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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: Antiviral Res. 2017 Jul 22;145:175–183. doi: 10.1016/j.antiviral.2017.07.011

Dual-targeted anti-TB/anti-HIV heterodimers

Liudmila Alexandrova 1, Sonia Zicari 2, Elena Matyugina 1, Anastasia Khandazhinskaya 1,*, Tatiana Smirnova 3, Sofia Andreevskaya 3, Larisa Chernousova 3, Christophe Vanpouille 2,*, Sergei Kochetkov 1, Leonid Margolis 2
PMCID: PMC5576140  NIHMSID: NIHMS900608  PMID: 28743447

Abstract

HIV and M. tuberculosis are two intersecting epidemics making the search for new dual action drugs against both pathogens extremely important. Here, we report on the synthesis and suppressive activities of five dual-targeted HIV/TB compounds. These compounds constitute heterodimers of AZT, as anti-HIV molecules, and 5-arylaminouracil derivatives or 2′-deoxyuridine monophosphates analogs, as anti-TB molecules. We found that these compounds inhibit the growth of M. tuberculosis and suppress the replication of HIV in human cell cultures and human lymphoid tissues ex vivo. We identified one particular heterodimer that inhibited most potently both HIV and the drug-resistant TB strain MS −115. This compound demonstrated low toxicity and had no cytostatic effect on cells in culture. This is a leading candidate for future development and further in vivo testing.

Keywords: HIV, TB, heterodimers, human tissues

1. Introduction

Infection by HIV and M. tuberculosis are among the main causes of death from infectious diseases worldwide. According to 2015 WHO estimates, 2.1 million people became newly infected with HIV and 1.1 million people died from AIDS-related illnesses. In 2015, among the causes of death from infectious diseases, tuberculosis (TB) ranked above HIV/AIDS with an estimated 10.4 million new cases and 1.8 million deaths [1].

HIV and TB epidemics fully intersect. On one hand, the risk of death in TB/HIV-coinfected individuals is twice that of HIV-infected individuals without TB, even when CD4 cell counts and antiretroviral therapy are taken into account [2]. On the other hand, HIV infection is the strongest known risk factor for progressing to TB disease for individuals with latent TB infection. Also, people living with HIV are 17–22 times more likely to develop TB than people without HIV. Apparently, M. tuberculosis and HIV act synergistically, resulting in a decline of immunological functions and death if untreated [3].

The synergy between TB and HIV makes the search for new combined dual action drugs extremely important. In general, combining two active compounds into one molecule has a long history [4–8]. In particular, we reported earlier on the synthesis of hybrid antivirals by linking two nucleoside reverse transcriptase inhibitors (NRTI) of HIV [9].

Here, we describe new anti-TB/anti-HIV compounds that are based on heterodimerization of our recently designed anti-TB 5-arylaminouracil derivatives [10] (as well as of 2′-deoxyuridine monophosphates and their analogs [11] with the anti-HIV NRTI, 3′-azido-3′-deoxythymidine (AZT)). We report here on the synthesis of five heterodimers. These compounds inhibit the growth of M. tuberculosis and suppress the replication of HIV in human cell cultures and human lymphoid tissues ex vivo. Four of the synthesized compounds proved to be of low cytotoxity in these systems.

2. Materials and methods

2.1. Chemical synthesis

Chemical synthesis and spectroscopic characterizations are described in the supplementary data file. Briefly, in this work, five new hybrid molecules were synthesized; three of them (1–3) are the conjugates of AZT and 5-(4-decyl-1,2,3-triazol-1-yl-methyl)-2′-deoxyuridine (dUR) (Scheme 1) and the other two (4 and 5) were conjugates of AZT and modified 5′-norcarbocyclic analogues of 2′,3′-dideoxy-2′,3′-didehydrouridine (Scheme 2).

Scheme 1.

Scheme 1

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Scheme 2.

Scheme 2

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A solution of dUR and 1,1′-carbonyldiimidazole in DMF was heated for 18 h at 37°C, and then AZT was added to give a mixture of products 1–3. The reaction mixture was then evaporated and chromatographed on silica gel with a gradient concentration of ethanol in chloroform (Scheme 1).

For the synthesis of heterodimers 4 and 5, the previously described method of selective N-3 alkylation was used [5]. The reaction of azidothymidine and dibromopropane in the presence of potassium carbonate gave intermediate 6 (15% yield), to which the second key component 7 or 8 was further added (Scheme 2). The yields of the heterodimers 4 and 5 on the second stage were 60% and 65%, respectively. Structures and purities of the synthesized compounds 1–5 were confirmed from 1H and 13C NMR spectroscopy, high-resolution mass spectroscopy, and TLC.

5′-O-[AZT-carbonyl]-5-[(4-decyl-1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine (1), 3′-O-[AZT-carbonyl]-5-[(4-decyl-1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine (2), and 3′,5′-di-O-[AZT-carbonyl]-5-[(4-decyl-1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine (3)

N,N′-carbonyldiimidazole (CDI, 95 mg, 0.59 mmol) was added to a solution of 5-(4-decyl-1,2,3-triazol-1-yl)-2′-deoxyuridine (dUR, 130 mg, 0.28 mmol) in DMF (3 mL), and the mixture was kept for 18 h at 37° C; then 3′-azido-3′-deoxythymidine (AZT, 309 mg, 1.16 mmol) was added and the mixture was kept for 18 h at 37° C. The mixture containing products 1, 2, and 3 was evaporated under vacuum; the residue was dissolved in chloroform (2 mL), applied onto a silica gel column (20×250 mm), and eluted with a gradient of ethanol in chloroform (0 → 7%) to give crude products 1–3. We performed final purification of the isolated products with preparative TLC on silica gel plates (20×20×2 mm) using ethyl acetate/ethanol/hexane (9:1:1, v/v) as an eluent to provide the desired compounds.

