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Measurement of Human Cytochrome P450 Enzyme Induction Based on Mesalazine and Mosapride Citrate Treatments Using a Luminescent Assay.

Kim YH, Bae YJ, Kim HS, Cha HJ, Yun JS, Shin JS, Seong WK, Lee YM, Han KM - Biomol Ther (Seoul) (2015)

Bottom Line: The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities.Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods.Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates.

View Article: PubMed Central - PubMed

Affiliation: Pharmacological Research Division, Toxicological and Research Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug safety, Cheongju 363-700.

ABSTRACT
Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their inter-assay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.

No MeSH data available.


Related in: MedlinePlus

The potential CYP induction ability of mosapride citrate (8.69, 86.9, and 869 ng/ml) in 3 hepatocyte cell lines measured using a luminometer. *p<0.05 and **p<0.01 versus each corresponding control.
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f4-bt-23-486: The potential CYP induction ability of mosapride citrate (8.69, 86.9, and 869 ng/ml) in 3 hepatocyte cell lines measured using a luminometer. *p<0.05 and **p<0.01 versus each corresponding control.

Mentions: We also performed CYP interaction studies using mosapride citrate, based on its Cmax value. The Cmax of mosapride citrate is 86.9 ng/mL (Kim et al., 2012). The results showed that CYP1A2 and CYP3A4 activities were induced by 869 ng/mL mosapride citrate (Fig. 4). The activity of CYP1A2 increased 3.66 and 2.81-fold in HMC478 and HMC493 cultures, respectively, while the activity of CYP3A4 increased 2.10, 4.64, and 4.28-fold in each of the 3 donor cell lines. In addition, CYP2B6 activity was also induced by mosapride citrate (Fig. 4). The fold-inductions doubled at Cmax and 10-fold of Cmax, with the exception of HMC424 hepatocytes. HMC424 treated with 869 ng/mL mosapride citrate showed a 5.46-fold increase, while HMC478 treated with 86.9 and 869 ng/mL mosapride citrate showed 3.14 and, 10.05-fold increases, respectively. Furthermore, the CYP2B6 activities of HMC493 were 2.63 and 8.12-fold increased after 8.69 and 869 ng/mL mosapride citrate treatments, respectively. On the other hand, CYP2C9 activity was not significantly changed by mosapride citrate treatment (Fig. 4).


Measurement of Human Cytochrome P450 Enzyme Induction Based on Mesalazine and Mosapride Citrate Treatments Using a Luminescent Assay.

Kim YH, Bae YJ, Kim HS, Cha HJ, Yun JS, Shin JS, Seong WK, Lee YM, Han KM - Biomol Ther (Seoul) (2015)

The potential CYP induction ability of mosapride citrate (8.69, 86.9, and 869 ng/ml) in 3 hepatocyte cell lines measured using a luminometer. *p<0.05 and **p<0.01 versus each corresponding control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556210&req=5

f4-bt-23-486: The potential CYP induction ability of mosapride citrate (8.69, 86.9, and 869 ng/ml) in 3 hepatocyte cell lines measured using a luminometer. *p<0.05 and **p<0.01 versus each corresponding control.
Mentions: We also performed CYP interaction studies using mosapride citrate, based on its Cmax value. The Cmax of mosapride citrate is 86.9 ng/mL (Kim et al., 2012). The results showed that CYP1A2 and CYP3A4 activities were induced by 869 ng/mL mosapride citrate (Fig. 4). The activity of CYP1A2 increased 3.66 and 2.81-fold in HMC478 and HMC493 cultures, respectively, while the activity of CYP3A4 increased 2.10, 4.64, and 4.28-fold in each of the 3 donor cell lines. In addition, CYP2B6 activity was also induced by mosapride citrate (Fig. 4). The fold-inductions doubled at Cmax and 10-fold of Cmax, with the exception of HMC424 hepatocytes. HMC424 treated with 869 ng/mL mosapride citrate showed a 5.46-fold increase, while HMC478 treated with 86.9 and 869 ng/mL mosapride citrate showed 3.14 and, 10.05-fold increases, respectively. Furthermore, the CYP2B6 activities of HMC493 were 2.63 and 8.12-fold increased after 8.69 and 869 ng/mL mosapride citrate treatments, respectively. On the other hand, CYP2C9 activity was not significantly changed by mosapride citrate treatment (Fig. 4).

Bottom Line: The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities.Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods.Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates.

View Article: PubMed Central - PubMed

Affiliation: Pharmacological Research Division, Toxicological and Research Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug safety, Cheongju 363-700.

ABSTRACT
Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their inter-assay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.

No MeSH data available.


Related in: MedlinePlus