Ergosterol biosynthesis inhibitors for the Ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease: 20 years specific treatment of Chagas disease: 20 years

after, does the promise holds?after, does the promise holds?

Julio A. Urbina, Ph.D.Julio A. Urbina, Ph.D.Instituto Venezolano de Investigaciones CientifícasInstituto Venezolano de Investigaciones Cientifícas

Caracas, VenezuelaCaracas, Venezuela

International Symposium on the Centenary of Chagas Disease DiscoveryInternational Symposium on the Centenary of Chagas Disease DiscoveryRio de Janeiro, Brazil, July 8Rio de Janeiro, Brazil, July 8thth-10-10thth, 2009, 2009

Current specific chemotherapy of Chagas disease and Current specific chemotherapy of Chagas disease and its limitationsits limitations

Drugs currently available (nifurtimox, Lampit®, Bayer and Drugs currently available (nifurtimox, Lampit®, Bayer and benznidazole, Rochagan®, Roche) were developed benznidazole, Rochagan®, Roche) were developed empiricallyempirically in in 1960s and 70s1960s and 70s

Both drugs are Both drugs are active in the acute and early chronic phaseactive in the acute and early chronic phase (60-80%) of (60-80%) of the disease, but efficacy varies with the geographical area, due to the disease, but efficacy varies with the geographical area, due to natural drug-resistantnatural drug-resistant T. cruzi T. cruzi strainsstrains

These compounds have,These compounds have, low antiparasitic efficacy in the established low antiparasitic efficacy in the established chronic phasechronic phase (≤20%), which is the most common clinical presentation, (≤20%), which is the most common clinical presentation, and can lead to irreversible GI tract and heart lesions. However, and can lead to irreversible GI tract and heart lesions. However, several studies have shown that these drugs may several studies have shown that these drugs may slow the progression slow the progression of the diseaseof the disease, probably by reducing the parasite load, probably by reducing the parasite load

The drugs may induce The drugs may induce adverse side effectsadverse side effects, whose frequency , whose frequency increases with the age of the patient and increases with the age of the patient and can lead to treatment can lead to treatment discontinuationdiscontinuation

Antiparasitic activity is Antiparasitic activity is inextricably linked to host toxicityinextricably linked to host toxicity, as both result , as both result from the generation of highly reactive free radicals by the drugsfrom the generation of highly reactive free radicals by the drugs

A ruthenium complex of benznidazole, with higher A ruthenium complex of benznidazole, with higher solubility and activity, in vitro and in vivo solubility and activity, in vitro and in vivo

((Silva et al. 2008. JMC 51, 4104-4114)Silva et al. 2008. JMC 51, 4104-4114)

Ergosterol biosynthesis inhibitors for the specific Ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease: strategy and basic findingstreatment of Chagas disease: strategy and basic findings

Trypanosoma cruzi, Trypanosoma cruzi, as fungi and yeasts, has an strict as fungi and yeasts, has an strict requirement of requirement of 24-alkyl sterols24-alkyl sterols (ergosterol and phytosterol (ergosterol and phytosterol analogs) for survival and analogs) for survival and cannot use the abundant supply of cannot use the abundant supply of cholesterol found in mammalianscholesterol found in mammalians

The ergosterol biosynthesis pathway has been The ergosterol biosynthesis pathway has been chemically chemically validated, at several steps, as a chemotherapeutic target in validated, at several steps, as a chemotherapeutic target in this organismthis organism

Ergosterol biosynthesis inhibitors (EBI) are Ergosterol biosynthesis inhibitors (EBI) are very potent anti-very potent anti-T. T. cruzi cruzi agents in vitroagents in vitro and combinations of EBI that act at and combinations of EBI that act at different steps of the pathway have different steps of the pathway have synergistic effects, in vitro synergistic effects, in vitro and in vivoand in vivo

