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R. Scott Obach - One of the best experts on this subject based on the ideXlab platform.
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Addressing the challenges of low Clearance in drug research
The AAPS journal, 2015Co-Authors: R. Scott ObachAbstract:As a result of high-throughput ADME screening, early metabolite identification, and exploration of novel chemical entities, low-Intrinsic-Clearance compounds continue to increase in drug discovery portfolios. Currently available in vitro tools have limited resolution below a certain Intrinsic Clearance value, which can lead to overestimation of Clearance and dose and underestimation of half-life. Significant advances have been made in recent years and novel approaches have been developed to address the challenges of low Clearance in drug discovery, such as the hepatocyte relay method, use of qNMR-based standards of biosynthesized drug metabolites to permit monitoring metabolite formation, coculture hepatocyte systems, and the time depending modeling approach. Future development in the field will enable faster, more precise, and lower cost profiling of the properties of low-Clearance compounds for Intrinsic Clearance, metabolite identification, and reaction phenotyping.
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In vitro-in vivo correlation for Intrinsic Clearance for CP-409,092 and sumatriptan: a case study to predict the in vivo Clearance for compounds metabolized by monoamine oxidase.
Xenobiotica; the fate of foreign compounds in biological systems, 2011Co-Authors: Amin Kamel, Kevin Colizza, Mithat Gunduz, Shawn Harriman, R. Scott ObachAbstract:Oxidative deamination of the GABAA partial agonist CP-409,092 and sumatriptan represents a major metabolic pathway and seems to play an important role for the Clearance of these two compounds.Similar to sumatriptan, human mitochondrial incubations with deprenyl and clorgyline, probe inhibitors of monoamine oxidase B and monoamine oxidase A (MAO-B and MAO-A), respectively, showed that CP-409,092 was metabolized to a large extent by the enzyme MAO-A.The metabolism of CP-409,092 and sumatriptan was therefore studied in human liver mitochondria and in vitro Intrinsic Clearance (CLint) values were determined and compared to the corresponding in vivo oral Clearance (CLPO) values. The overall objective was to determine whether an in vitro-in vivo correlation (IVIVC) could be described for compounds cleared by MAO-A.The Intrinsic Clearance, CLint, of CP-409,092 was approximately 4-fold greater than that of sumatriptan (CLint, values were calculated as 0.008 and 0.002 ml/mg/min for CP-409,092 and sumatriptan, resp...
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In Vitro-In Vivo Correlation for Intrinsic Clearance for Drugs Metabolized by Human Aldehyde Oxidase
Drug metabolism and disposition: the biological fate of chemicals, 2010Co-Authors: Michael Zientek, Ying Jiang, Kuresh Youdim, R. Scott ObachAbstract:The ability to predict in vivo Clearance from in vitro Intrinsic Clearance for compounds metabolized by aldehyde oxidase has not been demonstrated. To date, there is no established scaling method for predicting aldehyde oxidase-mediated Clearance using in vitro or animal data. This challenge is exacerbated by the fact that rats and dogs, two of the laboratory animal species commonly used to develop in vitro-in vivo correlations of Clearance, differ from humans with regard to expression of aldehyde oxidase. The objective of this investigation was to develop an in vitro-in vivo correlation of Intrinsic Clearance for aldehyde oxidase, using 11 drugs known to be metabolized by this enzyme. The set consisted of methotrexate, XK-469, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]pyrimidine (RS-8359), zaleplon, 6-deoxypenciclovir, zoniporide, O(6)-benzylguanine, N-[(2'-dimethylamino)ethyl]acridine-4-carboxamide (DACA), carbazeran, PF-4217903, and PF-945863. These compounds were assayed using two in vitro systems (pooled human liver cytosol and liver S-9 fractions) to calculate scaled unbound Intrinsic Clearance, and they were then compared with calculated in vivo unbound Intrinsic Clearance. The investigation provided a relative scale that can be used for in vitro-in vivo correlation of aldehyde oxidase Clearance and suggests limits as to when a potential new drug candidate that is metabolized by this enzyme will possess acceptable human Clearance, or when structural modification is required to reduce aldehyde oxidase catalyzed metabolism.
