**4.1 Reaction phenotyping studies**

Reaction phenotyping, also known as enzyme mapping, helps determine the enzymes involved in the metabolism of a specific drug. The data from these studies are essential in identifying potential drug interactions with common co-medications. Further, these studies help in anticipating possible pharmacokinetic changes caused by genetic polymorphisms in certain enzymes. Understanding the role of a specific enzyme involved in the metabolism of a drug is vital in the following conditions: 1) Identifying potential DDI with concomitant medications that may be inhibitors or inducers of the same enzymes [43]. 2) Establishing the metabolism of a drug by an enzyme that exhibits genetic polymorphism may result in significant inter-individual variability [44]. 3) Determining the formation of pharmacologically active metabolites [45]. 4) Deducing the extent of drug metabolism and the generation of significant metabolites [45].

In general, *in vitro* reaction phenotyping studies helps identify and characterize the formation of significant metabolites in drug metabolism at the preclinical stage. Toxicological studies assess the safety of these metabolites. In addition to toxicological considerations, detecting any pharmacological effects of major metabolites is also essential. Human radiolabel mass balance studies before phase III trials unravel main elimination pathways and systemic metabolite exposure. Data from the human radiolabel mass balance and the *in vitro* studies confirm metabolic pathways and the enzymes responsible for the drug metabolism. The CYP and non-CYP enzymes that contribute to ≥25% of drug elimination should be uncovered. The *in vivo* contribution is assessed by interaction with a potent, selective inhibitor or pharmacogenetic studies to decipher elimination pathways [46]. Assessment of reaction phenotyping uses approaches such as recombinantly expressed enzymes and correlation analysis. Recombinantly expressed enzymes use scaling methods like the relative activity factor (RAF) or the intersystem extrapolation factor (ISEF) for interpreting the relative contribution of the individual enzymes. The correlation analysis utilizes pooled HLM obtained from at least 10 donors for testing the activity toward respective probe substrates [47].

#### **4.2 Enzyme inhibition studies**

Enzyme inhibition experiments evaluate known CYP enzyme inhibitors on the metabolism of a drug by either pooled HLM or individual CYP isoforms. The usage of selective chemical inhibitors allows easy illustration of the metabolic pathways. To prevent false results, careful estimation of the drug and inhibitor concentrations for incubation is a must. Higher inhibitor concentrations exhibit non-selective chemical inhibition. For instance, quinidine and ketoconazole at <1 μM concentration act as selective CYP2D6 and CYP3A4 inhibitors. Although, at higher concentrations, these drugs inhibit other CYP isoforms as well. Chemical CYP inhibition is categorized into two types: reversible (could be competitive inhibition or non-competitive inhibition) and irreversible inhibition. In irreversible inhibition ("mechanism-based inhibition" or "suicide inhibition"), the CYP enzyme metabolizes the drug into a reactive metabolite that firmly binds to the enzyme's active site leading to a prolonged inactivation [48, 49]. These studies can be conducted before or after carrying out the cDNAexpressed recombinant CYP enzyme studies. They impart extra proof to assist the cDNA-expressed recombinant CYPs study results. Further, they may also provide a direction to these studies for the active isoform identification.

**Figure 3.**

*Workflow to assess enzyme inhibition using human liver microsomes.*

**Figure 3** demonstrates the protocol for the CYP inhibition study. The procedure involves incubating the drug with liver microsomes in the presence and absence of selective inhibitors at 37°C for 30 min [40]. The following inhibitors against the isoforms and their concentrations are recommended: furafylline (CYP1A2; 0.1, 1, 10 μM), 8- glitazones or quercetin (CYP2C8; 0.5, 1, 10 μM), quinidine (CYP2D6, 0.5, 1, 10 μM), sulphaphenazole (CYP2C19; 5, 20, 100 μM), methoxypsoralen (CYP2A6; 0.1, 1, 10 μM), troleandomycin (TAO; CYP3A, 0.5, 1, 10 μM), clomethiazole (CYP2E1, 0.1,1,10 μM) [40, 50]. Methanol (< 1% (v/v) of the entire mixture) is used to dissolve the inhibitors before adding them to the incubation mixture. Prior to drug addition, inhibitors undergo preincubation at 37°C, with NADPH and microsomes, reaching a final concentration of 10 mM. A positive control is carried out in the presence of the drug with 1% methanol in the incubation mixture, whereas a blank control lacks the drug. Thus, the control values are employed to successfully determine the percentage of inhibition observed in the metabolite generation.

Conventionally, *in vitro* CYP enzyme inhibition studies were conducted using HLM, for which isoform-specific substrates were incubated along with the investigational drug. At the end of the incubation, the formation of the metabolite is monitored by analytical techniques like high-performance liquid chromatography (HPLC), liquid chromatography coupled with mass spectrometry (LC-/MS), or fluorescence and this procedure is repeated for at least three different concentrations of that drug [51, 52]. Identification of many drug molecules in the drug discovery process is possible using combinatorial chemistry approaches and high throughput screening techniques.

#### **4.3 Drug metabolite profiling**

Metabolite profiling refers to the relative quantification, identification, and characterization of the number of metabolites formed in the biological matrices. These studies help researchers structurally and chemically modify the drug to increase its efficacy, reduce its toxicity, and facilitate the synthesis of a molecule with enhanced therapeutic activity [53–56]. The FDA guidance "Safety Testing of Drug Metabolites" states that the metabolic drug profile must be determined by *in vitro* and *in vivo* models at various phases of the drug development. *In vitro* metabolite profiling of a

drug can be performed using liver microsomes, hepatocytes or liver slices collected from humans or animals [57]. The regulatory bodies (ICH, EMEA, and FDA) recommend studying *in-vitro* and *in-vivo* hepatic drug metabolismFDA guidance mentions the importance of metabolism studies via the kidney and the gastrointestinal tract as most orally administered medications interact with the gastrointestinal enzymes [58, 59]. Other regulatory authorities lay minimal emphasis on extra-hepatic metabolism studies.

Using a single high concentration of drug or a series of concentrations produces a high concentration of metabolites that meets the demands of quantification. A concentration of 50 μM or concentrations of 5, 50, and 500 μM can be chosen for novel metabolites. Concentration should be higher than or equal to the Km value (Michaelis constant) recorded for the CYP substrates to generate metabolites in measurable amounts. A positive control having testosterone or phenacetin should be included for measuring the formation of 6β-hydroxytestosterone acetaminophen metabolites. A negative control without NADPH for each test compound helps determine the sources of metabolites other than oxidative metabolism (e.g., carboxylesterases, nonenzymatic metabolite formation, substrate impurities) [11].