However, commercially available EBI (ketoconazole, However, commercially available EBI (ketoconazole, itraconazole, terbinafine) itraconazole, terbinafine) are unable to cure are unable to cure T. cruzi T. cruzi infections in humans or experimental animals, infections in humans or experimental animals, probably due toprobably due to inadequate PK properties for this applicationinadequate PK properties for this application

Novel ergosterol biosynthesis inhibitors for the Novel ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease.1. specific treatment of Chagas disease.1.

lanosterol C14lanosterol C14 demethylase (CYP51) inhibitors demethylase (CYP51) inhibitors

New triazole derivatives, New triazole derivatives, originally originally developed as systemic antifungal developed as systemic antifungal agentsagents

Potent intrinsic anti-Potent intrinsic anti-T. cruzi T. cruzi activity activity and and special pharmacokinetic special pharmacokinetic properties (long half life, large properties (long half life, large volumes of distribution)volumes of distribution)

Can cure Can cure both acute and chronic both acute and chronic T. T. cruzi cruzi infectionsinfections in several animal in several animal modelsmodels

Poised for clinical development for the Poised for clinical development for the treatment of human Chagas disease treatment of human Chagas disease in short termin short term

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

Posaconazole oral suspension Posaconazole oral suspension (Noxafil®) (Noxafil®) was approved in 2005 by the European was approved in 2005 by the European

Union and Australia for theUnion and Australia for the treatment of treatment of refractory invasive fungal infections (rIFI)refractory invasive fungal infections (rIFI)

and in 2006 by the US Federal Drug and in 2006 by the US Federal Drug Administration for theAdministration for the prophylaxis of prophylaxis of

invasive aspergillosis and candidiasisinvasive aspergillosis and candidiasis and and for thefor the treatment of oropharyngeal treatment of oropharyngeal

candidiasis (OPC), including infections candidiasis (OPC), including infections refractory to fluconazole and/or itraconazolerefractory to fluconazole and/or itraconazole

Posaconazole is among the most potent and selective Posaconazole is among the most potent and selective anti-Trypanosoma cruzi agents in vitro (Urbina et al. anti-Trypanosoma cruzi agents in vitro (Urbina et al.

1998. AAC 42, 1771 )1998. AAC 42, 1771 )

MIC = 0.3 nM

MIC = 30 nM

POSA

KETO

Posaconazole-Posaconazole-induced growth induced growth

inhibition is inhibition is associated with the associated with the

complete depletion of complete depletion of T. cruzi-endogenous T. cruzi-endogenous sterols (Urbina et al. sterols (Urbina et al. 1998. AAC 42, 1771 )1998. AAC 42, 1771 )

Posaconazole-induced elimination of intracellular Posaconazole-induced elimination of intracellular amastigotes leads to cytoskeleton reassembly in amastigotes leads to cytoskeleton reassembly in

primary cultures of cardiomyocytes (Silva et al. 2006. primary cultures of cardiomyocytes (Silva et al. 2006. IJAA 27, 530 )IJAA 27, 530 )

A DCB 2µm

A B DC2µm

ActinActin

-tubulin-tubulin

uninfecteduninfected untreateduntreated POS-72hPOS-72h POS-96hPOS-96h

uninfecteduninfected untreateduntreated POS-48hPOS-48h POS-168hPOS-168h

Posaconazole is active against acute infections by Posaconazole is active against acute infections by nitrofuran- and nitroimidazole-resistant T. cruzi strains, nitrofuran- and nitroimidazole-resistant T. cruzi strains,

even if the host is immunosuppressed (Molina et al. even if the host is immunosuppressed (Molina et al. 2000. AAC 44, 150)2000. AAC 44, 150)

The anti-T. cruzi activity of posaconazole in a murine The anti-T. cruzi activity of posaconazole in a murine model of acute Chagas disease is less dependent on IFN-model of acute Chagas disease is less dependent on IFN-

than that of benznidazole. I. Parasitemia and survivalthan that of benznidazole. I. Parasitemia and survival (Ferraz et al., 2007. AAC 51, 1359) (Ferraz et al., 2007. AAC 51, 1359)

