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Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichia coli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 2',2'-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar K(m) and V(max) for Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower K(m) and 6-fold lower V(max) for Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P < 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in K(m) (P < 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.
Assuntos
Antimetabólitos Antineoplásicos/metabolismo , Citidina Desaminase/metabolismo , Desoxicitidina Quinase/metabolismo , Desoxicitidina/análogos & derivados , Farmacogenética , Biotransformação , Catálise , Citarabina/metabolismo , Citidina Desaminase/genética , Desoxicitidina/metabolismo , Desoxicitidina Quinase/genética , Variação Genética , Genótipo , Humanos , Cinética , Modelos Biológicos , Dinâmica não Linear , Fenótipo , Fosforilação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , GencitabinaRESUMO
Cryopreserved human hepatocytes suspended in human plasma (HHSHP) have previously provided accurate CYP3A drug-drug interaction (DDI) predictions from a single IC(50) that captures both reversible and time-dependent inhibition. The goal of this study was to compare the accuracy of DDI predictions by a protein-free human hepatocyte system combined with the fraction unbound in plasma for inhibitor(s) with those obtained with protein-containing incubations. Seventeen CYP3A, CYP2C9, or CYP2D6 inhibitors were incubated with hepatocytes in human plasma or hepatocyte maintenance medium (HMM) for 20 min over a range of concentrations after which midazolam 1'-hydroxylation, diclofenac 4'-hydroxylation or (R)-bufuralol 1'-hydroxylation were used to quantify the corresponding cytochrome P450 (P450) catalytic activities. Two methods were used to predict the human exposure ratio of the victim drug in the presence and absence of inhibitor. The HMM K(i, app) values were combined with the free average systemic plasma concentration ("free [I] with HMM K(i, app)") and the plasma K(i, app) values were combined with the total average systemic plasma concentration ("total [I] with plasma K(i, app)"). Of 63 clinical DDI studies, the total [I] with plasma K(i, app) method predicted 89% of cases within 2-fold of the reported interaction whereas the free [I] with HMM K(i, app) method predicted only 59%. There was a general underprediction by the free [I] with HMM K(i, app) method, which is consistent with an underestimation of in vitro inhibition potency in this system. In conclusion, the HHSHP system proved to be a simple, accurate predictor of DDIs for three major P450s and superior to the protein-free approach.
Assuntos
Técnicas de Cultura de Células/métodos , Criopreservação , Meios de Cultura , Sistema Enzimático do Citocromo P-450/fisiologia , Hepatócitos , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Meios de Cultura Livres de Soro , Citocromo P-450 CYP2C9 , Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Diclofenaco/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Etanolaminas/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Hidroxilação , Midazolam/farmacologia , Plasma , Valor Preditivo dos Testes , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
AIM: The most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin. METHODS: The metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4'-hydroxylation (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1'-hydroxylation/testosterone 6ß-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences. RESULTS: The samples of the two ethnic groups were not significantly different in cytochrome-b(5) concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A. CONCLUSIONS: These data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins.
Assuntos
Povo Asiático , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/enzimologia , População Branca , Adulto , Idoso , Cromatografia Líquida , Citocromos b5/metabolismo , Feminino , Humanos , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Análise de Regressão , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Cryopreserved human hepatocytes suspended in human plasma (HHSHP) represent an integrated metabolic environment for predicting drug-drug interactions (DDIs). In this study, 13 CYP3A reversible and/or time-dependent inhibitors (TDIs) were incubated with HHSHP for 20 min over a range of concentrations after which midazolam 1'-hydroxylation was used to measure CYP3A activity. This single incubation time method yielded IC(50) values for the 13 inhibitors. For each CYP3A inhibitor-victim drug pair, the IC(50) value was combined with total average plasma concentration of the inhibitor in humans, fraction of the victim drug cleared by CYP3A, and intestinal availability of the victim drug to predict the ratio of plasma area under the curve of the victim drug in the presence and absence of inhibitor. Of 52 clinical DDI studies using these 13 inhibitors identified in the literature, 85% were predicted by this method within 2-fold of the observed change, and all were predicted within 3-fold. Subsequent studies to determine mechanism (reversible and time-dependent inhibitors) were performed by using a range of incubation periods and inhibitor concentrations. This system differentiated among reversible inhibitors, TDIs, and the combination of both. When the reversible and inactivation parameters were incorporated into predictive models, 65% of 52 clinical DDIs were predicted within 2-fold of the observed changes and 88% were within 3-fold. Thus, HHSHP produced accurate DDI predictions with a simple IC(50) determined at a single incubation time regardless of the inhibition mechanism; further if needed, the mechanism(s) of inhibition can be identified.
