Butt, Ummara (2018) Using fatty acids to combat blindness in newborns. (PhD thesis), Kingston University, .
Abstract
The bacterial species 'Neisseria gonorrhoeae' ('N. gonorrhoea') and 'Staphylococcus aureus' ('S. aureus') are amongst the main microorganisms that cause ophthamia neonatorum, an infection of the eye that occurs when infants are born through an infected birth canal. The current treatment that involves the use of various antibiotics such as ciprofloxacin, cephalosporin, ceftriaxone and cefotaxime is becoming more ineffective due to the antibiotic resistance in 'N. gonorrhoea'. Fatty acids (FAs) are used by many organisms as defence mechanism against virulent bacteria. The high safety profile and broad spectrum of activity make them potential alternatives to currently used topical antibiotics for the treatment of eye infections in neonates. In this study, a design of experiment approach (DOE) was used to optimise the quantification of five fatty acids namely; lauric acid (LA), tridecanoic acid (TA), myristoleic acid (MOA), palmitoleic acid (POA), [alpha]-linolenic acid (ALA). a gas chromatographic method (GC-FID) was developed and validated according to ICH guidelines for the identification and quantification of fatty acids and monocaprin. Different approaches were investigated to avoid the fatty acids interaction with the calcium ions (Ca[sup]2+) in simulated tear fluid. One approach was the conversion of fatty acids into the esters forms. The prepared esters were characterised by nuclear magnetic resonance (NMR) spectroscopy and attenuated total reflectance infra-red (ATR) spectroscopy. These ester derivatives were tested against 'N.gonorrhea' using the disc diffusion method. Second approach was the selection of three calcium chelators, including ethylene diamine tetra acetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), dimercaptosuccinic acid (DMSA) to avoid the fatty acids interaction with the calcium ions. The antimicrobial effect of these chelating agents and the influence of these chelating agents ont he antimicrobial activity of fatty acids were investigated by using the disc diffusion method. The preformulation phase including saturation solubility studies were carried out ti select the suitable excipients. Pseudo-ternary phase diagrams were constructed to evaluate microemulsion (ME) regions. The prepared microemulsions were evaluated regarding their particle size, zeta potential, stability, viscosity, pH, self-diffusion NMR, and antimicrobial efficacy. Finally, prepared MEs were tested for ocular toxicity/ irritation potential using the bovine corneal opacity and permeability (BCOP) test and the hen egg chorioallantoic membrane (HET-CAM) test. The results showed that DOE can help to optimise a method to derivatise fatty acids and analyse them by GC-FID. The results of GC method validation showed that the developed GC-FID method is linear (coefficient of variation (R[sup]2) values were higher than 0.99), precise and accurate (highest recovery values and the lowest intra-day and inter-day variation values) for all FAs. Formation of fatty acid methyl esters (FAMEs) and ethyl esters (FAEEs) were confirmed by ATR and NMR amalyses without the formation of any artefacts. Some of the FA esters showed strong antimicrobial effect against 'N. gonorrhoea' but the chelating agents (EDTA, DTPA, and DMSA) showed no antimicrobial activity against 'N. gonorrhoea'. However, these chelators especially EDTA caused a significant increase in the antimicrobial activity of all fatty acids. Nine FA based microemulsions of [alpha]-linoleic acid with tween 80, cremophor EL, transcutol P and PEG 400 were formed using the phase titration method. Polarised light microscopy, pH, droplet size, zeta potential, and self-diffusion NMR were able to successfully characterise the ME formulations. FA based microemulsions were found to have a strong antimicrobial effect against 'N. gonorrhoea' and 'S. aureus'. Moreover, all ME formulations did not show any strong irritant effect. It was concluded that the [alpha]-linolenic acid based microemulsions could be used for potential treatment of opthalmia neonatorum.
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