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Can in vitro metabolism-dependent covalent binding data in liver microsomes distinguish hepatotoxic from nonhepatotoxic drugs? An analysis of 18 drugs with consideration of Intrinsic Clearance and daily dose.
Chemical research in toxicology, 2008Co-Authors: R. Scott Obach, Amit S. Kalgutkar, John R. Soglia, Sabrina X. ZhaoAbstract:In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro Intrinsic Clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the approach still would falsely identify some agents as potentially hepatotoxic.
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Three-Dimensional Quantitative Structure Activity Relationship Computational Approaches for Prediction of Human In Vitro Intrinsic Clearance
The Journal of pharmacology and experimental therapeutics, 2000Co-Authors: Sean Ekins, R. Scott ObachAbstract:Future alternatives to the presently accepted in vitro paradigm of prediction of Intrinsic Clearance, which could be used earlier in the drug discovery process, would potentially accelerate efforts to identify better drug candidates with more favorable metabolic profiles and less likelihood of failure with regard to human pharmacokinetic attributes. In this study we describe two computational methods for modeling human microsomal and hepatocyte Intrinsic Clearance data derived from our laboratory and the literature, which utilize pharmacophore features or descriptors derived from molecular structure. Human microsomal Intrinsic Clearance data generated for 26 known therapeutic drugs were used to build computational models using commercially available software (Catalyst and Cerius(2)), after first converting the data to hepatocyte Intrinsic Clearance. The best Catalyst pharmacophore model gave an r of 0.77 for the observed versus predicted Clearance. This pharmacophore was described by one hydrogen bond acceptor, two hydrophobic features, and one ring aromatic feature essential to discriminate between high and low Intrinsic Clearance. The Cerius(2) quantitative structure activity relationship (QSAR) model gave an r(2) = 0.68 for the observed versus predicted Clearance and a cross-validated r(2) (q(2)) of 0.42. Similarly, literature data for human hepatocyte Intrinsic Clearance for 18 therapeutic drugs were also used to generate two separate models using the same computational approaches. The best Catalyst pharmacophore model gave an improved r of 0.87 and was described by two hydrogen bond acceptors, one hydrophobe, and 1 positive ionizable feature. The Cerius(2) QSAR gave an r(2) of 0.88 and a q(2) of 0.79. Each of these models was then used as a test set for prediction of the Intrinsic Clearance data in the other data set, with variable successes. These present models represent a preliminary application of QSAR software to modeling and prediction of human in vitro Intrinsic Clearance.
Robert T. Mingoia - One of the best experts on this subject based on the ideXlab platform.
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Intra- and Interlaboratory Reliability of a Cryopreserved Trout Hepatocyte Assay for the Prediction of Chemical Bioaccumulation Potential
Environmental science & technology, 2014Co-Authors: Kellie A. Fay, John W. Nichols, Diane L. Nabb, Robert T. Mingoia, Ina Goeritz, Alex D. Hoffman, Barbra D. Ferrell, Heather M. Peterson, Helmut Segner, Xing HanAbstract:Measured rates of Intrinsic Clearance determined using cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of improving modeled bioaccumulation predictions for fish. To date, however, the intra- and interlaboratory reliability of this procedure has not been determined. In the present study, three laboratories determined in vitro Intrinsic Clearance of six reference compounds (benzo[a]pyrene, 4-nonylphenol, di-tert-butyl phenol, fenthion, methoxychlor and o-terphenyl) by conducting substrate depletion experiments with cryopreserved trout hepatocytes from a single source. O-terphenyl was excluded from the final analysis due to nonfirst-order depletion kinetics and significant loss from denatured controls. For the other five compounds, intralaboratory variability (% CV) in measured in vitro Intrinsic Clearance values ranged from 4.1 to 30%, while interlaboratory variability ranged from 27 to 61%. Predicted bioconcentration factors based on in vitro Clearance values exhibited a reduced level of interlaboratory variability (5.3-38% CV). The results of this study demonstrate that cryopreserved trout hepatocytes can be used to reliably obtain in vitro Intrinsic Clearance of xenobiotics, which provides support for the application of this in vitro method in a weight-of-evidence approach to chemical bioaccumulation assessment.
