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Toxicology Letters 202 (2011) 129–132

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

rug–drug interactions by azole antifungals: Beyond a dogma of CYP3A4 enzymectivity inhibition

denek Dvorak ∗

epartment of Cell Biology and Genetics, Faculty of Science, Palacky University, Slechtitelu 11, 783 71 Olomouc, Czech Republic

r t i c l e i n f o

rticle history:eceived 30 December 2010eceived in revised form 26 January 2011ccepted 28 January 2011vailable online 17 February 2011

eywords:zolesytochrome CYP3A4regnane X receptorlucocorticoid receptor

a b s t r a c t

Azoles antifungals are widespread used drugs for various medicinal indications. These drugs are wellknown for their numerous drug–drug interactions, which are believed to occur via inhibition of CYP3A4enzymatic activity and consequently altering pharmacokinetic of co-administered drugs. In the currentcommunication a complex view on the molecular interactions between azoles antimycotics and CYP3A4 ispresented. Beside inhibition of CYP3A4 catalytic activity, azoles influence transcriptional activity of preg-nane X receptor (PXR) and consequently expression of drug-metabolizing enzymes, including CYP3A4.Interactions between azoles and PXR occur by multiple mechanisms, including modulation of ligand-dependent activation of PXR (agonism, antagonism) or affecting recruitment of PXR co-activators SRC-1(steroid receptor co-activator 1) and HNF4� (hepatocyte nuclear factor 4�). Miconazole and ketoconazoleare antagonists of glucocorticoid receptor (GR), therefore these drugs inhibit GR-mediated expression of

rug interactions PXR and drug metabolizing cytochromes P450. In addition, PXR and GR are key regulators of interme-diary metabolism (e.g. carbohydrate, lipids or bile acids homeostasis) and many other cellular functions(e.g. immune response), hence, the interactions between azoles and PXR/GR are of broader physiologi-cal importance. In conclusion, while inhibition of CYP3A4 enzymatic activity by azoles is considered asprimary cause of azoles drug–drug interactions, the effects of azoles on PXR and GR should be taken inaccount. Apart from CYP3A4, azoles influence the expression and activity of others drug-metabolizing

cytochromes P450.

. Azoles antifungals

Azole antimycotics are large group of antifungal drugs, compris-ng imidazoles (e.g. ketoconazole and miconazole), triazoles (e.g.traconazole and fluconazole) and thiazoles (e.g. abafungin). Theserugs are used to treat primary fungal infections, but also as pro-hylactic or supporting treatment of immune suppressed patientsperioperative states, anticancer chemotherapy, HIV, organ trans-lants etc.). The mechanism of action of azoles is inhibition of fungalytochrome P450 CYP51A1 (lanosterol-14a-demethylase) that cat-lyzes conversion of lanosterol to ergosterol (Heeres et al.).

There are numerous reports on the drug–drug interactions

aused by azoles; e.g. with bortezomib (Venkatakrishnan et al.,009), bosutinib (Dvorak and PavekAbbas et al.), statins (Moldent al., 2008), venlafaxine (Lindh et al., 2003), tricyclic antidepres-ants (Spina et al., 1997b), carbamazepine (Spina et al., 1997a),

Abbreviations: CAR, constitutive androstane receptor; GR, glucocorticoid recep-or; HNF4�, hepatocyte nuclear factor 4 alpha; PXR, pregnane X receptor; RXR�,etinoid X receptor alpha; SRC-1, steroid receptor co-activator 1.∗ Tel.: +420 58 5634903; fax: +420 58 5634905.

