**2.** *In vitro* **test systems to conduct metabolic stability**

Metabolic stability study can be conducted by incubating the test compound with the appropriate metabolic model (e.g., liver microsomes, hepatocytes, S9 fractions) and analysis of incubation mixture by suitable analytical techniques like LC–MS/MS [39–41].

Microsomes and hepatocytes are the commonly used *in vitro* systems to conduct metabolic stability. Other systems used are S9 fractions, cytosol, recombinant expressed enzymes, and cell lines.

#### **2.1 Liver microsomes**

Microsomes obtained from different species (e.g., human liver microsomes, HLM; rat liver microsomes, RLM; mouse liver microsomes, MLM; dog liver microsomes, DLM, or monkey liver microsomes, MnLM) are used in the metabolic stability studies [42]. The most popular *in vitro* model is human liver microsomes. Alternatively, MLM is a good preliminary tool that the results obtained correlate well with the results obtained with HLM [43, 44]. Liver microsomes are subcellular fractions derived from the smooth endoplasmic reticulum of liver cells. Homogenization of the liver and then differential centrifugation is performed to prepare the liver microsomes [9, 45]. Phase I oxidation is evaluated by the addition of a cofactor like nicotinamide adenine dinucleotide phosphate (NADP). Glucuronidation is also studied by liver microsomes with the addition of uridine diphosphate glucuronic acid (UDPGA). Various metabolizing enzymes like cytochrome P450s (CYP), flavin monooxygenases (FMO), epoxide hydrolase and carboxyl esterases, and UDP glucuronyl transferases are present in the HLM preparation. Hence, they are commonly used to study the metabolic fate of drugs [46].

Metabolic stability assays are generally performed by incubating the compound with liver microsomes and depletion of a drug during incubation is measured by HPLC or LC–MS. In the metabolic stability assay, the incubation mixture consists of a test compound (which is dissolved in potassium phosphate buffer, if not soluble in phosphate buffer dissolved in acetonitrile, methanol, or DMSO and the final organic solvent concentration in the assay should always be ≤1% for acetonitrile and methanol or ≤ 0.2% for DMSO), NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) or NADPH regenerating system, potassium phosphate buffer and liver microsomes. Protein concentration usually does not exceed 2 mg/ mL to prevent too much nonspecific binding. In the first step, the mixture containing the test compound, buffer, and microsomes is pre-incubated at 37°C for 15 minutes before the addition of NADPH. The obtained mixture is incubated at 37°C for several time intervals (*e.g*., 15, 30, and 60 min). Incubation time is generally not more than 60 min for optimal conditions for enzymatic activity. At the predetermined time points, the reaction is quenched by the addition of ice-cold acetonitrile or methanol. The samples are vortexed and centrifuged; the supernatant is collected and analyzed by HPLC or LC–MS/MS. In the case of control samples, NADPH is replaced by potassium phosphate buffer [2, 24, 47–52].

The extent of metabolism (as substrate depletion) is calculated by using the following Equation [53].

$$\text{rate of depletion} \left(\text{pmol} / \text{min} / \text{mg}\right) = \frac{\left(\Delta \text{C} \ge 1000\right)}{\left(\text{B} \ge \text{T}\right)}\tag{1}$$

where ΔC = [concentration (or peak area) at 0 min] – [concentration (or peak area) at time T (nmol/mL or μM],

B is the microsome protein concentration (mg/mL),

T is the incubation time (min).

and 1000 is the conversion factor from nmol to pmol.

#### **2.2 Hepatocytes**

Phase I and Phase II drug metabolism is studied by using hepatocytes [9], which shows the heterogeneity of CYP expression in the human liver [54]. The metabolic profile of the number of drugs analyzed by cultured hepatocytes [55, 56] and suspensions of primary hepatocytes, and shown good *in vitro*– *in vivo* correlation [57–59]. A decrease in CYP expression is observed with cultured hepatocytes and thereby loss of liver specific functions. Phase I and Phase II enzyme activity is retained with cryopreserved hepatocytes. The disadvantage of hepatocytes may be an inter-individual variation that can be overcome by using mixtures of hepatocytes from different donors. HepatoPac is the new strategy used to create stable *in vitro* liver models that enable long-term hepatic metabolism and toxicity studies. It is a micropatterned hepatocyte-fibroblast co-culture system that can be used for continuous incubation of up to 7 days [2, 24, 28, 38, 60, 61].

