Cyclobenzaprine raises ROS levels in Leishmania infantum and reduces parasite burden in Infected mice

11 p.-5 fig. Ferreira Cunha-Júnior, Edézio et al.Background The leishmanicidal action of tricyclic antidepressants has been studied and evidences have pointed that their action is linked to inhibition of trypanothione reductase, a key enzyme in the redox metabolism of pathogenic trypanosomes. Cyclobenzaprine (CBP) is a tricyclic structurally related to the antidepressant amitriptyline, differing only by the presence of a double bond in the central ring. This paper describes the effect of CBP in experimental visceral leishmaniasis, its inhibitory effect in trypanothione reductase and the potential immunomodulatory activity.Methodology/Principal Findings In vitro antileishmanial activity was determined in promastigotes and in L. infantum-infected macrophages. For in vivo studies, L. infantum-infected BALB/c mice were treated with CBP by oral gavage for five days and the parasite load was estimated. Trypanothione reductase activity was assessed in the soluble fraction of promastigotes of L. infantum. For evaluation of cytokines, L. infantum-infected macrophages were co-cultured with BALB/c splenocytes and treated with CBP for 48 h. The supernatant was analyzed for IL-6, IL-10, MCP-1, IFN-γ and TNF-α. CBP demonstrated an IC50 of 14.5±1.1μM and an IC90 of 74.5±1.2 μM in promastigotes and an IC50 of 12.6±1.05 μM and an IC90 of 28.7±1.3 μM in intracellular amastigotes. CBP also reduced the parasite load in L. infantum-infected mice by 40.4±10.3% and 66.7±10.5% in spleen at 24.64 and 49.28 mg/kg, respectively and by 85.6±5.0 and 89.3±4.8% in liver at 24.64 and 49.28mg/kg, after a short-term treatment. CBP inhibited the trypanothione reductase activity with a Ki of 86 ± 7.7 μM and increased the ROS production in promastigotes. CBP inhibited in 53% the production of IL-6 in infected macrophages coculture.Conclusion/Significance To the best of our knowledge, this study is the first report of the in vivo antileishmanial activity of the FDA-approved drug CBP. Modulation of immune response and induction of oxidative stress in parasite seem to contribute to this efficacy.This work was supported by Programa Estratégico de Apoio à Pesquisa em Saúde,FIOCRUZ/Conselho Nacional de Desenvolvimento Científico e Tecnológico - www.cnpq.br, (PAPES/ CNPq 407680/2012-8 to ECTS and 407590/2012-9 to EEAA), Fundação de Apoio a Pesquisa do Estado do Rio de Janeiro ± www.faperj.br (Fellow and grant E-26/010.001828/2016 to EFCJ), Conselho Nacional de Desenvolvimento CientõÂfico e TecnoloÂgico (CNPq/Universal grant 470627/2013-1 to EEAA), São Paulo Research Foundation - www.fapesp.br (FAPESP 2015/23403-9 to AGT),Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad FEDER - www.idi.mineco.gob.es/ (SAF2015-65740-R) and Subdirección General de Redes y Centros de Investigación Cooperativa-FEDER -www.isciii.es/ (RD12/0018/0007) (to LR).Peer reviewe

