(T0930-10-68) Investigating the Mechanism of Synergism of Pyrazinamide When Administered in Combination with Spectinamide 1599 in Murine TB Models

Purpose: For treatment of tuberculosis (TB), combination therapy is a promising strategy for enhancing treatment efficacy, shortening treatment duration, and overcoming the problem of drug resistance. Pyrazinamide (PZA) is an indispensable component of first line tuberculosis therapy. In vivo studies of PZA in combination with an investigational agent, spectinamide 1599, in murine models of TB have demonstrated substantial in vivo synergy in antimycobacterial acivity between 1599 and PZA [1, 2] but the mechanism of the synergistic interaction between these two drugs is currently unknown. The aim of the current study is to investigate whether pharmacokinetic interactions at the host organism or macrophage level are the driving mechanism behind the observed synergistic interaction between 1599 and PZA.

Methods: The role of alveolar macrophages in phagocytosis and persistence of Mycobacterium tuberculosis (Mtb) is well documented. As one of the potential mechanisms for the observed synergy, we hypothesized that 1599 and PZA may interact at the macrophage level, whereby 1599 co-administration increases the intracellular concentration of PZA or vice-versa. To address this question, murine alveolar macrophages (MH-S cells) that were procured from ATCC were grown to 5 x 10⁵ cells /mL in each well of a 24-well plate. Uptake studies were performed at different concentrations of 1599 (100 µM to 400 µM) and PZA (200 µM to 2 mM) alone (controls) and in the combination (combo). The selected concentrations were based on the 1599 and PZA concentrations encountered in in vivo studies at therapeutic doses. The cell-to-medium concentration ratio (K_p) was calculated by measuring the intracellular and media concentrations of the drugs using LC-MS/MS, and the results obtained from the 3 independent experiments were statistically analysed. To assess a potental in vivo pharmacokinetic interaction between 1599 and pyrazinamide, a five-day consecutive multiple dose study was conducted in BALB/c mice. Using destructive sampling design with n=3 mice for each time point, 1599 (200 mg/Kg) and PZA (150 mg/Kg) were dosed daily by subcutaneous injection and by oral gavage, respectively, for the control groups (1599 only and PZA only) and combo group (both 1599&PZA). Dose selection was based on the dose levels tested in the previously performed efficacy studies that indicated synergistic effects. At each predetermined timepoint (predose and 0.08, 0.25, 0.5, 1, 3, 8, 24 hr after dosing) on the 5^th study day plasma, lungs, and BALF (bronchioalveolar lavage fluid) samples were collected. The collected samples were processed and assayed for 1599, PZA and its metabolite pyrazinoic acid (POA) concentrations by LC-MS/MS. The urea method was used to derive epithelial lining fluid (ELF) concentrations from concentratiosn measured in BALF. Areas under the concentration-time curves (AUC) in each sampled matrix were calculated and statistically compared using T-test.

Results: The measured Kp values from the uptake experiments perfomed in MH-S cells indicated that the intra cellular accumulation of 1599 and PZA in macrophages was neither increased nor decreased in the presence of the other.The in vivo study indicated that PZA decreased the 1599 plasma AUC ( 391 µg h/mL vs. 323 µg h/mL, 1.3-fold, P< 0.05) and increased the ELF AUC (244 µg h/g vs. 573 µg h/g) as well as the ELF-to-plasma AUC ratio (2.9-fold, P< 0.05). The effect of 1599 on PZA followed a similar but less pronounced pattern as the effect of PZA on 1599, i.e. 1599 modestly decreased the PZA plasma AUC (456.4 µg h/mL vs. 433.3 µg h/mL, 1.05-fold, P< 0.05) and increased the ELF AUC (3641 µg h/g vs. 3968 µg h/g) and ELF-to-plasma AUC ratio (1.14-fold, P< 0.05). Both drugs showed no significant effect on the lung-to-plasma AUC ratios. Similar to the parent compound PZA, the metabolite POA also displayed significantly less plasma exposure (AUC) in the presence of 1599 (120 µg h/mL vs. 94 µg h/mL, 1.3-fold, P< 0.05) like the parent compound PZA, and no significant differences were observed for lung-to-plasma and ELF-to-plasma AUC ratios

Conclusion: The significant enhancement in the exposure of both drugs, 1599 and PZA, in the ELF when they are co-administered is a potential source for the observed synergistic efficacy in Mtb infected mice. The underlying mechanism for the higher exposures in the ELF and lower exposures in the plasma are currently unknown and are further being investigated.

References: 1. Robertson, G.T., et al., Spectinamides are effective partner agents for the treatment of tuberculosis in multiple mouse infection models. J Antimicrob Chemother, 2017. 72(3): p. 770-777.
2. Gonzalez-Juarrero, M., et al., Preclinical Evaluation of Inhalational Spectinamide-1599 Therapy against Tuberculosis. ACS Infect Dis, 2021. 7(10): p. 2850-2863.