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Liver microsomes and S9 from rainbow trout (Oncorhynchus mykiss): comparison of basal-level enzyme activities with rat and determination of xenobiotic Intrinsic Clearance in support of bioaccumulation assessment.
Environmental toxicology and chemistry, 2008Co-Authors: Xing Han, Diane L. Nabb, Ching-hui Yang, Suzanne I. Snajdr, Robert T. MingoiaAbstract:Metabolism plays an important role in bioaccumulation of xenobiotics in fish. The applicability of trout liver microsomes and S9 fraction in bioaccumulation assessment of xenobiotics in fish was investigated in the present study. Basal-level activities of 7-ethoxyresorufin-O-dealkylase, testosterone 6beta-hydroxylase, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase in trout liver microsomes and S9 were significantly lower than those in rat liver microsomes and S9. The in vitro-to- in vivo scaling factors, which are the values of liver microsomal and S9 protein contents per unit weight of trout liver, were determined to be 38.4 +/- 5.1 (mean +/- standard deviation throughout) and 95.9 +/- 11.9 mg/g, respectively. Intrinsic Clearance (CL(int)) values for a number of reference compounds obtained from trout liver S9 were lower than those from trout liver microsomes. After correction with the scaling factors, trout liver microsomes and S9 provided equivalent prediction of trout hepatic Clearance (CL(H)) using the well-stirred liver model, but their CL(H) values were significantly lower than those obtained from freshly isolated trout hepatocytes. Consequently, trout liver microsomes and S9 showed poorer prediction of the bioconcentration factors of the reference compounds compared with trout hepatocytes. Unit conversion revealed that CL(int) values obtained from trout liver microsomes and S9 were 6.3 to 22.4% of those from trout hepatocytes, which explained, to a large extent, the differences in their CL(H) and bioconcentration factor prediction.
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Xenobiotic Intrinsic Clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss): determination of trout hepatocellularity, optimization of cell concentrations and comparison of serum and serum-free incubations.
Aquatic toxicology (Amsterdam Netherlands), 2008Co-Authors: Xing Han, Diane L. Nabb, Robert T. Mingoia, Ching-hui Yang, Suzanne I. Snajdr, Robert A. HokeAbstract:Abstract Metabolism plays an important role in bioaccumulation of xenobiotics in fish. In vitro determination of xenobiotic Intrinsic Clearance (CL int ) in trout hepatocytes and subsequent extrapolation to in vivo hepatic Clearance (CL H ) using the “well-stirred” liver model greatly improved our current practice of bioaccumulation assessment [Han, X., Nabb, D.L., Mingoia, R.T., Yang, C.H., 2007. Determination of xenobiotic Intrinsic Clearance in freshly isolated hepatocytes from rainbow trout ( Oncorhynchus mykiss ) and rat and its application in bioaccumulation assessment. Environ. Sci. Technol. 41, 3269–3276]. In an effort to further optimize this approach, we experimentally obtained the value of trout hepatocellularity ( H T ), a key scaling factor in the “well-stirred” liver model. H T was determined to be (540 ± 12) × 10 6 cells/g liver for male trout. We also investigated the potential effect of different cell concentrations on the determination of CL int values of molinate, 4,4-bis(dimethylamino)benzophenone, 4-nonylphenol, 2,4-di- tert -butylphenol, and benzo(a)pyrene. Linear relationships were established between Clearance rates and cell concentrations at 1 × 10 6 , 2 × 10 6 , 5 × 10 6 , and 10 × 10 6 cells/mL. This suggests that under our experimental conditions, CL int determination was independent of hepatocyte concentrations. In order to better understand the “in vitro binding” effect in in vitro-to-in vivo scaling, we obtained CL int values for the above-mentioned compounds in trout hepatocytes that were suspended in trout serum. Incubations in serum, in general, resulted relatively larger prediction of CL H values. Our findings suggest that in bioaccumulation assessment, the traditional medium incubation method offers a conservative estimate on fish metabolism of xenobiotics and the serum incubation approach could be used for certain classes of compounds that are of challenge for in silico prediction of their plasma and in vitro binding properties.
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Determination of xenobiotic Intrinsic Clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat and its application in bioaccumulation assessment.
