RESUMO
Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83%, for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97%, for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9. 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug-drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential.
Assuntos
Criopreservação , Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas/fisiologia , Fígado , Preservação de Órgãos , Testes de Toxicidade/métodos , Adulto , Idoso , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cães , Relação Dose-Resposta a Droga , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/fisiologia , Formazans/metabolismo , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Coelhos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Sais de Tetrazólio/metabolismoRESUMO
Cytochrome P450 (CYP) inhibition and induction are the key mechanisms in drug-drug interactions. Aside from clinical studies, primary human hepatocytes may represent the most appropriate experimental system for the evaluation of CYP induction in humans. A consensus of an international panel on the present status and future research directions in the application of primary human hepatocytes in the evaluation of CYP-induction is presented here. The following observations are concluded to be generally true: (1) Human hepatocytes isolated from both biopsy samples and transplantable livers are suitable for induction studies. (2) Hormonally-defined media can be used for the evaluation of CYP induction. (3) Isozyme-selective induction of CYP1A and 3A by known inducers are observed. (4) Reproducibility of induction could be improved by using hepatocytes plated as confluent cultures. (5) Induction could be observed for hepatocytes treated at 1-3 days after culturing. (6) Treatment duration of 2 days in general leads to near maximal induction. (7) In general, there is a good qualitative correlation between human hepatocyte results in vitro and clinical observations in vivo. (8) When the same inducers were evaluated in independent laboratories, similar data were generally observed. We conclude that primary human hepatocytes represent an appropriate model for mechanistic evaluation of CYP induction and as a screening tool for CYP induction potential of xenobiotics. A set of data acceptance criteria are proposed: (1) Positive response should be observed with concurrent positive control chemicals; (2) reproducible observation should be observed with multiple human donors; (3) for negative response, the doses used should not be cytotoxic; and (4) replicate treatment and/or multiple dose treatment should be performed to allow statistical analysis. Future studies should include the further development of on: (1) The inducibility of CYP isozymes other than CYP1A and 3A, and phase II enzymes; (2) further development of culturing condition to allow optimal gene expression; (3) evaluation of the involvement of nonparenchymal cells on CYP induction of parenchymal cells; (4) the and validation of quantitative approaches to extrapolate in vitro data to in vivo data; (5) evaluation of possible individual variations and potential genetic polymorphism in inducibility; (6) further definition of species differences in CYP induction; (7) development of a 'normal' human hepatocyte cell line for CYP induction studies; (8) improvement of cryopreservation procedure of human hepatocytes; (9) definition of the molecular mechanisms of CYP induction; and (10) evaluation of the induction of phase II metabolic pathways.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Isoenzimas/biossíntese , Fígado/citologia , Fígado/enzimologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Indução Enzimática , HumanosRESUMO
In our laboratory, primary human hepatocytes are being investigated as an in vitro experimental system for the evaluation of pharmacokinetic drug-drug interactions. Our study here represents the first reported study that directly compares the cytochrome P450 isozyme 3A (CYP3A) induction potential of three antimicrobials derived from rifamycin B, namely, rifampin, rifapentine and rifabutin. Two endpoints of CYP3A activity, testosterone 6 beta-hydroxylation and midazolam 1-hydroxylation have been used. Results obtained with hepatocytes from four different human donors show consistently that rifampin and rifapentine are potent inducers of CYP3A, while a significantly lower induction potential is observed for rifabutin. The relative induction potency of the three antimicrobials (rifampin > rifapentine >> rifabutin) is consistent with the available human in vivo data. For CYP1A measured as ethoxyresorufin O-deethylase activity, CYP2C8/9 measured as tolbutamide 4-hydroxylation activity, CYP2D6 measured as dextromethorphan O-demethylation, and AZT glucuronidation, there is either no effect or, where induction is found to be statistically significant in these other endpoints, the maximum induction values are consistently < 100% of the control. Our results suggest that CYP3A is the major CYP induced by these rifamycin B derivatives. These studies illustrate the application of human hepatocytes in the evaluation of the structure-activity relationships in CYP induction for this class of chemicals and as an in vitro screen for drug-drug interaction potential via CYP induction.
