Thi Minh Nguyet Nguyen 1, Jordana Galizia 2, Dominique Ndjogou 3, Alfonso Mendoza-Losana 4, Stewart T Cole 5,6, Santiago Ramón-García 2,7, Nicolas Willand 3, Ilse Dubbelboer 1, Ulrika Simonsson 1
1 Department of Pharmaceutical Biosciences, Uppsala University (Uppsala, Sweden ), 2 Department of Microbiology, University of Zaragoza (Zaragoza, Spain ), 3 Univ. Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for Living Systems (Lille, France), 4 Department of Bioengineering, Universidad Carlos III de Madrid (Madrid, Spain), 5 Innovative Medicines for Tuberculosis (iM4TB) (Lausanne, Switzerland ), 6 Institut Pasteur (Paris, France), 7 Research & Development Agency of Aragón (ARAID) Foundation (Zaragoza , Spain )
Introduction/Objectives: Mycobacterium tuberculosis undergoes metabolic and physiological adaptations to survive in the diverse microenvironments of granulomas and cavities within the immune system in the human body. These adaptations result in varying susceptibility to antibiotic therapies, leading to prolonged treatment durations or the need for multi-drug therapy.1 Understanding the heterogeneity of bacterial growth and its impact on antibiotic response is therefore crucial. This study aimed to evaluate bacterial growth, the exposure-response relationships, and early clinical efficacy predictions of PBTZ169 through time-kill experiments conducted in vitro across different broth media.
Methods: Efficacy of PBTZ169 was evaluated in time-kill assay (TKA) experiments against log-phase cultures of Mycobacterium tuberculosis H37Rv, which exhibited a minimum inhibitory concentration of 1.2 ng/mL. Testing concentrations for PBTZ169 ranged from 0.6 to 96 ng/mL. Two different broth media were evaluated: 1) 7H9 medium supplemented with 0.5% glycerol and 10% OADC, and 2) 7H9 medium supplemented with 0.085% NaCl, 0.5% BSA, 0.004% cholesterol and 0.05% tyloxapol. Samples for bacterial counts were collected on days 1, 4, 7, 11, 14, and 21 post-inoculation, and colony-forming units (CFUs) as well as the most probable number (MPN) were assessed by plating serial dilutions onto Middlebrook 7H10 agar plates followed by manual counting. Each experiment was performed in triplicate The lower limits of quantification were 400 CFU/mL for CFU and 200 MPN/mL for MPN. Drug quantification in the media was performed using LC-MS/MS, with a LOQ of 0.14 ng/mL.
The modeling workflow involved two main steps. Initially, exposure-response relationships for PBTZ169 were established using multistate tuberculosis pharmacometrics model, informed by TKA experiments in glycerol and cholesterol-supplemented media.2 In the model, drug concentrations served as the drivers of effect. Lastly, the early bactericidal activities (EBA) of PBTZ169 based on the final exposure-response models were predicted, incorporating translational factors including human population pharmacokinetic model (based on phase 1 study data), lung distribution model based on a PK study in rats, and variability in mycobacterial susceptibility in humans to account for the differences between in vitro and humans.3 The in vitro data were modeled in NONMEM 7.5.1, and EBA predictions were simulated using the “deSolve” (version 1.4) package in R (version 4.2.3).
Results: A higher rate constant of bacterial growth was observed in cholesterol (0.346 days⁻¹) than in glycerol (0.199 days⁻¹). However, the maximal bacterial load was higher in glycerol (71.5 x 10⁶ mL⁻¹) than in cholesterol (13.2 x 10⁶ mL⁻¹). While there was a clear difference in bacterial growth between the two media, the drug effects of PBTZ169 were similar. The drug effects on fast-multiplying bacteria were described by a linear function for growth inhibition and an on/off function for the killing. Drug killing effect on slow-multiplying subpopulation was described by a linear function. The killing of the slow-multiplying subpopulation by PBTZ169 was higher in cholesterol than in glycerol. Other drug effect parameters were independent of growth medium. Consequently, the early efficacy prediction of PBTZ169 was higher in cholesterol than in glycerol. With a higher dose of PBTZ169, the predicted early efficacy increased most significantly in EBA0-2 days in cholesterol.
Conclusions: The bacterial growth and antibacterial effects of PBTZ169 were different between glycerol and cholesterol, highlighting the importance of adequate choice of the media being used in vitro experiments.
Acknowledgement: This work has received support from the Innovative Medicines Initiatives 2 Joint Undertaking (grant No 853989).
References:
1. Lenaerts A, Barry CE, Dartois V. Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses. Immunol Rev 2015; 264: 288–307.
2. Clewe O, Aulin L, Hu Y, Coates ARM, Simonsson USH. A multistate tuberculosis pharmacometric model: A framework for studying anti-tubercular drug effects in vitro. J Antimicrob Chemother 2016; 71: 964–74.
3. Wicha SG, Clewe O, Svensson RJ, et al. Forecasting Clinical Dose–Response From Preclinical Studies in Tuberculosis Research: Translational Predictions With Rifampicin. Clin Pharmacol Ther 2018; 104: 1208–18.
Reference: PAGE 34 (2026) Abstr 12007 [www.page-meeting.org/?abstract=12007]
Poster: Drug/Disease Modelling - Infection