Abstract

MATERIALS AND METHODS

RESULTS

Effects of NNRTIs, NRTIs, and PIs on HIV-1 replication and HIV-1 RT activity.

In the combination experiments described below, the ratios of the compounds used were based on the IC₅₀ of each compound determined in the HIV-1 RT enzyme assay and the HIV-1 (RF) yield reduction assay. For example, in the experiments in which the ratio of the compounds is 1:1, the first dilution contains each compound at its respective IC₅₀. Likewise, for the 3:1 and 1:3 ratios, the first dilution contains one compound at its IC₅₀ and the other at one-third the concentration of its IC₅₀.

We established the IC₅₀s of the individual compounds under investigation in both the HIV-1 RT enzyme assay and the yield reduction assay (Table ​(Table1).1). All compounds inhibited HIV-1 RT activity and HIV-1 (RF) replication in dose-dependent manners.

TABLE 1.

Potencies and conformity parameters for NNRTIs, NRTIs, and NFV against HIV-1 RT activity and HIV-1 (RF) replication^a

Compound group and compound	HIV-1 (RF) replication		HIV-1 RT activity
	IC50 (μM)b	r2	IC50 (μM)b	r2
NNRTIs
Efavirenz	0.0007 ± 0.0004	0.966	0.041 ± 0.012	0.981
DPC082	0.0004 ± 0.0001	0.966	0.054 ± 0.020	0.993
DPC083	0.0003 ± 0.0001	0.950	0.041 ± 0.0009	0.976
DPC961	0.0004 ± 0.0003	0.926	0.024 ± 0.004	0.973
DPC963	0.0004 ± 0.0002	0.967	0.019 ± 0.003	0.979
NVP	0.018 ± 0.003	0.938	2.34 ± 0.21	0.985
NRTIs
AZT	0.022 ± 0.014	0.973	0.0029 ± 0.0004	0.990
3TC	0.153 ± 0.023	0.987	1.45 ± 0.53	0.991
PI, NFV	0.005 ± 0.001	0.959	NDc	ND

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^aAll values are derived from a minimum of 10 independent experiments for each compound in each assay.

^bValues are means ± standard deviations.

^cND, not determined.

DISCUSSION

Efavirenz, DPC082, DPC083, DPC961, and DPC963 are highly potent and selective inhibitors of HIV-1 RT activity and HIV-1 replication in cell culture (6). However, because of the rapid selection of drug-resistant variants and the greater success of multidrug regimens in the clinic, these compounds would not be used as initial monotherapy but instead would be used as part of multidrug combinations. The success of a multidrug regimen in the clinic is dependent not only on pharmacokinetic and pharmacodynamic parameters but also on any effect that the drugs that make up the regimen may have on each other at the enzymatic and cellular levels.

In vitro drug interaction studies have shown that NNRTIs can act synergistically with an NRTI to inhibit HIV-1 RT activity and/or HIV-1 replication (1, 3, 4). In clinical studies, combinations of an NNRTI with one or more NRTIs result in significant improvements in patients' clinical markers (7, 9, 26). However, the apparent efficacies of the combinations in the clinical setting cannot be necessarily attributed to synergistic effects at the enzymatic or cellular level since other factors such as pharmacological synergy, decreased selection of drug-resistant variants, and genotypic changes in the virus population in the presence of multiple drugs may also affect their potencies.

