RESUMO
Previous use of a mechanistic static model to accurately quantify the increased rosuvastatin exposure due to drug-drug interaction (DDI) with coadministered atazanavir underpredicted the magnitude of area under the plasma concentration-time curve ratio (AUCR) based on inhibition of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1. To reconcile the disconnect between predicted and clinical AUCR, atazanavir and other protease inhibitors (darunavir, lopinavir and ritonavir) were evaluated as inhibitors of BCRP, OATP1B1, OATP1B3, sodium taurocholate cotransporting polypeptide (NTCP) and organic anion transporter (OAT) 3. None of the drugs inhibited OAT3, nor did darunavir and ritonavir inhibit OATP1B3 or NTCP. All drugs inhibited BCRP-mediated estrone 3-sulfate transport or OATP1B1-mediated estradiol 17ß-D-glucuronide transport with the same rank order of inhibitory potency (lopinavir>ritonavir>atazanavir>>darunavir) and mean IC50 values ranging from 15.5 ± 2.80 µM to 143 ± 14.7 µM or 0.220 ± 0.0655 µM to 9.53 ± 2.50 µM, respectively. Atazanavir and lopinavir also inhibited OATP1B3- or NTCP-mediated transport with a mean IC50 of 1.86 ± 0.500 µM or 65.6 ± 10.7 µM and 5.04 ± 0.0950 µM or 20.3 ± 2.13 µM, respectively. Following integration of a combined hepatic transport component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above for atazanavir, the newly predicted rosuvastatin AUCR reconciled with the clinically observed AUCR confirming additional minor involvement of OATP1B3 and NTCP inhibition in its DDI. The predictions for the other protease inhibitors confirmed inhibition of intestinal BCRP and hepatic OATP1B1 as the principal pathways involved in their clinical DDI with rosuvastatin.
Assuntos
Inibidores de Proteases , Ritonavir , Rosuvastatina Cálcica , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Lopinavir , Darunavir , Sulfato de Atazanavir/metabolismo , Proteínas de Neoplasias/metabolismo , Interações MedicamentosasRESUMO
INTRODUCTION: Transporters are significant in dictating drug pharmacokinetics, thus inhibition of transporter function can alter drug concentrations resulting in drug-drug interactions (DDIs). Because they can impact drug toxicity, transporter DDIs are a regulatory concern for which prediction of clinical effect from in vitro data is critical to understanding risk. AREA COVERED: The authors propose in vitro strategies to assist mitigating/removing transporter DDI risk during development by frontloading specific studies, or managing patient risk in the clinic. An overview of clinically relevant drug transporters and observed DDIs is provided, alongside presentation of key considerations/recommendations for in vitro study design evaluating drugs as inhibitors or substrates. Guidance on identifying critical co-medications, clinically relevant disposition pathways, and using mechanistic static equations for quantitative prediction of DDI is compiled. EXPERT OPINION: The strategies provided will facilitate project teams to study the right transporter at the right time to minimize development risks associated with DDIs. To truly alleviate or manage clinical risk, the industry will benefit from moving away from current qualitative basic static equation approaches to transporter DDI hazard assessment towards adopting the use of mechanistic models to enable quantitative DDI prediction, thereby contextualizing risk to ascertain whether a transporter DDI is simply pharmacokinetic or clinically significant requiring intervention.
Assuntos
Proteínas de Membrana Transportadoras , Modelos Biológicos , Humanos , Interações Medicamentosas , Proteínas de Membrana Transportadoras/metabolismo , Descoberta de DrogasRESUMO
A previous attempt to accurately quantify the increased simvastatin acid exposure due to drug-drug interaction (DDI) with coadministered telithromycin, using a mechanistic static model, substantially underpredicted the magnitude of the area under the plasma concentration-time curve ratio (AUCR) based on reversible inhibition of CYP3A4 and organic anion transporting polypeptide 1B1 (OATP1B1). To reconcile this disconnect between predicted and clinically observed AUCR, telithromycin was evaluated as a time-dependent inhibitor of CYP3A4 in vitro, as well as an inhibitor of OATP1B1. Telithromycin inhibited OATP1B1-mediated [3H]-estradiol 17ß-d-glucuronide (0.02 µM) transport with a mean IC50 of 12.0 ± 1.45 µM and was determined by IC50 shift and kinetic analyses to be a competitive reversible inhibitor of CYP3A4-mediated midazolam1- hydroxylation with a mean absolute inhibition constant (Ki) value of 3.65 ± 0.531 µM. The 2.83-fold shift in IC50 (10.4-3.68 µM) after a 30-minute metabolic preincubation confirmed telithromycin as a time-dependent inhibitor of CYP3A4; the mean inhibitor concentration that causes half-maximal inactivation of enzyme (KI) and maximal rate of inactivation of enzyme (kinact) values determined for inactivation were 1.05 ± 0.226 µM and 0.02772 ± 0.00272 min-1, respectively. After the integration of an enzyme time-dependent inhibition component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above, the newly predicted simvastatin acid AUCR (10.8 or 5.4) resulting from perturbation of its critical disposition pathways matched the clinically observed AUCR (10.8 or 4.3) after coadministration, or staggered administration, with telithromycin, respectively. These results indicate the time-dependent inhibition of CYP3A4 by telithromycin as the primary driver underlying its clinical DDI with simvastatin acid.
