In vitro and in vivo Evaluation of Synergism between Anti-Tubercular Spectinamides and Non-Classical Tuberculosis Antibiotics

Abstract

Spectinamides are new semi-synthetic spectinomycin derivatives with potent anti-tubercular activity. The reported synergism of the precursor spectinomycin with other antibiotics prompted us to examine whether spectinamides sensitize M. tuberculosis to other antibiotics not traditionally used in the treatment of tuberculosis to potentially expand therapeutic options for MDR/XDR Tuberculosis. Whole cell synergy checkerboard screens were performed using the laboratory strain M. tuberculosis H37Rv, lead spectinamide 1599, and a broad panel of 27 antibiotics. In vitro, 1599 synergized with 11 drugs from 6 antibiotic classes. The observed synergy was tested against clinical isolates confirming synergy with Clarithromycin, Doxycycline and Clindamycin, combinations of which were taken forward for in vivo efficacy determination. Co-administration of 1599 and clarithromycin provided additional bacterial killing in a mouse model of acute tuberculosis infection, but not in a chronic infection model. Further studies indicated that mismatched drug exposure profiles likely permitted induction of phenotypic clarithromycin resistance and subsequent loss of synergism. These studies highlight the importance of validating in vitro synergism and the challenge of matching drug exposures to obtain a synergistic outcome in vivo. Results from this study indicate that a 1599 clarithromycin combination is potentially viable, providing the drug exposures can be carefully monitored.

Figure 1. Efficacy of 1599 combinations in acute and chronic infection models.

Log₁₀ reduction in bacteria in lungs was determined by calculating the difference between bacillary loads in organs from the untreated group and treated groups. Mean log10 CFU reductions per lung ± the standard error of mean (SEM) are presented. (a) Murine model of acute tuberculosis infection where low-dose aerosol infected gamma interferon knock-out mice were treated twice a day for 9 consecutive days beginning 14 days post-infection. (b) Murine model of chronic tuberculosis infection where low-dose aerosol infected BALB/c mice received treatments once daily for 5/7 days for 28 days beginning 41 days post-infection. Abbreviations: Clar, clarithromycin; Clind, clindamycin; Doxy, doxycycline.

Figure 2. Pharmacokinetic modeling.

Free predicted 1599 plasma concentrations during efficacy trials. The MIC for 1599, 1 μg/mL was used to defined the in vivo therapeutic concentration (blue line). Predicted plasma concentrations are shown in (a) for BID and (b) for QD dosing. (c) shows the maximum consecutive time of free 1599 concentrations below the MIC for each dosing regimen.