Assessing the effectiveness of AS-48 in experimental mice models of Chagas' disease.

OBJECTIVES: We report the in vivo trypanocidal activity of the bacteriocin AS-48 (lacking toxicity), which is produced by Enterococcus faecalis, against the flagellated protozoan Trypanosoma cruzi, the aetiological agent of Chagas' disease. METHODS: We determined the in vivo activity of AS-48 against the T. cruzi Arequipa strain in BALB/c mice (in both acute and chronic phases of Chagas' disease). We evaluated the parasitaemia, the reactivation of parasitaemia after immunosuppression and the nested parasites in the chronic phase by PCR in target tissues. RESULTS: AS-48 reduced the parasitaemia profile in acute infection and showed a noteworthy reduction in the parasitic load in chronic infection after immunosuppression according to the results obtained by PCR (double-checking to demonstrate cure). CONCLUSIONS: AS-48 is a promising alternative that provides a step forward in the development of a new therapy against Chagas' disease.

Repositioning of leishmanicidal [1,2,3]Triazolo[1,5-a]pyridinium salts for Chagas disease treatment: Trypanosoma cruzi cell death involving mitochondrial membrane depolarisation and Fe-SOD inhibition.

Trypanosomatidae is a family of unicellular parasites belonging to the phylum Euglenozoa, which are causative agents in high impact human diseases such as Leishmaniasis, Chagas disease and African sleeping sickness. The impact on human health and local economies, together with a lack of satisfactory chemotherapeutic treatments and effective vaccines, justifies stringent research efforts to search for new disease therapies. Here, we present in vitro trypanocidal activity data and mode of action data, repositioning leishmanicidal [1,2,3]Triazolo[1,5-a]pyridinium salts against Trypanosoma cruzi, the aetiological agent of Chagas disease. This disease is one of the most neglected tropical diseases and is a major public health issue in Central and South America. The disease affects approximately 6-7 million people and is widespread due to increased migratory movements. We screened a suite of leishmanicidal [1,2,3]Triazolo[1,5-a]pyridinium salt compounds, of which compounds 13, 20 and 21 were identified as trypanocidal drugs. These compounds caused cell death in a mitochondrion-dependent manner through a bioenergetic collapse. Moreover, compounds 13 and 20 showed a remarkable inhibition of iron superoxide dismutase activity of T. cruzi, a key enzyme in the protection from the damage produced by oxidative stress.

Effective Tetradentate Compound Complexes against Leishmania spp. that Act on Critical Enzymatic Pathways of These Parasites.

The spectrum and efficacy of available antileishmanial drugs is limited. In the present work we evaluated in vitro the antiproliferative activity of 11 compounds based on tetradentate polyamines compounds against three Leishmania species (L. braziliensis, L. donovani and L. infantum) and the possible mechanism of action. We identified six compounds (3, 5, 6, 7, 8 and 10) effective against all three Leishmania spp both on extracellular and intracellular forms. These six most active leishmanicidal compounds also prevent the infection of host cells. Nevertheless, only compound 7 is targeted against the Leishmania SOD. Meanwhile, on the glucose metabolism the tested compounds have a species-specific effect on Leishmania spp.: L. braziliensis was affected mainly by 10 and 8, L. donovani by 7, and L. infantum by 5 and 3. Finally, the cellular ultrastructure was mainly damaged by 11 in the three Leishmania spp. studied. These identified antileishmania candidates constitute a good alternative treatment and will be further studied.

In vitro and in vivo identification of tetradentated polyamine complexes as highly efficient metallodrugs against Trypanosoma cruzi.

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), a series of tetraamine-based compounds was prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by PCR and reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels by (1)H NMR and TEM studies. Finally, as tetraamines are potentially capable of casuing oxidative damage in the parasites, the study was completed by assessing their activity as potential iron superoxide dismutase (Fe-SOD) and trypanothione reductase (TR) inhibitors. High-selectivity indexes observed in vitro were the basis of promoting three of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Tetraamines 2 and 3 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression and curative rates of 33 and 50%, respectively. Tetraamine 3 turned out to be a great inhibitor of Fe-SOD and TR. The high anti-parasitic activity and low toxicity render these tetraamines appropriate molecules for the development of an affordable anti-Chagas agent.

Effective anti-leishmanial activity of minimalist squaramide-based compounds.

