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. 2019 Jun 19;220(2):285-293.
doi: 10.1093/infdis/jiz105.

Piperazine-Derivative MMV665917: An Effective Drug in the Diarrheic Piglet Model of Cryptosporidium hominis

Affiliations

Piperazine-Derivative MMV665917: An Effective Drug in the Diarrheic Piglet Model of Cryptosporidium hominis

Sangun Lee et al. J Infect Dis. .

Abstract

Background: Cryptosporidiosis, an enteric protozoon, causes substantial morbidity and mortality associated with diarrhea in children <2 years old in low- to middle-income countries. There is no vaccine and treatments are inadequate. A piperazine-based compound, MMV665917, has in vitro and in vivo efficacy against Cryptosporidium parvum. In this study, we evaluated the efficacy of MMV665917 in gnotobiotic piglets experimentally infected with Cryptosporidium hominis, the species responsible for >75% of diarrhea reported in these children.

Methods: Gnotobiotic piglets were orally challenged with C hominis oocysts, and oral treatment with MMV665917 was commenced 3 days after challenge. Oocyst excretion and diarrhea severity were observed daily, and mucosal colonization and lesions were recorded after necropsy.

Results: MMV665917 significantly reduced fecal oocyst excretion, parasite colonization and damage to the intestinal mucosa, and peak diarrheal symptoms, compared with infected untreated controls. A dose of 20 mg/kg twice daily for 7 days was more effective than 10 mg/kg. There were no signs of organ toxicity at either dose, but 20 mg/kg was associated with slightly elevated blood cholesterol and monocytes at euthanasia.

Conclusions: These results demonstrate the effectiveness of this drug against C hominis. Piperazine-derivative MMV665917 may potentially be used to treat human cryptosporidiosis; however, further investigations are required.

Keywords: Cryptosporidium hominis; MMV665917; animal model; gnotobiotic pigs.

