RESUMO
INTRODUCTION: The key role of transporter biology in both the manifestation and treatment of disease is now firmly established. Experiences of sub-optimal drug exposure due to drug-transporter interplay have supported incorporation of studies aimed at understanding the interactions between compounds and drug transporters much earlier in drug discovery. While drug transporters can impact the most pivotal pharmacokinetic parameter with respect to human dose and exposure projections, clearance, at a renal or hepatobiliary level, the latter will form the focus of this perspective. AREAS COVERED: A synopsis of guidelines on which transporters to study together with an overview of the currently available toolkit is presented. A perspective on when to conduct studies with various hepatic transporters is also provided together with structural "alerts" which should prompt early investigation. EXPERT OPINION: Great progress has been made in individual laboratories and via consortia to understand the role of drug transporters in disease, drug disposition, drug-drug interactions and toxicity. A systematic analysis of the value posed by the available approaches and an inter-lab comparison now seems warranted. The emerging ability to use physico-chemical properties to guide future screening cascades promises to revolutionise the efficiency of early drug discovery.
Assuntos
Desenho de Fármacos , Fígado/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Transporte Biológico , Descoberta de Drogas/métodos , Interações Medicamentosas , Humanos , Proteínas de Membrana Transportadoras/metabolismo , FarmacocinéticaRESUMO
1. Organic anion transporting polypeptide 1B1 plays a pivotal role in the disposition of many anionic drugs. Significant overlap in substrate specificity between individual OATP isoforms has hampered the identification of the relative importance of individual isoforms for hepatic uptake of xenobiotics. 2. The present study focused on the use of siRNA technology to decrease OATP1B1 selectively in human hepatocytes. Following delivery of siRNA by the novel lipid, AtuFECT01, mRNA expression of OATP1B1 was reduced by 94%-98% with no significant toxicity. Off-target effects were also shown to be minimal as evidenced by the expression of common drug metabolizing enzymes, transporters, nuclear receptors and associated co-regulators. Uptake of estrone-3-sulfate (5 nM) by OATP1B1 was reduced by 82%-95%. This methodology was subsequently used to assess the relative contribution of OATP1B1 uptake in human hepatocytes for olmesartan (42%-62%), valsartan (28%-81%), rosuvastatin (64%-72%), pitavastatin (84%-98%) and lopinavir (64%-89%). These data are consistent with previous values obtained using a relative activity factor approach. 3. The siRNA approach provides a robust and reproducible method for assessing the relative contribution of OATP1B1 to hepatic uptake of new chemical entities. The technique also has potential utility in facilitating detailed characterization of drug-drug interactions involving hepatic drug transporters.
Assuntos
Hepatócitos/efeitos dos fármacos , Transportadores de Ânions Orgânicos/metabolismo , RNA Interferente Pequeno , Xenobióticos/farmacocinética , Sequência de Bases , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Estrona/análogos & derivados , Estrona/farmacocinética , Feminino , Fluorbenzenos/farmacocinética , Hepatócitos/metabolismo , Humanos , Imidazóis/farmacocinética , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Dados de Sequência Molecular , Transportadores de Ânions Orgânicos/genética , Pirimidinas/farmacocinética , Quinolinas/farmacocinética , RNA Interferente Pequeno/administração & dosagem , Rosuvastatina Cálcica , Sulfonamidas/farmacocinética , Tetrazóis/farmacocinética , Valina/análogos & derivados , Valina/farmacocinética , ValsartanaRESUMO
