**Abstract**

Metabolic stability of a compound is an important factor to be considered during the early stages of drug discovery. If the compound has poor metabolic stability, it never becomes a drug even though it has promising pharmacological characteristics. For example, a drug is quickly metabolized in the body; it does not have sufficient *in vivo* exposure levels and leads to the production of toxic, non-active or active metabolites. A drug is slowly metabolized in the body it could remain longer periods in the body and lead to unwanted adverse reactions, toxicity or may cause drug interactions. Metabolic stability assay is performed to understand the susceptibility of the compound to undergo biotransformation in the body. Intrinsic clearance of the compound is measured by metabolic stability assays. Different *in vitro* test systems including liver microsomes, hepatocytes, S9 fractions, cytosol, recombinant expressed enzymes, and cell lines are used to investigate the metabolic stability of drugs. Metabolite profiling is a vital part of the drug discovery process and LC–MS plays a vital role. The development of highresolution (HR) MS technologies with improved mass accuracy, in conjunction with novel data processing techniques, has significantly improved the metabolite detection and identification process. HR-MS based data acquisition (ion intensitydependent acquisition, accurate-mass inclusion list-dependent acquisition, isotope pattern-dependent acquisition, pseudo neutral loss-dependent acquisition, and mass defect-dependent acquisition) and data mining techniques (extracted ion chromatogram, product ion filter, mass defect filter, isotope pattern filter, neutral loss filter, background subtraction, and control sample comparison) facilitate the drug metabolite identification process.

**Keywords:** metabolic stability, *in vitro* test systems, LC–MS, data acquisition and data mining techniques

#### **1. Introduction**

Drug metabolism is a process by which xenobiotics such as drugs are easily removed from the body by converting them into more polar derivatives and pharmacologically inactive. Nevertheless, sometimes metabolism makes the compound less soluble, toxic or pharmacologically active. Therefore, information on the metabolism of new drug candidate is important to know the possible toxicity and

to circumvent failures in drug development. Bioavailability, half-life and clearance of a drug molecule are dependent on the rate of drug metabolism; these parameters define the dose and dosing frequency. A drug is difficult to develop, or market if the dose or dosing frequency is too high.

Drug metabolic reactions are two types, phase I and phase II biotransformation reactions. Hydrolysis, reduction, and oxidation are the phase I reactions catalyzed by cytochrome P450 (CYP) and flavin-containing monooxygenases (FMO). Phase II reactions are also called conjugation reactions in which metabolites produced in the phase I reactions may undergo glucuronide conjugation, glutathione conjugation, sulfoconjugation, amino acid conjugation, acetylation, and methylation. These reactions are catalyzed by enzymes like Uridine 5′-diphospho (UDP) glucuronyl transferases (UGTs) or sulfotransferases (SULTs), glutathione *S*– transferases (GSTs), *N*–acetyltransferases (NATs), and methyltransferases [1–5]. *In vivo* pharmacokinetics are predicted by using *in vitro* metabolic stability studies in the early stages of drug discovery and development. Metabolic profile evaluation is also an important issue in this field [6–8].

Susceptibility of a chemical compound to biotransformation is known as metabolic stability and is articulated as intrinsic clearance (CLint) and *in vitro* half-life (*t*1/2). Intrinsic clearance (CLint) is the ability of the liver to remove or metabolize the drug in the absence of flow restrictions and drug binding to cells, or proteins in the blood. *t*1/2 is defined as the time required for 50% elimination of the parent compound. Different models are used to predict additional indices like hepatic clearance (CLH), *in vivo t*1/2, and bioavailability. Hepatic clearance (CLH) is the most important parameter during drug development because most drugs are metabolized in the liver tissue [2, 9–13].

Metabolic stability of new drug molecule is assessed by *in vitro* techniques and then scaled to *in vivo* using scaling factors. When metabolic stability is performed with liver microsomes, *in vitro* half-life (t1/2) can be determined from the slope of the linear regression of the percentage of drug remaining against time. Microsomal intrinsic clearance (CLint, micr) can be determined using the equation ln2/*t*1/2 × [volume of incubation medium (μL)/microsomal protein in incubation (mg)] and the expressed units are μL min−1 mg−1. *In vivo* intrinsic(hepatic)clearance is estimated from liver microsomal data using the equation CLint = CLint,micr× (mg microsome g−1 liver) × [liver mass (g)/body mass (kg)] and is expressed in the units of mL min−1 kg−1. Scaling factors: 45 mg of microsomal protein per gram of liver tissue (humans, mice, rats, dogs, monkeys/value is applied to all species) and 26 g, 32 g, 30 g, 40 g and 87 g of liver tissue per kilogram of body weight is used for humans, monkeys, dogs, rats, and mice, respectively [2, 14–21].

McNaney *et al.* classified compounds based on their CLint values, compounds with CLint value above 15 mL min−1 kg−1 are called low clearance compounds, compounds with CLint value between 15 and 45 mL min−1 kg−1 are called intermediate clearance compounds and compounds with CLint values above 45 mL min−1 kg−1 are called as high clearance compounds [12]. High CLint and low *in vitro t*1/2 values indicate that the compound is rapidly metabolized and *in vivo* bioavailability of compound will be low. Hence, *in vitro t*1/2 values can be used for the classification of compounds; for example in the case of human CYP3A4 supersomes, compounds with *in vitro t*1/2value less than 10 min are classified as short *in vitro t*1/2 compounds, compounds with *in vitro t*1/2 value between 10 to 30 min are classified as moderate *t*1/2 agents and long *t*1/2 compounds are the compounds with *in vitro t*1/2 value greater than 30 min [22].

A new chemical entity, which is suitable as a drug candidate must maintain adequate concentration at the site of action and could be slowly removed from the body to make sure of its action. High metabolic stability, high clearance values, and active or toxic metabolites formation are the biggest challenges during the

#### In vitro *Metabolic Stability of Drugs and Applications of LC-MS in Metabolite Profiling DOI: http://dx.doi.org/10.5772/intechopen.99762*

drug discovery and development stages [23]. Compounds which have high clearance values are quickly removed from the body and show short duration of action. Conversely, compounds with low clearance values will show prolonged half-life and long duration of action, and so dosing will be reduced [24–28]. An important step in the drug discovery process is the identification of compounds with suitable metabolic profiles [29–31]; hence, the study of the chemical structure of molecule and identification of "soft spots" liable for biotransformation is required. Metabolic properties of the compound are improved by modification, or removal of the soft spots in the molecule [32, 33].

It is very important to carry out the metabolic stability of new molecules during the early stages of drug discovery to learn the metabolic characteristics. Even though some molecules pass in the *in vitro* level tests by showing promising results, they fail in the pharmacological and toxicological results at the *in vivo* level [24].

*In vivo* animal studies give important information regarding the metabolism of new chemical compounds, but these are costly, require more time, and are not suitable to test a large number of compounds. Hence, *in vitro* tests are used initially for the selection of compounds, and then a suitable animal model will be used in the drug development stages to determine the metabolic characteristics of selected compounds [23, 34–36]. During the drug discovery process, performance of metabolic stability by *in vitro* models is preferable compared with the animal models because the number of compounds to be tested is large and the amount of compound available for testing is small. Data from the *in vitro* metabolic studies will be useful for the targeted synthesis of compounds with required metabolic profiles and hence reduces the cost and time [2, 7, 37, 38].
