In vitro and in vivo activity of the chloroaryl-substituted imidazole viniconazole against Trypanosoma cruzi

Chagas disease (CD) is caused by the intracellular protozoan parasite Trypanosoma cruzi and affects more than 10 million people in poor areas of Latin America. There is an urgent need for alternative drugs with better safety, broader efficacy, lower costs and shorter time of administration. Thus the biological activity of viniconazole, a chloroaryl-substituted imidazole was investigated using in vitro and in vivo screening models of T. cruzi infection. Ultrastructural findings demonstrated that the most frequent cellular damage was associated with plasma membrane (blebs and shedding events), Golgi (swelling aspects) and the appearance of large numbers of vacuoles suggesting an autophagic process. Our data demonstrated that although this compound is effective against bloodstream and intracellular forms (16 and 24μ m, respectively) in vitro, it does not present in vivo efficacy. Due to the urgent need for novel agents against T. cruzi, the screening of natural and synthetic products must be further supported with the aim of finding more selective and affordable drugs for C

Evaluation of Arylimidamides DB1955 and DB1960 as Candidates against Visceral Leishmaniasis and Chagas' Disease: In Vivo Efficacy, Acute Toxicity, Pharmacokinetics, and Toxicology Studies

Arylimidamides (AIAs) have shown outstanding in vitro potency against intracellular kinetoplastid parasites, and the AIA 2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan dihydrochloride (DB766) displayed good in vivo efficacy in rodent models of visceral leishmaniasis (VL) and Chagas' disease. In an attempt to further increase the solubility and in vivo antikinetoplastid potential of DB766, the mesylate salt of this compound and that of the closely related AIA 2,5-bis[2-(2-cyclopentyloxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride (DB1852) were prepared. These two mesylate salts, designated DB1960 and DB1955, respectively, exhibited dose-dependent activity in the murine model of VL, with DB1960 inhibiting liver parasitemia by 51% at an oral dose of 100 mg/kg/day × 5 and DB1955 reducing liver parasitemia by 57% when given by the same dosing regimen. In a murine Trypanosoma cruzi infection model, DB1960 decreased the peak parasitemia levels that occurred at 8 days postinfection by 46% when given orally at 100 mg/kg/day × 5, while DB1955 had no effect on peak parasitemia levels when administered by the same dosing regimen. Distribution studies revealed that these compounds accumulated to micromolar levels in the liver, spleen, and kidneys but to a lesser extent in the heart, brain, and plasma. A 5-day repeat-dose toxicology study with DB1960 and DB1955 was also conducted with female BALB/c mice, with the compounds administered orally at 100, 200, and 500 mg/kg/day. In the high-dose groups, DB1960 caused changes in serum chemistry, with statistically significant increases in serum blood urea nitrogen, lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase levels, and a 21% decrease in body weight was observed in this group. These changes were consistent with microscopic findings in the livers and kidneys of the treated animals. The incidences of observed clinical signs (hunched posture, tachypnea, tremors, and ruffled fur) were more frequent in DB1960-treated groups than in those treated with DB1955. However, histopathological examination of tissue samples indicated that both compounds had adverse effects at all dose levels.This work was supported by a grant from the Bill and Melinda Gates Foundation, contract N01-AI-60011 with SRI International from the National Institute of Allergy and Infectious Diseases, FIOCRUZ, and by Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) PPSUS, APQ1, and Pensa-Rio (16/2009-E-26/110-313/2010), Conselho Nacional Desenvolvimento científico e Tecnológico (CNPq), PDTIS/FIOCRUZ, and PROEP. We thank the other members of the Consortium for Parasitic Drug Development for helpful discussions

Identification of Pyrazolo[3,4-e][1,4]thiazepin based CYP51 inhibitors as potential Chagas disease therapeutic alternative: In vitro and in vivo evaluation, binding mode prediction and SAR exploration