Assuntos
Antibióticos Antituberculose/farmacologia , Antituberculosos/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Isoenzimas/biossíntese , Isoenzimas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Rifabutina/farmacologia , Rifampina/análogos & derivados , Rifampina/farmacologia , Antibióticos Antituberculose/metabolismo , Antituberculosos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Indução Enzimática/efeitos dos fármacos , Glucuronatos/metabolismo , Humanos , Hidroxilação , Isoenzimas/metabolismo , Fígado/citologia , Rifabutina/metabolismo , Rifampina/metabolismo , Relação Estrutura-Atividade , Testosterona/metabolismo , Fatores de Tempo , Zidovudina/metabolismoRESUMO
Hepatic enzyme induction is often evoked as the cause for a variety of effects in animal studies, e.g. hepatic hypertrophy and secondary thyroid neoplasms in rodents. In clinical practice, enzyme induction often enhances drug clearance and may result in reduced efficacy. For example, carbamazepine or rifampin treatment induces P450 3A in humans, and as a result, dramatically reduces the efficacy of midazolam or cyclosporine. Due to species differences in substrate specificity and the regulation of various drug-metabolizing enzymes, assessing enzyme induction in human tissues is a desirable goal. Since induction often occurs as a result of increased synthesis of mRNA coding for a particular enzyme, induction may be quantified by measuring specific mRNA levels. We describe an approach to quantifying mRNA levels using reverse transcriptase-polymerase chain reaction (RT-PCR). This approach makes use of either radiolabeled PCR primers or fluorimetric quantification of product and does not require the synthesis of a competitor RNA or DNA molecule. Thus, Cyp2B1/2 mRNA can be shown to be induced 17-fold in the H4IIE rat hepatoma cell line. Likewise, Cyp3A and Cyp2A6 mRNAs can be shown to be induced in primary human hepatocytes cultured on collagen-coated plates and treated with rifampin for 72 h. By contrast, mRNA levels for Cyp1A1 and Cyp2E1 were not increased by rifampin treatment. This report demonstrates the potential of this approach for examining a number of mRNAs from drug-treated cultured cells, as a means of assessing metabolic enzyme induction.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , DNA Complementar/metabolismo , Isoenzimas/biossíntese , Fígado/enzimologia , Reação em Cadeia da Polimerase/métodos , Adulto , Animais , Antibióticos Antituberculose/farmacologia , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Indução Enzimática , Feminino , Humanos , Isoenzimas/genética , Fígado/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Rifampina/farmacologia , Sensibilidade e Especificidade , Transcrição GênicaRESUMO
An in vitro technique for the determination of radiopharmaceutical metabolism has been developed using isolated hepatocytes. Radioiodinated N-isopropyl-p-iodoamphetamine (IMP; iofetamine, USP) was employed a model tracer in these studies because its labeled metabolites are well-characterized. Hepatocytes isolated from the rat and human produced labeled metabolites in vitro for up to 4 h in a manner similar to that reported for humans in vivo. Identical metabolites were generated by all cell types investigated, but the rate of metabolism differed (rat >> human; female > male; fresh > frozen). The utility of this methodology for the preclinical evaluation of potential radiopharmaceuticals is described.
Assuntos
Anfetaminas/metabolismo , Radioisótopos do Iodo/metabolismo , Fígado/metabolismo , Adulto , Animais , Criopreservação , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Iofetamina , Fígado/citologia , Masculino , Ratos , Ratos Endogâmicos F344 , Fatores Sexuais , SolventesRESUMO
The Salmonella mutagenicity assay was used to compare the mutagenic activity of used crankcase oil (UCO) from diesel and spark-ignition (gasoline) engine passenger cars. UCO samples were obtained during periodic oil changes from 9 spark-ignition and 10 diesel-powered vehicles. Five samples of unused motor oil were also tested. Direct tests of UCO did not detect mutagenic activity in Salmonella typhimurium strain TA-98. Therefore, an extraction procedure was used to concentrate the mutagens and remove interfering chemicals. Extracts were tested both with and without Aroclor-1254-induced rat liver homogenate fraction (S-9). Dose-dependent mutagenicity with and without S-9 was observed in both diesel and spark-ignition engine UCO extracts. Mutagenic activity was also found in unused oil extracts, but it was lower than that in UCO extracts and generally required addition of S-9. The mutagenic potency of diesel UCO extracts was similar to that of gasoline UCO extracts, both with and without addition of S-9. This indicated that potential health risks associated with disposal, handling, and recycling of diesel UCO may not be significantly different from those of UCO from gasoline engines.