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. 2012 Dec 1;206(11):1706–1714. doi: 10.1093/infdis/jis602

HIV Nonnucleoside Reverse Transcriptase Inhibitors and Trimethoprim-Sulfamethoxazole Inhibit Plasmodium Liver Stages

Charlotte V Hobbs 1,a, Tatiana Voza 2,b, Patricia De La Vega 3, Jillian Vanvliet 1, Solomon Conteh 1, Scott R Penzak 4, Michael P Fay 5, Nicole Anders 6, Tiina Ilmet 7, Yonghua Li 7, William Borkowsky 7, Urszula Krzych 3, Patrick E Duffy 1, Photini Sinnis 8,b
PMCID: PMC3488198  PMID: 23125449

Abstract

Background. Although nonnucleoside reverse transcriptase inhibitors (NNRTIs) are usually part of first-line treatment regimens for human immunodeficiency virus (HIV), their activity on Plasmodium liver stages remains unexplored. Additionally, trimethoprim-sulfamethoxazole (TMP-SMX), used for opportunistic infection prophylaxis in HIV-exposed infants and HIV-infected patients, reduces clinical episodes of malaria; however, TMP-SMX effect on Plasmodium liver stages requires further study.

Methods. We characterized NNRTI and TMP-SMX effects on Plasmodium liver stages in vivo using Plasmodium yoelii. On the basis of these results, we conducted in vitro studies assessing TMP-SMX effects on the rodent parasites P. yoelii and Plasmodium berghei and on the human malaria parasite Plasmodium falciparum.

Results. Our data showed NNRTI treatment modestly reduced P. yoelii liver stage parasite burden and minimally extended prepatent period. TMP-SMX administration significantly reduced liver stage parasite burden, preventing development of patent parasitemia in vivo. TMP-SMX inhibited development of rodent and P. falciparum liver stage parasites in vitro.

Conclusions. NNRTIs modestly affect liver stage Plasmodium parasites, whereas TMP-SMX prevents patent parasitemia. Because drugs that inhibit liver stages target parasites when they are present in lower numbers, these results may have implications for eradication efforts. Understanding HIV drug effects on Plasmodium liver stages will aid in optimizing treatment regimens for HIV-exposed and HIV-infected infected patients in malaria-endemic areas.

Human immunodeficiency virus (HIV) infection and Plasmodium falciparum malaria overlap geographically, especially in sub-Saharan Africa. Studies suggest that, in coinfected patients, each disease exacerbates the other [1]. We have previously shown that HIV protease inhibitors (HIV PIs) inhibit Plasmodium liver stage development [2]. In contrast to HIV PIs, the effect of nonnucleoside reverse transcriptase inhibitors (NNRTIs) on Plasmodium liver stages remains uncharacterized. The World Health Organization (WHO) recommends HIV management with combination antiretroviral therapy (ART), generally including an NNRTI and 2 nucleoside reverse transcriptase inhibitors (NRTIs), or second-line therapy including an HIV PI and 2 NRTIs [3, 4]. Because these drugs are used in HIV-infected patients in malaria-endemic areas, effects of various ART components on Plasmodium requires further investigation.

Separately, trimethoprim-sulfamethoxazole (TMP-SMX), when used for opportunistic infection prophylaxis in HIV-exposed infants and HIV-infected patients [5, 6], reduces clinical malaria [1]. However, the effect of TMP-SMX on Plasmodium liver stages requires further evaluation as it is unclear whether TMP-SMX liver stage effect contributes to the reduction of clinical malaria episodes observed in studies. This will have implications for eradication.

