The Activity of Moxifloxacin against Acid-Phase and Non-Replicative Persister Phenotype Phase Mycobacterium tuberculosis in a Hollow Fiber Infection Model

23 A major goal for improving tuberculosis therapy is to identify drug regimens with 24 improved efficacy and shorter treatment durations. Shorter therapies improve patient 25 adherence to the antibiotic regimens that, in turn, decreases resistance emergence. 26 M. tuberculosis exists in multiple metabolic states. At the initiation of therapy, the 27 bulk of the population is in Log-Phase growth. Consequently, it is logical to focus initial 28 therapy on these organisms. Moxifloxacin has good early bactericidal activity against 29 Log-Phase growth bacteria and is a logical component of initial therapy. It would be 30 optimal if this agent also possessed activity against Acid-Phase and Non-Replicative 31 Persister (NRP) Phenotype organisms. We studied multiple exposures to moxifloxacin 32 (equivalent to 200 mg-800 mg daily) in our Hollow Fiber Infection Model against strain 33 H37Rv in Acid-Phase and against strain 18b in streptomycin starvation, which is a 34 model for NRP-Phase organisms. 35 Moxifloxacin possesses good activity against Acid-Phase organisms, generating 36 from 3.75 Log10(CFU/ml) cell kill (200 mg daily) to 5.16 Log10(CFU/ml) cell kill (800 mg 37 daily) over the 28 days of the experiment. 38 Moxifloxacin also has activity against the streptomycin-starved strain 18b. The 39 400-800 mg daily regimens achieved extinction at day 28, while the no-treatment control 40 still had 1.96 Log10(CFU/ml) culturable. The lowest dose (200 mg daily) still had 0.7 41 Log10(CFU/ml) measurable at day 28 ,a net kill of 1.26 Log10(CFU/ml). 42 Moxifloxacin is an attractive agent for early therapy, as it possesses activity 43 against three metabolic states of M. tuberculosis. 44 on M arch 2, 2020 by gest httpaac.asm .rg/ D ow nladed fom Introduction 45 Mycobacterium tuberculosis infects 25% of the world’s population. In 2016, there 46 were 10.4 million new cases of tuberculosis, which caused 1.7 million deaths. We have 47 also seen the rise of Multiply-Drug Resistant (MDR) M. tuberculosis and eXtremely Drug 48 Resistant (XDR) M. tuberculosis. These isolates are associated with a markedly 49 increased cost of therapy as well as an increased rate of death. 50 Resistance emergence is attributable to multiple causes, but the prolonged 51 duration of therapy makes attaining excellent adherence difficult, with a subsequent 52 increase in the rate of resistance emergence among poorly adherent patients. 53 Attainment of inadequate drug concentration-time profiles also has a negative impact on 54 resistance emergence. Yet another factor influencing the likelihood of resistance 55 emergence is bacterial burden, with very high burdens being associated with increasing 56 likelihood of resistance emergence. Finally, all these and other factors interact with each 57 other to inflate the rate of emergence of resistance. 58 M. tuberculosis exists in multiple metabolic states. Some of the best 59 characterized are Log-Phase growth, Acid-Phase growth and Non-Replicative Persister 60 (NRP) phenotype state. Bacterial burden interacts with these states. It is felt that the 61 slowly growing acid phase M. tuberculosis and very slowly or non-growing organisms in 62 NRP state are hardest to kill and contribute substantially to the long therapy duration 63 required to cure tuberculosis. Larger overall burdens will mostly consist of Log-Phase 64 organisms, but this will also drive larger Acid-Phase and NRP-Phase populations. 65 Further, resistance occurs mostly (but not exclusively) in Log-Phase populations. At 66 therapy initiation, it would be optimal to employ a regimen with excellent bactericidal 67 on M arch 2, 2020 by gest httpaac.asm .rg/ D ow nladed fom activity against Log-Phase organisms, but also have substantial activity against Acid68 Phase and NRP-Phase organism populations. As one of the agents for initial therapy, 69 we decided to examine moxifloxacin. Our group examined this fluoroquinolone 70 previously in the Hollow Fiber Infection Model (HFIM). In those evaluations, it was 71 clear that moxifloxacin possesses excellent early Log-kill. Indeed, in the REMox trial, 72 the arm that replaced isoniazid with moxifloxacin cleared the sputum significantly faster 73 than the control arm. Consequently, this agent is attractive for regimen inclusion at 74 therapy initiation. It would be even more attractive should it also possess activity against 75 Acid-Phase organisms and NRP-Phase organisms. It was the purpose of these studies 76 to examine moxifloxacin’s activity against M. tuberculosis in these metabolic states. 77 Results 78 MIC and Mutational Frequency to Resistance: The mutational frequency to 79 resistance was 1/10 CFU for H37Rv in Acid-Phase and 1/10 CFU for strain 18b in 80 streptomycin starvation (NRP-Phase). The selection plates here contained 75 mg/L of 81 streptomycin to allow organism growth. Henceforth, we will refer to NRP-Phase 82 organisms as strain 18b organisms in streptomycin starvation (ss-18b – this isolate is a 83 streptomycin auxotroph). 