The general procedure for a metabolic stability study is to prepare a hepatocyte suspension containing 106 cells/mL and incubate for 10 min (37°C, 5% CO2). Test compound solution is added to the cells and again incubated. At the predetermined time points (e.g., 15, 30, 60, and 90 min) reactions are stopped by the addition of ice-cold methanol or acetonitrile. Control samples are also prepared without hepatocytes. Supernatants are collected and analyzed for parent molecule and its metabolites by HPLC or LC–MS [43, 44, 60, 62–65].

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

#### **2.3 Recombinant expressed enzymes**

Recombinant expressed enzymes are the sources of specific P450 isoenzymes. The advantages are simplicity of the method and a single enzyme can be used for the study. In cases of low metabolism, recombinant enzymes can be used at high concentrations to increase metabolic activity for use in metabolic stability screening and in inhibitory potential evaluation. Recombinant enzymes can also be used in the confirmation of reaction phenotyping studies. Conversely, the absence of the remaining phase I and phase II enzymes can be considered as a disadvantage of this type of system [2, 66].

The assay procedure consists of a test compound solution, recombinant P450 isoenzyme, potassium phosphate buffer, and magnesium chloride. The mixture is pre-incubated for 15 min at 37°C and then the metabolic reaction is started by the addition of NADPH. The incubation is continued for different time points. The reaction is ended at each timepoint by the addition of ice cold acetonitrile or methanol and centrifuge the samples. Supernatants are collected and analyzed by HPLC or LC–MS [67, 68].

#### **2.4 Cytosol**

Cytosolic fraction is an *in vitro* model that has not been used commonly for biotransformation studies. The cytosol is produced by differential centrifugation of whole liver homogenate. Soluble enzymes of phase II, such as N-acetyltransferases (NAT), sulfotransferases (SULT), glutathione S-transferase (GST), carboxylesterase, diamine oxidase, soluble epoxide hydrolase, alcohol dehydrogenase, and xanthine oxidase are expressed in the cytosolic fraction, but the aforementioned initial three enzymes are expressed at higher concentration. This *in vitro* model requires cofactors like acetyl CoA (acetyl coenzyme A), acetyl CoA-regenerating system and dithiothreitol for NAT, 3′-phosphoadenosyl-5′-phosphosulfate (PAPS) for SULT, and glutathione for GST activity. The biotransformation by NAT, GST, or SULT can be studied separately or in combination depending on the cofactors added. The main disadvantage of this model is the lack of UGT and hence glucuronidation cannot be studied [69].

#### **2.5 S9 fractions**

S9 fraction preparations contain both cytosolic and microsomal fractions and as a result express a wide variety of metabolic enzymes-CYP, FMO, carboxylesterases, epoxide hydrolases, UGT, SULT, methyl transferases, acetyltransferases, GST, and others. This *in vitro* model can be used for metabolic, mutagenicity, and toxicity studies. The addition of co-factor is required for enzyme activity. The main advantage of S9 fraction over microsomes and cytosolic fraction is a more complete depiction of the metabolic profile due to the existence of phase I and phase II enzymes. In some cases, S9 fractions produce metabolites that are not formed by either microsomes or cytosol alone. However, the disadvantage of S9 fraction is overall lower enzyme activity compared with the microsomes and cytosol, thus it may leave some metabolites unnoticed [69, 70].

#### **2.6 Cell lines**

Cell lines are less popular than other described models due to de-differentiated cellular characteristics and absence of complete expression of all families of metabolic enzymes. The sources of cell lines are primary tumors of liver parenchyma. Currently, available cell lines include Hep G2, Hep3B, BC2, C3A, etc. Among them, the Hep G2 cell line is commonly used for biotransformation studies. Compared with the freshly isolated human hepatocytes, the metabolic activity of liver cell lines is generally low. Pretreatment of cell lines with various metabolic enzyme inducers partly reduces the problem of low activity. Even though, the induced activity is still below the enzymatic activity of freshly isolated human hepatocytes. An appropriate culture medium is required for the liver cell lines and the composition of the culture medium notably influences the metabolic activity. The described *in vitro* model is easy to culture and has steady enzyme concentration. Conversely, the lack or low expression of most important phase I and phase II drug metabolizing enzymes limits the application of this *in vitro* model. In addition, it is difficult to investigate individual enzymes due to their low expression level [70].
