Pig hepatocytes as an in vitro model to study the regulation of human CYP3A4: prediction of drug-drug interactions with 17α-ethynylestradiol

Olsen, Anette Kristensen; Hansen, Kristian T.; Friis, Christian

doi:10.1016/s0009-2797(97)00077-x

Cited by 41 publications

(31 citation statements)

References 25 publications

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“…The same applies for inducers, as for inhibitors, they are very rarely specific towards one enzyme, but are able to induce several enzymes [28]. It is therefore of importance, when studying the induction at the protein level, to know the specificity of the antibodies used for immunoblotting and to use species specific antibodies, if available, or to use antibodies specific to species closely related.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to CYP2A the CYP3A activity remained constant during the first 72 hrs. of incubation, and the activity could be induced by rifampicin 3-4 times [28,6] , a potent inducer of CYP3A4 in human hepatocyte cultures [29]. Rifampicin also induces both the 1-and 4-hydroxylation of midazolam, whereas dexamethasone, another inducer of human CYP3A4, could induce neither the testosterone hydroxylation nor the midazolam hydroxylation [2,30,12].…”

Section: Cyp3amentioning

confidence: 99%

“…Rifampicin also induces both the 1-and 4-hydroxylation of midazolam, whereas dexamethasone, another inducer of human CYP3A4, could induce neither the testosterone hydroxylation nor the midazolam hydroxylation [2,30,12]. Phenobarbital, phenytoin, and carbamazepine were also able to induce hydroxylation of testosterone [1,28]. Induction with phenobarbital showed that CYP3A could be enhanced both in vitro and in vivo and at both enzyme activity level, apoprotein level, and at mRNA level [3].…”

Section: Cyp3amentioning

confidence: 99%

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Porcine Cytochrome P450 and Metabolism

Skaanild¹

2006

CPD

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The pig and especially the minipig are becoming increasingly used as a test animal both in pharmacological and toxicological testing of new compounds. The minipig is used because of its size, it is easy to handle and less test substrate is required. When using an animal species for testing it is of importance to know if the test animal's posses the same abilities to metabolize drugs as humans. Some of the P450 enzymes have been characterized in the pig regarding substrate specificity, inhibition and regulation. The porcine enzymes CYP1A, CYP2A and CYP3A all metabolize the same test substrates as the human enzymes, whereas the enzymes CYP2B, CYP2D, and CYP2E in pig on the other hand seem to be different from the human enzymes concerning metabolism of the well know test substrates. Some of the porcine enzymes have been sequenced i.e.CYP1A, CYP2A, CYP2B, CYP2D, CYP2E and CYP3A and not surprisingly the porcine CYPs that metabolize the human test substrates are about 75% identical in cDNA sequences.What is needed is inhibitory antibodies against each of the porcine enzymes, in order to test whether a test compound is metabolized by one or the other enzyme. Until now chemical inhibitors have been used, but they are rarely 100% specific. Anti-human inhibitory antibodies have also been used, but they may not recognize the porcine enzyme and therefore will not inhibit the reaction. Antibodies for immunoblotting would also make it possible to estimate how much of the total P450 the individual enzymes comprise. From what is known about the porcine P450, it can be concluded that the pig seems to be a good test species if CYP1A, CYP2A or CYP3A are involved in the metabolism of the test compound, depending on the contribution of other enzymes in competing pathways.

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Section: Discussionmentioning

confidence: 99%

Section: Cyp3amentioning

confidence: 99%

Section: Cyp3amentioning

confidence: 99%

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Porcine Cytochrome P450 and Metabolism

Skaanild¹

2006

CPD

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“…Four enzymes in the CYP3A family have been cloned from pigs (domestic and minipig) [CYP3A22, CYP3A29 (Nissen et al, 1998), CYP3A39, and CYP3A46], with 75 to 78% amino acid identity to human CYP3A4 and 82 to 84% nucleotide similarity (Sakuma et al, 2004). Enterocytes, hepatocytes, and liver and intestinal microsomes from pigs have been used to study drug metabolism, and, even though the CYP3A enzymes are not identical to CYP3A4, similar activity and a corresponding rate of metabolism have been observed for substrates such as testosterone and tacrolimus (Olsen et al, 1997;Skaanild and Friis, 1997;Bader et al, 2000). Inhibition of CYP3A-mediated metabolism by ketoconazole has been reported in pig intestinal microsomes (Lampen et al, 1996).…”

Section: Lundahl Et Almentioning

confidence: 99%

In Vivo Investigation in Pigs of Intestinal Absorption, Hepatobiliary Disposition, and Metabolism of the 5α-Reductase Inhibitor Finasteride and the Effects of Coadministered Ketoconazole