Plasmodium parasites have a complex life cycle. The female Anopheles mosquito infects the mammalian host with sporozoites, the infective form of the parasite, which travel to the liver. There they invade hepatocytes and develop into liver stages, or exoerythrocytic forms (EEFs). These events constitute an asymptomatic period of infection and a time when parasite numbers are low. Infected hepatocytes release merozoites, which invade erythrocytes, initiating the phase of infection responsible for all clinical symptoms of malaria [7]. Most antimalarials target this symptomatic asexual blood stage. However, there is a need for drugs that target liver stages: preventing malaria infection by targeting liver stages will impact transmission and contribute to malaria eradication efforts. Here, we describe our investigations of NNRTIs (efavirenz, etravirine, and nevirapine) and TMP-SMX effects on liver stages of malaria parasites.

METHODS

Mice

Female Swiss Webster mice, aged 4–6 weeks and weighing 20–25 g, were purchased from Taconic or the National Institutes of Health (NIH). Mice experiments were performed at the National Institute of Allergy and Infectious Diseases (NIAID)/NIH and at New York University (NYU) in accordance with the guidelines of NIAID/NIH and NYU Institutional Animal Care and Use Committees.

Parasites and Mosquitoes

Anopheles stephensi mosquitoes were fed on mice infected with P. yoelii (17XNL) or Plasmodium berghei (ANKA), and sporozoites were harvested on days 14–18 (P. yoelii) or days 18–21 (P. berghei) by mosquito dissection. For P. falciparum (NF54), mosquitoes were membrane-fed on blood cultures containing mature gametocytes, and sporozoites were harvested 14–18 days later.

Drugs

NNRTIs were purchased from institutional pharmacies and used in their commercially available forms or were obtained from the NIH AIDS Research and Reference Reagent Program. TMP-SMX was used either in its commercially available suspension form or was reconstituted from analytical standard drug powder (Sigma) in a 1:5 TMP-SMX ratio, based on the TMP weight component. Either phosphate-buffered saline (PBS) or Oraplus was used as drug vehicle (Paddock Laboratories Inc, Minneapolis, MN). For pharmacokinetic studies, internal standards were provided as follows: efavirenz by Dr. David Meyers, Johns Hopkins University School of Medicine; etravirine, TMP, and SMX, by Toronto Research Chemicals (North York, Ontario, Canada); and nevirapine by NIH AIDS Research and Reference Reagent Program.

Drug Dose Determination and Application

For all in vivo studies, mice received drug 6 hours prior to infection and then BID (twice per day) the next day. BID dosing was employed to maximize overall drug exposure due to the short half-lives of these drugs in mice. Hepatotoxicity of drugs was assessed by measuring serum alanine transaminase (ALT) in uninfected, treated mice. Drug dosing was derived using a mouse-dosing equivalent regimen adjusting for differences in surface area–to–body weight ratio between mice and humans for in vivo studies [8], and on available published data for NNRTIs [9–14] and TMP-SMX [15–18] for both in vivo and in vitro studies. For in vitro experiments, TMP-SMX doses were based on published data of concentrations achieved in children and adults receiving prophylaxis regimens [15, 18].

Pharmacokinetics of NNRTIs and TMP-SMX for Rodent Studies

Serum samples were collected from 3 mice per time point at the following postdose time points: 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 12, and 24 hours. Blood was obtained by cardiac puncture, spun down for serum, frozen at −80°C, and shipped on dry ice for analysis using ultraperformance liquid chromatography–mass spectrometry (LC-MS). Geometric mean concentrations were calculated using 3 plasma concentrations per time point. Calibrators and quality controls were prepared using purchased mouse serum (Pel-Freez Biologicals, Rogers, AR). For all drugs, the method was validated over a range of 25–6400 ng/mL.

For each NNRTI sample preparation, serum (50 µL), internal standard (50 µL), and methanol (600 µL) were added to a 12 × 75 mm borosilicate glass tube. Following the addition of methanol, the samples were vortexed briefly and centrifuged. Subsequently, 10 µL of supernatant was injected onto a Waters ACQUITY UPLC BEH C8 analytical 1.7 µm column (Milford, MA). The LC-MS system used was a Waters ACQUITY UPLC interfaced with an AB Sciex API4000 mass spectrometer (Foster City, CA). Intra- and interday precision was <12.2%, with intra- and interday accuracy ranging from 93.3% to 108.5% [19].

