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Evaluation of 4β-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice

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Abstract

Cytochrome P450 3A (CYP3A) enzymes metabolize approximately half of all drugs on the market. Since the endogenous compounds 4β-hydroxycholesterol (4β-HC) and 25-hydroxycholesterol (25-HC) are generated from cholesterol via CYP3A enzymes, we examined whether the plasma levels of 4β-HC and 25-HC reflect hepatic CYP3A4 activity by using a CYP3A-humanized mouse model, in which the function of endogenous Cyp3a was genetically replaced by human CYP3A. CYP3A-humanized mice have great advantages for evaluation of the relationship between hepatic CYP3A protein levels and plasma and hepatic levels of 4β-HC and 25-HC. Levels of CYP3A4 protein in the liver microsomes of CYP3A-humanized mice were increased by treatment with pregnenolone-16α-carbonitrile, a CYP3A inducer. Hepatic and plasma levels of 4β-HC and 25-HC normalized by cholesterol were significantly correlated with hepatic CYP3A4 protein levels. In addition, in vitro studies using human liver microsomes showed that the formation of 4β-HC was strongly inhibited by a CYP3A inhibitor, while the inhibitory effect of the CYP3A inhibition on the formation of 25-HC was weak. These results suggested that CYP3A mainly contributed to the formation of 4β-HC in human liver microsomes, whereas other factors may be involved in the formation of 25-HC. In conclusion, the in vivo studies using CYP3A-humanized mice suggest that plasma 4β-HC and 25-HC levels reflect hepatic CYP3A4 activity. Furthermore, taking the results of in vitro studies using human liver microsomes into consideration, 4β-HC is a more reliable biomarker of hepatic CYP3A activity.

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References

  1. Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther. 1994;270(1):414–23.

    PubMed  CAS  Google Scholar 

  2. Thummel KE, Wilkinson GR. In vitro and in vivo drug interactions involving human CYP3A. Annu Rev Pharmacol Toxicol. 1998;38(1):389–430. https://doi.org/10.1146/annurev.pharmtox.38.1.389.

    Article  PubMed  CAS  Google Scholar 

  3. Baker AL, Kotake AN, Schoeller DA. Clinical utility of breath tests for the assessment of hepatic function. Semin Liver Dis. 1983;3(4):318–29. https://doi.org/10.1055/s-2008-1040784.

    Article  PubMed  CAS  Google Scholar 

  4. Watkins PB, Murray SA, Winkelman LG, Heuman DM, Wrighton SA, Guzelian PS. Erythromycin breath test as an assay of glucocorticoid-inducible liver cytochromes P-450. Studies in rats and patients. J Clin Invest. 1989;83(2):688–97. https://doi.org/10.1172/JCI113933.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Watkins PB, Hamilton TA, Annesley TM, Ellis CN, Kolars JC, Voorhees JJ. The erythromycin breath test as a predictor of cyclosporine blood levels. Clin Pharmacol Ther. 1990;48(2):120–9.

    Article  PubMed  CAS  Google Scholar 

  6. Thummel KE, Shen DD, Podoll TD, Kunze KL, Trager WF, Hartwell PS, et al. Use of midazolam as a human cytochrome P450 3A probe: I. In vitro-in vivo correlations in liver transplant patients. J Pharmacol Exp Ther. 1994;271(1):549–56.

    PubMed  CAS  Google Scholar 

  7. Thummel KE, Shen DD, Podoll TD, Kunze KL, Trager WF, Bacchi CE, et al. Use of midazolam as a human cytochrome P450 3A probe: II. Characterization of inter- and intraindividual hepatic CYP3A variability after liver transplantation. J Pharmacol Exp Ther. 1994;271(1):557–66.

    PubMed  CAS  Google Scholar 

  8. Thummel KE, O'Shea D, Paine MF, Shen DD, Kunze KL, Perkins JD, et al. Oral first-pass elimination of midazolam involves both gastrointestinal and hepatic CYP3A-mediated metabolism. Clin Pharmacol Ther. 1996;59(5):491–502. https://doi.org/10.1016/S0009-9236(96)90177-0.

    Article  PubMed  CAS  Google Scholar 

  9. Kinirons MT, O'Shea D, Downing TE, Fitzwilliam AT, Joellenbeck L, Groopman JD, et al. Absence of correlations among three putative in vivo probes of human cytochrome P4503A activity in young healthy men. Clin Pharmacol Ther 1993;54(6):621–629.

  10. Bienvenu T, Rey E, Pons G, d'Athis P, Olive G. A simple non-invasive procedure for the investigation of cytochrome P-450 IIIA dependent enzymes in humans. Int J Clin Pharmacol Ther Toxicol 1991;29(11):441–445.

