Mass spectrometric imaging for Tuberculosis drug development.

Doctoral Degree. University of KwaZulu-Natal, Westville.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

The analysis of alcohol content in hand sanitisers (in the Durban region) using gas chromatography-mass spectrometry during the COVID-19 pandemic

The COVID-19 pandemic has resulted in an unprecedented surge in the demand for alcohol-based hand sanitisers (ABHS). The Centre for Disease Control  (CDC) and World Health Organisation (WHO) recommend alcohol, i.e., isopropanol or ethanol, at a 60-95% concentration in ABHS for sufficient antiviral  protection. Consumers need to be vigilant of substandard hand sanitisers being marketed to the public. The frequent exposure of microorganisms to  alcohol concentrations below the recommended range for infection prevention may lead to resistant mutations, and above the range may be ineffective.  Therefore, this study aimed to verify the stated alcohol content in hand sanitisers from their respective labels. We analysed 50 hand sanitiser samples  available to our region in Durban, KwaZulu-Natal, South Africa, using a Shimadzu GC-MS-QP2010 Ultra equipped with a Zebron ZB-wax capillary column.  The hand sanitisers analysed had a range of 44–93% alcohol content. The data from our study also revealed that 32% (16) of hand sanitisers did not  adhere to the stated alcohol indicated on their labels. 16% (8) contained >80% and 12% (6) contained <60%, while 6% (3) of the ABHS contained  1-propanol and ethyl acetate as contaminants, respectively. This study clearly emphasises manufacturers’ exploitation of the pandemic and the need for  stricter guidelines and regulations for consistency amongst ABHS manufacturers. The public should also be more alert to the % alcohol stated (ideal    range 60-80%) on the sanitizer bottle and note one needs to rub their hands together until it feels dry

Synthesis, in vitro evaluation, and 68Ga-radiolabeling of CDP1 toward PET/CT imaging of bacterial infection

Bacterial infections are a major concern in the human health sector due to poor diagnosis and development of multidrug-resistant strains. PET/CT provides a means for the non-invasive detection and localization of the infectious foci; however, the radiotracers available are either cumbersome to prepare or their exact contribution toward the imaging is not yet established. Human antimicrobial peptides are of interest for development as PET radiotracers as they are an integral component of the immune system, non-immunogenic toward the recipient, and show selectivity toward pathogens such as bacteria. Herein we report on the potential of LL37, a human cathelicidin antimicrobial peptide, as a radiotracer for bacterial imaging. Bifunctional chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid was utilized to functionalize the antimicrobial peptide, which in turn was capable of chelating gallium. The synthesized natGa-CDP1 showed bacterial selectivity and low affinity toward hepatic cells, which are favorable characteristics for further preclinical application.The Department of Science and Technology, University of KwaZulu Natal, National Research Foundation and Aspen Pharmacare.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1747-0285http://wileyonlinelibrary.com/journal/cbdd2018-10-30hj2018Nuclear Medicin

A comparison of mycotoxin contamination of premium and grocery brands of pelleted cat food in South Africa

Contamination with mycotoxins is of concern to pet owners and veterinary practitioners owing to their ability to cause disease and exacerbate the pathological changes associated with other diseases. Currently, there is a lack of information regarding the mycotoxin content of common premium brand (PB) and grocery brand (GB) cat feeds. Therefore, we undertook to determine the mycobiota content of feed samples, from both categories (n = 6 each), and measured the levels of aflatoxin (AF), fumonisin (FB), ochratoxin A (OTA) and zearalenone (ZEA) by high performance liquid chromatographic analysis. There were high concentrations of mycotoxins in both categories of feed, regardless of the notion that PBs are of a higher quality. The concentration of these toxins may contribute to the development of related pathologies in felines

Advances in spatial mass spectrometry enable in-depth neuropharmacodynamics

Mass spectrometry imaging (MSI) is a powerful technique that combines the abil-ity of microscopy to provide spatial information about multiple molecular species with the specificity of mass spectrometry (MS) for unlabeled mapping of analytes in diverse biological tissues. Initial pharmacological applications focused on drug distributions in different organs, including the compartmentalized brain. However, recent technological advances in instrumentation, software, and chemical tools have allowed its use in quantitative spatial omics. It now enables visualization of distributions of diverse molecules at high lateral resolution in studies of the pharmacokinetic and neuropharmacodynamic effects of drugs on functional biomolecules. Therefore, it has become a versatile technique with a multitude of applications that have transformed neuropharmacological re-search and enabled research into brain physiology at unprecedented resolution, as described in this review

On-Tissue Chemical Derivatization for Comprehensive Mapping of Brain Carboxyl and Aldehyde Metabolites by MALDI-MS Imaging

The visualization of small metabolites by MALDI mass spectrometry imaging in brain tissue sections is challenging due to low detection sensitivity and high background interference. We present an on-tissue chemical derivatization MALDI mass spectrometry imaging approach for the comprehensive mapping of carboxyls and aldehydes in brain tissue sections. In this approach, the AMPP (1-(4-(aminomethyl)phenyl)pyridin-1-ium chloride) derivatization reagent is used for the covalent charge-tagging of molecules containing carboxylic acid (in the presence of peptide coupling reagents) and aldehydes. This includes free fatty acids and the associated metabolites, fatty aldehydes, dipeptides, neurotoxic reactive aldehydes, amino acids, neurotransmitters and associated metabolites, as well as tricarboxylic acid cycle metabolites. We performed sensitive ultrahigh mass resolution MALDI-MS detection and imaging of various carboxyl-and aldehyde containing endogenous metabolites simultaneously in rodent brain tissue sections. We verified the AMPP-derivatized metabolites by tandem MS for structural elucidation. This approach allowed us to image numerous aldehydes and carboxyls, including certain metabolites which had been undetectable in brain tissue sections. We also demonstrated the application of on-tissue derivatization to carboxyls and aldehydes in coronal brain tissue sections of a nonhuman primate Parkinson's disease model. Our methodology provides a powerful tool for the sensitive, simultaneous spatial molecular imaging of numerous aldehydes and carboxylic acids during pathological states, including neurodegeneration, in brain tissue