RESUMO
Pharmacokinetic (PK) variability in cancer clinical trials may be due to heterogeneous populations and identifying sources of variability is important. Use of healthy subjects in clinical pharmacology studies together with detailed knowledge of the characteristics of patients with cancer can allow for quick identification and quantification of factors affecting PK variability. PK data and sources of variability of 40 marketed molecularly targeted oncology therapeutics were compiled from regulatory approval documents covering an 18-year period (1999-2017). Variability in PK parameters was compared and contributors to variability were identified. The results show that PK variability was ~ 16% higher for peak plasma concentration (Cmax ) and area under the concentration time curve (AUC) in patients with cancer compared with healthy subjects. Several factors were identified as major contributors to variability including hepatic/renal impairment and cytochrome P450 inhibition/induction. Lower PK variability in healthy subjects may represent an opportunity to perform rapid and robust pharmacological and PK assessments to inform subsequent studies in the development of new cancer therapies.
Assuntos
Antineoplásicos/farmacocinética , Neoplasias/tratamento farmacológico , Administração Oral , Adulto , Fatores Etários , Antineoplásicos/administração & dosagem , Área Sob a Curva , Variação Biológica da População , Índice de Massa Corporal , Aprovação de Drogas , Europa (Continente) , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Neoplasias/sangue , Estados Unidos , United States Food and Drug AdministrationAssuntos
Pesquisa Biomédica/organização & administração , Prática Clínica Baseada em Evidências/organização & administração , Sistemas de Informação , Projetos de Pesquisa , Biomarcadores , Pesquisa Biomédica/normas , Coleta de Dados/métodos , Tomada de Decisões , Indústria Farmacêutica/organização & administração , Registros Eletrônicos de Saúde/organização & administração , Prática Clínica Baseada em Evidências/normas , Humanos , Dispositivos Eletrônicos VestíveisRESUMO
Many approved drugs demonstrate different pharmacokinetics, pharmacodynamics, and/or safety across racial and ethnic groups. The primary objective of the current study was to summarize the racial and ethnic makeup of cancer clinical drug trials using cancer drugs approved by the U.S. Food and Drug Administration (FDA) between January 1, 2010, and July 31, 2016. In clinical studies used for FDA approvals, 82.3% of participants identified as white, 10.2% as Asian, 2.3% as black, and 4.7% as Hispanic. Black participants made up 7.7% of U.S. and Canadian cancer clinical drug trials and 2.6% of global cancer clinical drug trials while Asian participants made up 13.5% of global cancer clinical drug trials but only 1.8% of U.S. and Canadian cancer clinical drug trials. The current study indicates that although cancer clinical drug trials have become more inclusive of Asian participants, other racial and ethnic minority groups remain under-represented. This may result in an inadequate understanding of drug safety and efficacy in many racial and ethnic populations.
Assuntos
Ensaios Clínicos como Assunto , Neoplasias/epidemiologia , Humanos , Neoplasias/tratamento farmacológicoRESUMO
The aim of personalized medicine is to offer the right treatment to the right person at the right dose, thus maximizing efficacy and minimizing toxicity for each individual patient. Pharmacogenomic approaches attempt to refine the aim of personalized medicine by utilizing an individual's germline and somatic DNA signatures to guide treatment. In this review, we highlight the current use of pharmacogenomic based biomarker information in drug labeling. We also present several case studies on the implementation of pharmacogenomic strategies in drug discovery and development. Lastly, we comment on current challenges to implementing pharmacogenomic based testing in the clinic.
