Chagas Disease Discovery (1909

Presented by DNDi at ASTMH 2009

Drugs for Neglected Diseases Iniciative & Universidade Federal de Ouro Preto, Brazil

Combination of Benznidazole and Nifurtimox plus

Posaconazole enhances activity against Trypanosoma cruzi

Maria Terezinha Bahia, André Talvani, Shing Chang, Isabela Ribeiro

Neglected populations Future

New Chemical Entities ‐ Target Product Profile Old drugs with new Chemotherapy strategies Specific anti‐ T. cruzi treatment Improvements in Case Management –Heart Disease Present (2009) Vector control/eradication programmes

Description of main clinical manifestations

Trypanosoma cruzi identification

Past (1909) Chagas disease discovery (1909 – 2009)

1 Presented by DNDi at ASTMH 2009

Evaluating combination treatment…

Combination with registered compounds (Benznidazole/Nifurtimox)

Aims: (i) improvement of efficacy (ii) improvement of safety and tolerability (iii) reduction of the dose and duration of the therapeutic regimen (iv) Potential impact on resistance development to each individual compounds from the combinations

Starting point:

Evaluation of combination therapy Nifurtimox/Benznidazole + Azole compounds « Reduction in time, costs and risks »

Current available drugs

• Benznidazole and Nifurtimox: nitroheterocyclic drugs

• Parasiticidal activity: conversion of reactive intermediates within the parasite that generate superoxide, which causes oxidative damage to components of the parasite

• Compounds activated within the parasite, probably by a Type 1 Mitochondrial Nitroreductase enzyme

Wilkinson et al. PNAS 2008; 105:5022‐7 Paulino et al. Mini Rev Med Chem. 2005

2 Presented by DNDi at ASTMH 2009

Trypanosoma cruzi Ergosterol Biosynthesis Pathway

Azole class of compounds: Posaconazole, Ravuconazole

E.G. Hankins et al. / Molecular & Biochemical Parasitology 144 (2005) 68–75

Combination of nitroheterocyclic compounds Benznidazole and Nifurtimox plus Posaconazole

Swiss mice inoculated with 5.0 x 103 blood trypomastigotes of T. cruzi Y strain Treatment initiated 4 days post‐infection with confirmed parasitaemia 7 consecutive days with each single drug by gavage

Therapeutic dose (n=6) Y strain

Half of the therapeutic dose (n=6) Y strain

One forth of the therapeutic dose (n=6) Y strain

3 Presented by DNDi at ASTMH 2009

Mice infected with Y strain of Trypanosoma cruzi treated with different doses of Benznidazole, Nifurtimox and Posaconazole

Dose Parasitemia suppression Parasitemia Parasitemia peak x (dose+SD) reactivation (day) 103 * Benznidazole 100mg 1.33+0.52 5 10.0 (17th) 50 mg 1.83+0.75 1 84.6 (16th) 25 mg ND ‐ 246.6 (8th) Nifurtimox 50 mg 1.0+0.0 5 8.3 (16th) 25 mg ND ‐ 30.6 (8th) 12.5 mg ND ‐ 396.6 (8th) Posaconazole Ps 20 mg 1.33+0..51 12 11.3 (24th) Ps 10 mg 1.5+0.54 11 49.3 (25th) Ps 5.0 mg 1.17+0.41 11 318.6 (26th) No treated control group Control ‐ ‐900.6 (8th)

Nifurtimox

Maximum parasitemia levels of the Y strain 7 aabc 6 infected mice treated with different 5 4 doses of Benznidazole, Nifurtimox and 3 Posaconazole 2 Log -Log Parasitemia 1 0 Nfx 50 mg Nfx 25 mg Nfx 12.5 mg

Benznidazole Posaconazole

a ab b a ab b 7 7 6 6 5 5 4 4 3 3 2 2 Log -Log Parasitemia 1 -Log Parasitemia 1 0 0 Bz - 100 mg Bz - 50 mg Bz - 25 mg Ps - 20 mg Ps - 10 mg Ps - 5 mg

4 Presented by DNDi at ASTMH 2009

Nifurtimox 100 Survival rates of the Y strain infected 80

mice treated with different doses of 60

Benznidazole, Nifurtimox and 40 Survival % Survival Posaconazole 20 0 0 5 10 15 20 25 30 Days after infection Nfx - 50 mg Nfx - 25 mg Nfx - 12.5 mg IC

