Preclinical Studies of Amixicile, a Systemic Therapeutic Developed for Treatment of Clostridium difficile Infections That Also Shows Efficacy against Helicobacter pylori

Abstract

dAmixicile 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 M (NTZ was toxic above 10 M); 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 · g/ml (30 mg/kg dose) to 328 h · g/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 g/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.

Synthesis and Antimicrobial Evaluation of Amixicile-Based Inhibitors of the Pyruvate-Ferredoxin Oxidoreductases of Anaerobic Bacteria and Epsilonproteobacteria

“…1), that retained both potency and selectivity for PFOR targets and possessed good pharmacokinetic properties (10,11,13,14,16). In preclinical studies, amixicile showed equivalence with vancomycin and other mainline therapeutics in the treatment of Clostridium difficile infections (CDI) and, similarly, with metronidazole in the treatment of Helicobacter pylori infections in mouse models (14,15). Importantly, amixicile did not accumulate in the mouse cecum or alter the gut microbiome of healthy animals (15).…”

“…Unlike many nitro-containing drugs, the nitro group of NTZ, a weak acid, is not susceptible to nitroreduction or otherwise chemically modified (11-13). Conceptually, therapeutics that target the function of a vitamin cofactor, itself a small molecule, are unlikely to be amenable to mutation-based drug resistance (11,14,15).…”

“…In preclinical studies, amixicile showed equivalence with vancomycin and other mainline therapeutics in the treatment of Clostridium difficile infections (CDI) and, similarly, with metronidazole in the treatment of Helicobacter pylori infections in mouse models (14,15). Importantly, amixicile did not accumulate in the mouse cecum or alter the gut microbiome of healthy animals (15). Based on serum binding, it has been suggested that amixicile most likely concentrates in areas of mucosal inflammation via serum leakage, where it is active locally against offending susceptible microbes (15).…”

“…Unlike many nitro-containing drugs, the nitro group of NTZ, a weak acid, is not susceptible to nitroreduction or otherwise chemically modified (11-13). Conceptually, therapeutics that target the function of a vitamin cofactor, itself a small molecule, are unlikely to be amenable to mutation-based drug resistance (11,14,15).NTZ is largely retained in the intestine, where it is used for the treatment of infections caused by Cryptosporidium and Giardia (16). Based on several in vitro studies (17, 18) and recognizing the potential for a systemic derivative, we chemically interrogated the NTZ scaffold, and from ϳ350 derived analogues, identified an attractive candidate, amixicile (Fig.…”

Role of Serum Amyloid A, Granulocyte-Macrophage Colony-Stimulating Factor, and Bone Marrow Granulocyte-Monocyte Precursor Expansion in Segmented Filamentous Bacterium-Mediated Protection from Entamoeba histolytica

“…SFB and E. histolytica colonization and mouse gene expression were measured by realtime PCR in stool and cecal lysate. For SFB colonization, qPCR with Sybr green was performed, and data were normalized to expression of a conserved eubacterial 16S RNA gene (EUB) (19). Primer concentrations, annealing temperatures, and cycle numbers were optimized for each primer pair.…”