ControlControl(C57BL/6)(C57BL/6)

IFN-IFN-KOKO

IL-12IL-12KOKO

LT CD4+, LT CD8+ or LB cells have different effects on the LT CD4+, LT CD8+ or LB cells have different effects on the anti-T. cruzi activity of posaconazole and benznidazole in a anti-T. cruzi activity of posaconazole and benznidazole in a murine model of acute Chagas disease. I. Parasitemia and murine model of acute Chagas disease. I. Parasitemia and

survival (Ferraz et al., 2009. AAC 53, 174)survival (Ferraz et al., 2009. AAC 53, 174)

Control(C57BL/6)

LTCD4+ KO

LTCD8+ KO

LB KO

Posaconazole is more effective than benznidazole in the Posaconazole is more effective than benznidazole in the induction of parasite clearance in a murine model of induction of parasite clearance in a murine model of

acute Chagas disease (Olivieri et al., submitted)acute Chagas disease (Olivieri et al., submitted)

Posaconazole is more effective than benznidazole in Posaconazole is more effective than benznidazole in preventing cardiac damage in a murine model of acute preventing cardiac damage in a murine model of acute

Chagas disease (Olivieri et al., submitted)Chagas disease (Olivieri et al., submitted)

C Tc

TcBZ

TcPOS

Posaconazole is active against chronic infections by Posaconazole is active against chronic infections by nitrofuran- and nitroimidazole-resistant T. cruzi strains. nitrofuran- and nitroimidazole-resistant T. cruzi strains.

(Molina et al. 2000. AAC 44, 150)(Molina et al. 2000. AAC 44, 150)

CL Y

Co

lom

bia

na

Control

Posaconazole (5mg/kg/day)

0.000.10

0.20

0.30

0.40

0.50

0.60

Pa

ras

ito

log

ica

l cu

re

(da

y 1

91)

Control

Benznidazole(100 mg/kg/day)

Posaconazole(5 mg/kg/day)

Posaconazole(20 mg/kg/day)

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

Based on these findings Based on these findings a clinical development a clinical development program of posaconazole for the treatment of program of posaconazole for the treatment of chronic Chagas disease is being advanced by chronic Chagas disease is being advanced by

Schering PloughSchering Plough. . The first clinical studies (Phase II) are planned The first clinical studies (Phase II) are planned

for the for the second semester of 2009 and will second semester of 2009 and will include several Latin American countries and include several Latin American countries and

possibly Spain possibly Spain

Ravuconazole has a simpler molecular structure than Ravuconazole has a simpler molecular structure than posaconazole, with comparable in vitro anti-T. cruzi posaconazole, with comparable in vitro anti-T. cruzi

activity and a remarkable PK profile in humansactivity and a remarkable PK profile in humans Ravuconazole is a potent, broad-Ravuconazole is a potent, broad-

spectrum, antifungal triazole currently in spectrum, antifungal triazole currently in phase II clinical trials (Eisai Co., Japan) phase II clinical trials (Eisai Co., Japan) for the treatment of for the treatment of invasive fungal invasive fungal infectionsinfections

The compound has potent intrinsic anti-The compound has potent intrinsic anti-T. cruzi T. cruzi activity activity (MIC against cultured (MIC against cultured intracellular amastigotes: 0.5-1 nM)intracellular amastigotes: 0.5-1 nM)

Its activity in animal models of acute and Its activity in animal models of acute and chronic Chagas disease is suppressive, chronic Chagas disease is suppressive, rather than curative, probably due to rather than curative, probably due to inadequate pharmacokinetic propertiesinadequate pharmacokinetic properties

The compound remains a candidate for The compound remains a candidate for the treatment of human Chagas disease the treatment of human Chagas disease due its due its remarkable PK profile in man, remarkable PK profile in man, terminal half life of 4-8 days and large terminal half life of 4-8 days and large (>1000 L) volume of distribution)(>1000 L) volume of distribution)

