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1.
Drug Metab Dispos ; 42(8): 1301-13, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24855184

RESUMEN

Organic anion transporting polypeptide (Oatp) 1a/1b knockout and OATP1B1 and -1B3 humanized mouse models are promising tools for studying the roles of these transporters in drug disposition. Detailed characterization of these models will help to better understand their utility for predicting clinical outcomes. To advance this approach, we carried out a comprehensive analysis of these mouse lines by evaluating the compensatory changes in mRNA expression, quantifying the amounts of OATP1B1 and -1B3 protein by liquid chromatography-tandem mass spectrometry, and studying the active uptake in isolated hepatocytes and the pharmacokinetics of some prototypical substrates including statins. Major outcomes from these studies were 1) mostly moderate compensatory changes in only a few genes involved in drug metabolism and disposition, 2) a robust hepatic expression of OATP1B1 and -1B3 proteins in the respective humanized mouse models, and 3) functional activities of the human transporters in hepatocytes isolated from the humanized models with several substrates tested in vitro and with pravastatin in vivo. However, the expression of OATP1B1 and -1B3 in the humanized models did not significantly alter liver or plasma concentrations of rosuvastatin and pitavastatin compared with Oatp1a/1b knockout controls under the conditions used in our studies. Hence, although the humanized OATP1B1 and -1B3 mice showed in vitro and/or in vivo functional activity with some statins, further characterization of these models is required to define their potential use and limitations in the prediction of drug disposition and drug-drug interactions in humans.


Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Transportadores de Anión Orgánico/metabolismo , Animales , Interacciones Farmacológicas/fisiología , Fluorobencenos/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/enzimología , Hígado/metabolismo , Transportador 1 de Anión Orgánico Específico del Hígado , Masculino , Ratones , Pravastatina/metabolismo , Pirimidinas/metabolismo , ARN Mensajero/genética , Rosuvastatina Cálcica , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos , Sulfonamidas/metabolismo
2.
Pharmacol Rev ; 63(2): 390-410, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21490128

RESUMEN

CYP3A is one of the most important drug-metabolizing enzymes, determining the first-pass metabolism, oral bioavailability, and elimination of many drugs. It is also an important determinant of variable drug exposure and is involved in many drug-drug interactions. Recent studies with CYP3A knockout and transgenic mice have yielded a number of key insights that are important to consider during drug discovery and development. For instance, studies with tissue-specific CYP3A-transgenic mice have highlighted the importance of intestinal CYP3A-dependent metabolism. They also revealed that intestinal CYP3A plays an important role in the regulation of various drug-handling systems in the liver. Intestinal CYP3A activity can thus have far-reaching pharmacological effects. Besides CYP3A, the active drug efflux transporter P-glycoprotein also has a strong effect on the pharmacokinetics of numerous drugs. CYP3A and P-glycoprotein have an extensive overlap in their substrate spectrum. It has been hypothesized that for many drugs, the combined activity of CYP3A and P-glycoprotein makes for efficient intestinal first-pass metabolism of orally administered drugs as a result of a potentially synergistic collaboration. However, there is only limited in vitro and in vivo evidence for this hypothesis. There has also been some confusion in the field about what synergy actually means in this case. Our recent studies with Cyp3a/P-glycoprotein combination knockout mice have provided further insights into the CYP3A-P-glycoprotein interplay. We here present our view of the status of the synergy hypothesis and an attempt to clarify the existing confusion about synergy. We hope that this will facilitate further critical testing of the hypothesis and improve communication among researchers. Above all, the recent findings and insights into the interplay between CYP3A and P-glycoprotein may have implications for improving oral drug bioavailability and reducing adverse side effects.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Citocromo P-450 CYP3A/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Disponibilidad Biológica , Diseño de Fármacos , Interacciones Farmacológicas , Sinergismo Farmacológico , Interacciones Alimento-Droga , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Preparaciones Farmacéuticas/administración & dosificación
3.
Mol Pharmacol ; 82(4): 636-44, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22767610

