RESUMEN
Human 3D liver microtissues/spheroids are powerful in vitro models to study drug-induced liver injury (DILI) but the small number of cells per spheroid limits the models' usefulness to study drug metabolism. In this work, we scale up the number of spheroids on both a plate and a standardized organ-chip platform by factor 100 using a basic method which requires only limited technical expertise. We successfully generated up to 100 spheroids using polymer-coated microwells in a 96-well plate (= liver-plate) or organ-chip (= liver-chip). Liver-chips display a comparable cellular CYP3A4 activity, viability, and biomarker expression as liver spheroids for at least one week, while liver-plate cultures display an overall reduced hepatic functionality. To prove its applicability to drug discovery and development, the liver-chip was used to test selected reference compounds. The test system could discriminate toxicity of the DILI-positive compound tolcapone from its less hepatotoxic structural analogue entacapone, using biochemical and morphological readouts. Following incubation with diclofenac, the liver-chips had an increased metabolite formation compared to standard spheroid cultures. In summary, we generated a human liver-chip model using a standardized organ-chip platform which combines up to 100 spheroids and can be used for the evaluation of both drug safety and metabolism.
RESUMEN
Early assessment of metabolism pathways of new chemical entities guides the understanding of drug-drug interactions. Selective enzyme inhibitors are indispensable in CYP reaction phenotyping. The most commonly applied CYP2C19 inhibitor, omeprazole, lacks selectivity. Two promising alternatives, (+)-N-3-benzylnirvanol and (-)-N-3-benzylphenobarbital, are already used as CYP2C19 inhibitors in some in vitro studies with suspended human hepatocytes. However, a full validation proving their suitability in terms of CYP and non-CYP selectivity has not been presented in literature. The present study provides a thorough comparison between omeprazole, (+)-N-3-benzylnirvanol, and (-)-N-3-benzylphenobarbital in terms of potency and selectivity and shows the superiority of (-)-N-3-benzylphenobarbital as a CYP2C19 inhibitor in suspended human hepatocytes. Furthermore, we evaluated the application of (-)-N-3-benzylphenobarbital to predict the in vivo contribution of CYP2C19 to drug metabolism [fraction metabolized (fm) of CYP2C19, fmCYP2C19]. A set of 10 clinically used CYP2C19 substrates with reported in vivo fmCYP2C19 data was evaluated. fmCYP2C19, which was predicted using data from suspended human hepatocyte incubations, underestimated the in vivo fmCYP2C19 The use of a different hepatocyte batch with a different CYP3A4/CYP2C19 activity ratio showed the impact of intrinsic CYP activities on the determination of fmCYP2C19 Overall, this study confirms the selective CYP2C19 inhibition by (-)-N-3-benzylphenobarbital over other CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4) and clinically relevant non-CYP enzymes [aldehyde oxidase, flavin-containing monooxygenase 3, N-acetyltransferase 2, uridine diphosphate glucuronosyltransferase (UGT) 1A1, UGT1A4, UGT2B7, UGT2B15] in suspended human hepatocytes. (-)-N-3-benzylphenobarbital is therefore the preferred CYP2C19 inhibitor to assess fmCYP2C19 in suspended human hepatocytes in comparison with omeprazole and (+)-N-3-benzylnirvanol. SIGNIFICANCE STATEMENT: (-)-N-3-Benzylphenobarbital is a more potent and selective inhibitor of CYP2C19 in suspended human hepatocytes than omeprazole and (+)-N-3-benzylnirvanol. (-)-N-3-Benzylphenobarbital can be used to predict the fraction metabolized by CYP2C19 in suspended human hepatocytes.
