Tissue distribution of an anti-TB drug, TBA-354 in rats via mass spectrometric investigations.

The paper demonstrates the potential of tandem liquid chromatography mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) techniques in evaluating the fundamental in vivo pharmacokinetics and tissue distribution properties of a bicyclic nitroimidazole derivative, TBA-354. A validated LC-MS/MS method was used to quantify TBA-354 in rat plasma, lung, and brain homogenate samples. Concentration-time profiles of plasma (ng/mL) and lung homogenate (ng/g) for TBA-354 after a single dose of 20 mg/kg to rats via i.p.

Brain concentration-time profile for TBA-354 after a single dose of 20 mg/kg to the rats via i.p.

1.1. Introduction

MALDI-MSI

MALDI-MSI is one of the emerging techniques that favors the mapping of a wide range of compounds (large non-volatile biomolecules, e.g. peptides, proteins and smaller molecules such as drugs and their metabolites) directly from tissue samples [87] . Research continues to improve MALDI-MSI experiments with regard to image resolution improvement [94, 95], sample preparation optimization [96], and instrument sensitivity [97]. The first step involves uniformly applying the MALDI matrices to the tissue sample.

Spray coating is a technique normally used in MSI to achieve uniform matrix application to the tissue sample [99, 100].

Figure 1.4. The standard workflow of MALDI-MSI experiments [105].

1.2. Aims and objectives of the study

Outline of this thesis

1.4. References

Mugunthan, G., et al., Synthesis and screening of galactose-linked nitroimidazoles and triazoles against Mycobacterium tuberculosis. Shobo, A., et al., MALDI MSI and LC-MS/MS: Towards preclinical determination of the neurotoxic potential of fluoroquinolones. Baijnath, S., et al., Evidence for the presence of clofazimine and its distribution in the healthy mouse brain.

Upton, A.M., et al., In Vitro and In Vivo Activities of the Nitroimidazole TBA-354 against Mycobacterium tuberculosis. Jallapally, A., et al., 2-Butyl-4-chloroimidazole-based substituted piperazine-thiosemicarbazone hybrids as potent inhibitors of Mycobacterium tuberculosis. Ashtekar, D.R., et al., In vitro and in vivo activities of the nitroimidazole CGI 17341 against Mycobacterium tuberculosis.

Manjunatha, U.H., et al., Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis. Carroll, D.I., et al., Subpicogram detection system for gas phase analysis based on atmospheric pressure ionization (API) mass spectrometry. Munyeza, C.F., et al., Rapid and widespread distribution of doxycycline in rat brain: a mass spectrometric imaging study.

Nordhoff, E., et al., Sample preparation protocols for MALDI-MS of peptides and oligonucleotides using prestructured sample supports. Leinweber, B.D., et al., Enhanced MALDI-TOF imaging yields increased protein signals at high molecular mass.

1.1. Highlights

2.2. Abstract

2.3. Introduction

The toxicity of TB drug candidates in clinical development has been one of the biggest obstacles in TB research. The Tuberculosis Society recently announced the withdrawal of TBA-354 from phase 1 clinical trials due to participants showing mild signs of neurotoxicity [15, 16]. In drug development studies, it is important to understand the drug's pharmacokinetics and distribution in various biological systems.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is one of the main analytical techniques used to quantify the total drug concentration in target organs, but it has the disadvantage of not being able to provide spatial information on drug distribution. In assessing the safety of a drug, knowledge of its specific location is an important aspect in drug development, and molecular imaging techniques are commonly used to assess the specific localization of a drug. Well-established techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) are commonly used for molecular imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an emerging technique used for direct analysis of compounds from tissue samples without radioactive labeling [ 19 , 20 ]. Previously, we have successfully used MSI to investigate the distribution of several anti-TB drugs in the brain [21–24], including pretomanid [13]. Therefore, in this study we focused on developing mass spectrometry methods to understand the preclinical pharmacokinetics and spatial distribution of TBA-354 via a validated quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and matrix-assisted laser desorption. /ionization-mass spectrometry imaging (MALDI-MSI), respectively.

Figure 2.1. Chemical structures of TBA-354 (A: target analyte) and pretomanid (B: internal standard)

2.4. Materials and methods

A 100 µg/ml stock solution of TBA-354 and the internal standard (IS) were prepared separately in MeOH and stored at -20 oC where they were found to be stable. For calibration standards, 100 µL blanks of untreated Sprague-Dawley rats were added to working standard solutions of TBA-354 (50 µL) and IS (50 µL). During sample preparation, 100 µL of IS was added to 100 µL of biological sample to obtain 250 ng/mL and vortexed for 1 min, followed by addition of 800 µL of ACN (for lung and brain homogenates) or MeOH (for plasma homogenate). extract target analytes while inducing protein precipitation from biological samples.

Selection of SPE cartridges was based on target analyte recoveries after filtration. Experiments performed to assess the specificity and selectivity of the method involved comparison of chromatograms from six different sets of blank biological samples obtained from six different sources. Each blank biological sample was tested under the LC-MS/MS conditions mentioned above to assess whether there were any interfering peaks in the retention times of TBA-354 and IS.

