**3. Drug metabolic pathways**

80 Chromatography – The Most Versatile Method of Chemical Analysis

toxic substances is also frequently investigated [4].

are important in drug-induced toxicity [7].

molecular modification of the active agent itself [9].

drug is extensively metabolized [1]. Interindividual differences in drug metabolism also lead to the research of factors that affect drug metabolism [2, 3]. Moreover, a metabolism of

In early discovery, drug metabolism input provides a basis for choosing chemical structures and lead compounds with desirable drug metabolism and pharmacokinetic (DMPK) or safety profiles [5, 6]. It is the fact that the shift of the rate of drug attrition from 40% in 1990 to 10% in 2000 was due to increased efforts in applying DMPK principles for drug development. Beside traditional drug metabolism research that focuses on absorption, distribution, metabolism and excretion *in vitro* and *in vivo* studies, the knowledge about pharmacogenetics, pharmacogenomics and transporters brought many advances in drug metabolism research [5]. For the feasibility to successfully monitor the drug metabolism, suitable bioanalytical methods have to be developed and validated. Studies of metabolic fate of drugs in living systems may be divided into three areas: 1) elucidation of biotransformation pathways, 2) determination of pharmacokinetics of the parent drug and/or its primary metabolites and 3) identification of chemically-reactive metabolites that

Metabolism is a process of biotransformation when drugs are transformed into a different chemical form by enzymatic reactions. Mainly, metabolism increases drug hydrophilicity and decreases the toxicity and activity of most drugs. On the other hand, the biotransformation reactions could lead to bioactivation of drugs in which case the metabolite is more toxic and/or more active than the parent drug (reactive metabolite formation) [8]. The mechanism of bioactivation of drugs may be classified into following categories: biotransformation to stable but toxic metabolites, biotransformation to electrophiles, biotransformation to free radicals and formation of reactive oxygen metabolites. Additionally, bioactivations are also the transformations of a prodrug, promoiety or bioprecursor prodrug to a more effective metabolite [9]. Prodrug approach is commonly used in order to overcome the poor bioavailability of the active form of the drug. In case when prodrug consists of two pharmacologically active drugs that are coupled together in a single molecule it is called promoiety. Another type of prodrug is a bioprecursor drug which does not contain a carrier or promoiety, but results from a

There are several factors influencing drug metabolism such as genetic, physiologic, pharmacodynamic and environmental factors. CYP2D6, CYP2C19, CYP2C9, CYP3A4, CYP3A5 are enzymes that are responsible for metabolism of many marketed drugs and are also highly polymorphic [10]. Many non-cytochrome P450 drug metabolizing enzymes also play important role in the metabolism of a variety of drugs. Among them polymorphisms of thiopurine methyltransferase (TPMT), butyrylcholinesterase, N-acetyltransferase (NAT) and UDP-glucuronosyltransferase (UGT) influence the metabolism of drugs [11]. Different physiological factors such as age, sex, disease state, pregnancy, exercise, circadian rhythm and starvation lead to the impaired metabolism among subjects and should be taken into consideration when evaluating the drug metabolism. Dose, frequency, route of Drugs are metabolized by different reactions that are classified into two groups: phase I and phase II. Phase I reactions include oxidation, reduction and hydrolysis. The function of phase I reactions is to introduce a new functional group within a molecule, to modify an existing functional group or to expose a functional group that is a substrate for phase II reactions. Phase I reactions are responsible for enhancement of drugs' hydrophilicity and consequently facilitate the excretion. Phase II reactions represent conjugating reactions and mainly further increase the hydrophilicity and facilitate the excretion of metabolites from the body [10]. Enzymes that catalyze phase I reactions include microsomal monooxygenases (cytochrome P450, flavin-dependent monooxygenase) and peroxidases, cytosolic and mitochondrial oxidases, reductases and hydrolytic enzymes. Cytochrome P450 enzymes may catalyze aliphatic hydroxylation, N-, O-, S-dealkylation, oxidative dehalogenation, epoxidation [6]. The participation (%) of hepatic CYP450 isoforms in the metabolism of clinically important drugs is as follows: 3A4/5 (36%), 1A1 (3%), 1A2 (8%), 2B6 (3%), 2C8/9 (17%), 2C18/19 (8%), 2D6 (21%), 2E1 (4%) [10]. Flavin-dependent monooxygenase, a flavoprotein, is a microsomal monooxygenase that is not dependent on cytochrome P450. It is capable of oxidizing nucleophilic nitrogen and sulfur atoms [6, 10]. Other typical phase I oxidation enzymes are monoamineoxidase (MAO), diamineoxidase (DAO), cyclooxygenase (COX), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), molybdenum hydroxylase (include aldehyde oxidase, xanthine oxidase and xanthine dehydrogenase). In addition to promoting oxidative metabolism, cytochrome P450 enzymes may also catalyze reductive biotransformation reactions for the reduction of azo and nitro compounds to primary amines [10, 12]. Hydrolytic enzymes that consist of non-specific esterases and amidases are also a member of phase I enzymes of metabolism [6, 10].

Phase I reactions may be followed by phase II reactions; however preceding phase I reactions are not a prerequisite. Phase II enzymes are highly capable of polarizing lipophilic drugs through conjugation with a polar substrate that facilitates excretion [13]. Contrary to phase I reactions, phase II reactions demand energy to drive the reaction. Energy is usually consumed to generate a cofactor or an activated intermediate then utilized as co-substrate [6]. Phase II reactions are catalysed by UDPglucuronosyltransferases (UGT), sulfotransferases (SULT), N-acetyltransferases (NAT), glutathione-S-transferases (GST) and methyltranserases [6, 10, 13]. Of the conjugating reactions glucuronidation, which catalyzes the transfer of glucuronic acid to aliphatic and aromatic compounds, is the most important. UGTs are able to form O-, N- and S-

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

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 83

When performing the experiment with supersomes, the experiment with control nontransfected supersomes should be conducted. A NADPH regenerating system (NRS), which consists of β-NADPH, glucose-6-phosphate and glucose-6-phosphate dehydrogenase, or NADPH is required in the incubation for the evaluation of CYP activity and uridine diphospoglucuronic acid (UDPGA) has to be added as a cofactor when evaluating UGT

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

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-

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

enzyme activity [14-16].

[14-18, 20].

[14-16, 18].

16].

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