RESUMEN

We investigated the interactions of the anticancer drug vinorelbine with drug efflux transporters and cytochrome P450 3A drug-metabolizing enzymes. Vinorelbine was transported by human multidrug-resistance associated protein (MRP) 2, and Mrp2 knockout mice displayed increased vinorelbine plasma exposure after oral administration, suggesting that Mrp2 limits the intestinal uptake of vinorelbine. Using P-glycoprotein (P-gp), Cyp3a-, and P-gp/Cyp3a knockout mice, we found that the absence of P-gp or Cyp3a resulted in increased vinorelbine plasma exposure, both after oral and intravenous administration. Surprisingly, P-gp/Cyp3a knockout mice displayed markedly lower vinorelbine plasma concentrations than wild-type mice upon intravenous administration but higher concentrations upon oral administration. This could be explained by highly increased formation of 4'-O-deacetylvinorelbine, an active vinorelbine metabolite, especially in P-gp/Cyp3a knockout plasma. Using wild-type and Cyp3a knockout liver microsomes, we found that 4'-O-deacetylvinorelbine formation was 4-fold increased in Cyp3a knockout liver and was not mediated by Cyp3a or other cytochrome P450 enzymes. In vitro incubation of vinorelbine with plasma revealed that vinorelbine deacetylation in Cyp3a and especially in P-gp/Cyp3a knockout mice but not in P-gp-deficient mice was strongly up-regulated. Metabolite formation in microsomes and plasma could be completely inhibited with the nonspecific carboxylesterase (CES) inhibitor bis(4-nitrophenyl) phosphate and partly with the CES2-specific inhibitor loperamide, indicating that carboxylesterase Ces2a, which was appropriately up-regulated in Cyp3a and especially in P-gp/Cyp3a knockout liver was responsible for the 4'O-deacetylvinorelbine formation. Such compensatory up-regulation can complicate the interpretation of knockout mouse data. Nonetheless, P-gp, Mrp2, Cyp3a, and Ces2a clearly restricted vinorelbine availability in mice. Variation in activity of their human homologs may also affect vinorelbine pharmacokinetics in patients.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacocinética , Carboxilesterasa/metabolismo , Citocromo P-450 CYP3A/metabolismo , Vinblastina/análogos & derivados , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Administración Oral , Animales , Disponibilidad Biológica , Transporte Biológico , Carboxilesterasa/antagonistas & inhibidores , Citocromo P-450 CYP3A/genética , Perros , Femenino , Humanos , Técnicas In Vitro , Loperamida/farmacología , Células de Riñón Canino Madin Darby , Ratones , Ratones Noqueados , Microsomas Hepáticos/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Nitrofenoles/farmacología , Regulación hacia Arriba , Vinblastina/metabolismo , Vinblastina/farmacocinética , Vinorelbina , Miembro 4 de la Subfamilia B de Casete de Unión a ATP
4.
Trends Pharmacol Sci ; 30(5): 223-7, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19328560

RESUMEN

Cytochrome P450 3A (CYP3A) enzymes metabolize a wide variety of xenobiotics including many drugs. Because CYP3A is localized in both the liver and intestine, it can make a major contribution to the presystemic elimination of substrate drugs after oral administration ('first-pass metabolism'). However, assessments of the relative importance of intestinal versus hepatic CYP3A-mediated first-pass metabolism have been difficult to make and are subject to extensive discussion. To assess systematically the relative contributions of the intestine and liver to first-pass metabolism in vivo, Cyp3a knockout mice expressing human CYP3A4 in the liver or intestine have recently been generated. Analysis of these mice, together with previous observations in humans, substantiates that intestinal CYP3A4 can operate independently of the liver as a highly efficient metabolic barrier during the uptake of various drugs from the intestine. We expect that the insights obtained with these mouse models will contribute to the development of better oral drugs and treatment regimens.


Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Citocromo P-450 CYP3A/genética , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos
5.
Int J Cancer ; 127(12): 2959-64, 2010 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-21351274

RESUMEN

Docetaxel is one of the most widely used anticancer drugs. A major problem with docetaxel treatment, however, is the considerable interpatient variability in docetaxel exposure. Another disadvantage of the drug is that it has a very low oral bioavailability and can, therefore, only be administered intravenously. The drug-metabolizing enzyme CYP3A and the drug transporter MDR1 (P-glycoprotein) are major determinants of docetaxel pharmacokinetics. In vitro studies have indicated that docetaxel is also a substrate for the drug transporter MRP2, but the in vivo importance of MRP2 for docetaxel is currently unknown. We, therefore, investigated the role of MRP2 in the pharmacokinetics of docetaxel by utilizing Mrp2(-/-) mice. We also generated and characterized Cyp3a/Mdr1a/b/Mrp2(-/-) combination knockout mice to get more insight into how these drug-handling systems work together in determining docetaxel pharmacokinetics. The systemic exposure in Mrp2(-/-) mice was not significantly different from wild-type, after either oral or intravenous administration. Strikingly, however, in Cyp3a/Mdr1a/b/Mrp2(-/-) mice, systemic docetaxel exposure was increased 166-fold after oral administration when compared with wild-type mice, and 2.3-fold when compared with Cyp3a/Mdr1a/b(-/-) mice. Interestingly, this 166-fold increase was disproportionate compared with that for the separate Cyp3a (12-fold) or Mdr1a/b/Mrp2 (4-fold) knockouts. The oral bioavailability was increased to 73% in the Cyp3a/Mdr1a/b/Mrp2(-/-) strain, versus only 10% in wild-type mice. Our data thus indicate that in the absence of CYP3A and Mdr1a/b activity, Mrp2 has a marked impact on docetaxel pharmacokinetics. These findings could have important implications for improving the oral bioavailability and reducing the variability in docetaxel exposure.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Transportadoras de Casetes de Unión a ATP/fisiología , Sistema Enzimático del Citocromo P-450/fisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Taxoides/farmacocinética , Subfamilia B de Transportador de Casetes de Unión a ATP , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Disponibilidad Biológica , Citocromo P-450 CYP3A , Docetaxel , Masculino , Ratones , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Taxoides/farmacología , Distribución Tisular
6.
J Clin Invest ; 117(11): 3583-92, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17975676

