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Effect of HIV protease inhibitors on New World Leishmania
Parasitology International 61 (2012) 538–544
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Parasitology International journal homepage: www.elsevier.com/locate/parint
Effect of HIV protease inhibitors on New World Leishmania Izabel G. Demarchi a, Thaís G.V. Silveira a, Izabel C.P. Ferreira b, Maria V.C. Lonardoni a,⁎ a b
Departamento de Análises Clínicas e Biomedicina, Universidade Estadual de Maringá, PR, Brazil Departamento de Farmácia e Farmacologia, Universidade Estadual de Maringá, PR, Brazil
a r t i c l e
i n f o
Article history: Received 13 December 2011 Received in revised form 23 April 2012 Accepted 25 April 2012 Available online 2 May 2012 Keywords: Antiretroviral HAART Cutaneous leishmaniasis Leishmania HIV protease inhibitors
a b s t r a c t The incidence of HIV/Leishmania co-infection decreases after antiretroviral drug therapy; therefore, the in vitro and in vivo activity of three antiretroviral drugs against Leishmania (Viannia) braziliensis and L. (L.) amazonensis was evaluated. Different concentrations of indinavir (IDV), atazanavir (ATV), and ritonavir (RTV) were added to promastigote cultures, and the 50% inhibitory concentration (IC50) was determined. IDV and RTV were also evaluated against intracellular amastigotes, and the Infection Index determined. BALB/c mice, infected with L. (L.) amazonensis in the left footpad, were treated orally with IDV and RTV for 30 days, and monitored by measuring the footpad thickness and parasite load of regional lymph nodes and spleen. For promastigotes, IDV exhibited an IC50 value of 100 μM against L.(L.) amazonensis. The RTV IC50 for L. (L.) amazonensis and L. (V.) braziliensis were 40 and 2.3 μM, respectively, and the ATV IC50 for L. (V.) braziliensis was 266 μM. For intracellular amastigotes, IDV (25, 50, and 100 μM) significantly decreased the Infection Index of L. (L.) amazonensis (56.8%, 47.9%, and 65.0%) and L. (V.) braziliensis (37.8%, 48.7%, and 43.2%). RTV (12.5, 25, and 50 μM) decreased the infection index of L. (L.) amazonensis by 26.3%, 42.4%, and 44.0%, and that of L. (V.) braziliensis by 27.6%, 37.3%, and 39.2%. Antiretroviral-treated mice had a significant reduction in footpad thickness after the third week of IDV and after the fifth week of RTV treatment. However, there was no reduction in parasite load. These results suggest that IDV and RTV have anti-Leishmania activity, but only in higher concentrations. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The World Health Organization estimates that 33.3 million of the world's people are infected by human immunodeficiency virus (HIV), and each year 2.6 million people are newly infected [1]. At least one third of virus carriers live in endemic areas for leishmaniases [2]. Leishmaniases affect around 350 million people in risk areas, most of them in developing countries [3]. Cutaneous leishmaniasis (CL) is one of the most common diseases affecting humans in developing countries, and manifests itself through cutaneous involvement. The main etiological agents of American cutaneous leishmaniasis(ACL) in Brazil are Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis; ACL manifests itself through cutaneous, mucosal, or disseminated types [3–5]. Leishmaniases cause serious diseases in HIV carriers, and treatment is difficult and often ineffective. New therapeutic strategies are urgently required [6]. Amphotericin B is a well-known antileishmanial drug and has been used when ACL treatment with pentavalent antimonies fails [4]. Lipid formulations of amphotericin B (AMB) have been employed
⁎ Corresponding author at: Av Colombo, 5790, CEP 87020-900, Maringá, Paraná, Brazil. Tel.: +55 44 30114878; fax: +55 44 30114805. E-mail addresses: [email protected] (I.G. Demarchi), [email protected] (T.GV. Silveira), [email protected] (I.CP. Ferreira), [email protected] (M.VC. Lonardoni). 1383-5769/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2012.04.006
successfully in the treatment of the cutaneous and mucosal forms of ACL, both in immune-competent patients and in those with any type of immunosuppression, including HIV-infected people [7–9]. Leishmaniases may modify disease progression by HIV, and HIVcaused immunodepression facilitates the dissemination of Leishmania [4]. Both HIV and Leishmania multiply in macrophages, and the cellular immune response becomes crucial in infection control [10]. The introduction of highly active antiretroviral therapy (HAART) has resulted in a decrease in opportunistic diseases, including the incidence of HIV/ Leishmania co-infection [11]. Protease inhibitors employed in the treatment of acquired immunodeficiency syndrome (AIDS) affect the aspartic protease enzyme of HIV. It is believed that, in addition to blocking virus multiplication, this pathway may also affect the growth of other microorganisms [12,13], such as Leishmania sp. [14–16], Trypanosoma cruzi [17], Toxoplasma gondii [18,19], Plasmodium spp. [20–22], Giardia duodenalis and Trichomonas vaginalis [23], Crypstosporidium parvum [24], Candida albicans, and Pneumocystiscarinii [11]. However, their biochemical target is unknown. Proteasomes are essential for the multiplication, differentiation, and intracellular survival of Leishmania parasites [25–31]. Recent in vitro studies with Old World Leishmania showed that proteasomes of promastigote and amastigote forms may be the targets of antiretroviral protease inhibitors (HIVPrIs) [16,32–34]. Some studies have reported aspartic proteinase activity in extracts from these parasites
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[31,33,34]. Santos et al. [35] observed that HIV aspartyl peptidase inhibitors impaired the growth of Leishmania amazonensis, influenced the parasite ultrastructure, and decreased the activity of aspartyl peptidases. White et al. [16] showed that HIVPrIs affect Ddi1 protein, the only member of the aspartic proteinase family that is present in Leishmania major, and suggested that the proteasomes are a potential target for the HIVPrIs in these parasites. Few studies on the activity of antiretrovirals in New World Leishmania have investigated the effect on L. (L.) amazonensis [35,36]. The effects of these drugs on the course of infection caused by L. (V.) braziliensis are not known. In this study, we investigated the efficacy of three HIVPrIs (ritonavir, indinavir and atazanavir) on the multiplication of promastigotes; the efficacy of ritonavir and indinavir on intracellular amastigotes of L. (V.) braziliensis and L. (L.) amazonensis, and on an experimental infection by L. (L.) amazonensis in BALB/c mice. 2. Materials and methods 2.1. Chemicals Indinavir (Merck & Co., Inc., Rahway, NJ, USA) and ritonavir (Abbott Laboratories, North Chicago, IL, USA) were donated by the manufacturers. Amphotericin B (Anforicin, Cristalia Laboratory, São Paulo, SP, Brazil) and atazanavir (Reyataz, Bristol-Myers Squibb, Princeton, NJ, USA) were donated by the Paraná State Secretariat of Health.
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drugs and DMSO. The results were evaluated by linear regression of the inhibition percentage [38]. 2.4. Macrophage viability assay Four days after intraperitoneal inoculation of 1 ml of thioglycolate broth, macrophages from the peritoneal cavity of BALB/c mice were obtained according to Lonardoni et al. [39]. Briefly, the peritoneal cavity was washed with 10 to 12 ml sterile RPMI 1640 (Invitrogen) and cell suspensions were adjusted to 1 × 10 6 macrophages/ml. Next, 500 μl was distributed on 13 mm-diameter sterile glass coverslips (Glastecnica, São Paulo, SP, Brazil), placed in 24-well culture plates (TPP, Switzerland). The plates were incubated for 1 h at 25 °C, and non-adherent cells were removed by three sterile PBS washings. The adherent cells were incubated in RPMI 1640 supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 μg/ml). The cultures were treated with different concentrations of indinavir (25, 50, and 100 μM), ritonavir (12.5, 25, and 50 μM), or amphotericin B (0.025, 0.05, and 0.1 μM) diluted in DMSO. The final concentration of DMSO did not exceed 0.005%, and no cytotoxic effect on the macrophages was observed. Cultures not treated with drugs or the diluent were used as control. The plates were maintained at 37 °C in a humid atmosphere containing 5% CO2 for 24 h, and then the coverslips were stained with 1% Trypan Blue (Sigma-Aldrich) and examined microscopically. All tests were done in triplicate, and the results are expressed as percentage viability.