Assuntos
Farmacogenética , Medicina de Precisão , Descoberta de Drogas , Rotulagem de Medicamentos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Cytochrome P450 (CYP) 26A1 and 26B1 are heme-containing enzymes responsible for metabolizing all-trans retinoic acid (at-RA). No crystal structures have been solved, and therefore homology models that provide structural information are extremely valuable for the development of inhibitors of cytochrome P450 family 26 (CYP26). The objectives of this study were to use homology models of CYP26A1 and CYP26B1 to characterize substrate binding characteristics, to compare structural aspects of their active sites, and to support the role of CYP26 in the metabolism of xenobiotics. Each model was verified by dockingat-RA in the active site and comparing the results to known metabolic profiles ofat-RA. The models were then used to predict the metabolic sites of tazarotenic acid with results verified by in vitro metabolite identification experiments. The CYP26A1 and CYP26B1 homology models predicted that the benzothiopyranyl moiety of tazarotenic acid would be oriented toward the heme of each enzyme and suggested that tazarotenic acid would be a substrate of CYP26A1 and CYP26B1. Metabolite identification experiments indicated that CYP26A1 and CYP26B1 oxidatively metabolized tazarotenic acid on the predicted moiety, with in vitro rates of metabolite formation by CYP26A1 and CYP26B1 being the highest across a panel of enzymes. Molecular analysis of the active sites estimated the active-site volumes of CYP26A1 and CYP26B1 to be 918 Å(3)and 977 Å(3), respectively. Overall, the homology models presented herein describe the enzyme characteristics leading to the metabolism of tazarotenic acid by CYP26A1 and CYP26B1 and support a potential role for the CYP26 enzymes in the metabolism of xenobiotics.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Nicotínicos/metabolismo , Xenobióticos/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/química , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Preparações Farmacêuticas/metabolismo , Receptores do Ácido Retinoico/agonistas , Ácido Retinoico 4 Hidroxilase , Especificidade por Substrato , Tretinoína/metabolismoRESUMO
PURPOSE: In this work, we assessed the ability of fluorophotometry to measure the vitreal pharmacokinetics (PK) of fluorescently-labeled ranibizumab in the rabbit after intravitreal injection. We compared these values to those obtained using enzyme-linked immunosorbent assays (ELISA). Data obtained in this study were also compared to historical ranibizumab ocular PK data, either measured in-house or previously published. METHODS: Three individual in vivo studies were performed in New Zealand White rabbits to assess the feasibility of using fluorophotometry to measure rabbit ocular PK of ranibizumab; explore the dynamic range of dosing fluorescently-labeled ranibizumab; and directly compare ranibizumab concentrations and calculated PK parameters measured by vitreal fluorophotometry to those measured using ELISA. RESULTS: In direct comparisons between fluorophotometry and ELISA, the calculated clearance (CL) values were 0.26 and 0.21 mL/day, the volumes of distribution at steady state (Vss) were 0.80 and 0.94 mL, the half-lives (t1/2) were 3.1 and 2.9 days and the dose normalized areas under the curve (AUC/D) were 4.7 and 3.9 µg·day/mL/µg, respectively. These values fell within the ranges of 0.13 to 0.44 mL/day for CL, 0.5 to 1.8 mL for Vss, 2.8 to 3.5 days for t1/2, and 2.3 to 7.9 µg·day/mL/µg for AUC/D that have been either measured previously in-house or published elsewhere. CONCLUSIONS: Although not suitable for measuring retinal concentrations, fluorophotometry is a valuable, noninvasive method to measure vitreous concentrations of protein therapeutics after intravitreal injection.
Assuntos
Fluorofotometria , Fatores Imunológicos/farmacocinética , Ranibizumab/farmacocinética , Corpo Vítreo/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Meia-Vida , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/análise , Injeções Intravítreas , Masculino , Coelhos , Ranibizumab/administração & dosagem , Ranibizumab/análise , Corpo Vítreo/químicaRESUMO
Iron is a trace element important for the proper folding and function of various proteins. Physiological regulation of iron stores is of critical importance for RBC production and antimicrobial defense. Hepcidin is a key regulator of iron levels within the body. Under conditions of iron deficiency, hepcidin expression is reduced to promote increased iron uptake from the diet and release from cells, whereas during conditions of iron excess, induction of hepcidin restricts iron uptake and movement within the body. The cytokine IL-6 is well established as an important inducer of hepcidin. The presence of this cytokine during inflammatory states can induce hepcidin production, iron deficiency, and anemia. In this study, we show that IL-22 also influences hepcidin production in vivo. Injection of mice with exogenous mouse IgG1 Fc fused to the N terminus of mouse IL-22 (Fc-IL-22), an IL-22R agonist with prolonged and enhanced functional potency, induced hepcidin production, with a subsequent decrease in circulating serum iron and hemoglobin levels and a concomitant increase in iron accumulation within the spleen. This response was independent of IL-6 and was attenuated in the absence of the IL-22R-associated signaling kinase, Tyk2. Ab-mediated blockade of hepcidin partially reversed the effects on iron biology caused by IL-22R stimulation. Taken together, these data suggest that exogenous IL-22 regulates hepcidin production to physiologically influence iron usage.