For TMP and SMX sample preparations, 30 µL of plasma and 30 µL of internal standard spiking solution were added to a 96-well plate and mixed. Next, 50 µL of this was transferred to an Agilent Captivia 0.45-µm protein precipitation filter plate (Santa Clara, CA). Then, 450 µL of 150 acetonitrile (ACN) was added to each sample and mixed. Vacuum was applied and samples were collected into a 96-well plate, which was placed under a nitrogen stream to evaporate the samples. Samples were then reconstituted using 500 µL of ACN:H2O (50:50). The plate was heat sealed, and 5 µL was injected onto the LC-MS system. Intra- and interday precision was <12.8%, with intra- and interday accuracy ranging from 95% to 103.4% [19].

Pharmacokinetic parameters for NNRTIs, TMP, and SMX were determined using noncompartmental methods with WinNonlin Professional software (version 5.0; Pharsight Corporation, Mountain View, CA). Maximum serum drug concentrations (Cmax) and time to reach those maximum concentrations (Tmax) were determined by visual inspection of the concentration-time profiles. The elimination rate constant (λz) was estimated as the absolute value of the slope of a linear regression of a natural logarithm of concentration vs time using a geometric mean of at least 3 points on the line. Half-life (T1/2) was calculated as “ln (2)/λz.” Area under the concentration versus time curve (AUC) from 0 hours to the last quantifiable concentration (AUC0-last) was determined using the linear trapezoidal rule. Apparent oral clearance (CL/F) was estimated as dose divided by AUC.

Quantification of Liver Stage Parasite Burden for Rodent Malaria Parasites In Vivo

Mice dosed as outlined above were injected intravenously with 104 P. yoelii sporozoites. Livers were harvested at 40 hours after sporozoite infection because parasites may emerge into the bloodstream at this point [20]. From livers, total RNA was isolated using TRI reagent (Molecular Research Center). Liver parasite burden was quantified as described elsewhere [21]. Briefly, reverse transcription was performed using 1 µg of total RNA and random hexamers. Quantitative polymerase chain reaction (PCR) was performed with the QuantiTect SYBR Green PCR Kit (Qiagen), using primers specific for the 18S ribosomal RNA (rRNA) sequence of P. yoelii [21]. Ten-fold dilutions of a plasmid construct containing part of the P. yoelii 18S sequence were used to create a standard curve. Experiments were performed 4 times with 4–5 mice per group per experiment.

Time to Detection of Patent Parasitemia for Rodent Malaria Parasites In Vivo

Mice were dosed as outlined above and injected intravenously with 103 P. yoelii sporozoites on day 0 as previously described [22], or with 2 × 106 parasite-infected red blood cells (iRBCs) on day 2. This inoculum and timing approximates the blood stage inoculum and timing that would result from an infection with 103 sporozoites, assuming that 50% of the inoculum successfully infected and matured to the next life cycle stage [23] and that each EEF produced between 103 and 104 hepatic merozoites [24]. From day 3, mice were monitored for the presence of erythrocytic-stage parasites by Giemsa-stained blood smears. Experiments were performed at least twice with 4–5 mice per group per experiment.

Development Assay for Rodent Malaria Liver Stages (EEFs) In Vitro

8 × 104 to 1 × 105P. yoelii or P. berghei sporozoites were added to monolayers of Hep G2 cells or Hepa1-6 cells at 37°C. After 1 hour, cells were washed and incubated with the indicated concentrations of TMP-SMX prepared in dimethyl sulfoxide (DMSO), or DMSO only, in Dulbecco Modified Eagle medium (DMEM; PABA-free) culture medium, with medium and drug replenished daily. After 48 hours (the time required for parasite liver stage development for these species [20]), cells were fixed and stained with a monoclonal antibody specific for Plasmodium HSP70 [25], followed by a fluorophore-conjugated secondary antibody. Samples were mounted with VectaShield (Vector Laboratories, Burlingame, CA) with DAPI nuclear stain (Sigma-Aldrich, St. Louis, MO). EEFs were counted with a Leica inverted laser-scanning confocal microscope (model TCS SP2 AOBS). Randomly chosen EEFs (9–12 per experimental well) were measured with Leica LCS Lite software (version 2.61, Build 1538). Experiments were run in duplicates per condition, and three independent experiments were performed.