2.2. Culture and maintenance of Leishmania L. (V.) braziliensis (MHOM/BR/1987/M11272) isolated from a patient from northwestern Paraná and identified at the Instituto Evandro Chagas (Belém, PA, Brazil) and L. (L.) amazonensis (MHOM/BR/1977/ LTB0016) were maintained by inoculation of 1 × 107 parasites in the left footpad of hamsters aged between 30 and 40 days. For isolation of the parasites, the hamsters were euthanized with ketamine (Agner União, Embu-Guaçu, SP, Brazil) and xylazine (Coopazine ®, Intervet Schering-Plough, Cotia, SP, Brazil). The sections of the lymph nodes were inoculated in culture medium 199 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal calf serum (Invitrogen), 1% human urine, 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and antibiotics (100 UI/ml penicillin and 100 μg/ml streptomycin, both obtained from Sigma-Aldrich), and incubated at 25 °C. The parasites were maintained by weekly transfers in supplemented medium 199. 2.3. Activity of HIV protease inhibitors against Leishmania spp. promastigote forms Antiretroviral activity against Leishmania was evaluated according to Ferreira et al. [37]. Briefly, promastigotes of L. (L.) amazonensis and L. (V.) braziliensis were cultured in Schneider's insect medium (Sigma-Aldrich), pH 7.2, supplemented with 10% FCS (v/v) and 2 mM L-glutamine until they reached the logarithmic growth phase. The drugs were dissolved in dimethylsulfoxide (1.6% v/v DMSO in the first well; obtained from Sigma-Aldrich) and diluted in series in a ratio of two, starting from ½, on a cell culture plate with 96 wells (TPP® test plate, Switzerland). The HIVPrIs (indinavir, ritonavir and atazanavir) were tested in concentrations from 0.75 to 400 μM, and amphotericin B from 0.01 to 17 μM (positive controls). The concentration of DMSO used had no effect on the parasites. Next, 100 μl of the suspension containing 4 × 10 6 parasites/ml was distributed in each well of the culture plate. After 24 h at 25 °C, an aliquot of each well was added to a solution containing 10% eosin and 2% formalin, and the parasites were counted in a Neubauer chamber. All tests were performed in triplicate. The inhibitory concentration that caused a 50% decrease in survival (IC50) of promastigotes was calculated. Values were compared to those obtained in control cultures without
2.5. Effect of HIV protease-inhibiting drugs on amastigote forms of Leishmania spp Establishing and plating of macrophages were performed as described in the previous section. Macrophages were infected by promastigote forms in the proportion of six parasites for each macrophage. The cultures were incubated for 6 h at 37 °C in an atmosphere of 5% CO2. The supernatant was then removed. The drugs dissolved in DMSO and diluted in supplemented RPMI 1640 medium were added to cultures at concentrations of 25, 50, and 100 μM (indinavir), 12.5, 25, and 50 μM (ritonavir), and 0.025, 0.05, and 0.1 μM (Amphotericin B). After 24 h at 37 °C in a 5% CO2 atmosphere, the coverslips were removed, washed with PBS, stained with Fast Panoptic reagent (Laborclin, Pinhais, PR, Brazil) and analyzed under an optical microscope. At least 200 cells were counted to calculate the Infection Index, which was determined from the percentage of infected macrophages multiplied by the mean number of parasites per macrophage. 2.6. Effects of HIV protease-inhibiting drugs in mice experimentally infected by L. (L.) amazonensis BALB/c mice have been used extensively to study the immune response to and the effect of drugs on species of Leishmania. Strains of L. braziliensis differ in causing cutaneous lesions of BALB/c mice [40,41] and it is difficult to use a mouse model to evaluate the effect of drugs on L. braziliensis. The strain of L. (V.) braziliensis tested in this study, isolated from a patient, did not establish an infection or lesions in mice, and did not develop evenly and progressively in hamsters. Since a suitable experimental model for this strain of L. (V.) braziliensis is not yet established, the experiments to evaluate the HIVPrIs effect in vivo were performed only with L. (L.) amazonensis as previously described [42]. Briefly, female BALB/c mice aged 9–10 weeks were infected with L. (L.) amazonensis 5 × 105promastigotes/40 μl PBS in the left footpad, and 40 μl PBS was inoculated in the right footpad. 30 days post-infection, the mice were divided into groups and treated by oral gavage for 30 consecutive days. Group A was treated with indinavir (8 mg/kg/day), and group B with ritonavir (2 mg/kg/day). The doses were used according to the information provided by the manufacturers. The controls were composed of animals (group C) treated with
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amphotericin B (5 mg/kg/day), a drug used to treat cases of HIV/ Leishmania co-infection [4]; and animals that received no treatment (group D). Another group of animals that were not infected and were not treated (group E) was followed. After infection, the vertical thickness of the footpad of each animal was measured weekly, with a pachymeter (Vernier Calipers, Japan). The results were given as the difference between the measurements of the infected and the uninfected footpad, in millimeters (mm). Mice were weighed weekly. The load of Leishmania was evaluated according to methodology by Buffet et al. [43], with modifications. Animals were euthanized with ketamine and xylazine, and the popliteal lymph nodes draining the infected footpad and the spleen were aseptically removed and weighed. Each organ was macerated in supplemented medium 199. Next, 225 μl of supplemented medium 199 was distributed in 96well culture plates, and 75 μl of each suspension, in duplicate, was added to the first well. Suspensions were diluted in series in a ratio of 4, starting from ¼. After 5 days at 25 °C, the cultures were observed in an inverted light microscope. Each well was analyzed for promastigote forms, and was scored as positive when at least one parasite was found. The plates were kept for another 7 days and then re-examined. The parasite load (number of parasites/gram of tissue) was calculated as follows: the geometric mean of the reciprocal of the positive dilution from each duplicate was divided by the weight of the organ. The value obtained was multiplied by the reciprocal of the fraction of the homogenized organ added to the first well. 2.7. Ethical aspects This investigation was approved by the Ethics Committee on the use of Experimental Animals of the State University of Maringá (warrant no. 056/2007).
Table 1 Effect of HIV protease-inhibiting drugs on the in vitro growth of promastigote forms of L. (L.) amazonensis and L. (V.) braziliensis after 24 h incubation. Drugs
Indinavir Ritonavir Atazanavir Amphotericin B
L. (L.) amazonensis
L. (V). braziliensis
IC50 (μM)
S.D.
IC50 (μM)
S.D.
100 40 266 b0
0.3 1.9 1.5 0
400 2.3 400 b0
0 0.1 0 0
(IC50: 50% inhibitory concentration; μM: micromolar; S.D.: standard deviation of the mean).
At the lowest concentration, the results showed that treatment with indinavir (25 μM) decreased the Infection Index of macrophage infection of L. (V.) braziliensis by 37.8%, and ritonavir (12.5 μM) by 27.6% (p b 0.05). Amphotericin B reduced the Infection Index by 79.2% (0.025 μM) (Fig. 1B).