Assuntos
Inibidores do Citocromo P-450 CYP3A , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Inibidores Enzimáticos/metabolismo , Hepatócitos/metabolismo , Midazolam/metabolismo , Área Sob a Curva , Citocromo P-450 CYP3A/sangue , Relação Dose-Resposta a Droga , Humanos , Hidroxilação , Plasma , Fatores de TempoRESUMO
The carboxylesterases (CESs) are a family of serine hydrolases that hydrolyze compounds containing an ester, amide, or thioester. In humans, two dominant forms, CES1 and CES2, are highly expressed in organs of first-pass metabolism and play an important role in xenobiotic metabolism. The current study was conducted to better understand species-related differences in substrate selectivity and tissue expression of these enzymes. To elucidate potential similarities and differences among these enzymes, a series of 4-nitrophenyl esters and a series of gemcitabine prodrugs were evaluated using enzyme kinetics as substrates of expressed and purified CESs from beagle dog, cynomolgus monkey, and human genes. For the substrates examined, human and monkey CES2 more efficiently catalyzed hydrolysis compared with CES1, whereas CES1 was the more efficient enzyme in dog. Quantitative real-time polymerase chain reaction and Western blot analyses indicate that the pattern of CES tissue expression in monkey is similar to that of human, but the CES expression in dog is unique, with no detectable expression of CES in the intestine. Loperamide, a selective human CES2 inhibitor, was also found to be a CES2-selective inhibitor in both dog and monkey. This is the first study to examine substrate specificity among dog, human, and monkey CESs.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Animais , Sequência de Bases , Western Blotting , Hidrolases de Éster Carboxílico/genética , Cães , Humanos , Macaca fascicularis , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Especificidade por SubstratoRESUMO
Inhibition of a drug-metabolizing enzyme by the reversible interaction of a drug with the enzyme, thus decreasing the metabolism of another drug, is a major cause of clinically significant drug-drug interactions. This chapter defines the four reversible mechanisms of inhibition exhibited by drugs: competitive, noncompetitive, uncompetitive, and mixed competitive/noncompetitive. An in vitro procedure to determine the potential of a drug to be a reversible inhibitor is also provided. Finally, a number of examples of clinically significant drug-drug interactions resulting from reversible inhibition are described.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Algoritmos , Ligação Competitiva , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/farmacologia , Interações Medicamentosas , Humanos , Concentração Inibidora 50 , CinéticaRESUMO
Large species differences in the expression of carboxylesterases (CE) have been described, but the interrelationships of CEs across species are not well characterized. In the current analyses, sequences with genomic structures similar to human CEs were found in piscine, avian, and mammalian genomes. Analyses of these genes suggest that four CE groups existed prior to mammalian divergence, with another form occurring after eutherian-marsupial divergence, yielding five distinct mammalian CE groups. The CE1 and CE2 groupings appear to have undergone extensive gene duplication in species with herbivorous and omnivorous diets underscoring the potential importance of these two groups in xenobiotic metabolism. However, CE3, CE4, and CE5 have remained at one gene per species in almost all observed cases. In avian and piscine genomes, only two CE groupings each were observed in the currently available sequence data. Finally, this study presents considerations for a broader phylogenetic-based nomenclature that could encompass other serine hydrolases in addition to the CEs.