We found that efavirenz, DPC083, DPC961, and DPC963 acted synergistically to inhibit RT activity when they were used in combination with the NRTIs AZT-TP or 3TC-TP in the HIV-1 RT enzyme assay. The synergism seen with the combination of efavirenz and AZT-TP in the RT assay agreed with data previously published by Carroll et al. (3). Moreover, we also observed that the synergism of these combinations increased (as determined by decreased CIs) as the levels of inhibition of RT activity or HIV-1 replication increased. Similar results were obtained with the quinazolinone derivatives of efavirenz and AZT-TP. Furthermore, combinations of the four NNRTIs and 3TC-TP also acted synergistically to inhibit HIV-1 RT activity. Although the mechanism for the synergy seen by use of these NNRTIs plus AZT and 3TC is not known, it is conceivable that the interaction of the NNRTI with the RT at the NNRTI binding site, which has been shown to cause conformational distortion of the catalytic aspartate triad (8), may allow improved incorporation of the NRTI into the growing DNA molecule, leading to more efficient chain termination or decreased rates of phosphorolytic removal of the terminator. Any and all of these effects would result in apparent synergy in the overall level of inhibition observed.

In the virus replication assay, all NNRTI-NRTI combinations acted additively to slightly synergistically. Since the combinations were less synergistic in the cell-based system than in the enzyme system, the cellular environment must, in some way, affect the interaction of these two classes of compounds. One possible explanation for this observation is that the cellular environment mitigates some of the molecular rearrangement in the HIV-1 RT that occurs in the “test tube” environment. Alternately, the cell could affect the intracellular concentrations of drug such that the concentrations or ratios are in the range at which they act more additively to inhibit enzyme function. This could be through alteration of the amount of drug taken up by the cell, the intracellular half-life, or the rate of elimination.

Since it is not possible to determine the effect that combinations of NNRTIs and PIs have on each other's potencies in a single enzyme assay, these combinations were analyzed only by the yield reduction assay. As expected, we found that the combinations of the NNRTIs efavirenz, DPC082, DPC083, DPC963, and DPC963 and the PI NFV acted additively to synergistically to inhibit HIV-1 (RF) replication in MT-2 cells. Synergy between inhibitors of the HIV-1 RT and protease may be the result of effects on reduced virus levels in later rounds of infection in in vitro assays caused by agents that act early instead of late in the virus replication cycle.

We found that the combination of efavirenz and NVP was the only NNRTI-NNRTI combination that consistently acted strongly antagonistically in both the HIV-1 RT enzyme assay and the virus replication assay. Since antagonism was seen in both assays, we can assume that the effect observed occurred at the level of the enzyme and was not a result of an effect on a cellular process. It is possible that this antagonism could be a result of very different affinities of the HIV-1 RT enzyme for the two compounds. A similar explanation has been forwarded to explain the antagonism seen with the combination of AZT and d4T. In vitro, mitogen-activated thymidine kinase, which is responsible for converting these two compounds to their monophosphate forms inside the cell, has a 600-fold greater affinity for AZT than d4T (12). This results in a very low conversion of d4T to d4T-TP and a lower than expected level of activated AZT, leading to a weaker total antiviral effect than that of either compound alone.

We observed that efavirenz in combination with any of the related NNRTIs acted antagonistically in the yield reduction assay, while it acted additively to slightly antagonistically in the RT enzyme assay. While it is predictable that these combinations should have acted additively to inhibit HIV-1 RT in the enzyme assay, it is not easily explained why these compounds acted antagonistically in the cellular environment. Since there was no change in the expected and observed potencies of the drug combinations in the RT enzyme assay, the antagonism seen in the virus assay must be a consequence of a cellular process that influences the potencies of these compounds. As with the NNRTI-NRTI combinations in the virus replication assay, we speculate that these drug combinations may affect such cellular processes that influence compound uptake, localization, intracellular half-life, and/or elimination. For example, NNRTIs of the benzoxazenone and quinazolinone classes are highly protein bound (6); equilibration with nonspecific intracellular binding sites may be altered with multiple competing inhibitors.

