Kinetics of diazepam metabolism in rat hepatic microsomes and hepatocytes and their use in predicting in vivo hepatic clearance

Zomorodi, Katayoun; Carlile, David; Houston, J. Brian

doi:10.3109/00498259509046662

Cited by 42 publications

(19 citation statements)

References 12 publications

(6 reference statements)

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“…Substrate concentrations for depletion incubations were chosen based on published and in-house K m values (Zomorodi et al, 1995).…”

Section: Methodsmentioning

confidence: 99%

“…This metabolite, in theory, may compete with the parent for binding to the same P450, resulting in inhibition of the parent metabolism. Examples of compounds in the literature believed to demonstrate this phenomenon both in vitro and in vivo include phenytoin (Gerber et al, 1971;Ashley and Levy, 1972;Ashforth et al, 1995) and diazepam (Savenije-Chapel et al, 1985;Zomorodi et al, 1995;Carlile et al, 1997). This phenomenon will be addressed in more detail in a subsequent paper.…”

Section: Jones and Houstonmentioning

confidence: 99%

See 1 more Smart Citation

Substrate Depletion Approach for Determining in Vitro Metabolic Clearance: Time Dependencies in Hepatocyte and Microsomal Incubations

Jones¹,

Houston²

2004

Drug Metab Dispos

167

121

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ABSTRACT:The substrate depletion method is a popular approach used for the measurement of in vitro intrinsic clearance (CL int ). However, the incubation conditions used in these studies can vary, the consequences of which have not been systematically explored. Initial substrate depletion incubations using rat microsomes and hepatocytes were performed for eight benzodiazepines: alprazolam, clobazam, clonazepam, chlordiazepoxide, diazepam, flunitrazepam, midazolam, and triazolam. Subsequent predictions of in vivo CL int (ranging from 3 to 200 ml/min) and hepatic clearance (CL H ) (ranging from 0.3 to 15 ml/min) demonstrated that the general predictive ability of this approach was similar to that of the traditional metabolite formation method. A more detailed study of the substrate depletion profiles and CL int estimates indicated that the concentration of enzyme used is of particular importance.The metabolism of triazolam, clonazepam, and diazepam was monoexponential at all cell densities using hepatocytes; however, with microsomes, biphasic depletion was apparent, particularly at higher microsomal protein concentrations (2-5 mg/ml). Enzyme activity studies indicated that enzyme loss was more pronounced in the microsomal system (ranged from 8 to 65% activity after a 1-h incubation) compared with the hepatocyte system (approximately 100% activity after a 1-h incubation). For clonazepam (a low clearance substrate), these biphasic profiles could be explained by loss of enzyme activity. To ensure accurate predictions of in vivo CL int and CL H when using the substrate depletion approach, based on the results obtained for this class of drugs, it is recommended that low enzyme concentrations and short incubation times are used whenever possible.The in vitro measurement of intrinsic clearance (CL int ) using hepatic microsomes and/or hepatocytes is frequently used in both academia and the pharmaceutical industry to estimate the in vivo metabolic stability of new drug entities in both rat and human (Houston, 1994;Iwatsubo et al., 1997;Obach, 1999;McGinnity and Riley, 2001). The results of these assays are scaled and modeled, as described in eq. 1 for the well stirred liver model, to estimate in vivo hepatic clearance (CL H ).where Q H is the hepatic blood flow, SF represents the milligrams of microsomal protein or million cells per gram of liver multiplied by the grams of liver weight; f u b is the unbound fraction of drug in the blood, and f u inc is the unbound fraction in the incubation matrix.Traditionally, the metabolite formation method has been used for measurement of in vitro CL int , where the initial rate of metabolite production using hepatic microsomes or hepatocytes is measured over a range of substrate concentrations under linear conditions with respect to protein concentration/cell density and time (Madan et al., 2002;Houston et al., 2003). In general, short incubation times and low enzyme (protein) concentrations are used in these studies, since compliance with the Michaelis-Menten equation assumes less t...

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“…Substrate concentrations for depletion incubations were chosen based on published and in-house K m values (Zomorodi et al, 1995).…”

Section: Methodsmentioning

confidence: 99%

Section: Jones and Houstonmentioning

confidence: 99%

Substrate Depletion Approach for Determining in Vitro Metabolic Clearance: Time Dependencies in Hepatocyte and Microsomal Incubations

Jones¹,

Houston²

2004

Drug Metab Dispos

167

121

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“…Following those reports, many results were published and consistently indicated that the in vivo hepatic (or total body) clearance could be reasonably well predicted from the metabolic (intrinsic) clearance determined in the in vitro system on the basis of existing pharmacokinetic premises (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Most studies in early years used rat liver microsomes and/or rat hepatocytes for the prediction of clearance due to the feasibility of in vitro experiments and limited availability of human samples (9)(10)(11)(12)(13)(14)(15)(16)(17)21,24).…”

Section: Prediction Of Hepatic Clearance: From Liver Microsomes To Hementioning

confidence: 99%

“…Most studies in early years used rat liver microsomes and/or rat hepatocytes for the prediction of clearance due to the feasibility of in vitro experiments and limited availability of human samples (9)(10)(11)(12)(13)(14)(15)(16)(17)21,24). The prediction strategy established in rat has been extended to that for human as the in vitro samples (liver microsomes, liver slices, and freshly isolated hepatocytes) from human have become prevalent since early 1990s (18)(19)(20)22,23,(25)(26)(27).…”

