Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study

Abstract

Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.).

Keywords: CRS3123; Clostridium difficile; Gram-positive bacteria; antimicrobial agents; glucuronidation; pharmacokinetics.

Copyright © 2017 American Society for Microbiology.

Figures

FIG 1

Composite of estimated concentrations of CRS3123 following five escalating doses. The concentrations were determined using the parent drug as the standard, but the concentrations depicted are for three molecular species combined, the parent drug and two of the three glucuronide metabolites that coelute with the parent drug. The parent drug constitutes an unknown percentage of the depicted concentrations. Black, cohort A, 100 mg; red, cohort B, 200 mg; blue, cohort C, 400 mg; green, cohort D, 800 mg; orange, cohort E, 1,200 mg. Note that absorption was not dose proportional and that relative bioavailability after the 1,200-mg dose was less than that after the 800-mg dose.

FIG 2

LC/MS chromatograms of urine (A) and plasma (B) that show that CRS3123 was metabolized after absorption and excreted as two molecular species. Such modification was not present in the CRS3123 standard chromatogram (C). The parent drug eluted at 2.87 min and a metabolite at 3.08 min. Mass spectral analysis showed that the mass of the slower-eluting molecule (3.08 min.) corresponded to an addition of 176 mass units, the mass of a hexuronide substitution, to CRS3123. As glucuronic acid is the only plausible hexuronide, an increase of 176 mass units could be attributed to the addition of a glucuronide to the CRS3123 molecule. Cleavage fragments at masses 554, 556, and 558, attributable to 0,2A cross-ring cleavage, confirmed the glucuronate moiety. All clinical samples contained the second peak (A and B), which was not present in the standard (C).

FIG 2

LC/MS chromatograms of urine (A) and plasma (B) that show that CRS3123 was metabolized after absorption and excreted as two molecular species. Such modification was not present in the CRS3123 standard chromatogram (C). The parent drug eluted at 2.87 min and a metabolite at 3.08 min. Mass spectral analysis showed that the mass of the slower-eluting molecule (3.08 min.) corresponded to an addition of 176 mass units, the mass of a hexuronide substitution, to CRS3123. As glucuronic acid is the only plausible hexuronide, an increase of 176 mass units could be attributed to the addition of a glucuronide to the CRS3123 molecule. Cleavage fragments at masses 554, 556, and 558, attributable to 0,2A cross-ring cleavage, confirmed the glucuronate moiety. All clinical samples contained the second peak (A and B), which was not present in the standard (C).

FIG 3

Structure of CRS3123. The three putative reaction sites for the enzymatic conversion of CRS3123 to N-glucuronide by conjugation with UDP-glucuronate are indicated by the numbers 1, 2, and 3 (circled). 1 and 2 are secondary aliphatic amines, and 3 is a heterocyclic amine. The formation of glucuronides could be expected at these sites.

FIG 4

LC/MS/MS spectra of a urine specimen with use of the modified LC program summarized in Table 3. The specimen was chosen for its available volume. The three panels are spectra from three different MRM transitions. (Top) MRM transition between m/z 514 and 290.9 product ion. The mass of CRS3123, 514 Da, is within the range of this MRM transition, and it produces a peak at 3.52 min. Three additional peaks are found at 2.52 (glucuronide A), 5.14 (glucuronide C), and 5.34 (glucuronide B) minutes. Glucuronides A and C coeluted with the parent compound, as illustrated in Fig. 1 and 2. (Middle) MRM transition between m/z 690.3 and 514.0 product ion. Since CRS3123 has no mass of >514 Da, a peak representing the parent drug is absent, but peaks representing glucuronides A (2.55 min), C (5.12 min), and B (5.33 min), which have nominal masses of 690 Da, are present. (Bottom) MRM transition between m/z 690.3 and 556.0. Glucuronide B is present at 5.34 min. The presence of the parent compound in the top panel shows that glucuronidation is not required for renal clearance.

FIG 5

Composite of glucuronide B concentrations in plasma (A) and comparison with the concentration of the three molecular species, combined, that coelute as the “parent drug” (B). (A) Comparative mean glucuronide B concentrations by protocol time for cohorts A to E. The concentrations were determined using the parent drug as standard; since the mass of glucuronide B is 176 mass units greater than that of the parent drug, use of the parent drug as the standard overestimates the concentrations of the glucuronide. Black, cohort A, 100 mg; red, cohort B, 200 mg; blue, cohort C, 400 mg; green, cohort D, 800 mg; orange, cohort E, 1,200 mg. Note that the concentrations of glucuronide B paralleled those of the parent drug in Fig. 1. (B) Mean concentrations of parent drug and glucuronide B, over time, in cohort E. Note that glucuronidation began with absorption and the peak concentration of glucuronide B coincided with that of the parent drug.