Assuntos
Inibidores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Cetolídeos/farmacologia , Sinvastatina/análogos & derivados , Antibacterianos , Área Sob a Curva , Interações Medicamentosas , Células HEK293 , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Microssomos Hepáticos , Sinvastatina/farmacologia , Sinvastatina/uso terapêutico , Fatores de TempoRESUMO
Metformin is a common co-medication for many diseases and the victim of clinical drug-drug interactions (DDIs) perpetrated by cimetidine, trimethoprim and pyrimethamine, resulting in decreased active renal clearance due to inhibition of organic cation transport proteins and increased plasma exposure of metformin. To understand whether area under the plasma concentration-time curve (AUC) increases relate to absorption, in vitro inhibitory potencies of these drugs against metformin transport by human organic cation transporter (OCT) 1, and the apical to basolateral absorptive permeability of metformin across Caco-2 cells in the presence of therapeutic intestinal concentrations of cimetidine, trimethoprim or pyrimethamine, were determined. Whilst all inhibited OCT1, none enhanced metformin's absorptive permeability (~0.5 × 10-6 cm/sec) suggesting that DDI AUC changes are not related to absorption. Subsequently, to understand whether inhibition of renal transporters are responsible for AUC increases, in vitro inhibitory potencies against metformin transport by human OCT2, multidrug and toxin extrusion (MATE) 1 and MATE2-K were determined. Ensuing IC50 values were incorporated into mechanistic static equations, alongside unbound maximal plasma concentration and transporter fraction excreted values, in order to calculate theoretical increases in metformin AUC due to inhibition by cimetidine, trimethoprim or pyrimethamine. Calculated theoretical fold-increases in metformin exposure confirmed solitary inhibition of renal MATE1 to be the likely mechanism underlying the observed exposure changes in clinical DDIs. Interestingly, clinically observed increases in metformin AUC were predicted more closely when the renal transporter fraction excreted value derived from oral metformin administration, rather than intravenous, was utilized in theoretical calculations, likely reflecting the "flip-flop" pharmacokinetic profile of the drug.
Assuntos
Cimetidina/farmacologia , Metformina/farmacologia , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Pirimetamina/farmacologia , Trimetoprima/farmacologia , Área Sob a Curva , Células CACO-2 , Interações Medicamentosas , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Fator 1 de Transcrição de Octâmero/metabolismoRESUMO
The intestinal efflux transporter breast cancer resistance protein (BCRP) restricts the absorption of rosuvastatin. Of the transporters important to rosuvastatin disposition, fostamatinib inhibited BCRP (IC50 = 50 nM) and organic anion-transporting polypeptide 1B1 (OATP1B1; IC50 > 10 µM), but not organic anion transporter 3, in vitro, predicting a drug-drug interaction (DDI) in vivo through inhibition of BCRP only. Consequently, a clinical interaction study between fostamatinib and rosuvastatin was performed (and reported elsewhere). This confirmed the critical role BCRP plays in statin absorption, as inhibition by fostamatinib resulted in a significant 1.96-fold and 1.88-fold increase in rosuvastatin area under the plasma concentration-time curve (AUC) and Cmax, respectively. An in vitro BCRP inhibition assay, using polarized Caco-2 cells and rosuvastatin as probe substrate, was subsequently validated with literature inhibitors and used to determine BCRP inhibitory potencies (IC50) of the perpetrator drugs eltrombopag, darunavir, lopinavir, clopidogrel, ezetimibe, fenofibrate, and fluconazole. OATP1B1 inhibition was also determined using human embryonic kidney 293-OATP1B1 cells versus estradiol 17ß-glucuronide. Calculated parameters of maximum enterocyte concentration [Igut max], maximum