Calibration curves for quantification of TBA-354 in rat biological samples were constructed by plotting analyte and IS peak area ratios against theoretical analyte concentrations using 1/x2 weighted linear regression. According to EMA guidelines, the mean concentration should be within ± 15% of the nominal concentration value for the QC sample, except for the LLOQ, which should be within ± 20% of the nominal concentration value [25]. The average peak ratio of the analyte for each spiked sample was then compared to the average peak ratio of the analyte standard solution at the same concentration.

The stability of a drug in biological samples depends on the chemical and physical properties of the drug, storage conditions and matrix effect [26]. Rat brain sectioning was done using a Leica Microsystems CM1100 (Wetzlar, Germany) cryostat set at -20 ◦C.

2.5. Results and discussion

The detection of TBA-354 (retention time = 6.5 min) and IS (retention time = 5.9 min) was highly selective and there were no interfering endogenous substances during the retention time. The results confirmed that the method is selective for the analysis of TBA-354 in rat plasma lung and brain homogenates. According to the stability study performed, TBA-354 is stable in any of the conditions investigated as the average concentration at each QC level was within ±15% of the nominal concentration.

The LC-MS/MS method was validated and then applied to the investigation of plasma pharmacokinetics, lung and brain distribution of TBA-354 after i.p. PK and tissue distribution parameters for TBA-354 in plasma, lung and brain homogenates are summarized in Table 2.4. However, after 24.00 hours, traces of TBA-354 were detected, but the concentrations were below the limit of quantification.

TBA-354 was able to enter the central nervous system (CNS) and cross the blood-brain barrier (BBB) ​​and was detected after 0.25 hours in the brain. This study demonstrates the use of MSI to visualize and localize traces of TBA-354 that were able to penetrate the BBB and enter the brain, where it exerts its neurotoxic effects. Images representing 0.50 to 6.00 h showed a gradual increase in the m/z 437.1 ion distributed mostly in the neocortex.

Using MSI to investigate the localization of the drug in rat brain, the parent ion (m/z 437.1) was detected in the sections obtained from 0.50 to 8.00 hours after dosing. From the results, it shows that the imbalance between an anti-TB activity and the toxicity of TBA-354 is unacceptable, which is an unfortunate circumstance that led to the termination of this drug's clinical trials.

Figure 2.2. MRM chromatograms and mass spectrums for TBA-354 (500 ng/mL) and IS (250 ng/mL) in plasma samples

2.6. Conclusions

2.7. Acknowledgements

2.8. Funding

2.9. Transparency declarations

2.10. References

Bratkowska, D., et al., Determination of the antitubercular drug PA-824 in rat plasma, lung, and brain tissue by mass spectrometry coupled to liquid chromatography: Application to a pharmacokinetic study. Chan, K., et al., MALDI mass spectrometry imaging of gangliosides in rat brain using ionic liquid matrix. Shobo, A., et al., Visualization of time-dependent distribution of rifampicin in rat brain using MALDI MSI and quantitative LCMS/MS.

Kour, G., et al., Development and validation of a highly sensitive LC–MS/MS-ESI method for quantification of IIIM-019—A novel nitroimidazole derivative with promising action against tuberculosis: Application to drug development.

3.1. General discussion and conclusions

The study of drug distribution in tissues is important in the development of drugs, the evaluation of their localization at target sites and the understanding of their toxic effects due to accumulation. The technique allows direct estimation of the spatial localization of known and unknown compounds without any prior labeling. MSI can be customized to produce high-quality results while maintaining the spatial resolution of target analytes.

We have realized that the distribution of a drug in the brain tissue section is often non-uniform, therefore the conclusion of the study based on the results obtained from a single brain section is often not sufficient. Quantification of the total concentration of the drug in the brain using LC/MS/MS is necessary to support spatial imaging results. Nevertheless, in a drug distribution study, it is rational to expect the MSI results to be relative to the quantitative LC-MS/MS results, especially when examining the time-dependent distribution of a drug.

To support this claim, MSI results were compared with quantitative LC-MS/MS results for the same time points and showed good intercorrelation. The MSI results we obtained showed that the drug has high BBB permeability and accumulates in the neocortical regions of the brain, which explains the reason why the drug shows certain neurotoxic signs during the clinical trial as reported by the TB alliance [8]. Imaging experiments helped us to understand the localization and accumulation of TBA-354 in different regions of the brain.

In conclusion, this study has proven that LC-MS/MS is an accurate and highly sensitive technique that can be used in preclinical studies for the quantification of TBA-354 in biological samples. This demonstrates that in future drug development studies, LC-MS/MS and MSI techniques can be used complementary for preclinical quantitative and drug distribution studies in the brain, allowing deeper understanding and potentially predicting possible neurotoxic side effects.

3.2. References

Supporting information for chapter 2