Assuntos
Hepcidinas/fisiologia , Interleucinas/fisiologia , Ferro/metabolismo , Sequência de Aminoácidos , Anemia Ferropriva/sangue , Anemia Ferropriva/induzido quimicamente , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Hepcidinas/antagonistas & inibidores , Hepcidinas/biossíntese , Hepcidinas/genética , Hepcidinas/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Interleucina-6/fisiologia , Interleucinas/genética , Interleucinas/farmacologia , Interleucinas/toxicidade , Ferro/sangue , Deficiências de Ferro , Síndrome de Job/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de IgG/deficiência , Receptores de Interleucina/agonistas , Receptores de Interleucina/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Baço/metabolismo , Baço/patologia , TYK2 Quinase/deficiência , TYK2 Quinase/metabolismo , Interleucina 22RESUMO
Drug-drug interactions (DDIs) between therapeutic proteins (TPs) and small-molecule drugs have recently drawn the attention of regulatory agencies, the pharmaceutical industry, and academia. TP-DDIs are mainly caused by proinflammatory cytokine or cytokine modulator-mediated effects on the expression of cytochrome P450 enzymes. To build consensus among industry and regulatory agencies on expectations and challenges in this area, a working group was initiated to review the preclinical state of the art. This white paper represents the observations and recommendations of the working group on the value of in vitro human hepatocyte studies for the prediction of clinical TP-DDI. The white paper was developed following a "Workshop on Recent Advances in the Investigation of Therapeutic Protein Drug-Drug Interactions: Preclinical and Clinical Approaches" held at the Food and Drug Administration White Oak Conference Center on June 4 and 5, 2012. Results of a workshop poll, cross-laboratory data comparisons, and the overall recommendations of the in vitro working group are presented herein. The working group observed that evaluation of TP-DDI for anticytokine monoclonal antibodies is currently best accomplished with a clinical study in patients with inflammatory disease. Treatment-induced changes in appropriate biomarkers in phase 2 and 3 studies may indicate the potential for a clinically measurable treatment effect on cytochrome P450 enzymes. Cytokine-mediated DDIs observed with anti-inflammatory TPs cannot currently be predicted using in vitro data. Future success in predicting clinical TP-DDIs will require an understanding of disease biology, physiologically relevant in vitro systems, and more examples of well conducted clinical TP-DDI trials.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Proteínas/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Avaliação Pré-Clínica de Medicamentos , Indústria Farmacêutica , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Proteínas/farmacologia , Estados Unidos , United States Food and Drug AdministrationRESUMO
Previous experiments performed in recombinant systems have suggested that protein-protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein-protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein-protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.
Assuntos
Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Hepatócitos/metabolismo , Propofol/metabolismo , RNA Interferente Pequeno/genética , Regulação para Baixo , Glucuronosiltransferase/genética , Células HEK293 , Humanos , Técnicas In Vitro , Isoenzimas/genética , Isoenzimas/metabolismo , UDP-Glucuronosiltransferase 1ARESUMO
Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4ß,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4ß,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4ß,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4ß-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.