3.2. Activity in vivo of HIV protease inhibitors The in vivo effect of antiretroviral drugs was evaluated in the experimental infection of BALB/c mice by L. (L.) amazonensis. Antiretroviral drugs were administered orally for 1 month, and the animals were then followed for 10 weeks. The lesion thickness of mice treated with indinavir (8 mg/kg/day) and amphotericin B (5 mg/kg/day) decreased from the third week post-treatment when compared with the untreated control group (p b 0.05). Animals treated with ritonavir (2 mg/kg/day) showed a regression of infection only after the fifth week (p b 0.05) (Fig. 2). Animals treated with an antiretroviral or
2.8. Statistical analyses All results were first analyzed by the Shapiro–Wilk test for normality. Results with a normal distribution were analyzed by Student's t test, and the others were analyzed by the Mann–Whitney test. The software Statistic 6.0 was used, and differences were considered significant at the 5% level. 3. Results 3.1. In vitro antileishmanial activity Both indinavir and ritonavir showed an antiproliferative effect on Leishmania sp. promastigotes, to different degrees. Ritonavir inhibited the growth of promastigotes of both Leishmania species studied. Indinavir showed an inhibitory effect only against L. (L.) amazonensis, in which the IC50 of 100 μM was 2.5 times higher than the IC50 of ritonavir, and 100 times higher than that of amphotericin B. Atazanavir showed a very high IC50, and because of this, only indinavir and ritonavir were assessed for their antiproliferative activity on intracellular amastigote forms of Leishmania spp. As expected, the reference drug amphotericin B inhibited the proliferation of both Leishmania species (Table 1). The drug concentrations tested showed no effect on the viability of the macrophages. In higher concentrations, the percentage of viable cells for amphotericin B (0.1 μM) was 95.0%, indinavir (100 μM) 82.8%, and ritonavir (50 μM) 70.2%. Since the HIVPrs did not show toxicity to macrophages, we tested its activity on Leishmania spp. amastigote forms. Indinavir and ritonavir also inhibited the multiplication of Leishmania spp. amastigotes in mouse peritoneal macrophages. Indinavir, ritonavir, and amphotericin B significantly reduced (pb 0.05) the infection index of L. (L.) amazonensis, to the lowest concentrations investigated, respectively 25 μM (56.8%), 12.5 μM (26.3%), and 0.025 μM (57.9%) (Fig. 1A).
Fig. 1. Activity of HIV protease inhibitors on amastigote forms of L. (L.) amazonensis (A) and L. (V.) braziliensis (B). Peritoneal macrophages of BALB/c mice (5 × 105) were infected by promastigote forms of Leishmania spp. (6 promastigotes: 1 macrophage) for 6 h at 37 °C in an atmosphere of 5% CO2. After 24 h, the drugs were added in cultures and incubated in an atmosphere of 5% CO2. The Infection Index was calculated. *p b 0.05 with regard to non-treated controls.
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amphotericin B showed no differences in body weight. Fig. 3 shows the footpad of animals that were treated with the drugs, not treated, or uninfected. Indinavir- and amphotericin B-treated animals showed slight healing at the margin of the lesions, and a decrease in lesion thickness. In contrast, ritonavir-treated mice failed to show any healing in the lesion, although the footpad did decrease in thickness. Also, the weight of lymph nodes of indinavir- and amphotericin Btreated animals was significantly less than that of non-treated animals; however, there was no significant reduction in spleen weight. This analysis showed that the parasite load in the regional lymph nodes and the spleen was lower in indinavir-, ritonavir- and amphotericin B-treated animals than in untreated ones, although these differences were not statistically significant (Table 2).
4. Discussion We investigated the effect of HIV protease inhibitors on New World Leishmania. The antiretroviral drugs showed a dose-dependent inhibitory effect on the growth of promastigotes of L. (L.) amazonensis and L. (V.) braziliensis, and inhibited the intracellular amastigote proliferation in macrophages. To our knowledge, this is the first investigation of the effects of HIV protease inhibitors on L. (V.) braziliensis, the species involved in mucocutaneous leishmaniasis. The studies conducted with mice infected by L. (L.) amazonensis showed that HIVPrIs reduced the thickness of the lesion and healing at the margin of the lesions. Numerous studies suggest that protease inhibitors affect HIV opportunistic diseases [18,23,27,44]. The antiretroviral drugs, especially HIVPrIs, have a strong effect on controlling opportunistic diseases in HIV-carrier patients, and on inhibiting the growth of certain pathogenic microorganisms [17]. This activity is partly attributed to the reconstitution of the patients’ immunological system, which triggers an increase in lymphocyte T CD4+ and CD8+ and a decrease in HIV viral load. Furthermore, some studies suggest that HIVPrIs can inhibit aspartyl protease and cysteine protease of parasites [14–16,25,34,45–47]. Since the HIVPrIs ritonavir and nelfinavir also affect other important microorganisms such as Toxoplasma gondii, it has been suggested that aspartic proteinase could be involved in the replication of protozoans and that its inhibition affects parasite survival [19]. Ritonavir
Fig. 2. Course of the experimental infection in BALB/c mice infected with Leishmania (Leishmania) amazonensis after treatment with HIV protease inhibitors. Animals were infected with 1.2 × 107 promastigotes of L. (L.) amazonensis and treated orally with 8 mg/kg of indinavir, 2 mg/kg of ritonavir, or 5 mg/kg of amphotericin B. Treatment started the 4th week after infection and continued for 30 days (arrow). Each dot represents the thickness of the infected footpad minus that of the uninfected contralateral footpad ± S.E.M. (n = 10). *p b 0.05 for indinavir and amphotericin B; **p b 0.05 for ritonavir with regard to the non-treated control group.
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and indinavir, and to a lesser extent saquinavir inhibited the activities of aspartic proteases secreted by Candida albicans [44,48,49]. The employment of protease-inhibiting antiretroviral drugs on HIV is well known and well documented, although little is known of the effect of these drugs on HIV/Leishmania co-infections. This investigation demonstrated that ritonavir and indinavir have a dose-dependent antileishmanial activity against promastigote forms of L. (L.) amazonensis and L. (V.) braziliensis. However, the antiproliferative activities of these drugs differ between these two species. While the ritonavir IC50 was 40 μM for Leishmania (L.) amazonensis, for L. (V.) braziliensis it was approximately 17 times lower (2.3 μM), showing that L.(V.) braziliensis is more sensitive than L. (L.) amazonensis to this HIVPrI; whereas indinavir and atazanavir showed a higher IC50 for both species. A difference in sensitivity to HIVPrIs between the species of Leishmania has been reported. Savoia et al. [14] showed that the 50% lethal dose of indinavir was 8.3 μM on promastigotes of L. major, and in lower concentrations on L. infantum [14]. Santos et al. [35] observed that indinavir inhibits the growth of L. amazonensis at five-fold higher concentrations (500 μM) than detected here. This difference in IC50 between the studies might be a function of the different assays employed. Trudel et al. [15] reported that ritonavir inhibits Leishmania donovani promastigotes in mononuclear cells, as well as in phorbolmyristate acetate-differentiated THP-1 macrophages and monocyte-derived macrophages (MDMs). They noted that the concentrations of 12.5 and 25 μM of ritonavir inhibited, respectively, 48.9% and 93.2% of promastigote growth in THP1, and 30.9% and 51.2% in promastigotes in MDMs. Our results suggest that the inhibitory effect of ritonavir is stronger in L. (V.) braziliensis than in L. (L.) amazonensis promastigotes. Valdivieso et al. [34] found both an antiproliferative effect on Leishmania mexicana promastigotes and axenic amastigotes, and a dose-dependent inhibition of Leishmania promastigotes aspartyl-proteinase activity by HIVPrIs, leading them to suggest that this activity is the target of the drug. White et al. [16] showed that the Leishmania major Ddi1-like protein, the only aspartic proteinase encoded by the Leishmania genome, is susceptible to the action of HIVPrIs and showed greater sensitivity to indinavir than to ritonavir. These authors described significant differences in the sequence of this protein in Leishmania species, mainly L. braziliensis, which has an N terminus and a number of short insertions in relation to the other Leishmania sp. This sequence was also found in L. infantum, L. major, and Leishmania mexicana, but not in L. amazonensis. Since our study showed that indinavir and ritonavir inhibited L. (L.) amazonensis, it is suggested that these HIVPrIs may act by mechanisms that do not involve the Ddi1 protein. It remains to be clarified whether the variability in the sequence of this protein is sufficient to explain the differences in the inhibitor sensitivity of the parasites, or whether other factors, for instance cellular uptake, might be involved [16]. Indinavir and ritonavir decreased infection of mice peritoneal macrophages by L. (L.) amazonensis and L. (V.) braziliensis. Ritonavir in amounts of 12.5 and 25 μM inhibited the intracellular proliferation of amastigotes of L. (L.) amazonensis (26.3% and 42.4%) and L. (V.) braziliensis (37.3% and 27.6%). Our results concord with the findings of Trudel et al. [15], since they noted that the concentrations of 12.5 and 25 μM of ritonavir inhibited, respectively, 26.3% and 43.7% of the growth of amastigotes of L. donovani in MDMs. We suggest that this inhibitory effect on amastigotes may result from the action of HIVPrIs on conserved enzyme systems, necessary for the intracellular survival of Leishmania. The present study found that the dose of ritonavir required to inhibit amastigote proliferation is five times higher than that required to inhibit promastigotes of Leishmania sp. Variability in the activity of HIVPrIs against different parasite forms has been described; this variability could result from its action through different uptake systems [34], and from the different molecules (carbohydrates and enzymes) [50,51,16] that are present in the promastigote and amastigote forms. Thus, it remains to be clarified whether the variability of the antiproliferative activity of HIVPrI on different forms and species of Leishmania might occur
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Fig. 3. Left footpad of BALB/c mice infected by L. (L.) amazonensis. (A) Ulcerated lesion (of the control group) 30 days after infection; (B) ulcerated lesion of the control group 60 days after infection; (C) treated with 8 mg/kg/day indinavir; (D) treated with 2 mg/kg/day ritonavir; (E) treated with 5 mg/kg/day amphotericin B; F) animal uninfected by Leishmania. Groups c, d, e were treated orally for 30 days.
