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

ABSTRACT BOOK 50th World Conference on Lung Health of the International Union Against Tuberculosis and Lung Disease (The Union)

The International Journal of Tuberculosis and Lung Disease is an official journal of The Union. The Journal’s main aim is the continuing education of physicians and other health personnel, and the dissemination of the most up-to-date infor mation in the field of tuberculosis and lung health. It publishes original articles and commissioned reviews not only on the clinical and biological and epidemiological aspects, but also—and more importantly—on community aspects: fundamental research and the elaboration, implementation and assessment of field projects and action programmes for tuberculosis control and the promo tion of lung health. The Journal welcomes articles submitted on all aspects of lung health, including public health-related issues such as training programmes, cost-benefit analysis, legislation, epidemiology, intervention studies and health systems research

Pharmaceutical Particulates and Membranes for Delivery of Drugs and Bioactive Molecules

This book is a collection of papers published in the Special Issue of Pharmaceutics, entitled "Pharmaceutical Particulates and Membranes for Delivery of Drugs and Bioactive Molecules". A drug release profile is a consequential factor for nanoparticle application, directly related to drug stability and therapeutic results, as well as formulation development. Pharmaceutical particulates of different sizes and shapes (e.g., liposomes, oil-in-water emulsions, polymeric nano- and microspheres, metallic nanoparticles (NPs) such as gold, silver and iron oxide crystals, and core-shell hybrid NPs) offer many diagnostic and therapeutic applications. Membranes are also extensively utilized in many applications. They are especially beneficial to the distribution of macromolecular drugs and biopharmaceutical drugs (peptides, proteins, antibodies, oligonucleotides, plasmids, and viruses) with physicochemical and pharmacokinetic vulnerability. The delivery of drugs and bioactive molecules using particulates and membranes has gained a great deal of attention for various applications, such as the treatment of secondary infections, cancer treatment, skin regeneration, orthopaedic applications, and antimicrobial drug delivery. In addition, several production techniques have been utilized for the fabrication of particulates and membranes in the last decade, which include lyophilisation, micro-emulsion, nano-spray dryer, nano-electrospinning, slip casting and 3D printers. Therefore, pharmaceutical particulates and membranes possess excellent prospects to deliver drugs and bioactive molecules with the potential to improve new delivery strategies like sustained and controlled release

Evaluation of PD-L1 expression in various formalin-fixed paraffin embedded tumour tissue samples using SP263, SP142 and QR1 antibody clones

Background & objectives: Cancer cells can avoid immune destruction through the inhibitory ligand PD-L1. PD-1 is a surface cell receptor, part of the immunoglobulin family. Its ligand PD-L1 is expressed by tumour cells and stromal tumour infltrating lymphocytes (TIL). Methods: Forty-four cancer cases were included in this study (24 triple-negative breast cancers (TNBC), 10 non-small cell lung cancer (NSCLC) and 10 malignant melanoma cases). Three clones of monoclonal primary antibodies were compared: QR1 (Quartett), SP 142 and SP263 (Ventana). For visualization, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform for immunohistochemical staining Ventana BenchMark GX. Results: Comparing the sensitivity of two different clones on same tissue samples from TNBC, we found that the QR1 clone gave higher percentage of positive cells than clone SP142, but there was no statistically significant difference. Comparing the sensitivity of two different clones on same tissue samples from malignant melanoma, the SP263 clone gave higher percentage of positive cells than the QR1 clone, but again the difference was not statistically significant. Comparing the sensitivity of two different clones on same tissue samples from NSCLC, we found higher percentage of positive cells using the QR1 clone in comparison with the SP142 clone, but once again, the difference was not statistically significant. Conclusion: The three different antibody clones from two manufacturers Ventana and Quartett, gave comparable results with no statistically significant difference in staining intensity/ percentage of positive tumour and/or immune cells. Therefore, different PD-L1 clones from different manufacturers can potentially be used to evaluate the PD- L1 status in different tumour tissues. Due to the serious implications of the PD-L1 analysis in further treatment decisions for cancer patients, every antibody clone, staining protocol and evaluation process should be carefully and meticulously validated

Fleas of fleas: The potential role of bacteriophages in Salmonella diversity and pathogenicity.

Non-typhoidal salmonellosis is an important foodborne and zoonotic infection, that causes significant global public health concern. Diverse serovars are multidrug-resistant and encode several virulence indicators, however, little is known on the role prophages play in driving these characteristics. Here, we extracted prophages from 75 Salmonella genomes, which represent the 15 most important serovars in the United Kingdom. We analysed the genomes of the intact prophages for the presence of virulence factors which were associated with; diversity, evolution and pathogenicity of Salmonella and to establish their genomic relationships. We identified 615 prophage elements from the Salmonella genomes, from which 195 prophages are intact, 332 being incomplete while 88 are questionable. The average prophage carriage was found to be more prevalent in S. Heidelberg, S. Inverness and S. Newport (10.2-11.6 prophages/strain), compared to S. Infantis, S. Stanley, S. Typhimurium and S. Virchow (8.2-9 prophages/strain) and S. Agona, S. Braenderup, S. Bovismorbificans, S. Choleraesuis, S. Dublin, and S. Java (6-7.8 prophages/strain), and S. Javiana and S. Enteritidis (5.8 prophages/strain). Cumulatively, 2760 virulence factors were detected from the intact prophages and associated with cellular functionality being linked to effector delivery/secretion system (73%), adherence (22%), magnesium uptake (2.7%), resistance to antimicrobial peptides (0.94%), stress/survival (0.4%), exotoxins (0.32%) and antivirulence (0.18%). Close and distant clusters were formed among the prophage genomes suggesting different lineages and associations with bacteriophages of other Enterobacteriaceae. We show that diverse repertoire of Salmonella prophages are associated with numerous virulence factors, and may contribute to diversity, pathogenicity and success of specific serovars