**1. Introduction**

The evolution of a new drug entity proceeds through a preclinical screening stage, during which the pharmacological and toxicological properties are scrutinized [1]. After oral administration, the drug gets absorbed and reaches the liver through the portal circulation for its metabolism. Cytochrome P450 (CYP450) enzymes are responsible for metabolizing most of the drugs in Phase I metabolism. However, flavin-containing monooxygenase (FMO) and enzymatic or nonenzymatic hydrolysis are also involved in the drug's metabolism but to a lesser extent. Phase II metabolism results in the production of metabolites conjugated to different chemical moieties like glucuronide, sulfate, glutathione, glycine, and acetate [2].

In 2016, the U.S. Food and Drug Administration (FDA) issued regulations for determining the safety and evaluation of drug metabolites for their toxicity in nonclinical species. These guidelines also provide a recommendation for identifying and characterizing drug metabolites. *In vivo* clinical metabolism studies involve the screening of biological matrices such as serum, urine, feces, and hair for identification of metabolites, whereas *in vitro* drug metabolism studies using human liver microsomes (HLM), human hepatocytes (either fresh or cryopreserved), and recombinant expression of cytochrome P450 enzymes (supersomes) in determining the human metabolic pathways [3, 4]. Comprehensive studies for Phase I and Phase II metabolism involve extensive HLM and human hepatocytes [5]. HLM helps determine the activity of drug metabolizing enzymes, CYPs and UGTs, present in the liver conveniently and straightforwardly [6, 7]. Further, it offers numerous advantages: high throughput screening, ease of storage, economic, repeatable, and simple usage with higher chances of clinical success [8]. Characterization of drug metabolic properties, assessing metabolic stability, and identifying metabolites are essential in determining the safety and success of clinical development [9].

### **2. Drug metabolism: a brief background**

The concept of drug metabolism emerged around the mid-19th century but flourished in the 20th century [10]. Metabolism of most pharmaceutical drugs occurs in the liver. In drug metabolism, enzymes convert drugs to highly polar metabolites to facilitate excretion from the body. Drug metabolism helps assess the oral bioavailability, elimination half-life, and clearance of the body's drug substance. The deduced parameters help decide the dose adjustment and the drug substance's administration frequency [11]. The drug concentration should always reside within the therapeutic window, i.e., between the minimum effective concentration (MEC) and the maximum safety concentration (MSC), to avoid therapeutic failure and adverse effects [12]. CYPs being abundant in the liver, metabolize the majority of drugs [12]. Furthermore, CYPs regulate the biotransformation of endogenous as well as exogenous compounds [13]. Among all the CYP isoforms, CYP3A4 contributes to the metabolism of more than 50% of the marketed drugs [14, 15].

Drug metabolism reactions are divided into Phase I, Phase II, and Phase III reactions. Phase I reactions result in oxidation, reduction, and hydrolysis. The Phase I enzyme families include the CYP superfamily, flavin-containing monooxygenases (FMO), monoamine oxidases, alcohol or aldehyde dehydrogenases, reductases, esterases, amidases, and epoxide hydrolases. Phase II reactions lead to the addition or conjugation of highly polar groups to the drug molecule after Phase I reactions. Occasionally, direct Phase II reactions occur when susceptible functional groups are present on the molecule without being preceded by Phase I reactions. Common Phase II reactions include glucuronidation, sulphation, methylation, N-acetylation, and glutathione conjugation [16]. Phase III metabolism occurs through the elimination of drug molecules through the efflux pump [12]. The primary objective of drug metabolism is to eliminate the drug from the body by converting the lipophilic centers to hydrophilic centers, thus making them water-soluble for easy elimination through the kidney [17, 18]. Sometimes, metabolism may result in the conversion of a drug into a toxic metabolite. On the contrary, metabolism also converts an inactive drug (prodrug) to its active metabolite for achieving the desired medicinal results [18]. Many metabolites of known drugs like desloratadine (parent drug- loratadine), oxazepam (parent drug- diazepam), and cetirizine (parent drug- hydroxyzine) have been found to possess equivalent or enhanced therapeutic activity than the parent drug [19]. Similarly, the discovery of paracetamol was precious as it replaced the use of phenacetin, a toxic parent moiety. Hence, the metabolite's activity plays a significant part in bioequivalence studies [20].

