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Ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease: 20 years after, does the promise holds? Julio A. Urbina, Ph.D. Instituto.

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Presentation on theme: "Ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease: 20 years after, does the promise holds? Julio A. Urbina, Ph.D. Instituto."— Presentation transcript:

1 Ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease: 20 years after, does the promise holds? Julio A. Urbina, Ph.D. Instituto Venezolano de Investigaciones Cientifícas Caracas, Venezuela International Symposium on the Centenary of Chagas Disease Discovery Rio de Janeiro, Brazil, July 8th-10th, 2009

2 Current specific chemotherapy of Chagas disease and its limitations
Drugs currently available (nifurtimox, Lampit®, Bayer and benznidazole, Rochagan®, Roche) were developed empirically in 1960s and 70s Both drugs are active in the acute and early chronic phase (60-80%) of the disease, but efficacy varies with the geographical area, due to natural drug-resistant T. cruzi strains These compounds have, low antiparasitic efficacy in the established chronic phase (≤20%), which is the most common clinical presentation, and can lead to irreversible GI tract and heart lesions. However, several studies have shown that these drugs may slow the progression of the disease, probably by reducing the parasite load The drugs may induce adverse side effects, whose frequency increases with the age of the patient and can lead to treatment discontinuation Antiparasitic activity is inextricably linked to host toxicity, as both result from the generation of highly reactive free radicals by the drugs

3 A ruthenium complex of benznidazole, with higher solubility and activity, in vitro and in vivo (Silva et al JMC 51, )

4 Ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease: strategy and basic findings Trypanosoma cruzi, as fungi and yeasts, has an strict requirement of 24-alkyl sterols (ergosterol and phytosterol analogs) for survival and cannot use the abundant supply of cholesterol found in mammalians The ergosterol biosynthesis pathway has been chemically validated, at several steps, as a chemotherapeutic target in this organism Ergosterol biosynthesis inhibitors (EBI) are very potent anti-T. cruzi agents in vitro and combinations of EBI that act at different steps of the pathway have synergistic effects, in vitro and in vivo However, commercially available EBI (ketoconazole, itraconazole, terbinafine) are unable to cure T. cruzi infections in humans or experimental animals, probably due to inadequate PK properties for this application

5 Novel ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease.1. lanosterol C14a demethylase (CYP51) inhibitors New triazole derivatives, originally developed as systemic antifungal agents Potent intrinsic anti-T. cruzi activity and special pharmacokinetic properties (long half life, large volumes of distribution) Can cure both acute and chronic T. cruzi infections in several animal models Poised for clinical development for the treatment of human Chagas disease in short term

6 Posaconazole is a fourth generation antifungal triazole, based on the structure of itraconazole

7 Posaconazole oral suspension (Noxafil®) was approved in 2005 by the European Union and Australia for the treatment of refractory invasive fungal infections (rIFI) and in 2006 by the US Federal Drug Administration for the prophylaxis of invasive aspergillosis and candidiasis and for the treatment of oropharyngeal candidiasis (OPC), including infections refractory to fluconazole and/or itraconazole

8 Posaconazole is among the most potent and selective anti-Trypanosoma cruzi agents in vitro (Urbina et al AAC 42, 1771 ) POSA MIC = 0.3 nM KETO MIC = 30 nM

9 Posaconazole-induced growth inhibition is associated with the complete depletion of T. cruzi-endogenous sterols (Urbina et al AAC 42, 1771 )

10 Posaconazole-induced elimination of intracellular amastigotes leads to cytoskeleton reassembly in primary cultures of cardiomyocytes (Silva et al IJAA 27, 530 ) uninfected untreated POS-72h POS-96h A D C B 2µm Actin A B D C 2µm a-tubulin uninfected untreated POS-48h POS-168h

11 Posaconazole is active against acute infections by nitrofuran- and nitroimidazole-resistant T. cruzi strains, even if the host is immunosuppressed (Molina et al AAC 44, 150)

12 The anti-T. cruzi activity of posaconazole in a murine model of acute Chagas disease is less dependent on IFN-g than that of benznidazole. I. Parasitemia and survival (Ferraz et al., AAC 51, 1359) Control (C57BL/6) IFN-g KO IL-12 KO