84 The moxifloxacin MIC was 0.25 mg/L for Acid-Phase H37Rv and was 0.125 mg/L 85 for strain 18b grown in the presence of streptomycin. When in ss-18b state, the MIC 86 could not be determined. 87 Moxifloxacin Activity Against Acid-phase M. tuberculosis H37Rv: The activity 88 of 200, 400, 600 or 800 mg exposure equivalents of moxifloxacin administered daily are 89 on M arch 2, 2020 by gest httpaac.asm .rg/ D ow nladed fom shown in Figure 1. The reduction of bacterial burden from baseline (day zero) to day 28 90 ranged from 3.75 Log10(CFU/ml) [200 mg daily] to 5.16 Log10(CFU/ml) [800 mg daily]. 91 The effect of each moxifloxacin regimen on the subpopulation of M. 92 tuberculosis with reduced susceptibilities to moxifloxacin was assessed by quantitatively 93 culturing bacterial suspensions collected from the HFIM onto agar supplemented with 94 3.0 x MIC of moxifloxacin. No less-susceptible isolates were recovered from any arm at 95 any time point. This is consistent with the mutational frequency to resistance (1/10 96 CFU) and the initial bacterial burden (5.56 – 5.86 Log10(CFU/ml)). 97 Mathematical Modeling of Acid-phase Study Data: Both system outputs (drug 98 concentration-time profile and total bacterial burden) were modeled simultaneously for 99 all regimens using a variant of the model we previously described. As in the cited 100 paper, we also included a natural death rate term (KNat_Death [hr]) because of the overall 101 lack of increase in the bacterial burden over time in the untreated control. 102 The fit of the model to the data was acceptable when the predicted-observed 103 plots and measures of bias and precision were examined. The pre-Bayesian 104 (population) and Bayesian (individual) regressions are shown for both outputs in Figure 105 2, panels A-D. For the pre-Bayesian regression, the mean or median parameter vector 106 was used to generate the predicted values in the Predicted-Observed plot. For the 107 Bayesian analysis, the Bayesian parameter estimates for each Hollow Fiber arm were 108 used to generate the predicted values. It was expected that the Bayesian estimates 109 would perform better in terms of measured Bias and Precision relative to the pre110 Bayesian estimates. 111 on M arch 2, 2020 by gest httpaac.asm .rg/ D ow nladed fom The mean and median parameter vectors and their standard deviations are 112 displayed in Table 1. When the Bayesian model parameters were employed for 113 simulation, the bacterial kill attributable to 800 mg of moxifloxacin daily was 4.940 114 Log10(CFU/ml) relative to the No-Treatment Control. 115 In Table 1 the growth rate constant (Kg) from both the Mean and Median 116 parameter vector is low (0.0350 and 0.0204 hr), indicating the slow growth of M. 117 tuberculosis in this metabolic state. The estimates of the Kill rate constant (Kkill) are 10118 20 times that of the growth rate constant. Finally, it is of note that the natural death rate 119 constant estimates (Knat-death) are of the same magnitude as the growth rate constants, 120 explaining the overall lack of growth in the no-treatment control over the 28 days of the 121 experiment. 122 Activity Against ss-18b M. tuberculosis: 123 The activity of 200, 400, 600 and 800 mg exposure equivalents of moxifloxacin 124 administered once-daily is shown in Figure 3. The 400, 600 and 800 mg daily doses 125 mediated a 1.96 Log10(CFU/ml) kill at day 28 relative to the no-treatment control, when 126 eradication was achieved. The decline in the untreated control is due to the substantial 127 natural death rate for M. tuberculosis in this metabolic state in the HFIM. The substantial 128 decline in the no-treatment control was greater than seen previously in static in vitro 129 experiments, but consistent with previous experiments in the HFIM where the 130 restricted replication rate was induced by Wayne-Hayes Level II anaerobiosis. 131 The effect of each moxifloxacin regimen on the less-susceptible M. tuberculosis 132 subpopulation was determined by quantitatively culturing an aliquot of the bacterial 133 suspensions onto agar supplemented with 3.0 x MIC of moxifloxacin. All arms had some 134 on M arch 2, 2020 by gest httpaac.asm .rg/ D ow nladed fom moxifloxacin-less susceptible isolates recovered at baseline. This is a function of the 135 baseline bacterial burden (6.47 – 6.65 Log10(CFU/ml) relative to the inverse of the 136 mutational frequency to resistance (1/10 Log10(CFU)). This is consistent with the 137 observed recovery of 0.6 – 0.72 Log10(CFU/ml) of less susceptible organisms at 138 baseline. We were unable to recover resistant organisms from all arms after 2-4 days. 139 This is also consistent with the mutational frequency to resistance value and the decline 140 in the no-treatment control over this time frame (6.53 Log10(CFU/ml) at baseline and 141 5.62 Log10(CFU/ml) at day 4). Isolates recovered from the antimicrobial-containing 142 plates had elevated moxifloxacin MIC