Biological effects of trans, trans-farnesol in Leishmania amazonensis

IntroductionFarnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. Candida albicans predominantly secretes the trans, trans-farnesol (t, t-FOH) isomer, known for its role in regulating the virulence of various fungi species and modulating morphological transition processes. Notably, the evolutionary divergence in sterol biosynthesis between fungi, including Candida albicans, and trypanosomatids resulted in the synthesis of sterols with the ergostane skeleton, distinct from cholesterol. This study aims to assess the impact of exogenously added trans, trans-farnesol on the proliferative ability of Leishmania amazonensis and to identify its presence in the lipid secretome of the parasite.MethodsThe study involved the addition of exogenous trans, trans-farnesol to evaluate its interference with the proliferation of L. amazonensis promastigotes. Proliferation, cell cycle, DNA fragmentation, and mitochondrial functionality were assessed as indicators of the effects of trans, trans-farnesol. Additionally, lipid secretome analysis was conducted, focusing on the detection of trans, trans-farnesol and related products derived from the precursor, farnesyl pyrophosphate. In silico analysis was employed to identify the sequence for the farnesene synthase gene responsible for producing these isoprenoids in the Leishmania genome.ResultsExogenously added trans, trans-farnesol was found to interfere with the proliferation of L. amazonensis promastigotes, inhibiting the cell cycle without causing DNA fragmentation or loss of mitochondrial functionality. Despite the absence of trans, trans-farnesol in the culture supernatant, other products derived from farnesyl pyrophosphate, specifically α-farnesene and β-farnesene, were detected starting on the fourth day of culture, continuing to increase until the tenth day. Furthermore, the identification of the farnesene synthase gene in the Leishmania genome through in silico analysis provided insights into the enzymatic basis of isoprenoid production.DiscussionThe findings collectively offer the first insights into the mechanism of action of farnesol on L. amazonensis. While trans, trans-farnesol was not detected in the lipid secretome, the presence of α-farnesene and β-farnesene suggests alternative pathways or modifications in the isoprenoid metabolism of the parasite. The inhibitory effects on proliferation and cell cycle without inducing DNA fragmentation or mitochondrial dysfunction raise questions about the specific targets and pathways affected by exogenous trans, trans-farnesol. The identification of the farnesene synthase gene provides a molecular basis for understanding the synthesis of related isoprenoids in Leishmania. Further exploration of these mechanisms may contribute to the development of novel therapeutic strategies against Leishmania infections

The New Pyrazolyltetrazole Derivative MSN20 Is Effective via Oral Delivery against Cutaneous Leishmaniasis

Made available in DSpace on 2015-06-24T12:26:34Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) viviane_faloesetal_IOC_2014.pdf: 562560 bytes, checksum: f5760906474844701b123c68b008f7b4 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.UNIFEL. Instituto de Física e Química. Laboratório de Síntese de Moléculas Bioativas.Itajubá, MG, Brasil.Universidade Federal Fluminense. Instituto de Química. Departamento de Química Orgânica. Programa de Pós-Graduação em Química. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.An orally delivered, safe and effective treatment for leishmaniasis is an unmet medical need. Azoles and the pyrazolylpyrimidine allopurinol present leishmanicidal activity, but their clinical efficacies are variable. Here, we describe the activity of the new pyrazolyltetrazole hybrid, 5-[5-amino-1-(4′-methoxyphenyl)1H-pyrazole-4-yl]1H-tetrazole (MSN20). MSN20 showed a 50% inhibitory concentration (IC50) of 22.3 μM against amastigotes of Leishmania amazonensis and reduced significantly the parasite load in infected mice, suggesting its utility as a lead compound for the development of an oral treatment for leishmaniasis

Antileishmanial activity of amides from Piper amalago and synthetic analogs

Two natural amides isolated from the chloroform extract of Piper amalago L., Piperaceae, leaves, a hydrogenated derivative and seven synthetic analogs were tested against the promastigote and intracellular amastigote forms of Leishmania amazonensis. The antileishmanial activity was evaluated in terms of growth inhibitory concentration for 50% of protozoa (IC50). The cytotoxicity toward the J774A1 macrophages was evaluated in terms of the cytotoxic concentrations for 50% of macrophages (CC50). The ability to induce nitric oxide production was also investigated for all compounds. The saturated amide 7-(1,3-benzodioxol-5-yl)-1-(1-pyrrolidinyl)-1-heptanone was obtained by hydrogenation of the natural compound N-[7-(3',4'-methylenedioxyphenyl)-2(Z),4(Z)-heptadienoyl]pyrrolidine. Synthetic amides were prepared by addition of the appropriate amine to the corresponding acyl chloride. The natural compound, N-[7-(3',4'-methylenedioxyphenyl)-2(E),4(E)-heptadienoyl]pyrrolidine, was the most active of all tested compounds against the promastigote and intracellular amastigote forms with IC50 values of 15 µM and 14.5 µM, respectively. None of the compounds modulated the production of nitric oxide

Oral effectiveness of PMIC4, a novel hydroxyethylpiperazine analogue, in Leishmania amazonensis

