**2. Background of drug metabolism**

The study of the metabolic fate of drugs is an essential and important part of the drug development process. During drug evaluation the research of drug metabolism is of high importance especially when metabolites are pharmacologically active or toxic or when a

© 2012 Roškar and Trdan Lušin, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Roškar and Trdan Lušin, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 toxic substances is also frequently investigated [4].

Analytical Methods for Quantification of Drug Metabolites in Biological Samples 81

administration, tissue distribution and protein binding of the drug affect its metabolism. Moreover, environmental factors such as environmental chemicals, co-administered drugs, tobacco, smoking, alcohol drinking and dietary constituents may change not only the kinetics of enzyme reaction but also the whole pattern of metabolism, thereby altering the bioavailability, pharmacokinetics, pharmacologic activity or the toxicity of the drug [10, 11].

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

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-

**3. Drug metabolic pathways** 

metabolism [6, 10].

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 are important in drug-induced toxicity [7].

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 molecular modification of the active agent itself [9].

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 administration, tissue distribution and protein binding of the drug affect its metabolism. Moreover, environmental factors such as environmental chemicals, co-administered drugs, tobacco, smoking, alcohol drinking and dietary constituents may change not only the kinetics of enzyme reaction but also the whole pattern of metabolism, thereby altering the bioavailability, pharmacokinetics, pharmacologic activity or the toxicity of the drug [10, 11].
