GT160-246, a toxin binding polymer for treatment of Clostridium difficile colitis

Abstract

GT160-246, a high-molecular-weight soluble anionic polymer, was tested in vitro and in vivo for neutralization of Clostridium difficile toxin A and B activities. Five milligrams of GT160-246 per ml neutralized toxin-mediated inhibition of protein synthesis in Vero cells induced by 5 ng of toxin A per ml or 1.25 ng of toxin B per ml. In ligated rat ileal loops, 1 mg of GT160-246 neutralized fluid accumulation caused by 5 microg of toxin A. At doses as high as 80 mg/loop, cholestyramine provided incomplete neutralization of fluid accumulation caused by 5 microg of toxin A. GT160-246 protected 80% of the hamsters from mortality caused by infection with C. difficile, whereas cholestyramine protected only 10% of animals. Treatment of C. difficile-infected hamsters with metronidazole initially protected 100% of the hamsters from mortality, but upon removal of treatment, 80% of the hamsters had relapses and died. In contrast, removal of GT160-246 treatment did not result in disease relapse in the hamsters. GT160-246 showed no antimicrobial activity in tests with a panel of 16 aerobic bacteria and yeast and 22 anaerobic bacteria and did not interfere with the in vitro activities of most antibiotics. GT160-246 offers a novel, nonantimicrobial treatment of C. difficile disease in humans.

FIG. 1

Effects of GT160-246 on [³H]leucine incorporation by Vero cells in culture. Serial dilutions of C. difficile toxin A or toxin B were incubated with medium or GT160-246 (5 mg/ml) for 1 h at room temperature and then added to Vero cell monolayers. The cells were pulsed with [³H]leucine, and the level of incorporation was measured after 1 h of incubation. (A) Effect of toxin A with either medium alone or GT160-246. (B) Effect of toxin B in the presence of medium alone or GT160-246. Data represent the means for triplicate wells at each concentration. Error bars represent the standard errors of the means.

FIG. 2

Effect of GT160-246 or cholestyramine on the enterotoxic activity of toxin A in rat ileal loops. (A) Fluid accumulation induced by 5 μg of toxin A was measured in the presence of 0.25, 0.5, 1, 2.5, or 5 mg of GT160-246. Fluid accumulation was also measured in loops receiving PBS alone (control) or 5 mg of GT160-246 alone. (B) Inhibition of toxin A-mediated increases in intestinal permeability in rat intestinal loops by GT160-246. The permeability of ligated ileal loops to [³H]mannitol following treatment with 5 μg of toxin A was measured in the presence of 0.25, 0.5, 1, 2.5, or 5 mg of GT160-246. The permeability to mannitol was also measured in loops receiving PBS alone (control) or 5 mg of GT160–246 alone. (C) Fluid accumulation induced by 5 μg of toxin A was measured in the presence of 1.0, 2.5, 5.0, 10, 20, 40, or 80 mg of cholestyramine. Fluid accumulation was also measured in loops receiving PBS alone (control), 10 mg of cholestyramine alone, or 80 mg of cholestyramine alone. (D) Permeability of ligated ileal loops to [³H]mannitol following treatment with 5 μg of toxin A was measured in the presence of 1.0, 2.5, 5.0, 10, 20, 40, or 80 mg of cholestyramine. Permeability to mannitol was also measured in loops receiving PBS alone (control), 10 mg of cholestyramine alone, or 80 mg of cholestyramine alone. The number of loops in each treatment group is shown on each column. Error bars represent the standard errors of the means. Data were analyzed by analysis of variance and Dunnett's posttest to compare treatments to toxin control. ∗, P < 0.05.

FIG. 3

GT160-246 treatment of hamsters with C. difficile colitis. Hamsters were infected with C. difficile on day −1 and received 10 mg of clindamycin subcutaneously on day 0. Hamsters received saline alone (n = 10) or GT160-246 at 500 mg/kg/day (n = 10), 1,000 mg/kg/day (n = 10), or 1,500 mg/kg/day (n = 10) orally on days 1 through 7. Hamsters were observed for clinical signs of disease and mortality through day 14.

FIG. 4

Metronidazole treatment of hamsters with C. difficile colitis. Hamsters were infected with C. difficile on day −1 and received 10 mg of clindamycin per kg subcutaneously on day 0. Hamsters received 210 mg of metronidazole per kg per day orally (n = 5) on days 1 through 5. Hamsters were observed for morbidity and mortality through day 14.

FIG. 5

GT160-246 pretreatment protects hamsters from mortality from C. difficile colitis. (A) Hamsters were infected with C. difficile on day −1 and received 10 mg of clindamycin per kg subcutaneously on day 0. Hamsters were treated with saline (n = 10), 1,000 mg of GT160-246 per kg per day (n = 10), or 1,000 mg of cholestyramine per kg per day (n = 10) orally on days −2 through day 4. They were observed for morbidity and mortality through day 7. (B) Hamsters were treated with saline (n = 10) or GT160-246 at 500 mg/kg/day (n = 10) or 1,000 mg/kg/day (n = 10) orally from day −2 through day 4. They were observed for development of morbidity and mortality through day 15.

FIG. 6

GT160-246 treatment prevents histological damage caused by C. difficile. Representative histological sections from ceca of each treatment group are shown. (A) Noninfected hamster. (B) Noninfected hamster administered GT160-246. (C) C. difficile-infected hamster. (D) C. difficile-infected hamster treated prophylactically with 1,000 mg of GT160–246 per kg per day orally.