Induction of Liver and Intestinal Cytochrome P450 (CYP) Enzymes in Male and Female Cynomolgus Monkey

Published:  15 October 2008

The objective of the present study was to determine the effects of various prototypical inducers on the activity and mRNA expression of multiple CYP enzymes in monkey liver and intestine.

Male and female Cynomolgus monkeys were dosed orally (by nasogastric intubation) once daily for four days with saline (vehicle control), β-naphthoflavone (BNF, 50 mg/kg/day), phenobarbital (PB, 25 mg/kg/day), rifampin (RIF, 25 mg/kg/day), pyrazole (200 mg/kg/day) or omeprazole (OMP, 50 mg/kg/day). Microsomal samples from liver and intestine were analyzed for total cytochrome P450 content, cytochrome b5 content, NADPH-cytochrome c reductase activity, 7-ethoxyresorufin O-dealkylation (CYP1A1/2), testosterone 16α-hydroxylation and testosterone 16β-hydroxylation (CYP2B17), 4-nitrophenol hydroxylase (CYP2E1), testosterone 2α-hydroxylation and testosterone 6β-hydroxylation (CYP3A8). CYP mRNA levels in liver were determined for CYP1A1, 1A2, 2A24, 2B6, 2C8, 2C43, 2C75, 2E1, 3A8 and 4A11 by RT-PCR. Given the amount of data collected on this project, the primary focus will be CYP mRNA data.

Treatment of monkeys with BNF caused significant increases in liver CYP1A1 and CYP1A2 mRNA levels, and little or no increase (< 4-fold) in the mRNA levels of the other enzymes examined. Omeprazole caused increases in liver CYP1A1 and 1A2 mRNA levels, but as a CYP1A mRNA inducer OMP was less effective than BNF. Treatment with BNF caused an increase in CYP1A1 mRNA levels (66- and 240-fold in males and females) and CYP1A2 mRNA levels (51- and 1600-fold in males and females). Treatment with OMP caused an increase in CYP1A1 mRNA levels (2.2- and 5.8-fold in males and females) and CYP1A2 mRNA levels (14.9- and 173-fold in males and females). Similarly, BNF and OMP caused increases in liver microsomal CYP1A1/2 activity. Treatment with BNF caused significant increases in CYP1A activity in both liver and intestine. The increase in liver CYP1A activity by BNF was similar between male and female monkeys (11- and 9.4-fold, respectively) whereas induction of intestinal CYP1A activity by BNF was much lower in male than female monkeys (2.7- and 19-fold, respectively). Though to a smaller extent compared to BNF, OMP also caused increases in microsomal CYP1A1/2 activity (3.7- and 4.3-fold in the liver) and (1.5 and 4.3-fold in intestine) for male and female monkeys, respectively. Treatment with PB caused a large increase in liver CYP2B6 mRNA levels (21.6- and 52.5-fold in males and females, respectively), and caused less pronounced increases in CYP2A24 (5.6- and 7.3-fold), 2C8 (7.3- and 6.5-fold), 2C43 (6.4- and 5.7-fold), 2C75 (6.2- and 7.0-fold), and 3A8 (2.6- and 6.0-fold) mRNA levels in males and females, respectively. PB also caused an increase in liver microsomal CYP2B17 and CYP3A8 activity (3.2- and 2.2-fold, respectively). Rifampin treatment caused an increase in liver CYP2B6 (3.6- and 8.2-fold), 2C8 (8.2- and 7.5-fold), 2C43 (6.9- and 8.1-fold), 2C75 (6.7- and 8.6-fold) and 3A8 (5.9- and 5.8-fold) mRNA levels in males and females, respectively. Except for a small increase (3.5-fold in CYP2B6 and 2.7-fold in CYP4A mRNA levels), pyrazole did not cause an increase in the liver mRNA levels of any of the CYP enzymes examined. Results from this study indicate that BNF and OMP are in vivo inducers of CYP1A1 and CYP1A2 in monkeys, while PB and rifampin are inducers of CYP2B, 2C and CYP3A enzymes.