5′-O-[AZT-carbonyl]-5-[(4-decyl-1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine (1)

5′-O-[AZT-carbonyl]-5-[(4-decyl-1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine (1) was obtained as a white solid (31 mg, 15%). UV: λmax 264.2 nm (19 215). 1H-NMR (DMSO-d6):11.53 (1H, s, 3NHdUR), 11.30 (1H, s, 3NHAZT), 7.89 (1H, s, CHdUR), 7.72 (1H, s, H6dUR), 7.43 (1H, s, H6AZT), 6.16 (1Н, dd, J6.7, 6.8, H1′dUR), 6.11 (1Н, t, J6.5, H1′AZT), 5.45 (1H, d, J4.6, 3′OHdUR), 5.12 (2H, s, 5CH2OdUR), 4.48–4.24 (6H, m, H3′AZT + H3′dUR + H5′AZT + H5′dUR), 3.97 (1H, m, H4′AZT + H4′dUR), 2.55 (2Н, m, CH2C9H19 dUR), 2.46–2.19 (4H, m, H2′AZT + H2′dUR), 1.75 (3H, s, 5CH3 AZT), 1.55 (2H, m, CH2CH2C6H17 dUR), 1.23 (14H, m, C2H4(CH2)7CH3 dUR), 0.85 (3Н, dd, J6.7, 6.8, CH3 dUR). 13C NMR (DMSO-d6): δ 163.52, 162,41, 154.04, 150. 25, 150.023, 146.69, 140.77, 135.86, 121, 55, 109.86, 108.19, 84.67, 84.61, 83.70, 80.39, 80.31, 69.95, 67.75, 67.06, 59.84, 45.67, 38.77, 35.52, 31.19, 28.90, 28.68, 28.64, 28.59, 24.92, 22.00, 13.85, 11.93. MS (ESI): calculated for C33H46N10O10 743.3471 [M+H]+, found 743.3467, calculated for C33H46N10O10 765.3291.4040 [M+Na]+, found 765.3274.

3′-O-[AZT-carbonyl]-5-[(4-decyl-1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine (2)

3′-O-[AZT-carbonyl]-5-[(4-decyl-1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine (2) was obtained as a white solid (25 mg, 12%). UV: λmax 264.2 nm (19 220). 1H-NMR (DMSO-d6):11.57 (1H, s, 3NHdUR), 11.32 (1H, s, 3NHAZT), 8.12 (1H, s, CHdUR), 7.74 (1H, s, H6dUR), 7.48 (1H, s, H6AZT), 6.13 (2Н, m, H1′AZT + H-1′dUR), 5.19 (2H, m, H3′dUR + 5′OHdUR), 5.12 (2H, m, 5CH2OdUR), 4.48 (1H, m, H3′AZT), 4.35 (2H, m, H5′AZT), 4.09 (1H, m, H4′AZT), 4.02 (1H, m, H4′dUR), 3.63 (2H, m, H-5′dUR), 2.56 (2Н, m, CH2C9H19 dUR), 2.46–2.34 (4H, m, H2′AZT + H2′dUR), 1.80 (3H s, 5CH3 AZT), 1.55 (2H, m, CH2CH2C6H17 dUR), 1.24 (14H, m, C2H4(CH2)7CH3 dUR), 0.85 (3Н, dd, J6.7, 7.0, CH3 dUR). 13C NMR (DMSO-d6): δ163.62, 162,48, 153.56, 150. 33, 150.13, 146.80, 140.49, 135.99, 121, 63, 109.89, 108.24, 84.67, 84.44, 83.79, 80.39, 78.76, 66.94, 61.22, 59.72, 45.83, 36.86, 35.66, 31.25, 28.94, 28.74, 28.64, 28.62, 24.98, 22.0513.87, 12.07. MS (ESI): calculated for C33H46N10O10 743.3471 [M+H]+, found 743.3481, calculated for C33H46N10O10 765.3291.4040 [M+Na]+ found 765.3271.

3′,5′-di-O-[AZT-carbonyl]-5-[(4-decyl-1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine (3)

3′,5′-di-O-[AZT-carbonyl]-5-[(4-decyl-1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine (3) was obtained as a white solid (61mg, 21%). UV: λmax 264.2 nm (28 800). 1H-NMR (DMSO-d6): 11.61, 11.32, 11.31 (3H, 3s, 3NH dUR +(3NHAZT)2), 7.97 (1H, s, CHdUR), 7.73 (1H, s H6dUR), 7.48, 7.43 (2H, 2s, (H6AZT)2), 6.13 (3H, m, (H1′AZT)2 +H1′dUR), 5.12 (2H, s, CH2OdUR), 4.47 (2H, m, (H3′AZT)2), 4.35 (5H, m, (H5′AZT)2 + H4′dUR), 4.01 (2H, m, (H-4′AZT)2), 3.31 (2H, M, H5′dUR), 2.57 (2Н, T, J7.62, CH2C9H19 dUR), 2.12 (2H, M, H2′ dUR), 1.80–1.78 (6H, 2s, (5CH3 AZT)2), 1.52 (2H, m, CH2CH2C6H17 dUR), 1.23(14H, m, C2H4(CH2)7CH3 dUR), 0.85 (3Н, T, J6.9, CH3.dUR). 13C NMR (DMSO-d6): δ 164.07, 162,938, 154.44, 153.99, 151.37, 150. 80, 150.53, 147.26, 141.45, 138.12, 136.52, 136.39, 122.09, 110.39, 109.969, 109.92, 85.53, 85.16, 84.46, 84.28, 83.89, 80.84, 78.07, 67.86, 67.70, 66.82, 60.64, 60.41, 60.22, 46.20, 36.11, 31.74, 2945, 2923, 29.13, 25.47, 22.54, 14.36, 12.59, 12.45. MS (ESI): calculated for C44H57N15O15 1036.4221 [M+H]+, found 1036.4231, calculated for C44H57N15O15 1058.4040 [M+Na]+ found 1058.4051.

3′-Azido-3-N-(3-bromopropyl)-2′,3′-dideoxythymidine (6)

To a solution of AZT (1 eq, 500 mg, 1.87 mM) in acetone were added K2CO3 (2 eq, 516 mg, 3.74 mM) and 3-bromopropane (2 eq, 378 mkL, 3.74 mM). The reaction mixture was refluxed for 4 h. After evaporation, the residue was purified by column chromatography on silica gel and eluted with hexane: ethyl acetate (1:1), with a yield of 6 (83%) as an off-white powder. 1H NMR (CDCl3): 7.35–7.34 (1Н, s, H6), 6.06–6.03 (1H, m, H1′), 4.39–4.37 (1H, m, H4′), 4.08–4.05 (2H, t, 5′CH2), 3.97–3.95 (2H, t, CH2-N), 3.84–3.79 (1H, s, OH), 3.42–3.39 (2H, t, CH2-Br), 2.54–2.52 (1H, m, H2′b), 2.44–2.41 (2 H, m, H2′a, H3′), 2.22–2.18 (2H, m, CH2), 1.92 (1H, s, CH3). 13C NMR (CDCl3): 163.37, 150.99, 134.96, 110.66, 87.67, 84.68, 62.21, 60.11, 40.50, 37.52, 31.06, 30.45, 13.38.