Environmental science & technology, 2007Co-Authors: Xing Han, Diane L. Nabb, Robert T. Mingoia, Ching-hui YangAbstract:Bioaccumulation in fish depends on the dynamics of various processes that involve fish uptake, storage, and elimination of xenobiotics. Elimination via fish biotransformation is a primary process that can be evaluated in an in vitro system to improve the performance of the prediction of xenobiotic bioaccumulation potentials. In this study, values of Intrinsic Clearance (CLint) of seven reference compounds (atrazine, molinate, 4,4-bis(dimethylamino)-benzophenone, 4-nonylphenol, 2,4-di-tert-butylphenol, trifluralin, benzo(a)pyrene) in hepatocytes freshly isolated from rainbow trout and rat were determined using a substrate depletion approach. Atrazine was metabolized in rat hepatocytes with a CLint value of 3.81 +/- 1.96 mL/h/ 10(6) cells, whereas in trout hepatocytes, the Clearance was not significant until very high cell concentration was used and the rate was estimated to be approximately 0.002 mL/h/10(6) cells. Intrinsic Clearance values for all other compounds were 5.5-78.5-fold lower in trout hepatocytes than those in rat hepatocytes. Trout hepatic Clearance (CL(H)) values were extrapolated from the CLint values using a "well-stirred" liver model. Biotransformation rate constants (kMET) of the compounds in trout were subsequently estimated and used as inputs to a kinetic model for the prediction of bioconcentration factors (BCF) in fish. Compared to the BCF values predicted without consideration of fish biotransformation, the inclusion of estimated kMET values significantly improved fish BCF predictions for the reference compounds. This study demonstrates a framework for future bioaccumulation assessment of xenobiotics using combined information of the physical-chemical properties of the compounds and the biotransformation potentials of the compounds in fish.
Diane L. Nabb - One of the best experts on this subject based on the ideXlab platform.
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reliability of in vitro methods used to measure Intrinsic Clearance of hydrophobic organic chemicals by rainbow trout results of an international ring trial
Toxicological Sciences, 2018Co-Authors: John W. Nichols, Kellie A. Fay, Mary Jo Bernhard, Ina Bischof, John W. Davis, Marlies Halder, Karla Johanning, Heike Laue, Diane L. Nabb, Christian SchlechtriemAbstract:In vitro assays are widely employed to obtain Intrinsic Clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure Intrinsic Clearance in rainbow trout. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was characterized as the percent coefficient of variation (CV) in measured in vitro Intrinsic Clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells) for each chemical and test system. Mean intralaboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, whereas interlaboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. When CLIN VITRO, INT values were extrapolated to in vivo Intrinsic Clearance estimates (CLIN VIVO, INT; l/d/kg fish), both assays yielded similar levels of activity (<4-fold difference for all chemicals). Hepatic Clearance rates (CLH; l/d/kg fish) calculated using data from both assays exhibited even better agreement. These findings show that both assays are highly reliable and suggest that either may be used to inform chemical bioaccumulation assessments for fish. This study highlights several issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays.