Lundahl¹,

Hedeland²,

Bondesson³

et al. 2011

Drug Metab Dispos

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ABSTRACT:The overall aim of this detailed investigation of the pharmacokinetics (PK) and metabolism of finasteride in pigs was to improve understanding of in vivo PK for this drug and its metabolites. Specific aims were to examine the effects of ketoconazole coadministration on the PK in three plasma compartments (the portal, hepatic, and femoral veins), bile, and urine and to use these data to study in detail the intestinal absorption and the liver extraction ratio and apply a semiphysiological based PK model to the data. The pigs received an intrajejunal dose of finasteride (0.8 mg/kg) either alone (n ‫؍‬ 5) or together with ketoconazole (10 mg/kg) (n ‫؍‬ 5) or an intravenous dose (0.2 mg/kg) (n ‫؍‬ 3). Plasma, bile, and urine (collected from 0 to 6 h) were analyzed with ultraperformance liquid chromatography-tandem mass spectrometry. Ketoconazole increased the bioavailability of finasteride from 0.36 ؎ 0.23 to 0.91 ؎ 0.1 (p < 0.05) and the terminal half-life from 1.6 ؎ 0.4 to 4.0 ؎ 1.1 h (p < 0.05). From deconvolution, it was found that the absorption rate from the intestine to the portal vein was rapid, and the product of the fraction absorbed and the fraction that escaped gut wall metabolism was high (f a ⅐ F G ϳ1). Interestingly, the apparent absorption rate constant (k a ) to the femoral vein was lower than that to the portal vein, probably because of binding and distribution within the liver. The liver extraction ratio was time-dependent and varied with the two routes of administration. After intrajejunal administration, from 1 to 6 h, the liver extraction ratio was significantly (p < 0.05) reduced by ketoconazole treatment from intermediate (0.41 ؎ 0.21) to low (0.21 ؎ 0.10).

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“…35-40 kg. The pigs were feed commercial chow and provided with water ad libitum.The pigs were euthanized by use of a captive bolt pistol, and the hepatocytes were isolated using a two step collagenase perfusion technique according to Olsen et al (1997). The isolated hepatocytes had approximately 70% viability as assessed by tryphan blue exclusion before plating.…”

mentioning

confidence: 99%

Expression Changes of CYP2A and CYP3A in Microsomes from Pig Liver and Cultured Hepatocytes

Skaanild¹,

Friis²

2000

Pharmacology & Toxicology

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The P450 enzymes of the liver are responsible for the metabolism of a wide range of chemical compounds, and hepatocytes are used in pharmacological and toxicological in vitro tests. Thus, it is important to know how stable these enzymes are in culture. We measured the activity of CYP2A and CYP3A in microsomes isolated from both pig liver and primary pig hepatocyte cultures, together with the apoprotein concentration using Western blotting. The CYP2A activity and apoprotein concentration decreased rapidly; only about 5 percent remained after 48 hr incubation, whereas the CYP3A activity and apoprotein concentration was constant. CYP3A was induced 3 times after exposure to rifampicin, whereas neither rifampicin nor pyrazole could induce CYP2A. The hepatocytes were also incubated with varying concentration of FCS and autologous serum, however without effect on the stability of CYP2A, nor did different concentrations of growth hormone and testosterone added to the cultures have any effect.The P450 enzymes are involved in the oxidation of a wide range of compounds including drugs, steroids, vitamins etc. (Nelson et al. 1996). The action of P450 may lead to eventual detoxification of the substrate, either direct or through the subsequent action of phase 2 enzymes. However the activity of P450 can also produce toxic metabolites. Because of these unique functions of the liver, cultured hepatocytes and microsomes from hepatocytes and livers have been used for toxicological and metabolic studies in vitro. Several studies show a decrease in enzyme activity of some of the cytochrome P450 isoenzymes. These declines have been observed in both human (George et al. 1996;Vandenbranden et al. 1998) and rat (Liddle et al. 1992) primary hepatocytes cultures. It is therefore essential to establish how pig hepatocytes P450 react in cultures, especially if pig hepatocytes are used as a substitute for i.e. human hepatocytes in toxicological and pharmacological in vitro studies. It is important to know if the P450 isoenzymes remain active and in the same proportions in microsomes isolated from liver and cultured hepatocytes. Since preliminary studies in our laboratory indicate that CYP2A in pig hepatocytes decrease very fast after start of culture, the objectives of this study were: 1) to measure and compare the activity and the immunochemical level of two isoenzymes CYP2A and CYP3A in microsomes from livers, from freshly isolated hepatocytes and from hepatocytes cultured up to 48 hr, 2) to investigate the inducibility of CYP2A and CYP3A with classical inducers, 3) to analyze if serum contains ''stimulating factors'' for the activity of the two isoenzymes, and 4) Author for correspondence: Mette T. Skaanild, The Royal Veterinary and Agricultural University, Department of Pharmacology and Pathobiology, Ridebanevej 9, DK-1870 Frederiksberg C, Denmark (fax π45 35 35 35 14, e-mail mts/kvl.dk). the effect of growth hormone and testosterone exposure was also studied. Materials and MethodsThe experiment was performed using female Dani...

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Pig hepatocytes as an in vitro model to study the regulation of human CYP3A4: prediction of drug-drug interactions with 17α-ethynylestradiol

Cited by 41 publications

References 25 publications

Porcine Cytochrome P450 and Metabolism

Porcine Cytochrome P450 and Metabolism

In Vivo Investigation in Pigs of Intestinal Absorption, Hepatobiliary Disposition, and Metabolism of the 5α-Reductase Inhibitor Finasteride and the Effects of Coadministered Ketoconazole

Expression Changes of CYP2A and CYP3A in Microsomes from Pig Liver and Cultured Hepatocytes

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