American trypanosomiasis or Chagas disease (CD) is a vector borne pathology caused by the parasite Trypanosoma cruzi (T. cruzi), which remains a serious global health problem. The current available treatment for CD is limited to two nitroderivatives with limited efficacy and several side effects. The rational design of ergosterol synthetic route inhibitors (e.g. CYP51 inhibitors) represents a promising strategy for fungi and trypanosomatids, exhibiting excellent anti-T.cruzi activity in pre-clinical assays. In the present work, we evaluate through different approaches (molecular docking, structure activity relationships, CYP51 inhibitory assay, and phenotypic screenings in vitro and in vivo) the potency and selectivity of a novel CYP51 inhibitor (compound 1) and its analogues against T.cruzi infection. Regarding anti-parasitic effect, compound 1 was active in vitro with EC50 3.86 and 4.00 μM upon intracellular (Tulahuen strain) and bloodstream forms (Y strain), respectively. In vivo assays showed that compound 1 reduced in 43% the parasitemia peak but, unfortunately failed to promote animal survival. In order to promote an enhancement at the potency and pharmacological properties, 17 new analogues were purchased and screened in vitro. Our findings demonstrated that five compounds were active against intracellular forms, highlighting compounds 1e and 1f, with EC50 2.20 and 2.70 μM, respectively, and selectivity indices (SI) = 50 and 36, respectively. Against bloodstream trypomastigotes, compound 1f reached an EC50 value of 20.62 μM, in a similar range to Benznidazole, but with low SI (3). Although improved the solubility of compound 1, the analogue 1f did not enhance the potency in vitro neither promote better in vivo efficacy against mouse model of acute T.cruzi infection arguing for the synthesis of novel pyrazolo[3,4-e][1,4]thiazepin derivatives aiming to contribute for alternative therapies for CD

In Vitro and In Vivo Investigation of the Efficacy of Arylimidamide DB1831 and Its Mesylated Salt Form - DB1965 - against Trypanosoma cruzi Infection

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA - DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T.cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC50 value/48 h of 5–40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T.cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T.cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole

Evaluation of Arylimidamides DB1955 and DB1960 as Candidates against Visceral Leishmaniasis and Chagas' Disease: In Vivo Efficacy, Acute Toxicity, Pharmacokinetics, and Toxicology Studies

ABSTRACT Arylimidamides (AIAs) have shown outstanding in vitro potency against intracellular kinetoplastid parasites, and the AIA 2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan dihydrochloride (DB766) displayed good in vivo efficacy in rodent models of visceral leishmaniasis (VL) and Chagas' disease. In an attempt to further increase the solubility and in vivo antikinetoplastid potential of DB766, the mesylate salt of this compound and that of the closely related AIA 2,5-bis[2-(2-cyclopentyloxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride (DB1852) were prepared. These two mesylate salts, designated DB1960 and DB1955, respectively, exhibited dose-dependent activity in the murine model of VL, with DB1960 inhibiting liver parasitemia by 51% at an oral dose of 100 mg/kg/day × 5 and DB1955 reducing liver parasitemia by 57% when given by the same dosing regimen. In a murine Trypanosoma cruzi infection model, DB1960 decreased the peak parasitemia levels that occurred at 8 days postinfection by 46% when given orally at 100 mg/kg/day × 5, while DB1955 had no effect on peak parasitemia levels when administered by the same dosing regimen. Distribution studies revealed that these compounds accumulated to micromolar levels in the liver, spleen, and kidneys but to a lesser extent in the heart, brain, and plasma. A 5-day repeat-dose toxicology study with DB1960 and DB1955 was also conducted with female BALB/c mice, with the compounds administered orally at 100, 200, and 500 mg/kg/day. In the high-dose groups, DB1960 caused changes in serum chemistry, with statistically significant increases in serum blood urea nitrogen, lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase levels, and a 21% decrease in body weight was observed in this group. These changes were consistent with microscopic findings in the livers and kidneys of the treated animals. The incidences of observed clinical signs (hunched posture, tachypnea, tremors, and ruffled fur) were more frequent in DB1960-treated groups than in those treated with DB1955. However, histopathological examination of tissue samples indicated that both compounds had adverse effects at all dose levels