unbound hepatic inlet concentration, transporter fraction excreted value, and determined IC50 value were incorporated into mechanistic static equations to compute theoretical increases in rosuvastatin AUC due to inhibition of BCRP and/or OATP1B1. Calculated theoretical increases in exposure correctly predicted the clinically observed changes in rosuvastatin exposure and suggested intestinal BCRP inhibition (not OATP1B1) to be the mechanism underlying the DDIs with these drugs. In conclusion, solitary inhibition of the intestinal BCRP transporter can result in clinically significant DDIs with rosuvastatin, causing up to a maximum 2-fold increase in exposure, which may warrant statin dose adjustment in clinical practice.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/efeitos dos fármacos , Interações Medicamentosas/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Oxazinas/metabolismo , Piridinas/metabolismo , Rosuvastatina Cálcica/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Aminopiridinas , Área Sob a Curva , Células CACO-2 , Células HEK293 , Humanos , Absorção Intestinal/efeitos dos fármacos , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Proteínas de Membrana Transportadoras/metabolismo , Morfolinas , Transportadores de Ânions Orgânicos/metabolismo , PirimidinasRESUMO
PURPOSE: Fostamatinib, a spleen tyrosine kinase inhibitor and prodrug of the active metabolite R406, is being developed as an anti-inflammatory drug for several indications for which polypharmacy is likely. Digoxin, indicated for congestive cardiac failure, may be used for certain supraventricular dysrhythmias. The studies reported herein examined whether fostamatinib and R406 are inhibitors of P-glycoprotein (P-gp) in vitro and evaluated the effect of fostamatinib on the pharmacokinetic parameters of digoxin to understand drug-drug interaction (DDI) potential in the clinic. METHODS: Inhibition of P-gp-mediated digoxin transport by fostamatinib and R406 was determined across Caco-2 cell monolayers. Apparent permeability of digoxin was determined and used to calculate efflux ratios and percentage inhibition. Half maximal inhibitory concentrations (IC50) and theoretical gastrointestinal concentration [I2] (dose in moles per 250 mL) were calculated to gauge clinical DDI potential. In a subsequent Phase I study, the plasma concentration-time profiles and resulting pharmacokinetic parameters were examined across 2 treatment periods: (1) oral digoxin loading dose of 0.25 mg BID on day 1 and 0.25 mg once daily on days 2 to 8, and (2) oral digoxin 0.25 mg once daily and oral fostamatinib 100 mg BID on days 9 to 15. FINDINGS: Fostamatinib (but not R406) was determined to be a P-gp inhibitor in vitro (IC50 = 3.2 µM). On the basis of a theoretical gastrointestinal concentration (I2)/IC50 ratio of 216 ([I2] = 691 µM), predictions indicated the potential for absorption-based DDI in vivo through inhibition of intestinal P-gp. In the clinical study, when digoxin was co-administered with fostamatinib, digoxin levels were higher before dosing and throughout the dosing interval, and an increase in exposure to digoxin was observed. Co-administration led to a 1.70-fold increase in digoxin maximum plasma concentration at steady state (Cmax,ss) versus digoxin administration alone (2.18 vs 1.32 ng/mL). Median digoxin time of Cmax was earlier when digoxin was co-administered with fostamatinib (1.00 vs 1.48 hours). The digoxin AUC during the dosing interval at steady state was increased 1.37-fold with co-administration. No severe or serious adverse events or deaths were reported. IMPLICATIONS: Fostamatinib was confirmed to be a P-gp inhibitor in vitro and in vivo, and a DDI with digoxin was apparent. Co-administration of digoxin and fostamatinib was generally well tolerated. However, continued review of digoxin response and dose is advisable should these agents be prescribed concomitantly. ClinicalTrials.gov identifier: NCT01355354.
