Potent synergistic anti-human immunodeficiency virus (HIV) effects using combinations of the CCR5 inhibitor aplaviroc with other anti-HIV drugs

Abstract

Aplaviroc (AVC), an experimental CCR5 inhibitor, potently blocks in vitro the infection of R5-tropic human immunodeficiency virus type 1 (R5-HIV-1) at subnanomolar 50% inhibitory concentrations. Although maraviroc is presently clinically available, further studies are required to determine the role of CCR5 inhibitors in combinations with other drugs. Here we determined anti-HIV-1 activity using combinations of AVC with various anti-HIV-1 agents, including four U.S. Food and Drug Administration-approved drugs, two CCR5 inhibitors (TAK779 and SCH-C) and two CXCR4 inhibitors (AMD3100 and TE14011). Combination effects were defined as synergistic or antagonistic when the activity of drug A combined with B was statistically greater or less, respectively, than the additive effects of drugs A and A combined and drugs B and B combined by using the Combo method, described in this paper, which provides (i) a flexible choice of interaction models and (ii) the use of nonparametric statistical methods. Synergistic effects against R5-HIV-1(Ba-L) and a 50:50 mixture of R5-HIV-1(Ba-L) and X4-HIV-1(ERS104pre) (HIV-1(Ba-L/104pre)) were seen when AVC was combined with zidovudine, nevirapine, indinavir, or enfuvirtide. Mild synergism and additivity were observed when AVC was combined with TAK779 and SCH-C, respectively. We also observed more potent synergism against HIV-1(Ba-L/104pre) when AVC was combined with AMD3100 or TE14011. The data demonstrate a tendency toward greater synergism with AVC plus either of the two CXCR4 inhibitors compared to the synergism obtained with combinations of AVC and other drugs, suggesting that the development of effective CXCR4 inhibitors may be important for increasing the efficacies of CCR5 inhibitors.

FIG. 1.

Dose-response curves of single and combined drug assays. Three representative dose-response curves are shown. (A) Dose-response curve with the same-drug combination (AVC-AVC). PHA-PBMCs were exposed to R5-HIV-1_Ba-L and cultured in the presence of AVC alone or AVC-AVC over 7 days. AVC was serially diluted threefold to give concentrations in the range of 0.1 to 24.3 nM. The percent inhibition values were determined on the basis of the amounts of p24 Gag proteins in the culture supernatants. (B) AVC was combined with ZDV at a fixed ratio (1:11), and the assay was conducted as described above for panel A. (C) AVC (concentration range, 0.3 to 72.9 nM) was combined with AMD3100 at a 1:11 ratio. PHA-PBMCs were exposed to a 50:50 mixture of R5-HIV_Ba-L and X4-HIV-1_ERS104pre and cultured in the presence of AVC alone, AMD3100 alone, or AVC-AMD3100. All assays were performed on 5 to 10 different occasions, and all the values shown represent the arithmetic means ± 1 standard deviation.

FIG. 2.

Effects of same-drug combinations. The serially diluted anti-HIV-1 agents AVC (A), ZDV (B), NVP (C), IDV (D), and ENF (E) were combined with the same agent diluted under the same conditions; PHA-PBMCs were exposed to R5-HIV-1_Ba-L and cultured in the presence of the drugs combined. The combination effects (percent synergy values on the vertical z axis) were determined on the basis of the Bliss independence method. In the 3-D graphs, obtained on the basis of the method of Prichard et al. (27, 28, 29), the average percent synergy values at each concentration derived from 10 experiments were plotted. The hatched area represents synergism (percent synergy values, >0), while the open area represents additivity or antagonism (percent synergy values, ≤0). Numbers in parentheses represent the average percent synergy values (±1 standard deviation). The x and y axes indicate the concentrations of the drug tested (nM). All assays were performed in duplicate, and each experiment was independently conducted 10 times.

FIG. 3.

Effects of AVC in combination with other anti-HIV-1 agents against R5-HIV-1_Ba-L. Drug combination assays were conducted, and the %synergy^mean values (the mean of the nine percent synergy values from each set of the data) are shown in three settings: (i) AVC-AVC, (ii) test agent (to be combined with AVC)-test agent, and (iii) AVC-test agent (A to D). The AVC-AVC combination and the test agent-test agent combination were tested on 10 different occasions, while the AVC-test agent combination assay was done on 5 different occasions. The differences in the %synergy^mean values between the three settings were analyzed by using the Wilcoxon rank sum test. The short bars indicate the arithmetic means. The combination effects are also shown in 3-D graphs, as determined on the basis of the method of Prichard et al. (see the legend to Fig. 2).

FIG. 4.

Effects of AVC in combination with other CCR5 inhibitors. The effects of AVC in combination with SCH-C (A) or TAK779 (B) when they were exposed to R5-HIV-1_Ba-L are shown. No significant synergism was seen when AVC was combined with SCH-C or TAK779 compared with that seen with AVC-AVC. There was a trend toward antagonism when AVC-SCH-C with SCH-C-SCH-C and a trend toward synergism when AVC-TAK779 was compared with TAK779-TAK779. When these data were examined by the method of Prichard et al. (27, 29), AVC-SCH-C showed a mixed pattern but with an inclination toward antagonism (C), while AVC-TAK779 showed a mixed pattern but with an inclination toward synergy (D).

FIG. 5.

Effects of AVC in combination with other anti-HIV-1 agents against a 50:50 mixture of R5-HIV-1_Ba-L and X4-HIV-1_ERS104pre. PHA-PBMCs were exposed to a 50:50 mixture of R5-HIV-1_Ba-L and X4-HIV-1_ERS104pre and cultured in the presence of AVC in combination with AMD3100 (A), TE14011 (B), ENF (C), ZDV (D), NVP (E), or IDV (F) for 7 days, and the amounts of p24 Gag proteins in the culture supernatants were determined. Differences in the %synergy^mean values between AVC-AVC, test agent-test agent, and AVC-test agent were examined by using the Wilcoxon rank sum test. A statistically significant difference, or a strong trend, was observed for all combinations except between IDV-IDV and AVC-IDV (P = 0.2). The short bars indicate the arithmetic means obtained. The average percent synergy values for the AVC-test agent combinations were also plotted in 3-D graphs, as determined on the basis of the method of Prichard et al. (see the legend to Fig. 2) (G to L). Assays with AVC in combination with each drug and the same-drug combination were performed 5 times and 10 times, respectively. All assays were conducted in duplicate.