RESUMO
Agonism of GPR119 is viewed as a potential therapeutic approach for the treatment of type II diabetes and other elements of metabolic syndrome. During progression of a previously disclosed candidate 1 through mice toxicity studies, we observed tonic-clonic convulsions in several mice at high doses. An in vitro hippocampal brain slice assay was used to assess the seizure liability of subsequent compounds, leading to the identification of an aryl sulfone as a replacement for the 3-cyano pyridyl group. Subsequent optimization to improve the overall profile, specifically with regard to hERG activity, led to alkyl sulfone 16. This compound did not cause tonic-clonic convulsions in mice, had a good pharmacokinetic profile, and displayed in vivo efficacy in murine models. Importantly, it was shown to be effective in wild-type (WT) but not GPR119 knockout (KO) animals, consistent with the pharmacology observed being due to agonism of GPR119.
Assuntos
Epilepsia Tônico-Clônica/prevenção & controle , Oxidiazóis/farmacocinética , Pirimidinas/farmacocinética , Receptores Acoplados a Proteínas G/agonistas , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cães , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Feminino , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/uso terapêutico , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxidiazóis/química , Oxidiazóis/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
A series of conformationally restricted GPR119 agonists were prepared based around a 3,8-diazabicyclo[3.2.1]octane scaffold. Examples were found to have markedly different pharmacology in mouse and human despite similar levels of binding to the receptor. This highlights the large effects on GPCR phamacology that can result from small structural changes in the ligand, together with inter-species differences between receptors.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Heterocíclicos com 2 Anéis/química , Pirimidinas/química , Receptores Acoplados a Proteínas G/agonistas , Animais , Disponibilidade Biológica , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Permeabilidade da Membrana Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Cães , Meia-Vida , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/farmacocinética , Humanos , Células Madin Darby de Rim Canino , Camundongos , Ligação Proteica , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-AtividadeRESUMO
G protein coupled receptor 119 (GPR119) is viewed as an attractive target for the treatment of type 2 diabetes and other elements of the metabolic syndrome. During a program toward discovering agonists of GPR119, we herein describe optimization of an initial lead compound, 2, into a development candidate, 42. A key challenge in this program of work was the insolubility of the lead compound. Small-molecule crystallography was utilized to understand the intermolecular interactions in the solid state and resulted in a switch from an aryl sulphone to a 3-cyanopyridyl motif. The compound was shown to be effective in wild-type but not knockout animals, confirming that the biological effects were due to GPR119 agonism.
Assuntos
Oxidiazóis/síntese química , Piridinas/síntese química , Receptores Acoplados a Proteínas G/agonistas , Animais , Disponibilidade Biológica , Carbamatos/síntese química , Carbamatos/química , Carbamatos/farmacologia , Cristalografia por Raios X , Cães , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Oxidiazóis/química , Oxidiazóis/farmacologia , Piperidinas/síntese química , Piperidinas/química , Piperidinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequenas , Solubilidade , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonas/síntese química , Sulfonas/química , Sulfonas/farmacologiaRESUMO
GPR119 is increasingly seen as an attractive target for the treatment of type II diabetes and other elements of the metabolic syndrome. During a programme aimed at developing agonists of the GPR119 receptor, we identified compounds that were potent with reduced hERG liabilities, that had good pharmacokinetic properties and that displayed excellent glucose-lowering effects in vivo. However, further profiling in a GPR119 knock-out (KO) mouse model revealed that the biological effects were not exclusively due to GPR119 agonism, highlighting the value of transgenic animals in drug discovery programs.