[AZT]-N3-propyl-N3-[1-N-[4′-(o,o-dimethylbenzyl)cyclopent-2′-enyl]uracil] (4)

To a solution of (6) in dry acetonitrile (62 mg, 0.16 mM) were added K2CO3 (40 mg, 0.3 mM) and 1-N-[4′-(o,o-dimethylbenzyl)cyclopent-2′-enyl]uracil (48 mg, 0.14 mM). The reaction mixture was refluxed for 4 h. After evaporation, the residue was purified by column chromatography on silica gel and eluted with chloroform: methanol (98:2); yield of 4 (75%) as a yellow powder. 1H NMR (CDCl3): 7.59 (2 H, s, 2×H6), 7.31, 7.27, 7.19 (3H, s, Ph), 6.39–6.38 (1H, m, H1′), 6.03–6.0 (2H, m, H1′AZT, H2′), 5.86–5.84 (1H, m, H3′), 5.73–5.71 (2H, m, H5Ura, H4′), 4.39–4.37 (1H, m, H3′AZT), 4.01–4.00 (5H, m, 2×CH2, H4′AZT), 3.94–3.92 (2H, m, 5′CH2AZT), 3.09–3.05 (1H, m, H5′a), 2.76 (1H, s, OH), 2.59–2.56 (1H, m, H2′aAZT), 2.01 (6H, s, 2CH3Bz), 2.03–2.00 (1H, m, H2′bAZT), 1.89 (1H, s, CH3), 1.84–1.82 (1H, m, H5′b). 13C NMR (CDCl3): 166.33, 163.41, 162.80, 151.68, 150.99, 138.69, 138.40, 136.61, 135.14, 135.02, 134.99, 134.47, 129.78, 127.44, 110.46, 102.44, 87.81, 84.74, 62.06, 59.99, 59,46, 39.34, 39.26, 37.77, 37.39, 29.84, 26.35, 21.31, 13.41. HRMS:found m/z 656.2436; calculated for C31H35N7O8 [M+Na]+ 656.2439.

[AZT]-N3-propyl-N3-[1-N-(4′-hydroxycyclopent-2′-enyl)-5-(p-butoxyphenylamino)]uracil (5)

[AZT]-N3-propyl-N3-[1-N-(4′-hydroxycyclopent-2′-enyl)-5-(p-butoxyphenylamino)]uracil (5) was obtained by the method described for (4) using 1-N-(4′-hydroxycyclopent-2′-enyl)-5-(p-butoxyphenylamino)uracil with 68% yield as a pale yellow powder. 1H NMR (CDCl3): 7.03 (H, s, H6AZT), 6.91 (H, s, H6Ura), 6.82–6.80 (4H, m, Ph), 6.15–6.14 (1H, m, H1′), 6.02–5.99 (H, m, H1′AZT), 5.81–5.80 (1H, m, H2′), 5.79–5.74 (1H, m, H3′), 5.33–5.32 (1H, m, H4′), 4.78–4.77 (1H, m, H4′AZT), 4.40–4.38 (1H, m, H3′AZT), 4.05–4.03 (5H, m, 2×CH2), 3.97–3.92 (4H, m, 5′CH2AZT, CH2α), 3.79–3.74 (2H, m, 2×OH), 2.82–2.79 (1H, m, H5′a), 2.59–2.56 (1H, m, H2′aAZT), 2.39–2.38 (1H, m, H2′bAZT), 2.05–2.03 (2H, m, CH2), 1.90 (1H, s, CH3), 1.85–1.82 (3H, m, CH2β, H5′b), 1.76–1.74 (2H, m, CH2γ), 1.53–1.51 (1H, m, CH3). 13C NMR (CDCl3): 163.34, 160.54, 154.61, 151.04, 149.33, 138.95, 135.11, 131.93, 121.26, 120.66, 116.77, 115.65, 110.48, 87.95, 87.67, 84.68, 75.05, 68.27, 62.19, 62.04, 61.91, 59.90, 39.75, 39.63, 39.18, 37.22, 31.51, 29.78, 26.42, 19.34, 13.93, 13.34. HRMS:found m/z 687.2857; calculated for C32H40N8O8 [M+Na]+ 687.2861.

2.2. Stability of compounds synthesized

Chemical stability at pH 2.2, 7.4, and 9.0 as well as stability in human blood serum were evaluated according to the standard procedures [12, 13]. We performed HPLC-HRMS analysis of the samples using an Agilent 1260 chromatograph equipped with an Agilent Poroshell-120 column (3.0 × 50 mm; 2.7 μm) and a Phenomenex SecurityGuard Ultra C-18 precolumn and used a Bruker Daltonics micrOTOF-Q II mass-spectrometer for detection. The column was eluted in a gradient of acetonitrile (A) in water (B). Gradient parameters: 100% B for 2 min; 10% → 20% A for 2 min, 20% → 30% A for 2 min, 30% → 40% A for 2 min, 40% → 50% A for 2 min, 50% → 60% A for 2 min, 60% → 70% A for 2 min, 70% → 80% A for 2 min, 80% → 90% A for 2 min, 90% → 100% A for 2 min; 100% A for 5 min, 100% → 0% A for 5 min. Flow rate was set at 0.130 ml/min.

2.3. Anti-HIV-1 activity

HIV-1 inhibition by compounds (1–5) was assessed in MT-4 cell cultures and in human tissue ex vivo.

2.3.1. MT-4 cell cultures

Human T lymphocytes MT-4 (obtained from American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. We performed HIV-1 infection of MT-4 by incubating 100 μL of MT-4 at 107 cells/mL with 100 μL of X4LAI.04 containing 5 ng of p24gag in a vial for 1 h 30 min at 37°C. After incubation, infected cell suspensions were washed with PBS and resuspended in 1 mL of medium. We first transferred 100 μL of infected MT-4 cells to a 12-well plate and then mixed them with 900 μL of medium containing the test compound at an appropriate dilution. MT-4 cells were incubated at 37°C and cultured for 3 days.

