Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans.
SummaryPhysiologically based pharmacokinetic models for bumped kinase inhibitors suggest that in vivo anticryptosporidial efficacy is associated with gastrointestinal concentrations of drug.
There is need for a more efficient cell-based assay amenable to high-throughput drug screening against Giardia lamblia. Here, we report the development of a screening method utilizing G. lamblia engineered to express red-shifted firefly luciferase. Parasite growth and replication were quantified using D-luciferin as a substrate in a bioluminescent read-out plateform. This assay was validated for reproducibility and reliability against the Medicines for Malaria Venture (MMV) Pathogen Box compounds. For G. lamblia, forty-three compounds showed ≥ 75% inhibition of parasite growth in the initial screen (16 μM), with fifteen showing ≥ 95% inhibition. The Pathogen Box was also screened against Nanoluciferase expressing (Nluc) C. parvum, yielding 85 compounds with ≥ 75% parasite growth inhibition at 10 μM, with six showing ≥ 95% inhibition. A representative set of seven compounds with activity against both parasites were further analyzed to determine the effective concentration that causes 50% growth inhibition (EC50) and cytotoxicity against mammalian HepG2 cells. Four of the seven compounds were previously known to be effective in treating either Giardia or Cryptosporidium. The remaining three shared no obvious chemical similarity with any previously characterized anti-parasite diarrheal drugs and offer new medicinal chemistry opportunities for therapeutic development. These results suggest that the bioluminescent assays are suitable for large-scale screening of chemical libraries against both C. parvum and G. lamblia.
Cryptosporidium is a leading cause of pediatric diarrhea worldwide. Currently there is neither a vaccine nor a consistently effective drug available for this disease. Selective 5-aminopyrazole-4carboxamide-based bumped-kinase inhibitors (BKIs) are effective in both in vitro and in vivo models of Cryptosporidium parvum. Potential cardiotoxicity in some BKIs led to the continued exploration of the 5-aminopyrazole-4-carboxamide scaffold to find safe and effective drug candidates for Cryptosporidium. A series of newly designed BKIs were tested for efficacy against C. parvum using in vitro and in vivo (mouse infection model) assays, and safety issues. Compound 6 (BKI 1708) was found to be efficacious at 8 mg/kg dosed once daily (QD) for 5 days with no observable signs of toxicity up to 200 mg/kg dosed QD for 7 days. Compound 15 (BKI 1770) was found to be efficacious at 30 mg/kg dosed twice daily (BID) for 5 days with no observable signs of toxicity up to 300 mg/kg dosed QD for 7 days. Compounds 6 and 15 are promising pre-clinical leads for cryptosporidiosis therapy with acceptable safety parameters and efficacy in the mouse model of cryptosporidiosis.
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