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Reliability of In Vitro Methods Used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial
Toxicological sciences : an official journal of the Society of Toxicology, 2018Co-Authors: John W. Nichols, Kellie A. Fay, Mary Jo Bernhard, Ina Bischof, John W. Davis, Marlies Halder, Karla Johanning, Heike Laue, Diane L. NabbAbstract:In vitro assays are widely employed to obtain Intrinsic Clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure Intrinsic Clearance in rainbow trout. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was characterized as the percent coefficient of variation (CV) in measured in vitro Intrinsic Clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells) for each chemical and test system. Mean intralaboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, whereas interlaboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. When CLIN VITRO, INT values were extrapolated to in vivo Intrinsic Clearance estimates (CLIN VIVO, INT; l/d/kg fish), both assays yielded similar levels of activity (
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Intra- and Interlaboratory Reliability of a Cryopreserved Trout Hepatocyte Assay for the Prediction of Chemical Bioaccumulation Potential
Environmental science & technology, 2014Co-Authors: Kellie A. Fay, John W. Nichols, Diane L. Nabb, Robert T. Mingoia, Ina Goeritz, Alex D. Hoffman, Barbra D. Ferrell, Heather M. Peterson, Helmut Segner, Xing HanAbstract:Measured rates of Intrinsic Clearance determined using cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of improving modeled bioaccumulation predictions for fish. To date, however, the intra- and interlaboratory reliability of this procedure has not been determined. In the present study, three laboratories determined in vitro Intrinsic Clearance of six reference compounds (benzo[a]pyrene, 4-nonylphenol, di-tert-butyl phenol, fenthion, methoxychlor and o-terphenyl) by conducting substrate depletion experiments with cryopreserved trout hepatocytes from a single source. O-terphenyl was excluded from the final analysis due to nonfirst-order depletion kinetics and significant loss from denatured controls. For the other five compounds, intralaboratory variability (% CV) in measured in vitro Intrinsic Clearance values ranged from 4.1 to 30%, while interlaboratory variability ranged from 27 to 61%. Predicted bioconcentration factors based on in vitro Clearance values exhibited a reduced level of interlaboratory variability (5.3-38% CV). The results of this study demonstrate that cryopreserved trout hepatocytes can be used to reliably obtain in vitro Intrinsic Clearance of xenobiotics, which provides support for the application of this in vitro method in a weight-of-evidence approach to chemical bioaccumulation assessment.
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Liver microsomes and S9 from rainbow trout (Oncorhynchus mykiss): comparison of basal-level enzyme activities with rat and determination of xenobiotic Intrinsic Clearance in support of bioaccumulation assessment.
Environmental toxicology and chemistry, 2008Co-Authors: Xing Han, Diane L. Nabb, Ching-hui Yang, Suzanne I. Snajdr, Robert T. MingoiaAbstract:Metabolism plays an important role in bioaccumulation of xenobiotics in fish. The applicability of trout liver microsomes and S9 fraction in bioaccumulation assessment of xenobiotics in fish was investigated in the present study. Basal-level activities of 7-ethoxyresorufin-O-dealkylase, testosterone 6beta-hydroxylase, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase in trout liver microsomes and S9 were significantly lower than those in rat liver microsomes and S9. The in vitro-to- in vivo scaling factors, which are the values of liver microsomal and S9 protein contents per unit weight of trout liver, were determined to be 38.4 +/- 5.1 (mean +/- standard deviation throughout) and 95.9 +/- 11.9 mg/g, respectively. Intrinsic Clearance (CL(int)) values for a number of reference compounds obtained from trout liver S9 were lower than those from trout liver microsomes. After correction with the scaling factors, trout liver microsomes and S9 provided equivalent prediction of trout hepatic Clearance (CL(H)) using the well-stirred liver model, but their CL(H) values were significantly lower than those obtained from freshly isolated trout hepatocytes. Consequently, trout liver microsomes and S9 showed poorer prediction of the bioconcentration factors of the reference compounds compared with trout hepatocytes. Unit conversion revealed that CL(int) values obtained from trout liver microsomes and S9 were 6.3 to 22.4% of those from trout hepatocytes, which explained, to a large extent, the differences in their CL(H) and bioconcentration factor prediction.
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Xenobiotic Intrinsic Clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss): determination of trout hepatocellularity, optimization of cell concentrations and comparison of serum and serum-free incubations.
