Efforts to develop effective rectal microbicides have been considerably delayed relative to those for vaginal microbicides, where several compounds are or will be tested in phase III clinical trials (http://www.microbicide.org
). Furthermore, of the compounds evaluated here, PMPA is already in phase II/IIB and UC-781 and TMC120 in phase I clinical trials as formulated vaginal microbicides. While much of the technology developed for vaginal microbicides may be important for the development of effective rectal microbicides, it is not clear whether the same formulations will provide protection against both routes of HIV transmission. Indeed, anatomical differences, including the number and accessibility of target cells, the dynamics of drug absorption, and the surface area, make it highly likely that effective prevention of HIV transmission via RAI (56
) will require the design of rectum-specific formulations.
Previous studies have demonstrated that the individual drugs evaluated here (the NRTI PMPA and the two NNRTIs UC-781 and TMC120) exhibit dose-dependent activity against R5 and X4 isolates (19
) in cellular and cervical explant models. In this study, we have evaluated, side by side, their activities alone and in dual combinations in two cellular models and in colorectal explants. The order of potency and the range of IC50
s for all three compounds were the same in all models tested (TZM-bl cells, PBMCs, and colorectal tissue) against a panel of HIV-1 clade B isolates. TMC120 was more active than UC-781, with IC50
s in the nanomolar order, and PMPA was the least potent, with IC50
s in the μM range. To assess the combinatorial activity (synergy/additivity/antagonism) of drugs, it was not possible to use the Chou-Talalay equation (15
) included in the Calcusyn analysis software. In order for this equation to be applied correctly, the slopes of all the titration curves compared must be parallel. This was not possible to achieve, due to donor-to-donor variation in the explant model and the use of RTI-resistant isolates. Hence, to provide a quantitative indication of the potential increase or decrease in activity, we chose a concept similar to “dose reduction” (14
) and calculated the reductions in the IC50
, and/or IC90
of one drug when it was used in combination with another drug. While this method does not provide a numerical indication (combination index) of the combinatorial effects, it does allow the classification of combinations as “positive” or “negative.” Double combinations of the three compounds in NRTI-NNRTI or NNRTI-NNRTI mixtures were more potent than the individual compounds titrated alone in both cellular and tissue models. Importantly, none of the compounds lost activity when titrated in a combination. Moreover, the IC50
of each compound was reduced when it was included in any of the dual combinations tested (Tables and ; see also Table S2 in the supplemental material). These results are particularly encouraging, considering that the range of concentrations used in the in vitro models are significantly lower than those already included in vaginal gels tested in clinical trials.
The remarkably similar results obtained with TZM-bl cells, activated PBMCs, and colorectal tissue prove the value of the reporter cell line in the screening of candidate microbicides, providing a sensitive and cost-effective tool for quickly assessing drug activity alone and in combination. Recently, TZM-bl cells have been shown to be contaminated with replication-competent gamma-retroviruses, most likely an ecotropic murine leukemia virus (MLV) (74
). The presence of contaminating ecotropic MLV would not substantially affect our results or conclusions. MLV does not encode a viral transactivator required for reporter readout and hence TZM-bl cells demonstrate very low background reporter expression. Furthermore, MLV does not interfere with HIV entry and/or transactivation as evidenced by the high reporter expression following HIV infection. Moreover, endogenous MLV reverse transcriptase activity in TZM-bl cells is highly unlikely to have influenced the activity of RT inhibitors against HIV-1, as reflected by similar activity of the evaluated drugs in TZM-bl cells, PBMCs, and explant cultures.
In addition to determining the efficacies of individual drugs and combinations against wild-type virus, assessment of activity against resistant strains is of critical importance. NNRTI resistance is increasingly prevalent in HIV-infected populations in the developed and developing worlds. Furthermore, different clades may develop resistance more or less rapidly against NRTIs (10
) and NNRTIs. Hence, combinations of compounds that inhibit the virus by different mechanisms may provide more-effective coverage against rectal transmission where resistant isolates are already prevalent.
To this end, dual combinations of PMPA, UC-781, and TMC120 were tested against NNRTI- and NRTI-resistant isolates. In the TZM-bl assay, the NNRTI-resistant isolate A17 was fully resistant to UC-781 and partially resistant to TMC120 (Fig. ). Not surprisingly, the combination of UC-781 with TMC120 was no better than TMC120 alone. A combination of PMPA with either UC-781 or TMC120 restored activity against the NNRTI escape mutant A17. However, neither combination was more active than PMPA alone against this virus. While this would be expected for UC-781, it is interesting that the low activity of TMC120 against this virus did not enhance the activity of PMPA when these drugs were used in combination. We cannot exclude the possibility that, in formulations where the dose of TMC120 could be increased, the titration curve of TMC120 might reach higher levels of inhibition against A17 alone or in combination with PMPA.