Development Assay for P. falciparum Liver Stages (EEFs) In Vitro

This assay was carried out as described elsewhere [25, 26]. Briefly, P. falciparum sporozoites were isolated from mosquitoes day 14 after the infective blood meal using the Ozaki method [27]. Then, 5.5 × 104 to 7.5 × 104 sporozoites per well were seeded onto monolayers of HC04 cells and incubated in the presence of TMP-SMX prepared in DMSO, or DMSO only, in DMEM (PABA-free) culture medium, with media and drug and replenished daily. On day 4, EEFs were fixed and stained with a monoclonal antibody specific for Plasmodium HSP70 [25, 28], followed by a fluorophore-conjugated secondary antibody. Samples were mounted with Vecta Shield with DAPI nuclear stain. EEFs were counted with an epifluorescence microscope (model Olympus BX50 or BX51). Due to technical limitations of the assay, P. falciparum EEF sizes were not measured. Experiments were run in duplicates per condition, and 2 independent experiments were performed.

Statistical Analysis

For liver parasite burden, the Paule and Mandel random effects meta-analysis method was used to estimate ratios for treated: control. Then, 95% confidence intervals (CIs) were calculated based on t-tests of log transformed values, and 2-tailed P values are reported [29]. For prepatent period analysis, hazard ratios (HRs) were estimated using proportional hazards model using a generalized linear model with the complementary log link [30]. Because none of the mice receiving TMP-SMX developed patent parasitemia, to estimate an upper bound on the HR, we set the time to patency for 1 mouse equal to 4 days and used the resulting upper 95% confidence limit. All other analyses were performed using Kruskal–Wallis with Dunn's post-test on combined experiments to identify statistically significant differences between treated mice and control at the P < .05 level. Statistical analyses were performed using R (version 2.13.0) or GraphPad Prism software (version 5).

RESULTS

NNRTIs Modestly Reduce Plasmodium Liver Stage Parasite Burden

Mice were dosed 6 hours prior to sporozoite infection, and then BID the next day, to increase overall drug exposure (AUC; Supplementary Table 1). This dosing did not result in hepatotoxicity (Supplementary Figure 1). Efavirenz and nevirapine were tested at single doses in experiments in vivo because pharmacokinetic studies demonstrated levels closer to clinical relevance [11, 15, 18, 31, 32], whereas etravirine was tested at high and lower doses as high-dose etravirine resulted in concentrations above clinical relevance. In separate experiments, drug vehicle (PBS and Oraplus) effect on liver stage parasite burden was assessed, and no effect was observed (data not shown). NNRTIs had only a modest effect on reduction of liver parasite burden. Nonetheless, with the exception of the low-dose etravirine, these reductions were statistically significant (Figure 1).

Figure 1.

Figure 1.

