Pharmacologic Comparison of Clinical Neutral Endopeptidase Inhibitors in a Rat Model of Acute Secretory Diarrhea

Abstract

Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil-treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy.

Fig. 1.

Effect of NEP inhibitor treatments on castor oil–induced experimental diarrhea. Rats were given a single oral treatment of vehicle (10:10:80 ethanol/kolliphor EL/water) or NEP inhibitor 30 minutes before castor oil administration as described in Materials and Methods. (A–C) Time course of cumulative stool weight measured in independent studies of racecadotril and other indicated NEP inhibitors. (D and F) Total area under the time course curves (AUC) calculated for each treatment from the stool accumulation data. Data are expressed as the mean ± S.E.M. (n = 10/group; owing to gavage injury, one animal was terminated early from both the vehicle and racecadotril groups shown in (A); these were excluded from the analysis). *P < 0.05 versus vehicle. ^#P < 0.05 versus racecadotril (two-tailed T test).

Fig. 2.

Dose-dependent efficacy of select NEP inhibitors in experimental diarrhea. (A) Diarrhea measured in rats given a single oral treatment with vehicle (10:10:80 ethanol/kolliphor EL/water), 100 mg/kg racecadotril, or varying doses of sacubitril 30 minutes before castor oil administration. Data are expressed as the mean ± S.E.M (n = 8/group). *P < 0.05 versus vehicle. (B) Dose effect of sacubitril treatment on NEP enzyme activity measured in lung tissue extracts, with racecadotril comparator. Values are shown for each animal in the treatment group; mean is indicated by the horizontal line. (C) Diarrhea measured in rats given a single oral treatment with 100 mg/kg racecadotril or varying doses of candoxatril 30 minutes before castor oil administration. Data are expressed as the mean ± S.E.M. (n = 8/group). *P < 0.05 versus vehicle, ^#P < 0.05 versus racecadotril. (D) Dose effect of candoxatril treatment on NEP enzyme activity measured in lung tissue extracts made from each animal, with racecadotril comparator.

Fig. 3.

PK and PD comparison of sacubitril and racecadotril. Vehicle (10:10:80 ethanol/kolliphor EL/water) (), 100 mg/kg racecadotril (), or 100 mg/kg sacubitril () was administered to normal healthy rats by oral gavage. At each time point, samples were collected from each indicated organ tissue for measurement of NEP enzyme activity. (E) At the same time points, the active metabolites of racecadotril (thiorphan, ) and sacubitril (LBQ657, ) were determined in plasma samples as described in Materials and Methods. Data are expressed as the mean ± S.E.M of values from four rats.

Fig. 4.

Effect of compound predosing interval on protection from castor oil–mediated diarrhea. (A) Diarrhea measured in rats given a single oral treatment with 100 mg/kg racecadotril or 100 mg/kg sacubitril 90 minutes before castor oil administration. (B) Diarrhea measured in rats given a single oral treatment with 100 mg/kg racecadotril or 100 mg/kg sacubitril 150 minutes before castor oil administration. (C) Diarrhea measured in rats treated with sacubitril at 30 minutes, 240 minutes, or both 30 and 240 minutes before castor oil administration. Data are expressed as the mean ± S.E.M. [n = 8/group; one animal was terminated early from the vehicle group in (C) owing to gavage injury, and it was excluded from the analysis]. *P < 0.05 versus vehicle.

Fig. 5.

NEP inhibitor treatments do not delay intestinal transit. (A) Effects of loperamide and sacubitril treatments on gastrointestinal transit as evaluated using the charcoal meal test in rats. (B) Effects of loperamide, racecadotril, and candoxatril treatments on gastrointestinal transit in the same model. Animals received test agents 30 minutes before administration of the charcoal meal and were sacrificed 20 minutes later. Vehicle was 5% ethanol in water. The racecadotril formulation also contained 0.2% Tween 20 for solubility. Data are expressed as the mean ± S.E.M. of values from five rats. *P < 0.05 versus vehicle.