Efficacy of Novel Pyrazolone Phosphodiesterase Inhibitors in Experimental Mouse Models of Trypanosoma cruzi

Abstract

Pyrazolones are heterocyclic compounds with interesting biological properties. Some derivatives inhibit phosphodiesterases (PDEs) and thereby increase the cellular concentration of cyclic AMP (cAMP), which plays a vital role in the control of metabolism in eukaryotic cells, including the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease (CD), a major Neglected Tropical Disease. In vitro phenotypic screening identified a 4-bromophenyl-dihydropyrazole dimer as anti-T. cruzi hit and 17 novel pyrazolone analogues with variations on the phenyl ring were investigated in a panel of phenotypic laboratory models. Potent activity against the intracellular forms (Tulahuen and Y strains) was obtained with EC50 values within the 0.17 - 3.3 μM range. Although most were not active against bloodstream trypomastigotes, an altered morphology and loss of infectivity were observed. Pretreatment of the mammalian host cells with pyrazolones did not interfere with infection and proliferation, showing that the drug activity was not the result of changes to host cell metabolism. The pyrazolone NPD-227 increased the intracellular cAMP levels and was able to sterilize of T. cruzi-infected cell cultures. Thus, due to its high potency and selectivity in vitro, and its additive interaction with benznidazole (Bz), NPD-227 was next assessed in the acute mouse model. Oral dosing for 5 days of NPD-227 at 10 mg/kg + BZ at 10 mg/kg not only reduced parasitaemia (>87 %) but also protected against mortality (>83% survival), hence demonstrating superiority to the monotherapy schemes. These data support these pyrazolone molecules as potential novel therapeutic alternatives for Chagas disease.

Perspectives for a new drug candidate for Chagas disease therapy

“… 46 Ultrastructural analysis of drug-treated bloodstream trypomastigotes showed cellular changes consistent with autophagy and osmotic stress, mechanisms already previously linked to cAMP signaling as also observed for other PDE inhibitors. 42 , 43 , 44 , 45 , 46 Compound 9 raised both cellular and supernatant cAMP levels, confirming inhibition of T. cruzi PDE(s).…”

“…A greater anti-parasitic effect was observed against intracellular amastigotes (EC 50 values ranging from 0.17 to 3.3 µM on Tulahuen and Y strains) as compared to bloodstream trypomastigotes (Y strain). 44 Previous validation studies performed by the measurement of cAMP levels showed that untreated amastigotes have higher ability to efflux this second messenger than untreated bloodstream forms, which may influence, at least in part, the different susceptibility profile of trypomastigotes and amastigotes, as observed with the exposure of some PDE inhibitors. 42 , 44 The pyrazoles NPD-227 altered the morphology of bloodstream trypomastigotes inducing a rounding effect that impaired their invasion into the cardiac host cells, denoting that although not able to induce a rapid lysis, this pyrazole reduced the parasite infective fitness.…”

“… 44 Previous validation studies performed by the measurement of cAMP levels showed that untreated amastigotes have higher ability to efflux this second messenger than untreated bloodstream forms, which may influence, at least in part, the different susceptibility profile of trypomastigotes and amastigotes, as observed with the exposure of some PDE inhibitors. 42 , 44 The pyrazoles NPD-227 altered the morphology of bloodstream trypomastigotes inducing a rounding effect that impaired their invasion into the cardiac host cells, denoting that although not able to induce a rapid lysis, this pyrazole reduced the parasite infective fitness. 44 The rounding effect induced by NPD-227 in bloodstream forms may be related to an osmo-deregulation, an effect already reported during PDEC inhibition in T. cruzi 45 As noticed with other classes of PDE inhibitors such as phthalazinones, 42 pyrazolones caused profounds insults in Golgi, flagellar pocket, and plasma membrane of T. cruzi likewise suggestive of osmotic stress attributed to increased cAMP levels that was observed in treated parasites, validating the targeting of at least one PDE parasite isoforms.…”

“… 42 , 44 The pyrazoles NPD-227 altered the morphology of bloodstream trypomastigotes inducing a rounding effect that impaired their invasion into the cardiac host cells, denoting that although not able to induce a rapid lysis, this pyrazole reduced the parasite infective fitness. 44 The rounding effect induced by NPD-227 in bloodstream forms may be related to an osmo-deregulation, an effect already reported during PDEC inhibition in T. cruzi 45 As noticed with other classes of PDE inhibitors such as phthalazinones, 42 pyrazolones caused profounds insults in Golgi, flagellar pocket, and plasma membrane of T. cruzi likewise suggestive of osmotic stress attributed to increased cAMP levels that was observed in treated parasites, validating the targeting of at least one PDE parasite isoforms. 45 Due to the high potency and selectivity in vitro and its additive interaction with Bz, the pyrazolone NPD-227 was further investigated in experimental mouse model of T. cruzi (Y strain) of acute infection.…”

Experimental Combination Therapy with Amiodarone and Low-Dose Benznidazole in a Mouse Model of Trypanosoma cruzi Acute Infection

“…The combined therapy group, Bz25/AMD50, received 25 mg Bz/kg/day plus 50 mg AMD/kg/day. The dose of 25 Bz mg/kg is well established as a suboptimal dose ( 60 ), which corresponds to a one-quarter Bz optimal dose (100 mg Bz/kg/day) ( 63 ). A dose of 50 mg/kg/day of AMD was chosen based on the estimated body surface area of mice and the average body surface area of an adult human and is considered equivalent to the clinical dosage for an adult human (300 mg/kg/day) ( 64 ).…”