In Vivo Evaluation of Leishmanicidal Activity of Benzophenone Derivatives by qPCR

In many tropical areas, infections by Leishmania species are endemic and include visceral leishmaniasis (VL), which is often fatal if untreated. Outside India, VL treatment and control are based on long-term administration of highly toxic pentavalent antimonials. Previously, we described the synthesis and in vitro leishmanicidal activity of a series of nine benzophenone derivatives with low toxicity towards murine macrophages. Here in, we report the in vivo evaluation of the most promising active compounds of that series in an experimental model of established VL by L. (L). infantum chagasi in hamsters. Importantly, parasite DNA (amastigote form) quantification in infected tissues was performed by real time PCR, for improved detection accuracy and speed. Compounds 2-Hydroxy-4O-(3,3-dimethyl)-allylbenzophenone (LFQM-117 (1)), 4-O-(3,3-Dimethyl)-allylbenzophenone (LFQM-120 (2)) and 4,4′-Di-methoxybenzophenone (LFQM-121 (3)) were administered as oral suspensions (50 mg/kg/day) for 10 days, after 50 days of parasite inoculation, and their efficacy was compared to pentavalent antimonial Glucantime (GLU). Compound 1 significantly reduced the number of parasites in the spleen (1.64 × 102 amastigotes/g, vs. 1.16 × 106 amastigotes/g in the untreated control), while compound 2 significantly reduced (p<0.05) the number of parasites in the liver (1.28 × 104 amastigotes/g, vs. 1.76 × 105 amastigotes/g in the untreated control) of infected animals. Glucantime was the most effective in the treatment of infected animals (1.15 × 101 and 3.20 × 102 amastigotes/g in the spleen and liver, respectively), but with higher toxicity then the most active compounds LFQM-117 (1) and LFQM-120 (2).


Introduction
Leishmaniasis are zoonosis caused by flagellated protozoan parasites from the genus Leishmania and are transmitted to humans by the bite of infected female phlebotomine sandflies. This group of diseases occurs mainly in tropical and subtropical regions of the planet, being endemic mostly in developing countries. Visceral Leishmaniasis (VL) is responsible for approximately 50,000 human deaths annually, with 90% of new cases of visceral leishmaniasis occurring in Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan [1,2]. VL produces a prolonged and irregular fever, anemia, and hepatosplenomegaly. The disease affects mainly the liver, spleen and bone marrow, but can also affects the lymph nodes, kidney, intestines, lungs and skin. If untreated, VL can be fatal. In the New World, VL (termed ' American VL') is caused by the species L. (L.) infantum chagasi, which infects both adults and children, and is transmitted by sand flies from the genus Lutzomyia [3]. Outside India, where resistance to pentavalent antimony compounds has emerged, treatment and control of VL are based on Glucantime and Pentostam use, which have limitations like long-term administration, high toxicity and unsafety for use in pregnant patients [4]. Benzophenone derivatives have shown promising in vitro leishmanicidal activity and a number of prototype candidates are under evaluation as lead compounds for the development of new drugs against leishmaniasis. In a recent study, we reported a series of nine alkyl-substituted benzophenone derivatives with increased leishmanicidal activity against promastigote form of L. amazonensis and reduced toxicity towards murine macrophages [5]. Our results suggested that the increased lipophilicity of these compounds could facilitate membrane permeation for drug entry into the parasite cells.
More recently, these benzophenone derivatives were further evaluated in vitro against amastigote form of L. amazonensis and compounds LFQM-117 (1), LFQM-120 (2) and LFQM-121 (3) showed the best relationship between their potency (IC50) and safety (SI), considering their effects towards amastigote forms and murine macrophages, respectively [6]. Despite their promising activity and low toxicity in vitro, these compounds have not been tested in vivo to evaluate their effectiveness in controlling or treating the disease in animal models of experimental leishmaniasis. Then, here in, we report the evaluation of the most active and promising hydroxy-alkylbenzophenone derivatives 1-3 described previously [5,6], in an in vivo model of leishmaniasis by L. (L.) infantum chagasi in hamsters. To improve treatment monitoring, the detection and quantification of amastigote forms was performed by quantitative real-time PCR (qPCR). When compared to conventional PCR, qPCR represents a more efficient, sensitive and robust technique for the detection of amastigotes RNA in various tissues (spleen, skin, lymph, blood and bone marrow) and in different clinical groups [7][8][9]. In addition, qPCR is currently considered the most reliable method to evaluate the in vivo activity of compounds against visceral leishmaniasis, allowing effective disease monitoring, with increased speed and accuracy compared with standard methods [10,11].

Parasites and animals
Leishmania (L.) infantum chagasi (strain MHOM/BR/1972/BH46) promastigotes were maintained in M-199 medium supplemented with 10% calf serum and 0.25% hemin, at 24°C. Amastigotes were maintained by passaging in female golden hamsters (Mesocricetus auratus). Animals were kept in sterile absorbent material boxes, with food and water ad libitum, and in natural light/dark cycle. All experimental procedures involving animals were approved by the Research Ethics Commission of the Federal University of Alfenas (project number 394/2012), and were performed according to the Guide for the Care and Use of Laboratory Animals [14]. For experimental infections, the spleen of infected animals was removed and macerated using a tissue grinder and the number of amastigotes was determined as described previously.