Interestingly, a recent study claimed that the combination of efavirenz and NVP acted synergistically to inhibit HIV-1 replication in fresh peripheral blood mononuclear cells (15). We do not know the reason for such a disconnect in their results and those that we report here, but it may be because vastly different biological models (i.e., clinical isolates in fresh peripheral blood mononuclear cells versus HIV-1 [RF] in MT-2 cells) and different experimental procedures (e.g., the method used to treat the virus with drug, the method used to detect viable virus, and the model used to calculate CIs) were used in the two studies. Others have previously shown that the concentrations and ratios of compounds used can greatly affect the interactions of two compounds used in combination in vitro (12; D. P. Merrill, T.-C. Chou, and M. S. Hirsch, Reply, J. Infect. Dis. 174:672, 1996). In our own studies with two NNRTIs used in combination, we found that by greatly altering the compound ratios, we could affect the final CIs (data not shown).

To attempt to make our model as relevant as possible, we have incorporated the following. First, we have set up our compound concentrations and ratios to be what we believe are biologically relevant. For instance, in our 1:1 ratio, the initial dilution contains each compound at it IC₅₀, whereas the 1:3 and 3:1 ratios contain one compound at its IC₅₀ and the other compound at one-third its IC₅₀. In all three cases, the initial dilution is then serially diluted 32-fold by use of 2-fold dilutions. Second, we have determined that our results are in agreement with those that have been published previously. For example, with the combination of efavirenz and AZT-TP, we have obtained results similar to those obtained by Carroll et al. (3) (Table ​(Table2);2); and we found that the combination of AZT and d4T acted additively in the HIV-1 RT enzyme assay and antagonistically in the virus replication assay (data not shown), in agreement with the results of Hoggard et al. (13) and Zhu et al. (28). Finally, we tested efavirenz in combination with itself and found that it acted, as expected, additively to inhibit both HIV-1 RT function in the enzyme assay and HIV-1 replication in the yield reduction assay (data not shown).

It is unknown if our in vitro observations of the antagonistic action of efavirenz plus another NNRTI would translate to antagonistic action in the clinical situation; however, there is precedence for these in vitro models to be predictive of the clinical situation (11, 13). The best example is the combination of AZT and d4T. Although there is some disagreement in the literature whether this combination acts additively or antagonistically in an in vitro cell-based system to inhibit HIV-1 replication (13, 20, 28), this combination has been shown to interact antagonistically with the cellular thymidine kinase (13). The different results seen in the two HIV-1 replication systems are believed to be due more to differences in the concentrations and the ratios of the compounds tested than to a difference in the cell types or virus strains used (12; Merrill et al., reply). In the clinic, the combination of AZT and d4T was found to be inferior to d4T alone (11). HIV-1-infected patients who were treated with the combination showed greater declines in CD4-cell count and lesser decreases in plasma HIV-1 RNA levels from the baseline levels than patients who were treated with d4T. Thus, in this case, the in vitro model was predictive of the clinical outcome.

The results of this study suggest that the effectiveness of antiretroviral treatment regimens that include efavirenz or one of the related quinazolinone NNRTIs in combination with NRTIs or PIs should not be adversely affected. However, antiretroviral combination regimens that may contain efavirenz together with another NNRTI may result in less than expected antiviral activity. This may be especially true when drug-naive HIV-1 is the target virus population. Interestingly, HIV-1-infected individuals who were undergoing cotreatment with efavirenz and NVP had lower levels of exposure to efavirenz than HIV-1-infected individuals who were treated only with efavirenz (29). Nevertheless, results from a preliminary clinical study in which efavirenz, NVP, and didanosine were used in combination to treat HIV-1-infected individuals showed that a double NNRTI and NRTI combination may be as effective as a regimen containing an NNRTI, a PI, and an NRTI (W. Jordan, R. Jefferson, F. Yemofio, L. Tolber, V. Conlan, H. Carroll, D. C. Green, A. Green, and R. Green, Abstr. XIII Int. AIDS Conf., 2000). Additional studies to determine the clinical effectiveness of dual NNRTI therapy are ongoing (clinical study 2NN, sponsored by Boehringer Ingleheim).

Acknowledgments

REFERENCES