RESUMEN

Cytochrome P450 3A (CYP3A) enzymes constitute an important detoxification system that contributes to primary metabolism of more than half of all prescribed medications. To investigate the physiological and pharmacological roles of CYP3A, we generated Cyp3a-knockout (Cyp3a-/-) mice lacking all functional Cyp3a genes. Cyp3a-/- mice were viable, fertile, and without marked physiological abnormalities. However, these mice exhibited severely impaired detoxification capacity when exposed to the chemotherapeutic agent docetaxel, displaying higher exposure levels in response to both oral and intravenous administration. These mice also demonstrated increased sensitivity to docetaxel toxicity, suggesting a primary role for Cyp3a in xenobiotic detoxification. To determine the relative importance of intestinal versus hepatic Cyp3a in first-pass metabolism, we generated transgenic Cyp3a-/- mice expressing human CYP3A4 in either the intestine or the liver. Expression of CYP3A4 in the intestine dramatically decreased absorption of docetaxel into the bloodstream, while hepatic expression aided systemic docetaxel clearance. These results suggest that CYP3A expression determines impairment of drug absorption and efficient systemic clearance in a tissue-specific manner. The genetic models used in this study provide powerful tools to further study CYP3A-mediated xenobiotic metabolism, as well as interactions between CYP3A and other detoxification systems.


Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Ratones Noqueados , Xenobióticos/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Docetaxel , Humanos , Ratones , Ratones Transgénicos , Microsomas/metabolismo , Fenotipo , Taxoides/administración & dosificación , Taxoides/farmacocinética
7.
FASEB J ; 23(1): 224-31, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18794335

RESUMEN

CYP3A4 is an important xenobiotic metabolizing enzyme. We previously found that CYP2C55 is highly up-regulated in Cyp3a(-/-) mice. Here, we have further investigated the mechanism of regulation of CYP2C55 and other detoxifying systems in Cyp3a(-/-) mice. Induction studies with prototypical inducers demonstrated an important role for the nuclear receptors PXR and CAR in the up-regulation of CYP2C55. Subsequent diet-switch experiments revealed that food-derived xenobiotics are primarily responsible for the increased induction of CYP2C55, as well as of several other primary detoxifying systems in Cyp3a(-/-) mice. Our data suggest that CYP3A normally metabolizes food-derived activators of PXR and/or CAR, explaining the increased levels of such activators in Cyp3a(-/-) mice and subsequent up-regulation of a range of detoxifying systems. Interestingly, our studies with tissue-specific CYP3A4 transgenic Cyp3a(-/-) mice revealed that not only hepatic but also intestinal expression of CYP3A4 could reduce the hepatic expression of detoxifying systems to near wild-type levels. Apparently, intestinal CYP3A4 can limit the hepatic exposure to food-derived activators of nuclear receptors, thereby regulating the expression of a range of detoxifying systems in the liver. This broad biological effect further emphasizes the importance of intestinal CYP3A activity and could have profound implications for the prediction of drug exposure.


