Archives of Toxicology

, Volume 93, Issue 7, pp 1917–1926 | Cite as

The Toll-like receptor agonist imiquimod is metabolized by aryl hydrocarbon receptor-regulated cytochrome P450 enzymes in human keratinocytes and mouse liver

  • Melina Mescher
  • Julia Tigges
  • Katharina M. Rolfes
  • Anna L. Shen
  • Jeremiah S. Yee
  • Christian Vogeley
  • Jean Krutmann
  • Christopher A. Bradfield
  • Dieter Lang
  • Thomas Haarmann-StemmannEmail author
Molecular Toxicology


The Toll-like receptor 7 agonist imiquimod (IMQ) is an approved drug for the topical treatment of various skin diseases that, in addition, is currently tested in multiple clinical trials for the immunotherapy of various types of cancers. As all of these trials include application of IMQ to the skin and evidence exists that exposure to environmental pollutants, i.e., tobacco smoke, affects its therapeutic efficacy, the current study aims to elucidate the cutaneous metabolism of the drug. Treatment of human keratinocytes with 2.5 µM benzo[a]pyrene (BaP), a tobacco smoke constituent and aryl hydrocarbon receptor (AHR) agonist, for 24 h induced cytochrome P450 (CYP) 1A enzyme activity. The addition of IMQ 30 min prior measurement resulted in a dose-dependent inhibition of CYP1A activity, indicating that IMQ is either a substrate or inhibitor of CYP1A isoforms. Incubation of 21 recombinant human CYP enzymes with 0.5 µM IMQ and subsequent LC–MS analyses, in fact, identified CYP1A1 and CYP1A2 as being predominantly responsible for IMQ metabolism. Accordingly, treatment of keratinocytes with BaP accelerated IMQ clearance and the associated formation of monohydroxylated IMQ metabolites. A co-incubation with 5 µM 7-hydroxyflavone, a potent inhibitor of human CYP1A isoforms, abolished basal as well as BaP-induced IMQ metabolism. Further studies with hepatic microsomes from CD-1 as well as solvent- and β-naphthoflavone-treated CYP1A1/CYP1A2 double knock-out and respective control mice confirmed the critical contribution of CYP1A isoforms to IMQ metabolism. Hence, an exposure to life style-related, dietary, and environmental AHR ligands may affect the pharmacokinetics and, thus, treatment efficacy of IMQ.


Aryl hydrocarbon receptor Cytochrome P450 Imiquimod Immunotherapy Psoriasis 



We thank Daniel W. Nebert for generously providing CYP1A1/CYP1A2 double KO mice, and Franziska Weigner, Melanie Scheinpflug, Ragnhild Wirth, and Diane Schmiegelt for technical support. Research in the THS lab is supported by DFG grant HA 7346/2–1 and the Juergen Manchot Foundation. The CAB lab thanks NIEHS grant ES028377.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

204_2019_2488_MOESM1_ESM.docx (506 kb)
Supplementary file1 (DOCX 507 kb)