Novel T. cruzi-CYP 51 inhibitors with remarkable Novel T. cruzi-CYP 51 inhibitors with remarkable potency and selectivity against the parasite,potency and selectivity against the parasite,

in vitro and in vivo in vitro and in vivo Potent Potent T. cruziT. cruzi-CYP 51 inhibitors -CYP 51 inhibitors

serendipitously discovered in the serendipitously discovered in the search of search of T. cruziT. cruzi-specific -specific protein protein farnesyltransferase (PFT) inhibitorsfarnesyltransferase (PFT) inhibitors. . Further optimization has led to Further optimization has led to compounds with remarkable potency compounds with remarkable potency (sub-nanomolar MIC) and 10(sub-nanomolar MIC) and 1044-fold -fold selectivity against intracellular selectivity against intracellular amastigotes amastigotes in vitro, devoid of anti-in vitro, devoid of anti-PFT activity. PFT activity. The compounds are also The compounds are also active in vivo active in vivo (Kraus et al. 2009. JMC (Kraus et al. 2009. JMC 52, 1639; Suryadevara et al. 2009. 52, 1639; Suryadevara et al. 2009. ibid 52: 3703)ibid 52: 3703)

A novel series of A novel series of T. cruziT. cruzi-CYP 51 -CYP 51 inhibitors inhibitors identified in a screen against identified in a screen against Mycobacterium tuberculosis Mycobacterium tuberculosis CYP 51. CYP 51. Active in vitro and in vivo against Active in vitro and in vivo against T. T. cruzicruzi ((Chen et al. 2009. PLoS Negl Chen et al. 2009. PLoS Negl Trop Dis 3: e372)Trop Dis 3: e372)

Novel ergosterol biosynthesis inhibitors for the specific Novel ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease. 2: Amiodarone as an anti-treatment of Chagas disease. 2: Amiodarone as an anti-

T. cruzi agent (T. cruzi agent (Benaim et al. 2006. JMC 16, 569-577)Benaim et al. 2006. JMC 16, 569-577)

Amiodarone is the Amiodarone is the most frequently most frequently used antiarrhythmic agent used in the used antiarrhythmic agent used in the management of chronic Chagas management of chronic Chagas disease patients with cardiac disease patients with cardiac compromisecompromise

The mechanism of action involves a The mechanism of action involves a direct direct blockade of Cablockade of Ca2+2+ plasmalemal plasmalemal channels, a shift of the inactivation channels, a shift of the inactivation potential to more negative values and potential to more negative values and an increase of the refractory periodan increase of the refractory period

The compound has unusual The compound has unusual pharmacokinetic properties: pharmacokinetic properties: tissue tissue levels are 100 to 1000-fold higher than levels are 100 to 1000-fold higher than those in sera and the terminal those in sera and the terminal elimination half life is ≥20 dayselimination half life is ≥20 days

Recent work has shown that this Recent work has shown that this compound also has compound also has broad-spectrum broad-spectrum antifungal activity, which is mediated antifungal activity, which is mediated by interference with the cells’ Caby interference with the cells’ Ca2+2+ homeostasishomeostasis

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

Effects of amiodarone on free CaEffects of amiodarone on free Ca2+ 2+ levels in T. cruzi levels in T. cruzi amastigotesamastigotes

Rhod-2 Rhodamine 123 Merge

C

A

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

Table 2. Free sterols present in Trypanosoma cruzi epimastigotes (EP

stock) grown in the absence or presence of posaconazole, amiodarone or

their combinationa

Sterolc Control Posaconazole12.5 nM

Amiodarone12.5 µM

Posaconazole12.5 nM+

Amiodarone12.5 µM

Exogenous Cholesterol 31.2 46.1 63.4 78.8

Endogenous, 14-desmethyl:

Ergosterol 15.3 5.1 5.1 n.d.d

24-ethyl-5,7,22-cholesta-trien–3b-ol 15.4 12.2 6.7 n.d.

Ergosta-8,24(24’)-dien-3b-ol 6.5 13.7 10.2 n.d.