Benznidazole Posaconazole

100 100

80 80

60 60

40 40 Survival % Survival % Survival 20 20

0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Days after infection Days after infection

Bz - 50 mg Bz - 100 mg Bz - 25 mg IC Ps 20 mg Ps 10 mg Ps 5.0 mg IC

Drug Combination experiments

Drug combinations:

Benznidazole (Bz) plus Posaconazole (Ps) 25 and 50 mg/kg.day of Bz + 5 and 10 mg mg/kg/day of Ps

Nifurtimox (Nfx) plus Posaconazole (Ps) 12.5 and 25 mg/kg.day of Nfx + 5 and 10 mg mg/kg.day of Ps

5 Presented by DNDi at ASTMH 2009

Benznidazole plus Posaconazole treatment

Treatment Parasitemia Parasitemia Parasitemia Survival rate (%) scheme suppression reactivation peak (n=6) (dose+SD) (day)

Bz/Ps 1.4+0.55 10 21.600 (22nd) 100% (50/10mg) Bz/Ps (50/5mg) 1.33+0.52 10 60.000 (22nd) 100% Bz/Ps 1.67+0.52 10 40.166 100% (25/10mg) (26th) Bz/Ps (25/5mg) 1.16+0.41 7 11.133 100% (27th) Bz 100mg 1.33+0.52 5 10.000 100% (17th) Ps 20 mg 1.33+0..51 12 11.333 100% (24th)

Benznidazole plus Posaconazole treatment Parasitaemia 6

Log - Parasitemia 1

g g g g g g

m m m m m m 0 0 0 5 0 5 0 2 1 s 1 s 1 - s s - P P s P / P / z / / P g g B g g m m m m 0 5 0 5 5 2 5 z 2 z z B z B B B

Maximum parasitemia – log parasite/0.1 mL of blood

6 Presented by DNDi at ASTMH 2009

Nifurtimox plus Posaconazole treatment

Treatment Parasitemia Parasitemia Parasitemia Survival rate scheme suppression reactivation peak (%) (n=6) (dose+SD) (day)

Nfx /Ps 1.5+0.55 11 63.333 100% (25/10mg) (27th) Nfx /Ps 1.33+0.52 11 27.333 100% (25/5mg) (27th) Nfx /Ps 1.0+0.0 10 68.666 84% (12.5/10mg) (27th) Nfx /Ps 1.16+0.41 9 68.666 100% (12.5/5mg) (27th) Ps 20 mg 1.33+0.51 12 11.333 100% (24th) Nfx 50 mg 1.0+0.0 5 8.333 100% (16th)

Nifurtimox plus Posaconazole treatment Parasitaemia

Log-Parasitemia 1

g mg m 0 mg - 2 10 mg Ps Nfx - 50 mg g + Ps 5 + Ps g + Ps 5 m g m m 25 2.5 x 1 f x Nfx 25 mg + Ps N10 mg 12.5 f fx N N

Maximum parasitemia – log parasite/0.1 mL of blood

7 Presented by DNDi at ASTMH 2009

Drug combination experiments Conclusions and future directions

9The combination of Ps and Bz or Nfx was significantly more efficacious against T. cruzi than the same dose of each drugs alone. 9Need for confirmation of findings in experimental models with prolonged exposure and immunosuppression for assessment of cure 9 Alternative combination regimens for Chagas disease should be further investigated

“A one‐shot inexpensive, nontoxic drug to be used in individual cases as well as for preventing Chagas disease transmission is still a vague dream.”

Brener Z: Chemotherapy of Trypanosoma cruzi infection”

Advances in Pharmacology and Chemotherapy, 13: 2‐40, 1975.

Prof. Z Brener (1928-2002)

8 Presented by DNDi at ASTMH 2009

Collaborators:

Post-graduated students: Isabel Mayer DNDi: F. Alves Livia Figueiredo B. Bourdin Ivo Caldas R. Don Sergio Caldas E. Torreele

Graduated students: Andre Gravel Alvaro Neto Tassiane Martins Technicians: Vivian Figueiredo Hidelmagna Silva Maira Azevedo