Assuntos
Digoxina , Oxazinas , Piridinas , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Aminopiridinas , Células CACO-2 , Digoxina/administração & dosagem , Digoxina/sangue , Digoxina/farmacocinética , Interações Medicamentosas , Feminino , Humanos , Morfolinas , Oxazinas/administração & dosagem , Oxazinas/sangue , Oxazinas/farmacocinética , Piridinas/administração & dosagem , Piridinas/sangue , Piridinas/farmacocinética , PirimidinasRESUMO
PURPOSE: Systemic exposure to rosuvastatin is approximately double that of Caucasians in Asian subjects. We investigated whether this pattern of increased exposure exists for other statins. METHODS: Plasma exposure following single-dose rosuvastatin 20 mg, atorvastatin 40 mg or simvastatin 40 mg was studied in Chinese, Japanese and Caucasian subjects. Plasma concentrations were determined using LC-MS methods. Impact of polymorphisms in SLCO1B1 (T521>C and A388>G) and in ABCG2 (C421>A) on exposure to rosuvastatin, atorvastatin, simvastatin and simvastatin acid was assessed. RESULTS: Relative to Caucasians, geometric mean area under the curve from time zero to time of last quantifiable concentration was 86 % (90 % confidence interval (CI), 51-130 %) and 55 % (26-91 %) higher for rosuvastatin in Chinese and Japanese subjects, respectively, 53 % (25-88 %) and 69 % (37-108 %) higher for atorvastatin, 23 % (0-52 %) and 12 % (-0.9-39 %) higher for simvastatin and 28 % (5-56 %) and 34 % (10-64 %) higher for simvastatin acid. Geometric mean maximum drug concentration was also proportionally higher for each statin. Polymorphisms in SLCO1B1 T521>C or ABCG2 C421>A were associated with higher exposure to rosuvastatin, atorvastatin and simvastatin acid (but not simvastatin) within a population, but only the ABCG2 C421>A polymorphism contributed towards between-population exposure differences. In individuals carrying wild-type alleles for both SLCO1B1 and ABCG2, area under the plasma concentration-time curve (AUC) still appeared to be higher for rosuvastatin, atorvastatin and simvastatin acid in Chinese and Japanese subjects compared with Caucasians, respectively. CONCLUSION: Increased exposure to statins in Asian subjects versus Caucasians may represent a more general class phenomenon than previously recognized.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Povo Asiático/genética , Atorvastatina/farmacocinética , Proteínas de Neoplasias/genética , Transportadores de Ânions Orgânicos/genética , Rosuvastatina Cálcica/farmacocinética , Sinvastatina/análogos & derivados , Sinvastatina/farmacocinética , População Branca/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Adolescente , Adulto , Idoso , Atorvastatina/efeitos adversos , Atorvastatina/sangue , Feminino , Genótipo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/sangue , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Rosuvastatina Cálcica/efeitos adversos , Rosuvastatina Cálcica/sangue , Sinvastatina/efeitos adversos , Sinvastatina/sangue , Adulto JovemRESUMO
PURPOSE: Systemic exposure to rosuvastatin in Asian subjects living in Japan or Singapore is approximately twice that observed in Caucasian subjects in Western countries or in Singapore. This study was conducted to determine whether pharmacokinetic differences exist among the most populous Asian subgroups and Caucasian subjects in the USA. METHOD: Rosuvastatin pharmacokinetics was studied in Chinese, Filipino, Asian-Indian, Korean, Vietnamese, Japanese and Caucasian subjects residing in California. Plasma concentrations of rosuvastatin and metabolites after a single 20-mg dose were determined by mass spectrometric detection. The influence of polymorphisms in SLCO1B1 (T521>C [Val174Ala] and A388>G [Asn130Asp]) and in ABCG2 (C421>A [Gln141Lys]) on exposure to rosuvastatin was also assessed. RESULTS: The average rosuvastatin area under the curve from time zero to time of last quantifiable concentration was between 64 and 84 % higher, and maximum drug concentration was between 70 and 98 % higher in East Asian subgroups compared with Caucasians. Data for Asian-Indians was intermediate to these two ethnic groups at 26 and 29 %, respectively. Similar increases in exposure to N-desmethyl rosuvastatin and rosuvastatin lactone were observed. Rosuvastatin exposure was higher in subjects carrying the SLCO1B1 521C allele compared with that in non-carriers of this allele. Similarly, exposure was higher in subjects carrying the ABCG2 421A allele compared with that in non-carriers. CONCLUSION: Plasma exposure to rosuvastatin and its metabolites was significantly higher in Asian populations residing in the USA compared with Caucasian subjects living in the same environment. This study suggests that polymorphisms in the SLCO1B1 and ABCG2 genes contribute to the variability in rosuvastatin exposure.
Assuntos
Povo Asiático/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Rosuvastatina Cálcica/farmacocinética , População Branca/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Alelos , Feminino , Genótipo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/sangue , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Proteínas de Neoplasias/genética , Transportadores de Ânions Orgânicos/genética , Polimorfismo de Nucleotídeo Único/genética , Rosuvastatina Cálcica/efeitos adversos , Rosuvastatina Cálcica/sangue , Estados UnidosRESUMO
Bringing a new drug to market is costly in terms of capital and time investments, and any development issues encountered during late-stage clinical trials can often be the result of in vitro-in vivo extrapolations (IVIVE) not accurately reflecting clinical outcome. In the discipline of drug metabolism and pharmacokinetics (DMPK), current in vitro cellular methods do not provide the 3D structure and function of organs found in vivo; therefore, new dynamic methods need to be established to aid improvement of IVIVE. In this review, we highlight the importance of model progression into dynamic systems for use within drug development, focusing on devices developed currently in the areas of the liver and blood-brain barrier (BBB), and the potential to develop models for other organ systems, such as the kidney. We discuss the development of dynamic 3D bioreactor-based systems as in vitro models for use in DMPK studies.
