Exploring Metabolic Stability: In Vitro Metabolic Models for Slowly Metabolized Compounds

In vitro metabolic stability studies have been widely used in drug discovery, including determining chemical sequences, guiding structural modifications, establishing in vitro-in vivo correlations (IVIVC), and predicting appropriate dosages for human dosing. With effective drug design strategies, medicinal chemists have successfully optimized the structure to efficiently block the metabolic site, resulting in an increasing number of compounds that exhibit low clearance ^1-3. These molecules are referred to as slowly metabolized compounds (also known as low-clearance compounds) which need prolonged incubation periods and robust enzyme activity systems in in vitro studies, making in vitro research extremely challenging. To address this challenge, this blog provides appropriate model selection methods based on the characteristics of slowly metabolized compounds or the research objectives.

What are the main in vitro metabolic stability study models for slowly metabolized compounds?

Liver microsomes and suspension hepatocytes are the most commonly used models for studying metabolic stability metabolized by CYP450 and non-CYP enzymes(in hepatocytes) in vitro. However, traditional in vitro metabolism study models have limitations in predicting the clearance rate of these compounds and their metabolites ⁴.

Current primary study methods for slowly metabolized compounds ^3-7 are as follows:

• Plated hepatocyte incubation method: The incubation time can be extended up to 48 h, and even up to 1 week with normal enzyme activity by adding HepExtend culture factor.

• Suspension hepatocyte relay method: The incubation time can be extended by 20 h or more using suspension hepatocytes.

• Hepatocyte and stromal cell co-incubation system: The incubation time can be extended up to 72 h based on two currently well-established and commercial models: HμREL and HepatoPac.

• HepaRG Incubation System: Using human hepatocellular carcinoma cell lines, the incubation time can be extended to 24 h.

The significance of the plated hepatocyte incubation method and suspension hepatocyte relay method

Among the above four methods, the plated hepatocyte method and suspension hepatocyte relay method are relatively convenient and flexible. The plated hepatocyte method offers the advantage of intact transporter expression, while the suspension hepatocyte relay method provides flexibility in species selection. Both methods exhibit good in vitro-in vivo correlation (IVIVC) and are easily accessible.

(1)  Plated hepatocytes incubation method

• Hepatocyte metabolic stability study procedure: Cell resuscitation, cell counting, cell plating, and incubation.

Validation of plated hepatocyte method

Hepatocyte culture methods: Monolayer culture and "Sandwich" culture ⁸.

(2)  Suspension hepatocyte relay method ³

• Study principles and protocol

This technique facilitates the evaluation of slowly metabolized compounds over a period of up to 20 hours. Because freshly hepatocyte suspension is used every 4 hours, the cells maintain their metabolic capacity throughout the entire study process.

By applying the two in vitro metabolic stability study models established by WuXi AppTec DMPK, more accurate in vitro clearance data of slowly metabolized compounds can be obtained, which can further predict in vivo clearance rates and provide guidance for clinical dosage and dosing frequency. We will consistently investigate other research methods on slowly metabolized compounds, including the co-culture method and other models. We will continue to focus on new research on in vitro metabolic methods and explore additional models to facilitate drug development.

If you want to learn more details about the strategies for slowly metabolized compounds, please read the article now.

Committed to accelerating drug discovery and development, we offer a full range of discovery screening, preclinical development, clinical drug metabolism, and pharmacokinetic (DMPK) platforms and services. With research facilities in the United States (New Jersey) and China (Shanghai, Suzhou, Nanjing, and Nantong), 1,000+ scientists, and over fifteen years of experience in Investigational New Drug (IND) application, our DMPK team at WuXi AppTec are serving 1,500+ global clients, and have successfully supported 1,200+ IND applications.  

Talk to a WuXi AppTec expert today to get the support you need to achieve your drug development goals.