In vitro and in vivo activity of the chloroaryl-substituted imidazole viniconazole against Trypanosoma cruzi

SUMMARY Chagas disease (CD) is caused by the intracellular protozoan parasite Trypanosoma cruzi and affects more than 10 million people in poor areas of Latin America. There is an urgent need for alternative drugs with better safety, broader efficacy, lower costs and shorter time of administration. Thus the biological activity of viniconazole, a chloroaryl-substituted imidazole was investigated using in vitro and in vivo screening models of T. cruzi infection. Ultrastructural findings demonstrated that the most frequent cellular damage was associated with plasma membrane (blebs and shedding events), Golgi (swelling aspects) and the appearance of large numbers of vacuoles suggesting an autophagic process. Our data demonstrated that although this compound is effective against bloodstream and intracellular forms (16 and 24 μ m, respectively) in vitro, it does not present in vivo efficacy. Due to the urgent need for novel agents against T. cruzi, the screening of natural and synthetic products must be further supported with the aim of finding more selective and affordable drugs for CD

CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds

Submitted by Sandra Infurna ([email protected]) on 2016-09-06T16:08:23Z No. of bitstreams: 1 cristiane_silva_etal_IOC_2012.pdf: 948085 bytes, checksum: 167f4d98e683569fad019aa30b70196b (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-09-06T16:23:08Z (GMT) No. of bitstreams: 1 cristiane_silva_etal_IOC_2012.pdf: 948085 bytes, checksum: 167f4d98e683569fad019aa30b70196b (MD5)Made available in DSpace on 2016-09-06T16:23:08Z (GMT). No. of bitstreams: 1 cristiane_silva_etal_IOC_2012.pdf: 948085 bytes, checksum: 167f4d98e683569fad019aa30b70196b (MD5) Previous issue date: 2012Vanderbilt University. School of Medicine. Department of Biochemistry. Nashville, TN, USA.Vanderbilt University. Vanderbilt Institute of Chemical Biology. Department of Chemistry. Nashville, TN, USA.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 Biologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, Brasil.Vanderbilt University. School of Medicine. Department of Medicine. Vanderbilt Ingram Cancer Center. Nashville, TN, USA.Vanderbilt University. School of Medicine. Department of Biochemistry. Nashville, TN, USA.Vanderbilt University. Vanderbilt Institute of Chemical Biology. Department of Chemistry. Nashville, TN, USA.Vanderbilt University. School of Medicine. Department of Biochemistry. Nashville, TN, USA.CYP51 (sterol 14α-demethylase) is a cytochrome P450 enzyme essential for sterol biosynthesis and the primary target for clinical and agricultural antifungal azoles. The azoles that are currently in clinical use for systemic fungal infections represent modifications of two basic scaffolds, ketoconazole and fluconazole, all of them being selected based on their antiparasitic activity in cellular experiments. By studying direct inhibition of CYP51 activity across phylogeny including human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum, we identified three novel protozoa-specific inhibitory scaffolds, their inhibitory potency correlating well with antiprotozoan activity. VNI scaffold (carboxamide containing β-phenyl-imidazoles) is the most promising among them: killing T. cruzi amastigotes at low nanomolar concentration, it is also easy to synthesize and nontoxic. Oral administration of VNI (up to 400 mg/kg) neither leads to mortality nor reveals significant side effects up to 48 h post treatment using an experimental mouse model of acute toxicity. Trypanosomatidae CYP51 crystal structures determined in the ligand-free state and complexed with several azole inhibitors as well as a substrate analog revealed high rigidity of the CYP51 substrate binding cavity, which must be essential for the enzyme strict substrate specificity and functional conservation. Explaining profound potency of the VNI inhibitory scaffold, the structures also outline guidelines for its further development. First steps of the VNI scaffold optimization have been undertaken; the results presented here support the notion that CYP51 structure-based rational design of more efficient, pathogen-specific inhibitors represents a highly promising direction

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