Metabolism, in vitro

Microsomes Can be frozen and used on demand Contains major drug metabolizing enzymes Straighforward to use Limited enzyme complement Closed system so may not be representative of in vivo situation 

Hepatocytes Culture methods available Full enzyme complement - contain enzymes and enzyme cofactors not present in subcellular fractions Full enzyme complement - contain enzymes and enzyme cofactors not present in subcellular fractions Can be used quantitatively Useful for prediction of Phase 1 and 11 metabolism Require fresh tissue No cell-cell contact Need collagenase digestion Handling difficult Closed system so may not be representative of in vivo situation 

Liver slices Easy to prepare Cell-cell contact No use of proteolytic enzymes All cell types present Phase 1 and 11 metabolic processes accessible Require fresh tissue Necrosis of slice centre Long-term culture difficult Quantitative studies variable 

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Walker, C.H. (1981). The correlation between in vivo and in vitro metabolism of pesticides in vertebrates. Progress in Pesticide Biochemistry 1, 247-286. 

Obach RS, Baxter JG, Liston TE, Silber BM, Jones BC, MacIntyre F, Ranee DJ, Wastall P. The prediction of human pharmacokinetic parameters from precl-inical and in vitro metabolism data. J Pharmacol Exp Ther 1997 Oct 283(l) 46-58... 

In vitro metabolism of pH]PbTx-3 was studi in isolated rat hepatocytes (25). Hepatocyte monolayers cultured in 6-well plates containing 1 ml modified Williams E medium were incubated with 0.1 fig radiolabeled toxin at 37 C for 24 hr. The... 

By applying an extension of the clearance concept 30, 31), in vitro metabolism was used to predict in vivo toxin elimination. Hepatocytes were incubated with 0.5 to 10 pg unlabeled PbTx-3 containing 0.1 pg radiolabeled toxin as tracer. Disappearance of parent compound and the appearance of metabolites were measured by HPLC equipped with a Radiomatic isotope detector. (1.6 nmol/min/g liver)... 

Data from both in vivo and in vitro systems showed PbTx-3 to have an intermediate extraction ratio, indicating in vivo clearance of PbTx-3 was equally dependent upon liver blood flow and the activity of toxin-metabolizing enzymes. Studies on the effects of varying flow rates and metabolism on the total body clearance of PbTx-3 are planned. Finally, comparison of in vivo metabolism data to those derived from in vitro metabolism in isolated perfused livers and isolated hepatocytes suggested that in vitro systems accurately reflect in vivo metabolic processes and can be used to predict the toxicokinetic parameters of PbTx-3. 

Magee, P.N. in de Serres, F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. (eds), In Vitro Metabolic Activation in Mutagenesis Testing , Elsevier/North Holland Biomedical Press, Amsterdam, 1976 pp. 213-216. 

The second study was performed using either cytosolic or microsomal fractions from rat liver as the in vitro metabolic mammal models [238]. The studied compound, benzofuroxan (128, Fig. 20), is metabolized to o-quinone dioxime and 2,3-diaminophenazine (Scheme 4). 

Yeum, K. J., Y. C. Leekim et al. (1994). In vitro metabolism of beta-carotene by lipoxygenase and human stomach mucosal homogenates. FASEB J. 8(4) A192-A192. 

Among common in vitro metabolizing systems, liver microsomes and liver S9 fractions are used more often in metabolite synthesis than other systems. The majority of drug metabolism is mediated by CYPs [8]. Liver microsomes contain a high concentration of CYPs and other... 

Unfortunately, when clearance is largely metabolic and low, allometry can significantly over-predict the human value [71]. Recent investigations have attempted to address this by combining allometric approaches with in vitro metabolism data [5],... 

T. E., Silber, M., Jones, B. C., MacIntyre, F., Rance, D. J., Wastall, P., The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data, J. Pharmacol. Exp. Ther. 1997,... 

Davit, B., Reynolds, K., Yuan, R., Ajayi, F., Conner, D. et al., FDA evaluations using in vitro metabolism to predict and interpret in vivo metabolic drug-drug interactions impact on labeling, J. Clin. Pharmacol. 