2.3.2. Human tissue culture ex vivo

Human tonsillar tissues were obtained from patients undergoing routine tonsillectomy at the Children’s National Medical Center (Washington DC) under an IRB-approved protocol. Tissues were dissected into 2- to 3-mm3 blocks and placed onto collagen sponge gels in culture medium at the air–liquid interface as described earlier [14]. Human tonsillar tissues (27 blocks of tissue from each of n donors for each experimental condition) were pretreated with compounds (1–5) overnight and then infected with a prototypical X4 variant of HIV-1 (HIV-1 X4LAI.04) (Rush University Virology Quality Assurance Laboratory, Chicago, IL). The tissue culture was kept for 12 days, and drugs were replenished after each medium change (every 3 days).

2.3.3. Evaluation of antiviral activity of compounds (1–5)

We measured p24gag using a bead-based Luminex assay as described earlier [15]. We then evaluated the antiviral activity of each compound by measuring inhibition of human HIV-1 replication in MT-4 cell cultures and in human lymphoid tissues. For each compound, in MT-4 cell cultures or in lymphoid tissue ex vivo, HIV-1 inhibition at each single concentration was defined by the following formula: inhibition = (1−Rcompounds/RCtl)×100, where Rcompounds and RCtl are the amounts of p24 accumulated in the medium in compound-treated cultures and in untreated cultures, respectively. We calculated the EC50 values (with 95% confidence interval (CI)) by fitting the data points to a sigmoidal dose–response curve, using Prism software, (version 6.0; GraphPad). The EC50 (50% effective concentration) is defined as the compound concentration required for inhibition of viral replication by 50%.

2.4 Cell toxicity

Cell toxicity was assessed both in MT-4 cell cultures and in human lymphoid tissue.

2.4.1. Viability assay in MT-4 cell cultures

We performed viability assays (cytotoxicity and cytopathicity) in MT-4 cell cultures with the Nucleocounter automated cell counting system (ChemoMetec). We determined the numbers of total cells and dead cells in control (untreated) cultures and in cultures treated with heterodimers (1–5) using a propidium iodide–based assay according to the manufacturer’s protocol. We collected and analyzed data using Nucleoview software (version 1.0; ChemoMetec).

2.4.2. Viability assay in human lymphoid tissue

To assess the cytotoxicity of heterodimers in human tonsillar tissues, we isolated cells from the compound-treated and untreated tissue blocks and stained them for surface markers. Each cell suspension was divided in two fractions and used for two different stainings. The first fraction was stained with a combination of the following fluorescence-labeled monoclonal antibodies: anti–CD3-QD655, anti–CD4-QD605, anti–CD8-QD800, anti–CD45RA-FITC, and anti–CCR7-PE-Cy7. The second fraction was stained with the following antibodies: anti–CD3-QD655, anti–CD4-QD605, anti–CD8-QD800, anti–CD25-APC, anti–CD38-PE, anti–CD95-PE-Cy7, and anti–HLA-DR-BV570 (Caltag Laboratories, CA; B&D Biosciences, CA). Data were acquired and analyzed on a NovoCyte flow cytometer (AceaBisociences, Inc). We quantified cell depletion using Trucount beads (Becton Dickinson) for volumetric control and normalized cell numbers by tissue-block weights.

2.5. Anti-TB activity

Two strains of M. tuberculosis, H37Rv and MS-115, were used for testing anti-TB activity of the synthesized heterodimers. The former strain is sensitive to standard anti-TB drugs, while the latter was MDR-resistant [10, 16]. The cultures were checked for the absence of contamination by nonspecific microflora. Anti-TB activity was measured as described earlier [16] with each concentration of the test compounds, and the control samples were tested in duplicate.

3. Results

3.1. Heterodimer synthesis

In this work, five new hybrid molecules were synthesized; three of them (1–3) are the conjugates of AZT and 5-(4-decyl-1,2,3-triazol-1-yl-methyl)-2′-deoxyuridine (dUR) (Scheme 1) and the other two (4 and 5) were conjugates of AZT and modified 5′-norcarbocyclic analogues of 2′,3′-dideoxy-2′,3′-didehydrouridine (Scheme 2).

A solution of dUR and 1,1′-carbonyldiimidazole in DMF was heated for 18 h at 37°C, and then AZT was added to give a mixture of products 1–3. The reaction mixture was then evaporated and chromatographed on silica gel with a gradient concentration of ethanol in chloroform (Scheme 1).

For the synthesis of heterodimers 4 and 5, the previously described method of selective N-3 alkylation was used [5]. The reaction of azidothymidine and dibromopropane in the presence of potassium carbonate gave intermediate 6 (15% yield), to which the second key component 7 or 8 was further added (Scheme 2). The yields of the heterodimers 4 and 5 on the second stage were 60% and 65%, respectively. Structures and purities of the synthesized compounds 1–5 were confirmed from 1H and 13C NMR spectroscopy, high-resolution mass spectroscopy, and TLC.

3.2 Stability of compounds synthesized

All the tested compounds were stable to chemical hydrolysis at рН 2.2 (glycine+HCl) and at рН 7.4 (PBS). Compounds 4 and 5 were also stable at рН 9.0 (glycine+NaOH); T1/2 for compounds 1–3 at the same conditions was about 14–15 h.

Similar T1/2 values for compounds 1–3 were obtained in human blood serum. Serum activity was assessed with the standard compounds: T1/2 for 3′,5′-diacetyl thymidine was 18 h; T1/2 for TMP was 20 min. For compounds 1 and 2, both AZT and dUR derivative were found as main products. For compound 3, the entire set of hydrolysis products was detected: 1, 2, AZT, and dUR. Compound 4 produced only slight debenzoylation at the carbocyclic fragment after 15 h in human blood serum (debenzoylated product retention time was 19.50 min); compound 5 was stable. Retention times were as follows: 1, 22.10 min; 2, 22.50 min; 3, 23.05 min; 4, 21.85 min; 5, 21.10 min; AZT, 13.75 min; and dUR, 20.80 min.

3.3. Heterodimers inhibited HIV-1 replication

The anti-HIV activities of all the heterodimers (1–5) were evaluated in MT-4 cell cultures and in ex vivo human lymphoid tissues. All the compounds inhibited X4Lai.04 replication in MT-4 cell cultures. HIV-1 inhibition was assessed at heterodimer concentrations ranging from 1 nM to 30 μM. HIV-1 inhibition was dose-dependent, with EC50 for heterodimers 1–3 being around 2 logs lower than that of heterodimers 4–5 (Table 1).

Table 1.