  1. Ahmad N, Mukhtar H (2004) Cytochrome p450: a target for drug development for skin diseases. J Invest Dermatol 123(3):417–425CrossRefPubMedGoogle Scholar
  2. Alvarez P, Jensen LE (2016) Imiquimod Treatment causes systemic disease in mice resembling generalized pustular psoriasis in an IL-1 and IL-36 dependent manner. Mediat Inflamm 2016:6756138CrossRefGoogle Scholar
  3. Baron JM, Holler D, Schiffer R et al (2001) Expression of multiple cytochrome p450 enzymes and multidrug resistance-associated transport proteins in human skin keratinocytes. J Invest Dermatol 116(4):541–548CrossRefPubMedGoogle Scholar
  4. Berghofer B, Frommer T, Haley G, Fink L, Bein G, Hackstein H (2006) TLR7 ligands induce higher IFN-alpha production in females. J Immunol 177(4):2088–2096CrossRefPubMedGoogle Scholar
  5. Brown VL, Atkins CL, Ghali L, Cerio R, Harwood CA, Proby CM (2005) Safety and efficacy of 5% imiquimod cream for the treatment of skin dysplasia in high-risk renal transplant recipients: randomized, double-blind, placebo-controlled trial. Arch Dermatol 141(8):985–993CrossRefGoogle Scholar
  6. Buters JT, Sakai S, Richter T et al (1999) Cytochrome P450 CYP1B1 determines susceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas. Proc Natl Acad Sci U S A 96(5):1977–1982CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cen X, Liu S, Cheng K (2018) The role of toll-like receptor in inflammation and tumor immunity. Front Pharmacol 9:878CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chang CY, Puga A (1998) Constitutive activation of the aromatic hydrocarbon receptor. Mol Cell Biol 18(1):525–535CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chiaro CR, Patel RD, Marcus CB, Perdew GH (2007) Evidence for an aryl hydrocarbon receptor-mediated cytochrome p450 autoregulatory pathway. Mol Pharmacol 72(5):1369–1379CrossRefPubMedGoogle Scholar
  10. Di Meglio P, Duarte JH, Ahlfors H et al (2014) Activation of the aryl hydrocarbon receptor dampens the severity of inflammatory skin conditions. Immunity 40(6):989–1001CrossRefPubMedPubMedCentralGoogle Scholar
  11. Dragin N, Uno S, Wang B, Dalton TP, Nebert DW (2007) Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line. Biochem Biophys Res Commun 359(3):635–642CrossRefPubMedPubMedCentralGoogle Scholar
  12. Flutter B, Nestle FO (2013) TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis. Eur J Immunol 43(12):3138–3146CrossRefPubMedGoogle Scholar
  13. Frauenstein K, Tigges J, Soshilov AA et al (2015) Activation of the aryl hydrocarbon receptor by the widely used Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2). Arch Toxicol 89(8):1329–1336CrossRefPubMedGoogle Scholar
  14. Geisse J, Caro I, Lindholm J, Golitz L, Stampone P, Owens M (2004) Imiquimod 5% cream for the treatment of superficial basal cell carcinoma: results from two phase III, randomized, vehicle-controlled studies. J Am Acad Dermatol 50(5):722–733CrossRefPubMedPubMedCentralGoogle Scholar
  15. Goldstein D, Hertzog P, Tomkinson E et al (1998) Administration of imiquimod, an interferon inducer, in asymptomatic human immunodeficiency virus-infected persons to determine safety and biologic response modification. J Infect Dis 178(3):858–861CrossRefPubMedGoogle Scholar
  16. Gotwals P, Cameron S, Cipolletta D et al (2017) Prospects for combining targeted and conventional cancer therapy with immunotherapy. Nat Rev Cancer 17(5):286–301CrossRefPubMedGoogle Scholar
  17. Hadley G, Derry S, Moore RA (2006) Imiquimod for actinic keratosis: systematic review and meta-analysis. J Invest Dermatol 126(6):1251–1255CrossRefGoogle Scholar
