Determination of a Human Hepatic Microsomal Scaling Factor for Predicting in Vivo Drug Clearance

Hakooz, Nancy; K, Ito; Rawden, Helen; Gill, Helen; Lemmers, L.G.; Boobis, Alan R.; Edwards, Robert J.; Carlile, David; Lake, Brian G.; Houston, J. Brian

doi:10.1007/s11095-006-9531-2

Cited by 64 publications

(53 citation statements)

References 22 publications

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“…For substrates displaying sigmoidal kinetics, the equivalent of CL int , the clearance at maximal activation (CL max ) was calculated (Witherow and Houston, 1999). Hepatocyte CL int (l/min/10 6 cells) and microsomal CL int (l/min/mg protein) were scaled to in vivo CL int (ml/min/kg) using a hepatocellularity of 120 ϫ 10 6 cells g Ϫ1 liver, a microsomal recovery value of 40 mg microsomal protein g Ϫ1 liver (Hakooz et al, 2006), and a human liver weight of 21.4 g liver kg Ϫ1 body weight. After normalizing both data sets to per gram of liver, the unbound CL int values from cryopreserved hepatocytes and human liver microsomes were compared.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Cryopreserved Human Hepatocytes as an Alternative in Vitro System to Microsomes for the Prediction of Metabolic Clearance

Griffin²,

2006

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ABSTRACT:Human liver microsomes have typically resulted in marked underprediction of in vivo human intrinsic clearance (CL int ); therefore, the utility of cryopreserved hepatocytes as an alternative in vitro system has become an important issue. In this study, 10 compounds (tolbutamide, diclofenac, S-warfarin, S-mephenytoin, dextromethorphan, bufuralol, quinidine, nifedipine, testosterone, and terfenadine) were selected as substrate probes for CYP2C9, 2C19, 2D6, and 3A4, and the kinetics of metabolite formation (n ‫؍‬ 14 pathways) were investigated in three individual lots of cryopreserved hepatocytes and in a pool of human liver microsomes. For the majority of the compounds, lower unbound K M or S 50 values were observed in hepatocytes compared with microsomes, on average by 50% over a 200-fold range (0.5-140 M). Expressed on an equivalent liver weight basis, a good correlation between microsomal and hepatocyte V max values was observed for most pathways greater than 5 orders of magnitude (0.16-216 nmol/min/g liver). Unbound hepatocyte CL int (CL int,u ) values, when scaled to the whole liver (range 0.38-4000 ml/min/kg), were on average 2.5-fold higher than microsomal CL int,u values, with the exception of tolbutamide and diclofenac, for which lower hepatocellular CL int,u values were observed. Hepatocyte predicted CL int values were compared with human in vivo CL int values, and to supplement our data, in vitro data from cryopreserved hepatocytes were collated from four other published sources. These data show that for 37 drugs, there is, on average, a 4.5-fold under-prediction of the in vivo CL int using cryopreserved hepatocytes, representing a significant reduction in prediction bias compared with human microsomes.Human liver microsomes have traditionally been the most commonly used in vitro system for the prediction of metabolic clearance, in particular, for new chemical entities within drug discovery programs in the pharmaceutical industry. However, the use of this system has typically resulted in an underestimation of clearance, as illustrated by a data set of 55 compounds in which a 9-fold under-prediction of the in vivo intrinsic clearance (CL int ) was observed . As a result of this, attention in recent years has been placed on the use of alternative in vitro systems for clearance prediction. Fresh human hepatocytes are envisaged as a potentially more accurate system because of the full complement of both phase I and phase II metabolizing enzymes, along with the presence of transporter proteins, which should result in drug concentrations within the hepatocyte that are equivalent to in vivo concentrations within the liver. However, the limited availability of fresh human tissue and the cost implications involved in the preparation of freshly isolated human hepatocytes have resulted in cryopreserved hepatocytes emerging as the favored alternative, which also have the added advantage of being readily available commercially and more convenient to use (Li et al., 1999).Two major issues concerning the use ...

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Section: Methodsmentioning

confidence: 99%

Evaluation of Cryopreserved Human Hepatocytes as an Alternative in Vitro System to Microsomes for the Prediction of Metabolic Clearance

Griffin²,

2006

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“…We postulate another reason that stems from the original method used to derive the standard MPPGL (40 mg/g liver tissue). Hakooz et al (2006) used total protein determination along with the P450 spectrum shift and cytochrome C reduction by P450 reductase to generate the relationship of MPPGL. This number is now used for scaling all liver microsomal content and has not been problematic because most research centers on drugs cleared by P450.…”

Section: Miyagi Et Almentioning

confidence: 99%

Neonatal Development of Hepatic UGT1A9: Implications of Pediatric Pharmacokinetics

Miyagi

Milne²,

Coughtrie³

et al. 2012

Drug Metab Dispos

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ABSTRACT:This article reports on the development of UDP-glucuronosyltransferase 1A9 (UGT1A9) in neonatal and pediatric liver. The substrate 4-methylumbelliferone (4MU) with specific inhibition by niflumic acid was used to define specific UGT1A9 activity. Subsequently, in silico pharmacokinetic (PK) and physiology-based pharmacokinetic (PBPK) modeling was used to determine UGT1A9 maturation and hepatic clearance. Modeled maximal enzyme activity was 27.9 nmol ⅐ min ؊1 ⅐ mg protein

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“…The CL int and CL max values were scaled to in vivo units using the scaling factors of 120 ϫ 10 6 hepatocytes/g of liver (Hakooz et al, 2006) and 21.4 g of liver/kg b.wt. (Davies and Morris, 1993).…”

Section: Donato Et Almentioning

confidence: 99%

Metabolite Formation Kinetics and Intrinsic Clearance of Phenacetin, Tolbutamide, Alprazolam, and Midazolam in Adenoviral Cytochrome P450-Transfected HepG2 Cells and Comparison with Hepatocytes and In Vivo

Donato¹,

Hallifax²,

Picazo³

et al. 2010

Drug Metab Dispos

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Determination of a Human Hepatic Microsomal Scaling Factor for Predicting in Vivo Drug Clearance

Abstract: A value for human liver microsomal scaling of 40 mg microsomal protein per gram liver has been established. The reason for underprediction previously reported for 52 different drug substrates was not the use of an incorrect value for the scaling factor.

Cited by 64 publications

References 22 publications

Evaluation of Cryopreserved Human Hepatocytes as an Alternative in Vitro System to Microsomes for the Prediction of Metabolic Clearance

Evaluation of Cryopreserved Human Hepatocytes as an Alternative in Vitro System to Microsomes for the Prediction of Metabolic Clearance

Neonatal Development of Hepatic UGT1A9: Implications of Pediatric Pharmacokinetics

Metabolite Formation Kinetics and Intrinsic Clearance of Phenacetin, Tolbutamide, Alprazolam, and Midazolam in Adenoviral Cytochrome P450-Transfected HepG2 Cells and Comparison with Hepatocytes and In Vivo

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