Validation of a semi-automated human hepatocyte assay for the determination and prediction of intrinsic clearance in discovery

Reddy, A. Rajasekhar; Heimbach, Tycho; Freiwald, Sascha; Smith, Danielle; Winters, R. Thomas; Michael, Steven; Surendran, Narayanan; Cai, Hongliang

doi:10.1016/j.jpba.2004.09.030

Cited by 29 publications

(16 citation statements)

References 16 publications

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“…Therefore activation of UGT enzymes provided no benefits with respect to predictivity for these reference drugs. For these compounds it is possible that the Lave et al (1997); 7, Ekins and Obach (2000); 8, Soars et al (2002); 9, Reddy et al (2005); 10, Blanchard et al (2006); and 11, Blanchard et al (2005).…”

Section: Discussionmentioning

confidence: 99%

Reliability of human cryopreserved hepatocytes and liver microsomes asin vitrosystems to predict metabolic clearance

2008

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A total of 110 drugs, selected to cover a range of physicochemical and pharmacokinetic properties, were used to explore standard approaches to the prediction of in vivo metabolic clearance using drugdepletion profiles from human liver microsomes (HLMs) and cyropreserved hepatocytes. A total of 41 drugs (37% of the compounds tested) showed measurable depletion rates using HLMs (depletion by 20% or more over the time course). The most reliable correlations in terms of bias (average fold error (AFE) ¼ 2.32) and precision (root mean square error (RMSE) ¼ 3501) were observed by comparing in vivo intrinsic clearance (CL int ), calculated using the parallel-tube model and incorporating the fraction unbound in blood, with in vitro CL int adjusted for microsomal binding. For these reference drugs, 29% of predictions were within two-fold of the observed values and 66% were within five-fold. Compared with HLMs, clearance predictions with cryopreserved hepatocytes (57 drugs) were of similar precision (RMSE ¼ 3608) but showed more bias (AFE ¼ 5.21) with 18% of predictions within two-fold of the observed values and 46% within five-fold. However, with a broad complement of drug-metabolizing enzymes, hepatocytes catalysed measurable CL int values for a greater proportion (52%) of the reference compounds and were particularly proficient at defining metabolic rates for drugs with predominantly phase 2 metabolic routes.

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Section: Discussionmentioning

confidence: 99%

Reliability of human cryopreserved hepatocytes and liver microsomes asin vitrosystems to predict metabolic clearance

2008

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“…Human hepatocytes in suspension cultures lasting several hours have been utilized in 96-well and 384-well formats for determining the metabolic clearance of drugs. 37,38 Further, Wolff et al have established a method for plated rat hepatocytes cultured for multiple days in 384-well format for high content screening (HCS) to monitor cellular functions. 39 However, no published reports have described employing cultured hepatocytes in 1536-well format.…”

Section: Introductionmentioning

confidence: 99%

Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

Moeller¹,

Shukla

Xia

2012

ASSAY and Drug Development Technologies

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Hepatotoxicity is a major concern for both drug development and toxicological evaluation of environmental chemicals. The assessment of compound-induced hepatotoxicity has traditionally relied on in vivo testing; however, it is being replaced by human in vitro models due to an emphasis on the reduction of animal testing and species-specific differences. Since most cell lines and hybridomas lack the full complement of enzymes at physiological levels found in the liver, primary hepatocytes are the gold standard to study liver toxicities in vitro due to the retention of most of their in vivo activities. Here, we optimized a cell viability assay using plateable cryopreserved human hepatocytes in a 1536-well-plate format. The assay was validated by deriving inhibitory concentration at 50% values for 12 known compounds, including tamoxifen, staurosporine, and phenylmercuric acetate, with regard to hepatotoxicity and general cytotoxicity using multiple hepatocyte donors. The assay performed well, and the cytotoxicity of these compounds was confirmed in comparison to HepG2 cells. This is the first study to report the reliability of using plateable cryopreserved human hepatocytes for cytotoxicity studies in a 1536-well-plate format. These results suggest that plateable cryopreserved human hepatocytes can be scaled up for screening a large compound library and may be amenable to other hepatocytic assays such as metabolic or drug safety studies.

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“…The metabolic kinetic study of CMDCK was subsequently conducted at an enzyme content of 0.5 mg/mL, a substrate concentration of 4 μmol/L and an incubation time of 30 min. As the key parameter for the in vitro-in vivo correlation, the intrinsic clearance (CL int ) for CMDCK was directly obtained from the in vitro T 1/2 [15,16] , based on the widely accepted well-stirred model. The hepatic clearance (CL h ) was then estimated using in vitro CL int data ( Table 2).…”

Section: Discussionmentioning

confidence: 99%

Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes

Zhuang¹,

Deng²,

Li³

et al. 2011

Acta Pharmacol Sin

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Aim:To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK), a novel anti-HIV agent, by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs). Methods: CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5. LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites. In the experiments with recombinant cytochrome P450 enzymes, specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism. Results: CMDCK was rapidly and extensively metabolized by HLMs. Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL·min -1 ·kg -1 , which was comparable to the mean human hepatic blood flow rate (20.7 mL·min -1 ·kg -1 ). The major metabolic pathway of CMDCK was oxidation, and a total of 14 metabolites were detected. CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism. Conclusion: CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites. CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.

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Validation of a semi-automated human hepatocyte assay for the determination and prediction of intrinsic clearance in discovery

Cited by 29 publications

References 16 publications

Reliability of human cryopreserved hepatocytes and liver microsomes asin vitrosystems to predict metabolic clearance

Reliability of human cryopreserved hepatocytes and liver microsomes asin vitrosystems to predict metabolic clearance

Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes

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