because these drugs act on different molecules that are essential for the survival of the intracellular and extracellular parasite forms. Inhibition of Leishmania sp. proliferation can be attributed to the inhibition of proteinases that are important for the differentiation and multiplication of these parasites. Santos et al. [35] showed that
Table 2 Effect of HIV protease inhibitors on the weight of organs and parasite load of BALB/c mice experimentally infected with Leishmania (Leishmania) amazonensis. Group
Organs
Weight (g)
Control
L S L S L S L S
0.102 0.156 0.061 0.146 0.071 0.161 0.059 0.152
Indinavir Ritonavir Amphotericin B
L, lymph node; S, spleen.
p
Parasite load
p
0.035 0.516 0.057 0.779 0.032 0.761
1852 × 103 ± 833 × 103 2545 ± 2382 1215 × 103 ± 907 × 103 2294 ± 2714 1078 × 103 ± 1209 × 103 569 ± 985 455 × 103 ± 407 × 103 0
0.421 0.910 0.413 0.255 0.059 0.138
treatment with HIV proteinase inhibitors (including indinavir) caused profound changes in the Leishmania ultrastructure that were suggestive of apoptotic death, and that an aspartyl peptidase may be the intracellular target of the inhibitors. Mukohup and coworkers reported that protease inhibition in Leishmania donovani by nonspecific inhibitors of HIV caused slow growth of the parasites, and they suggested that this effect might result from inhibition of the biosynthesis of macromolecules that are essential for parasite survival [52]. The HIV aspartyl-proteinase inhibitors demonstrate an antiproliferative effect on promastigote and axenic amastigote forms of Leishmania sp. Also, saquinavir mesylate and nelfinavir induced, in culture, a reduction in the percentage of co-infected HIV/Leishmania monocytes and amastigotes of Leishmania per macrophage [32]. Antiretroviral drugs may affect Leishmania through other mechanisms. Kumar et al. related that nelfinavir generates oxidative stress in Leishmania donovani amastigotes, through a mechanism of caspaseindependent apoptosis [32]. Protease inhibitors also stimulate the production of microbicidal molecules and reactive oxygen species by infected macrophages. The cytotoxic activity of nitric oxide (NO) on
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Leishmania is crucial in the course of the infection, since Leishmania infantum cysteine proteinase may undergo NO-mediated chemical modification, with concomitant loss of activity, thereby representing a potential target of the antiparasitic treatment [45,47]. When the effects of the antiretrovirals on L. (L.) amazonensis experimental infection of mice were evaluated, it was evident that indinavir-treated animals had a significant decrease in the infected footpad after the third week of treatment. A similar decrease in the infected footpad occurred only at the fifth week in ritonavir-treated mice. Studies on the animals' organ weight showed that the weight of popliteal lymph nodes of animals treated by antiretroviral drugs was lower than that of non-treated ones. Although the parasite load, which may reflect the activity of antiretroviral drugs in infection control, was lower in antiretroviral drug-treated animals, the decline was not statistically significant. These results can be attributed to a typical activity of these drugs in mice, leishmanistatic instead of leishmanicidal, perhaps because of the route used to administer the drugs (our study used the oral route), or the initiation of the treatment 30 days after infection. “Per os” administration has been successfully used for the evaluation of antiretroviral activities in microorganisms such as Crypstosporidium parvum and Plasmodium chabaudi [24,53]. However, it is unclear whether this is the most appropriate route of administration of HIVPrIs in mice infected with Leishmania spp. Although the antiretroviral doses were equivalent to those employed in humans, it may be questioned whether they were sufficient to inhibit the already-established Leishmania infection. Although the present study detected an inhibitory activity of antiretroviral drugs against L. (L.) amazonensis and L. (V.) braziliensis, the possibility of infection control by the parasites in co-infected patients should not be discarded. The action of HAART in HIV/Leishmania coinfection raises interesting prospects for the development of new drugs, based on this novel parasite proteinase family [16,34]. The real applicability of protease inhibitors for the treatment of American cutaneous leishmaniasis remains to be investigated. Most reports on the activity of antiretroviral drugs are related to visceral leishmaniasis. A 64.8% decrease in the prevalence of visceral leishmaniasis caused by L. infantum after antiretroviral therapy, which included saquinavir, indinavir, nelfinavir, or ritonavir in an HIV virus-infected population was reported in Spain [54]. In France, HIV/Leishmania co-infections were significantly reduced following HAART therapy with protease inhibitors [55]. The results showed that HIV protease inhibitors have an inhibitory effect on L. (L.) amazonensis and L. (V.) braziliensis. So far, we do not know how these drugs act on New World Leishmania. Further studies are needed to elucidate the mechanism of action of the HIV protease inhibitors on different species of Leishmania and on the production of chemical mediators involved in the activation of macrophages, in order to assess their potential for use in leishmaniases-HIV co-infections. Acknowledgements This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Merck Sharp &Dohme of Brazil, and Abbott Laboratory of Brazil Ltda. References [1] WHO, World Health Organization. Towards universal access: scaling up priority HIV/AIDS interventions in the health sector. progress report 2009. Geneva: WHO Publications; 2009. [2] Borges AS, Machado AA, Ferreira MS, de Castro Figueiredo JF, Silva GF, Cimerman S, et al. Concurrent leishmaniasis and human immunodeficiency virus (HIV) infection: a study of four cases. Revista da Sociedade Brasileira de Medicina Tropical 1999;32:713–9. [3] Brasil. Ministério da Saúde. Manual de recomendações para diagnostico, tratamento e acompanhamento de pacientes com a coinfecção Leishmania-HIV/Ministério da Saúde, Secretaria de Vigilância em Saúde Departamento de Vigilância Epidemiológica. Brasília: Ministério da Saúde; 2011. [4] Vianna G. Sobre uma espécie de Leishmania. Brasil-Médico 1912;25:411.