Assuntos
Hipoglicemiantes/química , Receptores Acoplados a Proteínas G/agonistas , Administração Oral , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-AtividadeRESUMO
Accurate assignment of the concentration of victim drug/inhibitor available at the enzyme active site, both in vivo and within an in vitro incubation, is an essential requirement in rationalizing and predicting drug-drug interactions. Inhibitor accumulation within the liver, whether as a result of active transport processes or intracellular binding, may best be accounted for using hepatocytes rather than hepatic microsomes to estimate in vitro inhibitory potency. The aims of this study were to compare K(i) values determined in rat liver microsomes and freshly isolated rat hepatocytes of four cytochrome P450 (P450) inhibitors (clarithromycin, enoxacin, nelfinavir, and saquinavir) with known hepatic transporter involvement and a range of uptake (cell/medium concentration ratios 20-3000) and clearance (10-1200 µl/min/10(6) cells) properties. Inhibition studies were performed using two well established P450 probe substrates (theophylline and midazolam). Comparison of unbound K(i) values showed marked differences between the two in vitro systems for inhibition of metabolism. In two cases (clarithromycin and enoxacin, both low-clearance drugs), inhibitory potency in hepatocytes markedly exceeded that in microsomes (10- to 20-fold), and this result was consistent with their high cell/medium concentration ratios. For nelfinavir and saquinavir (high-clearance, extensively metabolized drugs), the opposite trend was seen in the K(i) values: despite very high cell/medium concentration ratios, stronger inhibition was evident within microsomal preparations. Hence, the consequences of hepatic accumulation resulting from uptake transporters vary according to the clearance of the inhibitor. This study demonstrates that transporter-enzyme interplay can result in differences in inhibitory potency between microsomes and hepatocytes and hence drug-drug interaction predictions that are not always intuitive.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Hepatócitos/enzimologia , Microssomos Hepáticos/enzimologia , Animais , Separação Celular , Claritromicina/farmacologia , Interações Medicamentosas , Enoxacino/farmacologia , Masculino , Midazolam/metabolismo , Nelfinavir/farmacologia , Ratos , Ratos Sprague-Dawley , Saquinavir/farmacologia , Teofilina/metabolismoRESUMO
BACKGROUND: Approximately 50% of all patients with primary central nervous system lymphoma (PCNSL) are aged ≥65 years; however, this group is relatively understudied, and to the authors's knowledge, optimal treatment for older patients is not well defined. METHODS: This was a retrospective review of PCNSL patients aged ≥65 years who were treated at Memorial Sloan-Kettering Cancer Center between 1986 and 2008. A multivariate analysis of demographic and clinical variables on prognosis and receipt of treatment was performed. RESULTS: One hundred seventy-four patients between the ages of 65 and 89 years were identified; there was a slight predominance of women (52.9%). One hundred forty-eight patients were treated with chemotherapy at the time of diagnosis (98% with methotrexate-based therapy) and 31 of these patients also received whole-brain radiotherapy (WBRT). Sixteen patients received WBRT alone. A radiographic response to chemotherapy was noted in 76% of patients. Ninety patients developed disease progression after initial treatment; 74 received salvage therapy and 48% of these patients responded to salvage treatment. The median overall survival was 25 months (range, 18-33 months), and the 3-year survival rate was 36%. Approximately 20.1% of patients were alive for ≥11 years. WBRT was delivered more frequently before 1998, and patients with a history of prior malignancy were less likely to receive WBRT. Age and performance status were identified as the most important predictors of survival. Treatment-related neurotoxicity at 2 years was strongly associated with receipt of WBRT (P=.0002). CONCLUSIONS: PCNSL in the elderly remains sensitive to methotrexate-based chemotherapy and aggressive treatment may be warranted both at the time of diagnosis and disease recurrence.
