Preclinical Evaluations To Identify Optimal Linezolid Regimens for Tuberculosis Therapy

Abstract

Linezolid is an oxazolidinone with potent activity against Mycobacterium tuberculosis. Linezolid toxicity in patients correlates with the dose and duration of therapy. These toxicities are attributable to the inhibition of mitochondrial protein synthesis. Clinically relevant linezolid regimens were simulated in the in vitro hollow-fiber infection model (HFIM) system to identify the linezolid therapies that minimize toxicity, maximize antibacterial activity, and prevent drug resistance. Linezolid inhibited mitochondrial proteins in an exposure-dependent manner, with toxicity being driven by trough concentrations. Once-daily linezolid killed M. tuberculosis in an exposure-dependent manner. Further, 300 mg linezolid given every 12 hours generated more bacterial kill but more toxicity than 600 mg linezolid given once daily. None of the regimens prevented linezolid resistance. These findings show that with linezolid monotherapy, a clear tradeoff exists between antibacterial activity and toxicity. By identifying the pharmacokinetic parameters linked with toxicity and antibacterial activity, these data can provide guidance for clinical trials evaluating linezolid in multidrug antituberculosis regimens.

Importance: The emergence and spread of multidrug-resistant M. tuberculosis are a major threat to global public health. Linezolid is an oxazolidinone that is licensed for human use and has demonstrated potent activity against multidrug-resistant M. tuberculosis. However, long-term use of linezolid has shown to be toxic in patients, often resulting in thrombocytopenia. We examined therapeutic linezolid regimens in an in vitro model to characterize the exposure-toxicity relationship. The antibacterial activity against M. tuberculosis was also assessed for these regimens, including the amplification or suppression of resistant mutant subpopulations by the chosen regimen. Higher exposures of linezolid resulted in greater antibacterial activity, but with more toxicity and, for some regimens, increased resistant mutant subpopulation amplification, illustrating the trade-off between activity and toxicity. These findings can provide valuable insight for designing optimal dosage regimens for linezolid that are part of the long combination courses used to treat multidrug-resistant M. tuberculosis.

FIG 1

Cell growth of K562 cells in the HFIMS system during linezolid therapy. Linezolid exposure had a minimal effect on K562 cellular proliferation after 16 days in the HFIM system. At various time points, cells were harvested from the HFIM system, and live cells were enumerated using the trypan blue exclusion method.

FIG 2

OXPHOS mitochondrial protein levels for complexes 1, 3, 4, and 5 from K562 cells treated with linezolid in the HFIM system. The effects of linezolid on the production of OXPHOS protein complexes 1 (A), 3 (B), 4 (C), and 5 (D) in K562 cells harvested from the HFIM system were assessed. Cell pellets were lysed, and OXPHOS mitochondrial protein levels were quantified by ELISA.

FIG 3

ATP production and caspase 3 activation in K562 cells treated with linezolid in the HFIM system. The effects of linezolid exposure on the production of ATP (A) and caspase 3 activation (B) in K562 cells harvested from the HFIM system were assessed. ATP levels were quantified using a colorimetric ATP assay, and caspase 3 activation was assessed via a colorimetric caspase 3 assay.

FIG 4

Influence of linezolid exposure (24-h AUC) and trough concentration on mitochondrial toxicity. The area under the OXPHOS protein complex 4-time curve (AUC_OXPHOS4) illustrated in Fig. 2C was calculated for all hollow-fiber arms and plotted against the linezolid 24-h AUC exposures and trough concentrations achieved for each regimen in the HFIM system. Black circles correspond to the q24h regimens, and red squares represent the q12h regimens. An inhibitory sigmoid-E_max effect model was fitted to the data (blue line).

FIG 5

Antibacterial activity of linezolid on M. tuberculosis in the HFIM system. The antibacterial effect of linezolid on the total population of M. tuberculosis (A) and the linezolid-resistant M. tuberculosis subpopulation (B) was determined. Hollow-fiber experiments were performed as two independent studies, and the data are the combined results of both experiments. The data points represent the mean values from both experiments, and the error bars correspond to the standard error of the mean.

FIG 6

Influence of linezolid exposure on the amplification of linezolid-resistant bacterial mutants after 3 weeks of therapy. Hollow-fiber studies were performed twice as two independent experiments. The data points represent the geometric means from the two experiments for each linezolid exposure.