Pharmacokinetic-Pharmacodynamic modelling of intracellular Mycobacterium tuberculosis growth and kill rates is predictive of clinical treatment duration

Abstract

Tuberculosis (TB) treatment is long and complex, typically involving a combination of drugs taken for 6 months. Improved drug regimens to shorten and simplify treatment are urgently required, however a major challenge to TB drug development is the lack of predictive pre-clinical tools. To address this deficiency, we have adopted a new high-content imaging-based approach capable of defining the killing kinetics of first line anti-TB drugs against intracellular Mycobacterium tuberculosis (Mtb) residing inside macrophages. Through use of this pharmacokinetic-pharmacodynamic (PK-PD) approach we demonstrate that the killing dynamics of the intracellular Mtb sub-population is critical to predicting clinical TB treatment duration. Integrated modelling of intracellular Mtb killing alongside conventional extracellular Mtb killing data, generates the biphasic responses typical of those described clinically. Our model supports the hypothesis that the use of higher doses of rifampicin (35 mg/kg) will significantly reduce treatment duration. Our described PK-PD approach offers a much needed decision making tool for the identification and prioritisation of new therapies which have the potential to reduce TB treatment duration.

Figure 1

Automated work flow of image analysis in Harmony. THP-1 macrophages infected with GFP-H37Rv were imaged on the Operetta platform described in the Methods section. The images were subsequently analysed with Harmony software in the following work flow (a) Image capture from one field of view displaying brightfield (grey), Hoechst (blue), and GFP-H37Rv (green). (b) Image without brightfield, displaying Hoechst (blue) and GFP-H37Rv (green). (c) Identification of extracellular GFP-H37Rv, detected M. tuberculosis are highlighted in red, the nucleus of the macrophages is in grey. (d) Identification of the nucleus of macrophages, detected nuclei (pink). (e) Cytoplasm detection, cytoplasm in blue and yellow. (f) M. tuberculosis infected macrophage (outline in orange), intracellular M. tuberculosis highlighted in red.

Figure 2

Intracellular (macrophage) M. tuberculosis time-dependent kill. Panels display time-kill profiles of RIF (a), ETB (b), INH (c), and PZA (d). Data is mean ± S.D derived from multiple independent experiments (n ≥ 3) performed in triplicate.

Figure 3

Intracellular (macrophage) and extracellular M. tuberculosis time-kill relationships. (a) Simulated concentration–kill rate relationship based on parameters generated from analysing intracellular Mtb kill curves for RIF (black circles), ETB (black triangles), INH (black diamonds) and PZA (black squares). (b) shows the concentration–kill relationship for RIF intracellular (black circles) and extracellular (open circles). (c) show the concentration–kill relationship of ETB intracellularly (black triangles) and extracellularly (open triangles) and (d) for the concentration–kill relationship of INH intracellularly (black diamonds) and extracellularly (open diamonds).

Figure 4

PK-PD Monte-Carlo simulations of clinical Mtb response based on concentration-kill rate dynamics derived from in vitro models of intracellular and extracellular Mtb. (a) Predicted dynamics of total TB in patients receiving HRZE combination for 6 months based on extracellular kill rates only. (b) Predicted dynamics of total TB in patients receiving HRZE combination for 6 months based on intracellular kill rates only. (c) Predicted dynamics of total TB in patients receiving HRZE combination for 6 months based on the assumption that extracellular TB constitutes 95% and intracellular 5% of total TB (1-month view). (d) Predicted dynamics of total TB in patients receiving HRZE combination for 6 months based on the assumption that extracellular TB constitutes 95% and intracellular 5% of total TB (6-month view). (e) Probability of sputum sample conversion to Mtb culture-positive status over time as observed in TB patients receiving standard HRZE treatment in a previous clinical study (solid black line) compared to the predicted probabilities over time using our novel PK-PD model (dashed red line). Dashed green line shows our PK-PD prediction when using a standard HRZE regimen but with an elevated dose of RIF (35 mg/kg). The comparison assumes that the limit of detection for positive culture conversion is 10 CFU/mL when using Löwenstein–Jensen medium culture assays which have been implemented in the comparator clinical study. (f) Predicted dynamics of total TB in patients receiving HRZE with a high dose of RIF (35 mg/kg) for 3 months on the assumption that extracellular TB constitutes 95% and intracellular 5% of total TB (6-month view).