  18. Harvey RD, Morgan ET (2014) Cancer, inflammation, and therapy: effects on cytochrome p450-mediated drug metabolism and implications for novel immunotherapeutic agents. Clin Pharmacol Ther 96(4):449–457CrossRefPubMedGoogle Scholar
  19. Harvey G, Pontefract D, Hughes BR, Brinkmann D, Christie C (2019) Impact of smoking on imiquimod response in patients with vulval intraepithelial neoplasia. Clin Exp Dermatol. CrossRefPubMedGoogle Scholar
  20. Hecht SS (2003) Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer 3(10):733–744CrossRefPubMedGoogle Scholar
  21. Heikkinen AK, Susitaival P (2011) Severe systemic reaction to topical imiquimod. Acta Derm Venereol 91(5):594–595CrossRefPubMedGoogle Scholar
  22. Hemmi H, Kaisho T, Takeuchi O et al (2002) Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 3(2):196–200CrossRefPubMedGoogle Scholar
  23. IARC (2010) Some non-heterocyclic polycyclic aromatic hydrocarbons and some related exposures. IARC Monogr Eval Carcinog Risks Hum 92:1–853Google Scholar
  24. Kleiner HE, Vulimiri SV, Reed MJ, Uberecken A, DiGiovanni J (2002) Role of cytochrome P450 1a1 and 1b1 in the metabolic activation of 7,12-dimethylbenz[a]anthracene and the effects of naturally occurring furanocoumarins on skin tumor initiation. Chem Res Toxicol 15(2):226–235CrossRefPubMedGoogle Scholar
  25. Lang D, Radtke M, Bairlein M (2019) Highly variable expression of CYP1A1 in human liver and impact on pharmacokinetics of riociguat and granisetron in humans. Chem Res Toxicol. CrossRefPubMedGoogle Scholar
  26. Mescher M, Haarmann-Stemmann T (2018) Modulation of CYP1A1 metabolism: from adverse health effects to chemoprevention and therapeutic options. Pharmacol Ther 187:71–87CrossRefPubMedGoogle Scholar
  27. Murray IA, Patterson AD, Perdew GH (2014) Aryl hydrocarbon receptor ligands in cancer: friend and foe. Nat Rev Cancer 14(12):801–814CrossRefPubMedPubMedCentralGoogle Scholar
  28. Nakamura H, Ariyoshi N, Okada K, Nakasa H, Nakazawa K, Kitada M (2005) CYP1A1 is a major enzyme responsible for the metabolism of granisetron in human liver microsomes. Curr Drug Metab 6(5):469–480CrossRefPubMedGoogle Scholar
  29. Nebert DW (2017) Aryl hydrocarbon receptor (AHR): "pioneer member" of the basic-helix/loop/helix per-Arnt-sim (bHLH/PAS) family of "sensors" of foreign and endogenous signals. Prog Lipid Res 67:38–57CrossRefPubMedPubMedCentralGoogle Scholar
  30. Nebert DW, Wikvall K, Miller WL (2013) Human cytochromes P450 in health and disease. Philos Trans R Soc Lond B Biol Sci 368(1612):20120431CrossRefPubMedPubMedCentralGoogle Scholar
  31. Nelson AC, Huang W, Moody DE (2001) Variables in human liver microsome preparation: impact on the kinetics of l-alpha-acetylmethadol (LAAM) n-demethylation and dextromethorphan O-demethylation. Drug Metab Dispos 29(3):319–325PubMedGoogle Scholar
  32. Oesch F, Fabian E, Landsiedel R (2018) Xenobiotica-metabolizing enzymes in the skin of rat, mouse, pig, guinea pig, man, and in human skin models. Arch Toxicol 92(8):2411–2456CrossRefPubMedPubMedCentralGoogle Scholar
  33. O'Malley M, King AN, Conte M, Ellingrod VL, Ramnath N (2014) Effects of cigarette smoking on metabolism and effectiveness of systemic therapy for lung cancer. J Thorac Oncol 9(7):917–926CrossRefPubMedGoogle Scholar
  34. Phillips DH, Venitt S (2012) DNA and protein adducts in human tissues resulting from exposure to tobacco smoke. Int J Cancer 131(12):2733–2753CrossRefPubMedGoogle Scholar
  35. Piccardo MT, Stella A, Valerio F (2010) Is the smokers exposure to environmental tobacco smoke negligible? Environ Health 9:5CrossRefPubMedPubMedCentralGoogle Scholar