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Leishmania mexicana: identification and characterization of an aspartyl proteinase activity. Experimental Parasitology 2007;116:77–82. [32] Kumar P, Lodge R, Trudel N, Ouellet M, Ouellette M, Tremblay MJ. Nelfinavir, an HIV-1 protease inhibitor, induce oxidative stress-mediated, caspase-independent apoptosis in Leishmania amastigotes. PLoS Neglected Tropical Diseases 2010;4:e642. [33] Alves CR, Corte-Real S, Bourguignon SC, Chaves CS, Saraiva EM. Leishmania amazonensis: early proteinase activities during promastigote-amastigote differentiation in vitro. Experimental Parasitology 2005;109:38–48. [34] Valdivieso E, Rangel A, Moreno J, Saugar JM, Cañavate C, Alvar J, et al. Effects of HIV aspartyl-proteinase inhibitors on Leishmania sp. Experimental Parasitology 2010;126:557–63. [35] Santos LO, Marinho FA, Altoé EF, Vitório BS, Alves CR, Britto C, et al. 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Parasitology International journal homepage: www.elsevier.com/locate/parint
Effect of HIV protease inhibitors on New World Leishmania Izabel G. Demarchi a, Thaís G.V. Silveira a, Izabel C.P. Ferreira b, Maria V.C. Lonardoni a,⁎ a b
Departamento de Análises Clínicas e Biomedicina, Universidade Estadual de Maringá, PR, Brazil Departamento de Farmácia e Farmacologia, Universidade Estadual de Maringá, PR, Brazil
a r t i c l e
i n f o
Article history: Received 13 December 2011 Received in revised form 23 April 2012 Accepted 25 April 2012 Available online 2 May 2012 Keywords: Antiretroviral HAART Cutaneous leishmaniasis Leishmania HIV protease inhibitors
a b s t r a c t The incidence of HIV/Leishmania co-infection decreases after antiretroviral drug therapy; therefore, the in vitro and in vivo activity of three antiretroviral drugs against Leishmania (Viannia) braziliensis and L. (L.) amazonensis was evaluated. Different concentrations of indinavir (IDV), atazanavir (ATV), and ritonavir (RTV) were added to promastigote cultures, and the 50% inhibitory concentration (IC50) was determined. IDV and RTV were also evaluated against intracellular amastigotes, and the Infection Index determined. BALB/c mice, infected with L. (L.) amazonensis in the left footpad, were treated orally with IDV and RTV for 30 days, and monitored by measuring the footpad thickness and parasite load of regional lymph nodes and spleen. For promastigotes, IDV exhibited an IC50 value of 100 μM against L.(L.) amazonensis. The RTV IC50 for L. (L.) amazonensis and L. (V.) braziliensis were 40 and 2.3 μM, respectively, and the ATV IC50 for L. (V.) braziliensis was 266 μM. For intracellular amastigotes, IDV (25, 50, and 100 μM) significantly decreased the Infection Index of L. (L.) amazonensis (56.8%, 47.9%, and 65.0%) and L. (V.) braziliensis (37.8%, 48.7%, and 43.2%). RTV (12.5, 25, and 50 μM) decreased the infection index of L. (L.) amazonensis by 26.3%, 42.4%, and 44.0%, and that of L. (V.) braziliensis by 27.6%, 37.3%, and 39.2%. Antiretroviral-treated mice had a significant reduction in footpad thickness after the third week of IDV and after the fifth week of RTV treatment. However, there was no reduction in parasite load. These results suggest that IDV and RTV have anti-Leishmania activity, but only in higher concentrations. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The World Health Organization estimates that 33.3 million of the world's people are infected by human immunodeficiency virus (HIV), and each year 2.6 million people are newly infected [1]. At least one third of virus carriers live in endemic areas for leishmaniases [2]. Leishmaniases affect around 350 million people in risk areas, most of them in developing countries [3]. Cutaneous leishmaniasis (CL) is one of the most common diseases affecting humans in developing countries, and manifests itself through cutaneous involvement. The main etiological agents of American cutaneous leishmaniasis(ACL) in Brazil are Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis; ACL manifests itself through cutaneous, mucosal, or disseminated types [3–5]. Leishmaniases cause serious diseases in HIV carriers, and treatment is difficult and often ineffective. New therapeutic strategies are urgently required [6]. Amphotericin B is a well-known antileishmanial drug and has been used when ACL treatment with pentavalent antimonies fails [4]. Lipid formulations of amphotericin B (AMB) have been employed
⁎ Corresponding author at: Av Colombo, 5790, CEP 87020-900, Maringá, Paraná, Brazil. Tel.: +55 44 30114878; fax: +55 44 30114805. E-mail addresses: [email protected] (I.G. Demarchi), [email protected] (T.GV. Silveira), [email protected] (I.CP. Ferreira), [email protected] (M.VC. Lonardoni). 1383-5769/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2012.04.006
successfully in the treatment of the cutaneous and mucosal forms of ACL, both in immune-competent patients and in those with any type of immunosuppression, including HIV-infected people [7–9]. Leishmaniases may modify disease progression by HIV, and HIVcaused immunodepression facilitates the dissemination of Leishmania [4]. Both HIV and Leishmania multiply in macrophages, and the cellular immune response becomes crucial in infection control [10]. The introduction of highly active antiretroviral therapy (HAART) has resulted in a decrease in opportunistic diseases, including the incidence of HIV/ Leishmania co-infection [11]. Protease inhibitors employed in the treatment of acquired immunodeficiency syndrome (AIDS) affect the aspartic protease enzyme of HIV. It is believed that, in addition to blocking virus multiplication, this pathway may also affect the growth of other microorganisms [12,13], such as Leishmania sp. [14–16], Trypanosoma cruzi [17], Toxoplasma gondii [18,19], Plasmodium spp. [20–22], Giardia duodenalis and Trichomonas vaginalis [23], Crypstosporidium parvum [24], Candida albicans, and Pneumocystiscarinii [11]. However, their biochemical target is unknown. Proteasomes are essential for the multiplication, differentiation, and intracellular survival of Leishmania parasites [25–31]. Recent in vitro studies with Old World Leishmania showed that proteasomes of promastigote and amastigote forms may be the targets of antiretroviral protease inhibitors (HIVPrIs) [16,32–34]. Some studies have reported aspartic proteinase activity in extracts from these parasites
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[31,33,34]. Santos et al. [35] observed that HIV aspartyl peptidase inhibitors impaired the growth of Leishmania amazonensis, influenced the parasite ultrastructure, and decreased the activity of aspartyl peptidases. White et al. [16] showed that HIVPrIs affect Ddi1 protein, the only member of the aspartic proteinase family that is present in Leishmania major, and suggested that the proteasomes are a potential target for the HIVPrIs in these parasites. Few studies on the activity of antiretrovirals in New World Leishmania have investigated the effect on L. (L.) amazonensis [35,36]. The effects of these drugs on the course of infection caused by L. (V.) braziliensis are not known. In this study, we investigated the efficacy of three HIVPrIs (ritonavir, indinavir and atazanavir) on the multiplication of promastigotes; the efficacy of ritonavir and indinavir on intracellular amastigotes of L. (V.) braziliensis and L. (L.) amazonensis, and on an experimental infection by L. (L.) amazonensis in BALB/c mice. 2. Materials and methods 2.1. Chemicals Indinavir (Merck & Co., Inc., Rahway, NJ, USA) and ritonavir (Abbott Laboratories, North Chicago, IL, USA) were donated by the manufacturers. Amphotericin B (Anforicin, Cristalia Laboratory, São Paulo, SP, Brazil) and atazanavir (Reyataz, Bristol-Myers Squibb, Princeton, NJ, USA) were donated by the Paraná State Secretariat of Health.
