Clotilde Marín

In Vitro and in Vivo Trypanocidal Activity of Flavonoids from Delphinium staphisagria against Chagas Disease

The in vitro and in vivo trypanocidal activities of nine flavonoids (1-9) isolated from the aerial parts of Delphinium staphisagria have been studied in both the acute and chronic phases of Chagas disease. The antiproliferative activity of these substances against Trypanosoma cruzi (epimastigote, amastigote, and trypomastigote forms) in some cases exhibited more potent antitrypanosomatid activity and lower toxicity than the reference drug, benznidazole. Studies in vitro using ultrastructural analysis together with metabolism-excretion studies were also performed in order to identify the possible action mechanism of the compounds tested. Alterations mainly at the level of the mitochondria may explain metabolic changes in succinate and acetate production, perhaps due to the disturbance of the enzymes involved in sugar metabolism within the mitochondrion. In vivo studies provided results consistent with those observed in vitro. No signs of toxicity were detected in mice treated with the flavonoids tested, and the parasitic charge was significantly lower than in the control assay with benznidazole. The effects of these compounds were also demonstrated with the change in the anti-T. cruzi antibody levels during the chronic stage.

In vitro and in vivo antiparasital activity against Trypanosoma cruzi of three novel 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one-based complexes.

Conventional reactions of the versatile multidentate ligand 5-methyl-1,2,4-triazolo[1,5-a] pyrimidin-7(4H)-one (HmtpO) with metallic(II) perchlorate salts lead to three novel multidimensional complexes [Cu(HmtpO)(2)(H(2)O)(3)](ClO(4))(2)·H(2)O (1), {[Cu(HmtpO)(2)(H(2)O)(2)](ClO(4))(2) ·2HmtpO}(n) (2) and {[Co(HmtpO)(H(2)O)(3)](ClO(4))(2)·2H(2)O}(n) (3). We have tested the antiparasital activity in vitro and in vivo of the three new complexes against Trypanosoma cruzi showing very promising results and overcoming clearly the reference drug commonly used for the Chagas disease treatment, benznidazole.

In vitro leishmanicidal activity of imidazole- or pyrazole-based benzo[g]phthalazine derivatives against Leishmania infantum and Leishmania braziliensis species

OBJECTIVES To evaluate the in vitro leishmanicidal activity of imidazole-based (1-4) and pyrazole-based (5-6) benzo[g]phthalazine derivatives against Leishmania infantum and Leishmania braziliensis. METHODS The in vitro activity of compounds 1-6 was assayed on extracellular promastigote and axenic amastigote forms, and on intracellular amastigote forms of the parasites. Infectivity and cytotoxicity tests were performed on J774.2 macrophage cells using meglumine antimoniate (Glucantime) as the reference drug. The mechanisms of action were analysed by iron superoxide dismutase (Fe-SOD) and copper/zinc superoxide dismutase (CuZn-SOD) inhibition, metabolite excretion and transmission electronic microscopy (TEM). RESULTS Compounds 1-6 were more active and less toxic than meglumine antimoniate. Data on infection rates and amastigote mean numbers showed that 2, 4 and 6 were more active than 1, 3 and 5 in both L. infantum and L. braziliensis. The inhibitory effect of these compounds on the antioxidant enzyme Fe-SOD of promastigote forms of the parasites was remarkable, whereas inhibition of human CuZn-SOD was negligible. The ultrastructural alterations observed in treated promastigote forms confirmed the greater cell damage caused by the most active compounds 2, 4 and 6. The modifications observed by (1)H-NMR in the nature and amounts of catabolites excreted by the parasites after treatment with 1-6 suggested that the catabolic mechanisms could depend on the structure of the side chains linked to the benzo[g]phthalazine moiety. CONCLUSIONS All the compounds assayed were active in vitro against the two Leishmania species and were less toxic against mammalian cells than the reference drug, but the monosubstituted compounds were significantly more effective and less toxic than their disubstituted counterparts.

Antileishmaniasis activity of flavonoids from Consolida oliveriana.

A set of flavonoids from Consolida oliveriana, kaempferol (1), quercetin (2), trifolin (3), and acetyl hyperoside (5) and their O-acetyl derivatives (1a, 2a, 3a), and octa-O-acetylhyperoside (4) showed leishmanicidal activity against promastigote as well as amastigote forms of Leishmania spp. The cellular proliferation, metabolic, and ultrastructural studies showed that the acetylated compounds 2a, 3a, and 4 were highly active against Leishmania (V.) peruviana, while 2a as well as 4 were effective against Leishmania (V.) braziliensis. These compounds were not cytotoxic and are effective at similar concentrations up to or lower than the reference drugs (pentostam and glucantim).