Assuntos
Órgãos Bioartificiais/tendências , Reatores Biológicos , Modelos Biológicos , Tecnologia Farmacêutica/tendências , Animais , Pesquisa Biomédica , Humanos , Tecnologia Farmacêutica/métodosRESUMO
To support drug development, the drug-drug interaction potential (DDI) of an investigational drug (AZX) was assessed against the probe estradiol 17ß-glucuronide as well as against simvastatin acid, atorvastatin, pravastatin, pitavastatin, fluvastatin, rosuvastatin and estrone 3-sulfate. The inhibitory potentials of the OATP1B1 inhibitors rifamycin SV and gemfibrozil were assessed in parallel. Monolayer cellular uptake assays were used to determine inhibition of human OATP1B1. Apparent K(m) values for the OATP1B1-mediated transport of [(3)H] substrates were determined prior to their use as probes in inhibition studies, and ranged from 0.6 to 29 µM for statins. The K(m) of lipophilic simvastatin acid could not be determined due to its high passive permeability that masked OATP1B1 transport, and therefore this statin could not be used as a probe. Estrone 3-sulfate exhibited biphasic kinetics, whereas estradiol 17ß-glucuronide demonstrated simple Michaelis-Menton kinetics. AZX moderately inhibited OATP1B1-mediated transport of all statins (IC(50)=4.6-9.7 µM), except fluvastatin, of estradiol 17ß-glucuronide (IC(50)=5.3 µM), and weakly inhibited estrone 3-sulfate (IC(50)=79 µM). Rifamycin SV strongly, and gemfibrozil weakly, inhibited the OATP1B1-mediated transport of substrates. Estradiol 17ß-glucuronide was identified as a good surrogate probe for statins when assessing OATP1B1 inhibitory potential using this test system. Inhibition data was used to predict the likelihood of a clinical DDI, using current draft US FDA guidance and recommendations of the International Transporter Consortium. Predictions for AZX indicated the potential for an OATP1B1-mediated DDI in vivo and that a clinical interaction study is warranted to confirm whether AZX is an OATP1B1 inhibitor in the clinic.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Transportadores de Ânions Orgânicos/metabolismo , Linhagem Celular , Interações Medicamentosas , Estradiol/farmacologia , Estrona/análogos & derivados , Estrona/farmacologia , Genfibrozila/farmacologia , Glucuronídeos/farmacologia , Células HEK293 , Humanos , Cinética , Transportador 1 de Ânion Orgânico Específico do Fígado , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Permeabilidade , Rifamicinas/farmacologiaRESUMO
Breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) can play a role in the absorption, distribution, metabolism, and excretion of drugs, impacting on the potential for drug-drug interactions. This study has characterized insect cell- and mammalian cell-derived ABC-transporter-expressing membrane vesicle test systems and validated methodologies for evaluation of candidate drugs as substrates or inhibitors of BCRP or MRP2. Concentration-dependent uptake of BCRP ([³H]oestrone 3-sulfate, [³H]methotrexate, [³H]rosuvastatin) and MRP2 ([³H]oestradiol 17ß-glucuronide, [³H]pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC50) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined. The apparent K(m) for probes [³H]oestrone 3-sulfate and [³H]oestradiol 17ß-glucuronide was determined in insect cell vesicles to be 7.4 ± 1.7 and 105 ± 8.3 µM, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC50 values of 0.74 ± 0.18 and 36 ± 6.1 µM, respectively. All other inhibitors exhibited concentration-dependent inhibition. There was no significant difference in parameters generated between test systems. On the basis of the validation results, acceptance criteria to identify substrates/inhibitors of BCRP and MRP2 were determined for insect cell vesicles. The approach builds on earlier validations to support drug registration and extends from those cell-based systems to encompass assay formats using membrane vesicles.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bioensaio/métodos , Interações Medicamentosas , Controle de Medicamentos e Entorpecentes , Membranas Artificiais , Proteínas de Neoplasias/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Soluções Tampão , Aprovação de Drogas , Estabilidade de Medicamentos , Insetos , Cinética , Controle de Qualidade , Reprodutibilidade dos Testes , Fatores de Tempo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Methotrexate is the most commonly used drug to treat rheumatoid arthritis. Since clinical efficacy studies in rheumatoid arthritis are conducted on a background of methotrexate therapy, it is necessary to assess the potential of rheumatoid arthritis candidate drugs to perpetrate a drug-drug interaction (DDI) with methotrexate during development. Consequently, we need to identify the regulatory in vitro studies required to facilitate this assessment. We therefore reviewed the literature to ascertain the methotrexate disposition pathways implicated with known DDIs. Experiments were conducted to confirm that methotrexate was identified as a substrate for these pathways in our laboratory. The literature indicated active renal elimination (mediated by the human transporters OAT1, OAT3, MRP2 and BCRP) to be the principal pathway for methotrexate DDI risk. With the exception of MRP2, methotrexate was confirmed as a substrate of these transporters using oocyte and membrane vesicle test systems. A rheumatoid arthritis candidate drug (AZD9056) and sulfasalazine