Assuntos
Calcifediol/metabolismo , Citocromo P-450 CYP3A/biossíntese , Fígado/metabolismo , Osteomalacia/induzido quimicamente , Adulto , Linhagem Celular , Indução Enzimática , Feminino , Humanos , Hidroxilação , Masculino , Pessoa de Meia-Idade , Osteomalacia/enzimologia , Osteomalacia/metabolismo , Rifampina/farmacologia , Adulto JovemRESUMO
There is considerable evidence that pregnancy changes the disposition of drugs in an enzyme- and gestational stage-specific manner. On the basis of probe drug studies, the activity of CYP3A4 and CYP2D6 increases and CYP1A2 decreases during human pregnancy. However, no studies of CYP2B6 activity during human pregnancy have been conducted. In rodent models and in HepG2 cells, CYP2B enzymes have been shown to be regulated by estradiol. Because estradiol concentrations increase by â¼50-fold during human pregnancy, it was hypothesized that the increasing estradiol concentrations during human pregnancy would result in induction of CYP2B6 activity. Hepatocytes from three female donors were treated with estradiol, and the EC(50) and E(max) were measured for CYP2B6 mRNA and bupropion hydroxylation activity. The measured values were used to predict the magnitude of CYP2B6 induction during human pregnancy. At 100 nM total estradiol, a concentration achievable during the third trimester of pregnancy, CYP2B6 activity was predicted to increase by 1.5-3-fold, based on increased CYP2B6 activity and mRNA. When the E(max) and EC(50) values were compared with those for carbamazepine and rifampin, estradiol was found to be as potent an inducer of CYP2B6 as rifampin and carbamazepine. These data suggest that, during human pregnancy, the increasing estradiol concentrations will result in increased clearance of drugs that have CYP2B6-mediated clearance pathways. This could in part explain the observed increase in methadone clearance during pregnancy.
Assuntos
Analgésicos Opioides/metabolismo , Hidrocarboneto de Aril Hidroxilases/biossíntese , Estradiol/farmacologia , Hepatócitos/efeitos dos fármacos , Metadona/metabolismo , Oxirredutases N-Desmetilantes/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Biotransformação , Bupropiona/metabolismo , Carbamazepina/farmacologia , Células Cultivadas , Citocromo P-450 CYP2B6 , Relação Dose-Resposta a Droga , Indução Enzimática , Feminino , Hepatócitos/enzimologia , Humanos , Hidroxilação , Taxa de Depuração Metabólica , Oxirredutases N-Desmetilantes/genética , Gravidez , Cultura Primária de Células , RNA Mensageiro/biossíntese , Rifampina/farmacologia , Especificidade por Substrato , Regulação para CimaRESUMO
Carnosic acid is a phenolic diterpene isolated from rosemary (Rosmarinus officinalis), which may have anticancer, antiadipogenic, and anti-inflammatory properties. Recently, carnosic acid was shown to prevent weight gain and hepatic steatosis in a mouse model of obesity and type II diabetes. Based on these results, carnosic acid has been suggested as a potential treatment for obesity and nonalcoholic fatty liver disease; however, little is known about the safety of carnosic acid at doses needed to elicit a pharmacological effect. For this reason, hepatotoxicity and cytochrome P450 inhibition and induction studies were performed in primary human hepatocytes and microsomes. Measuring cellular ATP, carnosic acid showed a dose-dependent increase in hepatotoxicity with an EC(50) value of 94.8 ± 36.7 µM in three human hepatocyte donors without a concurrent increase in the apoptosis markers caspase-3/7. In human liver microsomes, carnosic acid did not exhibit significant time-dependent inhibition for any of the cytochrome P450 enzymes investigated, although it did inhibit CYP2C9- and CYP3A4-catalyzed reactions with K(i) values of 9.2 and 4.3 µM, respectively. Carnosic acid also induced CYP2B6 and CYP3A4 mRNA and enzyme activity in a dose-dependent manner. At 10 µM, carnosic acid increased CYP2B6 enzyme activity 61.6 and 49.3% in two donors compared with phenobarbital, and it increased CYP3A enzyme activity 82.6 and 142% compared with rifampicin. These results indicate the potential for drug interactions with carnosic acid and illustrate the need for an appropriate safety assessment before being used as a weight loss supplement.