Asunto(s)
Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Intestinos/enzimología , Hígado/enzimología , Animales , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 2 del Citocromo P450 , Dieta , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Noqueados , Receptor X de Pregnano , Receptores de Esteroides/genética
8.
Clin Cancer Res ; 15(7): 2344-51, 2009 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-19276246

RESUMEN

PURPOSE: Imatinib, a BCR-ABL tyrosine kinase inhibitor, is a substrate of the efflux transporters P-glycoprotein (P-gp; ABCB1) and ABCG2 (breast cancer resistance protein), and its brain accumulation is restricted by both transporters. For dasatinib, an inhibitor of SCR/BCR-ABL kinases, in vivo interactions with P-gp and ABCG2 are not fully established yet. EXPERIMENTAL DESIGN: We used Abcb1a/1b(-/-), Abcg2(-/-), and Abcb1a/1b;Abcg2(-/-) mice to establish the roles of P-gp and ABCG2 in the pharmacokinetics and brain accumulation of dasatinib. RESULTS: We found that oral uptake of dasatinib is limited by P-gp. Furthermore, relative brain accumulation, 6 hours after administration, was not affected by Abcg2 deficiency, but absence of P-gp resulted in a 3.6-fold increase after oral and 4.8-fold higher accumulation after i.p. administration. Abcb1a/1b;Abcg2(-/-) mice had the most pronounced increase in relative brain accumulation, which was 13.2-fold higher after oral and 22.7-fold increased after i.p. administration. Moreover, coadministration to wild-type mice of dasatinib with the dual P-gp and ABCG2 inhibitor elacridar resulted in a similar dasatinib brain accumulation as observed for Abcb1a/1b;Abcg2(-/-) mice. CONCLUSIONS: Brain accumulation of dasatinib is primarily restricted by P-gp, but Abcg2 can partly take over this protective function at the blood-brain barrier. Consequently, when both transporters are absent or inhibited, brain uptake of dasatinib is highly increased. These findings might be clinically relevant for patients with central nervous system Philadelphia chromosome-positive leukemia, as coadministration of an inhibitor of P-gp and ABCG2 with dasatinib might result in better therapeutic responses in these patients.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Acridinas/farmacología , Antineoplásicos/farmacocinética , Encéfalo/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/farmacocinética , Tetrahidroisoquinolinas/farmacología , Tiazoles/farmacocinética , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antineoplásicos/sangre , Antineoplásicos/metabolismo , Transporte Biológico , Línea Celular , Dasatinib , Perros , Ratones , Ratones Noqueados , Inhibidores de Proteínas Quinasas/sangre , Inhibidores de Proteínas Quinasas/metabolismo , Pirimidinas/sangre , Pirimidinas/metabolismo , Tiazoles/sangre , Tiazoles/metabolismo
9.
Drug Metab Dispos ; 37(12): 2305-13, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19752211

RESUMEN

CYP3A4 is an important determinant of drug-drug interactions. In this study, we evaluated whether cytochrome P450 3A knockout mice [Cyp3a(-/-)] and CYP3A4 transgenic (CYP3A4-Tg) mice can be used to study drug-drug interactions in the liver and intestine. Triazolam was used as a probe drug because it is a highly specific CYP3A substrate and not a P-glycoprotein substrate. Triazolam metabolism was profoundly reduced in Cyp3a(-/-) mice both in vitro and in vivo. In vitro studies revealed clear species differences in humans and mice, but triazolam metabolism in microsomes derived from CYP3A4-Tg "humanized" mice closely resembled that in human microsomes. It is interesting to note that studies with tissue-specific CYP3A4-Tg mice revealed that intestinal CYP3A4 has a major impact on oral triazolam exposure, whereas the effect of hepatic CYP3A4 was limited. To mimic a drug-drug interaction, we coadministered triazolam with the prototypical CYP3A inhibitor ketoconazole, which increased triazolam exposure in all the CYP3A-proficient mouse strains but not in Cyp3a(-/-) mice. We further found that the anticancer drug gefitinib is a potent stimulator of 1'-OH triazolam formation in vitro. It is noteworthy that we could also show in vivo stimulation of triazolam metabolism by gefitinib, resulting in a lower oral triazolam exposure. To our knowledge, this is the first in vivo example of direct stimulation of CYP3A4 activity after oral drug administration. Overall, this study illustrates how Cyp3a(-/-) and CYP3A4-Tg mice can be used to study drug-drug interactions. The data clarify that for drugs that are not P-glycoprotein substrates, intestinal metabolism also can be more important than hepatic metabolism after oral administration.


Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Intestinos/enzimología , Hígado/enzimología , Triazolam/farmacocinética , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Citocromo P-450 CYP3A/genética , Inhibidores del Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/deficiencia , Sistema Enzimático del Citocromo P-450/genética , Interacciones Farmacológicas , Activadores de Enzimas/administración & dosificación , Inhibidores Enzimáticos/administración & dosificación , Gefitinib , Humanos , Hidroxilación , Intestinos/efectos de los fármacos , Cetoconazol/administración & dosificación , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Microsomas/enzimología , Quinazolinas/administración & dosificación , Especificidad de la Especie , Especificidad por Sustrato , Triazolam/administración & dosificación
10.
Mol Pharmacol ; 73(3): 1029-36, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18156313

RESUMEN

The cytochrome P450 3A (CYP3A) enzymes represent one of the most important drug-metabolizing systems in humans. Recently, our group has generated cytochrome P450 3A knockout mice to study this drug-handling system in vivo. In the present study, we have characterized the Cyp3a knockout mice by studying the metabolism of midazolam, one of the most widely used probes to assess CYP3A activity. We expected that the midazolam metabolism would be severely reduced in the absence of CYP3A enzymes. We used hepatic and intestinal microsomal preparations from Cyp3a knockout and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo metabolite formation was determined after intravenous administration of midazolam. We were surprised to find that our results demonstrated that there is still marked midazolam metabolism in hepatic (but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found comparable amounts of midazolam as well as its major metabolites in plasma after intravenous administration in Cyp3a knockout mice compared with wild-type mice. These data suggested that other hepatic cytochrome P450 enzymes could take over the midazolam metabolism in Cyp3a knockout mice. We provide evidence that CYP2C enzymes, which were found to be up-regulated in Cyp3a knockout mice, are primarily responsible for this metabolism and that several but not all murine CYP2C enzymes are capable of metabolizing midazolam to its 1'-OH and/or 4-OH derivatives. These data illustrate interesting compensatory changes that may occur in Cyp3a knockout mice. Such flexible compensatory interplay between functionally related detoxifying systems is probably essential to their biological role in xenobiotic protection.


Asunto(s)
Ansiolíticos/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Midazolam/metabolismo , Animales , Citocromo P-450 CYP3A/genética , ADN Complementario/biosíntesis , Interpretación Estadística de Datos , Ratones , Ratones Noqueados , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Regulación hacia Arriba/efectos de los fármacos
11.
Drug Discov Today ; 23(12): 2023-2030, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-29928850

RESUMEN

Target concentration is typically not considered in drug discovery. However, if targets are expressed at relatively high concentrations and compounds have high affinity, such that most of the drug is bound to its target, in vitro screens can give unreliable information on compound affinity. In vivo, a similar situation will generate pharmacokinetic (PK) profiles that deviate greatly from those normally expected, owing to target binding affecting drug distribution and clearance. Such target-mediated drug disposition (TMDD) effects on small molecules have received little attention and might only become apparent during clinical trials, with the potential for data misinterpretation. TMDD also confounds human microdosing approaches by providing therapeutically unrepresentative PK profiles. Being aware of these phenomena will improve the likelihood of successful drug discovery and development.


Asunto(s)
Bibliotecas de Moléculas Pequeñas/farmacocinética , Animales , Ensayos Clínicos como Asunto , Sistemas de Liberación de Medicamentos/métodos , Humanos , Distribución Tisular/fisiología
12.
Curr Drug Metab ; 8(1): 59-77, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17266524

RESUMEN

Cytochrome P450 (CYP) 2D6 is one of the most important drug metabolizing enzymes and the rationalization and prediction of potential CYP2D6 substrates is therefore advantageous in the discovery and development of new drugs. Experimentally, the active site of CYP2D6 can be probed by site directed mutagenesis studies. Such studies can be designed from structural models of enzyme-substrate complexes. Modeling approaches can subsequently be used to rationalize the observed effect of mutations on metabolism and inhibition. The current paper will present the construction, refinement and validation of the CYP2D6 homology model used in our laboratory for the prediction and rationalisation of CYP2D6 substrate metabolism and CYP2D6-ligand interactions. The model could explain reported site-directed mutagenesis data (for example, mutation of E216 and D301). Furthermore, based on the model, new CYP2D6 mutants were constructed and studied in our lab, and also for these mutants a rationalization of experimentally observed characteristics could be achieved (I106E, F120A, T309V, F483A). CYP2D6-substrate interaction fingerprint analysis of docked substrates in our homology model suggests that several other active site residues are probably interacting with ligands as well, opening the way for further mutagenesis studies. Our homology model was found to agree with most of the details of the recently solved substrate-free CYP2D6 crystal structure [Rowland et al. J. Biol. Chem. 2006, 281, 7614-7622]. Structural differences between the homology model and crystal structure were the same differences observed between substrate-free and substrate-bound structures of other CYPs, suggesting that these conformational changes are required upon substrate binding. The CYP2D6 crystal structure further validates our homology modeling approach and shows that computational chemistry is a useful and valuable tool to provide models for substrate-bound complexes of CYPs which give insight into CYP-ligand interactions. This information is essential for successful pre-experimental virtual screening, as well as accurate hypothesis generation for in vitro studies in drug discovery and development.