Obach et al. [27] proposed a model to predict human bioavailability from a retrospective study of in vitro metabolism and in vivo animal pharmacokinetic (PK) data. While their model yielded acceptable predictions (within a factor of 2) for an expansive group of compounds, it relied extensively on in vivo animal PK data for interspecies scaling in order to estimate human PK parameters. Animal data are more time-consuming and costly to obtain than are permeability and metabolic clearance data hence, this approach may be limited to the later stages of discovery support when the numbers of compounds being evaluated are fewer. 

M. E., Veski, P., Lennernas, H., Friedrichsen, G., Steffansen, B., Stability and in vitro metabolism of dipeptide model prodrugs with affinity for the oligopeptide transporter, Eur. J. Pharm. Sci. 2000, 11, 43-50. 

Lin G, Cui Y-Y, Liu X-Q and Wang Z-T (2002b), Species differences in the in vitro metabolic activation of the hepatotoxic pyrrolizidine alkaloids clivorine , Chem Res Toxicol, 15, 1421-1428. 

Guengerich FP, Geiger LE, Hogy LL, et al. 1981. In vitro metabolism of acrylonitrile to 2-cyanoethylene oxide, reaction with glutathione, and irreversible binding to proteins and nucleic acids. Cancer Res 41 4925-4933. 

Tanii H, Hashimoto K. 1986. Influence of ethanol on the in vivo and in vitro metabolism of nitriles in mice. Arch Toxicol 58 171-176. 

The 8,9- and 10,11-dihydrodiols formed in the metabolism of BA and DMBA respectively are all highly enriched (>90%) in R,R enantiomers (Table III). Labeling experiments using molecular oxygen-18 in the in vitro metabolism of the respective parent compounds and subsequent mass spectral analyses of dihydrodiol metabolites and their acid-catalyzed dehydration products indicated that microsomal epoxide hydrolase-catalyzed hydration reactions occurred exclusively at the nonbenzylic carbons of the metabolically formed epoxide intermediates (unpublished results). These findings indicate that the 8,9- and 10,11-epoxide intermediates, formed in the metabolism of BA and DMBA respectively, contain predominantly the 8R,9S and 10S,11R enantiomer, respectively. These stereoselective epoxidation reactions are relatively insensitive to the cytochrome P-450 isozyme contents of different rat liver microsomal preparations (Table III). 

Nitrosoarenes are readily formed by the oxidation of primary N-hydroxy arylamines and several mechanisms appear to be involved. These include 1) the metal-catalyzed oxidation/reduction to nitrosoarenes, azoxyarenes and arylamines (144) 2) the 02-dependent, metal-catalyzed oxidation to nitrosoarenes (145) 3) the 02-dependent, hemoglobin-mediated co-oxidation to nitrosoarenes and methe-moglobin (146) and 4) the 0 2-dependent conversion of N-hydroxy arylamines to nitrosoarenes, nitrosophenols and nitroarenes (147,148). Each of these processes can involve intermediate nitroxide radicals, superoxide anion radicals, hydrogen peroxide and hydroxyl radicals, all of which have been observed in model systems (149,151). Although these radicals are electrophilic and have been suggested to result in DNA damage (151,152), a causal relationship has not yet been established. Nitrosoarenes, on the other hand, are readily formed in in vitro metabolic incubations (2,153) and have been shown to react covalently with lipids (154), proteins (28,155) and GSH (17,156-159). Nitrosoarenes are also readily reduced to N-hydroxy arylamines by ascorbic acid (17,160) and by reduced pyridine nucleotides (9,161). 

The In Vitro Metabolic Activation of Nitro Polycyclic Aromatic Hydrocarbons... 

The In vitro metabolic reduction of nitro PAHs was first reported in 1967 (124). Since then a number of similar investigations have been conducted however, most of these studies have been performed under only anaerobic conditions. The oxidative metabolism of nitro PAHs has been examined only recently, but as considered in the following sections, both reductive and oxidative metabolic pathways may be important in the metabolic activation of nitro PAHs. 

Dauterman, W.C. and W. Muecke. 1974. In vitro metabolism of atrazine by rat liver. Pestic. Biochem. Physiol. 4 212-219. 

Gill, S.J. 1980. In vitro metabolism of carbofuran by liver microsomes of the padifield fish Trichogaster pectoralis. Bull. Environ. Contam. Toxicol. 25 697-701. 

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