Assuntos
Abietanos/farmacologia , Adipogenia/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450 , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Extratos Vegetais/farmacologia , Abietanos/efeitos adversos , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Sistema Enzimático do Citocromo P-450/metabolismo , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Hepatócitos/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Extratos Vegetais/efeitos adversos , Rifampina/farmacologia , Redução de Peso/efeitos dos fármacosRESUMO
Exposure to cytokines can down-regulate hepatic cytochrome P450 enzymes. Accordingly, relief of inflammation by cytokinetargeted drug therapy has the potential to up-regulate cytochrome P450s and thereby increase clearance of co-administered drugs. This study examined the effects of the inflammatory cytokine, interleukin 1ß (IL-1ß), and IL-1ß/interleukin 6 (IL-6) combinations on drug metabolizing enzymes in human hepatocyte culture. Treatment of hepatocytes with IL-1ß revealed suppression of mRNA expression of several clinically important cytochrome P450 isoenzymes, with EC50 values that differed by isoenzyme. Suppression of CYP1A2 activity by IL-1ß could not be measured in 3 of 5 donors due to lack of response, and in the two remaining donors the average EC50 was 450 pg/mL. CYP3A activity had an EC50 of suppression of 416 ± 454 pg/mL. Measurable EC50s were obtained for all 5 donors for CYP2C8, 3A4, 3A5, 4A11 and IL-6R mRNA with fold differences which varied between 9.5-fold (CYP2C8) to 109-fold (CYP4A11). When hepatocytes were treated with IL-1ß and IL-6 in combination at concentrations which ranged from 1-100 pg/mL, IL-6 was the main determinant of increases in acute phase response marker mRNA and of decreases in CYP3A4 mRNA. There was no synergy between IL-1ß and IL-6 in the regulation of cytochrome P450 mRNA when dosed in combination, although the effects of the two cytokines in combination were additive in certain instances. These data indicate that IL-1ß and IL-6 both suppress cytochrome P450 mRNA and enzyme levels in vitro and that, at similar physiologically-relevant concentrations in vitro, IL-6 is more potent than IL-1ß.
Assuntos
Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Interleucina-1beta/administração & dosagem , Interleucina-6/administração & dosagem , Preparações Farmacêuticas/metabolismo , Técnicas de Cultura de Células , Combinação de Medicamentos , HumanosRESUMO
Changes in cytochrome P450 expression incurred by inflammatory disease were studied in a murine collagen antibody induced arthritis (CAIA) model and compared to bacterial lipopolysaccharide-treated mice and to cytochrome P450 changes in primary mouse hepatocytes following combination treatments with cytokines IL-1ß, IL-6, or TNFα. CAIA in female mice increased serum IL-1ß, IL-6 and hepatic serum amyloid A (SAA) mRNA and significantly altered cytochrome P450 mRNA and activity levels. Most cytochrome P450 isoforms were down-regulated, although some, such as Cyp3a13, were up-regulated. Cytokine effects on cytochrome P450 levels in mouse hepatocytes were compared at in vitro cytokine concentrations similar to those measured in CAIA mouse serum in vivo. In vivo and in vitro cytochrome P450 suppression by cytokines was congruent for some cytochrome P450 isoforms (Cyp1a2, Cyp2c29, and Cyp3a11) but not for others (cytochrome P450 oxidoreductase (POR) and Cyp2e1). In mouse hepatocytes, IL-6 and IL-1ß in combination in vitro caused a synergistic increase in SAA mRNA expression, but not in cytochrome P450 suppression. IL-1ß and IL-6 were equipotent in the suppression of cytochrome P450 gene expression, while TNFα caused mild suppression only at the highest concentrations used. TNFα in combination with IL-1ß, IL-6, or both had a protective effect against IL-1ß and IL-6-mediated cytochrome P450 suppression. When IL-1ß or IL-6 was combined with low concentrations of TNFα, several P450 isoforms were induced rather than suppressed. These data highlight the complexities of performing in vitro-in vivo comparisons using disease models for cytochrome P450 regulation by cytokines.