In order to evaluate the in vitro leishmanicidal activity of N,N'-Squaramides derivatives, compounds that feature both hydrogen bond donor and acceptor groups and are capable of multiple interactions with complementary sites, against Leishmania infantum, Leishmania braziliensis and Leishmania donovani a series of 18compounds was prepared and assayed on extracellular and intracellular parasite forms. Infectivity and cytotoxicity tests were performed on J774.2 macrophage cells using meglumine antimoniate (Glucantime) as the reference drug. Changes in metabolite excretion by 1H-NMR and the ultrastructural alterations occurring in the parasites treated using transmission electron microscopy (TEM), was analyzed. Compounds 1, 7, 11, 14 and 17 were the more active and less toxic. Infection rates showed that the order of effectiveness was 17 > 11 > 14 > 7 for both L. infantum and L. braziliensis and in the same way, the compound 1 for L. donovani. All these compounds have altered the typical structure of the promastigotes, glycosomes and mitochondria. These severe modifications by the compounds are the ultimate reasons for the alterations observed in the excretion products. The Squaramide 17 (3-(butylamino)-4-((3-(dimetilamino)propyl)(methyl)amino)cyclobut-3-en-1,2-dione) was clearly the most efficient of all compounds. The data appear to confirm that the severe modifications generated in organelles such as glycosomes or mitochondria by the compounds are the ultimate reasons for the alterations observed in the excretion products of all species. The activity, stability, low cost of starting materials, and straightforward synthesis make amino squaramides appropriate molecules for the development of an affordable anti-leishmanial agent.

Avian Plasmodium in invasive and native mosquitoes from southern Spain.

BACKGROUND: The emergence of diseases of public health concern is enhanced by factors associated with global change, such as the introduction of invasive species. The Asian tiger mosquito (Aedes albopictus), considered a competent vector of different viruses and parasites, has been successfully introduced into Europe in recent decades. Molecular screening of parasites in mosquitoes (i.e. molecular xenomonitoring) is essential to understand the potential role of different native and invasive mosquito species in the local circulation of vector-borne parasites affecting both humans and wildlife. METHODS: The presence of avian Plasmodium parasites was molecularly tested in mosquitoes trapped in five localities with different environmental characteristics in southern Spain from May to November 2022. The species analyzed included the native Culex pipiens and Culiseta longiareolata and the invasive Ae. albopictus. RESULTS: Avian Plasmodium DNA was only found in Cx. pipiens with 31 positive out of 165 mosquito pools tested. None of the Ae. albopictus or Cs. longiareolata pools were positive for avian malaria parasites. Overall, eight Plasmodium lineages were identified, including a new lineage described here. No significant differences in parasite prevalence were found between localities or sampling sessions. CONCLUSIONS: Unlike the invasive Ae. albopictus, Cx. pipiens plays a key role in the transmission of avian Plasmodium in southern Spain. However, due to the recent establishment of Ae. albopictus in the area, further research on the role of this species in the local transmission of vector-borne pathogens with different reservoirs is required.

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis.

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.

Leishmanicidal activity of nine novel flavonoids from Delphinium staphisagria.

OBJECTIVES: To evaluate the in vitro leishmanicidal activity of nine flavonoid derivatives from Delphinium staphisagria against L. infantum and L. braziliensis. DESIGN AND METHODS: The in vitro activity of compounds 1-9 was assayed on extracellular promastigote and axenic amastigote forms and on intracellular amastigote forms of the parasites. Infectivity and cytotoxicity tests were carried on J774.2 macrophage cells using Glucantime as the reference drug. The mechanisms of action were analysed performing metabolite excretion and transmission electronic microscope ultrastructural alteration studies. RESULTS: Nine flavonoids showed leishmanicidal activity against promastigote as well as amastigote forms of Leishmania infantum and L. braziliensis. These compounds were nontoxic to mammalian cells and were effective at similar concentrations up to or lower than that of the reference drug (Glucantime). The results showed that 2(″)-acetylpetiolaroside (compound 8) was clearly the most active. CONCLUSION: This study has demonstrated that flavonoid derivatives are active against L. infantum and L. braziliensis.

Biological characteristics of the Trypanosoma cruzi Arequipa strain make it a good model for Chagas disease drug discovery.

Trypanosoma cruzi, the causative agent of Chagas disease (CD), is a genuine parasite with tremendous genetic diversity and a complex life cycle. Scientists have studied this disease for more than 100 years, and CD drug discovery has been a mainstay due to the absence of an effective treatment. Technical advances in several areas have contributed to a better understanding of the complex biology and life cycle of this parasite, with the aim of designing the ideal profile of both drug and therapeutic options to treat CD. Here, we present the T. cruzi Arequipa strain (MHOM/Pe/2011/Arequipa) as an interesting model for CD drug discovery. We characterized acute-phase parasitaemia and chronic-phase tropism in BALB/c mice and determined the in vitro and in vivo benznidazole susceptibility profile of the different morphological forms of this strain. The tropism of this strain makes it an interesting model for the screening of new compounds with a potential anti-Chagas profile for the treatment of this disease.

5-Nitroindazole derivatives as potential therapeutic alternatives against Acanthamoeba castellanii.