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Figures

Figure 1.
Figure 1.
Fecal excretion of Cryptosporidium hominis oocysts after challenge. Piglets were inoculated orally with C hominis oocysts 2 days after birth and were treated with MMV665917 3 days postchallenge. Rectal swabs were processed for oocyst measurement. The plots represent mean ± standard error of the mean. The data of oocyst excretions between groups were compared by Dunn’s multiple comparisons test. *, P < .05. Cryptosporidium hominis-infected group (n = 8, closed circle [●くろまる]); C hominis-infected and MMV665917 (20 mg/kg)-treated group (n = 7, closed square ■しかく); Cryptosporidium hominis-infected and MMV665917 (10 mg/kg)-treated group (n = 3, closed triangle [▲さんかく]).
Figure 2.
Figure 2.
Quantification of Cryptosporidium deoxyribonucleic acid (DNA) in feces and gut contents: (A) Cryptosporidium DNA in feces after dosing with MMV665917, and (B) Cryptosporidium DNA in gut contents. Piglets were inoculated orally with Cryptosporidium hominis oocysts 2 days after birth and were treated with MMV665917 3 days postchallenge. Rectal swabs were processed for Cryptosporidium DNA measurement. Plot A represents mean ± standard error of the mean (SEM), and a scatter dot plot is shown in B (mean ± SEM). The data of Cryptosporidium DNA between groups were compared by Dunn’s multiple comparisons test (*, P < .05; **, P < .01). Cryptosporidium hominis-infected group (n = 8, closed circle [●くろまる]); C hominis-infected and MMV665917 (20 mg/kg)-treated group (n = 7, closed square [■しかく]); C hominis-infected and MMV665917 (10 mg/kg)-treated group (n = 3, closed triangle [▲さんかく]).
Figure 3.
Figure 3.
Daily diarrhea scores observed in piglets after oocyst challenge and treatments: (A) MMV665917 20 mg/kg; (B) MMV665917 10 mg/kg; (C) 1% hydroxypropyl methyl-cellulose (HPMC). Piglets were inoculated orally with Cryptosporidium hominis oocysts 2 days after birth. The clinical signs of cryptosporidiosis were monitored daily, and diarrheal symptom were scored as described in Methods. The plots represent mean ± standard error of the mean. The data of diarrhea score between groups were compared by Dunn’s multiple comparisons test and Mann-Whitney test on each day: *, P < .05; **, P < .01; ***, P < .001; ****, P < .0001. Cryptosporidium hominis-infected group (n = 8, closed circle [●くろまる]); C hominis-infected and MMV665917 (20 mg/kg)-treated group (n = 7, closed square [■しかく]); MMV665917 (20 mg/kg)-treated group (n = 4, open square [□しろいしかく]); C hominis-infected and MMV665917 (10 mg/kg)-treated group (n = 3, closed triangle [▲さんかく]); MMV665917 (10 mg/kg)-treated group (n = 2, open triangle [△しろさんかく]); 1% HPMC-treated group (n = 2, open rhombus [◊]); and nontreated control (n = 2, open circle [○しろまる]).
Figure 4.
Figure 4.
Microscopic examination of hematoxylin and eosin-stained sections magnified ×ばつ40 from the spiral colon. (A) No infection, No treatment; (B) Cryptosporidium hominis infection only; and (C) C hominis and MMV665917 20 mg/kg. Spiral colon is colonized by C hominis at the surface epithelium (arrows) and in a colonic gland (oval) (B), whereas no parasite forms are found in a C hominis-infected piglet that was treated with MMV665917 (C).
Figure 5.
Figure 5.
Mucosal colonization and lesion scores in piglets. (A) Colonization scores; (B) lesion scores. Mucosal colonization was scored from 0 to 5 (0 = no infection detected; 1 = 1%–20% epithelial surface infected; 2 = 21%–40% surface infected; 3 = 41%–60% surface infected; 4 = 61%–80% surface infected; 5 = 81%–100% surface infected) in 3 fields from each anatomic site: duodenum, jejunum, ileum, cecum, and spiral colon and then averaged. For each piglet, the final score is a sum of the averages (A). Mucosal lesion scores were generated for each site for changes in villi, epithelial cells, and inflammation. The following scores were used: normal = 0, equivocal = 2.5, mild = 5, moderate = 10, marked = 15. The final lesion score was a sum of scores from duodenum, jejunum, ileum, cecum, and colon for each piglet. The scatter plot (A and B) represents mean ± standard error of the mean. A Mann-Whitney test was conducted using GraphPad Prism 7.03: *, P < .05; **, p<.01; ***, P < .001. Cryptosporidium hominis-infected group (n = 8, closed circle [●くろまる]); C hominis-infected and MMV665917 (20 mg/kg)-treated group (n = 7, closed square [■しかく]); MMV665917 (20 mg/kg)-treated group (n = 4, open square [□しろいしかく]); C hominis-infected and MMV665917 (10 mg/kg)-treated group (n = 3, closed triangle [▲さんかく]; MMV665917 (10 mg/kg)-treated group (n = 2, open triangle [△しろさんかく]); 1% hydroxypropyl methyl-cellulose (HPMC)-treated group (n = 2, open rhombus [◊]); and nontreated control (n = 2, open circle [○しろまる]).
Figure 6.
Figure 6.
MMV665917 concentration in plasma and gut contents: (A) MMV665917 concentration in plasma after dosing with MMV665917 20 mg/kg or 10 mg/kg dose and (B) MMV665917 concentration in gut contents. Piglets were inoculated orally with Cryptosporidium hominis oocysts 2 days after birth and dosing with MMV665917 began 3 days postchallenge. Plasma samples were collected at 0 hour (before 1st dose), 2 hours (post 1st dose), 72 hours (12 hours post 6th dose), 74 hours (2 hours post 7th dose), 144 hours (12 hours post 12th dose), 146 hours (2 hours post 13th dose), and 216 hours (60 hours post 14th dose). Gut contents were also collected at 216 hours (60 hours post 14th dose). Unpaired t test was conducted using GraphPad Prism 7.03. EC90 = 5.0 (2.5 to 13.3) μM.
Figure 7.
Figure 7.
Alteration of serum biochemical and hematological data in piglets after MMV665917 treatment. Serum biochemical and hematological parameters were analyzed from blood samples of euthanized piglets. The scatter plots represent mean ± standard error of the mean. Tukey’s multiple comparisons test was conducted among 3 groups using GraphPad Prism 7.03. *, P < .05; **, P < .01. −MMV665917 (n = 12) is sum of Cryptosporidium hominis-infected group (n = 8), 1% hydroxypropyl methyl-cellulose-treated group (n = 2), and nontreated control (n = 2); +MMV665917 10 mg/kg (n = 5) is sum of C hominis-infected and MMV665917 (10 mg/kg)-treated group (n = 3) and MMV665917 (10 mg/kg)-treated group (n = 2); +MMV665917 20 mg/kg (n = 11) is sum of C hominis-infected and MMV665917 (20 mg/kg)-treated group (n = 7) and MMV665917 (20 mg/kg)-treated group (n = 4).

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