Arylimidamides have potential for chemoprophylaxis against blood-transmitted Chagas disease

Chagas disease (CD) affects over 6 million people worldwide and can be transmitted iatrogenically. Crystal violet (CV) was previously used for pathogen reduction but has harmful side-effects. In the present study, three arylimidamides (AIAs) and CV were used to sterilize mice blood samples experimentally contaminated with bloodstream trypomastigotes (BT) of Trypanosoma cruzi, at non hemolytic doses. All AIAs were not toxic to mouse blood cells until the highest tested concentration (96 µM). The previous treatment of BT with the AIAs impaired the infection establishment of cardiac cell cultures. In vivo assays showed that pre-incubation of mouse blood samples with the AIAs and CV (96 µM) significantly suppressed the parasitemia peak, but only the AIA DB1831 gave ≥90% animal survival, while vehicle treated samples reached 0%. Our findings support further studies regarding the potential use of AIAs for blood bank purposes

Combined Treatment of Heterocyclic Analogues and Benznidazole upon Trypanosoma cruzi In Vivo

Chagas disease caused by Trypanosoma cruzi is an important cause of mortality and morbidity in Latin America but no vaccines or safe chemotherapeutic agents are available. Combined therapy is envisioned as an ideal approach since it may enhance efficacy by acting upon different cellular targets, may reduce toxicity and minimize the risk of drug resistance. Therefore, we investigated the activity of benznidazole (Bz) in combination with the diamidine prodrug DB289 and in combination with the arylimidamide DB766 upon T. cruzi infection in vivo. The oral treatment of T.cruzi-infected mice with DB289 and Benznidazole (Bz) alone reduced the number of circulating parasites compared with untreated mice by about 70% and 90%, respectively. However, the combination of these two compounds decreased the parasitemia by 99% and protected against animal mortality by 100%, but without providing a parasitological cure. When Bz (p.o) was combined with DB766 (via ip route), at least a 99.5% decrease in parasitemia levels was observed. DB766+Bz also provided 100% protection against mice mortality while Bz alone provided about 87% protection. This combined therapy also reduced the tissular lesions induced by T. cruzi infection: Bz alone reduced GPT and CK plasma levels by about 12% and 78% compared to untreated mice group, the combination of Bz with DB766 resulted in a reduction of GPT and CK plasma levels of 56% and 91%. Cure assessment through hemocultive and PCR approaches showed that Bz did not provide a parasitological cure, however, DB766 alone or associated with Bz cured ≥13% of surviving animals

Estudos in vitro e in vivo da atividade biológica de fluorquinolonas, tiossemicarbazonas, diamidinas aromáticas e aromáticas e as sobre Trypanosoma cruzi

