Permeability Of The Conjunctiva And The Sclera

The recognition that noncorneal penetration may be a productive route of intraocular entry for some drugs has triggered extensive research to understand the barrier properties of the conjunctiva and the sclera and the physicochemical determinants of drug permeability across these membranes. In vitro and in situ permeability studies have proven to be a useful approach in estimating drug transport for in vivo conditions. In general, permeability characteristics of ocular membranes correlate well with the intraocular absorption of drugs.

A. Methodology

Four methods have been commonly used for measuring permeability across ocular membranes. The first method is based on the measurement of steady-state permeability of drugs across the isolated ocular membrane using a side-by-side diffusion cell or a modified two-chamber Ussing apparatus.

Accordingly, the membrane permeability coefficients may be calculated using the following equation:

where AC/At is the change in the concentration with time represented by the slope of the linear part of the amount of drug accumulated versus time curve, VR is the volume of the receiver chamber, A is the surface area of the mounted membrane, and CD is the initial concentration of the drug in the donor chamber. This method assumes sink conditions. As pointed out by Maurice (32) in his critique of such techniques, it is important to ensure that the in vivo characteristics of the membranes are maintained as closely as possible. This requires care during the dissection to avoid folding or damage to the surfaces of the membranes and preservation of the tissue during the course of the experiment using physiologically relevant buffer systems (e.g., oxygenated glutathione bicarbonate Ringers solution) to maintain tissue viability. It is also important to measure, confirm, and report the integrity of the barrier based on electrical properties, hydration level, and diffusion of marker substances. Another alert is interspecies differences and caution in overinterpreting data obtained from animals to humans.

The second method is in situ perfusion technique that enables quantitation of drug uptake in live animals. This method was originally utilized for measuring corneal permeation and uptake of drugs (110,111) and subsequently applied to study the conjunctival and scleral uptake of various compounds (10,11,16). The technique involves affixing a cylindrical well on the surface of the eye near the corneoscleral junction using surgical adhesives (Fig. 4). A drug solution is then placed either inside the well bathing the cornea or outside the well bathing the remainder of the con-junctival sac. The rate of mass flux in the system can be described by:

dCsVs/dt = Cls.Cs where Cs is the concentration of drug in the systeml, Vs is the volume of fluid in the reservoir and Cls is a clearance parameter describing the loss of drug from the system. This method can be utilized to study the effect of physical-chemical drug properties while avoiding some of the problems associated with in vitro studies on isolated membranes as previously described. The primary disadvantage is the difficulty in extracting a true permeability coefficient or mechanistic information from such experiments.

A third is a flow-through permeation chamber where the excised tissue is mounted horizontally method (43,46,112). A small volume of the drug solution is applied on the external surface of the membrane and the internal surface is perfused with a physiological receptor solution. In this method the flux across the membrane can be calculated using the following equation:

Figure 4 In situ corneal blocking technique.

where V is the volume of the receiver compartment, C is the concentration in the receiver compartment, S is the exposed surface area, J is the flux at the membrane surface on the receptor side, Q is the flow rate out of the receptor compartment, and t is the time. This method is appropriate for the measurement and prediction of transient transport across the ocular membrane simulating more realistic ocular drug delivery scenarios. For example, it has been shown that the predictions of transscleral transport based on steady-state measurements may significantly overpredict the amount of drug delivered into the eye because the lag time for transport across the sclera is similar or longer than the drug-sclera contact time from an eye drop (43). Drug binding to the sclera may also prolong the lag time. A disadvantage of non-steady-state experiments is that the data treatment and mathematics is complicated.

The fourth method is the use of cell culture and physical models. During the past decade advances in cell culture techniques have resulted in the development of a primary culture model of rabbit conjunctival epithe lial cells exhibiting tight barrier properties (112-115). There has been an attempt to develop physical models to describe the transport of molecules through the cornea and the sclera by taking into account the ultrastructure of these tissues (44). The use of intestinal tissues to predict ocular permeability and ex vivo models based on isolated perfused tissue has also been reported (118). These techniques may offer an alternative to the use of animals in research and an opportunity for gaining a greater mechanistic understanding of transport processes. However, the practical application and scope of these methods remain to be determined.

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