Ergosta-5,7-dien-3b-ol 9.5 3.6 4.1 n.d.

Ergosta-5,7,24(24’)-trien-3b-ol 8.5 n.d. n.d. n.d.

Ergosta-7,24(24’)-dien-3b-ol 7.6 3.2 3.0 n.d.

24-ethyl-5,7-cholesta-dien-3b-ol 6.0 7.5 7.5 n.d.

Endogenous, 14-methyl:

24-methylenedihydrolanosterol n.d. 3.2 n.d. 16.9

Lanosterol n.d. 5.5 n.d. 4.3

CFree sterols were isolated and purified from whole cells grown in the absence or presence of

the indicated drug concentrations for 96 h; they were analyzed by h igh resolution gas-liquid

chromatography coupled with mass spectrometry, as described in Materials and Methods.

dn.d., not detected.

Synergistic effects effects of amiodarone and Synergistic effects effects of amiodarone and posaconazole on posaconazole on

a murine model of acute Chagas diseasea murine model of acute Chagas disease

Combination therapy with amiodarone and itraconazole Combination therapy with amiodarone and itraconazole led to led to parasitological cure and clinical improvement in a parasitological cure and clinical improvement in a

patient with advanced chronic Chagas cardiomyopathypatient with advanced chronic Chagas cardiomyopathy(Paniz-Mondolfi et al. 2009. Chemother. 55, 228-233)(Paniz-Mondolfi et al. 2009. Chemother. 55, 228-233)

Combination therapy with amiodarone and itraconazole Combination therapy with amiodarone and itraconazole led to parasitological cure and clinical improvement in a led to parasitological cure and clinical improvement in a

patient with advanced chronic Chagas cardiomyopathypatient with advanced chronic Chagas cardiomyopathy(Paniz-Mondolfi et al. 2009. Chemother. 55, 228-233)(Paniz-Mondolfi et al. 2009. Chemother. 55, 228-233)

Novel ergosterol biosynthesis inhibitors for the Novel ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease. 2. Squalene specific treatment of Chagas disease. 2. Squalene

synthase inhibitors (Urbina et al. 2004. AAC 48, 2379)synthase inhibitors (Urbina et al. 2004. AAC 48, 2379)

Squalene synthase has been Squalene synthase has been chemically validated as a chemically validated as a chemotherapeutic targetchemotherapeutic target in in T. T. cruzi cruzi and and Leishmania spp. Leishmania spp. (Urbina el al. 2001. MBP 125, (Urbina el al. 2001. MBP 125, 35)35)

Novel Novel quinuclidine derivativesquinuclidine derivatives, , specific inhibitors of squalene specific inhibitors of squalene synthase (SQS), also in synthase (SQS), also in development as cholesterol-development as cholesterol-lowering agents, have lowering agents, have potent and potent and selective anti-selective anti-T. cruziT. cruzi activity in activity in vitrovitro, as well as, as well as oral activity in oral activity in murine models of Chagas murine models of Chagas diseasedisease

Further development as Further development as antiparasitic agents will require antiparasitic agents will require parasite-specificparasite-specific SQS inhibitors SQS inhibitors

T. cruzi SQS sequence and heterologous expression in T. cruzi SQS sequence and heterologous expression in E. coli (Sealey-Cardona et al. 2007. AAC 51, 2139)E. coli (Sealey-Cardona et al. 2007. AAC 51, 2139)

Selective inhibitors of T. cruzi SQS and amastigote Selective inhibitors of T. cruzi SQS and amastigote proliferation (Sealey-Cardona et al. 2007. AAC 51, 2139)proliferation (Sealey-Cardona et al. 2007. AAC 51, 2139)

Novel ergosterol biosynthesis inhibitors for the specific Novel ergosterol biosynthesis inhibitors for the specific treatment of Chagas disease. 4: Compounds with a dual treatment of Chagas disease. 4: Compounds with a dual

mechanism of action (mechanism of action (Gerpe et al. 2008. BMC 16, 569-577)Gerpe et al. 2008. BMC 16, 569-577)