In vivo testing of experimental compounds against Leishmania
Female golden hamsters that had been recently weaned (120 g) were infected intraperitoneally with 1 × 10 7 amastigotes of L. (L.) infantum chagasi (MHOM/BR/1972/BH46) and maintained in sterile absorbent material boxes, with water and food ad libitum. In the chronic phase (around 50 days of infection), animals were divided into 5 groups (n=4/group), and subjected, for 10 consecutive days, to one of the following treatments: 0.5% of carboxymethyl cellulose (vehicle) suspension, administered orally (untreated, or UTG group); 50 mg/kg/ day of Glucantime (GLU), by intraperitoneal injection (GLU group); 50 mg/kg/day of compounds 1, 2 or 3, administered orally as suspensions in 0.5% of carboxymethyl cellulose. After 10 days of treatment, animals were sacrificed in a CO 2 chamber, and a sample of the spleen and the liver (approximately 50 mg) was removed, weighed and used for total RNA extraction, as previously described [9].

DNA extraction
Standard curves of parasite DNA for use in quantitative real-time PCR (qPCR) experiments were produced as described previously [8]. Briefly, promastigotes from stationary phase cultures were harvested by centrifugation at 1000 g for 10 minutes, washed twice in PBS (pH 7.2), and counted in a hemocytometer. Then, parasites were re-suspended in PBS (pH 7.2) for a concentration of 1 × 10 8 cells/mL, and serially diluted (1:10) up to a concentration of 1 × 10 2 cells/mL (in triplicates). Lysis buffer (10 mM Tris-HCl, pH 8.0, containing 10 mM EDTA, 0.5% SDS, 0.01% N-Lauroylsarcosine sodium salt, 100 ug/mL Proteinase K) was then added to parasite suspensions, at a ratio of 1:4 (v/v), samples were mixed by vortexing and incubated at 56°C, for 40 minutes. DNA was extracted from lysed samples using the QIAamp DNA extraction Mini Kit (Qiagen), according to the manufacturer's instructions.

RNA extraction and cDNA synthesis
Fragments of liver and spleen (~50 mg; weighed using sterile and disposable surgical material) removed from treated mice were placed in sterile microfuge tubes and frozen immediately at -80°C in storage buffer (RNAlater). RNA extraction was performed 24 hours after fragment removal, using the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. RNA samples were frozen immediately after extraction. For reverse-transcription into cDNA, 1 µl of dNTPs mix (10 mM) and 1 µl of random primers (3 µg/µL) were added to 11 µl of RNA sample, and samples were incubated in a thermal cycler for approximately 5 minutes, at 65°C. Then, tubes were placed on ice for 20 s, and 2 µl of DTT (100 mM) and 4 µl 5x buffer (Tris-HCl 250 mM, pH 8.3, containing 375 mM KCl, 15 mM MgCl 2 ) were added, and samples were incubated again in the thermal cycler for 20 s, at 37°C. Finally, 1 µl (200 U/µL) M-MLV RT enzyme was added and samples were incubated for 50 minutes, for cDNA synthesis. The purity of the cDNA sample was confirmed by measuring the absorbance at 260/280 in a NanoDrop ND2000, and sample integrity was verified by agarose gel electrophoresis and PCR. Samples were frozen at -20° C for subsequent use in qPCR.