  11. Joellenbeck L, Qian Z, Zarba A, Groopman JD. Urinary 6beta-hydroxycortisol/cortisol ratios measured by high-performance liquid chromatography for use as a biomarker for the human cytochrome P-450 3A4. Cancer Epidemiol Biomark Prev. 1992;1(7):567–72.

    CAS  Google Scholar 

  12. Watkins PB, Turgeon DK, Saenger P, Lown KS, Kolars JC, Hamilton T, et al. Comparison of urinary 6-beta-cortisol and the erythromycin breath test as measures of hepatic P450IIIA (CYP3A) activity. Clin Pharmacol Ther. 1992;52(3):265–73.

    Article  PubMed  CAS  Google Scholar 

  13. Kinirons MT, O'Shea D, Kim RB, Groopman JD, Thummel KE, Wood AJ, et al. Failure of erythromycin breath test to correlate with midazolam clearance as a probe of cytochrome P4503A. Clin Pharmacol Ther. 1999;66(3):224–31. https://doi.org/10.1016/S0009-9236(99)70029-9.

    Article  PubMed  CAS  Google Scholar 

  14. Yasui-Furukori N, Kondo T, Kubota T, Otake H, Ohkubo T, Nagasaki T, et al. No correlations between the urinary ratio of 6beta-hydroxycortisol to free cortisol and pharmacokinetics of alprazolam. Eur J Clin Pharmacol. 2001;57(4):285–8.

    Article  PubMed  CAS  Google Scholar 

  15. Bodin K, Bretillon L, Aden Y, Bertilsson L, Broomé U, Einarsson C, et al. Antiepileptic drugs increase plasma levels of 4beta-hydroxycholesterol in humans: evidence for involvement of cytochrome p450 3A4. J Biol Chem. 2001;276(42):38685–9. https://doi.org/10.1074/jbc.M105127200.

    Article  PubMed  CAS  Google Scholar 

  16. Bodin K, Andersson U, Rystedt E, Ellis E, Norlin M, Pikuleva I, et al. Metabolism of 4beta-hydroxycholesterol in humans. J Biol Chem. 2002;277(35):31534–40. https://doi.org/10.1074/jbc.M201712200.

    Article  PubMed  CAS  Google Scholar 

  17. Wide K, Larsson H, Bertilsson L, Diczfalusy U. Time course of the increase in 4beta-hydroxycholesterol concentration during carbamazepine treatment of paediatric patients with epilepsy. Br J Clin Pharmacol. 2008;65(5):708–15. https://doi.org/10.1111/j.1365-2125.2007.03078.x.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Marschall HU, Wagner M, Zollner G, Fickert P, Diczfalusy U, Gumhold J, et al. Complementary stimulation of hepatobiliary transport and detoxification systems by rifampicin and ursodeoxycholic acid in humans. Gastroenterology. 2005;129(2):476–85. https://doi.org/10.1016/j.gastro.2005.05.009.

    Article  PubMed  Google Scholar 

  19. Niemi M, Kivistö KT, Diczfalusy U, Bodin K, Bertilsson L, Fromm MF, et al. Effect of SLCO1B1 polymorphism on induction of CYP3A4 by rifampicin. Pharmacogenet Genomics. 2006;16(8):565–8. https://doi.org/10.1097/01.fpc.0000215070.52212.0e.

    Article  PubMed  CAS  Google Scholar 

  20. Kanebratt KP, Diczfalusy U, Bäckström T, Sparve E, Bredberg E, Böttiger Y, et al. Cytochrome P450 induction by rifampicin in healthy subjects: determination using the Karolinska cocktail and the endogenous CYP3A4 marker 4beta-hydroxycholesterol. Clin Pharmacol Ther. 2008;84(5):589–94. https://doi.org/10.1038/clpt.2008.132.

    Article  PubMed  CAS  Google Scholar 

  21. Diczfalusy U, Kanebratt KP, Bredberg E, Andersson TB, Böttiger Y, Bertilsson L. 4beta-hydroxycholesterol as an endogenous marker for CYP3A4/5 activity. Stability and half-life of elimination after induction with rifampicin. Br J Clin Pharmacol 2009;67(1):38–43. doi:https://doi.org/10.1111/j.1365-2125.2008.03309.x.

  22. Goodenough AK, Onorato JM, Ouyang Z, Chang S, Rodrigues AD, Kasichayanula S, et al. Quantification of 4-beta-hydroxycholesterol in human plasma using automated sample preparation and LC-ESI-MS/MS analysis. Chem Res Toxicol. 2011;24(9):1575–85. https://doi.org/10.1021/tx2001898.