Aquatic toxicology (Amsterdam Netherlands), 2008Co-Authors: Xing Han, Diane L. Nabb, Robert T. Mingoia, Ching-hui Yang, Suzanne I. Snajdr, Robert A. HokeAbstract:Abstract Metabolism plays an important role in bioaccumulation of xenobiotics in fish. In vitro determination of xenobiotic Intrinsic Clearance (CL int ) in trout hepatocytes and subsequent extrapolation to in vivo hepatic Clearance (CL H ) using the “well-stirred” liver model greatly improved our current practice of bioaccumulation assessment [Han, X., Nabb, D.L., Mingoia, R.T., Yang, C.H., 2007. Determination of xenobiotic Intrinsic Clearance in freshly isolated hepatocytes from rainbow trout ( Oncorhynchus mykiss ) and rat and its application in bioaccumulation assessment. Environ. Sci. Technol. 41, 3269–3276]. In an effort to further optimize this approach, we experimentally obtained the value of trout hepatocellularity ( H T ), a key scaling factor in the “well-stirred” liver model. H T was determined to be (540 ± 12) × 10 6 cells/g liver for male trout. We also investigated the potential effect of different cell concentrations on the determination of CL int values of molinate, 4,4-bis(dimethylamino)benzophenone, 4-nonylphenol, 2,4-di- tert -butylphenol, and benzo(a)pyrene. Linear relationships were established between Clearance rates and cell concentrations at 1 × 10 6 , 2 × 10 6 , 5 × 10 6 , and 10 × 10 6 cells/mL. This suggests that under our experimental conditions, CL int determination was independent of hepatocyte concentrations. In order to better understand the “in vitro binding” effect in in vitro-to-in vivo scaling, we obtained CL int values for the above-mentioned compounds in trout hepatocytes that were suspended in trout serum. Incubations in serum, in general, resulted relatively larger prediction of CL H values. Our findings suggest that in bioaccumulation assessment, the traditional medium incubation method offers a conservative estimate on fish metabolism of xenobiotics and the serum incubation approach could be used for certain classes of compounds that are of challenge for in silico prediction of their plasma and in vitro binding properties.
Xing Han - One of the best experts on this subject based on the ideXlab platform.
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Intra- and Interlaboratory Reliability of a Cryopreserved Trout Hepatocyte Assay for the Prediction of Chemical Bioaccumulation Potential
Environmental science & technology, 2014Co-Authors: Kellie A. Fay, John W. Nichols, Diane L. Nabb, Robert T. Mingoia, Ina Goeritz, Alex D. Hoffman, Barbra D. Ferrell, Heather M. Peterson, Helmut Segner, Xing HanAbstract:Measured rates of Intrinsic Clearance determined using cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of improving modeled bioaccumulation predictions for fish. To date, however, the intra- and interlaboratory reliability of this procedure has not been determined. In the present study, three laboratories determined in vitro Intrinsic Clearance of six reference compounds (benzo[a]pyrene, 4-nonylphenol, di-tert-butyl phenol, fenthion, methoxychlor and o-terphenyl) by conducting substrate depletion experiments with cryopreserved trout hepatocytes from a single source. O-terphenyl was excluded from the final analysis due to nonfirst-order depletion kinetics and significant loss from denatured controls. For the other five compounds, intralaboratory variability (% CV) in measured in vitro Intrinsic Clearance values ranged from 4.1 to 30%, while interlaboratory variability ranged from 27 to 61%. Predicted bioconcentration factors based on in vitro Clearance values exhibited a reduced level of interlaboratory variability (5.3-38% CV). The results of this study demonstrate that cryopreserved trout hepatocytes can be used to reliably obtain in vitro Intrinsic Clearance of xenobiotics, which provides support for the application of this in vitro method in a weight-of-evidence approach to chemical bioaccumulation assessment.
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Liver microsomes and S9 from rainbow trout (Oncorhynchus mykiss): comparison of basal-level enzyme activities with rat and determination of xenobiotic Intrinsic Clearance in support of bioaccumulation assessment.
Environmental toxicology and chemistry, 2008Co-Authors: Xing Han, Diane L. Nabb, Ching-hui Yang, Suzanne I. Snajdr, Robert T. MingoiaAbstract:Metabolism plays an important role in bioaccumulation of xenobiotics in fish. The applicability of trout liver microsomes and S9 fraction in bioaccumulation assessment of xenobiotics in fish was investigated in the present study. Basal-level activities of 7-ethoxyresorufin-O-dealkylase, testosterone 6beta-hydroxylase, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase in trout liver microsomes and S9 were significantly lower than those in rat liver microsomes and S9. The in vitro-to- in vivo scaling factors, which are the values of liver microsomal and S9 protein contents per unit weight of trout liver, were determined to be 38.4 +/- 5.1 (mean +/- standard deviation throughout) and 95.9 +/- 11.9 mg/g, respectively. Intrinsic Clearance (CL(int)) values for a number of reference compounds obtained from trout liver S9 were lower than those from trout liver microsomes. After correction with the scaling factors, trout liver microsomes and S9 provided equivalent prediction of trout hepatic Clearance (CL(H)) using the well-stirred liver model, but their CL(H) values were significantly lower than those obtained from freshly isolated trout hepatocytes. Consequently, trout liver microsomes and S9 showed poorer prediction of the bioconcentration factors of the reference compounds compared with trout hepatocytes. Unit conversion revealed that CL(int) values obtained from trout liver microsomes and S9 were 6.3 to 22.4% of those from trout hepatocytes, which explained, to a large extent, the differences in their CL(H) and bioconcentration factor prediction.