Asunto(s)
Citocromo P-450 CYP2D6/química , Modelos Moleculares , Secuencia de Aminoácidos , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Homología Estructural de Proteína
14.
J Pharm Sci ; 100(6): 2508-15, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21491455

RESUMEN

Lopinavir, a human immunodeficiency virus protease inhibitor, has a very low oral bioavailability, which can be enhanced with a low dose of the CYPA4 inhibitor ritonavir. Our aim was to separately quantify the role of intestinal and hepatic cytochrome P450 3A (CYP3A4) expression on lopinavir disposition in a novel mouse model. Lopinavir and ritonavir were administered to mice selectively expressing human CYP3A4 in the intestine and/or liver. Using nonlinear mixed-effects modeling, we could separately quantify the effects of intestinal CYP3A4 expression, hepatic CYP3A4 expression, and the presence of ritonavir on both the absorption and elimination of lopinavir, which was previously not possible using noncompartmental methods. Intestinal, but not hepatic, CYP3A4-related first-pass metabolism was the major barrier for systemic entry of lopinavir. Relative oral bioavailability of lopinavir in mice expressing both hepatic and intestinal CYP3A4 was only 1.3% when compared with mice that were CYP3A deficient. In presence of ritonavir, relative bioavailability increased to 9.5% due to inhibiton of intestinal, but not due to inhibition of hepatic first-pass metabolism. Hepatic CYP3A4 related systemic clearance was inversely related to ritonavir exposure and not only hepatic but also intestinal CYP3A4 expression contributed to systemic clearance of lopinavir.


Asunto(s)
Citocromo P-450 CYP3A/fisiología , Inhibidores de la Proteasa del VIH/farmacocinética , Modelos Biológicos , Pirimidinonas/farmacocinética , Ritonavir/farmacología , Animales , Disponibilidad Biológica , Citocromo P-450 CYP3A/biosíntesis , Citocromo P-450 CYP3A/genética , Interacciones Farmacológicas , Inhibidores de la Proteasa del VIH/administración & dosificación , Inhibidores de la Proteasa del VIH/química , Humanos , Intestinos/enzimología , Hígado/enzimología , Lopinavir , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Noqueados , Pirimidinonas/administración & dosificación , Pirimidinonas/química , Ritonavir/administración & dosificación , Solubilidad , Distribución Tisular
15.
Mol Cancer Ther ; 9(2): 319-26, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20103600

RESUMEN

Sorafenib is a second-generation, orally active multikinase inhibitor that is approved for the treatment of patients with advanced renal cell carcinoma and patients with unresectable hepatocellular carcinoma. We studied active transport of sorafenib in MDCK-II cells expressing human P-glycoprotein (P-gp/ABCB1) or ABCG2 (breast cancer resistance protein) or murine Abcg2. Sorafenib was moderately transported by P-gp and more efficiently by ABCG2 and Abcg2. Because sorafenib is taken orally, we orally administered sorafenib to wild-type, Abcb1a/1b(-/-), Abcg2(-/-), and Abcb1a/1b;Abcg2(-/-) mice, completely lacking functional Abcb1a/1b, Abcg2, or both, respectively, and we studied plasma pharmacokinetics and brain accumulation. The systemic exposure on oral administration was not different among all strains. However, brain accumulation was 4.3-fold increased in Abcg2(-/-) mice and 9.3-fold increased in Abcb1a/1b;Abcg2(-/-) mice. Moreover, when wild-type mice were treated with sorafenib in combination with the dual P-gp and ABCG2 inhibitor elacridar, brain accumulation was similar to that observed for Abcb1a/1b;Abcg2(-/-) mice. These results show that the brain accumulation of sorafenib is primarily restricted by ABCG2. This contrasts with previous studies using shared ABCG2 and P-gp substrates, which all suggested that P-gp dominates at the blood-brain barrier, and that an effect of ABCG2 is only evident when both transporters are absent. Interestingly, for sorafenib, it is the other way around, that is, ABCG2, and not P-gp, plays the dominant role in restricting its brain accumulation. Clinically, our findings may be relevant for the treatment of renal cell carcinoma patients with central nervous system relapses, as a dual ABCG2 and P-gp inhibitor might improve the central nervous system entry and thereby the therapeutic efficacy of sorafenib.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Bencenosulfonatos/farmacocinética , Encéfalo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/metabolismo , Piridinas/farmacocinética , Animales , Antineoplásicos/farmacocinética , Transporte Biológico , Encéfalo/metabolismo , Línea Celular , Línea Celular Tumoral , Perros , Humanos , Ratones , Ratones Transgénicos , Modelos Químicos , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Sorafenib
16.
Eur J Pharm Sci ; 36(4-5): 565-71, 2009 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-19138736

