Microdialysis has been a valuable sampling technique in drug disposition and pharmacokinetics. The main feature of this sampling procedure is that it does not induce any changes in the physiological volume of the sampling compartment. In the eye, it is a tool that enables one to obtain a complete pharmacokinetic profile from a single subject, thus reducing the number of animals involved. However, implantation of the probe can lead to trauma and tissue injury, and this could lead to alteration in drug disposition, especially if the injury is severe or if the animal is not allowed to recover from the trauma. The eye is a specialized organ that provides a very small physiological sampling compartment for pharmacokinetic studies. Therefore, any alterations in the composition of the sampling compartment could lead to a change in drug disposition. Rittenhouse et al.
17 have demonstrated that the microdialysis probe increased the protein content in the aqueous humor by almost 30-fold, which led to alterations in the disposition of propranolol. In our study, we observed a similar, though smaller, effect on the protein content in the vitreous. Increased protein levels could lead to increased drug binding and hence form a depot in the vitreous leading to slower elimination of free drug, assuming the binding process is reversible. As the free drug is eliminated from the vitreous, there may be a shift in the binding equilibrium, and drug molecules bound to protein are released at the moment the vitreous is no longer saturated. The protein levels were elevated approximately three- to fourfold with time in rabbits in which no recovery period was allowed after the surgery. These levels may not affect disposition of compounds that do not exhibit sufficient protein binding. The protein concentration at 8 hours after probe implantation was 2.5-fold higher in comparison with that in the control eye. All experiments were performed well after the completion of 5 days of recovery, and hence the protein concentration would be close to baseline. As a result, protein concentrations were not determined after 5 days. GCV exhibits 1% to 2% plasma protein binding and thus the effect of the increased protein content on its vitreous elimination is not strong. However, if a highly protein bound molecule is the subject of investigation, appropriate recovery time should be allowed to prevent any artifactual alterations in its vitreous disposition.
Anesthesia has a suppressive effect on many physiological processes, including heart rate and respiratory rate. These effects can lead to slower blood flow and fluid exchange. The distribution of a compound in the vitreous is mainly governed by diffusion. However, convective forces also play an important role in this process. Convective forces develop due to the pressure differences between the anterior part of the eye and the retinal surface.
18 A change in the pressure gradient can affect these convective forces, in turn affecting the distribution of a compound. The anesthetic combination used in these studies has been shown to alter the IOP and thus may affect the distribution of the drug. In addition, any tissue uptake processes involved in the distribution of the drug may be slowed, leading to higher concentrations remaining in the vitreous chamber. These physiologic changes are probably the reasons why anesthesia leads to higher steady state levels. In addition, the intravitreous injection was made through the sclera and not through an extraocular muscle. The 50-μL injection may have transiently increased the IOP, leading to reflux of some of the injected drug through the needle hole in the sclera. This may have contributed in part to the lower AUCs obtained in the conscious group of animals. In our studies, anesthesia increased the exposure levels but did not have a noticeable effect on the elimination rate with the vitreous
t 1/2 comparable to that in the conscious group II. The vitreous
t 1/2 of GCV was short (3–6 hours), indicating a major transretinal route of elimination in addition to any contribution from the aqueous outflow pathways. Mannitol, a paracellular marker, exhibits an intravitreous
t 1/2 of 4 hours (Atluri and Mitra, unpublished results, 2001). The intravitreous
t 1/2 for GCV in group I animals was 6 to 7 hours, which is longer than that obtained for mannitol. This result indicates that GCV, like mannitol, may follow a similar passive paracellular route of elimination. However, the possibility of a facilitative mechanism contributing to the rapid elimination of GCV cannot be ruled out and is an area that needs further investigation.
GCV has been previously studied in the eye.
19 The reported intravitreous
t 1/2 was 7 hours for a 196-μg dose. In addition, Macha and Mitra
20 have reported the GCV
t 1/2 to be 7 hours, by using ocular microdialysis. The intravitreous
t 1/2 in the animal group that was kept anesthetized and not given a sufficient recovery period after probe implantation (group I) was determined to be 6 hours (±1.5 hours), which is similar to values reported in the literature. However, an important point to be noted is that the animal models in the case of Lopez et al.
19 were pigmented. It is well known that ocular pigmentation can affect the elimination of a drug from the vitreous. The
t 1/2 of GCV obtained in groups that were given a sufficient recovery period (groups II and III) is in the range of 4 hours, which is slightly lower. The rabbits used in the current study were albino rabbits, and hence the difference in the intravitreous
t 1/2 compared with that obtained by Lopez et al. could be attributed to the effect of ocular pigmentation. Determinations in the model without a sufficient recovery period (group I) generated a
t 1/2 for GCV which is almost equal to the one obtained by Lopez et al. The release of small amounts of protein somehow makes up for the absence of ocular pigmentation in this model. Anesthesia is not expected to alter drug diffusion across the retinal barrier, which may signal a similar rate of elimination.
The studies presented in this article indicate that the choice of the pharmacokinetic model for studying disposition in the eye has to be made carefully. This decision should be based on several factors, such as the purpose of the study, the molecule under investigation, and the various possible interactions between the analyte and ocular tissues and fluids. For example, if the study is a comparative study between two compounds, the anesthetized model may be suitable for short-term studies to generate important pharmacokinetic parameters provided that the protein-binding characteristics of the two compounds are similar. In general, if there are no time constraints, the conscious model presented herein would be most suitable for preclinical research because of the absence of variables such as anesthesia and elevated protein levels. Parameters obtained from this model would provide true pharmacokinetic and disposition data necessary for preclinical studies involving ophthalmic drugs.