To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate selectivity and inhibitory potential of over 60 compounds towards CYP3A4 and CYP3A5 were assessed using Escherichia coli recombinant cell lines. CYP3A4-mediated metabolism predominated for many of the compounds studied. However, a number of drugs gave similar CL(int) estimates using CYP3A5 compared with CYP3A4 including midazolam (CL(int) = 3.4 versus 3.3 microl min(-1) pmol(-1)). Significant CYP3A5-mediated metabolism was also observed for several drugs including mifepristone (CL(int) = 10.3 versus 2.4 microl min(-1) pmol(-1)), and ritonavir (CL(int) = 0.76 versus 0.47 microl min(-1) pmol(-1)). The majority of compounds studied showed a greater inhibitory potential (IC(50)) towards CYP3A4 compared with CYP3A5 (eightfold lower on average). A greater degree of time-dependent inhibition was also observed with CYP3A4 compared with CYP3A5. The range of compounds investigated in the present study extends significantly previous work and suggests that CYP3A5 may have a significant role in drug metabolism particularly in populations expressing high levels of CYP3A5 and/or on co-medications known to inhibit CYP3A4.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Inibidores Enzimáticos/farmacologia , Microssomos Hepáticos/enzimologia , Linhagem Celular , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Interações Medicamentosas , Escherichia coli/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Mifepristona/farmacologia , Ritonavir/farmacologia , Especificidade por Substrato , Fatores de TempoRESUMO
The glucuronidation of a number of commonly used hepatic uridine diphosphate glucuronosyltransferase drug substrates has been studied in human tissue microsomes. Prediction of in vivo hepatic drug glucuronidation from liver microsomal data yielded a consistent 10-fold under-prediction. Consideration of protein binding was observed to be pivotal when predicting in vivo glucuronidation for acid substrates. Studies using human intestinal microsomes demonstrated the majority of drugs to be extensively glucuronidated such that the intrinsic clearance (CL(int)) of ethinylestradiol (CL(int) = 1.3 microl/min/mg) was twice that obtained using human liver microsomes (CL(int) = 0.7 microl/min/mg). The potential extrahepatic in vivo glucuronidation was calculated for a range of drug substrates from human microsomal data. These results indicate the contribution of intestinal drug glucuronidation to systemic drug clearance to be much less than either hepatic or renal glucuronidation. Therefore, data obtained with intestinal microsomes may be misleading in the assessment of the contribution of this organ to systemic glucuronidation. The use of hepatocytes to assess metabolic stability for drugs predominantly metabolized by glucuronidation was also investigated. Metabolic clearances for a range of drugs obtained using fresh preparations of human hepatocytes predicted accurately hepatic clearance reported in vivo. The use of cryopreserved hepatocytes as an in vitro tool to predict in vivo metabolism was also assessed with an excellent correlation obtained for a number of extensively glucuronidated drugs (R(2) = 0.80, p < 0.001).
Assuntos
Glucuronídeos/metabolismo , Taxa de Depuração Metabólica , Preparações Farmacêuticas/metabolismo , Albuminas/farmacologia , Algoritmos , Animais , Peso Corporal/fisiologia , Diálise , Glucuronosiltransferase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Cinética , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Tamanho do Órgão/fisiologia , Valor Preditivo dos Testes , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
UDP-glucuronosyltransferases (UGTs) are a major family of enzymes catalyzing the transfer of glucuronic acid to a range of endogenous compounds and xenobiotics facilitating their elimination in either urine or bile. Although the dog is commonly used in drug metabolism studies, relatively little is known about the expression and activity of UGTs in this species. This report describes the molecular cloning and functional characterization of the first dog UGT, UGT1A6. The cloned protein is composed of 528 amino acids with the variable region demonstrating a 67-72% identity with the variable regions of mouse, rat, and human UGT1A6. The enzyme expressed stably in V79 cells predominantly catalyzed the glucuronidation of simple, planar phenols (e.g., for 1-naphthol, K(m) = 41 microM, V(max) = 0.07 nmol/min/mg protein), a class of compounds extensively glucuronidated by human UGT1A6. Based on sequence homology and common catalytic activity, this dog UGT1A protein appears to be the canine orthologue of human UGT1A6.