Assuntos
Neoplasias do Sistema Nervoso Central/terapia , Linfoma não Hodgkin/terapia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/mortalidade , Neoplasias do Sistema Nervoso Central/radioterapia , Terapia Combinada , Intervalo Livre de Doença , Feminino , Humanos , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/mortalidade , Linfoma não Hodgkin/radioterapia , Masculino , Taxa de Sobrevida , Resultado do TratamentoRESUMO
Predicting drug-drug interactions requires an assessment of the drug concentration available to the enzyme active site, both in vivo, and within an in vitro incubation. These predictions are confounded when the inhibitor accumulates within the liver, either as a result of active transport processes or intracellular binding (including lysosomal trapping). In theory, hepatocytes should provide a more accurate estimation of inhibitory potency compared with microsomes for those compounds that undergo hepatic accumulation. However, they are not routinely used for Ki determination and there is limited comparative information available. Therefore, the aims of this study were to compare Ki values determined in rat microsomes and freshly isolated hepatocytes using six cytochrome P450 inhibitors (miconazole, fluconazole, ketoconazole, quinine, fluoxetine, and fluvoxamine) with a range of uptake properties (cell-to-medium concentration ratios 4.2-6000). Inhibition studies were performed using four probe substrates for CYP2C, CYP2D, and CYP3A enzymes (tolbutamide and phenytoin, dextromethorphan and midazolam, respectively). Comparison of unbound Ki values (range 0.05-30 microM) showed good agreement between microsomes and hepatocytes for inhibition of 18 pathways of metabolism. In addition to this, there was no relationship between the cell-to-medium concentration ratios (covering over 3 orders of magnitude) and the microsomal to hepatocyte Ki ratio of these inhibitors. These data suggest that the hepatic accumulation of these inhibitors results from intracellular binding rather than the involvement of uptake transporters and indicate that microsomes and hepatocytes appear to be equivalent for determining the inhibitory potency of the six inhibitors investigated in the present study.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Hepatócitos/enzimologia , Microssomos Hepáticos/enzimologia , Animais , Catálise/efeitos dos fármacos , Células Cultivadas , Citocromo P-450 CYP2D6/metabolismo , Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Dextrometorfano/metabolismo , Dextrometorfano/farmacocinética , Dextrometorfano/farmacologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Fluconazol/metabolismo , Fluconazol/farmacocinética , Fluconazol/farmacologia , Fluoxetina/metabolismo , Fluoxetina/farmacocinética , Fluoxetina/farmacologia , Fluvoxamina/metabolismo , Fluvoxamina/farmacocinética , Fluvoxamina/farmacologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Cetoconazol/metabolismo , Cetoconazol/farmacocinética , Cetoconazol/farmacologia , Cinética , Masculino , Miconazol/metabolismo , Miconazol/farmacocinética , Miconazol/farmacologia , Microssomos Hepáticos/metabolismo , Midazolam/metabolismo , Midazolam/farmacocinética , Midazolam/farmacologia , Fenitoína/metabolismo , Fenitoína/farmacocinética , Fenitoína/farmacologia , Quinina/metabolismo , Quinina/farmacocinética , Quinina/farmacologia , Ratos , Ratos Sprague-Dawley , Tolbutamida/metabolismo , Tolbutamida/farmacocinética , Tolbutamida/farmacologiaRESUMO
This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.
Assuntos
Proteínas de Transporte/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Hepatócitos/citologia , Humanos , Hepatopatias/metabolismo , Taxa de Depuração Metabólica , Modelos Biológicos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/metabolismo , Xenobióticos/efeitos adversos , Xenobióticos/metabolismo , Xenobióticos/farmacocinéticaRESUMO
Human liver microsomes have typically resulted in marked underprediction of in vivo human intrinsic clearance (CL(int)); therefore, the utility of cryopreserved hepatocytes as an alternative in vitro system has become an important issue. In this study, 10 compounds (tolbutamide, diclofenac, S-warfarin, S-mephenytoin, dextromethorphan, bufuralol, quinidine, nifedipine, testosterone, and terfenadine) were selected as substrate probes for CYP2C9, 2C19, 2D6, and 3A4, and the kinetics of metabolite formation (n = 14 pathways) were investigated in three individual lots of cryopreserved hepatocytes and in a pool of human liver microsomes. For the majority of the compounds, lower unbound K(M) or S(50) values were observed in hepatocytes compared with microsomes, on average by 50% over a 200-fold range (0.5-140 microM). Expressed on an equivalent liver weight basis, a good correlation between microsomal and hepatocyte V(max) values was observed for most pathways greater than 5 orders of magnitude (0.16-216 nmol/min/g liver). Unbound hepatocyte CL(int) (CL(int,u)) values, when scaled to the whole liver (range 0.38-4000 ml/min/kg), were on average 2.5-fold higher than microsomal CL(int,u) values, with the exception of tolbutamide and diclofenac, for which lower hepatocellular CL(int,u) values were observed. Hepatocyte predicted CL(int) values were compared with human in vivo CL(int) values, and to supplement our data, in vitro data from cryopreserved hepatocytes were collated from four other published sources. These data show that for 37 drugs, there is, on average, a 4.5-fold under-prediction of the in vivo CL(int) using cryopreserved hepatocytes, representing a significant reduction in prediction bias compared with human microsomes.