Assuntos
Hidrolases de Éster Carboxílico/genética , Sequência de Aminoácidos , Animais , Aves/genética , Hidrolases de Éster Carboxílico/classificação , Sequência Consenso , Peixes/genética , Genômica , Humanos , Mamíferos/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
AIMS: To develop a population pharmacokinetic model to describe the pharmacokinetics of desipramine in healthy subjects, after oral administration of a 50mg dose. Additional objectives were to develop a semi-mechanistic population pharmacokinetic model for desipramine, which allowed simulation of CYP2D6-mediated inhibition, when using desipramine as a probe substrate, and to evaluate certain study design elements, such as duration of desipramine pharmacokinetic sampling, required sample size and optimal pharmacokinetic sampling schedule for intermediate, extensive and ultrarapid metabolizers of CYP2D6 substrates. RESULTS: The mean population estimates of the first order absorption rate constant (k(a) ), apparent clearance (CL/F) and apparent volume of distribution at steady state (V(ss) /F) were 0.15h(-1) , 111 lh(-1) and 2950 l, respectively. Further, using the proposed semi-mechanistic hepatic intrinsic clearance model with Bayesian inference, mean population desipramine hepatic intrinsic clearance was estimated to be 262 lh(-1) with between-subject variability of 84%. d-optimal PK sampling times for intermediate metabolizers were calculated to be approximately 0.25, 24, 75 and 200h. Similar sampling times were found for ultrarapid and extensive metabolizers except that the second d-optimal sample was earlier at 14 and 19h, respectively, compared with 24h for intermediate metabolizers. This difference in sampling times between the three genotypes can be attributed to the different intrinsic clearances and elimination rates. CONCLUSIONS: A two compartment population pharmacokinetic model best described desipramine disposition. The semi-mechanistic population model developed is suitable to describe the pharmacokinetic behaviour of desipramine for the dose routinely used in drug-drug interaction (DDI) studies. Based on this meta-analysis of seven trials, a sample size of 21 subjects in cross-over design is appropriate for assessing CYP2D6 interaction with novel compounds.
Assuntos
Inibidores do Citocromo P-450 CYP2D6 , Desipramina/farmacocinética , Inibidores Enzimáticos/farmacocinética , Modelos Biológicos , Administração Oral , Adulto , Teorema de Bayes , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
Vincristine is metabolized to one primary metabolite, M1, by cDNA-expressed CYP3A4 and CYP3A5 and by CYP3A enzymes in human liver microsomes. For both systems, CYP3A5 is predicted to mediate approximately 80% of the CYP3A metabolism for individuals with high CYP3A5 expression (at least one CYP3A5(*)1 allele). In the current study, the role of CYP3A5 was quantified in the metabolism of vincristine with human cryopreserved hepatocytes. The hepatocytes were genotyped for common CYP3A5 allelic variants (CYP3A5(*)3, CYP3A5(*)6, and CYP3A5(*)7) to predict CYP3A5 expression. For each hepatocyte preparation, the rates of vincristine depletion and metabolite formation were quantified. Whereas human hepatocytes with predicted low CYP3A5 expression did not detectably metabolize vincristine, human hepatocytes with predicted high CYP3A5 expression metabolized vincristine to one primary metabolite, M1. In paired experiments using cryopreserved hepatocytes from the same donor, vincristine was incubated with intact cells and cell lysates supplemented with NADPH. The rates of M1 formation were 4 to 69-fold higher for the cell lysates compared with the intact cells. For one representative donor, the intact cells had a 3-fold higher K(m) value and a 3-fold lower V(max) value for M1 formation compared with the cell lysates. Thus, the rate of M1 formation in the hepatocytes may be influenced by the rate of vincristine translocation across the plasma membrane. We conclude that genetically determined CYP3A5 expression in human cryopreserved hepatocytes plays a major role in vincristine metabolism.