    Article  PubMed  CAS  Google Scholar 

  23. Björkhem-Bergman L, Bäckström T, Nylén H, Rönquist-Nii Y, Bredberg E, Andersson TB, et al. Comparison of endogenous 4β-hydroxycholesterol with midazolam as markers for CYP3A4 induction by rifampicin. Drug Metab Dispos. 2013;41(8):1488–93. https://doi.org/10.1124/dmd.113.052316.

    Article  PubMed  CAS  Google Scholar 

  24. Dutreix C, Lorenzo S, Wang Y. Comparison of two endogenous biomarkers of CYP3A4 activity in a drug-drug interaction study between midostaurin and rifampicin. Eur J Clin Pharmacol. 2014;70(8):915–20. https://doi.org/10.1007/s00228-014-1675-0.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Leil TA, Kasichayanula S, Boulton DW, LaCcreta F. Evaluation of 4β-hydroxycholesterol as a clinical biomarker of CYP3A4 drug interactions using a Bayesian mechanism-based pharmacometric model. CPT Pharmacometrics Syst Pharmacol. 2014;(3, 6):e120. https://doi.org/10.1038/psp.2014.18.

  26. Kasichayanula S, Boulton DW, Luo WL, Rodrigues AD, Yang Z, Goodenough A, et al. Validation of 4β-hydroxycholesterol and evaluation of other endogenous biomarkers for the assessment of CYP3A activity in healthy subjects. Br J Clin Pharmacol. 2014;78(5):1122–34. https://doi.org/10.1111/bcp.12425.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Aubry AF, Dean B, Diczfalusy U, Goodenough A, Iffland A, McLeod J, et al. Recommendations on the development of a bioanalytical assay for 4β-hydroxycholesterol, an emerging endogenous biomarker of CYP3A activity. AAPS J. 2016;18(5):1056–66. https://doi.org/10.1208/s12248-016-9949-3.

    Article  PubMed  CAS  Google Scholar 

  28. Lütjohann D, Marinova M, Schneider B, Oldenburg J, von Bergmann K, Bieber T, et al. 4beta-hydroxycholesterol as a marker of CYP3A4 inhibition in vivo—effects of itraconazole in man. Int J Clin Pharmacol Ther. 2009;47(12):709–15.

    Article  PubMed  Google Scholar 

  29. Honda A, Miyazaki T, Ikegami T, Iwamoto J, Maeda T, Hirayama T, et al. Cholesterol 25-hydroxylation activity of CYP3A. J Lipid Res. 2011;52(8):1509–16. https://doi.org/10.1194/jlr.M014084.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Hashimoto M, Kobayashi K, Watanabe M, Kazuki Y, Takehara S, Inaba A, et al. Knockout of mouse Cyp3a gene enhances synthesis of cholesterol and bile acid in the liver. J Lipid Res. 2013;54(8):2060–8. https://doi.org/10.1194/jlr.M033464.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Kazuki Y, Kobayashi K, Aueviriyavit S, Oshima T, Kuroiwa Y, Tsukazaki Y, et al. Trans-chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans. Hum Mol Genet. 2013;22(3):578–92. https://doi.org/10.1093/hmg/dds468.

    Article  PubMed  CAS  Google Scholar 

  32. Diczfalusy U. On the formation and possible biological role of 25-hydroxycholesterol. Biochimie. 2013;95(3):455–60. https://doi.org/10.1016/j.biochi.2012.06.016.

    Article  PubMed  CAS  Google Scholar 

  33. Williamson BL, Purkayastha S, Hunter CL, Nuwaysir L, Hill J, Easterwood L, et al. Quantitative protein determination for CYP induction via LC-MS/MS. Proteomics. 2011;11(1):33–41. https://doi.org/10.1002/pmic.201000456.

    Article  PubMed  CAS  Google Scholar 

  34. Newton DJ, Wang RW, Lu AY. Cytochrome P450 inhibitors. Evaluation of specificities in the in vitro metabolism of therapeutic agents by human liver microsomes. Drug Metab Dispos. 1995;23(1):154–8.

    PubMed  CAS  Google Scholar 

  35. Honda A, Yamashita K, Miyazaki H, Shirai M, Ikegami T, Xu G, et al. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS. J Lipid Res. 2008;49(9):2063–73. https://doi.org/10.1194/jlr.D800017-JLR200.

    Article  PubMed  CAS  Google Scholar 

  36. Honda A, Yamashita K, Hara T, Ikegami T, Miyazaki T, Shirai M, et al. Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS. J Lipid Res. 2009;50(2):350–7. https://doi.org/10.1194/jlr.D800040-JLR200.

