RX-P873, a Novel Protein Synthesis Inhibitor, Accumulates in Human THP-1 Monocytes and Is Active against Intracellular Infections by Gram-Positive (Staphylococcus aureus) and Gram-Negative (Pseudomonas aeruginosa) Bacteria

Abstract

The pyrrolocytosine RX-P873, a new broad-spectrum antibiotic in preclinical development, inhibits protein synthesis at the translation step. The aims of this work were to study RX-P873's ability to accumulate in eukaryotic cells, together with its activity against extracellular and intracellular forms of infection by Staphylococcus aureus and Pseudomonas aeruginosa, using a pharmacodynamic approach allowing the determination of maximal relative efficacies (Emax values) and bacteriostatic concentrations (Cs values) on the basis of Hill equations of the concentration-response curves. RX-P873's apparent concentration in human THP-1 monocytes was about 6-fold higher than the extracellular one. In broth, MICs ranged from 0.125 to 0.5 mg/liter (S. aureus) and 2 to 8 mg/liter (P. aeruginosa), with no significant shift in these values against strains resistant to currently used antibiotics being noted. In concentration-dependent experiments, the pharmacodynamic profile of RX-P873 was not influenced by the resistance phenotype of the strains. Emax values (expressed as the decrease in the number of CFU from that in the initial inoculum) against S. aureus and P. aeruginosa reached more than 4 log units and 5 log units in broth, respectively, and 0.7 log unit and 2.7 log units in infected THP-1 cells, respectively, after 24 h. Cs values remained close to the MIC in all cases, making RX-P873 more potent than antibiotics to which the strains were resistant (moxifloxacin, vancomycin, and daptomycin for S. aureus; ciprofloxacin and ceftazidime for P. aeruginosa). Kill curves in broth showed that RX-P873 was more rapidly bactericidal against P. aeruginosa than against S. aureus. Taken together, these data suggest that RX-P873 may constitute a useful alternative for infections involving intracellular bacteria, especially Gram-negative species.

FIG 1

Chemical structure and ionization status of RX-P873. The graph shows the evolution of the proportions of the two major microspecies of the molecule in the range of pHs that it could face in biological environments, as well as log D values calculated at pH 5.5 and 7.4 (using Reaxys software).

FIG 2

Apparent cellular accumulation of RX-P873 in THP-1 cells incubated for 2 h with RX-P873 at increasing extracellular concentrations. The left axis shows the cellular concentration of the drug expressed in nanograms per milligram of cell protein (prot). The data were used to fit a linear regression with a slope of 30.0 ± 1.9 ng ml⁻¹/mg liter⁻¹ and an R² value of 0.9923. The right axis shows the apparent cellular accumulation factor calculated using a conversion factor of 5 μl/mg cell protein. All values are means ± standard deviations (SDs) from three independent determinations.

FIG 3

Intracellular activity of RX-P873 and selected comparators (linezolid [LZD], vancomycin [VAN], daptomycin [DAP], moxifloxacin [MXF] or ciprofloxacin [CIP], and ceftazidime [CAZ]) against different strains of S. aureus (left) or P. aeruginosa (right) determined after 24 h of incubation with increasing concentrations of each drug. The ordinate shows the change in the number of CFU (log scale) (Δlog₁₀ CFU) per milligram of cell protein compared to that in the initial inoculum. Solid horizontal line, apparent bacteriostatic effect; dotted horizontal line, limit of detection. All values are means ± standard errors of the means (SEMs) from 2 to 3 experiments performed in triplicate (when not visible, the SEMs are smaller than the size of the symbols).

FIG 4

Comparison of intracellular C_s (top) and E_max (bottom) values for RX-P873 and its comparators calculated from the sigmoidal regressions of the concentration-effect studies whose results are shown in Fig. 3 (Hill slope = 1 for all antibiotics tested except ciprofloxacin against PA256, because E_max could not be calculated using a Hill slope of 1; the highest concentration tested was too far from that allowing the plateau value to be reached). The horizontal lines with central squares superimposed on the C_s values indicate the MIC values. Statistical analyses were performed by 2-way analysis of variance with the Tukey multiple-comparison test considering the different strains for each antibiotic. Data with different letters are significantly different from one another (P < 0.05). nd, not determined.

FIG 5

(Left and middle) Extracellular (dotted line) and intracellular (solid line) activity of RX-P873 against different strains of S. aureus (left) or P. aeruginosa (middle) determined after 24 h of incubation with increasing concentrations of each drug. The ordinate shows the change in the number of CFU (log scale) per milligram of cell protein of broth of broth compared to that in the initial inoculum; concentrations are expressed as multiples of the MIC for each strain. A single sigmoidal regression was fit to the whole set of data obtained for the three independent strains of each bacterial species. Solid horizontal line, apparent bacteriostatic effect; dotted horizontal line, limit of detection; dashed vertical line, MIC. All values are means ± standard errors of the means (SEMs) from 2 to 3 experiments performed in triplicate (when not visible, the SEMs are smaller than the size of the symbols). (Right) Comparison of the bacteriostatic concentration (top) and maximal efficacy (bottom) of RX-P873 against intracellular or extracellular bacteria. Statistical analyses were performed by 2-way analysis of variance with the Tukey multiple-comparison test. Data with different letters are significantly different from one another (P < 0.05).

FIG 6

Influence of time on the rate and extent of activity of RX-P873 against extracellular S. aureus ATCC 25923 (left) or P. aeruginosa PAO1 (right) determined over 24 h of incubation. The ordinate shows the change in the number of CFU (log scale) per milliliter of broth compared to that in the initial inoculum; concentrations are expressed as multiples of the MIC for each strain. Solid horizontal line, apparent bacteriostatic effect; dotted horizontal line, limit of detection. All values are means ± standard deviations (SDs) from 3 independent determinations (when not visible, error bars are smaller than the size of the symbols).