Assuntos
Antineoplásicos Fitogênicos/metabolismo , Citocromo P-450 CYP3A/fisiologia , Hepatócitos/metabolismo , Vincristina/metabolismo , Criopreservação , Citocromo P-450 CYP3A/genética , Inibidores do Citocromo P-450 CYP3A , Genótipo , Humanos , Taxa de Depuração Metabólica , Esteroide Hidroxilases/metabolismoRESUMO
2-Acetoxy-5-(alpha-cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (prasugrel) is a novel thienopyridine prodrug with demonstrated inhibition of platelet aggregation and activation. The biotransformation of prasugrel to its active metabolite, 2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid (R-138727), requires ester bond hydrolysis, forming the thiolactone 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone(R-95913), followed by cytochrome P450-mediated metabolism to the active metabolite. The presumed role of the human liver- and intestinal-dominant carboxylesterases, hCE1 and hCE2, respectively, in the conversion of prasugrel to R-95913 was determined using expressed and purified enzymes. The hydrolysis of prasugrel is at least 25 times greater with hCE2 than hCE1. Hydrolysis of prasugrel by hCE1 showed Michaelis-Menten kinetics yielding an apparent K(m) of 9.25 microM and an apparent V(max) of 0.725 nmol product/min/microg protein. Hydrolysis of prasugrel by hCE2 showed a mixture of Hill kinetics at low substrate concentrations and substrate inhibition at high concentrations. At low concentrations, prasugrel hydrolysis by hCE2 yielded an apparent K(s) of 11.1 microM, an apparent V(max) of 19.0 nmol/min/microg, and an apparent Hill coefficient of 1.42, whereas at high concentrations, an apparent IC(50) of 76.5 microM was obtained. In humans, no in vivo evidence of inhibition exists. In vitro transport studies using the intestinal Caco-2 epithelial cell model showed a high in vivo absorption potential for prasugrel and rapid conversion to R-95913. In conclusion, the human carboxylesterases efficiently mediate the conversion of prasugrel to R-95913. These data help explain the rapid appearance of R-138727 in human plasma, where maximum concentrations are observed 0.5 h after a prasugrel p.o. dose, and the rapid onset of action of prasugrel.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Piperazinas/farmacocinética , Tiofenos/farmacocinética , Biotransformação , Células CACO-2 , Cromatografia Líquida , Humanos , Hidrólise , Cloridrato de Prasugrel , Pró-Fármacos , Antagonistas do Receptor Purinérgico P2 , Espectrometria de Massas em TandemRESUMO
Pharmacogenomic (PGx) research on the absorption, distribution, metabolism, and excretion (ADME) properties of drugs has begun to have impact for both drug development and utilization. To provide a cross-industry perspective on the utility of ADME PGx, the Pharmaceutical Research and Manufacturers of America (PhRMA) conducted a survey of major pharmaceutical companies on their PGx practices and applications during 2003-2005. This white paper summarizes and interprets the results of the survey, highlights the contributions and applications of PGx by industrial scientists as reflected by original research publications, and discusses changes in drug labels that improve drug utilization by inclusion of PGx information. In addition, the paper includes a brief review on the clinically relevant genetic variants of drug-metabolizing enzymes and transporters most relevant to the pharmaceutical industry.
Assuntos
Farmacogenética , Farmacocinética , Arilsulfotransferase/genética , Catecol O-Metiltransferase/genética , Sistema Enzimático do Citocromo P-450/genética , Desenho de Fármacos , Indústria Farmacêutica , Interações Medicamentosas , Genótipo , Glucuronosiltransferase/genética , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Polimorfismo GenéticoRESUMO
INTRODUCTION: The effects of buffer and substrate solvent conditions on in vitro activity of carboxylesterases (CE) have not been previously described. Therefore, it is unknown if the many different assay conditions used by various laboratories have a substantial impact on the activity of CE enzymes. METHODS: Three human CEs were expressed and purified, and the hydrolysis of 4-nitrophenyl butyrate was measured to assess enzyme activity. Four buffers (HEPES, potassium phosphate, sodium phosphate, and Tris) were evaluated for their effects on enzyme activity at concentrations ranging from 5 to 900 mM, as well as phosphate buffered saline. Five commonly used substrate-carrier solvents (acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol) ranging from 0.25 to 6% were also assessed for their effect on enzyme activity. RESULTS: The clearances for the CEs in HEPES, potassium phosphate, sodium phosphate, and Tris up to 100 mM were similar to the CE clearances obtained with phosphate buffered saline. Higher buffer concentrations resulted in differential activity of the CEs. All three CEs tolerated the substrate solvents up to 2% as indicated by little effect of solvent on catalytic activity. At substrate solvent concentrations above 2% the CE activities were found to gradually decrease. In general, CES3 displayed substantially lower activity than CES1 and CES2. DISCUSSION: In conclusion, any of the buffers examined up to 100 mM resulted in clearance values similar to that of phosphate buffered saline for the hydrolysis of 4-nitrophenyl butyrate by the human CEs. With regard to the substrate solvents tested, acetone, acetonitrile, or dimethyl sulfoxide appear to be well tolerated by the CEs up to 2% of the total reaction volume.