Anti-HIV activity of compounds 1–5 in MT-4 cell cultures and in human tissues ex vivo

Tonsil ex vivo MT-4
EC50 (μM) 95% confidence interval EC50 (μM) 95% confidence interval
1 1.32 0.37–4.73 0.39 0.24–0.65
2 1.28 0.10–15.95 0.07 0.05–0.10
3 0.14 0.08–0.25 0.08 0.04–0.15
4 20.14 1.10–368.3 19.77 12.51–31.22
5 1.29 0.35–4.77 12.94 10.29–16.26
AZT 0.003 0.001–0.16 0.03 0.021–0.043
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Similarly, we found that all compounds inhibited X4Lai.04 replication in tissues ex vivo in a dose-dependent manner. For each heterodimer, the anti-HIV-1 activity in tissues was tested at concentrations ranging from 1 pM to 30 μM. As described earlier (reviewed in [14]), the absolute levels of HIV replication, evaluated from measurements of p24gag accumulation in culture medium, were donor-dependent. Therefore for each donor, HIV-inhibition in the treated experimental condition was expressed as a percent of that in the donor-matched untreated condition. Although EC50s were different from those in MT-4 cell cultures (Table 1), heterodimers 1–3 were more potent than heterodimers 4–5 as observed in MT-4 cultures. EC50s in human tissues ranged from 0.14 to 0.5 μM for heterodimers 1–3 and from 1 to 6 μM for heterodimers 4–5, respectively (Table 1).

3.4. Heterodimers inhibited Mycobacterium tuberculosis

We studied anti-mycobacterial activities of the synthesized compounds using two M. tuberculosis strains: H37Rv, a laboratory strain sensitive to the five first-line anti-TB drugs (isoniazid, rifampicin, streptomycin, ethambutol, and pyrazinamide), and MS-115, a clinical MDR-resistant strain. We evaluated the anti-mycobacterial effects of the compounds as bacterial growth in enriched Middlebrook 7H9 liquid medium, using BACTEC MGIT960 automated system registration in the presence of different concentrations of compounds. The resistance of MS-115 was confirmed in the experiments in which this strain was cultured in media containing rifampicin (1 mg/mL) and isoniazid (0.1 mg/mL). We found that its growth was similar to that in the media without these compounds. Levofloxacin (1.5 mg/mL) was used as a positive control.

We found that compounds 1–3 and 5 exhibit anti-mycobacterial activity in the in vitro system. The minimal concentrations at which inhibition of bacterial growth was not less than 99% for both strains (MIC (99)) were determined (Table 2).

Table 2.

MIC (99) values for the compounds tested against M. tuberculosis strains

Compounds MIC (99), μg/mL
M.tuberculosis H37Rv M.tuberculosis MS-115
1 50 (67μM) 50 (67μM)
2 10 (13.5μM) 20 (27μM)
3 20 (19.3μM) 10 (9.7 μM)
5 >50 (75μM) 50 (75μM)
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Thus, the anti- M. tuberculosis activity was highest for compounds 2 and 3. For these compounds, the MIC (99) values against the sensitive laboratory strain of M. tuberculosis, H37Rv, were 10 μg/mL and 20 μg/mL, respectively, while those for the clinical MDR strain of M. tuberculosis, MS-115, were equal to 20 μg/mL and 10 μg/mL, respectively.

3.5. Heterodimers were not toxic for isolated cells or for tissue cultures

First, we tested the potential cell toxicity of our newly-synthetized heterodimers in human tissue ex vivo, which remains the model of choice to evaluate the potency and cytotoxicity of newly synthesized compounds [14] (Figure 2). Here, we tested the toxicity of each compound at two concentrations, 10 and 50 μM, with each experimental condition performed with 27 tissue blocks from each donor. After 12 days of culture, we isolated cells and assessed the viability of various lymphocyte subsets using flow cytometry. We evaluated the depletion of B cells (CD3−) and of various sets of T cells (CD3+): CD3+CD4+, CD3+CD8+, CD45RA+CCR7+ (naïve), CD45RA−CCR7+ (central memory, TCM), CD45RA−CCR7− (effector memory, TEM) and different state of their activation (CD25+, CD38+, CD95+, HLA-DR+). We assessed the cytotoxicity of each heterodimer by comparing the numbers of cells of each of the above-listed subsets in treated tissue blocks with those in donor-matched untreated tissue blocks. To account for size differences between tissue blocks, data were normalized by the tissue weight (Figure 2).

Figure 2. Cell toxicity of heterodimer-inoculated ex vivo-cultured human lymphoid tissues.

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

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Tissue blocks (27 from each individual human tonsil donor) were treated with heterodimers 1–5 at 10 and 50 μM. At day 12, cells were stained with anti–CD3-QD655, anti–CD4-QD605, anti–CD8-QD800, anti–CD45RA-FITC, and anti–CCR7-PE-Cy7 (stain 1) or stained with anti–CD3-QD655, anti–CD4-QD605, anti–CD8-QD800, CD25-APC, CD38-PE, CD95-PE-Cy7, and HLA-DR-BV570 (stain 2). We evaluated the cytopathicity of the heterodimers for different cell subsets by comparing the numbers of cells in treated tissue blocks with those in matched untreated control tissue blocks. To account for size differences in tissue blocks, we normalized the data by the weight of the tissues. Presented are means ± SEM (error bars) of six independent experiments performed with tissues from six different donors.

On day 12 of culture, the numbers of lymphocytes in the CD3−, CD3+, CD3+CD4+, CD3+CD8+, Tnaïve, TCM, TEM, and activated T cell subsets were not statistically different (P>0.05) in untreated tissues compared with donor-matched tissues treated with heterodimers 1–5 at a concentration of 10 μM. In contrast, when heterodimers were used at a concentration of 50 μM, mild cell depletion was observed (10–20% cell loss in treated versus untreated conditions). No particular heterodimer was more cytotoxic than others.

Next, heterodimers were used in the MT-4 cell culture, allowing the evaluation of both their cytotoxic and cytostatic effects. For each compound, the CC50 (concentration that reduced the viability of MT-4 by 50%) and the IC50 (concentration that inhibited MT-4 cell growth by 50%) were evaluated. As with what was observed in tissues, no significant cytotoxicity was observed for any of the five newly developed heterodimers (the CC50s ranged from 74 to >300 μM) (Table 3). Although heterodimers were not toxic, some of them slowed down cell growth, with IC50s ranging from 18 to 150 μM). Heterodimer 3 was the least toxic and the least cytostatic, with both CC50>300 μM and IC50 = 150 μM.

