Pharmacokinetics and Efficacy of the Benzothiazinone BTZ-043 against Tuberculous Mycobacteria inside Granulomas in the Guinea Pig Model

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's leading cause of mortality from a single bacterial pathogen. With increasing frequency, emergence of drug-resistant mycobacteria leads to failures of standard TB treatment regimens. Therefore, new anti-TB drugs are urgently required. BTZ-043 belongs to a novel class of nitrobenzothiazinones, which inhibit mycobacterial cell wall formation by covalent binding of an essential cysteine in the catalytic pocket of decaprenylphosphoryl-β-d-ribose oxidase (DprE1). Thus, the compound blocks the formation of decaprenylphosphoryl-β-d-arabinose, a precursor for the synthesis of arabinans. An excellent in vitro efficacy against M. tuberculosis has been demonstrated. Guinea pigs are an important small-animal model to study anti-TB drugs, as they are naturally susceptible to M. tuberculosis and develop human-like granulomas after infection. In the current study, dose-finding experiments were conducted to establish the appropriate oral dose of BTZ-043 for the guinea pig. Subsequently, it could be shown that the active compound was present at high concentrations in Mycobacterium bovis BCG-induced granulomas. To evaluate its therapeutic effect, guinea pigs were subcutaneously infected with virulent M. tuberculosis and treated with BTZ-043 for 4 weeks. BTZ-043-treated guinea pigs had reduced and less necrotic granulomas than vehicle-treated controls. In comparison to the vehicle controls a highly significant reduction of the bacterial burden was observed after BTZ-043 treatment at the site of infection and in the draining lymph node and spleen. Together, these findings indicate that BTZ-043 holds great promise as a new antimycobacterial drug.

Keywords: BTZ-043; MDR-TB; Mycobacterium bovis BCG; Mycobacterium tuberculosis; guinea pig; new antibiotics; treatment.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1

Plasma levels of BTZ-043 and its metabolite after oral BTZ-043 administration to guinea pigs. Four guinea pigs were given an oral dose of BTZ-043 (either 50 mg/kg, 200 mg/kg, or 400 mg/kg (all micronized) or 200 mg/kg (wet milled)). After 15 and 30 min and at 1, 2, 4, 8, and 24 h, blood was obtained from the vena saphena lateralis. The blood was centrifuged and plasma was separated into two samples, which were stabilized with ascorbic acid. After extraction, the samples were measured by mass spectrometry. The plasma levels of BTZ-043 (M0) and its metabolite M1 are shown for each time point as the group mean. Error bars represent the standard errors of the means.

FIG 2

After multidose application of BTZ-043, plasma levels remained elevated after 24 h. (A) Six guinea pigs were given repeated single daily oral doses of 400 mg/kg of BTZ-043 over 7 consecutive days. On the first and last day, blood was obtained from the vena saphena lateralis at 15 and 30 min and at 1, 2, 4, 8, and 24 h after administration. The blood was centrifuged and plasma was separated into two samples, which were stabilized with ascorbic acid. After extraction, the samples were measured by mass spectrometry. The group mean of the plasma levels from BTZ-043 (M0) and its metabolite (M1) for each time point is shown for day 1 (B) and day 7 (C). Error bars represent the standard errors of the means.

FIG 3

High levels of BTZ-043 are reached in BCG-induced granulomas. (A) Six guinea pigs were inoculated subcutaneously with 1 × 106 CFU of BCG Pasteur 1173. After 28 days, treatment with a daily oral dose of 400 mg/kg of BTZ-043 was started and continued for 7 consecutive days. At day 35, guinea pigs were euthanized and BCG-induced granulomas at the injection site were taken for analysis. (B) Parts of granulomas were cryopreserved and levels of BTZ-043 (M0) and its metabolite (M1) were measured via mass spectrometry. Individual levels and the means of M0 and M1 are shown for each of the six animals. Error bars represent the standard errors of the means. (C) For four treated animals, parts of granulomas were homogenized, serially diluted, and plated on 7H11 agar plates. After 3 weeks, CFU were counted. Results from two additional guinea pigs that were inoculated with BCG but not treated (nt) are depicted for comparison.

FIG 4

BTZ-043 treatment significantly reduced M. tuberculosis-induced pathology. (A) Guinea pigs (n = 6, each) were infected subcutaneously with 1 × 103 CFU of virulent M. tuberculosis strain H37Rv. After 14 days, treatment with a daily oral dose of 300 mg/kg of BTZ-043 was started and continued for 28 days. As a control, guinea pigs were treated with either vehicle solution or 60 mg/kg of isoniazid (INH). (B) After 4 weeks, guinea pigs were euthanized and necropsied. Granulomas at the infection site, the draining axillary lymph node, and spleen were macroscopically scored. Bars represent the group medians with ranges, and asterisks represent the level of significance as calculated by nonparametric Mann-Whitney U test (*, P ≤ 0.05; **, P ≤ 0.01).

FIG 5

BTZ-043 treatment significantly reduced M. tuberculosis-induced necrotic lesions. Six weeks after infection and after 4 weeks of treatment, BTZ-043-treated or control animals (vehicle or INH) were euthanized and dissected and organ samples were collected. Formalin-fixed, paraffin-embedded sections of the infection site granuloma (A), the axillary lymph node (B), and the spleen (C) were HE stained, whole-slide images were obtained, and absolute granuloma areas as well as area of caseous necrosis were measured. Representative microphotographs for the respective organ are shown on the left. In the middle, the quantitative analysis of the granulomatous area is shown for the three groups. Graphs on the right depict the quantitative analysis of necrotic lesions. Bars show group means, error bars indicate the standard errors of the means, and asterisks indicate the level of significance as calculated by nonparametric Mann-Whitney U test (*, P ≤ 0.05; **, P ≤ 0.01).

FIG 6

BTZ-043 treatment significantly reduced burden of virulent M. tuberculosis H37Rv. Six weeks after infection with virulent M. tuberculosis H37Rv, BTZ-043-treated or control animals (vehicle or INH) were euthanized and dissected and organ samples were obtained. Tissue samples of injection site granulomas (A), draining axillary lymph nodes (B), and the spleen (C) were homogenized and the number of CFU determined. Dots represent individual values for each animal. Black bars represent the group means and error bars the standard errors of the means. Asterisks show level of significance as calculated by nonparametric Mann-Whitney U test (**, P ≤ 0.01).