Liver microsomes human experimental systems

Several kinetic parameters can be measured on different experimental systems to account for the interaction of a compound with CYPs. For example when studying the metabolic stability of a compound, it could be measured in a recombinant CYP system, in human liver microsomes, in hepatocytes and so on. Each system increases in biological complexity. Although in the recombinant CYP system only the cytochrome under consideration is studied, in the case of the human liver microsomes, there is a pool of enzyme present that includes several CYPs, and finally in the hepatocyte cell system, metabolizing enzymes play an important role in the metabolic compound stability. In addition, transport systems are also present that could involve recirculation or other transport phenomena. The more complex the experimental system, the more difficult it is to extract information on the protein/ligand interaction, albeit it is closer to the in vivo real situation and therefore to the mechanism that is actually working in the body. 

Typical experimental procedures are as follows The test drug candidate is incubated with pooled human liver microsomes (e.g., 1 mg protein/mL) that were previously preincubated with ABT (1 or 2 mM) for 30 minutes at (37 1)°C in the presence of an NADPH-generating system. Incubations of the drug candidate in the absence of ABT serve as controls. For hepatocytes, suspensions of freshly isolated or cryopreserved hepatocytes (lx 106 cells/ mL) are preincubated with 100-pM ABT for 30 minutes in 0.25 mL of Krebs-Henseleit buffer or Waymouth s medium (without phenol red) supplemented with FBS (4.5%), insulin (5.6 pg/mL), glutamine (3.6 mM), sodium pyruvate (4.5 mM), and dexamethasone (0.9 pM) at the final concentrations indicated. After the preincubation, the drug candidate is added to the incubation and the rate of metabolism of the drug candidate is compared in hepatocytes or microsomes with and without ABT treatment. A marked difference in metabolism caused by ABT is evidence that CYP plays a prominent role in the metabolism of the drug candidate. 

In vitro experimental system rCYP human liver microsomes, or hepatocytes. 

The toxicokinetics of MTBE have been studied in animal models, primarily rodents. The information available to date on the biological fate of ETBE and TAME indicates that their kinetics are expected to be similar to those of MTBE. This has been confirmed experimentally in rodents, in in vitro systems using liver microsome homogenates, and also in studies with human volunteers inhaling these fuel oxygenates while at rest or during light exercise. 

Oxidative cellular damage by reactive oxygen species such as superoxide anion, hydroperoxy and hyassociated with various human chronic diseases, e.g. cancers, inflammation, arthritis, atherosclerosis and also with the process of ageing. Claims that diet and increased intake of nutrients exhibiting antioxidative activity have a preventative effect on chronic diseases have increased in recent years. In this context, polyphenolic compounds such as tannins, flavonoids, coumarins, lignans and caffeic acid derivatives, which are abundantly contained in a large number of medicinal plants, foods and beverages, are of particular interest for human health care because of the antioxidative properties widely found in plant phenolics. The antioxidative activity of tannins has been extensively studied in various in vitro and in vivo experimental systems and summarized in reviews [96, 97]. Such activity includes the inhibition of lipid peroxidation induced by NADPH-ADP and ascorbic acid-ADP in rat liver microsomes and mitochondria, respectively... 

Perhaps the most important contribution to error in use of expressed cytochrome P450s is in the interpretation and extrapolation of generated data. The increased metabolic rate per milligram protein or per nanomole P450 relative to human liver microsomes can be overinterpreted in some cases where experimental conditions are not appropriately controlled. For example, there is potential for false positive results in some cases, where the rates of metabolic turnover for one enzyme may overestimate the contribution predicted from a single enzyme, based on subsequent data from other experimental systems such as human liver microsomes with selective chemical inhibitors. 

Because clearance at the whole-body level often is determined by metabolism at the cellular level, it is possible to use a variety of human-derived in vitro systems to determine rates of metabolism. These systems include pure human enzymes (such as cytochrome P450 enzymes) (13) and human liver subcellular fractions (S9 and microsomes) (14). However, with enzymes and subcellular fractions, some information is lost because the whole-cell integration of subcellular processes has been disrupted. The use of cultured human hepatocytes retains the whole-cell integration at the expense of greater experimental complexity (15). Each system provides a different window on the metabolic processes, is relatively easy to use, and can be obtained from commercial sources. Rates and pathways of metabolism may be compared with a series of discovery compounds to identify those with the greatest relative metabolic stability or with a benchmark compound of known human PK characteristics to provide a more absolute estimate of hepatic metabolic clearance. 

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