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Xenobiotic Intrinsic Clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss): determination of trout hepatocellularity, optimization of cell concentrations and comparison of serum and serum-free incubations.
Aquatic toxicology (Amsterdam Netherlands), 2008Co-Authors: Xing Han, Diane L. Nabb, Robert T. Mingoia, Ching-hui Yang, Suzanne I. Snajdr, Robert A. HokeAbstract:Abstract Metabolism plays an important role in bioaccumulation of xenobiotics in fish. In vitro determination of xenobiotic Intrinsic Clearance (CL int ) in trout hepatocytes and subsequent extrapolation to in vivo hepatic Clearance (CL H ) using the “well-stirred” liver model greatly improved our current practice of bioaccumulation assessment [Han, X., Nabb, D.L., Mingoia, R.T., Yang, C.H., 2007. Determination of xenobiotic Intrinsic Clearance in freshly isolated hepatocytes from rainbow trout ( Oncorhynchus mykiss ) and rat and its application in bioaccumulation assessment. Environ. Sci. Technol. 41, 3269–3276]. In an effort to further optimize this approach, we experimentally obtained the value of trout hepatocellularity ( H T ), a key scaling factor in the “well-stirred” liver model. H T was determined to be (540 ± 12) × 10 6 cells/g liver for male trout. We also investigated the potential effect of different cell concentrations on the determination of CL int values of molinate, 4,4-bis(dimethylamino)benzophenone, 4-nonylphenol, 2,4-di- tert -butylphenol, and benzo(a)pyrene. Linear relationships were established between Clearance rates and cell concentrations at 1 × 10 6 , 2 × 10 6 , 5 × 10 6 , and 10 × 10 6 cells/mL. This suggests that under our experimental conditions, CL int determination was independent of hepatocyte concentrations. In order to better understand the “in vitro binding” effect in in vitro-to-in vivo scaling, we obtained CL int values for the above-mentioned compounds in trout hepatocytes that were suspended in trout serum. Incubations in serum, in general, resulted relatively larger prediction of CL H values. Our findings suggest that in bioaccumulation assessment, the traditional medium incubation method offers a conservative estimate on fish metabolism of xenobiotics and the serum incubation approach could be used for certain classes of compounds that are of challenge for in silico prediction of their plasma and in vitro binding properties.
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Determination of xenobiotic Intrinsic Clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat and its application in bioaccumulation assessment.
Environmental science & technology, 2007Co-Authors: Xing Han, Diane L. Nabb, Robert T. Mingoia, Ching-hui YangAbstract:Bioaccumulation in fish depends on the dynamics of various processes that involve fish uptake, storage, and elimination of xenobiotics. Elimination via fish biotransformation is a primary process that can be evaluated in an in vitro system to improve the performance of the prediction of xenobiotic bioaccumulation potentials. In this study, values of Intrinsic Clearance (CLint) of seven reference compounds (atrazine, molinate, 4,4-bis(dimethylamino)-benzophenone, 4-nonylphenol, 2,4-di-tert-butylphenol, trifluralin, benzo(a)pyrene) in hepatocytes freshly isolated from rainbow trout and rat were determined using a substrate depletion approach. Atrazine was metabolized in rat hepatocytes with a CLint value of 3.81 +/- 1.96 mL/h/ 10(6) cells, whereas in trout hepatocytes, the Clearance was not significant until very high cell concentration was used and the rate was estimated to be approximately 0.002 mL/h/10(6) cells. Intrinsic Clearance values for all other compounds were 5.5-78.5-fold lower in trout hepatocytes than those in rat hepatocytes. Trout hepatic Clearance (CL(H)) values were extrapolated from the CLint values using a "well-stirred" liver model. Biotransformation rate constants (kMET) of the compounds in trout were subsequently estimated and used as inputs to a kinetic model for the prediction of bioconcentration factors (BCF) in fish. Compared to the BCF values predicted without consideration of fish biotransformation, the inclusion of estimated kMET values significantly improved fish BCF predictions for the reference compounds. This study demonstrates a framework for future bioaccumulation assessment of xenobiotics using combined information of the physical-chemical properties of the compounds and the biotransformation potentials of the compounds in fish.