The K65R mutation is associated with NRTI resistance. Indeed, a low rate of K65R mutant emergence has been observed in clade B-infected subjects during tenofovir-based therapy (5
). Clinical trial data for the emergence of K65R mutants in subjects infected with other clades are less clear and require further study (18
). However, in vitro studies suggest that resistance might occur more quickly with clade C viral isolates (10
). When the NRTI-resistant isolate 71361-1, which carries the K65R point mutation in RT, was tested, the activities of all three combinations were better than that of each drug alone, the most potent combination being PMPA plus TMC120. Interestingly, the presence of an additional mutation in RT, M184V, partially restored sensitivity to PMPA. This resensitization has also been reported during therapy (67
), suggesting that the emergence of certain mutations in an infected partner might be an unplanned benefit for the development of microbicides for the prevention of rectal transmission. Again, all combinations were more active against the 71361-1 isolate than any single drug used alone.
Interestingly, neither of the two NRTI-resistant isolates containing the K65R mutation could establish productive infection in colorectal explants (Fig. ). Therefore, this resistance mutation appears, at least in vitro, to come with a fitness cost for the virus. It is currently unclear whether isolates containing the K65R mutation have a lower frequency of transmission via the rectal route than wild-type virus in vivo. However, were this to be the case, it might in itself reduce the transmission of resistant strains in populations where the use of RTI-based microbicides may become common. In contrast, while the NNRTI-resistant isolate A17 was able to replicate effectively in colorectal tissue, all combinations, both NRTI-NNRTI and NNRTI-NNRTI, were active against this virus. In summary, the combination of an NRTI with an NNRTI that provided the widest coverage against potential resistant isolates was PMPA plus TMC120, which demonstrated more-potent activity than PMPA plus UC-781 or UC-781 plus TMC120. Studies in progress will determine whether triple combinations of these drugs further improve activity.
These data suggest that dual NRTI-NNRTI combinations are likely to reduce the possibility that exposure to preexisting drug-resistant HIV (either NRTI or NNRTI resistant) from an infected partner might overcome a microbicide, even if only one of the two component drugs were active against the resistant isolate. Therefore, should a dual-RTI-based microbicide prevent infection with a resistant HIV isolate, there would be no further potential for the development of resistance in the exposed subject. The likely requirements that individuals be prescreened for HIV-1 infection prior to having access to an ARV rectal microbicide and that they undergo regular follow up testing should reduce the likelihood of the use of such products by seropositive individuals unaware of their status, thereby reducing the possibility of selecting for resistance in an infected individual. However, were an individual to become infected due to a lack of product efficacy or due to inconsistent use, the risk for the development of drug resistance is currently unknown. This is particularly hard to calculate given the likely rapid reemergence of the wild-type virus upon cessation of product use (23
), the time between infection and the initiation of therapy (often a period of years), the development of second-line therapy active against common ARV escape mutations, and the observation that ARV therapy still provides immunological and virological benefits by suppressing nonresistant virus (27
). Ultimately, the possible selection for drug resistance by the use of ARV-containing microbicides, and the clinical as well as epidemiological consequences, can be addressed only in clinical trials.
The inhibitory activities of PMPA, UC-781, and TMC120, tested alone and in combination, in the colorectal explant model provide key data on the potential tissue drug concentrations required to prevent rectal transmission of HIV. It will be important to determine whether such tissue concentrations equate to protection in vivo (79
). Key to this will be the development of combination formulations specifically targeted for rectal administration. As a first step toward this goal, we have recently demonstrated, in a simian immunodeficiency virus rectal challenge model, that protection in vivo was associated with the drug concentration in vivo and with ex vivo protection of colorectal explants (17
). Further work is needed to determine whether the drug concentrations shown to inhibit HIV replication in colorectal explants can provide a surrogate biomarker of in vivo efficacy. To this end, ex vivo challenge of rectal biopsy specimens is currently under evaluation in an ongoing phase I rectal microbicide trial of formulated UC-781 used at two different doses (0.1 and 0.25%) (J. Elliott, I. McGowan, A. Adler, E. J. Johnson, K. Tanner, D. Cho, T. Saunders, E. Khanukhova, C. Mauck, and P. Anton, presented at Microbicides 2008, New Delhi, India). Should this trial distinguish between the placebo formulation and the two different doses of drug, this may provide the first indication that such a strategy may have utility in wider microbicide trials.
In conclusion, this study supports the rationale for the further development of RTI combinations and the investigation of mixtures with other types of anti-HIV compounds, including protease inhibitors and entry/fusion inhibitors, as part of a comprehensive strategy to develop effective colorectal microbicides. Furthermore, our data suggest that some RTI escape mutations may come with a fitness cost for rectal mucosal transmission, an important issue that requires further investigation. This study further validates the use of the recently developed colorectal explant system as a tool for the preclinical evaluation of potential microbicides and as a model with which to assess the infectivities and replication capacities of wild-type and ARV-resistant viral isolates. Although these studies demonstrate in vitro that RTI combinations used prior to exposure to virus provide coverage against HIV-1 isolates containing RT escape mutants, the predictive value of inhibitory tissue concentrations in vitro can be validated only by detailed tissue pharmacokinetics, ex vivo challenge studies in nonhuman primate models, and human clinical trials.