Induction of CYP3A4, MRP1, and MDR1 protein expression was analyzed by In-Cell Western (ICW) assay.

<p>Top section in each panel is a respective representative immunoblot of CYP3A4, MRP-1 or MDR1. Bottom plot in each panel demonstrates respective mean CYP3A4, MRP-1 or MDR1 fluorescence signal intensities of triplicates and error bars represent SEM values. *p< 0.05 compared to control, ^#p<0.05 compared to EVG, and ^p<0.05 compared to EVG+COBI, ^$p<0.05 compared to EtOH. Values were mean ± SEM from 3 individual experiments. EVG, Elvitegravir; COBI, Cobicistat; EtOH, ethanol.</p

Intracellular elvitegravir concentration in the presence of MRP1 or MDR1 inhibitors and cobicistat + ethanol.

<p>EVG quantified from cell lysates using LC MS/MS after 24 hours treatment with indicated drugs. Data are expressed as mean ± SEM from 3 independent experiments. *p< 0.05 compared to EVG+COBI+EtOH (Control). EVG, Elvitegravir; COBI, Cobicistat; EtOH, ethanol.</p

Docking of elvitegravir (EVG), cobicistat (COBI) with the human CYP3A4.

<p>EVG docking in the (A) absence and (B) presence of ethanol. (C) Docking of COBI in the absence and presence of ethanol. Chemscore was used for scoring the ligand-CYP3A4 interactions. The simulated interactions are grouped into clusters: site-1, region 1–4 (see text for the details). CYP3A4 is shown in light green, EVG in yellow, COBI in magenta, heme of the P450 in red and alcohol in indigo. Black arrows show ethanol molecule.</p

Time-dependent stability studies of elvitegravir at low (0.040 μM), medium (0.75 μM) and high (3.50 μM) quality control concentrations in triplicates.

<p>The variations are expressed as a percentage change from freshly prepared quality controls.</p

MS/MS spectrum of elvitegravir (EVG) with proton adducts [M+H]⁺ in ESI-positive mode.

<p>The y-axis shows the intensity (CPS, count per second); the x-axis shows the mass to charge ratio (<i>m/z</i>,<i>Da</i>). The EVG structure was made using ChemDraw Ultra (version 6.0.1; CambridgeSoft.com). Red dotted lines on the structure indicates the fragmentation pattern of EVG.</p

Saturation kinetics of elvitegravir (EVG) in the human liver microsomes (HLM) in the absence and presence of 20 mM ethanol.

<p>The maximum enzyme velocity (V_max) and Michaelis-Menten constant (K_m) were calculated by fitting the data to Michaelis-Menten model and enzyme activity was reported as μmol/min/mg protein. Mean ± S.E.M values were calculated from the fitting of the curve. The results are representative of at least three independent experiments.</p

Matrix effect (ME), recovery (RE) and process efficiency (PE) of elvitegravir (EVG) (n = 6).

<p>Matrix effect (ME), recovery (RE) and process efficiency (PE) of elvitegravir (EVG) (n = 6).</p

Multiple reaction monitoring (MRM) chromatogram peaks of elvitegravir (EVG) and its corresponding internal standard (IS), ritonavir (RTV).

<p>(A) Extracted blank (y axis:0–520 CPS), (B) extracted IS (0.14 μM; y axis:0–2.3e⁶ CPS), (C) extracted lower limit of quantitation (LLOQ, 0.003 μM; y axis:0–2.0e⁶ CPS), (D) upper limit of quantitation (ULOQ, 1 μM; y axis:0–1.10e⁷ CPS), (E) blank after ULOQ (y axis:0–3400 CPS). The y-axis shows the intensity (CPS, count per second); the x-axis indicates the run time in minutes. PA, peak area: PAR, peak area ratio (analyte/IS).</p

Inter-day accuracy and precision of calibration standards (n>6) and quality control (QC) standards (n>6) of elvitegravir in human liver microsomes.

<p>Inter-day accuracy and precision of calibration standards (n>6) and quality control (QC) standards (n>6) of elvitegravir in human liver microsomes.</p

Inhibition of human recombinant CYP3A4 activity by elvitegravir (EVG) and/or cobicistat (COBI) in the presence and absence of 20 mM ethanol.

<p>Enzyme activity was determined by using vivid^® blue screening kit with baculosomes containing CYP3A4. Dose-response inhibition curve generated by plotting log EVG concentration on x-axis and CYP3A4 activity on y-axis. Data are expressed as the mean ± S.E.M. The results are representative of at least three independent experiments. The CYP3A4 inhibition by ethanol alone is presented in the inset. COBI C1 represents 4 μM and COBI C2 represents 0.04 μM.</p