Effects of Gender, Age and Ethnicity on Human Cytochrome P450 Activity

Published:  21 November 2006

It has been well documented that the expression of CYP enzymes is influenced by endogenous factors, such as genetic polymorphisms and hormone levels, and by exogenous factors, such as diet, exposure to drugs, alcohol consumption, cigarette smoking and various environmental factors (Parkinson, et al., 2004). For this study, we examined the effects of three endogenous factors and two exogenous factors on individual CYP activities in over 300 samples of human liver microsomes. The aim of this analysis was to evaluate whether the age, gender, or ethnicity of the donor should influence the selection of human liver microsomes for drug metabolism studies, as well as whether cigarette smoking and alcohol consumption are reliable indicators of elevated CYP1A2 and CYP2E1 activity, respectively...

Microsomes and S9 Prepared from Renal Tissue Yield High CYP, FMO and UGT Activities that are Stable…

Published:  31 October 2005

Microsomes and S9 Prepared from Renal Tissue Yield High CYP, FMO and UGT Activities that are Stable over Multiple Freeze/Thaw Cycles

We have developed procedures to prepare microsomes and S9 fraction from the kidneys of humans, Cynomolgus monkeys, Beagle dogs and Sprague Dawley rats, and we have assessed selected CYP, FMO and UGT activity in pooled and, in the case of humans, individual preparations. These microsomal samples were analyzed for their ability to catalyze the 12-hydroxylation of lauric acid (a marker of CYP4A activity), the N-oxygenation of benzydamine (a marker of FMO activity), the N-demethylation of benzydamine (a marker of CYP3A activity) and the glucuronidation of 4-methylumbelliferone (a reaction catalyzed by several forms of UDP-glucuronosyltransferase). We have also established that renal microsomes and S9 fraction can be subjected to as many as 10 freeze/thaw cycles with little or no loss of CYP4A, UGT, or NADPH-cytochrome c reductase activity. These results suggest that our method for processing kidney tissue is well suited to preserving the enzymatic activity of renal microsomes and S9 fraction, such that these subcellular fractions can be used to assess the Phase 1 and Phase 2 metabolism of drugs in the kidney.

Microsomes Prepared from Eluted Enterocytes Yield High Cytochrome P450 and UGT Activities that are…

Published:  25 August 2004

Microsomes Prepared from Eluted Enterocytes Yield High Cytochrome P450 and UGT Activities that are Stable over Multiple Freeze/Thaw Cycles

Enterocytes in the upper region of the small intestine play a significant role in the first-pass metabolism (pre-systemic clearance) of many orally ingested xenobiotics. For this reason, functionally active and stable intestinal subcellular fractions are required to assess the first-pass metabolism of drugs by cytochromes P450, UDP-glucuronosyltransferases and other drug-metabolizing enzymes. The present study summarizes enzymatic activity data from individual and pooled animal and human intestinal microsomes that were prepared from fresh duodenum/jejunum based on an enterocyte elution method with EDTA and various protease inhibitors. All samples were analyzed for their ability to catalyze testosterone 6ß-hydroxylation, 4-methylumbelliferone glucuronidation, and NADPH-cytochrome c reduction. Microsomes prepared from chemically eluted enterocytes had substantially greater CYP3A activity than those prepared from small intestinal samples subjected to mechanical scraping. Freezing/thawing small intestinal microsomes for up to 5 cycles did not cause significant loss of CYP3A, NADPH-cytochrome c reductase or UDP-glucuronosyltransferases (UGTs) activity. These results suggest that our elution method for processing small intestines is well suited to preserving microsomal enzymatic activities.

The Use of Immortalized Hepatocytes in Induction Studies

Published:  25 August 2004

Primary cultures of human hepatocytes are widely used to evaluate the cytochrome P450 (CYP) enzyme-inducing potential and/or toxicity of drug candidates. However, the availability of human hepatocytes for this purpose is limited and erratic, and there are large inter-individual differences in the magnitude of induction (due to differences in both "control" and "induced" CYP activities). Immortalized human hepatocytes, Fa2N-4, developed by Multicell Technologies, (Warwick, RI) have the potential to overcome these limitations. The cells, immortalized through transformation of human hepatocytes with SV40 T antigen, display in vitro cell morphology that closely resembles primary human hepatocytes (Figure 1). The Fa2N-4 cells retain many of the characteristics of primary hepatocytes, including the inducibility of multiple CYP mRNAs and enzyme activities (Mills et al., 2002; Morris et al., 2003; Czerwinski et al., 2003). In this study, we further characterized the ability of these cells to respond to enzyme-inducing xenobiotics. Additionally, we examined the toxicity profile of multiple enzyme inducers in primary hepatocytes and Fa2N-4 cells.