E-mail address: moulin@email.cz

378-4274/$ – see front matter © 2011 Published by Elsevier Ireland Ltd.oi:10.1016/j.toxlet.2011.01.027

© 2011 Published by Elsevier Ireland Ltd.

antivirotics (Kaeser et al., 2009) and many others (Gubbins andHeldenbrand). The majority of drug–drug interactions are causedby inhibition of drug-metabolizing enzymes. In case of azoles, theinhibition of CYP3A4 (but also CYP2C9) enzymatic activity is con-sidered as the main source of drug–drug interactions by azoles.Indeed, azole antifungals are potent inhibitors of human CYP3A4,as revealed by several studies (Lamb et al., 2000; Monostory et al.,2004; Sakaeda et al., 2005; Wang et al., 2007; Youdim et al., 2008)(Fig. 1(1)).

In the current communication, several other mechanismsby which azole antifungals influence human drug-metabolizingenzymes are described.

2. Pregnane X receptor

An orphan nuclear receptor, pregnane X receptor (PXR; NR1I2)is key transcriptional regulator of drug-metabolizing enzymes,including CYP3A4 (Lehmann et al., 1998). In its active form, PXR

is in complex with heterodimerization partner retinoid X receptoralpha (RXR�; NR2B1) (Pascussi et al., 2008) and with several co-activators, such as steroid receptor co-activator 1 (SRC-1) (Watkinset al., 2003) and hepatocyte nuclear factor 4 alpha (HNF4�) (Joveret al., 2001, 2009; Tirona and Kim, 2005).

130 Z. Dvorak / Toxicology Letters 202 (2011) 129–132

Fig. 1. Effects of azole antifungals on human CYP3A4. (1) azoles inhibit catalytic activity of CYP3A4 enzyme; (2) ketoconazole disrupts interaction between SRC-1 and PXR;( omotec anscri R in la

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3) ketoconazole disrupts interaction between HNF4� and PXR; (4) itraconazole prlotrimazole are the ligands for PXR and augment basal and rifampicin-mediated trnhibit/potentiate PXR-mediated induction of CYP3A4; (7) ketoconazole binds to PXre antagonists of GR.

The most of studies were carried out with ketoconazole. Con-icting data were obtained regarding the effects of ketoconazolebut also fluconazole and clotrimazole) on CYP3A4 expression.ome authors described that azoles induce CYP3A4 (Luo et al.,002; Svecova et al., 2008), whereas others observed inhibitionf xenobiotic-inducible expression of CYP3A4 (Wang et al., 2007).his behavior indicates partial agonism of ketoconazole againstXR. However, azoles also interfere with interactions betweenXR and its transcriptional co-activators. Huang et al. describedhat xenobiotic-mediated induction of CYP3A4 gene transcrip-ion was inhibited by ketoconazole. They found that ketoconazoleisrupted the interaction between PXR and SRC-1 (Huang et al.,007) (Fig. 1(2)). Lim et al. (2009) demonstrated that the inhi-ition of the interaction of PXR with HNF4� might be involved

n ketoconazole-mediated inhibition of CYP3A4 gene expressionFig. 1(3)). In our recent paper, we have observed additive andntagonistic interactions of azoles with rifampicin on PXR tran-criptional activity and CYP3A4 expression (Svecova et al., 2008).traconazole stimulated recruitment of SRC-1 to PXR, but it did notnteract with PXR ligand binding domain (Fig. 1(4)). Oxiconazole,conazole and miconazole were identified as potent PXR ligandsnd inducers of CYP3A4 (Fig. 1(5)). We observed that ketocona-ole suppressed rifampicin-mediated CYP3A4 transactivation andRC-1 recruitment to PXR, which is in accordance with data from

ther authors (Huang et al., 2007; Wang et al., 2007) (Fig. 1(6)). Inddition, ketoconazole diminished interaction of rifampicin withXR ligand binding domain (Fig. 1(7)). Interestingly, ketoconazolenduced CYP3A4 mRNA through CYP3A4 promoter response ele-

ents, albeit it is not an agonist of PXR (Svecova et al., 2008).

s interaction between SRC-1 and PXR; (5) oxiconazole, econazole, miconazole andiptional activity of PXR; (6) ketoconazole, miconazole, fluconazole and oxiconazoleigand binding domain; (8) GR up-regulates PXR; (9) ketoconazole and miconazole

Overall, azole antimycotics differentially affect PXR tran-scriptional activity, with consequences in CYP3A4 expression.Interactions between azoles and PXR-signaling pathway are cer-tainly the cause of drug–drug interactions. Importantly, PXR isimportant regulator of metabolism of carbohydrates, lipids, bileacids and cholesterol. Therefore, interactions between azoles andPXR are of broader clinical and physiological significance.