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drugs and DMSO. The results were evaluated by linear regression of the inhibition percentage [38]. 2.4. Macrophage viability assay Four days after intraperitoneal inoculation of 1 ml of thioglycolate broth, macrophages from the peritoneal cavity of BALB/c mice were obtained according to Lonardoni et al. [39]. Briefly, the peritoneal cavity was washed with 10 to 12 ml sterile RPMI 1640 (Invitrogen) and cell suspensions were adjusted to 1 × 10 6 macrophages/ml. Next, 500 μl was distributed on 13 mm-diameter sterile glass coverslips (Glastecnica, São Paulo, SP, Brazil), placed in 24-well culture plates (TPP, Switzerland). The plates were incubated for 1 h at 25 °C, and non-adherent cells were removed by three sterile PBS washings. The adherent cells were incubated in RPMI 1640 supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 μg/ml). The cultures were treated with different concentrations of indinavir (25, 50, and 100 μM), ritonavir (12.5, 25, and 50 μM), or amphotericin B (0.025, 0.05, and 0.1 μM) diluted in DMSO. The final concentration of DMSO did not exceed 0.005%, and no cytotoxic effect on the macrophages was observed. Cultures not treated with drugs or the diluent were used as control. The plates were maintained at 37 °C in a humid atmosphere containing 5% CO2 for 24 h, and then the coverslips were stained with 1% Trypan Blue (Sigma-Aldrich) and examined microscopically. All tests were done in triplicate, and the results are expressed as percentage viability.
2.2. Culture and maintenance of Leishmania L. (V.) braziliensis (MHOM/BR/1987/M11272) isolated from a patient from northwestern Paraná and identified at the Instituto Evandro Chagas (Belém, PA, Brazil) and L. (L.) amazonensis (MHOM/BR/1977/ LTB0016) were maintained by inoculation of 1 × 107 parasites in the left footpad of hamsters aged between 30 and 40 days. For isolation of the parasites, the hamsters were euthanized with ketamine (Agner União, Embu-Guaçu, SP, Brazil) and xylazine (Coopazine ®, Intervet Schering-Plough, Cotia, SP, Brazil). The sections of the lymph nodes were inoculated in culture medium 199 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal calf serum (Invitrogen), 1% human urine, 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and antibiotics (100 UI/ml penicillin and 100 μg/ml streptomycin, both obtained from Sigma-Aldrich), and incubated at 25 °C. The parasites were maintained by weekly transfers in supplemented medium 199. 2.3. Activity of HIV protease inhibitors against Leishmania spp. promastigote forms Antiretroviral activity against Leishmania was evaluated according to Ferreira et al. [37]. Briefly, promastigotes of L. (L.) amazonensis and L. (V.) braziliensis were cultured in Schneider's insect medium (Sigma-Aldrich), pH 7.2, supplemented with 10% FCS (v/v) and 2 mM L-glutamine until they reached the logarithmic growth phase. The drugs were dissolved in dimethylsulfoxide (1.6% v/v DMSO in the first well; obtained from Sigma-Aldrich) and diluted in series in a ratio of two, starting from ½, on a cell culture plate with 96 wells (TPP® test plate, Switzerland). The HIVPrIs (indinavir, ritonavir and atazanavir) were tested in concentrations from 0.75 to 400 μM, and amphotericin B from 0.01 to 17 μM (positive controls). The concentration of DMSO used had no effect on the parasites. Next, 100 μl of the suspension containing 4 × 10 6 parasites/ml was distributed in each well of the culture plate. After 24 h at 25 °C, an aliquot of each well was added to a solution containing 10% eosin and 2% formalin, and the parasites were counted in a Neubauer chamber. All tests were performed in triplicate. The inhibitory concentration that caused a 50% decrease in survival (IC50) of promastigotes was calculated. Values were compared to those obtained in control cultures without
2.5. Effect of HIV protease-inhibiting drugs on amastigote forms of Leishmania spp Establishing and plating of macrophages were performed as described in the previous section. Macrophages were infected by promastigote forms in the proportion of six parasites for each macrophage. The cultures were incubated for 6 h at 37 °C in an atmosphere of 5% CO2. The supernatant was then removed. The drugs dissolved in DMSO and diluted in supplemented RPMI 1640 medium were added to cultures at concentrations of 25, 50, and 100 μM (indinavir), 12.5, 25, and 50 μM (ritonavir), and 0.025, 0.05, and 0.1 μM (Amphotericin B). After 24 h at 37 °C in a 5% CO2 atmosphere, the coverslips were removed, washed with PBS, stained with Fast Panoptic reagent (Laborclin, Pinhais, PR, Brazil) and analyzed under an optical microscope. At least 200 cells were counted to calculate the Infection Index, which was determined from the percentage of infected macrophages multiplied by the mean number of parasites per macrophage. 2.6. Effects of HIV protease-inhibiting drugs in mice experimentally infected by L. (L.) amazonensis BALB/c mice have been used extensively to study the immune response to and the effect of drugs on species of Leishmania. Strains of L. braziliensis differ in causing cutaneous lesions of BALB/c mice [40,41] and it is difficult to use a mouse model to evaluate the effect of drugs on L. braziliensis. The strain of L. (V.) braziliensis tested in this study, isolated from a patient, did not establish an infection or lesions in mice, and did not develop evenly and progressively in hamsters. Since a suitable experimental model for this strain of L. (V.) braziliensis is not yet established, the experiments to evaluate the HIVPrIs effect in vivo were performed only with L. (L.) amazonensis as previously described [42]. Briefly, female BALB/c mice aged 9–10 weeks were infected with L. (L.) amazonensis 5 × 105promastigotes/40 μl PBS in the left footpad, and 40 μl PBS was inoculated in the right footpad. 30 days post-infection, the mice were divided into groups and treated by oral gavage for 30 consecutive days. Group A was treated with indinavir (8 mg/kg/day), and group B with ritonavir (2 mg/kg/day). The doses were used according to the information provided by the manufacturers. The controls were composed of animals (group C) treated with
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amphotericin B (5 mg/kg/day), a drug used to treat cases of HIV/ Leishmania co-infection [4]; and animals that received no treatment (group D). Another group of animals that were not infected and were not treated (group E) was followed. After infection, the vertical thickness of the footpad of each animal was measured weekly, with a pachymeter (Vernier Calipers, Japan). The results were given as the difference between the measurements of the infected and the uninfected footpad, in millimeters (mm). Mice were weighed weekly. The load of Leishmania was evaluated according to methodology by Buffet et al. [43], with modifications. Animals were euthanized with ketamine and xylazine, and the popliteal lymph nodes draining the infected footpad and the spleen were aseptically removed and weighed. Each organ was macerated in supplemented medium 199. Next, 225 μl of supplemented medium 199 was distributed in 96well culture plates, and 75 μl of each suspension, in duplicate, was added to the first well. Suspensions were diluted in series in a ratio of 4, starting from ¼. After 5 days at 25 °C, the cultures were observed in an inverted light microscope. Each well was analyzed for promastigote forms, and was scored as positive when at least one parasite was found. The plates were kept for another 7 days and then re-examined. The parasite load (number of parasites/gram of tissue) was calculated as follows: the geometric mean of the reciprocal of the positive dilution from each duplicate was divided by the weight of the organ. The value obtained was multiplied by the reciprocal of the fraction of the homogenized organ added to the first well. 2.7. Ethical aspects This investigation was approved by the Ethics Committee on the use of Experimental Animals of the State University of Maringá (warrant no. 056/2007).
Table 1 Effect of HIV protease-inhibiting drugs on the in vitro growth of promastigote forms of L. (L.) amazonensis and L. (V.) braziliensis after 24 h incubation. Drugs
Indinavir Ritonavir Atazanavir Amphotericin B
L. (L.) amazonensis
L. (V). braziliensis
IC50 (μM)
S.D.
IC50 (μM)
S.D.
100 40 266 b0
0.3 1.9 1.5 0
400 2.3 400 b0
0 0.1 0 0
(IC50: 50% inhibitory concentration; μM: micromolar; S.D.: standard deviation of the mean).
At the lowest concentration, the results showed that treatment with indinavir (25 μM) decreased the Infection Index of macrophage infection of L. (V.) braziliensis by 37.8%, and ritonavir (12.5 μM) by 27.6% (p b 0.05). Amphotericin B reduced the Infection Index by 79.2% (0.025 μM) (Fig. 1B).