Made available in DSpace on 2015-05-04T16:34:36Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) claudia_levyetal_IOC_2014.pdf: 620953 bytes, checksum: e1bae1d82f29d4be01678c3fa2ec7a2c (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Síntese de Fármacos. Rio de Janeiro, RJ, Brasil.Universidade Federal de Pelotas. Centro de Ciências Químicas, Farmacêuticas e de Alimentos. Laboratório de Química Aplicada à Bioativos. Pelotas, RS, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Síntese de Fármacos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Departamento de Síntese de Fármacos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ, Brasil.Pentavalent antimonials have saved the lives of thousands of Leishmania-infected patients more than seventy years but, unfortunately, they are highly toxic and require parenteral delivery. Therefore, the search for safer and orally delivered alternative is a need. This paper describes the antileishmanial properties of PMIC4, a novel hydroxyethylpiperazine analogue. PMIC4 showed potent activity against intracellular amastigotes of Leishmania amazonensis, with IC50 of 1.8 lM and selectivity index higher than 100-fold, calculated in relation to the toxicity on the host cell. Following laboratory animal welfare policies, we analyzed the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties and calculated the Lipinski’s rule of five of PMIC4 before proceeding to in vivo tests. PMIC4 satisfied Lipinski’s rule of five and presented high probability of human intestinal absorption, suggesting a good chance of druglikeness and oral bioavailability. For in vivo studies, PMIC4 was administered via intralesional injection (3.4 mg/kg/day, three times a week) or orally (34.0 mg/kg/day, five times a week) to L. amazonensisinfected BALB/c mice throughout the 98 day experiments. At the end of the treatment period, serum markers of toxicity were measured. When administered orally, PMIC4 controlled the lesions in L. amazonensis- infected BALB/c mice without altering serological markers of toxicity. These results demonstrate that PMIC4 is a promising molecular scaffold, orally effective against experimental leishmaniasis

Preclinical Studies Evaluating Subacute Toxicity and Therapeutic Efficacy of LQB-118 in Experimental Visceral Leishmaniasis

Submitted by Sandra Infurna ([email protected]) on 2017-02-09T14:30:16Z No. of bitstreams: 1 edesio_cunhajr_etal_IOC_2016.pdf: 2057722 bytes, checksum: b905a7232a9bd50caaeeaa9c5415f825 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-02-09T14:45:46Z (GMT) No. of bitstreams: 1 edesio_cunhajr_etal_IOC_2016.pdf: 2057722 bytes, checksum: b905a7232a9bd50caaeeaa9c5415f825 (MD5)Made available in DSpace on 2017-02-09T14:45:46Z (GMT). No. of bitstreams: 1 edesio_cunhajr_etal_IOC_2016.pdf: 2057722 bytes, checksum: b905a7232a9bd50caaeeaa9c5415f825 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ. Brasil.Universidade do Estado do Rio de Janeiro. Laboratório Imunol. Aplicada e Bioq. de Proteínas e Prod. Naturais. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ. Brasil.Universidade do Estado do Rio de Janeiro. Laboratório Imunol. Aplicada e Bioq. de Proteínas e Prod. Naturais. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Departamento de Patologia e Laboratórios. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Laboratório Imunol. Aplicada e Bioq. de Proteínas e Prod. Naturais. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Laboratório de Química. Macaé, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Pesquisas de Produtos Naturais. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Laboratório Imunol. Aplicada e Bioq. de Proteínas e Prod. Naturais. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ. Brasil.Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is the second major cause of death by parasites, after malaria. The arsenal of drugs against leishmaniasis is small, and each has a disadvantage in terms of toxicity, efficacy, price, or treatment regimen. Our group has focused on studying new drug candidates as alternatives to current treatments. The pterocarpanquinone LQB-118 was designed and synthesized based on molecular hybridization, and it exhibited antiprotozoal and anti-leukemic cell line activities. Our previous work demonstrated that LQB-118 was an effective treatment for experimental cutaneous leishmaniasis. In this study, we observed that treatment with 10 mg/kg of body weight/day LQB-118 orally inhibited the development of hepatosplenomegaly with a 99% reduction in parasite load. An in vivo toxicological analysis showed no change in the clinical, biochemical, or hematological parameters. Histologically, all of the analyzed organs were normal, with the exception of the liver, where focal points of necrosis with leukocytic infiltration were observed at treatment doses 5 times higher than the therapeutic dose; however, these changes were not accompanied by an increase in transaminases. Our findings indicate that LQB-118 is effective at treating different clinical forms of leishmaniasis and presents no relevant signs of toxicity at therapeutic doses; thus, this framework is demonstrated suitable for developing promising drug candidates for the oral treatment of leishmaniasis

Cyclobenzaprine Raises ROS Levels in Leishmania infantum and Reduces Parasite Burden in Infected Mice

