**4.2. Human liver microsomes (HLM)**

82 Chromatography – The Most Versatile Method of Chemical Analysis

by the transfer of methyl group from methionine [10].

**4. Models for evaluation of drug metabolism** 

studies will be presented.

herb-drug and food-drug interactions [15].

glucuronides and require uridine diphosphate glucuronic acid for glucuronide formation [6, 10]. SULT is the enzyme responsible for the formation of sulfate esters in the presence of co-substrate 3'-phospohoadenosine-5'-phosphosulfate (PAPS). Aromatic amines, hydrazines, sulfonamides and certain aliphatic amines are biotransformed to amides in a reaction catalyzed by N-acetyltransferase and utilize acetyl coencyme A as cofactor [6]. Another important conjugating reaction is a conjugation with glutathione which is present in many cells at high concentrations. Glutathione conjugation captures reactive electrophiles and transforms them to stable, often non-toxic tioethers [6]. Methylation is a process that results in a formation of O-, N- and S-methylated products

In this chapter different *in vitro* and *in vivo* models for the evaluation of drug metabolism are presented. Advantages and disadvantages of subcellular fractions (microsomes, recombinant enzymes, cytosolic liver fractions, liver S9 fraction), cellular fractions (isolated hepatocytes, immortal cell lines, liver slices, perfused liver), *in vivo* animal and human

Biotransformation occurs in liver, intestine, kidney, lungs, brain, nasal epithelium and skin. Since liver is the most important organ for drug metabolism [14, 15] the liver-based *in vitro* technologies for evaluation of drug metabolism are presented below. *In vitro* models that range from whole cell system to enzyme preparations are now increasingly applied for quantitative and qualitative assessment in preclinical drug development, post-approval routine checks, identification of metabolic determinant factors and prediction of drug-drug,

**4.1. Recombinant human CYP and UGT enzymes (supersomes, baculosomes)** 

resolve this problem a pore-forming agents such as alamethicin are used [14-18].

Recombinant human CYP and UGT enzymes have proven to be a useful tool in *in vitro* biotransformation studies. This *in vitro* model, referred to also as supersomes or baculosomes, is produced by transfection of insect cells with cDNA for human CYP and UGT by baculo virus, namely insect cells lack endogenous CYP and UGT activity. The advantage of this system is that enzyme activity of one single CYP or UGT isoform is expressed and therefore the assessment of individual metabolic enzyme and its contribution to the metabolic pathway could be performed. Additionaly, this *in vitro* system could be used also for the evaluation of drug-drug interactions. Moreover, due to availability of supersomes with different CYP and UGT genotypes, the influence of different polymorphisms on drug biotransformation could be estimated. Currently, all common human CYPs and UGTs co-expressed with NADPH-cytochrome P450 reductase are commercially available. The disadvantage of this *in vitro* model is the latency of glucuronidation because the active site of UGT is shielded behind a hydrophobic barrier. To HLM are vesicles of hepatocyte endoplasmic reticulum obtained by differential centrifugation of liver preparations (homogenates) from fresh human liver, liver slices, liver cell lines and primary hepatocytes. This subcellular fraction is a rich source of following enzymes: cytochrome P450s, flavin-monooxigenase (FMO), carboxyl esterases, epoxyde hydrolase and UGTs. Therefore, HLM are most frequently utilized *in vitro* model in drug metabolic profiling and drug interaction studies. Moreover, the influence of specific isoenzymes is studied using liver microsomes in the presence of specific inhibitors. There are interindividual variations in the activity of human liver microsomes; therefore they can be utilized also to study interindividual variability. In case of general estimation of drug metabolism, pooled microsomes from a large bank of individual liver tissues can be used to overcome the influence of interindividual variability. Microsomes from other human organs (intestine, kidney, lung) [19] are also available and are utilized to evaluate extrahepatic metabolism. Additionally, gender-specific microsomes are available for the estimation of gender-based discrepancies in drug biotransformation. In drug discovery process HLM are used for metabolite identification, evaluation of interspecies differences in drug metabolism, prediction of *in vivo* clearance, reaction phenotyping and metabolic pathway identification [14-18, 20].

NADPH or NRS is required in the incubation for the estimation of CYP activity. In order to evaluate the UGT activity UDPGA and alamethicin (pore-forming reagent) are required [14- 16].

The advantages of HLM are ease of use, low costs, best-characterized *in vitro* model for estimation of drug biotransformation, easy storage, appropriate for studying of interindividual and population-based variation, long term storage, provide qualitative estimations of *in vitro* drug metabolism, convenient tool for high throughput screening of compounds, appropriate for lead compound optimization studies and drug interaction studies. However, some disadvantages of HLM also exist. HLM are not appropriate for quantitative estimation of drug biotransformation because of absence of enzymes like NAT, GST and SULT and cofactors needed. This limits the expected metabolic competition and formation of some *in vivo* present metabolites. Another drawback is a very difficult assessment of the fraction of drug bound to plasma proteins versus to microsomes which is an important factor in the estimation of *in vivo* biotransformation [14-16, 18].
