies, radiolabeled molecules are still frequently required for the quantification of metabolites and to assess the retention and excretion of all drug related material without relying on structural information and MS ionization properties. In this perspective, we describe the activities of Isotope Chemistry at AstraZeneca and give a brief overview of different commonly used approaches for the preparation of (14)C- and (3)H-labeled drug candidates. Also various drug metabolism and pharmacokinetic studies utilizing radiolabeled drug candidates are presented with in-house examples where relevant. Finally, we outline strategic changes to our use of radiolabeled compounds in drug metabolism and pharmacokinetic studies, with an emphasis on delaying of in vivo studies employing radiolabeled drug molecules.
Asunto(s)
Preparaciones Farmacéuticas/metabolismo , Farmacocinética , Animales , Radioisótopos de Carbono/farmacocinética , Descubrimiento de Drogas , Humanos , Tritio/farmacocinéticaRESUMEN
In drug discovery, the central process of constructing and testing hypotheses, carefully conducting experiments and analysing the associated data for new findings and information is known as the design-make-test-analyse cycle. Each step relies heavily on the inputs and outputs of the other three components. In this article we report our efforts to improve and integrate all parts to enable smooth and rapid flow of high quality ideas. Key improvements include enhancing multi-disciplinary input into 'Design', increasing the use of knowledge and reducing cycle times in 'Make', providing parallel sets of relevant data within ten working days in 'Test' and maximising the learning in 'Analyse'.
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Diseño de Fármacos , Industria Farmacéutica , Preparaciones Farmacéuticas , HumanosRESUMEN
BACKGROUND: A high-throughput bioanalytical methodology for analysis and quantification of lipophilic pharmaceutical compounds in plasma using liquid-liquid extraction (LLE) was developed. RESULTS: A fast and robust alternative to the widely used protein precipitation of plasma samples is sometimes required in order to avoid matrix effects in MS detection. LLE is known to produce clean extracts and hence reduce levels of matrix components that cause ion suppression. The proposed sample preparation was automated LLE using 96-well plates and a Tecan GenMate 96-tips liquid handling robot. With direct injection of the organic phase (methyl tert-butyl ether: iso-hexane 50:50 v/v) onto a reversed-phase column and without evaporation of the organic phase and reconstitution of the sample, the LLE was no more time consuming than standard protein precipitation, furthermore, matrix effects were minimized. The small injection volume (5 µl) when used with lipophilic compounds and a rapid gradient elution made it possible to inject the organic phase with maintained chromatographic performance. Good chromatographic behavior was confirmed for eight commercially available lipophilic compounds. CONCLUSIONS: The proposed method of LLE with injection of the organic phase onto a reversed-phase column in LC-MS/MS is no more time consuming than standard protein precipitation, and matrix effects were minimized, thus making it suitable as a high-throughput bioanalytical methodology for use in drug discovery.
Asunto(s)
Extracción Líquido-Líquido/métodos , Plasma/química , Espectrometría de Masas en Tándem/métodos , Animales , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Extracción Líquido-Líquido/instrumentación , Estructura Molecular , Compuestos Orgánicos/química , Piperidinas/análisis , Pirazoles/análisis , Rimonabant , Sulfonas/análisis , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
Drug toxicity is a leading cause of attrition of candidate drugs during drug development as well as of withdrawal of drugs post-licensing due to adverse drug reactions in man. These adverse drug reactions cause a broad range of clinically severe conditions including both highly reproducible and dose dependent toxicities as well as relatively infrequent and idiosyncratic adverse events. The underlying risk factors can be split into two groups: (1) drug-related and (2) patient-related. The drug-related risk factors include metabolic factors that determine the propensity of a molecule to form toxic reactive metabolites (RMs), and the RM and non-RM mediated mechanisms which cause cell and tissue injury. Patient related risk factors may vary markedly between individuals, and encompass genetic and non-genetic processes, e.g. environmental, that influence the disposition of drugs and their metabolites, the nature of the adverse responses elicited and the resulting biological consequences. We describe a new strategy, which builds upon the strategies used currently within numerous pharmaceutical companies to avoid and minimize RM formation during drug discovery, and that is intended to reduce the likelihood that candidate drugs will cause toxicity in the human population. The new strategy addresses drug-related safety hazards, but not patient-related risk factors. A common target organ of toxicity is the liver and to decrease the likelihood that candidate drugs will cause liver toxicity (both non-idiosyncratic and idiosyncratic), we propose use of an in vitro Hepatic Liability Panel alongside in vitro methods for the detection of RMs. This will enable design and selection of compounds in discovery that have reduced propensity to cause liver toxicity. In vitro Hepatic Liability is assessed using toxicity assays that quantify: CYP 450 dependent and CYP 450 independent cell toxicity; mitochondrial impairment; and inhibition of the Bile Salt Export Pump. Prior to progression into development, a Hepatotoxicity Hazard Matrix combines data from the Hepatic Liability Panel with the Estimated RM Body Burden. The latter is defined as the level of covalent binding of radiolabelled drug to human hepatocyte proteins in vitro adjusted for the predicted human dose. We exemplify the potential value of this approach by consideration of the thiazolidinedione class of drugs.
Asunto(s)
Descubrimiento de Drogas , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Hígado/efectos de los fármacos , Medición de RiesgoRESUMEN
The high separation efficiency of capillary electrophoresis (CE), combined with the high selectivity and sensitivity of mass spectrometry (MS) detection offers the potential of unique resolving power and high-throughput capacity to the analysis and structural identification of complex mixtures. Recent advances in CE-MS interfaces and commercially available 96-capillary instruments have made the implementation of routine CE methods for drug screening feasible.:
RESUMEN
A high-throughput pKa screening method based on pressure-assisted capillary electrophoresis (CE) and mass spectrometry (MS) is presented. Effects of buffer type and ionic strength on sensitivity and pKa values were investigated. Influence of dimethyl sulfoxide (DMSO) concentration present in the sample on effective mobility measurement was examined. A series of ten volatile buffers, covering a pH range from 2.5 to 10.5 with the same ionic strength, was employed. The application of volatile background electrolytes resulted in significant signal increase as compared with commonly used non-volatile phosphate buffers. In general, the CE/MS system provided a ten-fold higher sensitivity than conventional UV detection. The newly developed CE/MS method offers high-throughput capacity by pooling a number of compounds into a single sample. Simultaneous measurement of more than 50 compounds was readily achieved in less than 150 min. The measured pKa values are consistent with the published data obtained from the CE/UV method and are also in good agreement with data generated by other methods. Other advantages of using CE/MS for pKa screening are illustrated with typical examples, including poorly soluble compounds and non-UV-absorbing compounds.
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Preparaciones Farmacéuticas/análisis , Algoritmos , Tampones (Química) , Fenómenos Químicos , Química Física , Dimetilsulfóxido , Evaluación Preclínica de Medicamentos , Electroforesis Capilar , Indicadores y Reactivos , Espectrometría de Masas , Dinámicas no Lineales , Estándares de Referencia , Solventes , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
Separation of enantiomers of a thiazolbenzenesulfonamide compound was performed on a Chiralpak AD column using subcritical fluid chromatography. Effects of alcohol modifier and temperature on the separations were studied. The results revealed that while the main adsorbing interactions were between the hydroxyl group of the analyte and the carbamate group of the stationary phase, chiral discrimination was achieved through an inclusion mechanism within the chiral cavity created along the amylose chains. Analogs and synthetic precursors of the thiazolbenzenesulfonamide studied were also investigated so as to understand the effect of functional groups and configuration of the analyte molecule upon chiral recognition.