Assuntos
Artrite Experimental/imunologia , Inibidores das Enzimas do Citocromo P-450 , Hepatócitos/citologia , Animais , Células Cultivadas , Feminino , Expressão Gênica , Hepatócitos/enzimologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4ß,25-dihydroxyvitamin D(3) [4ß,25(OH)(2)D(3)] dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4ß,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4ß,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4ß,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Vitamina D/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Microssomos Hepáticos/enzimologia , Espectrometria de Massas em TandemRESUMO
The cytokine-mediated suppression of hepatic drug-metabolizing enzymes by inflammatory disease and the relief of this suppression by successful disease treatment have recently become an issue in the development of drug interaction labels for new biological products. This study examined the effects of the inflammatory cytokine interleukin-6 (IL-6) on drug-metabolizing enzymes in human hepatocyte culture and the abrogation of these effects by a monoclonal antibody directed against IL-6. Treatment of human hepatocytes with IL-6 (n = 9 donors) revealed pan-suppression of mRNA of 10 major cytochrome P450 isoenzymes, but with EC(50) values that differed by isoenzyme. Some EC(50) values were above the range of clinically relevant serum concentrations of IL-6. Marker activities for CYP1A2 and CYP3A4 enzyme were similarly suppressed by IL-6 in both freshly isolated and cryopreserved hepatocytes. IL-6 suppressed induction of CYP1A2 enzyme activity by omeprazole and CYP3A4 enzyme activity by rifampicin but only at supraphysiological concentrations of IL-6. Glycosylated and nonglycosylated IL-6 did not significantly differ in their ability to suppress CYP1A2 and CYP3A4 enzyme activity. A monoclonal antibody directed against IL-6 abolished or partially blocked IL-6-mediated suppression of CYP1A2 and CYP3A4 enzyme activity, respectively. These data indicate that experimentation with IL-6 and anti-IL-6 monoclonal antibodies in human hepatocyte primary culture can quantitatively measure cytochrome P450 suppression and desuppression and determine EC(50) values for IL-6 against individual cytochrome P450 isoenzymes. However, the complex biology of inflammatory disease may not allow for quantitative in vitro-in vivo extrapolation of these simple in vitro data.
Assuntos
Anticorpos Monoclonais/farmacologia , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/metabolismo , Hepatócitos/efeitos dos fármacos , Interleucina-6/farmacologia , Preparações Farmacêuticas/metabolismo , Anticorpos Monoclonais/imunologia , Proteína C-Reativa/imunologia , Técnicas de Cultura de Células , Citocromo P-450 CYP1A2/biossíntese , Inibidores do Citocromo P-450 CYP1A2 , Citocromo P-450 CYP3A/biossíntese , Inibidores do Citocromo P-450 CYP3A , Células HEK293 , Hepatócitos/enzimologia , Hepatócitos/imunologia , Humanos , Interleucina-6/imunologia , Isoenzimas , Modelos Biológicos , Ligação Proteica , Receptores de Interleucina-6/biossíntese , Proteína Amiloide A Sérica/imunologia , Espectrometria de Massas em Tandem , Fatores de Tempo , TransfecçãoRESUMO
Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor characterized kinetically. We expressed the rCYP26A1 in baculovirus-infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion K(m) 9.4+/-3.3nM and V(max) 11.3+/-4.3pmolesmin(-1)pmoleP450(-1)) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sequência de Bases , Ciclo Celular/fisiologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Sistema Enzimático do Citocromo P-450/metabolismo , Primers do DNA , Amplificação de Genes , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Rim/embriologia , Rim/enzimologia , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ácido Retinoico 4 Hidroxilase , Tretinoína/metabolismoRESUMO
A common feature of many CYP2C9 ligands is their weak acidity. As revealed by crystallography, the structural basis for this behavior involves a charge-pairing interaction between an anionic moiety on the substrate and an active site R108 residue. In the present study we attempted to re-engineer CYP2C9 to better accept basic ligands by charge reversal at this key residue. We expressed and purified the R108E and R108E/D293N mutants and compared their ability with that of native CYP2C9 to interact with (S)-warfarin, diclofenac, pyrene, propranolol, and ibuprofen amine. As expected, the R108E mutant maintained all the native enzyme's pyrene 1-hydroxylation activity, but catalytic activity toward diclofenac and (S)-warfarin was abrogated. In contrast, the double mutant displayed much less selectivity in its behavior toward these control ligands. Neither of the mutants displayed significant enhancement of propranolol metabolism, and all three preparations exhibited a type II (inhibitor) rather than type I (substrate) spectrum with ibuprofen amine, although binding became progressively weaker with the single and double mutants. Collectively, these data underscore the importance of the amino acid at position 108 in the acid substrate selectivity of CYP2C9, highlight the accommodating nature of the CYP2C9 active site, and provide a cautionary note regarding facile re-engineering of these complex cytochrome P450 active sites.