Fumihiro Nakamori - One of the best experts on this subject based on the ideXlab platform.
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Correlation of Intrinsic in vitro and in vivo Clearance for drugs metabolized by hepatic UDP-glucuronosyltransferases in rats.
Drug metabolism and pharmacokinetics, 2011Co-Authors: Fumihiro Nakamori, Yoichi Naritomi, Shigeyuki Terashita, Masako Furutani, Fujiko Takamura, Hiroya Miura, Hidetsugu Murai, Toshio TeramuraAbstract:A method for quantitatively predicting the hepatic Clearance of drugs by UDP-glucuronosyltransferases (UGTs) from in vitro data has not yet been established. We examined the relationship between in vitro and in vivo Intrinsic Clearance by rat hepatic UGTs using 10 drugs. For these 10 drugs, the in vitro Intrinsic Clearance by UGTs (CL(int, in vitro)) measured using alamethicin-activated rat liver microsomes was in the range 0.10-4500 ml/min/kg. Microsomal binding (f(u, mic)) was determined to be in the range 0.29-0.95 and the unbound Intrinsic Clearance (CL(uint, in vitro)) to be in the range 0.11-9600 ml/min/kg. The contribution of rat hepatic glucuronidation to drug elimination was 12.0%-76.6% and in vivo Intrinsic Clearance by UGTs was 5.7-9000 ml/min/kg. To evaluate the discrepancy between the in vitro and in vivo values, a scaling factor was calculated (CL(int, in vivo)/CL(int, in vitro)); the values were found to be in the range 0.89-110. The average fold error of the scaling factor values incorporating f(u, mic) was closer to unity than that without f(u, mic). The scaling factor values incorporating f(u, mic) were
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correlation of Intrinsic in vitro and in vivo Clearance for drugs metabolized by hepatic udp glucuronosyltransferases in rats
Drug Metabolism and Pharmacokinetics, 2011Co-Authors: Yoichi Naritomi, Shigeyuki Terashita, Fumihiro Nakamori, Masako Furutani, Fujiko Takamura, Hiroya Miura, Hidetsugu Murai, Toshio TeramuraAbstract:Summary: A method for quantitatively predicting the hepatic Clearance of drugs by UDP-glucuronosyltransferases (UGTs) from in vitro data has not yet been established. We examined the relationship between in vitro and in vivo Intrinsic Clearance by rat hepatic UGTs using 10 drugs. For these 10 drugs, the in vitro Intrinsic Clearance by UGTs (CL int, in vitro ) measured using alamethicin-activated rat liver microsomes was in the range 0.10–4500 ml/min/kg. Microsomal binding (f u, mic ) was determined to be in the range 0.29–0.95 and the unbound Intrinsic Clearance (CL uint, in vitro ) to be in the range 0.11–9600 ml/min/kg. The contribution of rat hepatic glucuronidation to drug elimination was 12.0%–76.6% and in vivo Intrinsic Clearance by UGTs was 5.7–9000 ml/min/kg. To evaluate the discrepancy between the in vitro and in vivo values, a scaling factor was calculated (CL int, in vivo /CL int, in vitro ); the values were found to be in the range 0.89–110. The average fold error of the scaling factor values incorporating f u, mic was closer to unity than that without f u, mic . The scaling factor values incorporating f u, mic were in vitro and in vivo values. Thus, using alamethicin-activated liver microsomes, incorporating f u, mic into CL int, in vitro , and considering the contribution of glucuronidation may enable us to quantitatively predict in vivo hepatic glucuronidation from in vitro data.