Assuntos
Criopreservação , Hepatócitos/metabolismo , Taxa de Depuração Metabólica , Microssomos Hepáticos/metabolismo , Humanos , CinéticaRESUMO
BACKGROUND: Quantitative predictions of in vivo drug-drug interactions (DDIs) resulting from metabolic inhibition are commonly made based upon the inhibitor concentration at the enzyme active site [I] and the in vitro inhibition constant (K(i)). Previous studies have involved the use of various plasma inhibitor concentrations as surrogates for [I] along with K(i) values obtained from published literature. Although this approach has resulted in a high proportion of successful predictions, a number of falsely predicted interactions are also observed. OBJECTIVES: To focus on three issues that may influence the predictive value of the [I]/K(i) ratio approach: (i) the use of unbound K(i) (K(i,u)) values generated from standardised in vitro experiments compared with literature values; (ii) the selection of an appropriate [I]; and (iii) incorporation of the impact of intestinal metabolic inhibition for cytochrome P450 (CYP) 3A4 predictions. To this end we have selected eight inhibitors of CYP2C9, CYP2D6 and CYP3A4 and 18 victim drugs from a previous database analysis to allow prediction of 45 clinical DDI studies. METHODS: In vitro kinetic and inhibition studies were performed in human liver microsomes using prototypic probe substrates of CYP2C9 and CYP2D6, with various inhibitors (miconazole, sulfaphenazole, fluconazole, ketoconazole, quinidine, fluoxetine, fluvoxamine). The K(i) estimates obtained were corrected for non-specific microsomal binding, and the K(i,u) was incorporated into in vivo predictions using various [I] values. Predictions for CYP3A4 were based upon in vitro data obtained from a previous publication within our laboratory, and an assessment of the impact of the interaction in the gut wall is included. Predictions were validated against 45 in vivo studies and those within 2-fold of the in vivo ratio of area under the plasma concentration-time curve of the substrate, in the presence and absence of the inhibitor (AUC(i)/AUC) were considered successful. RESULTS: Predictions based upon the average systemic total plasma drug concentration ([I](av)) [incorporating the effects of parallel drug elimination pathways] and the K(i,u) value resulted in 91% of studies predicted to within 2-fold of the in vivo AUC(i)/AUC. This represents a 35% improvement in prediction accuracy compared with predictions based upon total K(i) values obtained from various published literature sources. A corresponding reduction in bias and an increase in precision were also observed compared with the use of other [I] surrogates (e.g. the total and new unbound maximum hepatic input plasma concentrations). No significant improvement in prediction accuracy was observed by incorporating consideration of gut wall inhibition for CYP3A4. CONCLUSION: DDI predictions based upon the use of K(i,u) data obtained under a set of optimal standardised conditions were significantly improved compared with predictions using in vitro data collated from various sources. The use of [I](av) as the [I] surrogate generated the most successful predictions as judged by several criteria. Incorporation of either plasma protein binding of inhibitor or gut wall CYP3A4 inhibition did not result in a general improvement of DDI predictions.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Algoritmos , Área Sob a Curva , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Citocromo P-450 CYP2C9 , Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Bases de Dados Factuais , Inibidores Enzimáticos/farmacologia , Previsões , Humanos , Técnicas In Vitro , Cinética , Microssomos/metabolismoRESUMO