Asunto(s)
Inhibidores del Citocromo P-450 CYP2C19/farmacología , Citocromo P-450 CYP2C19/metabolismo , Mefenitoína/análogos & derivados , Omeprazol/farmacología , Fenobarbital/análogos & derivados , Técnicas de Cultivo de Célula , Células Cultivadas , Hepatocitos , Humanos , Concentración 50 Inhibidora , Mefenitoína/farmacología , Fenobarbital/farmacologíaRESUMEN
Early determination of CYP3A4/5 contribution to the clearance of new chemical entities is critical to inform on the risk of drug-drug interactions with CYP3A inhibitors and inducers. Several in vitro approaches (recombinant P450 enzymes, correlation analysis, chemical and antibody inhibition in human liver microsomes) are available, but they are usually labor-intensive and/or suffer from specific limitations. In the present study, we have validated the use of azamulin as a specific CYP3A inhibitor in human hepatocytes. Azamulin (3 µM) was found to significantly inhibit CYP3A4/5 (>90%), whereas other P450 enzymes were not affected (less than 20% inhibition). Because human hepatocytes were used as a test system, the effect of azamulin on other key drug-metabolizing enzymes (aldehyde oxidase, carboxylesterase, UGT, flavin monooxygenase, and sulfotransferase) was also investigated. Apart from some UGTs showing minor inhibition (â¼20%-30%), none of these non-P450 enzymes were inhibited by azamulin. Use of CYP3A5-genotyped human hepatocyte batches in combination with CYP3cide demonstrated that azamulin (at 3 µM) inhibits both CYP3A4 and CYP3A5 enzymes. Finally, 11 compounds with known in vivo CYP3A4/5 contribution have been evaluated in this human hepatocyte assay. Results showed that the effect of azamulin on the in vitro intrinsic clearance of these known CYP3A4/5 substrates was predictive of the in vivo CYP3A4/5 contribution. Overall, the study showed that human hepatocytes treated with azamulin provide a fast and accurate estimation of CYP3A4/5 contribution in metabolic clearance of new chemical entities. SIGNIFICANCE STATEMENT: Accurate estimation of CYP3A4/5 contribution in drug clearance is essential to anticipate risk of drug-drug interactions and select the appropriate candidate for clinical development. The present study validated the use of azamulin as selective CYP3A4/5 inhibitor in suspended human hepatocytes and demonstrated that this novel approach provides a direct and accurate determination of the contribution of CYP3A4/5 (fraction metabolized by CYP3A4/5) in the metabolic clearance of new chemical entities.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/farmacología , Inhibidores del Citocromo P-450 CYP3A/farmacología , Citocromo P-450 CYP3A/metabolismo , Eliminación Hepatobiliar/efectos de los fármacos , Triazoles/farmacología , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Interacciones Farmacológicas , Hepatocitos , Humanos , Hígado/efectos de los fármacos , Hígado/enzimología , Microsomas Hepáticos , Cultivo Primario de CélulasRESUMEN
Metabolite quantification and profiling continues to grow in importance in today's drug development. The guidance provided by the 2008 FDA Metabolites in Safety Testing Guidance and the subsequent ICH M3(R2) Guidance (2009) has led to a more streamlined process to assess metabolite exposures in preclinical and clinical studies in industry. In addition, the European Bioanalysis Forum (EBF) identified an opportunity to refine the strategies on metabolite quantification considering the experience to date with their recommendation paper on the subject dating from 2010 and integrating the recent discussions on the tiered approach to bioanalytical method validation with focus on metabolite quantification. The current manuscript summarizes the discussion and recommendations from a recent EBF Focus Workshop into an updated recommendation for metabolite quantification in drug development.
