Tolerability of Mānuka Honey with CycloPower™ in Microemulsion on the ocular surface: Evaluation through gene quantification studies of MMP-9, IL-6 and MUC5AC

Abstract

Blepharitis is a chronic inflammatory disease of the eyelids and a common cause of ocular discomfort and irritation. The pathophysiological mechanisms of the disease are complex and the etiology uncertain, however current research suggests bacteria and inflammation are key factors. New Zealand Mānuka Honey has unique antibacterial and anti-inflammatory properties, therefore a lid treatment using Mānuka Honey with CycloPower™ in microemulsion (MHCPME) has been developed at the University of Auckland for the management of blepharitis. This study forms part of a larger investigation aiming to objectively evaluate tolerance of this novel agent on human eyes. RNA expression of three biomarkers of inflammation; MMP-9, IL-6 and MUC5AC, was evaluated using ocular surface tissue samples collected by impression cytology before and after a 2-week unilateral treatment period from 23 healthy adults. Preliminary studies were conducted to refine the methodology. Two impression cytology devices used for sampling at the ocular surface were deemed comparable in their RNA yield, but the EYEPRIM™ design assisted cell collection. The use of topical anesthetic did not appear to inhibit PCR applications. The PureLink® RNA Mini Kit yielded sufficient RNA with greater efficiency compared to the traditional Trizol method of RNA extraction and purification. A 5’-hydrolysis probe assay was superior to an intercalating dye assay for quantification of the genes due to improved specificity from a dual labeled probe. For the MHCPME tolerability trial conjunctival tissue samples were harvested using the EYEPRIM™ device with topical anesthetic and processed using the PureLink® RNA Mini Kit. Samples were tested for the presence of inhibitors before undergoing cDNA synthesis which was confirmed using a standard Beta-actin PCR. The Normfinder algorithm determined the most stable of six reference genes amongst the sample population (Beta-actin and B2M). The genes of interest were quantified relatively against the expression of these reference genes. MIQE guidelines were followed to ensure validity of the qPCR experiments in this prospective, randomized, investigator-masked, controlled paired eye trial. No significant difference post eyelid treatment was observed in the genes of interest, (p>0.05 in all cases). These results, together with favourable outcomes from the clinical component of the tolerability trial, indicated that the treatment is suitable to progress towards efficacy testing. Key words: Blepharitis, Dry Eye Disease, MGD, Inflammation, bacteria, impression cytology, real-time qPCR, Mānuka Honey, MMP-9, IL-6, MUC5AC