13 LT CD4+, LT CD8+ or LB cells have different effects on the anti-T
LT CD4+, LT CD8+ or LB cells have different effects on the anti-T. cruzi activity of posaconazole and benznidazole in a murine model of acute Chagas disease. I. Parasitemia and survival (Ferraz et al., AAC 53, 174) Control (C57BL/6) LTCD4+ KO LTCD8+ KO LB KO

14 Posaconazole is more effective than benznidazole in the induction of parasite clearance in a murine model of acute Chagas disease (Olivieri et al., submitted)

15 Posaconazole is more effective than benznidazole in preventing cardiac damage in a murine model of acute Chagas disease (Olivieri et al., submitted) C Tc TcBZ TcPOS

16 Posaconazole is active against chronic infections by nitrofuran- and nitroimidazole-resistant T. cruzi strains. (Molina et al AAC 44, 150)

17 Novel CYP51 inhibitors are active against nitroheterocycle-resistant T
Novel CYP51 inhibitors are active against nitroheterocycle-resistant T. cruzi I, T. cruzi II and hybrid strains

18 Based on these findings a clinical development program of posaconazole for the treatment of chronic Chagas disease is being advanced by Schering Plough. The first clinical studies (Phase II) are planned for the second semester of 2009 and will include several Latin American countries and possibly Spain

19 Ravuconazole has a simpler molecular structure than posaconazole, with comparable in vitro anti-T. cruzi activity and a remarkable PK profile in humans Ravuconazole is a potent, broad-spectrum, antifungal triazole currently in phase II clinical trials (Eisai Co., Japan) for the treatment of invasive fungal infections The compound has potent intrinsic anti-T. cruzi activity (MIC against cultured intracellular amastigotes: nM) Its activity in animal models of acute and chronic Chagas disease is suppressive, rather than curative, probably due to inadequate pharmacokinetic properties The compound remains a candidate for the treatment of human Chagas disease due its remarkable PK profile in man, terminal half life of 4-8 days and large (>1000 L) volume of distribution)

20 Novel T. cruzi-CYP 51 inhibitors with remarkable potency and selectivity against the parasite, in vitro and in vivo Potent T. cruzi-CYP 51 inhibitors serendipitously discovered in the search of T. cruzi-specific protein farnesyltransferase (PFT) inhibitors. Further optimization has led to compounds with remarkable potency (sub-nanomolar MIC) and 104-fold selectivity against intracellular amastigotes in vitro, devoid of anti-PFT activity. The compounds are also active in vivo (Kraus et al JMC 52, 1639; Suryadevara et al ibid 52: 3703) A novel series of T. cruzi-CYP 51 inhibitors identified in a screen against Mycobacterium tuberculosis CYP 51. Active in vitro and in vivo against T. cruzi (Chen et al PLoS Negl Trop Dis 3: e372)

21 Novel ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease. 2: Amiodarone as an anti-T. cruzi agent (Benaim et al JMC 16, ) Amiodarone is the most frequently used antiarrhythmic agent used in the management of chronic Chagas disease patients with cardiac compromise The mechanism of action involves a direct blockade of Ca2+ plasmalemal channels, a shift of the inactivation potential to more negative values and an increase of the refractory period The compound has unusual pharmacokinetic properties: tissue levels are 100 to 1000-fold higher than those in sera and the terminal elimination half life is ≥20 days Recent work has shown that this compound also has broad-spectrum antifungal activity, which is mediated by interference with the cells’ Ca2+ homeostasis

22 Antiproliferative synergism of amiodarone and posaconazole against T
Antiproliferative synergism of amiodarone and posaconazole against T. cruzi amastigotes

23 Effects of amiodarone on free Ca2+ levels in T. cruzi amastigotes
Rhod-2 Rhodamine 123 Merge C A

24 Effects of amiodarone and posaconazole on free sterol levels in T
Effects of amiodarone and posaconazole on free sterol levels in T. cruzi epimastigotes

25 Synergistic effects effects of amiodarone and posaconazole on a murine model of acute Chagas disease

26 Combination therapy with amiodarone and itraconazole led to parasitological cure and clinical improvement in a patient with advanced chronic Chagas cardiomyopathy (Paniz-Mondolfi et al Chemother. 55, )

