Novel in vitro hepatocyte metabolism model facilitates slow-metabolizing pharmacokinetic studies

  Since the drug metabolizing enzyme activities gradually decrease with the prolongation of in vitro incubation time, traditional in vitro metabolism models have limitations such as short incubation time, low metabolic conversion and high lower limit of detection (the quantitative lower limit of CLint assay is 2.5 μL /( min·106 cells)), which can not accurately predict CLint of slowly metabolized drugs^[1]. Meanwhile, hepatocytes have more enzymes required for oxidation, reduction, hydrolysis and dimerization reactions compared to liver microsomes, and in in vitro systems, many novel models tend to be optimized based on the hepatocyte system and further mimic the in vivo environment to prolong the retention of enzyme activity in hepatocytes, and the prolongation of the incubation time provides the possibility to monitor sufficient metabolic transformation of slow metabolizing drugs^[1,2].

  In view of this, IPHASE, as a leader in biological reagents for in vitro research, has pioneered the HepatoMax™ System and HepatoCon™ Media System based on the study of adherent primary hepatocytes and stromal cells of various genera, including human, monkey, canine, rat, and mouse, etc. The HepatoMax™ System and HepatoCon™ Media System were developed using the adherent primary human hepatocytes as an example, and both systems were set up for the determination of the activity and metabolic stability of specific enzyme types and the increase in response value of metabolites of the drug tolbutamide at 10 days and 7 days to verify the systems. Metabolite increase response value at 10 days and activity and metabolic stability of specific enzyme types at 7 days were determined to validate the systems.

• (1)The two systematic drug validation culture processes are as follows:

  IPHASE-HepatoMax™ is a co-culture system of frozen walled primary human hepatocytes and stromal cells. Primary human hepatocytes were resuscitated and incubated with stromal cells in HepatoP™ medium for 3 days to start the drug administration assay, and the metabolism of the drug was carried out in serum-free HepatoDo™ medium, which was replenished only with the drug without changing the medium. A total of 4, 72, 168 and 240 hours after drug administration, the value added of drug metabolites was determined for analysis.

 

  IPHASE-HepatoCon™, on the other hand, is a system for culturing frozen adherent primary human hepatocytes in HepatoCon™ medium. After resuscitating the primary human hepatocytes, the drug administration assay was performed after 3 days of culture in HepatoCon™ medium alone, and the subsequent time of administration, the culture system and the assay methodology were the same as those of the HepatoMax™ system.

 

  Meanwhile, to ensure the accuracy and rigor of the results, IPHASE performed a medium control (cell-free) and a stromal cell control at the same time, with three replications, and the results showed that the IPHASE-HepatoMax™ and IPHASE-HepatoCon™ systems produced a strong peak metabolite response from day 3 onwards, and that they both showed a strong metabolic turnover rate.

 

 

• (2)Drug metabolism is mainly dependent on the hepatocyte CYP450 enzyme system, and its activity can be measured to better examine.

  The IPHASE-HepatoMax™ and IPHASE-HepatoCon™ systems have good applicability.

  IPHASE set up four categories of medium control (no cells), HepatoMax™, HepatoCon™ and stromal cells. Using wall-adherent primary human hepatocytes as an example, the metabolite increase values of CYP1A2, CYP2B6 and CYP3A4 were verified at day 0, 3 and 7 after hepatocyte recovery for 3 days using finasteride, bupropion and midazolam as the substrates, respectively. Metabolite increase values showed that both IPHASE-HepatoMax™ and IPHASE-HepatoCon™ systems showed active enzyme activity by day 7, consistently promoting normal drug metabolism.

 

• (3)In addition to the validation of enzyme activity, good metabolic stability is also an important and indispensable step in validating the utility of in vitro models.

  IPHASE set up three categories of HepatoMax™, HepatoCon™ and stromal cells to determine the metabolic stability of Tolbutimide over a period of 80 hours using adherent primary human hepatocytes as an example, which were administered 3 days after resuscitation of primary hepatocytes, and the results showed that both the IPHASE-HepatoMax™ and IPHASE-HepatoCon™ systems both showed robust metabolic clearance activity within the longest value of the assay time.

 

  In summary, IPHASE, as a leader in in vitro bio-reagents, has developed IPHASE HepatoMax™ and IPHASE HepatoCon™ in vitro models based on wall-applied primary human, monkey, canine, rat and mouse hepatocytes and stromal cells of various genera, with excellent metabolic turnover, active enzyme activities and powerful metabolic clearance activities, which offer new choices for our customers to develop low-clearance drugs, and are an indispensable partner to realize drug discovery! The IPHASE HepatoMax™ and IPHASE HepatoCon™ in vitro models offer excellent metabolic turnover rates, active enzyme activities and strong metabolic clearance activities, providing new options for customers to develop low-clearance drugs, and are an important partner in drug discovery!

 

Reference:

[1] 阮婷婷,鞠武建,熊海伟,等.低清除率药物的代谢稳定性预测模型研究进展[J].中国药科大学学报, 2019(2):152-160.

[2] Di L, Trapa P, Obach R S, et al. A novel relay method for determining low-clearance values[J]. Drug metabolism and disposition, 2012, 40(9): 1860-1865.