#### In Vitro *Drug Metabolism Studies Using Human Liver Microsomes DOI: http://dx.doi.org/10.5772/intechopen.108246*

First-pass metabolism explains metabolism before a drug reaches systemic circulation. This term refers to orally administered drugs that undergo metabolism in the gut or the liver before reaching the systemic circulation. **Figure 1** illustrates the various barriers to the drug reaching systemic circulation by the first-pass metabolism. During the drug discovery and development phases, the drug's metabolic fate should be kept in mind. Several approaches are in use ranging from empirical data-driven approaches to mechanistic models to predict drug metabolism. The empirical data-driven approaches, such as machine learning, involve approximations and assumptions, thereby providing high-speed predictions with low precision. In contrast, the mechanistic models involve quantum mechanics or molecular dynamics for providing significantly high accuracy; however, they consume time and effort [21].

Factors affecting drug metabolism are categorized into inter-individual factors and intra-individual factors [22, 23]. Inter-individual factors such as genetic factors, species differences, health conditions, enzyme induction/inhibition by xenobiotics or environmental factors, nutritional differences, and behavioral and cultural differences vary across individuals. However, they are uniform throughout the life of the organism [22]. Intra-individual factors can change throughout the lifetime, and different

#### **Figure 1.**

*First-pass metabolism by CYP3A4 and/or transport by P-glycoprotein (P-gp) in the enterocytes of small intestine wall and then hepatocytes of the liver before reaching the systemic circulation. FGI: Intestinal availability, FH: Hepatic availability,Foral: Oral bioavailability, EGI: Intestinal extraction ratio, EH: Hepatic extraction ratio.*

endogenous and exogenous conditions control these factors, but the effect may be more significant under genetic influence. These can occur through the interaction of xenobiotics with transcription factors or xenobiotics with the drug-metabolizing enzymes. This direct interaction of xenobiotics with the drug-metabolizing enzymes causes the induction or inhibition of those enzymes [23]. Internal factors include age, pregnancy, hormones, sex, diseased state, genetics, and species. In contrast, external factors comprise the environment and diet (alcohol, tobacco, chemicals, and drugs) [24, 25]. Several studies reported that the reduction of rate and efficiency of the drug metabolism in the aging population is due to changes in the drug-metabolizing enzyme activity, variation in plasma protein binding, hepatic blood flow, and decrease in the liver mass, leading to the slowing down of excretion of few metabolized drugs [26]. The results of fasting on drug biotransformation are around 10–20%. This factor becomes crucial when a drug with a narrow therapeutic range is administered or when fasting produces an effect in combination with other factors [27].

*In vitro* systems help in mimicking and understanding the *in vivo* metabolism process. Of these, liver microsomes and hepatocytes are utilized to predict hepatic clearance [28]. HLM and suspended hepatocytes are the most common *in vitro* methods for determining metabolic stability [29]. They generate metabolites on a large scale for determining metabolic stability and profiling for comparison. The technological advancements have led to the generation of recombinantly expressed CYPs, slicing of the tissues, isolation of hepatocytes, and purification of the enzymes in a reproducible manner [30]. Immobilizing HLM on magnetizable beads coated with silica (HLM-MGBS) showed increased *in-vitro* metabolic efficiency [31]. The cellular or tissue models are used to assess the toxicity of the drug substance and its metabolites in cells or tissues [32, 33]. The placental toxicity of anticancer drugs was elucidated using placental tissue explants and trophoblast cell lines [34].