27 Combination therapy with amiodarone and itraconazole led to parasitological cure and clinical improvement in a patient with advanced chronic Chagas cardiomyopathy (Paniz-Mondolfi et al Chemother. 55, )

28 Novel ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease. 2. Squalene synthase inhibitors (Urbina et al AAC 48, 2379) Squalene synthase has been chemically validated as a chemotherapeutic target in T. cruzi and Leishmania spp. (Urbina el al MBP 125, 35) Novel quinuclidine derivatives, specific inhibitors of squalene synthase (SQS), also in development as cholesterol-lowering agents, have potent and selective anti-T. cruzi activity in vitro, as well as oral activity in murine models of Chagas disease Further development as antiparasitic agents will require parasite-specific SQS inhibitors

29 T. cruzi SQS sequence and heterologous expression in E
T. cruzi SQS sequence and heterologous expression in E. coli (Sealey-Cardona et al AAC 51, 2139)

30 Selective inhibitors of T
Selective inhibitors of T. cruzi SQS and amastigote proliferation (Sealey-Cardona et al AAC 51, 2139)

31 Novel ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease. 4: Compounds with a dual mechanism of action (Gerpe et al BMC 16, )

32 Anti-T. cruzi activity of heteroallyl-containing 5-nitrofurans in vitro (Gerpe et al BMC 16, )

33 Effects of heteroallyl-containing 5-nitrofurans on sterol composition and redox cycling of T. cruzi epimastigotes (Gerpe et al BMC 16, )

34 Conclusions-1 There is an urgent need for safer and more effective treatments for Chagas disease, particularly in its chronic stage. Several new approaches are being advanced, based on our increasing knowledge of the biochemistry and physiology of T. cruzi T. cruzi has an essential requirement of ergosterol and other 24-alkyl sterol for survival and growth and ergosterol biosynthesis inhibitors are potent antifungal and anti-trypanosomatid agents Novel triazole derivatives, specific inhibitors of the parasite’s lanosterol C14 demethylase (CYP51), have trypanocidal activity in vitro and can cure both acute and chronic infections in several murine models of the disease. Several of these compounds are poised to undergo clinical development for Chagas disease in the short term, posaconazole and ravuconazole being the most advanced candidates

35 Conclusions-2 Recent studies have shown that amiodarone, the most frequently used antiarrhythmic agent used in the management of chronic Chagas disease patients with cardiac compromise, also has anti-T. cruzi activity in vitro and in vivo and acts synergistically with posaconazole. This finding raises the notion that symptomatic treatment with amiodarone of Chagas disease patients could have the added benefit of lowering the parasite load and enhancing the efficacy of specific anti-T. cruzi drugs and this has been recently verified in a patient with advanced Chagas myocardiopathy Other ergosterol biosynthesis inhibitors that could enter clinical development in the next decade are specific squalene synthase inhibitors such as quinuclidine derivatives A recent development in this field is the discovery of anti-T. cruzi agents with a dual mechanism of action: inhibition of ergosterol biosynthesis at the level of squalene epoxidase and generation of oxidative stress by redox cycling of reduced nitrofuran radicals

36 Collaborators IVIC, Caracas, Venezuela:
Universidad de Los Andes, Merida, Venezuela: Renee Lira Juan Luis Concepción Aura Caldera Carlos Sanz-Rodriguez Imeria Odremán Nuñez Instituto de Parasitología y Biomedicina, Granada, Spain Universidad Central de Venezuela, Caracas, Venezuela: Dolores Gonzalez-Pacanowska Gilberto Payares Luis Miguel Ruiz Judith Molina Cristina Sanoja University of Dundee, United Kingdom Centro de Pesquisas “Rene Rachou”, FIOCRUZ, Belo Horizonte, Brazil: Ian Glbert Zigman Brener Alvaro Romanha Universidad de la Republica, Montevideo, Uruguay: Marcela Ferraz Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil: Hugo Cerecetto Mercedez Gonzales Alejandra Gerpe Mirian C.S. Pereira Luciena Ribeiro Garzoni Maria Nazareth Meirelles Tania Araujo-Jorge


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