Mass spectrometric imaging for Tuberculosis drug development.

Abstract

For many years, Tuberculosis (TB) has plagued the human race claiming millions, if not billions, of lives. With the advent of short-course chemotherapy TB has become a manageable disease, however in recent times Mycobacterium tuberculosis has developed resistance to a number of established and trusted antibiotics. This coupled with severe forms of extra-pulmonary TB, has placed significant emphasis on the development of new anti-TB agents. The drug development process is a long and costly affair, with less than 1% of new drugs reaching clinical trials. This is where molecular imaging, in particular mass spectrometry imaging (MSI), is fast becoming a promising tool in the evaluation of drug candidates. MSI can be used to streamline the drug development process by fast tracking areas of target identification, target quantification, pharmacokinetics, drug distribution and tissue localization. MSI possesses some distinct advantages in terms of sample preparation and the lack of the need for radiolabeling, making it the ideal technique for in vivo tissue drug distribution studies. The objectives of this study were to demonstrate the value of MSI in the development and evaluation of new and existing TB antibiotics, focusing on central nervous system (CNS) manifestations of the disease. In order to achieve these objectives, two of the most promising antimycobacterial agents, clofazimine (CFZ) and linezolid (LIN), were selected. Initially, the distribution of these agents in a healthy animal model was investigated, since these would represent the minimum tissue concentrations achievable. The single-dose study for both drugs were similar, in that there was poor penetration into the brain after a 100mg/kg dose in a healthy murine and rodent model, respectively. A four-week multiple dose study was conducted, each of the antibacterials showed excellent accumulation in the CNS, with preference to specific areas of the brain, demonstrating the neuroprotective potential of these drugs (Chapters 2 and 3). For the effective evaluation of anti-TB drugs, the lung has to be taken into consideration since this is the primary site of M.tb infections. However, the lung poses problems in terms of sample preparation for MSI. Since the lung is responsible for gaseous exchange, it is made up of a number of air-filled spaces that are kept “open” by a fine balance in pressure, inside and outside the lung. When this balance is disturbed, such as when the thoracic cavity is pierced, to collect tissue, the lung collapses. This results in distortion of tissue structure and subsequent distribution information can be misleading. For this reason, we evaluated various established cryoprotectants as lung inflation media. This inflation procedure would main structural integrity of the lung and provide accurate tissue distribution data. From the cryoprotective agents tested in this experiment we found that 10% DMSO was ideal, in terms of structural preservation and accurate drug distribution (Chapter 4). As part of this series of experiments other anti-bacterial agents were also evaluated, to demonstrate the value of MSI in drug development. These drugs also appear in the antibiotic pipeline; tetracyclines, tigecycline (TIG) and doxycycline (DOX), rifampicin (RIF), gatifloxacin (GAT) and pretomanid (PA-824). The findings were very interesting in that each agent displayed a unique pattern of distribution, this is due to the chemical nature of these drugs and their interaction with the blood-brain-barrier (BBB). In addition to this, we have demonstrated how MSI can be used to determine various aspects of drug-tissue interaction for drug development. MSI was used to prove that the chemical properties of a drug do not always govern its movement across the BBB. RIF is a large drug molecule that one would not expect to permeate the brain, however this experiment has demonstrated its time-dependent distribution in the brain (Chapter 5). The results show how the tetracyclines have widespread tissue distribution in the brain, which contributes to their efficacy in the treatment of brain damage (Chapters 6 and 7). This technique was also used to understand how GAT enters the brain and contributes to the proven neurotoxicity of the flouroquinolones (Chapter 8). In the final chapter, we showed how MSI can be used in the tissue evaluation of novel antibiotics, such as pretomanid (Chapter 9). These findings emphasize the need to evaluate the drug distribution of antibiotics, since pathogens manifest themselves in different areas of the brain and cause damage. This information will be invaluable in our pursuit of effective treatments to CNS diseases and disorders, allowing medical practitioners to develop more targeted treatment programmes.