  36. Reiter MJ, Testerman TL, Miller RL, Weeks CE, Tomai MA (1994) Cytokine induction in mice by the immunomodulator imiquimod. J Leukoc Biol 55(2):234–240CrossRefPubMedGoogle Scholar
  37. Rowe JM, Welsh C, Pena RN, Wolf CR, Brown K, Whitelaw CB (2008) Illuminating role of CYP1A1 in skin function. J Invest Dermatol 128(7):1866–1868CrossRefPubMedGoogle Scholar
  38. Safe S, Cheng Y, Jin UH (2017) The aryl hydrocarbon receptor (AhR) as a drug target for cancer chemotherapy. Curr Opin Toxicol 2:24–29CrossRefPubMedPubMedCentralGoogle Scholar
  39. Schmidt JV, Su GH, Reddy JK, Simon MC, Bradfield CA (1996) Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc Natl Acad Sci U S A 93(13):6731–6736CrossRefPubMedPubMedCentralGoogle Scholar
  40. Schön MP, Schön M (2007) Imiquimod: mode of action. Br J Dermatol 157(Suppl 2):8–13CrossRefPubMedGoogle Scholar
  41. Sesardic D, Boobis AR, Murray BP et al (1990) Furafylline is a potent and selective inhibitor of cytochrome P450IA2 in man. Br J Clin Pharmacol 29(6):651–663CrossRefPubMedPubMedCentralGoogle Scholar
  42. Shimizu Y, Nakatsuru Y, Ichinose M et al (2000) Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor. Proc Natl Acad Sci U S A 97(2):779–782CrossRefPubMedPubMedCentralGoogle Scholar
  43. Smith SH, Jayawickreme C, Rickard DJ et al (2017) Tapinarof Is a natural AhR agonist that resolves skin inflammation in mice and humans. J Invest Dermatol 137(10):2110–2119CrossRefPubMedGoogle Scholar
  44. Smith M, Garcia-Martinez E, Pitter MR et al (2018) Trial watch: toll-like receptor agonists in cancer immunotherapy. Oncoimmunology 7(12):e1526250CrossRefPubMedGoogle Scholar
  45. Tigges J, Weighardt H, Wolff S et al (2013) Aryl hydrocarbon receptor repressor (AhRR) function revisited: repression of CYP1 activity in human skin fibroblasts is not related to AhRR expression. J Invest Dermatol 133(1):87–96CrossRefPubMedGoogle Scholar
  46. Tigges J, Haarmann-Stemmann T, Vogel CF et al (2014) The new aryl hydrocarbon receptor antagonist E/Z-2-benzylindene-5,6-dimethoxy-3,3-dimethylindan-1-one protects against UVB-induced signal transduction. J Invest Dermatol 134(2):556–559CrossRefPubMedGoogle Scholar
  47. van der Fits L, Mourits S, Voerman JS et al (2009) Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. J Immunol 182(9):5836–5845CrossRefPubMedGoogle Scholar
  48. Wincent E, Bengtsson J, Mohammadi Bardbori A et al (2012) Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor. Proc Natl Acad Sci U S A 109(12):4479–4484CrossRefPubMedPubMedCentralGoogle Scholar
  49. Zanger UM, Schwab M (2013) Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 138(1):103–141CrossRefPubMedGoogle Scholar
  50. Zhai S, Dai R, Friedman FK, Vestal RE (1998) Comparative inhibition of human cytochromes P450 1A1 and 1A2 by flavonoids. Drug Metab Dispos 26(10):989–992PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Melina Mescher
    • 1
  • Julia Tigges
    • 1
  • Katharina M. Rolfes
    • 1
  • Anna L. Shen
    • 2
  • Jeremiah S. Yee
    • 2
  • Christian Vogeley
    • 1
  • Jean Krutmann
    • 1
    • 3
  • Christopher A. Bradfield
    • 2
  • Dieter Lang
    • 4
  • Thomas Haarmann-Stemmann
    • 1
    Email author
  1. 1.IUF - Leibniz Research Institute for Environmental MedicineDüsseldorfGermany
  2. 2.The McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public HealthUniversity of WisconsinMadisonUSA
  3. 3.Medical FacultyHeinrich-Heine-UniversityDüsseldorfGermany
  4. 4.Bayer AG, PharmaceuticalsDMPK Drug MetabolismWuppertalGermany

Personalised recommendations