Section: Prediction Of Hepatic Clearance: From Liver Microsomes To Hementioning

confidence: 99%

Prediction of Hepatic Clearance in Human From In Vitro Data for Successful Drug Development

Chiba

Ishii²,

Sugiyama

2009

AAPS J

192

168

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Abstract. The in vivo metabolic clearance in human has been successfully predicted by using in vitro data of metabolic stability in cryopreserved preparations of human hepatocytes. In the predictions by human hepatocytes, the systematic underpredictions of in vivo clearance have been commonly observed among different datasets. The regression-based scaling factor for the in vitro-to-in vivo extrapolation has mitigated discrepancy between in vitro prediction and in vivo observation. In addition to the elimination by metabolic degradation, the important roles of transporter-mediated hepatic uptake and canalicular excretion have been increasingly recognized as a rate-determining step in the hepatic clearance. It has been, therefore, proposed that the in vitro assessment should allow the evaluation of clearances for both transporter(s)-mediated uptake/excretion and metabolic degradation. This review first outlines the limited ability of subcellular fractions such as liver microsomes to predict hepatic clearance in vivo. It highlights the advantages of cryopreserved human hepatocytes as one of the versatile in vitro systems for the prediction of in vivo metabolic clearance in human at the early development stage. The following section discusses the mechanisms underlying the systematic underprediction of in vivo intrinsic clearance by hepatocytes. It leads to the proposal for the assessment of hepatic uptake clearance as one of the kinetically important determinants for accurate predictions of hepatic clearance in human. The judicious combination of advanced technologies and understandings for the drug disposition allows us to rationally optimize new chemical entities to the drug candidate with higher probability of success during the clinical development.

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“…To study the contribution of P450 isoforms in the metabolism of NC, a substrate depletion approach was adopted (Zomorodi et al, 1995). The enzyme concentrations as well as the incubation times were chosen according to the preliminary results demonstrating that the NC depletion was linear under these conditions (Jones and Houston, 2004), and the incubations were performed as in our previous study with some modifications .…”

Section: Methodsmentioning

confidence: 99%

The Contribution of Human OCT1, OCT3, and CYP3A4 to Nitidine Chloride–Induced Hepatocellular Toxicity

Yang

et al. 2014

Drug Metab Dispos

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Nitidine chloride (NC), a quaternary ammonium alkaloid, has numerous pharmacological effects, such as anticancer activity. However, it was found that NC also has hepatocellular toxicity. Because organic cation transporters 1 and 3 (OCT1 and OCT3) might mediate the influx of NC into hepatocytes, multidrug and toxin extrusion 1 (MATE1) probably mediates the efflux of NC from hepatocytes, while cytochrome P450 (P450) enzymes might contribute to NC metabolism, the present study was to evaluate the contribution of OCT1, OCT3, MATE1, and P450 enzymes to NCinduced hepatocellular toxicity. Our results showed that the uptake of NC in Madin-Darby canine kidney (MDCK) cells expressing human (h) OCT1 and OCT3 (MDCK-hOCT1 and MDCK-hOCT3) was significantly higher than that in mock cells; the hOCT1-and hOCT3-mediated uptake followed typical Michaelis-Menten kinetics.Meanwhile, NC was also a substrate of hMATE1, although its transport capacity was much lower than that of OCT1 NC-induced cytotoxicity in MDCK-hOCT1 or MDCK-hOCT3 cells was obviously higher than that in mock cells. Quinidine and (+)-tetrahydropalmatine [(+)-THP], OCT1 and OCT3 inhibitors, significantly reduced the uptake of NC in MDCK-hOCT1 cells, MDCK-hOCT3 cells, and rat primary hepatocytes, but only (+)-THP markedly attenuated the NCinduced toxicity. In addition, P450 enzymes, such as CYP3A4, mediated the metabolism of NC, and NC-induced toxicity in MDCKhOCT1/hCYP3A4 cells was lower than that in MDCK-hOCT1 cells. Our results indicated that NC is a substrate of hOCT1, hOCT3, and CYP3A4; that OCT1 and OCT3 mediate the uptake of NC in hepatocytes and subsequently cause hepatotoxicity; and that NC-induced toxicity could be attenuated by CYP3A4-mediated metabolism.

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Kinetics of diazepam metabolism in rat hepatic microsomes and hepatocytes and their use in predicting in vivo hepatic clearance

Cited by 42 publications

References 12 publications

Substrate Depletion Approach for Determining in Vitro Metabolic Clearance: Time Dependencies in Hepatocyte and Microsomal Incubations

Substrate Depletion Approach for Determining in Vitro Metabolic Clearance: Time Dependencies in Hepatocyte and Microsomal Incubations

Prediction of Hepatic Clearance in Human From In Vitro Data for Successful Drug Development

The Contribution of Human OCT1, OCT3, and CYP3A4 to Nitidine Chloride–Induced Hepatocellular Toxicity

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