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Nonnucleoside reverse transcriptase inhibitors (NNRTIs) and trimethoprim-sulfamethoxazole (TMP-SMX) reduce liver stage parasite burden. Mice were dosed 6 h before sporozoite infection and then twice (BID) the following day. Forty hours after sporozoite infection, livers were harvested and total RNA was isolated. Parasite burden in the liver was quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Shown is the ratio of the geometric means of parasite ribosomal RNA (rRNA) for treated: control mice with 95% confidence intervals (CIs). CIs for each experiment were estimated by t-test, with meta effect calculated using the random-effects meta-analysis method. Results from 4 experiments are combined with 4–5 mice per group per experiment. The ratios of the geometric means of parasite rRNA liver burden for treated control mice were: efavirenz, 0.55 (P = .027, 95% CI: .32, .93); high dose etravirine, 0.35 (P < .0001, 95% CI: .22, .54); low dose etravirine, 0.61 (P = .30, 95% CI: .24, 1.55); nevirapine, 0.48 (P = .003, 95% CI: .29,.78). Alternatively expressed, the ratios of fold change reflect that each treatment decreased liver stage parasite burden on average by a factor of 1.82 for efavirenz, 2.89 for high-dose etravirine, 1.63 for low-dose etravirine, and 2.09 for nevirapine. For TMP-SMX, the ratio of liver stage parasite burden for animals receiving TMP-SMX compared to controls was 0.058 (P = .000006, 95% CI: .016940, .199710); thus there was a 17.2-fold decrease in liver stage parasite burden in treated compared with control animals.

NNRTIs Have Minimal Effect on Prepatent Period

The real-time quantitative polymerase chain reaction (RT-qPCR) assay does not indicate whether drug treatment prevents blood stage infection as it is not sufficiently sensitive to detect a single EEF, the development of which would result in blood stage infection [33].Thus, assessing drug effect on liver stage parasites involves both assessing liver stage parasite burden using RT-qPCR, as well as prepatent period, or time to detection of a blood stage infection by Giemsa-stained smear. Time to detectable blood stage infection after sporozoite infection is called the prepatent period, and in rodent models, this can occur as early as day 3. To assess whether NNRTIs could block blood stage infection, we infected mice treated with the regimen outlined above with 103 P. yoelii sporozoites and assessed prepatent period. Efavirenz and high-dose etravirine led to prolongation of prepatent period by an average of 0.7 and 0.6 days, respectively. This modest effect was statistically significant (efavirenz HR = 0.20, 95% CI: .06, .62, P = .006; etravirine high-dose HR = 0.18, 95% CI: .05, .57, P = .004). Nevirapine did not significantly increase prepatent period (HR = 0.38, 95% CI: .12,1.16, P = .09; Table 1).

Table 1.

Effect of NNRTIs and TMP-SMX on Prepatent Period

Experiment Inoculuma Drug Regimenb Positive Total Mean Prepatent Period (days)
1 Sporozoites Nevirapine 5/5 3.6
Efavirenz 5/5 3.6
Etravirine 5/5 4.0
Control 5/5 3.4
2 Sporozoites Nevirapine 5/5 3.2
Efavirenz 5/5 4.2
Etravirine 5/5 4.0
Control 5/5 3.2
2 Sporozoites TMP-SMX 0/5 NA
Control 5/5 3.2
3 Sporozoites TMP-SMX 18 and 24 hc 0/5 NA
Control 5/5 3.0
4 Sporozoites TMP-SMX 0/5 NA
Control 5/5 3.6
3 iRBC TMP-SMX 5/5 3.0
Control 5/5 3.0
5 iRBC TMP-SMX 5/5 3.2
Control 5/5 3.0
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Abbreviations: NA, not applicable; NNRTIs, nonnucleoside reverse transcriptase inhibitors; iRBC, infected red blood cell; TMP-SMX, trimethoprim-sulfamethoxazole.

a 103 Plasmodium yoelii sporozoites on day 0 or 2 × 106 infected RBC on day 2 were injected intravenously.

b All dosing was 6 h prior and 18 and 24 h after sporozoite infection or −48, −36, and −24 h after iRBC infection, unless otherwise indicated.

c In experiments performed once, single doses of TMP-SMX at 6, 18, or 24 h, or 2 doses at 6 and 18 h, did not result in prolongation of the prepatent period. As noted here for experiment 3, 2 doses at 18 and 24 h also prevented parasitemia, but this was not the final regimen used in other experiments because it was less consistent with human prophylaxis dosing.

