Non-invasive detection of pharmacological drugs in the eye

Pharmacological measurements of drug distribution in tissue (such as the eye) are critically important in the development of therapeutic strategies for clinical treatments, e.g., of laser-induced injuries.¹ We hypothesize that a given drug's efficacy correlates with how much it accumulates in the eye tissue. To test this hypothesis, an objective measure of retinal drug concentration is required. Photoacoustic spectroscopy (PAS) has been investigated as a non-invasive technique to ascertain the correct dosage of a therapeutic agent used to treat the eye.² By using this technique, it may be possible to analyze drug penetration in animals or humans without sacrificing tissue or taking samples from patients.

Conventional biochemical measurements of tissue drug concentrations are based on the analysis of isolated tissue samples. Techniques used include high performance liquid chromatography (HPLC)³ and mass spectrometry.⁴ Although capable of great sensitivity and accuracy, these methods are better suited to acute animal studies rather than repeated measurements on living subjects over a long period, because of the necessary limitation on obtaining physical samples. This is particularly true in ophthalmic studies. These factors may make the eye an especially suitable target organ for the application of PAS, so long as there is clear optical access to the retina and the tissue has relatively low optical background.

PAS⁵ depends on the fact that every material or pharmacological agent has a specific spectral ‘signature’, such that its absorption uniquely varies from one wavelength to another (from ultraviolet through visible and into the infrared). When molecules absorb light, their temperature increases as do their volumes. That produces a propagating pressure wave that can be detected: PAS conversion enables the measurement of low concentrations of drugs in tissue.

In the experiment, the generated photoacoustic signal from retinal pigment epithelium (RPE) was used to measure light transmission through solutions of drugs. An ocular phantom was made of pieces of porcine eye fixed inside a glass cuvette and filled with saline or different concentrations of dyes or drugs. These included Trypan Blue, Rose Bengal, and Amphotericin B (AB), with concentrations in the range of 1–50μg/ml. A q-switched laser at 532nm was used for correlated concentration measurements, while a tunable optical parametric oscillator (OPO) laser system was employed to scan through different light wavelengths to determine their spectra (see Figure 1). Both laser systems produce 5–10ns pulses.

The optical optimization of laser fluence was set below the threshold damage of isolated retina ∼40mJ/cm². A non-contact and sensitive optical method—the photothermal deflection technique (PhDT)⁶—was used to record the photoacoustic signals as a refractive index gradient, the latter produced by a pressure or heat wave when laser radiation was absorbed by the sample. Detection was carried out by monitoring the deflection of a HeNe laser (probe) beam. The optical transmission of the pump laser through test solutions was measured by comparing the photoacoustic signals of saline to different concentrations of pharmacological drugs.

Figure 2 shows the experimental results of measurements of of an Amphotericin B solution as a function of concentration. As predicted theoretically, the results show the linear relation between logT and concentration. As shown in Figure 3, the OPO system and subsequent PAS spectra are in good agreement with the optical absorption spectra, with peaks occurring at the same wavelengths for Amphotericin B.

PAS is feasible in ocular phantoms incorporating ex vivo ocular tissue, and the implementation described here promises a good sensitivity (1–5μg/ml) range for in vivo pharmacokinetics. While not quite as sensitive as current HPLC methods, it is probably adequate for most clinical applications. The PhDT technique was able to record photoacoustic signals with high resolution, but it may be difficult to apply this technique to measurements in vivo. Therefore, different implementations of PAS detection will be investigated. These will include a custom pressure transducer embedded in a contact lens fitted to the eye, or an optical hydrophone: i.e., a transparent polymer film incorporated in the distal end of an optical fiber.

This work was supported through a grant from the Office of Naval Research and an unrestricted grant from Research to Prevent Blindness, Inc., to the Department of Ophthalmology, University of Texas Health Science Center at San Antonio.