RESUMEN

Budesonide, a glucocorticoid with a high first-pass metabolism, is used for the oral treatment of inflammatory bowel disease. Cytochrome P450 3A4 (CYP3A4) is an enzyme involved in the metabolism of numerous drugs, including budesonide. Since inhibition or induction of CYP3A4 is often the cause of drug-drug interactions we analyzed how budesonide affects the activity and expression of this enzyme. CYP3A4 activity was assessed by the metabolism of a luminogenic substrate (luciferin-benzylether) using recombinant human CYP3A4 protein. We observed no inhibition of the metabolism in the presence of budesonide at concentrations up to 25 microM. Induction experiments in human LS180 colon carcinoma cells showed an increased expression of CYP3A4 mRNA after budesonide treatment. Transactivation assays revealed that budesonide activates the CYP3A4 promoter via the pregnane X receptor (PXR). In mice, oral budesonide administration (25mg/kg) for 4 days induced the murine homolog Cyp3a11 in the intestine 3-fold, whereas liver expression was notably less influenced. In knockout mice devoid of PXR, budesonide-mediated inductions were reduced compared to wild-type mice. In conclusion, we could demonstrate that budesonide is not an efficient inhibitor but rather an inducer of CYP3A via a PXR-mediated mechanism. In vivo, however, oral budesonide administration to mice showed only modest gene induction, which occurred mainly in the intestine. Therefore, the risk for budesonide-mediated drug interactions seems to be low but cannot be ruled out entirely.


Asunto(s)
Budesonida/farmacología , Citocromo P-450 CYP3A/biosíntesis , Inducción Enzimática/efectos de los fármacos , Glucocorticoides/farmacología , Proteínas de la Membrana/biosíntesis , Receptores de Esteroides/fisiología , Animales , Línea Celular Tumoral , Citocromo P-450 CYP3A/genética , Interacciones Farmacológicas , Humanos , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Receptor X de Pregnano , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Esteroides/genética
17.
Clin Cancer Res ; 15(24): 7616-7623, 2009 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-19996204

RESUMEN

PURPOSE: Trabectedin (Yondelis, ET-743) is a novel anticancer drug with potent activity against various tumors. However, dose-limiting hepatotoxicity was observed during clinical trials. Because recent reports have suggested that cytochrome P450 3A (CYP3A), as well as the drug transporters ABCB1, ABCC2, and ABCC3 might protect against trabectedin-mediated hepatotoxicity, we investigated the individual and combined roles of these detoxifying systems. EXPERIMENTAL DESIGN: Madin-Darby canine kidney cells expressing ABCC2 and ABCC3 were used to study in vitro trabectedin transport. We investigated the hepatotoxicity of trabectedin, and the plasma and liver levels of this drug and its metabolites in mice deficient for CYP3A, Abcb1a/1b, Abcc2, and/or Abcc3 after i.v. trabectedin administration. RESULTS: Trabectedin was transported by ABCC2 but only modestly by ABCC3. Contrary to our expectation, absence of CYP3A resulted in only a marginal increase in hepatotoxicity. Some hepatotoxicity was observed in Abcc2(-/-) mice, but very little in Abcb1a/1b(-/-) and Abcc3(-/-) mice. Strikingly, severe hepatotoxicity was found in Abcb1a/1b/Abcc2(-/-) and Abcc2/Abcc3(-/-) mice. However, hepatotoxicity was drastically decreased in Cyp3a/Abcb1a/1b/Abcc2(-/-) compared with Abcb1a/1b/Abcc2(-/-) mice. This suggests that the formation of CYP3A-specific metabolites is an important prerequisite for trabectedin-mediated hepatotoxicity. Further studies revealed that there is increased accumulation of metabolites of trabectedin, but not of trabectedin itself, in the livers of mice that lack Abcc2 but are CYP3A proficient. CONCLUSIONS: Our data show that ABCB1, ABCC2, and ABCC3 have a profound and partially redundant function in protection from trabectedin-mediated hepatotoxicity, presumably by clearing the liver from hepatotoxic trabectedin metabolites that are primarily formed by CYP3A. (Clin Cancer Res 2009;15(24):7616-23).