Table 3.

Evaluation of cell toxicity in MT-4 cell cultures

MT-4 MT-4
CC50 95% confidence interval IC50 (μM) 95% confidence interval
1 89.87 43.63–185.1 29.19 22.18–38.41
2 112.6 59.32–213.7 33.19 20.48–53.80
3 >300 472.1 very wide
4 53.62 30.32–94.82 14.25 12.67–160.2
5 46.96 39.59–55.69 17.5 15.2–20.14
AZT > 100 115.5 69.83–191.1
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CC50s (concentrations that reduce the viability of the MT-4 by 50%) and IC50s (concentrations that inhibit MT-4 or CEM cell growth by 50%)

4. Discussion

TB remains a major cause of morbidity and mortality globally, with an estimated ten million people developing the disease in 2015 [1]. Although the great majority of individuals infected with M. tuberculosis do not develop active TB [17], this scenario is distinct for individuals who are coinfected with HIV [3]. HIV coinfection is the most important risk factor for developing active TB for at least two reasons: (i) HIV coinfection increases the susceptibility to primary infection or reinfection with M. tuberculosis and (ii) it also increases the risk of TB reactivation for patients with latent TB. The risk of TB reactivation rises as the CD4 T cell count declines [18–20].

Reciprocally, M. tuberculosis infection has a negative impact on the immune response to HIV, accelerating the progression from HIV infection to AIDS [21–23] (reviewed in [3, 17]). This explains, in part, why TB remains the leading cause of death among people living with HIV. The effect of TB on HIV disease progression is linked to increased immune activation [24] (reviewed in [3, 17]) and to increased expression of the CCR5 and CXCR4 coreceptors on CD4 T cells [25].

Therefore, in HIV/TB-coinfected individuals, M. tuberculosis and HIV potentiate one another by accelerating the deterioration of immunity. Without treatment, HIV and TB considerably shorten lifespan. Paradoxically, initiation of antiretroviral therapy (ART) to treat HIV infection in HIV/TB-coinfected individuals may also result in a clinical deterioration known as IRIS (immune reconstitution syndrome) [26]. TB-IRIS, which happens against the background of increased CD4 T-cell count and suppression of HIV-1 RNA level in persons receiving ART, is estimated to occur in 11 to 45% of patients coinfected with TB and HIV. Moreover, despite the consistent decrease in TB risk among persons who receive ART, this risk remains higher than that in HIV-uninfected persons, even after several years of treatment, suggesting that immune restoration is not complete (reviewed in [26]).

The immense burden placed by TB and HIV on coinfected individuals and on health care systems in general necessitates a search for new active compounds. Here, we report on the designing of dual-targeted compounds, hybrid molecules (heterodimers) made of two compounds respectively active against HIV and TB. Such a strategy to design heterodimer compounds was already implemented in the late 1990s by Imbach and his associates, who synthesized the first bis-nucleoside phosphates [4]. However, those heterodimers were relatively resistant to enzymatic hydrolysis by phosphodiesterases and thus too stable to release the active compounds. Other compounds combining two different anti-HIV agents were later synthesized [5–8]. Recently, we reported on the design and synthesis of 3TC-AZT phosphonate, which potently inhibited HIV replication in human tissue ex vivo [9].

Here, we synthesized five dual-targeted HIV/TB heterodimer compounds. Four of them (1–3, 5) inhibit both HIV and TB because these molecules are dimers of the anti-HIV agent AZT and, as the anti-TB agent, either 2′-deoxyuridine analogue (1–3) or 5′-norcarbocyclic uracil derivative (5). Compound (4) is a heterodimer of an anti-TB and two anti-HIV reverse transcriptase inhibitors of different types. Using Mycobacteria growth indicator tubes, we showed that the newly-synthetized compounds expressed anti-TB activity (compounds 1–3, 5).

The anti-HIV activity was assessed in two different models: MT-4 cell cultures and human tissues ex vivo. MT-4 cell cultures were used because of their typical high level of HIV replication. Human tissue blocks ex vivo (histocultures) were used because they uniquely reflect many important functional aspects of cell–cell interactions in vivo. The system of human lymphoid tissue ex vivo was used earlier for preclinical evaluation of microbicides [27, 28].

Using these two systems, we showed that all the tested compounds inhibited HIV replication. In particular, we found that compounds 1–3 were generally more potent that compounds 4–5. Since AZT was the anti-HIV agent in all five heterodimers, this suggests that the breakage of the heterodimers into active molecules was dependent on the nature of the linker as well as on the position of the connection between two components of the hybrid. Thus, the activity of the heterodimers seemed to be directly determined by the hydrolytic stability in this system. Heterodimer 3 was the compound with the most potent anti-HIV activity altogether because of the two AZT molecules included. Compounds 4 and 5 were less active than 1–3 in the MT-4 cell system, but in the tissue system the difference between activity levels was not so dramatic. It can be assumed that there are additional mechanisms for the hydrolysis of heterodimers 4–5 in lymphoid tissue.

Drug resistance is a major issue in the treatment of TB. Multidrug-resistant TB (MDR TB) is resistant to at least two of the best anti-TB drugs, isoniazid and rifampin. Commonly, HIV patients with drug-resistant TB have a reduced survival rate compared with those with drug-susceptible TB [29, 30]. Therefore, it is of critical importance that our dual-targeted compounds active against HIV were active not only against a sensitive TB strain but also against a clinical MDR strain, TB MS-115, that is resistant to the five first-line anti-TB drugs isoniazid, rifampicin, streptomycin, ethambutol, and pyrazinamide.

The concurrent treatment of TB and HIV is generally associated with a higher risk of adverse reactions than treatment of either infection alone, because of overlapping drug toxicities and drug–drug interactions between antiretroviral therapy and anti-TB therapy [26, 31]. Therefore, an efficient dual-targeted drug has obvious advantages. Nevertheless, we carefully addressed the potential toxicity of our dual-targeted compounds. We addressed this issue in human tissues ex vivo first, as this model does not require exogenous activation, retains the natural cytoarchitecture of tissue, and contains all cell types found in tissue in vivo, thus making this model an adequate tool to approximate cell toxicity in vivo [14]. To exclude potential depletion of even a small but important subset of cells, we addressed cytotoxity in 12 different cell subsets, and in none of them did we find cell loss at concentrations of compounds that suppressed both HIV and TB. Even when the compounds were used at 50 μM (i.e., between 10 and 350 times the EC50 depending on the compound), mild cell loss was only noticed for some heterodimers and for some selected subsets. The lack of general toxicity of the tested heterodimers was confirmed in MT-4 cell cultures. In these cultures, we also addressed the cytostaticity of the heterodimers. We found that none of the compounds at concentrations that suppress HIV replication arrested cell growth.