Asunto(s)
Descubrimiento de Drogas/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Europa (Continente) , Humanos , Metabolómica/métodosRESUMEN
Drug induced liver injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n=40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n=11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n=14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Drogas en Investigación/efectos adversos , Hepatocitos/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Biomarcadores/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Técnicas de Cocultivo , Perros , Impedancia Eléctrica , Glutatión/metabolismo , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
We identified the enzyme(s) involved in the hydrolysis of the ethyl ester prodrug CDP323 (C28H29BrN403) and characterized its transesterification in the presence of ethanol with special emphasis on the risks of drug-drug interaction. The hydrolysis of CDP323 was evaluated in vitro using human liver and intestinal microsomes and recombinant human carboxylesterases (hCES1 and 2) and was shown to be approximately 20-fold higher in human liver microsomes when compared with human intestinal microsomes and in hCES1 when compared with hCES2. Nonspecific inhibitors of carboxylesterases significantly inhibited the hydrolysis of CDP323 (>80% inhibition) while specific inhibitors of CES2, acetylcholine esterase, arylesterase, and butyrylcholinesterase did not impair the hydrolysis reaction. The effect of ethanol on the kinetic parameters for hydrolysis was investigated, demonstrating that at high concentration (2%), Michaelis-Menten constant (Km), maximum velocity (Vmax), and intrinsic clearance (CLint) for the formation of the hydrolyzed product were decreased (â¼40%). The use of deuterated ethanol allowed more mechanistic investigations of the transesterification mechanism and showed that the intrinsic clearance based on parent loss was not impaired in the presence of alcohol. Overall, our data demonstrate that CDP323 is mainly hydrolyzed by hCES1 and is prone to transesterification in the presence of ethanol. Transesterification mechanisms compete with hydrolysis without impairing the overall clearance of the ester prodrug. Based on in vitro results, the risk of a clinically significant drug-drug interaction with ethanol is anticipated to be low.
Asunto(s)
Esterificación/fisiología , Integrina alfa4beta1/antagonistas & inhibidores , Integrinas/antagonistas & inhibidores , Fenilalanina/análogos & derivados , Profármacos/metabolismo , Butirilcolinesterasa/metabolismo , Carboxilesterasa/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Interacciones Farmacológicas/fisiología , Ésteres/metabolismo , Etanol/metabolismo , Humanos , Hidrólisis , Mucosa Intestinal/metabolismo , Intestinos/enzimología , Cinética , Hígado/enzimología , Hígado/metabolismo , Microsomas/enzimología , Microsomas/metabolismo , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Naftiridinas , Fenilalanina/metabolismoRESUMEN
Metabolism data provided with reduced cycle time has become of increasing importance for the early evaluation of DMPK properties of drugs in discovery. In this regard, quadrupole time-of-flight hybrid mass spectrometers (Q-TOF) can provide very reliable metabolite identification via accurate mass measurement of ions and the consequent access to the elemental composition of the metabolite. However, due to their cost, they are often used for drug metabolism studies on later stage drug candidates or to address challenging metabolism questions. A new prototype, consisting of a five-channel multiplexed electrospray ionization (ESI) source on a Q-TOF with one channel used for lock-mass compound infusion, was evaluated for metabolite identification. The goal was to increase the sample throughput of a single ESI-MS system by a factor of 4, while maintaining efficient metabolite separation in high-performance liquid chromatography (HPLC) as well as adequate sensitivity and mass accuracy, and ultimately improve the speed and quality of metabolism studies supporting drug discovery. The analytical performance of the system was assessed by evaluating the sensitivity and mass accuracy (using real in vitro and in vivo samples), inter-channel differences in retention times, MS/UV response, and cross-talk among channels. The sensitivity using the multiplexed ESI source was on average 2-fold lower than with single ESI, correlating well with previous literature data. The mass accuracy was comparable to that obtained using single ESI in both MS and MS/MS modes, making the metabolite identification process using the multiplexed ESI source as reliable as with single ESI. Compound-dependent differences in ionization efficiencies were observed among channels, and were minimized by analyzing related samples on the same channel. Finally, the level of cross-talk among channels was acceptable (around 0.3%) and comparable to levels previously published for quantitative applications using multiplexed ESI. The paper also focuses on the advantages and disadvantages of this new approach compared to other approaches in the literature in the field of metabolite identification.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Preparaciones Farmacéuticas/metabolismo , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Bilis/química , Bilis/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Cetotifen/farmacocinética , Microsomas Hepáticos/metabolismo , Ratas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Terfenadina/farmacocinética , Verapamilo/farmacocinéticaRESUMEN
Collybia fusipes is a common root rot fungus in mature pedunculate oak forest, that causes drastic destruction of the tree root systems, especially in dry or mildly waterlogged soils. We wanted to check, under controlled conditions or in forest ecosystems, whether reduced O2 during saturation of the soil by water could interact with disease evolution. Susceptibility of waterlogged oak seedlings to C. fusipes was tested in a greenhouse and the survival of the pathogen in woody substrates was assessed in hydromorphic soils in a forest. A direct and detrimental effect of soil waterlogging on C. fusipes survival was evidenced both under controlled conditions and in forest stands. Growth of C. fusipes mycelium on agar media was monitored under low O2 mole fraction and compared to that of Armillaria mellea and Heterobasidion annosum. A drastic reduction in mycelial growth was evidenced in C. fusipes and H. annosum but not in A. mellea.