TMP-SMX Reduces Plasmodium Liver Stage Parasite Burden

We inoculated P. yoelii sporozoites into mice that had been given TMP-SMX 6 hours prior to infection and then BID the next day, dosing BID to maximize overall drug exposure (AUC; Supplementary Table 1). This regimen was not hepatotoxic (Supplementary Figure 1). Liver stage parasite burden in treated mice was reduced, on average, by 17.2-fold (Figure 1).

TMP-SMX Prevents Patent Parasitemia

To determine whether this significant decrease in liver parasite burden prevented blood stage infection, we inoculated 103 P. yoelii sporozoites into mice that were given the same TMP-SMX regimen outlined above. None of the mice treated with TMP-SMX developed a blood stage infection (Table 1; HR estimate <0.035, with 95% CI, P < .0001).

Because the last dose of TMP-SMX was administered approximately 20–24 hours before the release of hepatic merozoites into the bloodstream [20], and because the half-lives of TMP and SMX were 1.62 and 1.89 hours, respectively (Supplementary Table 1), it would be unlikely that the lack of patent parasitemia could be accounted for by a residual TMP-SMX effect on emerging blood stages. Nonetheless, because we know TMP-SMX affects asexual blood stages [34], we tested whether this lack of patent parasitemia was due to a residual TMP-SMX effect. For this, mice were dosed with time intervals as outlined for the liver stage experiments, but infection was carried out with 2 × 106 iRBCs 20–24 hours after the last dose of TMP-SMX. This inoculum and timing approximates the blood stage inoculum and timing that would result from an infection with 103 sporozoites, assuming that 50% of the inoculum successfully infected and matured to the next life cycle stage [23] and that each EEF produced between 103 and 104 hepatic merozoites [24]. There were no significant differences in the onset of blood stage parasitemia between mice that received TMP-SMX compared with those that did not (HR = 0.85, 95% CI: .32, 2.23, P = .73, Table 1), suggesting that the TMP-SMX dose given during liver stage development does not have a residual effect on emerging blood stage parasites.

TMP-SMX Inhibits Rodent Species Plasmodium Development In Vitro

Our in vivo data demonstrate that TMP-SMX has an inhibitory effect on Plasmodium liver stages development, sufficient to prevent blood stage infection. When parasite rRNA levels were measured, however, we did not see complete inhibition of liver stages. The lack of patent parasitemia without complete inhibition of liver stages would suggest a block at least in EEF development. To study this further, we performed in vitro EEF development assays, and EEF numbers were not significantly different between treated and control samples (P > .05, Figure 2A). However, EEF measurements revealed that treated parasites were significantly smaller compared to controls both in P. berghei (Figure 2B, P < .001) and P. yoelii (data not shown) at each dose indicated. We also observed a lack of parasite nuclear division after 48 hours (Figure 2C). All this suggests arrest of EEF development (Figure 2).

Figure 2.

Figure 2.

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Trimethoprim-sulfamethoxazole (TMP-SMX) treatment inhibits rodent Plasmodium exoerythrocytic forms (EEF) development in vitro. P. berghei or P. yoelii sporozoites were added to Hepa 1–6 or Hep G2 cell monolayers and allowed to invade for 1 h. Unattached sporozoites were washed away, TMP-SMX at the indicated concentrations (TMP-SMX at 6 µg/mL (TMP) and 30 µg/mL (SMX); at 9 µg/mL (TMP) and 45 µg/mL (SMX); and at 12 µg/mL (TMP) and 60 µg/mL (SMX) was added and liver stages were allowed to develop for 48 hours. Cells were then fixed with methanol and liver stages were stained with a monoclonal antibody specific for Plasmodium HSP70 (green), and nuclei were stained with DAPI (blue). A, B, Shown are the combined results from 2 independent experiments with horizontal lines indicating the mean. Statistics were performed using Kruskal–Wallis with Dunn's post-test on combined experiments to identify statistically significant differences between treated and control. Three stars indicate P < .001. A, No. of EEFs in untreated and treated wells (represented as number of EEFs/5 fields per experimental well). No significant differences in EEF nos. were observed between treated and control groups (P > .05). B, Diameters of EEFs from untreated and treated wells (represented as measurements of 9–12 EEFs per experimental well per condition). For EEF size, significant differences were found between treated and control groups at all 3 dosing levels (P < .001). Representative images are shown in panel C. Bars for top panels = 150 µm; bottom panels = 6 µm.