In conclusion, we successfully designed and synthesized five dual-targeted compounds that inhibit HIV and TB in vitro and ex vivo. The compounds described here are the first examples of nucleoside derivatives possessing both anti-HIV and anti-tuberculosis activities. Heterodimers 1–5 are depot drugs comprising two active components and having different rates of release: they are either rapidly (1–3) or slowly (4–5) hydrolyzable compounds. To optimize the combination of the active components and their binding linker, further research is needed, in particular the evaluation of the pharmacokinetic parameters of anti-HIV and anti-TB agents released in the blood of laboratory animals. We found that one particular heterodimer, compound 3, was the most potent in inhibiting HIV and the drug-resistant TB strain MS -115. This compound 3 was also not toxic and had no cytopathic effect on cell cultures.

Supplementary Material

supplement

Figure 1. Scheme of synthesis of heterodimers.

Figure 1

Figure 1

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A. Scheme 1: synthesis of heterodimers of AZT and 5-(4-decyl-1,2,3-triazol-1-yl-methyl)-2′-deoxyuridine.

B. Scheme 2: Synthesis of heterodimers of AZT and the 5′-modified norcarbocyclic analogues of 2 ′, 3′-dideoxy-2′, 3′-didehydrouridine.

Figure 3.

Figure 3

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Highlights.

  • We synthetized five heterodimers of an anti-TB molecule and an anti-HIV molecule.

  • These compounds inhibited growth of M. tuberculosis and replication of HIV in a cell line and in human lymphoid tissue.

  • One compound that combined the strongest anti-bacterial and anti-viral effects was neither cytopathic nor cytostatic.

  • This compound is a leading candidate for future development and in vivo testing.

Acknowledgments

The work of CV, SZ, and LM was supported by the NIH office of AIDS Research – Intramural-to-Russian (I-to-R) Program and the NICHD Intramural Program. The synthetic work of LA and EM was supported by the joint NIH–RFBR grant NIZ_aNo13-04-91441. The work of AK and SK, as well as all physico-chemical analysis, was supported by Russian Science Foundation project No14-50-00060. We thank the entire staff of the Department of Pathology of Children’s National Medical Center for their generous assistance in obtaining human tonsillar tissues.