Submitted by Tatiana Oliveira ([email protected]) on 2012-06-06T15:32:21Z No. of bitstreams: 1 denise_gj_batista_ioc_bp_0023_2009.pdf: 5405466 bytes, checksum: 0bb4fce7e31b0593bb33cff01fa3e688 (MD5)Made available in DSpace on 2012-06-06T15:32:21Z (GMT). No. of bitstreams: 1 denise_gj_batista_ioc_bp_0023_2009.pdf: 5405466 bytes, checksum: 0bb4fce7e31b0593bb33cff01fa3e688 (MD5) Previous issue date: 2009Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, BrasilNo centenario da descoberta da doenca de Chagas (DC), esta parasitose negligenciada causada pelo Trypanosoma cruzi e importante causa de mortalidade e morbidade na America Latina, sem vacinas ou agentes quimioterapicos satisfatorios. Esta tese objetivou analisar atividade, seletividade e mecanismos de acao sobre o T.cruzi de 04 classes de compostos (fluorquinolonas - FQ, tiossemicarbazonas - TS, diamidinas aromaticas - DA e arilimidamidas – AIA – uma nova classe a partir de DA) in vitro e in vivo. FQ como NOR e SPAR sao antibioticos com ampla acao bactericida, atuando sobre o DNA via toposoimerases. Embora SPAR e NOR nao tenham sido ativas, seus complexos metalicos de cobre-fenantrolina foram efetivos sobre tripomastigotas e formas intracelulares de T.cruzi, (IC50 1,62 a 4,65µM). TS sao agentes anti-tumorais e microbicidas que atuam sobre ribonucleotideo redutases e cisteina proteases. Todas TS apresentaram baixa toxicidade (>200µM) sobre celulas de mamiferos. Embora N4-metil-4-nitrobenzaldeido-tiossemicarbazona, N4-metill-4-nitroacetofenona-tiossemicarbazona e seus complexos metalicos de manganes (Mn) não tenham sido ativos, o complexo de Mn da N4-metil-4-nitrobenzofenona-tiossemicarbazona revelou-se moderadamente ativo sobre formas sanguineas (IC50 19µM). A terceira classe foi DA, ligantes de DNA com excelente atividade sobre diversos patogenos. Embora todas tenham baixa toxicidade sobre cardiomiocitos, somente tres (DB1645, DB1582 e DB1651) foram ativas contra formas sanguineas e intracelulares (IC50 entre 0,15 a 13,3µM), com atividade relacionada a curvatura das moleculas (sendo as moleculas lineares as nao efetivas). Todas DA localizam-se no nucleo e kDNA dos parasitos, com maior acumulo na ulti Analise in vitro da AIA DB766 sobre T.cruzi revelou: (i) excelente atividade sobre formas sanguineas (60nM) e intracelulares (25nM), mantendo otima acao mesmo quando o parasito e incubado com 96% de sangue de camundongo, sugerindo assim, um potencial uso profilatico, (ii) ação sobre diferentes cepas (peridomiciliares/silvestres), incluindo as naturalmente resistentes ao benznidazole (BZ), com superior eficacia que BZ, (iii) localizacao em compartimentos ricos em DNA, induzindo danos ultra-estruturais na mitocondria. Com base no excelente indice de seletividade (>533 e 714), ensaios foram conduzidos durante a infeccao aguda experimental por T.cruzi (cepas Y e Colombiana), em doses diarias ou alternadas =100mg/kg/dia da DB766 sozinha ou associada ao BZ, administradas via ip ou p.o. por ate 20 dias, com primeira dose no inicio da parasitemia. DB766 (25 e 50mg/kg/dia ip e 100mg/kg/dia p.o.) reduziu (>90%) a carga parasitaria (sangue e tecido cardiaco) e protegeu 90-100% contra mortalidade, com semelhante eficacia ao BZ. AIA reverteu inflamacao e alteracoes eletricas cardiacas e protegeu contra lesoes hepaticas e musculares induzidas pela infeccao. Doses sub-otimas de BZ associado com a DB289 (DA - pro-droga da DB75 – via p.o.) ou com a DB766 (ip) resultaram em =99% reducao de parasitemia e 100% de protecao sobre mortalidade. BZ (p.o)+DB766 (ip) resultou em cura parasitologica (2 em 15 animais) avaliada por hemocultivo e PCR. AIA foi a mais ativa e seletiva sobre T. cruzi, estimulando a continuidade de ensaios pre-clinicos com representantes desta classe visando identificacao de novos farmacos para a DCIn the centenary of the discovery of Chagas' disease (AD), this parasitosis caused by Trypanosoma cruzi neglected and important cause of morbidity and mortality in Latin America, no vaccines or chemotherapeutic agents satisfactory. This thesis aims to analyze activity, selectivity and mechanisms of action of T. cruzi on the 04 classes of compounds (fluoroquinolones - CF thiosemicarbazones - TS, aromatic diamidines - DA and arilimidamidas - AIA - a new class from DA) in vitro and in vivo. CF as NOR and SPAR are antibiotics with broad antibacterial action, acting on the DNA via toposoimerases. Although SPAR and NOR have not been active, their metallic complexes of copper-phenanthroline were effective on trypomastigotes and intracellular forms of T. cruzi (IC50 1.62 to 4.65 mM). TS are anti-tumor agents and microbicides that act on ribonucleotide reductase and cysteine ​​proteases. All TS showed low toxicity (> 200μM) on cells of mammals. Although N4-methyl-4-nitrobenzaldehyde thiosemicarbazone, N4-methyl-4-nitroacetophenone thiosemicarbazone and its metal complex of manganese (Mn) were not active, the complex of Mn N4-methyl-4-nitrobenzophenone thiosemicarbazone revealed was moderately active on blood forms (IC50 19μM). The third class was DA, DNA binders with excellent activity against many pathogens. Although all have low toxicity cardiomyocyte, only three (DB1645, DB1582 and DB1651) were active against blood forms and intracellular (IC 50 from 0.15 to 13.3 mM), activity related to the curvature of the molecules (the molecules being the non-linear effective). OF all located in the nucleus and kDNA from parasites, with greater accumulation in the ulti In vitro analysis of EIA DB766 on T.cruzi revealed: (i) excellent activity against bloodstream forms (60nm) and intracellular (25nm), while maintaining optimal action even when the parasite and incubated with 96% blood of mice, thus suggesting a potential prophylactic use, (ii) act on different strains (peridomestic / wild), including naturally resistant to benznidazole (BZ), with higher efficacy than BZ, (iii) location compartments rich DNA damage inducing ultrastructural the mitochondria . Based on the excellent selectivity index (> 533 and 714), tests were conducted during experimental acute infection by T. cruzi (Y and Colombian strains) in daily doses or alternate = 100mg/kg/day of DB766 alone or associated with BZ, administered ip or po for up to 20 days with the first dose at the onset of parasitaemia. DB766 (25 100mg/kg/day and 50mg/kg/day ip and po) reduced (> 90%) the parasite load (blood and cardiac tissue) and 90-100% protected against mortality, with similar efficacy to the BZ. EIA reversed inflammation and cardiac electrical changes and protected against liver injury induced by infection and muscle. Sub-optimal doses of BZ associated with DB289 (DA - pro-drug of DB75 - via po) or DB766 (ip) resulted in ≥ 99% reduction of parasitemia and 100% protective on mortality. BZ (po) + DB766 (ip) resulted in parasitological cure (2, 15 dogs) assessed by blood culture and PCR. EIA was the most active and selective against T. cruzi, encouraging the continuation of pre-clinical trials with representatives of this class in order to identification of new drugs for A

