In this series of projects, the value of MSI in the evaluation of new and promising anti-TB agents, have been demonstrated. Not only does this bode well for anti-mycobacterial agents this technique can also be applied to the evaluation of other anti-bacterial agents, especially in terms of tissue distribution and localization. This has great potential for streamlining the drug development process by either identifying or eliminating drug candidates during the early stages. This is possible due to some distinct advantages MSI has over other more commonly used molecular imaging modalities, such as PET and MRI. MSI can be seen as the future of molecular imaging and diagnostics, allowing for the high-throughput screening of a large sample size within a short period of time, requiring minimal sample preparation. In addition, this technique allows for the simultaneous identification of drugs, drug metabolites, bio- markers, receptors etc., whereas other modalities are only able to detect one target molecule per an instance. Even with this versatility, the specificity of MSI is unquestioned since the molecular mass and signature of a compound is used to identify targets. The sum of all the MSI experiments carried out during this period has yielded interesting results in that no two anti- bacterial agents have displayed the same CNS distribution. This proves that brain drug localization is highly preferential and is affected by the chemical properties of the drug and the physical properties of the BBB. Using these findings, we were able to show how MSI can be highly beneficial in the pre-clinical evaluation of anti-bacterial agents, specifically targeting extra-pulmonary-TB of the brain.
In Chapter 1, we are the first to provide evidence that CFZ does in fact enter the brain, using MSI. This was in vast contrast to the popular belief that the drug does not penetrate the BBB.
CFZ showed widespread distribution in the brain, with high concentrations in the lateral ventricles. This is especially significant since the lateral ventricles house the choroid plexus and is responsible for the production and circulation of CSF throughout the brain. In Chapter 2, the same experimental approach was used to demonstrate the neuroprotective potential of LIN. MSI findings showed that the drug showed the greatest deposition in the brain stem, which forms the entry point of systemic blood vessels. This is important since the CNS is not the primary site of bacterial infections, which normally occur systemically and are haematogenously spread to the brain. By forming a “sentinel” in the brain stem, LIN has the potential to prevent the entry of infectious agents into the CNS.
In order for one to effectively evaluate TB drugs, the lung distribution of potential anti- mycobacterials needed to be understood. However, we noted the difficulties associated with the preparation of lung tissue for MSI. This led to the conceptualization of Chapter 4. Herein lung inflation using cryoprotectants was used to preserve lung structure during the preparation stages, in order to provide more precise distribution data. Various established cryoprotectants, including commercially available agents were tested as potential inflation media. During the various stages in the preparation, challenges were encountered with the different agents, eventually it was found that a 10% DMSO solution was the most ideal cryoprotective agent, which preserves the structural integrity of the lung and provides accurate drug distribution data.
In Chapters 5, 6, 7 and 8, we have demonstrated how LC-MS and MSI can be used for the evaluation of a wide range of antibiotics focusing on their applicability for CNS investigations centered around drug distribution in the brain. The findings revealed very significant results in that each antibiotic displayed some unique patterns of distribution and localization. This highlights how the chemical properties of a drug strongly influences its interaction with the BBB and subsequently influences its distribution to the various structures in the brain. These chapters also demonstrated the versatility and many applications of MSI, especially in terms of understanding drug behavior in the CNS. The results help us understand why the tetracyclines have been so effective in the treatment of brain disorders, this can now be attributed to their widespread tissue distribution in CNS. MSI was also used to prove that RIF, a typically large drug molecule, is able to permeate the brain in a time-dependent manner. This despite the drug failing many of the rules that predict drug entry into the CNS. This technique can also be used to understand the neurotoxicity of certain classes of drugs such as the flouroquinolones and in this case, GAT. This technique was also used, with great effectiveness, in the evaluation of pretomanid, a drug currently under clinical trials and similarly be applied to the evaluation of other such antibiotics. These finding further emphasize how MSI can be used to streamline the drug development process and help in the selection and identification of promising candidates while eliminating less promising candidates.
These series of experiments will pave the way for future research that will further cement the role of MSI as an invaluable tool in the drug development process. Studies to be carried out will include the investigation of the distribution of the anti-mycobacterial agents in the brain of TB-infected animals (such as mice and rabbits). This will also allow us to optimize our
167 thereby providing a new sample preparation protocol to be conducted in diagnostic laboratories.
For future experiments, it will also be highly beneficial to study the effects of multiple doses on the distribution of PA-824, DOX, TIG, RIF and GAT, since antibiotics are not prescribed as single doses but rather as multiple dose treatment regimens.
With all of the recent technological advancements in MSI applications, it is only natural that MSI will become an important technique in drug discovery, especially since it can be applied for the analysis of a wide range of tissue samples. MSI has the potential to be used in all aspects of drug metabolism pharmacokinetics (DMPK) and absorption, distribution, metabolism and excretion (ADME), especially due to its ability to measure a wide range of analytes (ranging from small drug molecules and their metabolites, to large endogenous proteins and lipids).
A current trend in MSI experiments reveal that this technique can provide more detailed information on drug distribution at a cellular level, which would include MI of intracellular targets and drug interaction with these targets. However, in order to achieve this, higher resolution images of regions showing high levels of drug localization, will be acquired. Since this instrumentation is specialized and currently not accessible in South Africa, the next step of these projects will be carried out in collaboration with world leaders in the field of bio- molecular imaging which is currently ongoing.