Preclinical studies of amixicile, a systemic therapeutic developed for treatment of Clostridium difficile infections that also shows efficacy against Helicobacter pylori

Abstract

Amixicile shows efficacy in the treatment of Clostridium difficile infections (CDI) in a mouse model, with no recurrence of CDI. Since amixicile selectively inhibits the action of a B vitamin (thiamine pyrophosphate) cofactor of pyruvate:ferredoxin oxidoreductase (PFOR), it may both escape mutation-based drug resistance and spare beneficial probiotic gut bacteria that do not express this enzyme. Amixicile is a water-soluble derivative of nitazoxanide (NTZ), an antiparasitic therapeutic that also shows efficacy against CDI in humans. In comparative studies, amixicile showed no toxicity to hepatocytes at 200 μM (NTZ was toxic above 10 μM); was not metabolized by human, dog, or rat liver microsomes; showed equivalence or superiority to NTZ in cytochrome P450 assays; and did not activate efflux pumps (breast cancer resistance protein, P glycoprotein). A maximum dose (300 mg/kg) of amixicile given by the oral or intraperitoneal route was well tolerated by mice and rats. Plasma exposure (rats) based on the area under the plasma concentration-time curve was 79.3 h · μg/ml (30 mg/kg dose) to 328 h · μg/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 μg/ml, the time to the maximum concentration of the drug in serum was 0.5 to 1 h, and the half-life was 5.6 h. Amixicile did not concentrate in mouse feces or adversely affect gut populations of Bacteroides species, Firmicutes, segmented filamentous bacteria, or Lactobacillus species. Systemic bioavailability was demonstrated through eradication of Helicobacter pylori in a mouse infection model. In summary, the efficacy of amixicile in treating CDI and other infections, together with low toxicity, an absence of mutation-based drug resistance, and excellent drug metabolism and pharmacokinetic metrics, suggests a potential for broad application in the treatment of infections caused by PFOR-expressing microbial pathogens in addition to CDI.

FIG 1

Chemical structures of amixicile, NTZ, VPC16a1011, and VPC16b2031.

FIG 2

Time course of plasma amixicile concentrations following a single oral dose. (A) Male rats received amixicile at 30 or 100 mg/kg in methylcellulose by gavage. Each datum point represents the mean plasma amixicile concentration of up to three rats ± the SD. (B) Male mice received amixicile at 200 mg/kg in PBS by oral gavage, and at each time point, five mice were sacrificed for blood collection. The mean and SD are presented. The data collected were used to generate the PK information presented in Table 5.

FIG 3

Therapeutic efficacy of amixicile in mice. Mice were infected with the SS1 strain of H. pylori, and following 2 weeks to enable the infection to manifest itself, mice were divided into groups with one serving as an untreated control and the other receiving one or two doses of amixicile or MTZ of 20 mg/kg/day. One week later, animals were sacrificed and stomach material was collected for bacterial enumeration. The data are reported as CFU/g of stomach material. The mean and SD from five animals are presented. Asterisks indicate statistical significance (P < 0.001).

FIG 4

Effect of amixicile on mouse gut microflora. Eight mice per group received either PBS or amixicile at 30 mg/kg/day by oral gavage. Primers for Firmicutes, Bacteroides species, Lactobacillus species, and segmented filamentous bacteria (SFB) were quantified by 16S rRNA expression that had been normalized to the total bacteria. The mean and SD are presented.