**3. HLM: a best** *in vitro* **model to conduct high-throughput drug metabolism studies**

HLM are the subcellular fractions derived from the liver's endoplasmic reticulum obtained by differential high-speed centrifugation. **Figure 2** displays the steps involved in the preparation of HLM from the human liver. HLM contains various enzymes such as CYPs, flavin-monooxygenase (FMO), carboxylesterases, epoxide hydrolase, and UGTs, making it a preferred *in vitro* model for drug metabolism studies. Assessing interindividual variability is possible with HLM as the activity of the microsomes differs in different individuals. This interindividual variability can be minimized while performing general metabolism studies by pooling the microsomes from different individuals. Microsomes from other human organs (intestine, kidney, lung) are also available and are utilized to evaluate extra-hepatic metabolism [35]. HLM aids in various studies like metabolite identification and profiling, assessment of interspecies variations, estimation of *in vivo* clearance, reaction phenotyping, and elucidation of the metabolic pathways [5, 8, 30, 36–38]. Furthermore, gender-specific microsomes are utilized in studying gender-based disparities in drug metabolism studies.

NADPH or NADPH regenerating system (NRS) is essential for the incubation process, and while determining the UGT activity, UDGPA and alamethicin are the In Vitro *Drug Metabolism Studies Using Human Liver Microsomes DOI: http://dx.doi.org/10.5772/intechopen.108246*

#### **Figure 2.**

*Preparation of human liver microsomes. Microsome preparation buffer composition: 10 mM potassium phosphate buffer, pH 7.4, with 1.15% (w/v) potassium chloride. Homogenize the liver twice at 20,500 rpm, each time for 30 s, with a 30 s cooling period between bursts. Centrifuge the homogenate for 5 min at 1000* g*, 4°C, then increase to 10,000* g *for a further 10 min. Ultracentrifuge the S9 fraction for 60 min at 105,000* g*, 4°C.*

prerequisites [5, 8, 37]. HLM are preferred as they are simple, economical, easy to store for long-term usage, and offer high throughput screening. Nevertheless, HLM has few drawbacks as it is unsuitable for quantitative assessments in drug metabolism studies since it lacks *N*-acetyltransferase (NAT), glutathione-S-transferase (GST), sulfotransferase (SULT) enzymes, and other cofactors. This drawback restricts the competitiveness in metabolism as well as limits the generation of a few metabolites. It also fails to consider the percentage of drugs bound to plasma proteins instead of microsomes crucial for *in vivo* metabolism studies [5, 8, 36, 37].
