Clinical and microbiologic efficacy of the piperazine-based drug lead MMV665917 in the dairy calf cryptosporidiosis model

<div><p>Cryptosporidiosis causes life-threatening diarrhea in infants, but the best available treatment is only modestly efficacious. Rodents infected with relevant <i>Cryptosporidium</i> species do not develop diarrhea, which complicates drug development. <i>Cryptosporidium parvum</i> infection of dairy calves, however, causes an illness like that seen in infants. Here, the clinical and microbiologic anti-<i>Cryptosporidium</i> efficacy of the piperazine-based compound MMV665917 was demonstrated in neonatal calves. Oral administration of MMV665917 (22 mg/kg once daily) was begun two days after the onset of severe diarrhea and continued for seven days. Treatment resulted in prompt resolution of diarrhea, and reduced total fecal oocyst shedding by ~94%. MMV665917 was useful for treatment, rather than just prophylaxis, since it was safe and effective when administered well after the onset of diarrhea. Furthermore, even though all animals received intensive supportive care, there was a strong trend towards improved secondary health outcomes, including general health, appetite, and dehydration measures amongst treated animals. These data establish MMV665917 as an outstanding lead compound for <i>Cryptosporidium</i> drug development.</p></div

Criteria used for scoring clinical signs and symptoms, including fecal consistency, overall health, hydration, and appetite.

<p>Criteria used for scoring clinical signs and symptoms, including fecal consistency, overall health, hydration, and appetite.</p

Effect of MMV665917 on secondary health measures in <i>C</i>. <i>parvum</i> infected calves.

<p>Overall health, hydration, and appetite were assessed twice daily on days 4–11 after infection using standardized three point scores (1 = normal, 3 = severely abnormal). The area under the curve (AUC) for each metric was determined for each animal’s average daily score plotted vs. time using a baseline score of 1 (normal). Each graph shows a scatter plot of data for individual animals, along with means and 95% confidence intervals (n = 7 for control; n = 6 for MMV665917 treated). p values were determined using the unpaired, single-sided student’s t test. <b>(A)</b> Overall health scores. <b>(B)</b> Hydration scores. <b>(C)</b> Appetite scores.</p

Once daily MMV665917 reduces fecal parasite shedding in <i>C</i>. <i>parvum</i> infected calves.

<p>Newborn dairy calves were infected with the <i>C</i>. <i>parvum</i> Iowa isolate and treated orally with either MMV665917 22 mg/kg once daily or drug vehicle alone from days 4–10 after infection, following development of severe diarrhea. Fecal oocyst shedding was measured using qPCR. <b>(A)</b> Fecal oocyst shedding vs. time for control (black circles) or MMV665917 treated (open squares) calves. Data are the means and SEMs of the Log₁₀ (oocysts/gram fecal dry matter) (n = 7 for control; n = 6 for MMV665917 treated). Dashed line indicates the approximate qPCR assay limit of detection (LOD). <b>(B)</b> Scatter plot showing the area under the curve (AUC) for Log₁₀ transformed fecal oocyst shedding for individual animals. The lines indicate the means and 95% confidence intervals. p value was determined using the unpaired, single-sided student’s t test.</p

Pharmacokinetics of MMV665917 in dairy calves.

<p><b>(A)</b> Uninfected calves. The graph shows calf fecal (circles) and serum (squares) MMV665917 concentrations vs. time following a single oral dose of 22 mg/kg given to healthy newborn calves. Data points are the means and SDs (n = 2 calves). <b>(B)</b> <i>Cryptosporidium parvum</i> infected calves. The graph shows calf fecal (circles) and serum (squares) MMV665917 concentrations vs. time for calves administered 22 mg/kg once daily for seven days, beginning 4 days after infection with the <i>C</i>. <i>parvum</i> Iowa isolate (indicated by blue arrows). The mean and SD (n = 3 calves) are shown.</p

Once daily MMV665917 reduces diarrhea in <i>C</i>. <i>parvum</i> infected calves.

<p>Newborn dairy calves were infected with the <i>C</i>. <i>parvum</i> Iowa isolate and treated orally with either MMV665917 22 mg/kg once daily or drug vehicle alone from days 4–10 after infection, following development of severe diarrhea. Diarrhea was scored based on fecal consistency, with scores of 3 and 1 corresponding to severe diarrhea and normal, respectively. <b>(A)</b> Diarrhea scores vs. time for control (black circles) or MMV665917 treated (open squares) calves. Data are the means and SEMs (n = 7 for control; n = 6 for MMV665917 treated). <b>(B)</b> Scatter plot showing the area under the curve (AUC) for fecal score vs. time for individual animals. The lines indicate the means and 95% confidence intervals. p value was determined using the unpaired, single-sided student’s t test.</p