were subsequently assessed as inhibitors of OAT1, OAT3 and BCRP to determine their DDI potential towards methotrexate. AZD9056 was neither an inhibitor of OAT1 nor OAT3 and did not inhibit their transport of methotrexate. AZD9056 was an inhibitor of BCRP and weakly inhibited BCRP-mediated transport of methotrexate (IC(50)=92µM). Sulfasalazine inhibited methotrexate transport mediated by all transporters studied (IC(50)<5µM). Subsequent assessment of the in vitro data using [I]/IC(50) ratios indicated that both AZD9056 and sulfasalazine were unlikely to cause a DDI with methotrexate in vivo. In conclusion, to support rheumatoid arthritis drug development it is proposed that regulatory in vitro studies for OAT1, OAT3 and BCRP inhibition be routinely conducted to assess the potential for a transporter-mediated DDI with methotrexate in vivo.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antirreumáticos/farmacologia , Interações Medicamentosas , Metotrexato/farmacocinética , Proteínas de Neoplasias/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Artrite Reumatoide/tratamento farmacológico , Transporte Biológico/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Rim/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metotrexato/uso terapêutico , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Medição de Risco , Sulfassalazina/farmacologia , Xenopus laevisRESUMO
To support drug development and registration, Caco-2 cell monolayer assays have previously been set up and validated to determine whether candidate drugs are substrates or inhibitors of human P-glycoprotein (P-gp). In this study, the drug-drug interaction (DDI) potential of N-(1-{(3R)-3-(3,5-difluorophenyl)-3-[4-methanesulfonylphenyl]propyl}piperidin-4-yl)-N-ethyl-2-[4-methanesulfonylphenyl]acetamide (AZD5672) was assessed accordingly, and a subsequent clinical digoxin interaction study was performed. AZD5672 (1-500 µM) demonstrated concentration-dependent efflux across cell monolayers, which was abolished in the presence of ketoconazole and quinidine, identifying AZD5672 as a P-gp substrate. In addition, P-gp-mediated digoxin transport was inhibited in a concentration-dependent manner by AZD5672 (IC(50) = 32 µM). Assessment of the calculated theoretical gastrointestinal inhibitor concentration ([I(2)]) and predicted steady-state maximum total plasma inhibitor concentration ([I(1)]) indicated the potential for a DDI at the intestinal but not the systemic level after the predicted therapeutic dose of AZD5672 (100 mg). A clinical study was performed and the plasma pharmacokinetics [observed maximum plasma drug concentration (C(max)) and area under the plasma concentration versus time curve from 0 to 72 h postdose (AUC(0-72 h))] of orally dosed digoxin (0.5 mg) were found to be unaffected by coadministration of AZD5672 (50 mg) at steady state. In contrast, a 150-mg dose of AZD5672 significantly increased digoxin C(max) and AUC(0-72 h) by 1.82- and 1.33-fold, respectively. Concentration-time profile comparisons indicated that digoxin elimination was unchanged by AZD5672, and the interaction was most likely to have resulted from inhibition of intestinal P-gp leading to increased digoxin absorption. The observed dose-dependent clinically significant interaction was accurately predicted using calculated [I(2)] and in vitro P-gp inhibition data, confirming AZD5672 to be a P-gp inhibitor in vivo.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Benzenoacetamidas/farmacocinética , Interações Medicamentosas , Modelos Biológicos , Sulfonamidas/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Adolescente , Adulto , Benzenoacetamidas/administração & dosagem , Benzenoacetamidas/sangue , Transporte Biológico , Células CACO-2 , Digoxina/administração & dosagem , Digoxina/sangue , Digoxina/farmacologia , Desenho de Fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Especificidade por Substrato , Sulfonamidas/administração & dosagem , Sulfonamidas/sangue , Adulto JovemRESUMO
The development of a novel method for absolute quantification of proteins based on isotope-coded affinity tagging using ICAT reagents is described. The method exploits synthetic peptide standards to determine protein content at the femtomole level in biological samples. The approach is generally applicable to any subset of proteins, but is particularly appropriate for quantitative analysis of multiple, closely related isoforms, and for hydrophobic proteins that are poorly represented in 2-D gels. Relative and absolute quantification techniques are applied to an important group of microsomal metabolic enzymes, the cytochromes P450 (P450), which are critical in determining the disposition, safety and efficacy of drugs in man. Measurement of the P450 induction profile in response to chemicals is a fundamental aspect of drug safety evaluation and is currently achieved by low-throughput methods employing poorly discriminatory antibodies or substrates. Tagging technology is shown to supersede conventional methods for P450 profiling in terms of discriminatory power and throughput, exemplified by the simultaneous detection of distinct induction profiles for cyp2c subfamily members in response to phenobarbitone: cyp2c29 expression, but not cyp2c40 or cyp2c50, was induced threefold by treatment. This technology should abbreviate the drug development pathway, and provide a widely applicable, rapid means of quantifying proteins.