3. Glucocorticoid receptor

Glucocorticoid receptor (GR; NR3C1) belongs to the familyof steroid/thyroid receptors. It regulates many cellular functionssuch as homeostasis, cell differentiation, proliferation, growth anddevelopment, and host defense. At molecular level, after bindingof glucocorticoid hormone (e.g. cortisol and dexamethasone), GRtranslocates to nucleus, where it forms homodimer GR�/GR� thattriggers transcriptional response. GR plays crucial role in regula-tion of drug metabolism, since it up-regulates xenoreceptors PXR(Pascussi et al., 2000, 2001) and CAR (Pascussi et al., 2003) (con-stitutive androstane receptor; NR1I3) and consequently indirectlycontrols expression of PXR- and CAR-dependent genes (Dvorak andPavek) (Fig. 1(8)).

Recently, it was demonstrated that ketoconazole and micona-zole (but not fluconazole) are antagonists of human GR in primary

cultures of human hepatocytes (Fig. 1(9)). Consistently, these twoazoles down-regulated xenoreceptors PXR and CAR, and conse-quently PXR- and CAR-dependent genes, including CYP2B6, CYP2C9and CYP3A4 were diminished (Duret et al., 2006). Inhibition of GRtranscriptional activity certainly produces many side-physiological

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Z. Dvorak / Toxicology

ffects in addition to down-regulation of PXR and CAR. Fur-hermore, diminution of PXR and CAR will have consequencesn metabolism of carbohydrates, lipids, bile acids and choles-erol.

The role of GR in the regulation of drug-metabolizingytochromes P450 is very complex, comprising several mecha-isms. Apart from regulating the regulatory proteins (PXR, CAR,XRs etc.), GR is involved in direct (cis) and indirect (trans) tran-criptional regulation of P450s (Dvorak and Pavek). For instance,unctional GR response elements were identified in promoters ofuman CYP2C8 (Ferguson et al., 2005), CYP2C9 (Gerbal-Chaloint al., 2002), CYP2C19 (Chen et al., 2003) and CYP3A5 (Schuetzt al., 1996) genes. Trans-regulation of human CYP2A6 (Onicat al., 2008) and CYP3A4 (El-Sankary et al., 2002; Pavek et al.,007) by GR, was demonstrated, involving HNF4� in the pro-ess. Therefore, antagonizing GR by ketoconazole and miconazoleesults in down-regulation of drug-metabolizing cytochromes450 CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4 andYP3A5. Glucocorticoids also positively regulate CYP1A2 expres-ion, by mechanisms not yet understood (Monostory et al., 2009;rzal et al., 2008).

. Conclusion

Azoles antifungals are one of the most frequently used drugsn human pharmacotherapy. They are potent inhibitors of humanYP3A4. For these reasons, azoles are subject of tight control inerms of drug–drug interactions. The majority of clinical trials arearried out with respect to inhibitory potency of azoles againstYP3A4 enzyme. In the current contribution, it is showed, thatpart from inhibition of CYP3A4 catalytic activity, azoles influenceYP3A4 activity by multiple mechanisms, including antagonizingR and interfering with PXR transcriptional activity. The interactionf azoles with PXR and GR will certainly results in more com-lex side effects of azoles than in inhibition of CYP3A4-mediatedetabolism of co-administered drugs.

onflict of interest statement

The author declares no conflict of interest.

cknowledgements

Our laboratory is supported by the grants from the Czech Scien-ific Agency GACR503/10/0579 and GACR304/10/0149.

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