Assuntos
Hidrolases de Éster Carboxílico/química , Solventes/química , Acetona/química , Acetonitrilas/química , Soluções Tampão , Butiratos/química , Dimetil Sulfóxido/química , Etanol/química , HEPES/química , Humanos , Hidrólise , Metanol/química , Fosfatos/química , Compostos de Potássio/química , Proteínas Recombinantes/química , Trometamina/químicaRESUMO
BACKGROUND & AIMS: Comorbidities associated with nonalcoholic fatty liver often require therapy with medications (eg, statins) metabolized by cytochrome P-450 3A (CYP3A). There is significant interindividual variability in CYP3A expression. However, human studies that systematically examined the relationship between hepatic steatosis and hepatic CYP3A activity are lacking. METHODS: The relationship of hepatic CYP3A activity with several variables including hepatic steatosis, CYP3A4 protein content, CYP3A4 mRNA expression, CYP3A5 genotype, and its mRNA expression was investigated in human liver samples (n = 49). CYP3A activity was quantified from liver microsomes by using testosterone as a probe, and hepatic steatosis was defined to be present if >5% of hepatocytes had large globules of intracellular fat displacing the nucleus. RESULTS: The mean +/- standard error hepatic CYP3A activity of the study group was 3156 +/- 2794 pmol x min(-1) x mg(-1) of protein, and it was not associated with age, gender, medicinal use, CYP3A5 or pregnane xenobiotic receptor mRNA expression, or CYP3A5 genotype. Twenty-four liver samples with steatosis had significantly lower hepatic CYP3A activity than 25 liver samples without steatosis (1978 +/- 299 vs 4287 +/- 659 pmol x min(-1) x mg(-1) of protein; P = .003). This difference persisted even after controlling for relevant covariates in the multivariate analysis (P = .04). However, CYP3A4 protein content was not different between the 2 groups (6 +/- 1.3 vs 8.5 +/- 2.2 pmol/mg protein; P = .3). There was a significant negative relationship between severity of steatosis and hepatic CYP3A activity (P = .01). CONCLUSIONS: Hepatic steatosis is associated with decreased hepatic CYP3A activity in humans via post-translational mechanism. Further studies are needed to confirm our findings.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Adolescente , Adulto , Idoso , Biópsia por Agulha , Western Blotting , Estudos de Casos e Controles , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Feminino , Regulação da Expressão Gênica , Marcadores Genéticos/genética , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Probabilidade , Prognóstico , RNA Mensageiro/análise , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estudos de Amostragem , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
OBJECTIVES: Tadalafil was examined in vitro and in vivo for its ability to affect human cytochrome P450 (CYP) 3A-mediated metabolism. METHODS: Reversible and mechanism-based inhibition of CYP3A by tadalafil was examined in human liver microsomes. The ability of tadalafil to influence CYP3A activity was also examined in primary cultures of human hepatocytes. The effect of tadalafil on the pharmacokinetics of CYP3A probe substrates was evaluated in human volunteers before and after coadministration with either a single dose or multiple doses of tadalafil (10 or 20 mg). RESULTS: Negligible competitive inhibition of CYP3A was observed in vitro. Mechanism-based inhibition of CYP3A was detected, albeit with a low potency. In human hepatocytes, exposure to 1 micromol/L or greater of tadalafil resulted in increased CYP3A protein expression; however, as with a combined effect of induction and inhibition, a corresponding increase in CYP3A activity did not occur. The clinical pharmacokinetics of midazolam and lovastatin, probe substrates of CYP3A, were unaffected by up to 14 days of tadalafil administration (90% confidence intervals for the ratio of least squares means for the pharmacokinetic parameters of tadalafil were contained within the no-effect boundaries of 0.7 to 1.43). CONCLUSIONS: In vitro results suggested that tadalafil would have little effect on the pharmacokinetics of drugs metabolized by CYP3A. Clinical studies demonstrated that the pharmacokinetics of 2 different CYP3A substrates, midazolam and lovastatin, were virtually unchanged after tadalafil coadministration. Thus therapeutic concentrations of tadalafil do not produce clinically significant changes in the clearance of drugs metabolized by CYP3A.