Anti-T. cruzi activity of heteroallyl-containing Anti-T. cruzi activity of heteroallyl-containing 5-nitrofurans in vitro (5-nitrofurans in vitro (Gerpe et al. 2008. BMC 16, 569-577)Gerpe et al. 2008. BMC 16, 569-577)

Effects of heteroallyl-containing Effects of heteroallyl-containing 5-nitrofurans on sterol composition and redox cycling of T. 5-nitrofurans on sterol composition and redox cycling of T.

cruzi epimastigotes (cruzi epimastigotes (Gerpe et al. 2008. BMC 16, 569-577Gerpe et al. 2008. BMC 16, 569-577))

Conclusions-1Conclusions-1 There is an urgent need for safer and more effective There is an urgent need for safer and more effective

treatments for Chagas disease, particularly in its chronic treatments for Chagas disease, particularly in its chronic stage. Several new approaches are being advanced, based stage. Several new approaches are being advanced, based on our increasing knowledge of the biochemistry and on our increasing knowledge of the biochemistry and physiology of physiology of T. cruziT. cruzi

T. cruzi T. cruzi has an has an essential requirement of ergosterol and other essential requirement of ergosterol and other 24-alkyl24-alkyl sterol for survival and growth and ergosterol sterol for survival and growth and ergosterol biosynthesis inhibitors are potent antifungal and anti-biosynthesis inhibitors are potent antifungal and anti-trypanosomatid agentstrypanosomatid agents

Novel triazole derivativesNovel triazole derivatives, specific inhibitors of the parasite’s , specific inhibitors of the parasite’s lanosterol C14lanosterol C14 demethylase (CYP51), demethylase (CYP51), have trypanocidal have trypanocidal activity in vitro and can cure both acute and chronic infectionsactivity in vitro and can cure both acute and chronic infections in several murine models of the disease. Several of these in several murine models of the disease. Several of these compounds are poised to undergo clinical development for compounds are poised to undergo clinical development for Chagas disease in the short term, Chagas disease in the short term, posaconazole and posaconazole and ravuconazole ravuconazole being the most advanced candidates being the most advanced candidates

Conclusions-2Conclusions-2

Recent studies have shown that Recent studies have shown that amiodaroneamiodarone, , the the most frequently most frequently used antiarrhythmic agent used in the management of chronic used antiarrhythmic agent used in the management of chronic Chagas disease patients with cardiac compromise, also has Chagas disease patients with cardiac compromise, also has anti-anti-T. T. cruzi cruzi activity in vitro and in vivo and acts synergistically with activity in vitro and in vivo and acts synergistically with posaconazole. posaconazole. This finding raises the notion that symptomatic This finding raises the notion that symptomatic treatment with amiodarone of Chagas disease patients treatment with amiodarone of Chagas disease patients could have could have the added benefit of lowering the parasite load and enhancing the the added benefit of lowering the parasite load and enhancing the efficacy of specific anti-efficacy of specific anti-T. cruzi T. cruzi drugs and this has been recently drugs and this has been recently verified in a patient with advanced Chagas myocardiopathyverified in a patient with advanced Chagas myocardiopathy

Other ergosterol biosynthesis inhibitors that could enter clinical Other ergosterol biosynthesis inhibitors that could enter clinical development in the next decade are development in the next decade are specific squalene synthase specific squalene synthase inhibitorsinhibitors such as such as quinuclidine derivativesquinuclidine derivatives

A recent development in this field is the discovery of anti-A recent development in this field is the discovery of anti-T. cruziT. cruzi agents with agents with a dual mechanism of actiona dual mechanism of action: : inhibition of ergosterol inhibition of ergosterol biosynthesisbiosynthesis at the level of squalene epoxidase and at the level of squalene epoxidase and generation of generation of oxidative stressoxidative stress by redox cycling of reduced nitrofuran radicals by redox cycling of reduced nitrofuran radicals

CollaboratorsCollaborators