Assuntos
Ácidos/metabolismo , Álcalis/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Engenharia de Proteínas , Hidrocarboneto de Aril Hidroxilases/genética , Sequência de Bases , Citocromo P-450 CYP2C9 , Primers do DNA , Heme/metabolismo , Humanos , Mutagênese , Espectrofotometria Ultravioleta , Especificidade por SubstratoRESUMO
The increasing number of transgenic or gene knockout mouse models generated for use in drug metabolism studies has meant that a greater understanding of the function and substrate specificities of murine cytochromes P450 (P450s) has become essential, particularly with the recent advances in "humanized" mouse models. In this study, we have heterologously expressed nine murine P450s--Cyp1a1, Cyp1a2, Cyp1b1, Cyp2a4, Cyp2b20, Cyp2c29, Cyp2d22, Cyp2e1, and Cyp3a11--individually with human P450 oxidoreductase to generate functional monooxygenase systems in Escherichia coli. We have identified a suitable fluorogenic probe for each P450 and determined the apparent kinetic parameters. These probes have enabled the screening of a panel of 31 test compounds classified as "drugs," "natural compounds," "endogenous compounds," and "pesticides" by measurement of IC(50), thus allowing the comparison of binding affinities. Human P450s CYP2C9, CYP2D6, and CYP3A4 were also included in the study to enable direct comparisons to be made with the mouse enzymes. Although there were general similarities between human and mouse P450s, perhaps the most significant finding in this study was the observation that, despite 77% amino acid identity, Cyp2d22 and CYP2D6 were remarkably dissimilar in a range of enzymatic properties, with potentially serious implications for pharmacokinetic studies using CYP2D substrates. The data presented in this study provide a solid foundation with which to assess the degree of similarity (or difference) between mouse and human P450s involved in xenobiotic metabolism and can be used as a basis for further studies.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/farmacologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar , Humanos , Cinética , Ligantes , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
During human pregnancy, CYP2C9, CYP2C19, and CYP2D6 activities are altered. The aim of the current study was to determine if this phenomenon can be replicated in the rat, and to evaluate the mechanisms that contribute to the changes in Cyp2c and Cyp2d activity during pregnancy. The intrinsic clearance of dextromethorphan O-demethylation, a measure of Cyp2d2 activity, was decreased 80% at both days 9 and 19 of gestation when compared to non-pregnant controls. The decreased intrinsic clearance was a result of both decreased V(max) and increased K(m)-values at both days of gestation. Quantitative RT-PCR revealed that transcripts of Cyp2d2 and Cyp2d4 were significantly decreased at day 19 of pregnancy (p<0.05) when compared to day 9 and non-pregnant controls. The decrease in Cyp2d mRNA levels correlated with a decrease in several nuclear receptor mRNA levels (RARalpha, RXRalpha, HNF1 and HNF3beta) but not with the mRNA levels of nuclear receptors usually associated with regulation of P450 enzymes (PXR, CAR and HNF4alpha). In contrast, Cyp2c12 and Cyp2c6 transcription and protein expression were not significantly altered during rat pregnancy although the intrinsic clearance of Cyp2c6 mediated diclofenac 4'-hydroxylation was increased 2-fold on day 19 of gestation when compared to non-pregnant controls. The increase in intrinsic clearance was due to a decrease in the K(m)-value for 4'-hydroxydiclofenac formation. These data show that pregnancy significantly alters the expression and activity of drug metabolizing enzymes in an enzyme and gestational stage specific manner. These changes are likely to have toxicological and therapeutic implications.