Biological activity of three novel complexes with the ligand 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one against Leishmania spp.

OBJECTIVES We report on new 1,2,4-triazolo[1,5-a]pyrimidine complexes that have been developed and examined for both antiproliferative in vitro activity against Leishmania infantum and Leishmania braziliensis, and report their possible mechanism of action on L. infantum and L. braziliensis. RESULTS Antileishmanial effects are described for newly synthesized Cu(II) and Co(II) complexes containing 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one (HmtpO) as a ligand. These complexes display a wide structural diversity: (i) mononuclear unit, [Cu(HmtpO)2(H2O)3](ClO4)2·H2O (1); (ii) two-dimensional framework, {[Cu(HmtpO)2(H2O)2](ClO4)(2)·2HmtpO}n (2); and (iii) chains, {[Co(HmtpO)(H2O)3](ClO4)(2)·2H2O}n (3). Compounds 1 and 2 appeared to be the most active against L. infantum (IC50 20.0 and 24.4 μM, respectively), and compounds 1 and 3 the most active against L. braziliensis (IC50 22.1 and 23.5 μM, respectively), with IC50s similar to those of the reference drug, glucantime (18.0 μM for L. infantum and 25.6 μM for L. braziliensis). These compounds were not toxic towards J774.2 macrophages. IC25 decreased infection capacity and severely reduced the multiplication of intracellular amastigotes, following the trend 1>3>2 for L. infantum and 3>1>2 for L. braziliensis. These complexes had an effect on the energy metabolism of the parasites at the level of the NAD+/NADH balance and the organelle membranes, causing their degradation and cell death. CONCLUSIONS Cellular proliferation, metabolic and ultrastructural studies showed that the compounds 2>1>3 were highly active against L. infantum and L. braziliensis.

In Vivo Trypanosomicidal Activity of Imidazole- or Pyrazole-Based Benzo(g)phthalazine Derivatives against Acute and Chronic Phases of Chagas Disease

The in vivo trypanosomicidal activity of the imidazole-based benzo[g]phthalazine derivatives 1-4 and of the new related pyrazole-based compounds 5 and 6 has been studied in both the acute and chronic phases of Chagas disease. As a rule, compounds 1-6 were more active and less toxic than benznidazole in the two stages of the disease, and the monosubstituted derivatives 2, 4, and 6 were more effective than their disubstituted analogs. Feasible mechanisms of action of compounds 1-6 against the parasite have been explored by considering their inhibitory effect on the Fe-SOD enzyme, the nature of the excreted metabolites and the ultrastructural alterations produced. A complementary histopathological analysis has confirmed that the monosubstituted derivatives are less toxic than the reference drug, with the behavior of the imidazole-based compound 4 being especially noteworthy.

Copper (II) complexes of [1,2,4]triazolo [1,5-a]pyrimidine derivatives as potential anti-parasitic agents.

Anti-proliferative effects are described for newly synthesised copper (II) complexes of two triazolo-pyrimidine derivatives (1,2,4-triazolo-[1,5-a]pyrimidine, tp, and 5,7-dimethyl 1,2,4-triazolo-[1,5-a]pyrimidine, dmtp) against to Trypanosoma cruzi and Leishmania (Viannia) peruviana. Of the compounds assayed, those that presented the ligand tp and auxiliary ligand 1,10-phenanthroline (C24b, C49) were most highly active against to T. cruzi with IC(50) within the range of the reference drug benznidazole. These compounds, together with C35 were the most effective against L. (V.) peruviana with an IC(50) greater than that presented by reference drugs (Pentostam and Glucantim). These compounds were not toxic to the host cell. IC(25) diminished the infection capacity and severely reduced the multiplication of intracellular forms of T. cruzi, and L. (V.) peruviana. In the case of T. Cruzi, the transformation to trypomastigote was seriously depressed. Copper (II) complexes C24b, C49 and C35, acted on the energy metabolism of the parasites at the level of the NAD(+)/NADH balance and at the level of the organelle membranes, causing degradation and cell death.

Phthalazine derivatives containing imidazole rings behave as Fe-SOD inhibitors and show remarkable anti-T. cruzi activity in immunodeficient-mouse mode of infection.