Inhibition of Trypanosoma cruzi proline racemase affects host-parasite interactions and the outcome of in vitro infection

Proline racemase is an important enzyme of Trypanosoma cruzi and has been shown to be an effective mitogen for B cells, thus contributing to the parasite’s immune evasion and persistence in the human host. Recombinant epimastigote parasites overexpressing TcPRAC genes coding for proline racemase present an augmented ability to differentiate into metacyclic infective forms and subsequently penetrate host-cells in vitro. Here we demonstrate that both anti T. cruzi proline racemase antibodies and the specific proline racemase inhibitor pyrrole-2- carboxylic acid significantly affect parasite infection of Vero cells in vitro. This inhibitor also hampers T. cruzi intracellular differentiation

Discovery of pyrrolo[2,3-b]pyridine (1,7-dideazapurine) nucleoside analogues as anti-Trypanosoma cruzi agents

Trypanosoma cruzi is the causative pathogen of Chagas disease and the main culprit for cardiac-related mortality in Latin-America triggered by an infective agent. Incapable of synthesizing purines de novo, this parasite depends on acquisition and processing of host-derived purines, making purine (nucleoside) analogues a potential source of antitrypanosomal agents. In this respect, hitherto 7-deazaadenosine (tubercidin) analogues attracted most attention. Here, we investigated analogues with an additional nitrogen (N1) removed. Structure-activity relationship investigation showed that C7 modification afforded analogues with potent antitrypanosomal activity. Halogens and small, linear carbon-based substituents were preferred. Compound 11 proved most potent in vitro, showed full suppression of parasitemia in a mouse model of acute infection, and elicited 100% animal survival after oral dosing at 25 mg/kg b.i.d. for 5 and 15 days. Cyclophosphamide-induced immunosuppression led to recrudescence. Washout experiments demonstrated a lack of complete clearance of infected cell cultures, potentially explaining the in vivo results

N6-modification of 7-deazapurine nucleoside analogues as anti-Trypanosoma cruzi and anti-Leishmania agents : structure-activity relationship exploration and in vivo evaluation

Chagas disease and leishmaniasis are two poverty-related neglected tropical diseases that cause high mortality and morbidity. Current treatments suffer from severe limitations and novel, safer and more effective drugs are urgently needed. Both Trypanosoma cruzi and Leishmania are auxotrophic for purines and absolutely depend on uptake and assimilation of host purines. This led us to successfully explore purine nucleoside analogues as chemotherapeutic agents against these and other kinetoplastid infections. This study extensively explored the modification of the 6-amino group of tubercidin, a natural product with trypanocidal activity but unacceptable toxicity for clinical use. We found that mono-substitution of the amine with short alkyls elicits potent and selective antitrypanosomal and antileishmanial activity. The methyl analogue 15 displayed the best in vitro activity against both T. cruzi and L. infantum and high selectivity versus host cells. Oral administration for five consecutive days in an acute Chagas disease mouse model resulted in significantly reduced peak parasitemia levels (75, 89 and 96% with 12.5, 25 and 50 mg/kg/day, respectively). as well as increased animal survival rates with the lower doses (83 and 67% for 12.5 and 25mg/kg/day, respectively)

Activity of DB289 alone or in combination with benznidazole (Bz) upon <i>T. cruzi</i>-infection in mice.