Results and Discussion
Current therapy against visceral leishmaniasis, based on the use of pentavalent antimonials, is highly toxic and requires a long treatment regimen. Thus, testing of new compounds with leishmanicidal activity is of paramount importance to improve disease treatment and control. Recently, we described the synthesis of benzophenones derivatives with potent activity against Leishmania (L.) amazonensis in vitro [5,6]. In this work, we evaluated the anti-parasitic effect of the benzophenone derivatives 1-3 in vivo in hamsters with established infection (50 days post-inoculation) with L. (L.) infantum chagasi. For sensitive and accurate quantification of parasites in infected tissues, we used a quantitative reverse transcription PCR (qRT-PCR) assay based on the detection of the LINJ31 marker (Linj31-qPCR), which was used successfully for the quantification of live amastigotes of Leishmania (L.) infantum chagasi [4,9,15,16]. The use of RNA as a sample, rather than DNA, minimizes the detection of residual DNA from dead parasites [8,16]. Similar qPCR-based tests allow effective quantification of parasite genetic material from the spleen and liver of infected hamsters [4,9] and yield reliable estimates of the parasite burden per gram of tissue [9,17].
To confirm that the system allows linear detection of parasites in infected tissue samples, we produced a standard curve using serial dilutions of DNA from cultured promastigotes of the standard strain of L. (L.) infantum chagasi (MHOM/BR/1972/BH46) (Figure 1). The standard curve showed robust linearity and reproducibility (R 2 =0.9925, slope=-3.139, and P value<0.0001), with a strong correlation between Ct values and the number of parasites used to prepare DNA samples. We also determined the detection limit of the system (data not shown), which corresponded to a Ct value of 41.52. To evaluate the anti-parasitic effect of compounds 1-3, hamsters with established leishmanial infection (50 days after parasite inoculation) were treated for ten days with 50 mg/kg/day (in oral suspension) of each test compound, and the spleen and liver of infected animals were collected for RNA extraction, cDNA generation by reverse transcription and qPCR analysis (qRT-PCR). The number of amastigotes per gram of (spleen or liver) tissue ( Figure 2) was then calculated based on the linear regression parameters obtained from the standard curve (Figure 1).
In the untreated group (UTG; vehicle-treated), the average number of parasites was 1.16 and 0.176 × 10 6 per gram of tissue, in the spleen and liver, respectively, confirming that these animals had an established infection (clinical symptoms such as ascites and alopecia) with L. (L.) infantum chagasi. Treatment with compound LFQM-117 (1) reduced significantly the number of amastigotes in the spleen (to 1.64 × 10 2 /g) when compared with the UTG (p<0.05) (Figure 2). In contrast, treatment with compound LFQM-120 (2) reduced the number of amastigotes in the liver of infected hamsters (to 1.28 × 10 4 /g; p<0.05 vs. UTG), with only a moderate (albeit statistically significant; p<0.05) decrease in parasite numbers in the spleen (to 5.38 × 10 4 /g), when compared with the UTG group ( Figure 2) and compound LFQM-121 (3) did not show statistically significant differences in parasite numbers in the spleen or liver of infected animals ( Figure 2).
In addition, we observed a potent leishmanicidal activity towards promastigotes in vitro, with IC 50 values of 23.66 µM (1) 27.73 µM (2) and 88.02 µM (3) [5] and the best selectivity index in relation to amastigote form, which the values were 6.7 (1); 11.9 (2) and 7.3 (3) [6]. It seems that there is a close relationship between the increase of lipophilicity of the compounds with the absorption rates and action in  different routes [18,19]. The higher lipophilicity of compounds 1 and 2 in comparison to compound 3 (QlogP=5.29, 4.73 and 3.57, respectively) is expected to facilitate drug penetration into host cells and parasites, and may underlie the increased activity of these molecules both in vivo (in the liver and spleen, for compounds 2 and 1, respectively), and in vitro, towards promastigotes. Recently, de Almeida et al. observed that in vitro activities of compounds 1 and 2 against cysteine proteases from Leishmania were higher than that of compound 3, which may contribute to the higher in vivo activities of 1 and 2 in vivo.
To check the validity of current anti-leishmanial treatment, we also treated one group of infected animals with Glucantime (meglumine antimoniate) as reference compound, since this pentavalent antimonial represents the current first-line of treatment against VL [18]. In a recent study, Pinto et al. used a similar methodology to evaluate the leishmanicidal activity of H1 histamine receptor antagonists, drugs belonging to therapeutic classes such as antiallergics and anxiolytics. Unlike the present work, the standard drug used for the experimental treatment was Miltefosine.
Glucantime (50 mg/kg/day, intraperitoneal route) was the most effective compound at reducing the parasite burden in infected animals, since treatment with this drug led to a reduction in the number of amastigotes to 1.15 and 0.32 × 10 2 , in the spleen and liver, respectively ( Figure 2). Although the same dosage was used for the treatment with all compounds (50 mg/kg/day), Glucantime was administered intraperitoneal, while the test compounds 1, 2 and 3 were administered orally. Theoretically, soluble compounds are totally absorbed in intravenous route but this absorption rate does not apply to others routes. Thereby, different exposure routes can show different effects because the actually absorbed dose is very different from the administered dose. The nature of the compounds can lead different absorption rates being faster to compounds in solution than undissolved suspensions. Furthermore, the biotransformation process after oral administration is distinct from the other routes due the firstpass effect in intestine [20]. This difference in administration route may have contributed to the increased efficacy of Glucantime relative to the other compounds, and it remains possible that the anti-parasitic effect of compounds 1, 2 and 3 would be improved by increasing the concentration of compounds or changing the therapeutic route.

Conclusion
The higher activities of benzophenone derivatives 1 and 2 against L. (L.) infantum chagasi in vivo (in comparison to compound 3 were compatible to the previously observed in vitro leishmanicidal activity and may be improved by increasing the concentration of compounds in each dose, changing the therapeutic regimen by increasing the number of treatment days, treating two or more times a day, or a combination of them. The pentavalent antimonial Glucantime was the most effective in the treatment of hamsters infected with L. (L.) infantum chagasi, but this drug is known by its high toxicity, and despite less potent, benzophenone derivatives such as compounds 1 and 2 could be interesting prototypes for the development of new effective and safe drug candidates. Further studies are now required to explore different therapeutic regimen and toxicity, and additional immunological and pharmacological assays could clarify the mode of action of compounds these benzophenone derivatives against Leishmania.