Amixicile, a novel inhibitor of pyruvate: ferredoxin oxidoreductase, shows efficacy against Clostridium difficile in a mouse infection model

Abstract

Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-μg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.

Fig 1

Chemical structures of compounds used in this study and parent compound nitazoxanide. Synthesis of amixicile and other analogues can be found in text S1 in the supplemental material and in references and .

Fig 2

Experimental scheme for acute CDI mouse model. Details can be found in the text.

Fig 3

Mouse cytotoxicity and clinical score. (A) Weight loss. Analogues were screened at 200 mg/kg, and body weights were recorded daily. (B) Clinical score. Clinical scores were computed on a scale of 0 (normal) to 20 (death) and evaluated on weight loss, eyes/nose, coat, activity, posture, and diarrhea against a severity scale for each of 0 to 4 with 4 being worst. The clinical score matrix is presented in Table S2 in the supplemental material.

Fig 4

Mouse survival study. (A) Survival curve. Groups of 8 mice were infected on day 0 with 10⁴ CFU/100 μl and on day 1 received the indicated compounds at 20 mg/kg body weight/day for 5 days. Animals were followed for 12 days postinfection. The results are depicted as percent survivors. (B) The percent survivors in each group are tabulated at days 5 and 12 postinfection. N, number of survivors/group. (C) Body weights. The mean weights of animals (groups of 8 or survivors) were measured each day for 12 days.

Fig 5

Mouse head-to-head challenge with fidaxomicin and vancomycin. (A) Survival curves. The data are combined from two experiments, one of 4 animals per group and the other of 7 per group. All animals received 1 × 10⁵ CFU in 100 μl. Three animals experienced gavage-related deaths (one in each group) unrelated to CDI, so the denominator for the combined study is 10 (except for the uninfected-control and vancomycin-plus-amixicile groups, each with n = 6). All drugs were administered by gavage at 20 mg/kg body weight/day for 5 days. (B) The percent survivors are tabulated at days 5 and 14. N, number of survivors/group. (C) Body weights. The mean weights (groups of 6 to 10) were measured each day for 14 days. (D) Clinical scores are assessed as indicated in the text and detailed in Table S2 in the supplemental material.