<p>(A): Parasitemia curve of infected mice that were either not treated or treated at 5 and 8 dpi with 25 and 50 mg/kg/day DB289 and 100 mg/kg/day Bz. (B): Parasitemia curve, (C) Cumulative Mortality and (D) Ponderal Curve of infected mice treated or not for twenty daily doses with 25 mg/kg/day DB289 combined or not with 50 mg/kg/day.</p

Cure assessment of DB289 and DB766 combined or not with benznidazole (Bz) in murine model of acute <i>T. cruzi</i>-infection.¹

1<p>Swiss male mice weight 20 to 24 g inoculated with 10⁴ blood trypomastigotes (Y strain).</p><p>Treatment was initiated at 5° dpi followed by 20 daily doses.</p>2<p>Intraperitoneal – ip.</p>3<p>per oral – p.o.</p

Phenotypic evaluation and in silico ADMET properties of novel arylimidamides in acute mouse models of Trypanosoma cruzi infection

Submitted by Sandra Infurna ([email protected]) on 2017-07-13T16:48:02Z No. of bitstreams: 1 cristiana_silva_etal_IOC_2017.pdf: 757102 bytes, checksum: c6e1b124c629b18cadf5c0a3ada29ee8 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-07-13T17:00:16Z (GMT) No. of bitstreams: 1 cristiana_silva_etal_IOC_2017.pdf: 757102 bytes, checksum: c6e1b124c629b18cadf5c0a3ada29ee8 (MD5)Made available in DSpace on 2017-07-13T17:00:16Z (GMT). No. of bitstreams: 1 cristiana_silva_etal_IOC_2017.pdf: 757102 bytes, checksum: c6e1b124c629b18cadf5c0a3ada29ee8 (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanossomatídeos. Rio de Janeiro, RJ. Brasil.Augusta University. Department of Chemistry and Physics. Augusta, USA.Georgia State University. Department of Chemistry. Atlanta, GA, USA.Georgia State University. Department of Chemistry. Atlanta, GA, USA / Mansoura University. Faculty of Pharmacy. Department of Pharmaceutical Organic Chemistry. Mansoura, Egypt.Georgia State University. Department of Chemistry. Atlanta, GA, USA.Augusta University. Department of Chemistry and Physics. Augusta, USA.Georgia State University. Department of Chemistry. Atlanta, GA, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ. Brasil.Abstract: Arylimidamides (AIAs), previously termed as reversed amidines, present a broad spectrum of activity against intracellular microorganisms. In the present study, three novel AIAs were evaluated in a mouse model of Trypanosoma cruzi infection, which is the causative agent of Chagas disease. The bis-AIAs DB1957, DB1959 and DB1890 were chosen based on a previous screening of their scaffolds that revealed a very promising trypanocidal effect at nanomolar range against both the bloodstream trypomastigotes (BTs) and the intracellular forms of the parasite. This study focused on both mesylate salts DB1957 and DB1959 besides the hydrochloride salt DB1890. Our current data validate the high activity of these bis-AIA scaffolds that exhibited EC50 (drug concentration that reduces 50% of the number of the treated parasites) values ranging from 14 to 78 nM and 190 to 1,090 nM against bloodstream and intracellular forms, respectively, also presenting reasonable selectivity indexes and no mutagenicity profile predicted by in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET). Acute toxicity studies using murine models revealed that these AIAs presented only mild toxic effects such as reversible abdominal contractions and ruffled fur. Efficacy assays performed with Swiss mice infected with the Y strain revealed that the administration of DB1957 for 5 consecutive days, with the first dose given at parasitemia onset, reduced the number of BTs at the peak, ranging between 21 and 31% of decrease. DB1957 was able to provide 100% of animal survival, while untreated animals showed 70% of mortality rates. DB1959 and DB1890B did not reduce circulating parasitism but yielded .80% of survival rates