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

Abstract

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.

Keywords: Aryl hydrocarbon receptor; Cytochrome P450; Imiquimod; Immunotherapy; Psoriasis.

Figure 1.. IMQ reduces BaP-induced EROD activity in human keratinocytes.

Effect of IMQ (10 μM, 25 μM, 50 μM) and 7-HF (1 μM) on EROD activity in (a) NHEKs from three female donors of different age (29 y, 36 y and 60 y; each measured in triplicates), and (b) immortalized HaCaT keratinocytes (n = 3). To induce EROD activity, keratinocytes were treated for 24 hrs with 0.5 μM BaP. IMQ and 7-HF were added 30 min before EROD measurement to exclude potential effects on CYP1A expression. EROD activity is shown as fold of BaP-pretreated DMSO ctrl. (c) HaCaT keratincoytes were treated with 25 μM and 50 μM IMQ, 2.5 μM BaP and DMSO alone and in combination. After 16 hrs, RNA was isolated and reverse transcribed. Transcript numbers of CYP1A1 and CYP1B1 were determined by qPCR and normalized to β-actin. (n.d. = not detectable; n.s. = not significant; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005).

Figure 2.. IMQ is metabolized by human CYP1A isoforms.

(a) Microsomal preparations of 21 heterologously expressed human CYP isoforms were incubated with 0.5 μM IMQ for 15 min at 37°C. IMQ content was determined by LC/MS. (ins. Ctrl = control insect cell preparation; n = 2). (b) Formation of monohydroxylated IMQ metabolites (RT = retention time) by four of the 21 heterologously expressed human CYP isoforms. (c) HaCaT keratinocytes were pre-treated for 24 hrs with 0.5 μM BaP or solvent. Next, cells were treated with 0.5 μM IMQ alone and in combination with 5 μM 7-HF and 2 μM furafylline (Fura). After 6 hrs, monohydroxylated IMQ metabolites in the cell culture supernatants were determined by LC/MS (n = 3). (d) HaCaT keratinocytes were pre-treated for 24 hrs with 0.5 μM BaP or solvent. Subseqeuntly, cells were treated with 1 μM granisetron alone and in combination with 5 μM 7-HF, 2 μM furafylline (Fura) and 0.5 μM IMQ. The amount of 7-hydroxylated granisetron in the cell culture supernatants was assessed after 6 hrs by LC/MS (n ≥ 4). (n.d. = not detectable; n.s. = not significant; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005).

Figure 3:. IMQ metabolism in hepatic microsomes from CD-1 mice.

Hepatic microsomes from CD-1 mice were incubated with 1 μM IMQ in absence or presence of 5 μM 7-HF for 60 min at 37°C. (a) IMQ content and (b) monohydroxylated IMQ metabolites were measured by LC/MS over time (n = 3).

Figure 4:. IMQ clearance and metabolite formation in heaptic microsomes from male wild-type B6 and CYP1A-KO mice.

Male CYP1A-proficient B6 and CYP1A-KO mice were treated with BNF (80 mg/kg) or corn oil per gavage. Each treatment group consisted of 2 animals per genotype). After 6 hrs, mice were sacrificed and livers were snap-frozen. Hepatic microsomes were prepared and incubated with 1 μM IMQ in absence or presence of 5 μM 7-HF for 45 min at 37°C. Subseqeuntly, (a) IMQ content and (b) monohydroxylated IMQ metabolites were measured by LC/MS over time (n = 2, each consisting of 2 technical replicates).