TMP-SMX Reduces P. falciparum EEF Numbers In Vitro

Because our in vivo and in vitro data suggested that TMP-SMX significantly affected rodent Plasmodium liver stage development, we assessed the effect of TMP-SMX on the human parasite P. falciparum EEF development. TMP-SMX significantly reduced P. falciparum liver stage forms, but only at the highest dose of TMP-SMX used (Figure 3, P < .001).

Figure 3.

Figure 3.

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Trimethoprim-sulfamethoxazole (TMP-SMX) treatment reduces Plasmodium falciparum exoerythrocytic form (EEF) nos. in vitro. P. falciparum sporozoites were seeded onto monolayers of HC04 cells and incubated in the presence of TMP-SMX at the indicated concentrations: TMP-SMX at 6 µg/mL (TMP) and 30 µg/mL (SMX); at 9 µg/mL (TMP) and 45 µg/mL (SMX); and at 12 µg/mL (TMP) and 60 µg/mL (SMX). On day 4, cells were fixed and stained with a monoclonal antibody specific for Plasmodium HSP70 and counted on an epifluorescence microscope. Shown are the combined results from 2 independent experiments, with horizontal lines indicating the mean. Statistics were performed using Kruskal–Wallis with Dunn's post-test on combined experiments to identify statistically significant differences between treated and control. Three stars indicate P < .001. For EEF no., significant differences were found between treated and control groups at the highest TMP-SMX dose only (P < .001).

DISCUSSION

Our data demonstrate that all 3 NNRTIs tested modestly reduced liver stage parasite burden, although etravirine only reduced liver stage parasite burden at levels above clinically relevant concentrations. However, only efavirenz and etravirine increased time to patent parasitemia, and etravirine exerted effect again only at supratherapeutic concentrations. Thus, efavirenz appeared to be most potent at levels approaching clinical relevance. Overall, modest NNRTI antiparasite liver stage effect parallels previously published data showing that NNRTIs have modest effect against P. falciparum erythrocytic stages in vitro at concentrations above clinical relevance, or no effect at all [35, 36]. In contrast, the HIV PIs have an inhibitory effect on Plasmodium liver stages in rodents, even when serum levels are undetectable [2], and on asexual erythrocytic stages at levels both at and below clinical relevance [36, 37]. Our data showing modest NNRTI liver stage effect, however, may be underestimated because AUCs were lower in mice than what is reached in humans on standard dosing.

How NNRTIs exert these observed modest antiparasite effects remains to be elucidated. NNRTIs function by inhibiting the activity of HIV reverse transcriptase (RT) by binding to the enzyme and inducing a conformational change that inhibits its activity [38]. However, there is no known RT in Plasmodium. Recent studies have shown efavirenz incubation with human liver cells results in oxidant stress and mitochondrially mediated apoptosis, and that preincubation with antioxidants markedly decreases activation of these pro-apoptotic pathways [39, 40]. This implies that reactive oxygen species may play a role in efavirenz-induced toxicity, and it is possible that Plasmodium parasites may experience similar toxic effects, especially because it is known that Plasmodium is sensitive to oxidant stress [41]. However, this may not completely explain the observed effects as similar findings have not been observed with nevirapine [42], whose described mechanisms of hepatotoxicity include immune-mediated processes. In the case of etravirine, generally, minimal hepatotoxicity has been observed [10].

Our data also show that TMP-SMX significantly reduces liver stage parasite burden and prevents the development of patent parasitemia. Other antifolates have an effect on Plasmodium liver stages [33], but evaluation of TMP-SMX in combination, on liver stage parasites in vitro and in vivo, and at levels that approximate clinical relevance, has not been studied previously. Our data complement recently published studies that evaluate TMP or SMX effect, individually, on liver stages of P. yoelii in vitro [43]. Furthermore, our in vitro and in vivo rodent data demonstrate that TMP-SMX treatment arrests EEF development. These data are consistent with prior data showing that antifolates work during stages of rapid division [44].

