SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1243

Grove et al.

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Figure 4. Relative quantitation of brimonidine signal in ocular tissues. Regions of interest containing the cornea and the iris were
selected, and the intensity of the brimonidine signal in each region was extracted. Each bar graph represents the mean intensity ± SEM.
*p < 0.05 (two sections per eye were analyzed, n = 3 eyes per time point).

Discussion
In drug discovery and development, the distribution of a
drug after dosing is critical to understand efficacy, toxicity,
and metabolism. Tissue homogenate liquid chromatography-mass spectrometry (LC-MS) and autoradiography are
two commonly used techniques to determine this, but each
has its drawbacks. LC-MS analysis of homogenates provides quantitative data for the concentration of a compound
in dissected tissue; however, all spatial information within
the tissue is lost. As an imaging technique, autoradiography
preserves the tissue localization but requires a radiolabel
and so is often used only for select compounds later in the
development process. In addition, because total radioactivity is being detected, one cannot distinguish between parent
drug and metabolites. MALDI IMS has emerged as an ideal
way to determine the distribution of a drug after dosing.
Because no label is required, these types of imaging studies
can be done earlier in the drug discovery process. In addition, the mass spectrometric detection is able to differentiate both parent drug and metabolites. However, while
quantitation is routine for LC-MS and autoradiography
methods, this is more challenging with MALDI IMS, but
progress is being made in this area.5,6,22,23
In this study, we were able to detect unlabeled brimonidine in ocular tissues. The localization determined with
MALDI IMS in our study corresponds well with previous
radiolabel studies carried out by Acheampong et al.18,24 In
these studies, 14C-brimonidine was dosed in rabbit and
monkey eyes, and drug concentrations were determined
after dissecting apart different regions of the eye and measuring total radioactivity. They found that brimonidine was
rapidly absorbed into the eye after topical application,
resulting in high concentrations in the aqueous humor and

the iris-ciliary body, the presumed site of action for lowering intraocular pressure,24 and demonstrated that brimonidine could reach the back of the eye at concentrations
sufficient to activate α2-receptors in retina.18 The drug distribution determined using MALDI IMS in our study lines
up well with regions of the eye where high radioactivity
was measured including the cornea, anterior chamber, iris,
and some in the posterior segment of the eye. Furthermore,
these previous studies noted high levels of brimonidine in
pigmented tissues such as the iris, which was attributed to
drug binding to melanin. Binding of brimonidine to melanin
could act as a depot of drug and prolonged retention of the
drug in the eye. Our study observed accumulation in pigmented tissues such as the iris as well. A drawback of our
current MALDI IMS study is that it does not provide quantitative data for brimonidine in particular eye regions such
as these other works. In addition, the sensitivity is likely
lower as we did not detect any brimonidine metabolites.
Brimonidine has been prescribed because of its mechanism of lowering intraocular pressure. It has been found to
decrease intraocular pressure in two ways: reducing aqueous humor production and increasing aqueous humor outflow, which would presume its site of action is in the
aqueous humor and ciliary body.16,24 We were able to show
brimonidine localized to this area in the same time frame as
its intraocular pressure-lowering effect (~60 min after dosing).25 Other studies have suggested a second mechanism,
in the posterior portion of the eye, by which brimonidine
can treat glaucoma by protecting retinal ganglion cells from
damage. These studies showed that brimonidine could reach
the back of the eye to have this effect.16,18,26 We were also
able to suggest that some brimonidine reached the posterior
region of the eye, which was observed at both time points
studied (Fig. 3). This is in agreement with radiolabel studies

Table of Contents for the Digital Edition of SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017