A Novel Piperazine-Based Drug Lead for Cryptosporidiosis from the Medicines for Malaria Venture Open-Access Malaria Box

Abstract

Cryptosporidiosis causes life-threatening diarrhea in children under the age of 5 years and prolonged diarrhea in immunodeficient people, especially AIDS patients. The standard of care, nitazoxanide, is modestly effective in children and ineffective in immunocompromised individuals. In addition to the need for new drugs, better knowledge of drug properties that drive in vivo efficacy is needed to facilitate drug development. We report the identification of a piperazine-based lead compound for Cryptosporidium drug development, MMV665917, and a new pharmacodynamic method used for its characterization. The identification of MMV665917 from the Medicines for Malaria Venture Malaria Box was followed by dose-response studies, in vitro toxicity studies, and structure-activity relationship studies using commercial analogues. The potency of this compound against Cryptosporidium parvum Iowa and field isolates was comparable to that against Cryptosporidium hominis. Furthermore, unlike nitazoxanide, clofazimine, and paromomycin, MMV665917 appeared to be curative in a NOD SCID gamma mouse model of chronic cryptosporidiosis. MMV665917 was also efficacious in a gamma interferon knockout mouse model of acute cryptosporidiosis. To determine if efficacy in this mouse model of chronic infection might relate to whether compounds are parasiticidal or parasitistatic for C. parvum, we developed a novel in vitro parasite persistence assay. This assay suggested that MMV665917 was parasiticidal, unlike nitazoxanide, clofazimine, and paromomycin. The assay also enabled determination of the concentration of the compound required to maximize the rate of parasite elimination. This time-kill assay can be used to prioritize early-stage Cryptosporidium drug leads and may aid in planning in vivo efficacy experiments. Collectively, these results identify MMV665917 as a promising lead and establish a new method for characterizing potential anticryptosporidial agents.

Identification of a potent benzoxaborole drug candidate for treating cryptosporidiosis

“…Consistent with this hypothesis, C. parvum time-kill curve assays showed that the approved drug nitazoxanide, which is effective in immunocompetent adults but equivalent to a placebo in AIDS patients, is likely parasitistatic or very slow acting ( Fig. 3a) 19 . Time-kill curve data were referenced to the vehicle control for each time point in order to isolate the effect of compounds from the spontaneous decline in C. parvum numbers that occurs after 24 to 48 h of in vitro culture.…”

“…Efficacy in acute and established murine cryptosporidiosis. AN7973 efficacy was first tested in vivo using a NOD scid IL2rg null (NOD scid gamma (NSG)) mouse model, which is a model of established C. parvum infection 19 . Treatment was begun 7 days after infecting mice by oral gavage with the C. parvum Bunch Grass Farms Iowa isolate.…”

“…Various doses of AN7973 were tested using this model and the C. parvum University of Arizona Iowa isolate. Clofazimine, which is parasitistatic and lacks efficacy against Cryptosporidium in the NSG mouse 19 , was used as a positive control 17 . AN7973 demonstrated dose-dependent efficacy, including efficacy at a dose of 10 mg per kg once daily.…”

“…ARTICLE simply by providing alternative drugs or by facilitating development of drug combinations to reduce the likelihood that resistance will evolve. Towards this goal, phenotypic screening assays have been developed and a growing number of lead compounds have been reported [17][18][19]32,[48][49][50][51][52] . The phosphatidyl-inositol 4 kinase (PI4K) inhibitor KDU731, which was repurposed from a malaria drug development effort, is currently the most advanced compound in the Cryptosporidium pipeline 16 .…”

“…Its activity in dairy calves is controversial and at best marginal 14,15 . New anti-Cryptosporidium drugs are urgently needed, and several promising drug leads have been reported recently [16][17][18][19] .…”