Assuntos
Ansiolíticos/farmacocinética , Anticolesterolemiantes/farmacocinética , Carbolinas/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Lovastatina/farmacocinética , Microssomos Hepáticos/efeitos dos fármacos , Midazolam/farmacocinética , Adulto , Área Sob a Curva , Células Cultivadas , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Feminino , Humanos , Masculino , Taxa de Depuração Metabólica , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , TadalafilaRESUMO
Current regulatory guidances do not address specific study designs for in vitro and in vivo drug-drug interaction studies. There is a common desire by regulatory authorities and by industry sponsors to harmonize approaches to allow for a better assessment of the significance of findings across different studies and drugs. There is also a growing consensus for the standardization of cytochrome P450 (CYP) probe substrates, inhibitors, and inducers and for the development of classification systems to improve the communication of risk to health care providers and patients. While existing guidances cover mainly CYP-mediated drug interactions, the importance of other mechanisms, such as transporters, has been recognized more recently and should also be addressed. This paper was prepared by the Pharmaceutical Research and Manufacturers of America (PhRMA) Drug Metabolism and Clinical Pharmacology Technical Working Groups and represents the current industry position. The intent is to define a minimal best practice for in vitro and in vivo pharmacokinetic drug-drug interaction studies targeted to development (not discovery support) and to define a data package that can be expected by regulatory agencies in compound registration dossiers.
Assuntos
Interações Medicamentosas , Projetos de Pesquisa , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucuronídeos/metabolismo , Humanos , Técnicas In Vitro , Preparações Farmacêuticas/metabolismo , Fenótipo , Especificidade por SubstratoRESUMO
INTRODUCTION: Glucuronidation by the uridine diphosphate glucuronosyltransferases (UGTs) plays a pivotal role in the clearance mechanism of both xenobiotics and endobiotics. The detection of glucuronides at low micromolar concentrations is required to accurately model in vitro enzyme kinetics and in vivo pharmacokinetics. However, relatively few glucuronides are currently available as standards for developing liquid chromatography and mass spectroscopy (LC/MS) bioanalytical methods. METHODS: The glucuronidation capacity of hepatic microsomes prepared from rat (RLM), dog (DLM), monkey (MLM), and human (HLM) was examined for five xenobiotic substrates. In each case, glucuronide standards were produced using the enzyme source most efficient for the production of that specific glucuronide. RESULTS: Dog hepatic microsomes were used to produce glucuronides for anthraflavic acid (yield: 14 mg), buprenorphine (yield: 14 mg), and octylgallate (total yield: 13 mg), whereas propofol glucuronide (yield: 20 mg), and ethinylestradiol glucuronide (yield: 8 mg) were prepared using HLM. All glucuronides were characterized by LC/MS/MS and nuclear magnetic resonance (NMR) spectroscopy. DISCUSSION: The multimilligram quantities of glucuronide standards produced by this method have many applications throughout drug discovery and toxicology. In addition to allowing the quantification of glucuronide formation from in vitro and in vivo studies, the authentic standards produced could also be used to assess potential pharmacological or toxicological effects of metabolites.
Assuntos
Ácido Gálico/análogos & derivados , Glucuronídeos/biossíntese , Microssomos Hepáticos/metabolismo , Padrões de Referência , Xenobióticos/metabolismo , Animais , Antraquinonas/metabolismo , Buprenorfina/metabolismo , Cromatografia Líquida de Alta Pressão , Cães , Etinilestradiol/metabolismo , Ácido Gálico/metabolismo , Glucuronídeos/análise , Glucuronosiltransferase/metabolismo , Haplorrinos , Humanos , Espectroscopia de Ressonância Magnética , Propofol/metabolismo , RatosRESUMO
Inhibition of a drug-metabolizing enzyme by the reversible interaction of a drug with the enzyme, thus decreasing the metabolism of another drug, is a major cause of clinically significant drug-drug interactions. This chapter defines the four reversible mechanisms of inhibition exhibited by drugs: competitive, noncompetitive, uncompetitive, and mixed competitive/noncompetitive. An in vitro procedure to determine the potential of a drug to be a reversible inhibitor is also provided. Finally, a number of examples of clinically significant drug-drug interactions resulting from reversible inhibition are described.
