Comparison of Antifungal Azole Interactions with Adult Cytochrome P450 3A4 versus Neonatal Cytochrome P450 3A7

Abstract

Adult drug metabolism is dominated by cytochrome P450 3A4 (CYP3A4), which is often inhibited by antifungal azole drugs, resulting in potential alterations in drug metabolism and adverse drug/drug interactions. In the fetal and neonatal stages of life, the 87% identical cytochrome P450 3A7 (CYP3A7) is expressed but not CYP3A4. Azole antifungals developed for adults are also used in neonates, assuming they interact similarly with both enzymes, but systematic information is lacking. Herein a method was developed for generating recombinant purified CYP3A7. Thirteen different azoles were then evaluated for binding and inhibition of purified human CYP3A4 versus CYP3A7. All imidazole-containing azoles bound both enzymes via coordination to the heme iron and inhibited both with IC₅₀ values ranging from 180 nM for clotrimazole to the millimolar range for imidazole itself. Across this wide range of potencies, CYP3A4 was consistently inhibited more strongly than CYP3A7, with clotrimazole being the least selective (1.5-fold) inhibitor and econazole the most selective (12-fold). Observations for 1,2,4-triazole-containing azoles were more varied. Most bound to CYP3A4 via coordination to the heme iron, but several also demonstrated evidence of a distinct binding mode at low concentrations. However, only posaconazole inhibited CYP3A4. Of the triazoles, only posaconazole inhibited CYP3A7, again less potently than CYP3A4. Spectral evidence for binding was weak or nonexistent for all triazoles. Overall, although the details of binding interactions do vary, the same azole compounds inhibit both enzymes, albeit with weaker interactions with CYP3A7 compared with CYP3A4.

Fig. 1.

Purified human CYP3A7. (A) In the resting state, purified CYP3A7 has a Soret peak at 417 nm, consistent with water bound to the heme iron (blue). Addition of imidazole-containing compounds such as the oxiconazole shown here shifts the Soret peak to 423.5 nm, consistent with binding to the heme iron (red). (B) CYP3A7 reduced carbon-monoxide difference spectrum.

Fig. 2.

Representative IC₅₀ determinations and nonlinear regression fits for CYP3A4 (blue) and CYP3A7 (red). (A) Posaconazole is the only triazole demonstrating significant inhibition, with similar IC₅₀ values for both enzymes. (B) Clotrimazole shows similarly high potency against both enzymes. (C) Econazole is 12.5-fold more potent for CYP3A4 than for CYP3A7. (D) Ketoconazole is 7.7-fold more potent at inhibition of CYP3A4 than CYP3A7.

Fig. 3.

Difference spectra for imidazole-containing azole binding to CYP3A enzymes demonstrate spectral shifts associated with type II binding. (A) Econazole binds very tightly to CYP3A4 (0.1 µM in a 5 cm cuvette) and was fit to the tight-binding equation (inset). (B) Econazole did not bind as tightly to CYP3A7 (1 μM in a 1 cm cuvette) and was fit to the standard equation (inset). Spectra obtained with increasing concentrations (rainbow order) of econazole are shown. Not all spectra are shown for clarity. All other ligands containing an imidazole moiety showed very similar type II spectral changes (data not shown).

Fig. 4.

Difference spectra for triazole-containing ligands binding to CYP3A enzymes demonstrate varied binding modes for CYP3A4 and little/no binding to CYP3A7. (A) Posaconazole binding to CYP3A4 (1 µM) exhibits a typical type II shift. The associated ΔA_max vs. [ligand] data are fit to the standard equation (inset). (B) Letrozole binding to CYP3A4 (1 μM) demonstrates two distinct binding modes, starting with a reverse type II and transitioning to a typical type II spectrum. While all points are shown in the inset, ΔA_max values corresponding to the reverse type II spectra (red) are excluded, and only those associated with the type II spectra (black) were fit to the standard equation to determine the type II K_d (inset). (C) Posaconazole titration into CYP3A7 1 (micromolar) yields little or no evidence of binding. (D) Similarly, letrozole addition to CYP3A7 (1 µM) results in little evidence of binding. CYP3A7 spectra in (C and D) were obtained under the same conditions as the CYP3A4 spectra in (A and D) are shown on the same scale to facilitate comparison. Spectra obtained with increasing concentrations of ligand are shown in rainbow order. Not all spectra are shown for clarity.