Pharmacokinetics of CamSA, a potential prophylactic compound against Clostridioides difficile infections

Abstract

Clostridioides difficile infections (CDI) are the leading cause of nosocomial antibiotic-associated diarrhea. C. difficile produces dormant spores that serve as infectious agents. Bile salts in the gastrointestinal tract signal spores to germinate into toxin-producing cells. As spore germination is required for CDI onset, anti-germination compounds may serve as prophylactics. CamSA, a synthetic bile salt, was previously shown to inhibit C. difficile spore germination in vitro and in vivo. Unexpectedly, a single dose of CamSA was sufficient to offer multi-day protection from CDI in mice without any observable toxicity. To study this intriguing protection pattern, we examined the pharmacokinetic parameters of CamSA. CamSA was stable to the gut of antibiotic-treated mice but was extensively degraded by the microbiota of non-antibiotic-treated animals. Our data also suggest that CamSA's systemic absorption is minimal since it is retained primarily in the intestinal lumen and liver. CamSA shows weak interactions with CYP3A4, a P450 hepatic isozyme involved in drug metabolism and bile salt modification. Like other bile salts, CamSA seems to undergo enterohepatic circulation. We hypothesize that the cycling of CamSA between the liver and intestines serves as a slow-release mechanism that allows CamSA to be retained in the gastrointestinal tract for days. This model explains how a single CamSA dose can prevent murine CDI even though spores are present in the animal's intestine for up to four days post-challenge.

Keywords: Bile salts; CDI; Clostridioides difficile; Drug therapy; Intestine; Liver; Metabolism; Microbiome.

Figure 1.. Potential intestinal bacterial metabolism of CamSA.

Bile salt hydrolases expressed by the GI microbiota can hydrolyze CamSA’s amide bond, resulting in cholate and metanilic acid (mSA). CamSA could potentially also be dehydroxylated at C-7 and C-12 by the gut microbiota to yield 7-deoxy-CamSA and 12-deoxy-CamSA, respectively.

Figure 2.. Hydrolysis of CamSA by fecal bacteria.

CamSA is slowly hydrolyzed into cholate and mSA by the GI microbiota of naive mice but is stable against the microbiota of mice treated with an antibiotic cocktail. CamSA was incubated with feces collected from mice without antibiotic treatment (black bars), or feces from antibiotic-treated mice (white bars). The data represent the averages from three independent measurements, and error bars represent the standard deviations. Statistical significance was determined using matched-pair t-tests and a two-sample t-test assuming unequal variances. Family-wise error rate is set at 0.01. **P < 0.01; ***P < 0.001.

Figure 3.. Effect of potential CamSA metabolites on CDI severity in spore challenged mice.

CamSA could be deconjugated and/or dehydroxylated by the GI microbiota. To test for the effect of CamSA metabolites, CDI-susceptible mice were treated with DMSO (black bars), cholate/mSA mixtures (gray bars), 12-deoxy-CamSA (diagonal-dashed bars), 7-deoxy-CamSA (checkered bars), or intact CamSA (white bars). Clinical endpoint (dashed line) was set at > 6 in the CDI severity scale^,. Mice treated with 12-deoxy-CamSA or a mixture of cholate and mSA developed severe CDI signs (score > 6) similar to DMSO-treated animals and reached clinical endpoint 48 hours post-challenge. In contrast, mice treated with 7-deoxy-CamSA developed only moderate CDI signs. CamSA treated mice developed little to no signs of CDI. The data represent the averages from independent measurements, and error bars represent the standard deviations. Statistical significance was determined using two sample t-tests assuming unequal variances. ****P < 0.0001.

Figure 4.. Average CamSA and mSA recovery in feces.

Mouse feces was collected at predetermined time points following CamSA administration. (A) CamSA recovered from cpx mice, (B) CamSA recovered from non-cpx mice, and (C) mSA recovered from non-cpx mice. The data represents the averages from five independent samples (n=5) for each treatment group, and error bars represent the standard deviations. Statistical significance was determined using a paired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5.. Total CamSA and mSA recovered from individual animal feces.

Mouse feces was collected at predetermined time points following CamSA administration. (A) CamSA recovered from cpx mice. The average of this data is shown in Fig. 4A and shows synchrony in CamSA excretion between individual mice. (B) CamSA recovered from non-cpx mice. The maximum amount of CamSA recovered at a single time point is four-fold lower than for cpx mice. CamSA recovery is asynchronous between individual animals but show a similar excretion trend. (C) mSA recovered from non-cpx mice. The amount of mSA recovered from non-cpx mice is approximately 40-fold higher than the amount of CamSA recovered from the same animals, indicating CamSA degradation by the mouse microbiota. mSA recovery is asynchronous between individual animals but shows a similar excretion trend. Lines connecting dots were included to aid visualization of data from each time point and do not indicate concentrations between time points. Each colored line represents an individual mouse and no statistical significance tests are appropriate for these line graphs. Average of the data with corresponding statistical significance tests are shown in Fig. 4A, 4B and 4C.

Figure 6.. Total CamSA and mSA quantified from mouse organs.

Mice were sacrificed at 4, 12, 22, 32, and 120 hours post-CamSA administration, and (A) livers, (B) blood, (C) kidneys, and (D) intestines were collected at the time of sacrifice. Tissues were homogenized and extracted. CamSA (squares) and mSA (circles) from cpx (closed symbols) and non-cpx (open symbols, dashed lines) animals were quantified by HPLC-MS. Each time point represents a single animal and hence no statistical tests are appropriate. Lines connecting dots were included to aid visualization of data from each time point and do not indicate concentrations between time points.

Figure 7.. Effect of CamSA and taurocholate on CYP3A4 activity.

BACULOSOMES® expressing CYP3A4 were assayed for their ability to convert a non-fluorescent Vivid Substrate® into a highly fluorescent byproduct in the presence of (A) CamSA, (B) mSA, or (C) taurocholate. As a control, CYP3A4 was incubated with ketoconazole at a final concentration of 10 μM. At this concentration of ketoconazole, CYP3A4 activity was completely inhibited.

Figure 8.. Effect of cholestyramine on CamSA and mSA recovery in liver.

Mice that had been fed with 0% cholestyramine (n=4) or 4% cholestyramine (n=3) were sacrificed 22 hours post-CamSA administration, and their livers with attached gallbladders were collected. CamSA recovered from liver was quantified by HPLC-MS.