3.2. Activity in vivo of HIV protease inhibitors The in vivo effect of antiretroviral drugs was evaluated in the experimental infection of BALB/c mice by L. (L.) amazonensis. Antiretroviral drugs were administered orally for 1 month, and the animals were then followed for 10 weeks. The lesion thickness of mice treated with indinavir (8 mg/kg/day) and amphotericin B (5 mg/kg/day) decreased from the third week post-treatment when compared with the untreated control group (p b 0.05). Animals treated with ritonavir (2 mg/kg/day) showed a regression of infection only after the fifth week (p b 0.05) (Fig. 2). Animals treated with an antiretroviral or
2.8. Statistical analyses All results were first analyzed by the Shapiro–Wilk test for normality. Results with a normal distribution were analyzed by Student's t test, and the others were analyzed by the Mann–Whitney test. The software Statistic 6.0 was used, and differences were considered significant at the 5% level. 3. Results 3.1. In vitro antileishmanial activity Both indinavir and ritonavir showed an antiproliferative effect on Leishmania sp. promastigotes, to different degrees. Ritonavir inhibited the growth of promastigotes of both Leishmania species studied. Indinavir showed an inhibitory effect only against L. (L.) amazonensis, in which the IC50 of 100 μM was 2.5 times higher than the IC50 of ritonavir, and 100 times higher than that of amphotericin B. Atazanavir showed a very high IC50, and because of this, only indinavir and ritonavir were assessed for their antiproliferative activity on intracellular amastigote forms of Leishmania spp. As expected, the reference drug amphotericin B inhibited the proliferation of both Leishmania species (Table 1). The drug concentrations tested showed no effect on the viability of the macrophages. In higher concentrations, the percentage of viable cells for amphotericin B (0.1 μM) was 95.0%, indinavir (100 μM) 82.8%, and ritonavir (50 μM) 70.2%. Since the HIVPrs did not show toxicity to macrophages, we tested its activity on Leishmania spp. amastigote forms. Indinavir and ritonavir also inhibited the multiplication of Leishmania spp. amastigotes in mouse peritoneal macrophages. Indinavir, ritonavir, and amphotericin B significantly reduced (pb 0.05) the infection index of L. (L.) amazonensis, to the lowest concentrations investigated, respectively 25 μM (56.8%), 12.5 μM (26.3%), and 0.025 μM (57.9%) (Fig. 1A).
Fig. 1. Activity of HIV protease inhibitors on amastigote forms of L. (L.) amazonensis (A) and L. (V.) braziliensis (B). Peritoneal macrophages of BALB/c mice (5 × 105) were infected by promastigote forms of Leishmania spp. (6 promastigotes: 1 macrophage) for 6 h at 37 °C in an atmosphere of 5% CO2. After 24 h, the drugs were added in cultures and incubated in an atmosphere of 5% CO2. The Infection Index was calculated. *p b 0.05 with regard to non-treated controls.
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amphotericin B showed no differences in body weight. Fig. 3 shows the footpad of animals that were treated with the drugs, not treated, or uninfected. Indinavir- and amphotericin B-treated animals showed slight healing at the margin of the lesions, and a decrease in lesion thickness. In contrast, ritonavir-treated mice failed to show any healing in the lesion, although the footpad did decrease in thickness. Also, the weight of lymph nodes of indinavir- and amphotericin Btreated animals was significantly less than that of non-treated animals; however, there was no significant reduction in spleen weight. This analysis showed that the parasite load in the regional lymph nodes and the spleen was lower in indinavir-, ritonavir- and amphotericin B-treated animals than in untreated ones, although these differences were not statistically significant (Table 2).
4. Discussion We investigated the effect of HIV protease inhibitors on New World Leishmania. The antiretroviral drugs showed a dose-dependent inhibitory effect on the growth of promastigotes of L. (L.) amazonensis and L. (V.) braziliensis, and inhibited the intracellular amastigote proliferation in macrophages. To our knowledge, this is the first investigation of the effects of HIV protease inhibitors on L. (V.) braziliensis, the species involved in mucocutaneous leishmaniasis. The studies conducted with mice infected by L. (L.) amazonensis showed that HIVPrIs reduced the thickness of the lesion and healing at the margin of the lesions. Numerous studies suggest that protease inhibitors affect HIV opportunistic diseases [18,23,27,44]. The antiretroviral drugs, especially HIVPrIs, have a strong effect on controlling opportunistic diseases in HIV-carrier patients, and on inhibiting the growth of certain pathogenic microorganisms [17]. This activity is partly attributed to the reconstitution of the patients’ immunological system, which triggers an increase in lymphocyte T CD4+ and CD8+ and a decrease in HIV viral load. Furthermore, some studies suggest that HIVPrIs can inhibit aspartyl protease and cysteine protease of parasites [14–16,25,34,45–47]. Since the HIVPrIs ritonavir and nelfinavir also affect other important microorganisms such as Toxoplasma gondii, it has been suggested that aspartic proteinase could be involved in the replication of protozoans and that its inhibition affects parasite survival [19]. Ritonavir
Fig. 2. Course of the experimental infection in BALB/c mice infected with Leishmania (Leishmania) amazonensis after treatment with HIV protease inhibitors. Animals were infected with 1.2 × 107 promastigotes of L. (L.) amazonensis and treated orally with 8 mg/kg of indinavir, 2 mg/kg of ritonavir, or 5 mg/kg of amphotericin B. Treatment started the 4th week after infection and continued for 30 days (arrow). Each dot represents the thickness of the infected footpad minus that of the uninfected contralateral footpad ± S.E.M. (n = 10). *p b 0.05 for indinavir and amphotericin B; **p b 0.05 for ritonavir with regard to the non-treated control group.
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and indinavir, and to a lesser extent saquinavir inhibited the activities of aspartic proteases secreted by Candida albicans [44,48,49]. The employment of protease-inhibiting antiretroviral drugs on HIV is well known and well documented, although little is known of the effect of these drugs on HIV/Leishmania co-infections. This investigation demonstrated that ritonavir and indinavir have a dose-dependent antileishmanial activity against promastigote forms of L. (L.) amazonensis and L. (V.) braziliensis. However, the antiproliferative activities of these drugs differ between these two species. While the ritonavir IC50 was 40 μM for Leishmania (L.) amazonensis, for L. (V.) braziliensis it was approximately 17 times lower (2.3 μM), showing that L.(V.) braziliensis is more sensitive than L. (L.) amazonensis to this HIVPrI; whereas indinavir and atazanavir showed a higher IC50 for both species. A difference in sensitivity to HIVPrIs between the species of Leishmania has been reported. Savoia et al. [14] showed that the 50% lethal dose of indinavir was 8.3 μM on promastigotes of L. major, and in lower concentrations on L. infantum [14]. Santos et al. [35] observed that indinavir inhibits the growth of L. amazonensis at five-fold higher concentrations (500 μM) than detected here. This difference in IC50 between the studies might be a function of the different assays employed. Trudel et al. [15] reported that ritonavir inhibits Leishmania donovani promastigotes in mononuclear cells, as well as in phorbolmyristate acetate-differentiated THP-1 macrophages and monocyte-derived macrophages (MDMs). They noted that the concentrations of 12.5 and 25 μM of ritonavir inhibited, respectively, 48.9% and 93.2% of promastigote growth in THP1, and 30.9% and 51.2% in promastigotes in MDMs. Our results suggest that the inhibitory effect of ritonavir is stronger in L. (V.) braziliensis than in L. (L.) amazonensis promastigotes. Valdivieso et al. [34] found both an antiproliferative effect on Leishmania mexicana promastigotes and axenic amastigotes, and a dose-dependent inhibition of Leishmania promastigotes aspartyl-proteinase activity by HIVPrIs, leading them to suggest that this activity is the target of the drug. White et al. [16] showed that the Leishmania major Ddi1-like protein, the only aspartic proteinase encoded by the Leishmania genome, is susceptible to the action of HIVPrIs and showed greater sensitivity to indinavir than to ritonavir. These authors described significant differences in the sequence of this protein in Leishmania species, mainly L. braziliensis, which has an N terminus and a number of short insertions in relation to the other Leishmania sp. This sequence was also found in L. infantum, L. major, and Leishmania mexicana, but not in L. amazonensis. Since our study showed that indinavir and ritonavir inhibited L. (L.) amazonensis, it is suggested that these HIVPrIs may act by mechanisms that do not involve the Ddi1 protein. It remains to be clarified whether the variability in the sequence of this protein is sufficient to explain the differences in the inhibitor sensitivity of the parasites, or whether other factors, for instance cellular uptake, might be involved [16]. Indinavir and ritonavir decreased infection of mice peritoneal macrophages by L. (L.) amazonensis and L. (V.) braziliensis. Ritonavir in amounts of 12.5 and 25 μM inhibited the intracellular proliferation of amastigotes of L. (L.) amazonensis (26.3% and 42.4%) and L. (V.) braziliensis (37.3% and 27.6%). Our results concord with the findings of Trudel et al. [15], since they noted that the concentrations of 12.5 and 25 μM of ritonavir inhibited, respectively, 26.3% and 43.7% of the growth of amastigotes of L. donovani in MDMs. We suggest that this inhibitory effect on amastigotes may result from the action of HIVPrIs on conserved enzyme systems, necessary for the intracellular survival of Leishmania. The present study found that the dose of ritonavir required to inhibit amastigote proliferation is five times higher than that required to inhibit promastigotes of Leishmania sp. Variability in the activity of HIVPrIs against different parasite forms has been described; this variability could result from its action through different uptake systems [34], and from the different molecules (carbohydrates and enzymes) [50,51,16] that are present in the promastigote and amastigote forms. Thus, it remains to be clarified whether the variability of the antiproliferative activity of HIVPrI on different forms and species of Leishmania might occur
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Fig. 3. Left footpad of BALB/c mice infected by L. (L.) amazonensis. (A) Ulcerated lesion (of the control group) 30 days after infection; (B) ulcerated lesion of the control group 60 days after infection; (C) treated with 8 mg/kg/day indinavir; (D) treated with 2 mg/kg/day ritonavir; (E) treated with 5 mg/kg/day amphotericin B; F) animal uninfected by Leishmania. Groups c, d, e were treated orally for 30 days.