Submitted by Sandra Infurna ([email protected]) on 2017-05-04T11:55:40Z No. of bitstreams: 1 edesio_cunhajr_etal_IOC_2017.pdf: 1518221 bytes, checksum: 6be150919adf4e381a7886f6406acc7b (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-05-04T12:14:58Z (GMT) No. of bitstreams: 1 edesio_cunhajr_etal_IOC_2017.pdf: 1518221 bytes, checksum: 6be150919adf4e381a7886f6406acc7b (MD5)Made available in DSpace on 2017-05-04T12:14:58Z (GMT). No. of bitstreams: 1 edesio_cunhajr_etal_IOC_2017.pdf: 1518221 bytes, checksum: 6be150919adf4e381a7886f6406acc7b (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ. Brasil.Universidade de São Paulo. Instituto de Medicina Tropical. São Paulo, SP, Brasil / Instituto Adolfo Lutz. Centro de Parasitologia e Micologia. São Paulo, SP, Brasil.Instituto Adolfo Lutz. Centro de Parasitologia e Micologia. São Paulo, SP, Brasil.Universidade de São Paulo. Instituto de Medicina Tropical. São Paulo, SP, Brasil.Centro de Investigaciones BioloÂgicas (CSIC). Unidad Asociada Interacciones, Metabolismo y Bioanálisis CSIC-CEU. Madrid, Spain.Universidad CEU San Pablo. Faculty of Pharmacy. Center for Metabolomics and Bioanalysis (CEMBIO. Madrid, Spain.Centro de Investigaciones BioloÂgicas (CSIC). Unidad Asociada Interacciones, Metabolismo y Bioanálisis CSIC-CEU. Madrid, Spain.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ. Brasil.Instituto Adolfo Lutz. Centro de Parasitologia e Micologia. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ. Brasil.The leishmanicidal action of tricyclic antidepressants has been studied and evidences have pointed that their action is linked to inhibition of trypanothione reductase, a key enzyme in the redox metabolism of pathogenic trypanosomes. Cyclobenzaprine (CBP) is a tricyclic structurally related to the antidepressant amitriptyline, differing only by the presence of a double bond in the central ring. This paper describes the effect of CBP in experimental visceral leishmaniasis, its inhibitory effect in trypanothione reductase and the potential immunomodulatory activity

Monocyclic Nitro-heteroaryl Nitrones with Dual Mechanism of Activation:Synthesis and Antileishmanial Activity

5-Nitro-furan nitrones (1) and 5-nitro-thiophene nitrones (2) were synthesized in one step. Compounds 1a-c had the most potent leishmanicidal activity against intracellular amastigote forms of Leishmania amazonensis and L. infantum (from 0.019 to 2.76 μM), with excellent selectivity (from 39 to 5673). The comparison of the leishmanicidal activity in promastigotes of wild type L. donovani with those overexpressing nitroreductases NRT1 or NRT2 shows that 1a,b are activated by both, which could slow the development of resistance. Their redox potential (Eredox) obtained by cyclic voltammetry (-0.67 and -0.62 V) shows that the reduction of the nitro group is modulated by the nitrone group. Oral administration of 1b to mice infected by L. infantum reduced the parasite load on the spleen by 76.6 and 95.0% with doses of 50 and 100 mg/kg, respectively, administered twice a day, for 5 days. In the liver, the parasite load suppression was above 75% with either treatment. </p

<i>In vitro</i> activity of CBP.

<p>(A) Promastigotes of <i>L</i>. <i>infantum</i> were incubated with CBP for 72 h. The growth inhibition was measured using resazurin. (n = 3) (B) Peritoneal macrophages were infected with <i>L</i>. <i>infantum</i> and treated with CBP for 72 h. The slides were stained and the results were expressed as an infection index^# [II = % infected cells × (number of amastigotes/total number of macrophages)]. The inset shows representative photos from the slides. (n = 3).</p

Effect of CBP on TryR and ROS production.

<p>(a) <i>TryR assay</i>. Soluble extract of promastigotes of <i>L</i>. <i>infantum</i> using oxidized trypanothione as substrate and clomipramine (50 μM) as positive control (PC) of inhibition. Reading was initiated after adding 100 μM of DTNB at 410nm (n = 3). (b) <i>ROS production</i>. Promastigotes of <i>L</i>. <i>infantum</i> were incubated with CBP and ROS generation was measured with H₂DCFDA reagent. Antimycin A 10 μM was used as positive control (PC). (n = 3) *p<0.05, ** p<0.01 and ***p<0.001.</p