A novel fluorophotometer for measuring concentration and diffusion of autofluorescent ophthalmic medication in the human eye

Abstract

As society is aging, the frequency of age-related diseases is increasing. An area of age-related diseases that requires special attention is that of ocular diseases as they eventually cause blindness and therefore have a huge impact on the quality of life of the patient. Understanding the underlying cause of the disease and being able to treat it effectively is thus becoming increasingly important. To evaluate the effectiveness of a treatment, the behaviour of the medication in the body needs to be known or estimated. Monitoring the diffusion of medication is commonly referred to as pharmacokinetics, and the pharmacokinetics of ophthalmic medication are currently assessed by sacrificing animal eyes at given points in time after the application of the drug. This procedure does not allow for a high enough temporal resolution and incorporates a number of disadvantages such as the fact that the eyes are non-human and that the behaviour of the drug has to be extrapolated from the data obtained from different eyes. An instrument that can measure the pharmacokinetics of ophthalmic medication in a non-invasive manner will therefore increase the knowledge about the behaviour of the compound of interest considerably.

This thesis presents a novel ocular fluorophotometer which can measure the diffusion of inherently ophthalmic drugs with high temporal and axial resolution (0.6 s and 124 μm, respectively) and a sensitivity of 2 nM in terms of Fluorescein concentration. The design process of the instrument and its performance in terms of resolution and noise are discussed. Thorough consideration of the eye safety of the instrument is presented. The instrument is calibrated for both a fluorescence standard (Fluorescein sodium salt) and an ophthalmic drug for treating open angle glaucoma (Brimonidine) and it is shown that the concentration of a sample with high signal to noise ratio can be accurately recovered with an error below 20%. It is however found that for samples with less good signal to noise ratios the accuracy decreases significantly. The repeatability of the measurements is also assessed and found to be slightly worse than the accuracy with an error of below 23% within one week. The calibration in in vitro porcine eyes is also discussed and suggestions made for its improvement. Further measurements of diffusing fluorescent compounds are then presented and discussed. It is found that the diffusion can be monitored with high temporal resolution and that the concentration after complete diffusion is measured correctly. Finally, measurements of in vivo eyes of human volunteers are presented and discussed. The instrument is shown to be of a similar standard to a commercially available instrument, and it is believed that the instrument presented here has the potential to become the new gold standard ocular fluorophotometer.