Differential effects of ethanol on spectral binding and inhibition of cytochrome P450 3A4 with eight protease inhibitors antiretroviral drugs

Abstract

Background: Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver, which metabolizes approximately 50% of the marketed drugs including antiretroviral agents. CYP3A4 induction by ethanol and its impact on drug metabolism and toxicity is known. However, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known, except that we have recently shown that ethanol facilitates the binding of a protease inhibitor (PI), nelfinavir, with CYP3A4. The current study was designed to examine the effect of ethanol on spectral binding and inhibition of CYP3A4 with all currently used PIs that differ in physicochemical properties.

Methods: We performed type I and type II spectral binding with CYP3A4 at 0 and 20 mM ethanol and varying PIs' concentrations. We also performed CYP3A4 inhibition using 7-benzyloxy-4-trifluoromethylcoumarin substrate and NADPH at varying concentrations of PIs and ethanol.

Results: Atazanavir, lopinavir, saquinavir, and tipranavir showed type I spectral binding, whereas indinavir and ritonavir showed type II. However, amprenavir and darunavir did not show spectral binding with CYP3A4. Ethanol at 20 mM decreased the maximum spectral change (δA(max)) with type I lopinavir and saquinavir, but it did not alter δA(max) with other PIs. Ethanol did not alter spectral binding affinity (K(D)) and inhibition constant (IC(50)) of type I PIs. However, ethanol significantly decreased the IC(50) of type II PIs, indinavir and ritonavir, and markedly increased the IC(50) of amprenavir and darunavir.

Conclusions: Overall, our results suggest that ethanol differentially alters the binding and inhibition of CYP3A4 with the PIs that have different physicochemical properties. This study has clinical relevance because alcohol has been shown to alter the response to antiretroviral drugs, including PIs, in HIV-1-infected individuals.

Fig. 1

Ball and stick structures of type I, type II, and spectrally unbound protease inhibitors (PIs). The structures were created using ChemDraw Ultra 6.0.6 (PerkinElmer, Cambridge, MA).

Fig. 2

Spectral binding of CYP3A4 by type I PIs (left panel a) and type II PIs (right panel) in the absence (filled circles) and presence of 20 mM ethanol (open circles). The spectral binding was performed at varying concentrations of atazanavir, lopinavir, saquinavir, and tipranavir. The δA_max and K_D are presented in Table 1.

Fig. 3

Inhibition of CYP3A4 by type I and spectrally unbound PIs in the absence (filled circles) and presence of 20 mM ethanol (open circles). The inhibition was performed at varying concentrations of atazanavir, lopinavir, saquinavir, tipranavir, amprenavir, and darunavir using 7-BFC substrate in the standard NADPH reaction. The 100% CYP3A4 activity corresponds to approximately 3.5 nmoles/min/nmol P450. The IC₅₀ values are presented in Table 2A.

Fig. 4

Inhibition of CYP3A4 by type II PIs at 0 mM (filled circles), 5 mM (open circles), 10 mM (filled triangles), and 20 mM (open triangles) ethanol. The inhibition was performed at varying concentrations of indinavir and ritonavir using 7-BFC substrate in the standard NADPH reaction. The 100% CYP3A4 activity corresponds to approximately 3.5 nmoles/min/nmol P450. The IC₅₀ values are presented in Table 2B.