Assuntos
Marcadores de Afinidade , Sistema Enzimático do Citocromo P-450/análise , Perfilação da Expressão Gênica , Marcação por Isótopo , Sequência de Aminoácidos , Animais , Western Blotting , Sequência Conservada , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Sistema Enzimático do Citocromo P-450/metabolismo , Eletroforese em Gel Bidimensional , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Dados de Sequência Molecular , Fenobarbital/farmacologia , Homologia de Sequência de AminoácidosRESUMO
The transcription factor NF-E2-related factor 2 (Nrf2) plays an essential role in the mammalian response to chemical and oxidative stress through induction of hepatic phase II detoxification enzymes and regulation of glutathione (GSH). Enhanced liver damage in Nrf2-deficient mice treated with acetaminophen suggests a critical role for Nrf2; however, direct evidence for Nrf2 activation following acetaminophen exposure was previously lacking. We show that acetaminophen can initiate nuclear translocation of Nrf2 in vivo, with maximum levels reached after 1 hour, in a dose dependent manner, at doses below those causing overt liver damage. Furthermore, Nrf2 was shown to be functionally active, as assessed by the induction of epoxide hydrolase, heme oxygenase-1, and glutamate cysteine ligase gene expression. Increased nuclear Nrf2 was found to be associated with depletion of hepatic GSH. Activation of Nrf2 is considered to involve dissociation from a cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), through a redox-sensitive mechanism involving either GSH depletion or direct chemical interaction through Michael addition. To investigate acetaminophen-induced Nrf2 activation we compared the actions of 2 other GSH depleters, diethyl maleate (DEM) and buthionine sulphoximine (BSO), only 1 of which (DEM) can function as a Michael acceptor. For each compound, greater than 60% depletion of GSH was achieved; however, in the case of BSO, this depletion did not cause nuclear translocation of Nrf2. In conclusion, GSH depletion alone is insufficient for Nrf2 activation: a more direct interaction is required, possibly involving chemical modification of Nrf2 or Keap1, which is facilitated by the prior loss of GSH.
Assuntos
Acetaminofen/farmacologia , Analgésicos não Narcóticos/farmacologia , Proteínas de Ligação a DNA/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Transativadores/metabolismo , Animais , Northern Blotting , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Masculino , Maleatos/farmacologia , Camundongos , Camundongos Endogâmicos , Fator 2 Relacionado a NF-E2 , Transativadores/genéticaRESUMO
The transcription factor NF-E2-related factor 2 (Nrf2) plays an essential role in the mammalian response to chemical and oxidative stress through induction of hepatic phase II detoxification enzymes and regulation of glutathione (GSH). Enhanced liver damage in Nrf2-deficient mice treated with acetaminophen suggests a critical role for Nrf2; however, direct evidence for Nrf2 activation following acetaminophen exposure was previously lacking. We show that acetaminophen can initiate nuclear translocation of Nrf2 in vivo, with maximum levels reached after 1 hour, in a dose dependent manner, at doses below those causing overt liver damage. Furthermore, Nrf2 was shown to be functionally active, as assessed by the induction of epoxide hydrolase, heme oxygenase-1, and glutamate cysteine ligase gene expression. Increased nuclear Nrf2 was found to be associated with depletion of hepatic GSH. Activation of Nrf2 is considered to involve dissociation from a cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), through a redox-sensitive mechanism involving either GSH depletion or direct chemical interaction through Michael addition. To investigate acetaminophen-induced Nrf2 activation we compared the actions of 2 other GSH depleters, diethyl maleate (DEM) and buthionine sulphoximine (BSO), only 1 of which (DEM) can function as a Michael acceptor. For each compound, greater than 60% depletion of GSH was achieved; however, in the case of BSO, this depletion did not cause nuclear translocation of Nrf2. In conclusion, GSH depletion alone is insufficient for Nrf2 activation: a more direct interaction is required, possibly involving chemical modification of Nrf2 or Keap1, which is facilitated by the prior loss of GSH.