A series of new phthalazine derivatives 1-4 containing imidazole rings were prepared. The monoalkylamino substituted derivatives 2 and 4 were more active in vitro against T. cruzi and less toxic against Vero cells than both their disubstituted analogues and the reference drug benznidazole. Compounds 2 and 4 highly inhibited the antioxidant parasite enzyme Fe-SOD, and molecular modeling suggested that they interact with the H-bonding system of the iron atom moiety. In vivo tests on the acute phase of Chagas disease gave parasitemia inhibition values twice those of benznidazole, and a remarkable decrease in the reactivation of parasitemia was found in the chronic phase for immunodeficient mice. Glucose metabolism studies showed that compounds 1-4 did not affect the succinate pathway but originated important changes in the excretion of pyruvate metabolites. The morphological alterations found in epimastigotes treated with 1-4 confirmed extensive cytoplasm damage and a high mortality rate of parasites.

An Iron-Superoxide Dismutase Antigen-Based Serological Screening of Dogs Indicates Their Potential Role in the Transmission of Cutaneous Leishmaniasis and Trypanosomiasis in Yucatan, Mexico

An increasing number of studies have reported high infection rates for American cutaneous leishmaniasis in dogs, which have thus been proposed as the reservoir host. Canine leishmaniasis is widespread in different states in Mexico, where a number of Leishmania species have been isolated from dogs. In the present study, the detection of different Leishmania species is described in stray dogs from two localities, namely Tulum and Celestún on the Yucatan Peninsula (Mexico). The use of iron-superoxide dismutase excreted by the parasites as the antigen fraction and enzyme-linked immunosorbent assay and western blot tests allowed us to confirm the presence of at least three species of Leishmania (Le. mexicana, Le. braziliensis, and Le. panamensis), some of which are reported for the first time in this species. In addition to a high prevalence of Le. mexicana and Le. braziliensis, and to a lesser degree, Le. panamensis, there is a significant prevalence of Trypanosoma cruzi, suggesting that the dog may be a source of transmission of trypanosomiasis. However, a more thorough epidemiological study on the dog population, both wild as well as urban, of the Yucatan Peninsula will be required to design a control strategy for these diseases, paying particular attention to the population affected and even broadening the study to other Mexican states as well as neighboring countries. These results again confirm that iron-superoxide dismutase excreted by the different trypanosomatid species constitutes a good source of antigen for serodiagnosis in epidemiological studies.

Therapeutic Potential of New Pt(II) and Ru(III) Triazole-Pyrimidine Complexes against Leishmania donovani

We have already established an in vitro culture system using murine macrophages infected with Leishmania donovani in which the time course of parasite growth is determined quantitatively. We adopted this system for the screening of three triazole-pyrimidine derivatives that would ideally prove to be effective against L. donovani with no toxicity to the host cell. Amphotericin B deoxycholate was used as the standard drug and gave a IC50 value of 3.89 µg/ml. The three triazole-pyrimidine compounds assayed have been reported to be potent growth inhibitors of L. donovani promastigote and amastigote stages. Compounds SPIV and SPVI exhibited the highest toxicity for extracellular forms of parasites, with IC50 values of 19.95 and 21.61 µg/ml, respectively. The triazole-pyrimidine SPV, although to a lower degree, also showed pronounced effects against promastigote forms with IC50 of 33.14 µg/ml. Drug activity was higher against amastigote than against promastigote stages. The compounds SPIV and SPVI interfered with the synthesis of macromolecules, affecting primarily DNA at the lower concentration tested (5 µg/ml), while SPV also showed interference, though to a lesser extent, and at a higher concentration (15 µg/ml) the percentage of inhibition rose considerably. The synthesis or RNA and proteins was also depressed significantly by these compounds at administration rates of 15 µg/ml. Ultrastructural alterations were evident in the main organelles of L. donovani (nucleus, kinetoplast, mitochondria), after the addition of the three compounds at a concentration of 5 µg/ml, to the in vitro culture. The in vitro promastigote forms of L. donovani can degrade glucose to carbon dioxide, and part of the carbon skeleton of the glucose is excreted as end metabolites. The excretion of these metabolites, mainly acetate, was also inhibited by the three compounds assayed, suggesting that this could be due to a direct effect on some of the enzymes related to this fermentation pathway or to the inhibition exerted by the compounds on enzyme synthesis.