Nuevas perspectivas en el tratamiento de la leishmaniosisEvaluación de derivados hidroxámicos y (-)-¿-bisabolol

Resumen

Leishmaniasis is a neglected disease which occurs in 98 countries across five continents with 350 million people living at risk. Visceral leishmaniasis has been estimated at 300,000 new cases each year. Current drugs used in visceral leishmaniasis present several drawbacks including high toxicity and adverse effects, increasing resistance, high cost and variability in specificity according to geographical region. Pentavalent antimonials show high toxicity and yet they have been the first-line treatment for decades, whereas pentamidine, amphotericin B and paromomycin are second-line treatment drugs. The last drug to be added to the therapeutic arsenal for leishmaniasis is miltefosine: initially intended as an anticancer drug, it became the first oral treatment against leishmaniasis, but it is not exempt from problems as well. The therapeutic requirements for visceral leishmaniasis include an oral, safe, effective and low-cost treatment. Therefore, new approaches for treating leishmaniasis and other neglected diseases are urgently needed. Histone deacetylases (HDAC) inhibitors have proved effective in the treatment of cancer and several molecules have been approved for the treatment of cutaneous T-cell lymphoma and they are showing promising activity in laboratory models of malaria. Histone-modifying enzymes, such as HDAC, are essential for the modulation of chromatin structure, thus indirectly regulating of gene expression in eukaryotic species. Their relevance seems even higher in trypanosomatid parasites, organisms which lack canonical transcription regulation. On the other hand, interest in using natural products against parasitic diseases has raised. (-)-¿-Bisabolol is monocyclic sesquiterpene widely used in cosmetic and dermatologic preparations that is found in many essential oils of traditional medicine plants such as Matricaria chamomilla, where this sesquiterpene is found in up to 50% concentrations. This natural compound has shown anti-inflammatory, anti-irritant and microbicidal activities and it has been successfully evaluated against L. infantum promastigotes. In this scenario, the aim of this work was to put forward new treatment perspectives for leishmaniasis. In order to accomplish it, the objectives of the study were to screen new synthetic HDAC inhibitors and the natural compound (-)-¿-bisabolol, to characterize HDAC enzymes in Leishmania, to get an insight into the mechanism of action of (-)-¿-bisabolol, to evaluate in vivo the most active structures; and finally, to assess the potential effectiveness and safety of these molecules in a pilot clinical trial involving dogs with canine leishmaniasis (CanL) naturally acquired. For in vitro evaluation, Bone marrow derived macrophages were infected with L. infantum, L. donovani, L. tropica or L. major promastigotes, or Trypanosoma cruzi trypomastigotes and incubated with the compounds at different concentrations. Pentamidine isethionate and meglumine antimoniate were used as reference drug. Inhibitory Concentration 50% (IC50) and Cytotoxic Concentration 50% (CC50) were calculated and the compounds were selected upon these parameters and their selectivity indices (SI). The mitochondrial activity of Leishmania was evaluated during the treatment with (-)-¿-bisabolol, as well as its ability to induce nitric oxide synthesis in macrophages in vitro. HDAC enzymes were identifies through bioinformatics analysis, they were tagged by creating HDAC-GFP fusion protein gene constructs and transfected with them. The localization of the enzyme was assessed through fluorescence microscopy. Balb/c mice were infected intraperitoneally with stationary-phase promastigotes. They were treated with MTC-305, MDG or (-)-¿-bisabolol at different doses, meglumine antimoniate at 104 mg SbV/kg or a combination of each compound with meglumine antimoniate. The parasites loads in spleen and liver were evaluated through quantitative PCR (qPCR). For the pilot clinical trials, 22 dogs with naturally acquired CanL were selected upon clinical and parasitological eligibility criteria. With the consent of their owners, they were treated with MTC-305 (3,75 mg/kg subcutaneously) or (-)-¿-bisabolol (30 mg/kg through oral route) and Glucantime® was used as a control reference drug (100 mg/kg subcutaneously). A combination of MTC-305 (1,5 mg/kg) and Glucantime® was also evaluated. Their clinical state was evaluated by calculating the clinical score, their immunological profile was evaluated through the determination of total IgG, IgG1 and IgG2 titres by IFAT and the quantitation of their cytokine expression level by reverse transcriptase qPCR. Parasite loads were evaluated in bone marrow, popliteal lymph node, peripheral blood and hair through qPCR. Efficacy criteria were established , based on the ability of the treatment to decrease bone marrow, popliteous lymph node and/or peripheral blood parasite loads. Among other synthetic compounds, (-)-¿-bisabolol, MTC-305 and MDG proved to be innocuous to mammal cells and very active against intracellular amastigotes of L. infantum (IC50 = 57, 3,2 and 0,7 µM, respectively). They also proved to be active in an in vivo model of visceral leishmaniasis due to L. infantum as they reduced parasite load in spleen and liver by more than 70% and 80% respectively without showing toxicity. They were more effective than meglumine antimoniate at reducing spleen parasite load and at least as effective as this antimonial drug in the liver. The possibility of oral administration is an advantageous feature of (-)-¿-bisabolol as well. In the pilot clinical trial, MTC-305 was effective in the six dogs treated, reducing bone marrow parasite loads in at least 50% whereas meglumine antimoniate was effective in two of the 6 dogs. Therefore the treatment with MTC-305 was more effective than the reference treatment without showing relevant toxicity. The combination was effective in 5 out of 6 dogs, thus not improving MTC-305 treatment, and it did not cause relevant side effects on the dogs. (-)-¿-bisabolol was effective in 3 out of 4 dogs and improved their Th1 response by enhancing their gamma interferon expression levels throughout the treatment without showing relevant safety issues, therefore being at least as effective as the current standard. One HDAC enzyme could be characterized in L. mexicana: it turned out to be associated with mitochondria, as it showed a mitochondrial distribution pattern in the promastigote stage. For the first time, a set if HDAC inhibitors have been evaluated in a laboratory model of visceral leishmaniasis due to L. infantum with promising results that were confirmed in a pilot clinical trials in dogs with naturally acquired canine leishmaniasis. (-)-¿-bisabolol showed outstanding results in the laboratory model of visceral leishmaniasis as well and they were also assessed in the pilot clinical trial, which revealed an immunostimulatory effect. Its oral bioavailability is a crucial advantage in the treatment of this disease.