Assuntos
Inibidores Enzimáticos/farmacologia , Enzimas/metabolismo , Ligação Competitiva , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Preparações Farmacêuticas/metabolismoRESUMO
Arsenite is critical pharmacologically as a treatment for advanced stage blood cancer. However, environmental exposure to arsenic results in multiple diseases. Previous studies have shown that arsenic decreases expression of CYP3A, a critical drug metabolizing enzyme in human and rat liver. In addition, acute and chronic arsenic exposure in liver stimulates an inflammatory response. Our work has shown that arsenite decreases nuclear levels of RXRα the nuclear receptor that, as a heterodimer partner with PXR, transactivates the CYP3A gene. These results suggest that arsenite decreases transcription of CYP3A by decreasing RXRα. The present report shows that exposure to 5 µM arsenite decreased the activity of a rat CYP3A promoter luciferase reporter in HepG2 cells. The activity of a RARE-luciferase reporter, that is transactivated by the retinoic acid receptor (RAR)/RXRα, was also decreased. Previous studies have shown that arsenic in the concentration range of 2-5 µM affects CYP3A mRNA. When rifampicin-treated primary human hepatocyte cultures were exposed to arsenite concentrations as low as 50 nM, CYP3A mRNA was decreased. Treatment of primary human hepatocytes with the proteasome inhibitor MG132 increased RXRα suggesting the involvement of the proteasome pathway in regulation of RXRα. Finally, arsenic induces a pro-inflammatory response in liver. Surprisingly, we show that in hepatocytes arsenite decreases expression of two inflammatory mediators, TNF and VEGF, an effect that is not predicted from suppression of RXRα activity.
Assuntos
Arsenitos/toxicidade , Citocromo P-450 CYP3A/genética , Poluentes Ambientais/toxicidade , Hepatócitos/efeitos dos fármacos , Receptor X Retinoide alfa/metabolismo , Adulto , Células Cultivadas , Feminino , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Receptores de Esteroides/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Cytochrome P450 enzymes are responsible for metabolizing many endogenous and xenobiotic molecules encountered by the human body. It has been estimated that 75% of all drugs are metabolized by cytochrome P450 enzymes. Thus, predicting a compound's potential sites of metabolism (SOM) is highly advantageous early in the drug development process. We have combined molecular dynamics, AutoDock Vina docking, the neighboring atom type (NAT) reactivity model, and a solvent-accessible surface-area term to form a reactivity-accessibility model capable of predicting SOM for cytochrome P450 2C9 substrates. To investigate the importance of protein flexibility during the ligand-binding process, the results of SOM prediction using a static protein structure for docking were compared to SOM prediction using multiple protein structures in ensemble docking. The results reported here indicate that ensemble docking increases the number of ligands that can be docked in a bioactive conformation (ensemble: 96%, static: 85%) but only leads to a slight improvement (49% vs. 44%) in predicting an experimentally known SOM in the top-1 position for a ligand library of 75 CYP2C9 substrates. Using ensemble docking, the reactivity-accessibility model accurately predicts SOM in the top-1 ranked position for 49% of the ligand library and considering the top-3 predicted sites increases the prediction success rate to approximately 70% of the ligand library. Further classifying the substrate library according to K(m) values leads to an improvement in SOM prediction for substrates with low K(m) values (57% at top-1). While the current predictive power of the reactivity-accessibility model still leaves significant room for improvement, the results illustrate the usefulness of this method to identify key protein-ligand interactions and guide structural modifications of the ligand to increase its metabolic stability.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Hidrocarboneto de Aril Hidroxilases/química , Citocromo P-450 CYP2C9 , Humanos , Ligantes , Modelos Moleculares , Conformação ProteicaRESUMO
Ticlopidine, clopidogrel, and prasugrel are thienopyridine prodrugs that inhibit adenosine-5'-diphosphate (ADP)-mediated platelet aggregation in vivo. These compounds are converted to thiol-containing active metabolites through a corresponding thiolactone. The 3 compounds differ in their metabolic pathways to their active metabolites in humans. Whereas ticlopidine and clopidogrel are metabolized to their thiolactones in the liver by cytochromes P450, prasugrel proceeds to its thiolactone following hydrolysis by carboxylesterase 2 during absorption, and a portion of prasugrel's active metabolite is also formed by intestinal CYP3A. Both ticlopidine and clopidogrel are subject to major competing metabolic pathways to inactive metabolites. Thus, varying efficiencies in the formation of active metabolites affect observed effects on the onset of action and extent of inhibition of platelet aggregation (IPA). Knowledge of the CYP-dependent formation of ticlopidine and clopidogrel thiolactones helps explain some of the observed drug-drug interactions with these molecules and, more important, the role of CYP2C19 genetic polymorphism on the pharmacokinetics of and pharmacodynamic response to clopidogrel. The lack of drug interaction potential and the absence of CYP2C19 genetic effect result in a predictable response to thienopyridine antiplatelet therapy with prasugrel. Current literature shows that greater ADP-mediated IPA is associated with significantly better clinical outcomes for patients with acute coronary syndrome.