    Article  PubMed  CAS  Google Scholar 

  37. Schuetz EG, Schmid W, Schutz G, Brimer C, Yasuda K, Kamataki T, et al. The glucocorticoid receptor is essential for induction of cytochrome P-4502B by steroids but not for drug or steroid induction of CYP3A or P-450 reductase in mouse liver. Drug Metab Dispos 2000;28(3):268–278.

  38. Xu C, Wang X, Staudinger JL. Regulation of tissue-specific carboxylesterase expression by pregnane x receptor and constitutive androstane receptor. Drug Metab Dispos. 2009;37(7):1539–47. https://doi.org/10.1124/dmd.109.026989.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Hukkanen J, Puurunen J, Hyötyläinen T, Savolainen MJ, Ruokonen A, Morin-Papunen L, et al. The effect of atorvastatin treatment on serum oxysterol concentrations and cytochrome P450 3A4 activity. Br J Clin Pharmacol. 2015;80(3):473–9. https://doi.org/10.1111/bcp.12701.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. de Graan AJ, Sparreboom A, de Bruijn P, de Jonge E, van der Holt B, Wiemer EA, et al. 4β-hydroxycholesterol as an endogenous CYP3A marker in cancer patients treated with taxanes. Br J Clin Pharmacol. 2015;80(3):560–8. https://doi.org/10.1111/bcp.12707.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Kawakami H, Ohtsuki S, Kamiie J, Suzuki T, Abe T, Terasaki T. Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection. J Pharm Sci. 2011;100(1):341–52. https://doi.org/10.1002/jps.22255.

    Article  PubMed  CAS  Google Scholar 

  42. Lund EG, Kerr TA, Sakai J, Li WP, Russell DW. cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism. J Biol Chem. 1998;273(51):34316–27.

    Article  PubMed  CAS  Google Scholar 

  43. Li X, Pandak WM, Erickson SK, Ma Y, Yin L, Hylemon P, et al. Biosynthesis of the regulatory oxysterol, 5-cholesten-3beta,25-diol 3-sulfate, in hepatocytes. J Lipid Res. 2007;48(12):2587–96. https://doi.org/10.1194/jlr.M700301-JLR200.

    Article  PubMed  CAS  Google Scholar 

  44. Shinkyo R, Guengerich FP. Inhibition of human cytochrome P450 3A4 by cholesterol. J Biol Chem 2011;286(21):18426–18433. doi:https://doi.org/10.1074/jbc.M111.240457.

  45. Smith LL. Cholesterol autoxidation. New York: Plenum Press. 1981; https://doi.org/10.1007/978-1-4757-9691-9.

  46. Diczfalusy U, Björkhem I. Still another activity by the highly promiscuous enzyme CYP3A4: 25-hydroxylation of cholesterol. J Lipid Res 2011;52(8):1447–1449. doi: https://doi.org/10.1194/jlr.E017806.

  47. Gjestad C, Haslemo T, Andreassen OA, Molden E. 4β-Hydroxycholesterol level significantly correlates with steady-state serum concentration of the CYP3A4 substrate quetiapine in psychiatric patients. Br J Clin Pharmacol. 2017;83(11):2398–405. https://doi.org/10.1111/bcp.13341.

    Article  PubMed  CAS  Google Scholar 

  48. Gjestad C, Huynh DK, Haslemo T, Molden E. 4β-hydroxycholesterol correlates with dose but not steady-state concentration of carbamazepine: indication of intestinal CYP3A in biomarker formation? Br J Clin Pharmacol. 2016;81(2):269–76. https://doi.org/10.1111/bcp.12833.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by JSPS KAKENHI Grant Number 15K08067 (KK), the Regional Innovation Strategy Support Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (YK and MO) and the Funding Program for Next Generation World-Leading Researchers (NEXT Program) from the Japan Society for the Promotion of Science (JSPS) (YK). We thank Y Sumida, T Kurosaki and M Morimura at Tottori University for technical assistance.

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Authors and Affiliations

  1. Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan

    Shin-ichiro Nitta, Mari Hashimoto, Hidetaka Akita, Kan Chiba & Kaoru Kobayashi

  2. LSI Medience Corporation, 3-30-1 Shimura, Itabashi-ku, Tokyo, 174-8555, Japan

    Shin-ichiro Nitta & Hiraku Suzuki

  3. Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan

    Yasuhiro Kazuki

  4. Chromosome Engineering Research Center (CERC), Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan

    Yasuhiro Kazuki, Shoko Takehara & Mitsuo Oshimura

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  1. Shin-ichiro Nitta
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Nitta, Si., Hashimoto, M., Kazuki, Y. et al. Evaluation of 4β-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice. AAPS J 20, 61 (2018). https://doi.org/10.1208/s12248-018-0186-9

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