AIMS: Success of the quantitative prediction of drug-drug interactions via inhibition of CYP-mediated metabolism from the inhibitor concentration at the enzyme active site ([I]) and the in vitro inhibition constant (K(i)) is variable. The aim of this study was to examine the impact of the fraction of victim drug metabolized by a particular CYP (f(mCYP)) and the inhibitor absorption rate constant (k(a)) on prediction accuracy. METHODS: Drug-drug interaction studies involving inhibition of CYP2C9, CYP2D6 and CYP3A4 (n = 115) were investigated. Data on f(mCYP) for the probe substrates of each enzyme and k(a) values for the inhibitors were incorporated into in vivo predictions, alone or in combination, using either the maximum hepatic input or the average systemic plasma concentration as a surrogate for [I]. The success of prediction (AUC ratio predicted within twofold of in vivo value) was compared using nominal values of f(mCYP) = 1 and k(a) = 0.1 min(-1). RESULTS: The incorporation of f(mCYP) values into in vivo predictions using the hepatic input plasma concentration resulted in 84% of studies within twofold of in vivo value. The effect of k(a) values alone significantly reduced the number of over-predictions for CYP2D6 and CYP3A4; however, less precision was observed compared with the f(mCYP). The incorporation of both f(mCYP) and k(a) values resulted in 81% of studies within twofold of in vivo value. CONCLUSIONS: The incorporation of substrate and inhibitor-related information, namely f(mCYP) and k(a), markedly improved prediction of 115 interaction studies with CYP2C9, CYP2D6 and CYP3A4 in comparison with [I]/K(i) ratio alone.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/metabolismo , Área Sob a Curva , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Citocromo P-450 CYP2C9 , Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Interações Medicamentosas , Humanos , Valor Preditivo dos TestesRESUMO
AIMS: In theory, the magnitude of an in vivo drug-drug interaction arising from the inhibition of metabolic clearance can be predicted using the ratio of inhibitor concentration ([I]) to inhibition constant (K(i)). The aim of this study was to construct a database for the prediction of drug-drug interactions from in vitro data and to evaluate the use of the various estimates for the inhibitor concentrations in the term [I]/K(i). METHODS: One hundred and ninety-three in vivo drug-drug interaction studies involving inhibition of CYP3A4, CYP2D6 or CYP2C9 were collated from the literature together with in vitro K(i) values and pharmacokinetic parameters for inhibitors, to allow calculation of average/maximum systemic plasma concentration during the dosing interval and maximum hepatic input plasma concentration (both total and unbound concentration). The observed increase in AUC (decreased clearance) was plotted against the estimated [I]/K(i) ratio for qualitative zoning of the predictions. RESULTS: The incidence of false negative predictions (AUC ratio > 2, [I]/K(i) < 1) was largest using the average unbound plasma concentration and smallest using the hepatic input total plasma concentration of inhibitor for each of the CYP enzymes. Excluding mechanism-based inhibition, the use of total hepatic input concentration resulted in essentially no false negative predictions, though several false positive predictions (AUC ratio < 2, [I]/K(i) > 1) were found. The incidence of true positive predictions (AUC ratio > 2, [I]/K(i) > 1) was also highest using the total hepatic input concentration. CONCLUSIONS: The use of the total hepatic input concentration of inhibitor together with in vitro K(i) values was the most successful method for the categorization of putative CYP inhibitors and for identifying negative drug-drug interactions. However this approach should be considered as an initial discriminating screen, as it is empirical and requires subsequent mechanistic studies to provide a comprehensive evaluation of a positive result.