Asunto(s)
Agaricales/patogenicidad , Enfermedades de las Plantas/microbiología , Quercus/microbiología , Agaricales/crecimiento & desarrollo , Ecosistema , Oxígeno , Raíces de Plantas/microbiología , Suelo , AguaRESUMEN
In several studies of oak decline in Europe, a semi-papillate homothallic Phytophthora taxon was consistently isolated, together with other Phytophthora species, from rhizosphere soil samples. It was also found associated with necrotic fine roots and stem necroses of Fagus sylvatica and Alnus glutinosa. Due to morphological and physiological similarities, the semi-papillate isolates were previously identified as P. syringae by various authors. The morphology, physiology and pathogenicity against fine roots of Quercus robur, Q. petraea and F. sylvatica, bark of A. glutinosa, leaves of Ilex aquifolium and apple fruits of this Phytophthora species are described and compared with those of related and similar Phytophthora species, namely P. ilicis, P. psychrophila, P. quercina, P. citricola and P. syringae. The phylogenetic placement on the basis of ITS and mtDNA sequence data was also examined. Isolates of this taxon produce colonies with stellate to rosaceous growth patterns and limited aerial mycelium on various agar media. Antheridia are predominantly paragynous. In water culture catenulate hyphal swellings and semi-papillate caducous sporangia, that are usually limoniform, ellipsoid or ovoid, are formed abundandly, mostly in lax or dense sympodia. This taxon is a moderately slow growing, low temperature species with optimum and maximum temperatures around 20 and 25 degrees C, respectively. Tested isolates are moderately aggressive to fine roots of oaks and beech, highly aggressive to holly leaves and apple fruits, and slightly pathogenic to alder bark. Thirteen tested isolates had an identical and distinct ITS sequence which was more similar to that of P. ilicis and P. psychrophila than any other known taxa. On the basis of their unique combination of morphological characters, colony growth patterns, cardinal temperatures for growth, growth rates, pathogenicity to oaks, beech, alder, apple and holly, their host range, and ITS and mtDNA sequences the semi-papillate caducous Phytophthora isolates from oaks, beech and alder are clearly separated from related and similar Phytophthora spp., and described as a new species, P. pseudosyringae sp. nov.
