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Inhibitors and Trimethoprim-Sulfamethoxazole. Inhibit Plasmodium Liver Stages. Charlotte V. Hobbs,1,a Tatiana Voza,2,b Patricia De La Vega,3 Jillian Vanvliet ...
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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 and Photini Sinnis8,b 1

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

Received 3 April 2012; accepted 21 June 2012. Presented in part at the 60th American Society of Tropical Medicine and Hygiene Conference, 4–8 December 2012 (Late Breaker Oral Presentation, abstract 2251). a Formerly of New York University School of Medicine, Department of Pediatrics, Division of Infectious Disease and Immunology, New York. b Formerly of New York University, Department of Medical Parasitology. Correspondence: Charlotte V. Hobbs, MD, Laboratory of Malaria Immunology and Vaccinology, National Institutes of Health, National Institute of Allergy and Infectious Diseases, 12735 Twinbrook Pkwy, Rockville, MD 20852 (charlotte. [email protected]). The Journal of Infectious Diseases 2012;206:1706–14 Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2012. DOI: 10.1093/infdis/jis602

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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.

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NIH/NIAID/Laboratory of Malaria Immunology and Vaccinology, Rockville, Maryland; 2Department of Biological Sciences, New York City College of Technology/CUNY, Brooklyn, New York; 3Walter Reed Army Institute of Research, Department of Cellular Immunology, Malaria Vaccine Branch, Silver Spring, Maryland; 4NIH Clinical Center, Pharmacy Department, Clinical Pharmacokinetics Research Laboratory, Bethesda, Maryland; 5NIH/NIAID/ Biostatistics Research Branch, Rockville, Maryland; 6Johns Hopkins School of Medicine, Department of Medicine, Division of Clinical Pharmacology, Baltimore, Maryland; 7Department of Pediatrics, Division of Infectious Disease and Immunology, New York University School of Medicine, New York, and 8Johns Hopkins Bloomberg School of Public Health, Department of Molecular Microbiology and Immunology, Baltimore, Maryland

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].

METHODS

Pharmacokinetics of NNRTIs and TMP-SMX for Rodent Studies

Mice

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