Assuntos
Camundongos , Acetaminofen , Acetaminofen/farmacologia , Fator 2 Relacionado a NF-E2/farmacologia , Camundongos/fisiologiaRESUMO
Glutathione S-transferase Pi (GSTP) detoxifies electrophiles by catalyzing their conjugation with reduced glutathione. A second function of this protein in cell defense has recently been proposed that is related to its ability to interact with c-Jun N-terminal kinase (JNK). The present study aimed to determine whether this interaction results in increased constitutive JNK activity in the absence of GSTP in GstP1/P2(-/-) mice and whether such a phenomenon leads to the up-regulation of genes that are relevant to cell defense. We found a significant increase in constitutive JNK activity in the liver and lung of GstP1/P2-/- compared with GstP1/P2(+/+) mice. The greatest increase in constitutive JNK activity was observed in null liver and was accompanied by a significant increase in activator protein-1 DNA binding activity (8-fold) and in the mRNA levels for the antioxidant protein heme oxygenase-1 compared with wild type. Furthermore UDP-glucuronosyltransferase 1A6 mRNA levels were significantly higher in the livers of GstP1/P2(-/-) compared with GstP1/P2(+/+) mice, which correlated to a 2-fold increase in constitutive activity both in vitro and in vivo. There was no difference in the gene expression of other UDP-glucuronosyltransferase isoforms, manganese superoxide dismutase, microsomal epoxide hydrolase, or GSTA1 between GstP1/P2(-/-) and GstP1/P2(+/+) mice. Additionally there was no phenotypic difference in the induction of heme oxygenase-1 mRNA after acetaminophen administration. This study not only demonstrates the role of GSTP as a direct inhibitor of JNK in vivo but also its role in regulating the constitutive expression of specific downstream molecular targets of the JNK signaling pathway.
Assuntos
Regulação da Expressão Gênica , Glutationa Transferase/deficiência , Isoenzimas/deficiência , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Glucuronosiltransferase/genética , Glutationa S-Transferase pi , Glutationa Transferase/genética , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1 , Isoenzimas/genética , Proteínas Quinases JNK Ativadas por Mitógeno , Fígado/enzimologia , Pulmão/enzimologia , Proteínas de Membrana , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Superóxido Dismutase/genética , Fator de Transcrição AP-1/genéticaRESUMO
GST pi (GSTP) is a member of the glutathione S-transferase (EC 2.5.1.18; GST) family of enzymes that catalyse the conjugation of electrophilic species with reduced glutathione and thus play an important role in the detoxification of electrophilic metabolites. Deletion of GSTP in mice has previously been shown to lead to enhanced susceptibility to chemical-induced skin carcinoma, consistent with its known metabolic functions. A decreased susceptibility to paracetamol hepatotoxicity has also been observed, which has not been fully explained. One possibility is that deletion of the GSTP gene locus results in compensatory changes in other proteins involved in defence against chemical stress. We have therefore used complementary protein expression profiling techniques to perform a systematic comparison of the protein expression profiles of livers from GSTP null and wild-type mice. Analysis of liver proteins by two-dimensional electrophoresis confirmed the absence of GSTP in null mice whereas GSTP represented 3-5% of soluble protein in livers from wild-type animals. There was a high degree of quantitative and qualitative similarity in other liver proteins between GSTP null and wild-type mice. There was no evidence that the absence of GSTP in null animals resulted in enhanced expression of other GST isoforms in the null mice (GST alpha, 1.48%, GST mu, 1.68% of resolved proteins) compared with the wild-type animals (GST alpha, 1.50%, GST mu, 1.40%). In contrast, some members of the thiol specific antioxidant family of proteins, notably antioxidant protein 2 and thioredoxin peroxidases, were expressed at a higher level in the GSTP null mouse livers. These changes presumably reflect the recently described role of GSTP in cell signalling and may underlie the protection against paracetamol toxicity seen in these animals.