Asunto(s)
Magnoliopsida/microbiología , Phytophthora/clasificación , Enfermedades de las Plantas/microbiología , Quercus/microbiología , Árboles/microbiología , Alnus/microbiología , ADN Mitocondrial/análisis , ADN Espaciador Ribosómico/análisis , Fagus/microbiología , Filogenia , Phytophthora/genética , Phytophthora/patogenicidad , Phytophthora/fisiología , Raíces de Plantas/microbiología , Análisis de Secuencia de ADNRESUMEN
Quantitative bioanalysis by direct nanoelectrospray infusion coupled to tandem mass spectrometry has been achieved using an automated liquid sampler integrated with an array of microfabricated electrospray nozzles allowing rapid, serial sample introduction (1 min/ sample). Standard curves prepared in human plasma for verapamil (r2 = 0.999) and its metabolite norverapamil (r2 = 0.998) were linear over a range of 2.5-500 ng/ mL. Based on the observed precision and accuracy, a lower limit of quantitation of 5 ng/mL was assigned for both analytes. Sample preparation consisted of protein precipitation with an organic solvent containing the structural analogue gallopamil as an internal standard. Protein precipitation was selected both to maximize throughput and to test the robustness of direct nanoelectrospray infusion. Aliquots of supernatant (10 pL) were transferred to the back plane of the chip using disposable, conductive pipet tips for direct infusion at a flow rate of 300 nL/min. Electrospray ionization occurred from the etched nozzles (30-microm o.d.) on the front of the chip, initiated by a voltage applied to the liquid through the pipet tip. The chip was positioned near the API sampling orifice of a triple quadrupole mass spectrometer, which was operated in selected reaction monitoring mode. Results are presented that document the complete elimination of system carry-over, attributed to lack of a redundant fluid path. This technology offers potential advantages for MS-based screening applications in drug discovery by reducing the time for methods development and sample analysis.
Asunto(s)
Monitoreo de Drogas/métodos , Espectrometría de Masa por Ionización de Electrospray , Verapamilo/análogos & derivados , Verapamilo/sangre , Humanos , Preparaciones Farmacéuticas/sangre , Estándares de Referencia , Verapamilo/metabolismoRESUMEN
Phytophthora lateralis, a pathogen of Chamaecyparis lawsoniana (Port-Orford cedar or Lawson's cypress), was confirmed in France, but isolates from Germany identified as P. lateralis or "similar to" P. lateralis proved to be P. gonapodyides. Previously, P. lateralis was known only from western North America, where it has been destructive in nurseries, ornamental plantings, and the forest since its introduction about 1920 (1). Reports from other locations have proved to be misidentifications or impossible to confirm. In France, P. lateralis was isolated and identified from C. lawsoniana on two occasions (1996 and 1998) in different parts of the country, probably stemming from a single original infestation of young, potted, greenhouse-propagated cedars in a commercial nursery. German isolates were from an old culture collection and from irrigation water in a nursery growing a wide range of woody ornamentals. Identifications were confirmed by comparison (2) with authentic isolates. P. lateralis isolates from France and Oregon formed laterally proliferating, elongated obpyriform sporangia that under the conditions of our tests could be dislodged by agitation, leaving a short pedicel. Also, brown chlamydospores formed laterally on the hyphae or terminally on a short stalk and oospores were not formed on standard media. Radial growth was about 2 mm per day. In contrast, sporangia of German isolates and known P. gonapodyides isolates were similar. They exhibited nested pro liferation, were broader than P. lateralis sporangia, and were not readily dehiscent. Some P. gonapodyides isolates, including those from Germany, formed chlamydospores, but these were nearly all catenulate and not lateral, and isolates grew faster (3 to 4 mm per day). Pathogenicity was tested by stem inoculation of C. lawsoniana. P. lateralis from France and Oregon produced lesions averaging 4.7 cm after 2 months (range 2.0 to 8.1 cm, six replicates per isolate, five isolates) while the six replicates of the two German isolates averaged 1.2- and 1.6-cm lesion lengths. Furthermore, sequences of internal transcribed spacer (ITS) DNA from French and Oregon P. lateralis isolates were identical, while sequences of German isolates were similar to P. gonapodyides (J. Duncan and D. Cooke, personal communiation). P. lateralis is a dangerous pathogen of C. lawsoniana and is also pathogenic to Taxus spp. (1), although less aggressive on this host. If established, it would be a serious threat to the widespread ornamental plantings and scattered forest plantations of C. lawsoniana in Europe. References: (1) E. M. Hansen and K. J. Lewis. Compendium of Conifer Diseases. American Phytopathological Society, 1997. (2) C. M. Tucker and J. A. Milbrath. Mycologia 34:94, 1942.