Induction of UDP-Glucuronosyltransferases in Cultures of Fresh and Immortalized Human Hepatocytes

Published:  25 August 2004

Induction of UDP-Glucuronosyltransferases in Primary Cultures of Fresh and Immortalized Human Hepatocytes

We have examined the induction of UDP-glucuronosyltransferases (UGTs) in primary cultures of human hepatocytes and in immortalized human hepatocytes (Fa2N-4 cells) under conditions that result in the induction of cytochrome P450 (CYP) enzymes. Primary cultures of human hepatocytes were treated daily for three days with either vehicle (0.1% DMSO or saline) or one of several prototypical human CYP inducers, namely b-naphthoflavone, omeprazole, phenobarbital, rifampin and isoniazid. Microsomes prepared from the primary hepatocyte cultures were analyzed by LC/MS/MS for thyroxine (T4) and triiodothyronine (T3) glucuronidation, and cell lysates were analyzed for UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 mRNA expression by the branched DNA (bDNA) assay. UGT1A1 mRNA levels increased (> 2 fold) following treatment of cultures with b-naphthoflavone, omeprazole, phenobarbital, rifampin or isoniazid. UGT1A6 mRNA expression was induced by b-naphthoflavone and isoniazid. UGT1A9 mRNA expression was induced by rifampin. UGT2B4 mRNA expression was induced by b-naphthoflavone and rifampin. UGT2B7 mRNA expression was induced by b-naphthoflavone and phenobarbital.

Fa2N-4 cells were treated for 3 days with DMSO (vehicle), omeprazole, phenobarbital, rifampin or 3-methylcholanthrene. Cells were analyzed for UGT1A1, 1A6, 1A9, 2B4 and 2B7 mRNA expression. Omeprazole induced UGT1A1 mRNA (8.8 fold) and UGT1A9 mRNA (4.4 fold) whereas b-naphthoflavone caused a 4.7-fold increase in UGT1A1 mRNA levels. The other inducers had little or no effect on UGT mRNA expression in Fa2N-4 cells. Microsomal UGT activity toward triiodothyronine and thyroxine was not induced in immortalized human hepatocytes treated with b-naphthoflavone or rifampin. Although statistically different from samples treated with vehicle, treatment of cultured human hepatocytes with rifampin caused less than a 2-fold increase in UGT activity toward T3. These results suggest that human UGTs are not as highly inducible as certain human CYP enzymes, nor as inducible as certain UGTs in rats.

Cytochrome P450 3A4 and 1A2 Induction in Immortalized and Primary Cultures of Human Hepatocytes

Published:  25 August 2004

Cytochrome P450 3A4 and 1A2 Induction in the Immortalized Hepatocyte Line, Fa2N-4, and Comparison with Primary Cultures of Human Hepatocytes

Primary cultures of human hepatocytes are the gold standard for evaluating induction of cytochrome P450 (CYP) enzymes by new molecular entities. Recently, immortalized human hepatocytes, known as Fa2N-4 cells, have been shown to express CYP3A4 and CYP1A2. Additionally, these cells exhibited enzyme induction in response to treatment with agonists of nuclear receptors (Mills et al., 2004). To establish the Fa2N-4 cells as a tool for determining CYP3A4 and CYP1A2 induction, we measured the cells' response to drugs previously evaluated in multiple primary hepatocyte cultures (Luo et al., 2002). Midazolam 1'-hydroxylase and phenacetin O-dealkylase activity were monitored using LC/MS/MS methods to determine the effects of the drugs on expression of CYP3A4 and CYP1A2, respectively. Toxic effects of the compounds were monitored with lactate dehydrogenase (LDH) leakage into the culture media. The magnitude of CYP3A4 and CYP1A2 induction in Fa2N-4 cells agrees closely with primary cultures of human hepatocytes. The comparison of the CYP response in the two cell culture systems validates immortalized hepatocytes as a preclinical tool for assessment of CYP3A4 and CYP1A2 induction.

Additional Posters Are Available Upon Request

Published:  24 August 2004

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