because these drugs act on different molecules that are essential for the survival of the intracellular and extracellular parasite forms. Inhibition of Leishmania sp. proliferation can be attributed to the inhibition of proteinases that are important for the differentiation and multiplication of these parasites. Santos et al. [35] showed that
Table 2 Effect of HIV protease inhibitors on the weight of organs and parasite load of BALB/c mice experimentally infected with Leishmania (Leishmania) amazonensis. Group
Organs
Weight (g)
Control
L S L S L S L S
0.102 0.156 0.061 0.146 0.071 0.161 0.059 0.152
Indinavir Ritonavir Amphotericin B
L, lymph node; S, spleen.
p
Parasite load
p
0.035 0.516 0.057 0.779 0.032 0.761
1852 × 103 ± 833 × 103 2545 ± 2382 1215 × 103 ± 907 × 103 2294 ± 2714 1078 × 103 ± 1209 × 103 569 ± 985 455 × 103 ± 407 × 103 0
0.421 0.910 0.413 0.255 0.059 0.138
treatment with HIV proteinase inhibitors (including indinavir) caused profound changes in the Leishmania ultrastructure that were suggestive of apoptotic death, and that an aspartyl peptidase may be the intracellular target of the inhibitors. Mukohup and coworkers reported that protease inhibition in Leishmania donovani by nonspecific inhibitors of HIV caused slow growth of the parasites, and they suggested that this effect might result from inhibition of the biosynthesis of macromolecules that are essential for parasite survival [52]. The HIV aspartyl-proteinase inhibitors demonstrate an antiproliferative effect on promastigote and axenic amastigote forms of Leishmania sp. Also, saquinavir mesylate and nelfinavir induced, in culture, a reduction in the percentage of co-infected HIV/Leishmania monocytes and amastigotes of Leishmania per macrophage [32]. Antiretroviral drugs may affect Leishmania through other mechanisms. Kumar et al. related that nelfinavir generates oxidative stress in Leishmania donovani amastigotes, through a mechanism of caspaseindependent apoptosis [32]. Protease inhibitors also stimulate the production of microbicidal molecules and reactive oxygen species by infected macrophages. The cytotoxic activity of nitric oxide (NO) on
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Leishmania is crucial in the course of the infection, since Leishmania infantum cysteine proteinase may undergo NO-mediated chemical modification, with concomitant loss of activity, thereby representing a potential target of the antiparasitic treatment [45,47]. When the effects of the antiretrovirals on L. (L.) amazonensis experimental infection of mice were evaluated, it was evident that indinavir-treated animals had a significant decrease in the infected footpad after the third week of treatment. A similar decrease in the infected footpad occurred only at the fifth week in ritonavir-treated mice. Studies on the animals' organ weight showed that the weight of popliteal lymph nodes of animals treated by antiretroviral drugs was lower than that of non-treated ones. Although the parasite load, which may reflect the activity of antiretroviral drugs in infection control, was lower in antiretroviral drug-treated animals, the decline was not statistically significant. These results can be attributed to a typical activity of these drugs in mice, leishmanistatic instead of leishmanicidal, perhaps because of the route used to administer the drugs (our study used the oral route), or the initiation of the treatment 30 days after infection. “Per os” administration has been successfully used for the evaluation of antiretroviral activities in microorganisms such as Crypstosporidium parvum and Plasmodium chabaudi [24,53]. However, it is unclear whether this is the most appropriate route of administration of HIVPrIs in mice infected with Leishmania spp. Although the antiretroviral doses were equivalent to those employed in humans, it may be questioned whether they were sufficient to inhibit the already-established Leishmania infection. Although the present study detected an inhibitory activity of antiretroviral drugs against L. (L.) amazonensis and L. (V.) braziliensis, the possibility of infection control by the parasites in co-infected patients should not be discarded. The action of HAART in HIV/Leishmania coinfection raises interesting prospects for the development of new drugs, based on this novel parasite proteinase family [16,34]. The real applicability of protease inhibitors for the treatment of American cutaneous leishmaniasis remains to be investigated. Most reports on the activity of antiretroviral drugs are related to visceral leishmaniasis. A 64.8% decrease in the prevalence of visceral leishmaniasis caused by L. infantum after antiretroviral therapy, which included saquinavir, indinavir, nelfinavir, or ritonavir in an HIV virus-infected population was reported in Spain [54]. In France, HIV/Leishmania co-infections were significantly reduced following HAART therapy with protease inhibitors [55]. The results showed that HIV protease inhibitors have an inhibitory effect on L. (L.) amazonensis and L. (V.) braziliensis. So far, we do not know how these drugs act on New World Leishmania. Further studies are needed to elucidate the mechanism of action of the HIV protease inhibitors on different species of Leishmania and on the production of chemical mediators involved in the activation of macrophages, in order to assess their potential for use in leishmaniases-HIV co-infections. Acknowledgements This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Merck Sharp &Dohme of Brazil, and Abbott Laboratory of Brazil Ltda. References [1] WHO, World Health Organization. Towards universal access: scaling up priority HIV/AIDS interventions in the health sector. progress report 2009. Geneva: WHO Publications; 2009. [2] Borges AS, Machado AA, Ferreira MS, de Castro Figueiredo JF, Silva GF, Cimerman S, et al. Concurrent leishmaniasis and human immunodeficiency virus (HIV) infection: a study of four cases. Revista da Sociedade Brasileira de Medicina Tropical 1999;32:713–9. [3] Brasil. Ministério da Saúde. Manual de recomendações para diagnostico, tratamento e acompanhamento de pacientes com a coinfecção Leishmania-HIV/Ministério da Saúde, Secretaria de Vigilância em Saúde Departamento de Vigilância Epidemiológica. Brasília: Ministério da Saúde; 2011. [4] Vianna G. Sobre uma espécie de Leishmania. Brasil-Médico 1912;25:411.
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