Amoebas of the genus Acanthamoeba are distributed worldwide, including species with a high pathogenic capacity for humans. In a similar way to what occurs with other parasitic protozoa, the available treatments show variable effectiveness in addition to high toxicity, which demands the development of new treatments. Positive results of 5-nitroindazole derivatives against several protozoa parasites suggest that these compounds may be a promising tool for the development of efficient antiparasitic drugs. In the present work we have evaluated the in vitro activity of ten 5-nitroindazole derivatives against Acanthamoeba castellanii trophozoites and cysts. To that end, AlamarBlue Assay Reagent® was used to determine the activity against trophozoites compared to the reference drug chlorhexidine digluconate. Cytotoxicity of the compounds was evaluated using Vero cells. The activity on cysts was evaluated by light microscopy and using a Neubauer chamber to quantifying cysts and presence of trophozoites, as an indication of cyst. Our results showed the effectiveness of the 5-nitroindazole derivatives tested against both trophozoites and cysts of A. castellani highlighting 5-nitroindazole derivative 8 which showed a 80% activity on cysts, which is higher than that of the reference drug. Moreover, 5-nitroindazole derivatives 8, 9 and 10 were more effective on trophozoites than the reference drug showing IC50 values lower than 5 µM. Taking together these results, these 5-nitroindazole derivatives specially compound 8, might be a promising alternative for the development of more efficient treatments against A. castellani infection.

Identification of Aryl Polyamines Derivatives as Anti-Trypanosoma cruzi Agents Targeting Iron Superoxide Dismutase.

Chagas disease (CD) is a tropical and potentially fatal infection caused by Trypanosoma cruzi. Although CD was limited to Latin America as a silent disease, CD has become widespread as a result of globalization. Currently, 6-8 million people are infected worldwide, and no effective treatment is available. Here, we identify new effective agents against T. cruzi. In short, 16 aryl polyamines were screened in vitro against different T. cruzi strains, and lead compounds were evaluated in vivo after oral administration in both the acute and chronic infections. The mode of action was also evaluated at the energetic level, and its high activity profile could be ascribed to a mitochondria-dependent bioenergetic collapse and redox stress by inhibition of the Fe-SOD enzyme. We present compound 15 as a potential compound that provides a step forward for the development of new agents to combat CD.

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.

Measuring outcomes in allergic rhinitis: psychometric characteristics of a Spanish version of the congestion quantifier seven-item test (CQ7).

BACKGROUND: No control tools for nasal congestion (NC) are currently available in Spanish. This study aimed to adapt and validate the Congestion Quantifier Seven Item Test (CQ7) for Spain. METHODS: CQ7 was adapted from English following international guidelines. The instrument was validated in an observational, prospective study in allergic rhinitis patients with NC (N = 166) and a control group without NC (N = 35). Participants completed the CQ7, MOS sleep questionnaire, and a measure of psychological well-being (PGWBI). Clinical data included NC severity rating, acoustic rhinometry, and total symptom score (TSS). Internal consistency was assessed using Cronbach's alpha and test-retest reliability using the intraclass correlation coefficient (ICC). Construct validity was tested by examining correlations with other outcome measures and ability to discriminate between groups classified by NC severity. Sensitivity and specificity were assessed using Area under the Receiver Operating Curve (AUC) and responsiveness over time using effect sizes (ES). RESULTS: Cronbach's alpha for the CQ7 was 0.92, and the ICC was 0.81, indicating good reliability. CQ7 correlated most strongly with the TSS (r = 0.60, p < 0.01), the PGWBI general health dimension (r = 0.56, p < 0.01), and the MOS Sleep scale 'sleep short of breath' dimension (r = 0.49, p < 0.01). Correlations with acoustic rhinometry were generally low. The instrument discriminated well between NC severity groups (ES 0.33-2.07) and AUC was 0.93, indicating excellent sensitivity and specificity. The measure was responsive to change (ES = 1.1) in patients reporting improvement in NC. CONCLUSIONS: The Spanish version of the CQ7 is appropriate for detecting, measuring, and monitoring NC in allergic rhinitis patients.

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.

Preclinical studies of toxicity and safety of the AS-48 bacteriocin.