Moreover, we have demonstrated that TMP-SMX in combination, and at levels based on clinical relevance, reduces P. falciparum EEF numbers. This is in contrast to the in vitro rodent malaria data, in which size, but not number, of EEFs was reduced. However, TMP-SMX effect is more pronounced for P. falciparum on EEF numbers than for rodent strains, possibly due to assay duration differences, parasite species drug sensitivity differences, or host cell line metabolic differences. For the first point, in contrast to the rodent malaria parasites that develop in 44–48 hours [20], P. falciparum takes approximately 7 days [45]; thus the in vitro assay is longer. This could permit the drug more time to exert a parasite killing effect, further reducing EEF numbers by the end of the assay. For the second point, different species of Plasmodium are differentially susceptible to the antifolates [44], so TMP-SMX may have a differential effect on rodent malarias as opposed to Plasmodium falciparum. For the third point, differences in host cell drug metabolism could also account for differential magnitude of drug effect. The HC04 and HepG2 cell lines used in this study were shown in a series of previous investigations to express a variety of Phase 1 and 2 drug metabolizing enzymes involved in TMP and/or SMX biotransformation and disposition [26, 46–48]; these include uridine diphosphate glucuronosyl transferase (UGT), cytochrome P450 isoforms, and a variety of uptake and efflux transport proteins [49]. Thus, the in vitro systems should reflect in vivo metabolic activity. However, it is possible that subtle drug metabolism differences exist. Reassuringly, TMP-SMX treatment of P. berghei–infected Hepa 1–6 cells, which are of mouse origin but which have also been well characterized [50], resulted in parallel findings to P. yoelii infection of Hep G2 cells. Clinical studies are warranted to validate these findings.

Given geographic overlap of HIV and malaria, and the copathogenic effects each disease has on the other [1], evaluation of HIV treatment regimens in the context of malaria is warranted. Our data support the need for clinical studies evaluating HIV PI-containing regimens, NNRTI-containing regimens, and TMP-SMX prophylaxis, each alone and in combination, for differential impact on malaria. Initiation of PI-based therapy in young HIV-infected patients may offer secondary prophylactic benefits for modifying malarial infection. In addition, if TMP-SMX effect on liver stage parasites is demonstrated at the clinical level, TMP-SMX prophylaxis for HIV-exposed and HIV-infected patients may offer additional benefit to eradication efforts in malaria-endemic areas.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Supplementary Data
supp_206_11_1706__index.html (428B, html)

Notes

Acknowledgments. We thank NIH AIDS Research and Reference Reagent Program. Kevin Spinno, Andre Laughinghouse, and Jean Noonan for their expert assistance with mosquito rearing; Sandra Gonzalez for maintenance of rodent parasite cycles; Lynn Lambert, Sachy Orr-Gonzalez, Katy Zeleski, Felix Santiago, Jade Basile, and Brittany Stokes for their support with animal work. The Department of Laboratory Medicine in the Clinical Center, for support with serum chemistries. Mosves Hovesepian, Robert Downes, and Karen Sillers for their assistance in obtaining drugs; Leonard Liebes for his help in serum drug level analysis; Jingyang Chen for his assistance with PCR assays; Johanna Daily, for helpful discussions; and Brian Kirmse, Marguerite Meitzler, Pragyan Acharya, Tejram Sahu, and Sunil Parikh for helpful discussions and review of this paper.

Financial support. This work was funded by the New York University Global Public Health Research Challenge Fund (P. S. and C. H.), NIH R01 AI056840 (P. S.), the NIH Division of Intramural Research, and the US Army Medical Research and Materiel Command.

 Potential conflicts of interest. All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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