18.
Cancer Res ; 69(23): 8996-9002, 2009 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-19920203

RESUMEN

Docetaxel is one of the most widely used anticancer drugs. A major problem with docetaxel treatment, however, is the considerable interpatient variability in docetaxel exposure. Another disadvantage of the drug is that it has a very low oral bioavailability and can therefore only be administered i.v. The drug-metabolizing enzyme cytochrome P450 3A (CYP3A) and the drug transporter P-glycoprotein (P-gp; MDR1) are considered to be major determinants of docetaxel pharmacokinetics. It has been hypothesized that CYP3A and P-gp work synergistically in limiting the systemic exposure to many orally ingested drugs. However, it has been difficult to examine this interplay in vivo. We therefore generated mice lacking all CYP3A and P-gp genes. Although missing two primary detoxification systems, Cyp3a/Mdr1a/1b(-/-) mice are viable, fertile, and without spontaneous abnormalities. When orally challenged with docetaxel, a disproportionate (>70-fold) increase in systemic exposure was observed compared with the increases in single Cyp3a(-/-) (12-fold) or Mdr1a/1b(-/-) (3-fold) mice. Unexpectedly, although CYP3A and P-gp collaborated extremely efficiently in lowering docetaxel exposure, their individual efficacy was not dependent on activity of the other protein. On reflection, this absence of functional synergism makes biological sense, as synergism would conflict with a robust detoxification defense. Importantly, the disproportionate increase in docetaxel exposure in Cyp3a/Mdr1a/1b(-/-) mice resulted in dramatically altered and lethal toxicity, with severe intestinal lesions as a major cause of death. Simultaneous inhibition of CYP3A/P-gp might thus be a highly effective strategy to improve oral drug bioavailability but with serious risks when applied to drugs with narrow therapeutic windows.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Citocromo P-450 CYP3A/deficiencia , Enfermedades Intestinales/inducido químicamente , Taxoides/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Administración Oral , Animales , Antineoplásicos/sangre , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Disponibilidad Biológica , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Docetaxel , Enfermedades Intestinales/sangre , Enfermedades Intestinales/metabolismo , Masculino , Ratones , Ratones Noqueados , Taxoides/sangre , Taxoides/toxicidad
19.
Antimicrob Agents Chemother ; 52(3): 1034-9, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18195061

RESUMEN

Salinomycin is a polyether organic anion that is extensively used as a coccidiostatic antibiotic in poultry and commonly fed to ruminant animals to improve feed efficiency. However, salinomycin also causes severe toxicity when accidentally fed to animals in high doses. In addition, humans are highly sensitive to salinomycin and severe toxicity has been reported. Multidrug efflux transporters like P-glycoprotein (P-gp), BCRP, and MRP2 are highly expressed in the intestine and can restrict the oral uptake and tissue penetration of xenobiotics. The purpose of this study was to investigate whether the anionic drug salinomycin is a substrate for one or more of these efflux pumps. Salinomycin was actively transported by human MDR1 P-gp expressed in polarized MDCK-II monolayers but not by the known organic anion transporters human MRP2 and murine Bcrp1. Using P-gp-deficient mice, we found a marked increase in plasma salinomycin concentrations after oral administration and decreased plasma clearance after intravenous administration. Furthermore, absence of P-gp resulted in significantly increased brain penetration. P-gp-deficient mice also displayed clearly increased susceptibility to salinomycin toxicity. Thus far, P-gp was thought to affect mainly hydrophobic, positively charged or neutral drugs in vivo. Our data show that P-gp can also be a major determinant of the pharmacokinetic behavior and toxicity of an organic anionic drug. Variation in P-gp activity might thus directly affect the effective exposure to salinomycin and possibly to other anionic drugs and toxin substrates. Individuals with reduced or absent P-gp activity could therefore be more susceptible to salinomycin toxicity.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacocinética , Antibacterianos/toxicidad , Encéfalo/metabolismo , Piranos/farmacocinética , Piranos/toxicidad , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antibacterianos/química , Disponibilidad Biológica , Transporte Biológico , Línea Celular , Perros , Humanos , Riñón/citología , Riñón/metabolismo , Masculino , Ratones , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Piranos/química , Piranos/metabolismo
20.
Arch Biochem Biophys ; 447(1): 53-8, 2006 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-16466686

RESUMEN

Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.


Asunto(s)
Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Escherichia coli/enzimología , Modelos Químicos , Modelos Moleculares , Sustitución de Aminoácidos , Simulación por Computador , Activación Enzimática , Escherichia coli/genética , Humanos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
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