The in vitro antimicrobial potency of the bacteriocin AS-48 is well documented, but its clinical application requires investigation, as its toxicity could be different in in vitro (haemolytic and antibacterial activity in blood and cytotoxicity towards normal human cell lines) and in vivo (e.g. mice and zebrafish embryos) models. Overall, the results obtained are promising. They reveal the negligible propensity of AS-48 to cause cell death or impede cell growth at therapeutic concentrations (up to 27 µM) and support the suitability of this peptide as a potential therapeutic agent against several microbial infections, due to its selectivity and potency at low concentrations (in the range of 0.3-8.9 µM). In addition, AS-48 exhibits low haemolytic activity in whole blood and does not induce nitrite accumulation in non-stimulated RAW macrophages, indicating a lack of pro-inflammatory effects. The unexpected heightened sensitivity of zebrafish embryos to AS-48 could be due to the low differentiation state of these cells. The low cytotoxicity of AS-48, the absence of lymphocyte proliferation in vivo after skin sensitization in mice, and the lack of toxicity in a murine model support the consideration of the broad spectrum antimicrobial peptide AS-48 as a promising therapeutic agent for the control of a vast array of microbial infections, in particular, those involved in skin and soft tissue diseases.

New polyamine drugs as more effective antichagas agents than benznidazole in both the acute and chronic phases.

Despite the continuous research effort that has been made in recent years to find ways to treat the potentially life threatening Chagas disease (CD), this remains the third most important infectious disease in Latin America. CD is an important public health problem affecting 6-7 million people. Since the need to search for new drugs for the treatment of DC persists, in this article we present a panel of new polyamines based on the tripodal structure of tris(2-aminomethyl)amine (tren) that can be prepared at low cost with high yields. Moreover, these polyamines present the characteristic of being water-soluble and resistant to the acidic pH values of stomach, which would allow their potential oral administration. In vitro and in vivo assays permitted to identify the compound with the tren moiety functionalized with one fluorene unit (7) as a potential antichagas agent. Compound 7 has broader spectrum of action, improved efficacy in acute and chronic phases of the disease and lower toxicity than the reference drug benznidazole. Finally, the action mechanisms studied at metabolic and mitochondrial levels shows that the trypanocidal activity of compound 7 could be related to its effect at the glycosomal level. Therefore, this work allowed us to select compound 7 as a promising candidate to perform preclinical evaluation studies.

Tetradentate polyamines as efficient metallodrugs for Chagas disease treatment in murine model.

A series of tetraamine-based compounds was prepared, and their trypanocidal effects against Trypanosoma cruzi and cytotoxicity were determined through the determination of IC50 values. In vivo assays were performed in mice, where parasitaemia levels were quantified by fresh blood examination and the assignment of a cure was determined by polymerase chain reaction and reactivation of blood parasitaemia levels after immunosuppression. The mechanisms of action were elucidated at metabolic and ultra-structural levels, by 1H NMR, Fe-SOD inhibition and TEM studies. The high-selectivity indexes observed in vitro were the basis of promoting one of the tested compounds to in vivo assays. Compound 6 induced a remarkable decrease in the reactivation of parasitaemia after immunosuppression and curative rates of 33%. The experiments allowed us to select compound 6 as a promising candidate for treating Chagas disease, but a further high-level study should be considered to obtain an improved efficiency.

In vitro activity of C20-diterpenoid alkaloid derivatives in promastigotes and intracellular amastigotes of Leishmania infantum.

The in vitro anti-proliferative effects are described of several atisine-type diterpenoid alkaloids against the protozoan parasite Leishmania infantum, which causes human visceral leishmaniasis and canine leishmaniasis in the Mediterranean basin, as well as human cutaneous leishmaniasis throughout the Mediterranean region. From a total of 43 compounds tested, including C19- and C20-diterpene alkaloids from several chemical classes, only 15,22-O-diacetyl-19-oxo-dihydroatisine, azitine and isoazitine were highly active against cultures of the parasite (promastigote form) with IC50 values within the range of the reference drug pentamidine-isothionate (7.39-12.80 mg/L for the test compounds, 11.32 mg/L for the positive control). These compounds were not toxic to the host cell. When treated with a dosage of 5 microg/mL of the active compounds (half of their IC50), the promastigote forms lost 80% of their infection capacity and the multiplication of extracellular forms of L. infantum was severely affected. The study showed that atisine-type C20-diterpenoid alkaloids exhibited promising anti-leishmanial properties with strong molecular selectivity. These might have implications for other intracellular pathogens- or phylogenetically related parasites, such as Trypanosoma spp.

An in vitro iron superoxide dismutase inhibitor decreases the parasitemia levels of Trypanosoma cruzi in BALB/c mouse model during acute phase.

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), two hydroxyphthalazine derivative compounds were prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels, by (1)H NMR and TEM studies. Finally, as these compounds are potentially capable of causing oxidative damage in the parasites, the study was completed, by assessing their activity as potential iron superoxide dismutase (Fe-SOD) inhibitors. High-selectivity indices observed in vitro were the basis of promoting one of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Compound 2 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression. Compound 2 turned out to be a great inhibitor of Fe-SOD. The high antiparasitic activity and low toxicity together with the modest costs for the starting materials render this compound an appropriate molecule for the development of an affordable anti-Chagas agent.