Effects of NO-1886 (Ibrolipim), a Lipoprotein Lipase-Promoting Agent, on Gene Induction of Cytochrome P450s, Carboxylesterases, and Sulfotransferases in Primary Cultures of Human Hepatocytes

Abstract

In the present study, the effects on expression of cytochrome P450 (CYP1A1, CYP1A2, CYP3A4 and CYP3A5), carboxylesterase (CES1 and CES2) and sulfotransferase (CHST1, CHST3, CHST4, CST, SULT2A1 and TPST2) mRNA in primary cultures of cryopreserved human hepatocytes were evaluated after exposure to NO-1886 (diethyl 4-[(4-bromo-2-cyanophenyl) carbamoyl] benzylphosphonate) for 48 hr at 2, 10, and 50 microM. Analysis was performed by RT-PCR in the presence of TaqMan probe. CYP1A1 and CYP1A2 mRNA levels after exposure to 50 microM omeprazole (positive control for CYP1As) were increased by 162 (p<0.001) and 37 times (p<0.001), respectively, compared with untreated controls. However, these mRNA levels were increased by 2 times or less after exposure to NO-1886. CYP3A4 and CYP3A5 mRNA levels after exposure to 50 microM rifampicin (positive control for CYP3As) were significantly increased by 5.8 (p<0.01) and 2.0 times (p<0.01), respectively, compared with untreated controls. The CYP3A4 mRNA level after exposure to 10 microM NO-1886 was increased by 1.3 times (p<0.05). Further, the CYP3A4 mRNA level after exposure to 50 microM NO-1886 was significantly increased by 3.6 times (p<0.001). However, the CYP3A5 mRNA level after exposure to 50 microM NO-1886 was not significantly increased. CES1 and CES2 mRNA levels after exposure to 50 microM NO-1886 were significantly increased by 1.4 (p<0.05) and 2.6 times (p<0.01), respectively, compared with untreated controls. CHST1, CST and SULT2A1 mRNA levels after exposure to 50 microM NO-1886 were significantly increased by 3.8 (p<0.001), 1.8 (p<0.01) and 4.4 times (p<0.01), respectively. CHST3, CHST4 and TPST2 mRNA levels after exposure to 50 microM NO-1886 were not significantly increased. This in vitro technique using primary cultured human hepatocytes is expected to be very useful for the preclinical evaluation of the induction of drug-metabolizing enzymes in humans.

Tissue-specific mRNA Expression Profiles of Human Solute Carrier Transporter Superfamilies

“…Table 5 lists the references for our studies concerning the measurement of these human mRNA targets by the TaqMan method. We have adopted the one-step RT-PCR method to measure the expression levels of every target gene related to the gene induction of drug-metabolizing enzymes in primary cultures of human hepatocytes 59,61,65,[67][68][69][70][71] and to the tissue-specific profiles of mRNA expression in human tissues, as described in our previous studies 9, [12][13][14]60,72) and the present study.…”

Tissue-Specific mRNA Expression Profiles of Human Toll-Like Receptors and Related Genes

“…The relative expression of each mRNA was calculated by the DCt method (where DCt is the value obtained by subtracting the Ct value of PPIA mRNA from the Ct value of the target mRNA), as employed in previous studies. [18][19][20] Specifically, the amount of target mRNA relative to PPIA mRNA is expressed as 2…”

Current Industrial Practices in Assessing CYP450 Enzyme Induction: Preclinical and Clinical

“…The drug metabolizing enzymes remain inducible after cryopreservation, and due to the significant variation in activities of drug metabolizing enzymes between individual human livers, certain lots of cryopreserved cells can generate results essentially indistinguishable from freshly isolated cells (35,46). mRNA, protein expression and activities of CYP1A2, 2B6, 2C9, 2E1 and 3A4 in cryopreserved hepatocytes are inducible by prototypical inducers (47), as is the activity of various UDP-glucuronosyltransferases, carboxylesterases, and sulfotransferases (48,49). The advantage cryopreserved cells offer over fresh isolates is that experiments can be planned ahead and are not dependent on the sporadic availability of fresh primary hepatocytes.…”