**4. Drug metabolism studies by HLM**

CYP activity changes in different species and this interspecies variation in drug metabolism can be estimated by investigating the *in vitro* drug metabolism in liver microsomes obtained from various species. The appropriate animal model for pharmacokinetics and toxicological studies can be determined by comparing the CYP metabolic profiles obtained from different species with the HLM. Microsomes can be acquired from the below-stated corporations: XenoTech LLC (www.xenotechllc.com), Human Biologics (www.humanbiologics.com), Cedra, Co. (www.cedracorp.com), BD Gentest (www.bdbiosciences.com) and Celsis International (www.celsis.com).

However, it is to be noted that the activity of the microsomes fluctuates among different batches and vendors. For instance, rat liver microsomes obtained from two different vendors demonstrated significant activity differences in the biotransformation of buspirone and loperamide. In contrast, the one obtained from the third vendor showed no activity. Furthermore, three batches obtained from the same supplier exhibited different activities in the biotransformation of buspirone and loperamide [39]. These differences were observed because of the innate differences in animals and varying preparation methods chosen by the vendors. Few vendors prepare liver microsomes by using phenylmethylsulfonylfluoride, while others use ethylenediaminetetraacetic acid (EDTA). Phenylmethylsulfonylfluoride inhibits trypsin-like proteases that are responsible for microsomal proteolytic degradation and certain carboxylesterases. On the other hand, EDTA chelates calcium and iron inhibiting both calcium-dependent phospholipases and lipid peroxidation. For each fresh batch, it is vital to examine the microsomal characterization data given by the vendor to check the CYP content, cytochrome b5, and NADPH-cytochrome c reductase activity.

For quantifying metabolites, drugs are incubated with microsomes with a low microsomal protein concentration, i.e., ≤0.5 mg/mL [40, 41]. This low concentration reduces the extent of protein binding to the drug. The final protein concentration of preparation is assessed by a Bradford protein assay or Lowry protein assay with bovine serum albumin as a standard. Storage of HLM at low temperatures (80°C) maintains the activity of CYP enzymes for an extended period [40]. Microsomes thawed and kept on ice for less than 2 hours can be re-frozen at 80°C for reuse as there will be insignificant loss of enzyme activity [42].

The drug concentration used *in vitro* studies is higher than that observed in blood in an animal study. When the *in vivo* drug concentrations are unknown, the final drug concentration is chosen from a range of 1–10 μM to simulate the *in vivo* conditions. Drug's incubating concentrations (0.5–15 μM) have a significant influence on the drug's stability consequences: higher concentrations of the drug are more stable in microsomes [39]. Thus, it is recommended to work with two different concentrations of a drug for understanding the concentration impact on its stability within the microsomes and additional matrices. Low concentrations of the drug are proposed for *in vitro* studies due to the following reasons: 1) The reaction between the test drug and enzyme follows a first-order reaction, i.e., the rate of the reaction is directly proportional to the concentration of the drug 2) Concentration of organic solvents always should be low as it reduces the microsomal activity.

The control groups should exclude the substrate, microsomes, NADPH, or the NRS from the incubation solution. Ice-cold organic solvent (e.g., acetonitrile or methanol) is used to quench the reaction. Incubation time of less than 2 h at 37°C is suggested for performing a stability study using microsomes [42]. In the extended incubation period, additional control group incubations need to be included to ensure the enzyme's activity and thermal degradation of the drug. When metabolite identification is difficult, the % of unchanged parent drug versus time will be recorded. Organic solvents employed for solubilizing lipophilic drugs inhibit CYP activity. DMSO concentrations of 0.2, 0.5, or 1% inhibit the CYP activity resulting in erroneous stability data of incubated drugs. DMSO specifically inhibits CYP2E1, and hence, it should be avoided for studies involving the CYP2E1 enzyme. Organic solvents like methanol, ethanol, acetonitrile, and PEG 400 also inhibit about 15–25% of CYP2E1, CYP3A4, CYP2D6, CYP2C9, and CYP2C19 activity. The permissible limits for the organic solvents in solubilizing the drugs while retaining the CYP activity are methanol <1.0%, acetonitrile <1.0% and DMSO <0.2% [11].
