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48th Annual ICAAC / IDSA 46th Annual Meeting
October 25-28, 2008
Washington, DC
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The New Antitubercular Drugs SQ109 and TMC207 Act Synergistically
In Vitro to Kill M. tuberculosis

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Reported by Jules Levin
ICAAC 2008
REDDY1, EINCK1, ANDRIES2 NACY1, 1Sequella, 2, L. EINCK , K. ANDRIESand C.A. NACY Sequella, Inc., Rockville, MD 20850 and V. M. REDDY Tibotec Pharmaceuticals Ltd., BVBA, Beers, Belgium
A number of initiatives to discover and develop new therapeutic agents that not only cure TB but also shorten the duration of treatment are ongoing (5, 13). SQ109 and TMC207 are among new drugs being developed for therapy of TB, and both are currently in human clinical trials. SQ109 is a NGeranyl(2 adamantyl) ethane1,2diamine discovered as best-in-class of a 63,000 combinatorial chemistry library of diamine antimicrobial agents (8,10). It is most active against M. tuberculosis(MTB) complex bacteria, and is as active on multidrug resistant (MDR) MTB as on drug sensitive strains. SQ109 inhibits cell wall biosynthesis, although the exact site of action is not yet known(7). TMC207 (also known as R207910) is a derivative of the diarylquinoline class of antimicrobial drugs. TMC207 is highly specific for Mycobacteria, and is equally effective against susceptible and MDR MTB (1). Both SQ109 and TMC207 are bactericidal. In combination drug studies with existing frontline antituberculars, SQ109 acts synergistically with isoniazid (INH) and rifampin (RIF) (3), whereas TMC207 is synergistic with pyrazinamide (PZA) (6). In this study we investigated, in vitro and in macrophages, the efficacy of combinations of TMC207 and SQ109 against MTB.
To determine the in vitroin vitro interaction of SQ109 and TM207, with and without RIF, for killing of MTB.
1. In these in vitro studies two- three-combination using SQ109, TMC207 and RIF, the combination SQ109+TMC207 was synergistic against the drug susceptible laboratory strain of MTB (H37Rv) or additive against the two clinical isolates of MTB. On the other hand, the combination TMC207+RIF combination was additive against both. Addition of RIF to the SQ109+TMC207 combination did not further improve efficacy of the two drugs.
2. TMC207+SQ109 combination killed MTB faster as determined by both RLU and GI estimation, and both methods were more sensitive than CFU determination. The apparent increased rate of killing by TMC207 in bioluminescence assay could be due to depletion of ATP by the drug. Since RLU depends on the amount of ATP within the bacteria, minor changes in the ATP levels in the bacteria reflect the RLU levels in the assay.
3. Increased rate of killing by the SQ109+TMC207 combination was also evident in BACTEC, which is the gold standard for susceptibility testing in MTB.
4. SQ109 combined with TMC207 enhanced the PAE of TMC207.
5. In macrophages, TMC207 was more effective at killing MTB than SQ109 or RIF. In combination, SQ109+TMC207 was superior to either TMC207+RIF or SQ109+RIF. It will be of interest to determine whether SQ109+TMC207 combination shows improved activity in vivo.
MIC against MTB (H37Rv):

By microdilution, the MIC of SQ109, TMC207 and RIF against H37Rv were 0.5, 0.25 and 0.0078 μg/against μg/respectively. By BACTEC method the MIC of these drugs against H37Rv were 0.25, 0.062 and 0.062 g/ml, respectively. The MIC of SQ109 and TMC207 were one or two dilutions lower by BACTEC compared to microdilution method; the reverse was true for RIF.
Two drug combinations: Combination of SQ109 with TMC207 was synergistic with H37Rv with Combination with ΩFIC two combination MTB8330 ΩFIC displayed ΩFIC of 0.375 in one experiment and 0.5 in the second. Against two clinical isolates of MTB, SQ109+TMC207 combination was additive displaying better activity against MTB8330 than MTB8642 with FIC 0.625 and 2.0, respectively. SQ109+TMC207 combination displayed additive effect against MSMG (FIC=1.0). SQ109+RIF combination, on the other hand, was synergistic against MTB (H37Rv) as well as MSMG with FIC of 0.094 and 0.132, respectively. In contrast TMC207+RIF combination had additive effects against H37Rv as well as two other clinical isolates of MTB with FIC of 2.0 against all the isolates. None of the combinations was antagonistic (Table 1).
Three drug combinations tested for interactions on MTB MTB-H37Rv: The two drugs (TMC207 and SQ109) were titrated in checkerboard titration in microtiter plates containing different concentrations of RIF: 0.5, 0.2, 0.1 and 0.05 x MIC. This study was designed to determine the lowest concentration (subMIC) of RIF that interacts with TMC207 and SQ109 to induce the greatest synergistic activity. Table 2 shows three drug combination data from one of the two experiments. RIF at 0.5 and0.2 MIC caused 2 fold decrease in MIC of SQ109 (Plate 1 & 2), but had no effect at 0.1 and 0.05 MIC (Plates 3 & 4). RIF did notcontribute to already existing synergy between SQ109 and TMC207 in combination (Plate 5).
Rate of killing of MTB: Rate of killing is the time required to kill ≥ 90% of initial organisms in the original culture at a given concentration of drug. By RLU, the drugs individually at 2x MIC ranked SQ109>RIF>>TMC207, but the drugs at 4x and 8x MIC ranked as follows: TMC207>>SQ109>RIF (Table 3). In drug combinations, SQ109+TMC207 was more effective, followed by TMC207+RIF and SQ109+RIF. Combinations of RIF with either of the drugs failed to enhance t he rate of killing significantly over the individual drugs.
By BACTEC method, the order of efficacy (90% kill) of drug combinations at 2x MIC was SQ109+RIF>>SQ109+TMC207>TMC207+RIF, and at higher concentrations, SQ109+RIF=SQ109+TMC207>TMC207+RIF.
The order of efficacy for reducing CFU count to ≦106 CFU (90% kill) for individual drugs was SQ109>RIF>TMC207 (Table 5); the order of drug combinations varied depending upon the day at which data was collected. At 4 days, the order was SQ109+TMC207=TMC207+RIF> SQ109+RIF; by 15 days, however, the SQ109-containing regimens were clearly better, and the order was SQ109+TMC207=SQ109+RIF>TMC207+RIF. In fact, however, the rate of kill of all the combinations was quite similar.
PAE of the drugs alone and in combination: The mean cumulative GI of drug free control and each drug (exposed for 2 hr) was plotted against time (Figure 1). Two hr exposure to SQ109 did not induce any PAE; PAE was 9 hr following 2 hr exposure to TMC207. INH, the positive control in this experiment, had a PAE of over 15 hr, which has been reported elsewhere (11). The PAE of the SQ109+TMC207 combination was 13.6 hr, in the same range as INH.
Determination of intracellular killing activity of the drug, alone or in combination.
When infected M M_ were exposed to drugs 4 days, TMC207 was clearly the best at elimination of intracellular bacteria, even at its MIC. When M_ were exposed to a single day of drugs, all three drugs, SQ109, TMC207, and RIF showed dose responses, with the order of activity TMC207>SQ109>>RIF (Table 6). Drug combinations had better activity than single drugs, SQ109+TMC207 was by far the best, and TMC207+RIF had the same activity as TMC207 by itself. Again, the combination of SQ109+RIF was substantially better than either drug alone (Table 7). When tested in combination at and below their MIC (Table 8) the order of activity was SQ1091+TMC207-1 > TMC207-1 > SQ109 0.5+TMC207-0.5 > SQ109-0.25+TMC207-0.25.