Assuntos
Glucuronosiltransferase/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , Cricetinae , Cães , Glucuronosiltransferase/metabolismo , Humanos , Microssomos Hepáticos/enzimologia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The in vitro glucuronidation of a range of structurally diverse chemicals has been studied in hepatic and renal microsomes from human donors and the beagle dog. These studies were undertaken to improve on the limited knowledge of glucuronidation by the dog and to assess its suitability as a model species for pharmacokinetic studies. In general, the compounds studied were glucuronidated severalfold more rapidly (based on intrinsic clearance estimates) by DLM than by HLM. Intrinsic clearance values for human UGT1A1 and UGT2B7 substrates were an order of magnitude higher in DLM than in HLM (e.g., gemfibrozil: 31 microl/min/mg versus 3.0 microl/min/mg; ketoprofen: 2.4 microl/min/mg versus 0.2 microl/min/mg). There were also drug-specific differences. HLM readily glucuronidated propofol (2.4 microl/min/mg) whereas DLM appeared unable to glucuronidate this drug directly. Regioselective differences in morphine glucuronidation were also apparent. Human kidney microsomes catalyzed the glucuronidation of many xenobiotics, although glucuronidation of the endobiotic bilirubin was not detectable in this tissue. In direct contrast, dog kidney microsomes glucuronidated bilirubin only (no glucuronidation of all other xenobiotics was detected). These preliminary studies indicated significant differences in the glucuronidation of xenobiotics by microsomes from the livers and kidneys of human and dog and should be confirmed using a larger panel of tissues from individual dogs. Early knowledge of the relative rates of in vitro glucuronidation, the UGTs responsible for drug glucuronidation, and their tissue distribution in different species could assist the design and analysis of preclinical pharmacokinetic and safety evaluation studies.
Assuntos
Glucuronídeos/biossíntese , Cetoprofeno/análogos & derivados , Rim/metabolismo , Microssomos Hepáticos/metabolismo , Adulto , Androstano-3,17-diol/análogos & derivados , Androstano-3,17-diol/biossíntese , Androstano-3,17-diol/metabolismo , Androstano-3,17-diol/farmacocinética , Animais , Cães , Feminino , Furosemida/análogos & derivados , Furosemida/metabolismo , Furosemida/farmacocinética , Genfibrozila/metabolismo , Genfibrozila/farmacocinética , Glucuronidase/metabolismo , Humanos , Cetoprofeno/metabolismo , Cetoprofeno/farmacocinética , Rim/enzimologia , Cinética , Masculino , Microssomos/enzimologia , Microssomos/metabolismo , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Modelos Animais , Propofol/metabolismo , Propofol/farmacocinética , Especificidade da EspécieRESUMO
1. The in vitro glucuronidation of a wide range of compounds has been studied in microsomes prepared from marmoset liver and kidney. These studies have been undertaken to evaluate the marmoset as a model species for drug glucuronidation and for comparison with conjugation by other species. 2. The compounds studied were glucuronidated by marmoset liver microsomes to varying extents (e.g. naproxen CLint 0.4 microl min(-1) mg(-1), 1-naphthol CLint 43 microl min(-1) mg(-1)) Both marmoset and rat liver microsomes glucuronidated morphine at the 3-position (marmoset CLint 19 microl min(-1) mg(-1), rat CLint 6.3 microl min(-1) mg(-1)) but glucuronidation at the 6-position was below, the level of radiodetection in both species. 3. Interestingly, marmoset liver microsomes were able to catalyse the glucuronidation of the tertiary amine imipramine to a significant extent (0.05 nmol min(-1) mg(-1)). However, no glucuronidation was detected by rat liver microsomes. 4. Conjugation of a range of substrates was detectable by marmoset kidney microsomes in contrast to rat kidney microsomes, which only catalysed the glucurondation of bilirubin and 1-naphthol (CLint 17 microl min(-1) mg(-1) and 18 microl min(-1) mg(-1), respectively). 5. This report and previous work in dog and human tissue microsomes suggest that the marmoset may be an alternative animal model for human drug glucuronidation, especially when the pathway of drug glucuronidation is known to differ between lower laboratory species and man.