We assessed the antimalarial activity of different HIV drugs. No activity was seen at concentrations up to 50 μM for all 5 nucleoside reverse transcriptase inhibitors tested, the nonnucleoside reverse transcriptase inhibitor nevirapine, the protease inhibitor darunavir, or the integrase inhibitor raltegravir. However, since nucleoside reverse transcriptase inhibitors function as prodrugs (35
), we cannot rule out the possibility that the drugs exert antimalarial activity after intracellular phosphorylation. Among nonnucleoside reverse transcriptase inhibitors, efavirenz and etravirine showed antimalarial activity, although their IC50
s were well above the levels of the drugs that circulate with standard therapy. The fusion inhibitor enfuvirtide and entry inhibitor maraviroc were also active although, as with the active nonnucleoside reverse transcriptase inhibitors, concentrations with meaningful antimalarial activity are probably not achieved with standard dosing. Consistent with older reports (22
), the most meaningful antimalarial activity was seen with HIV protease inhibitors. All tested protease inhibitors except darunavir showed antimalarial activity. Also consistent with older reports, lopinavir was the most potent of these agents. When provided in standard dosing with ritonavir, lopinavir circulates in the plasma at 10 to 19 μM, well above the IC50
s of 2 to 3 μM determined in this study and 1 to 5 μM seen in other studies using a variety of methods (1
). Serum from patients treated with lopinavir-ritonavir also exerted in vitro
antimalarial activity (36
). Thus, when administered in modern regimens to treat HIV infection, lopinavir is likely to exert clinically relevant antimalarial activity.
The antimalarial activities of different HIV protease inhibitors vary greatly. Indeed, among newer agents not previously studied, tipranavir had only modest activity, although due to high circulating concentrations of this drug, the activity may be clinically important. Darunavir was inactive at 50 μM. Thus, lopinavir remains the HIV protease inhibitor with the most promising antimalarial activity. This is a fortuitous result, as lopinavir-ritonavir is increasingly available in countries where malaria is endemic, and it is available in a heat-stable formulation (9
The antimalarial mechanism of action of HIV protease inhibitors is uncertain. Their action differs from that of the generic aspartic protease inhibitor pepstatin, as unlike pepstatin, they did not display synergy with cysteine protease inhibitors or enhanced activity against a cysteine protease knockout parasite (32
). These results suggest that, although lopinavir and ritonavir inhibited the food vacuole hemoglobinase plasmepsin II (32
), the HIV protease inhibitors may target other plasmodial proteases. P. falciparum
contains 10 aspartic protease genes, which encode 4 food vacuole hemoglobinases (plasmepsins I to IV [3
]), a protease that processes proteins for extracellular export (plasmepsin V [5
]), and other putative proteases with unknown functions. HIV protease inhibitors probably target plasmepsins, but this has not been confirmed, and it is unclear which plasmepsins are targeted or if all active protease inhibitors target the same enzymes.
We also evaluated the antimalarial activities of combinations of lopinavir and standard antimalarial agents. Interestingly, modest synergy was seen between lopinavir and lumefantrine. Synergy was not seen between lopinavir and any other tested antimalarial. Most combinations appeared to have additive effects. There was a trend toward antagonism between lopinavir and the aminoquinolines chloroquine, amodiaquine (studied as its active metabolite monodesethylamodiaquine), and piperaquine; for piperaquine and mefloquine, a trend toward antagonism was seen only for the chloroquine-resistant strain W2 and not the sensitive strain 3D7. Of interest, prior studies showed potentiation of chloroquine activity by HIV protease inhibitors (14
), although the results with lopinavir were modest. It is unclear whether differences in results between our groups are due to the different protease inhibitors studied, differences in methodology, or other factors. Other groups have reported synergy (29
) or antagonism (16
) between HIV protease inhibitors and artemisinins, although only older protease inhibitors and not lopinavir were studied; we found additive effects between lopinavir and dihydroartemisinin.
Our data and the results of other recent studies suggest that there are three means by which the use of lopinavir-ritonavir may have an impact upon the incidence of malaria. First, the antimalarial activity of lopinavir, with drug levels boosted by ritonavir, may kill erythrocytic parasites before infections progress to clinical illness. Second, after therapy for a prior infection, inhibition of cytochrome P450
3A4 by ritonavir may extend exposure to antimalarial drugs, as has been demonstrated for lumefantrine (12
), thereby prolonging the period during which a drug circulates at concentrations adequate to prevent new infections. Third, through synergistic effects, cocirculating antimalarials and antiretrovirals may prevent new infections more effectively than the antimalarials alone. For the last two described mechanisms, the evidence best supports an effect of lopinavir-ritonavir on the posttreatment prophylactic activity of lumefantrine. Therefore, it is of interest to evaluate the effect of lopinavir-ritonavir on those at high risk of malaria and on those treated for malaria in high-transmission areas with artemether-lumefantrine. Indeed, studies of the effects on malaria of treatment of HIV with lopinavir-ritonavir are under way. More broadly, as the use of antiretroviral therapy is increasing in countries where malaria is endemic, our results highlight the importance of evaluating the antimalarial effects of antiretroviral drugs, both in laboratory settings and in clinical trials.