Footnotes

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References

  • 1.Organization WH. Global tuberculosis report 2016. Geneva, Switzerland: World Health Organization; 2016. [Google Scholar]
  • 2.Suchindran S, Brouwer ES, Van Rie A. Is HIV infection a risk factor for multi-drug resistant tuberculosis? A systematic review. PLoS One. 2009;4:e5561. doi: 10.1371/journal.pone.0005561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Pawlowski A, Jansson M, Skold M, Rottenberg ME, Kallenius G. Tuberculosis and HIV co-infection. PLoS Pathog. 2012;8:e1002464. doi: 10.1371/journal.ppat.1002464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Puech F, Gosselin G, Balzarini J, Good SS, Rideout JL, De Clercq E, et al. Synthesis and biological evaluation of dinucleoside methylphosphonates of 3′-azido-3′-deoxythymidine and 2′, 3′-dideoxycytidine. Antiviral Res. 1990;14:11–23. doi: 10.1016/0166-3542(90)90062-c. [DOI] [PubMed] [Google Scholar]
  • 5.Velazquez S, Alvarez R, San-Felix A, Jimeno ML, De Clercq E, Balzarini J, et al. Synthesis and anti-HIV activity of [AZT]-[TSAO-T] and [AZT]-[HEPT] dimers as potential multifunctional inhibitors of HIV-1 reverse transcriptase. J Med Chem. 1995;38:1641–1649. doi: 10.1021/jm00010a008. [DOI] [PubMed] [Google Scholar]
  • 6.Velazquez S, Tunon V, Jimeno ML, Chamorro C, De Clercq E, Balzarini J, et al. Potential multifunctional inhibitors of HIV-1 reverse transcriptase. Novel [AZT]-[TSAO-T] and [d4T]-[TSAO-T] heterodimers modified in the linker and in the dideoxynucleoside region. J Med Chem. 1999;42:5188–5196. doi: 10.1021/jm991092+. [DOI] [PubMed] [Google Scholar]
  • 7.Pontikis R, Dolle V, Guillaumel J, Dechaux E, Note R, Nguyen CH, et al. Synthesis and evaluation of “AZT-HEPT”, “AZT-pyridinone”, and “ddC-HEPT” conjugates as inhibitors of HIV reverse transcriptase. J Med Chem. 2000;43:1927–1939. doi: 10.1021/jm991125l. [DOI] [PubMed] [Google Scholar]
  • 8.Laduree D, Sugeac E, Fossey C, Schmidt S, Laumond G, Aubertin AM. Synthesis of certain heterodimers expected as HIV-1 reverse transcriptase inhibitors. Nucleosides Nucleotides Nucleic Acids. 2003;22:873–875. doi: 10.1081/NCN-120022675. [DOI] [PubMed] [Google Scholar]
  • 9.Vanpouille C, Khandazhinskaya A, Karpenko I, Zicari S, Barreto-de-Souza V, Frolova S, et al. A new antiviral: chimeric 3TC-AZT phosphonate efficiently inhibits HIV-1 in human tissues ex vivo. Antiviral Res. 2014;109:125–131. doi: 10.1016/j.antiviral.2014.06.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Matyugina E, Novikov M, Babkov D, Ozerov A, Chernousova L, Andreevskaya S, et al. 5-Arylaminouracil Derivatives: New Inhibitors of Mycobacterium tuberculosis. Chem Biol Drug Des. 2015;86:1387–1396. doi: 10.1111/cbdd.12603. [DOI] [PubMed] [Google Scholar]
  • 11.Shmalenyuk ER, Chernousova LN, Karpenko IL, Kochetkov SN, Smirnova TG, Andreevskaya SN, et al. Inhibition of Mycobacterium tuberculosis strains H37Rv and MDR MS-115 by a new set of C5 modified pyrimidine nucleosides. Bioorg Med Chem. 2013;21:4874–4884. doi: 10.1016/j.bmc.2013.07.003. [DOI] [PubMed] [Google Scholar]
  • 12.Shirokova EA, Jasko MV, Khandazhinskaya AL, Ivanov AV, Yanvarev DV, Skoblov YS, et al. Uncharged AZT and D4T derivatives of phosphonoformic and phosphonoacetic acids as anti-HIV pronucleosides. J Med Chem. 2004;47:3606–3614. doi: 10.1021/jm0310176. [DOI] [PubMed] [Google Scholar]
  • 13.Khandazhinskaya AL, Yanvarev DV, Jasko MV, Shipitsin AV, Khalizev VA, Shram SI, et al. 5′-aminocarbonyl phosphonates as new zidovudine depot forms: antiviral properties, intracellular transformations, and pharmacokinetic parameters. Drug Metab Dispos. 2009;37:494–501. doi: 10.1124/dmd.108.022269. [DOI] [PubMed] [Google Scholar]
  • 14.Grivel JC, Margolis L. Use of human tissue explants to study human infectious agents. Nat Protoc. 2009;4:256–269. doi: 10.1038/nprot.2008.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Biancotto A, Brichacek B, Chen SS, Fitzgerald W, Lisco A, Vanpouille C, et al. A highly sensitive and dynamic immunofluorescent cytometric bead assay for the detection of HIV-1 p24. J Virol Methods. 2009;157:98–101. doi: 10.1016/j.jviromet.2008.11.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Matyugina ES, Andreevskaya SN, Smirnova TG, Khandazhinskaya AL. Carbocyclic analogues of inosine-5′-monophosphate: synthesis and biological activity. Acta Naturae. 2012;4:73–77. [PMC free article] [PubMed] [Google Scholar]
  • 17.Bruchfeld J, Correia-Neves M, Kallenius G. Tuberculosis and HIV Coinfection. Cold Spring Harb Perspect Med. 2015;5:a017871. doi: 10.1101/cshperspect.a017871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Moreno S, Baraia-Etxaburu J, Bouza E, Parras F, Perez-Tascon M, Miralles P, et al. Risk for developing tuberculosis among anergic patients infected with HIV. Ann Intern Med. 1993;119:194–198. doi: 10.7326/0003-4819-119-3-199308010-00003. [DOI] [PubMed] [Google Scholar]
  • 19.Antonucci G, Girardi E, Raviglione MC, Ippolito G. Risk factors for tuberculosis in HIV-infected persons. A prospective cohort study. The Gruppo Italiano di Studio Tubercolosi e AIDS (GISTA) JAMA. 1995;274:143–148. doi: 10.1001/jama.274.2.143. [DOI] [PubMed] [Google Scholar]
  • 20.Holmes CB, Wood R, Badri M, Zilber S, Wang B, Maartens G, et al. CD4 decline and incidence of opportunistic infections in Cape Town, South Africa: implications for prophylaxis and treatment. J Acquir Immune Defic Syndr. 2006;42:464–469. doi: 10.1097/01.qai.0000225729.79610.b7. [DOI] [PubMed] [Google Scholar]
  • 21.Whalen C, Horsburgh CR, Hom D, Lahart C, Simberkoff M, Ellner J. Accelerated course of human immunodeficiency virus infection after tuberculosis. Am J Respir Crit Care Med. 1995;151:129–135. doi: 10.1164/ajrccm.151.1.7812542. [DOI] [PubMed] [Google Scholar]
  • 22.Badri M, Ehrlich R, Wood R, Maartens G. Initiating co-trimoxazole prophylaxis in HIV-infected patients in Africa: an evaluation of the provisional WHO/UNAIDS recommendations. AIDS. 2001;15:1143–1148. doi: 10.1097/00002030-200106150-00009. [DOI] [PubMed] [Google Scholar]
  • 23.Lopez-Gatell H, Cole SR, Hessol NA, French AL, Greenblatt RM, Landesman S, et al. Effect of tuberculosis on the survival of women infected with human immunodeficiency virus. Am J Epidemiol. 2007;165:1134–1142. doi: 10.1093/aje/kwk116. [DOI] [PubMed] [Google Scholar]
  • 24.Vanham G, Edmonds K, Qing L, Hom D, Toossi Z, Jones B, et al. Generalized immune activation in pulmonary tuberculosis: co-activation with HIV infection. Clin Exp Immunol. 1996;103:30–34. doi: 10.1046/j.1365-2249.1996.907600.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Wolday D, Tegbaru B, Kassu A, Messele T, Coutinho R, van Baarle D, et al. Expression of chemokine receptors CCR5 and CXCR4 on CD4+ T cells and plasma chemokine levels during treatment of active tuberculosis in HIV-1-coinfected patients. J Acquir Immune Defic Syndr. 2005;39:265–271. doi: 10.1097/01.qai.0000163027.47147.2e. [DOI] [PubMed] [Google Scholar]
  • 26.Piggott DA, Karakousis PC. Timing of antiretroviral therapy for HIV in the setting of TB treatment. Clin Dev Immunol. 2011;2011:103917. doi: 10.1155/2011/103917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Lisco A, Vanpouille C, Tchesnokov EP, Grivel JC, Biancotto A, Brichacek B, et al. Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues. Cell Host Microbe. 2008;4:260–270. doi: 10.1016/j.chom.2008.07.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Andrei G, Lisco A, Vanpouille C, Introini A, Balestra E, van den Oord J, et al. Topical tenofovir, a microbicide effective against HIV, inhibits herpes simplex virus-2 replication. Cell Host Microbe. 2011;10:379–389. doi: 10.1016/j.chom.2011.08.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Chan ED, Iseman MD. Multidrug-resistant and extensively drug-resistant tuberculosis: a review. Curr Opin Infect Dis. 2008;21:587–595. doi: 10.1097/QCO.0b013e328319bce6. [DOI] [PubMed] [Google Scholar]
  • 30.Gunther G, Gomez GB, Lange C, Rupert S, van Leth F, Tbnet Availability, price and affordability of anti-tuberculosis drugs in Europe: a TBNET survey. Eur Respir J. 2015;45:1081–1088. doi: 10.1183/09031936.00124614. [DOI] [PubMed] [Google Scholar]
  • 31.Sterling TR, Pham PA, Chaisson RE. HIV infection-related tuberculosis: clinical manifestations and treatment. Clin Infect Dis. 2010;50(Suppl 3):S223–230. doi: 10.1086/651495. [DOI] [PubMed] [Google Scholar]

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